CHARACTERIZATION OF TYPE I AND TYPE III COLLAGENS IN HUMAN TISSUES

MICHAELABODE

Department of Clinical Chemistry

OULU 2000

OULUN YLIOPISTO, OULU 2000

CHARACTERIZATION OF TYPE I AND TYPE III COLLAGENS IN HUMAN TISSUES

MICHAELA BODE

Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 9 of the University Hospital of Oulu, on April 7th, 2000, at 12 noon.

Copyright © 2000Oulu University Library, 2000

OULU UNIVERSITY LIBRARYOULU 2000

ALSO AVAILABLE IN PRINTED FORMAT

Manuscript received 16 February 2000Accepted 18 February 2000

Communicated by Docent Kari Majamaa Docent Onni Niemelä

ISBN 951-42-5553-4

ISBN 951-42-5552-6ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

Bode, Michaela, Characterization of type I and type III collagens in human tissues.Department of Clinical Chemistry, University of Oulu, FIN-90014 University of Oulu,Finland2000Oulu, Finland(Manuscript received 14 February 2000)

Abstract

Fibrillar type I and III collagens are the major constituents of the extracellular matrix, providing thetissue with tensile strength and influencing cell attachment and migration. The amount of type IIIcollagen and the extent of its processing and cross-link maturation were studied in humanatherosclerotic plaques, abdominal aortic aneurysms, colon and ovarian cancer, and finally, colondiverticulosis, using a novel radioimmunoassay for the cross-linked aminoterminal telopeptide oftype III collagen. In addition, immunoassays for different structural domains of type I and type IIIcollagens, together with immunohistochemical methods, were applied.

In atherosclerotic plaques, the fully cross-linked type III collagen was the major collagen type.Type III collagen was completely processed, since the amount of type III pN-collagen wasnegligible. The amounts of free type I and III procollagen propeptides in the soluble fraction weresmall, indicating a low rate of collagen turnover. The proportion of type III collagen was lower inabdominal aortic aneurysms than in atherosclerotic aortic control samples. Furthermore, the amountof type III pN-collagen was significantly increased in aneurysms. Type I and III collagens werealso maturely cross-linked in colon diverticulosis, the only difference from normal colon tissuebeing the increased amount of the aminoterminal propeptide of type III procollagen in the solubletissue extract, indicating a slightly increased metabolic activity of type III collagen.

In malignant ovarian tumors, the cross-linking of type I and III collagens was defective. Asimilar trend was also seen for type I collagen in colon cancer. Even though procollagen synthesiswas increased in these malignancies, the total collagen content and the amounts of cross-linkedcollagens were decreased. The amount of type III pN-collagen was increased in malignant ovariantumors, whereas no such tendency was seen in colon cancer.

These findings suggest a wide variety of changes in the metabolism of type I and III collagensin diseases. Defective processing and cross-link maturation of these collagen types might result inimpaired fibril formation or increased susceptibility of collagens to proteolytic attack – both ofthem processes with a potential role in the pathogenesis of diseases.

Key words: aortic aneurysm, atherosclerosis, diverticulosis, neoplasms

Acknowledgements

This study was initiated at the Department of Medical Biochemistry in 1994, andcontinued at the Department of Clinical Chemistry. Professor Taina Pihlajaniemi, Headof the Department of Medical Biochemistry, University of Oulu, and Professor ArtoPakarinen, Chief Physician of the laboratory of the Oulu University Hospital, areacknowledged for the opportunity to work at these facilities.

I am deeply indebted to my supervisor, Professor Juha Risteli, for his guidance duringthese long years and his confidence in my project. I also wish to express my gratitude tomy co-supervisor, Docent Leila Risteli, Director of Learning and Research Services,University of Oulu, whose expert knowledge of collagen and help with the manuscriptshave been of great value.

Docent Onni Niemelä and Docent Kari Majamaa are acknowledged for their thoroughreviewing and valuable comments on the manuscript of this thesis.

I thank my collaborators, Professor Tatu Juvonen, Professor Frej Stenbäck, DocentTuomo Karttunen, Docent Jyrki Mäkelä, Docent Ylermi Soini, Jukka Melkko, M.D.,Ph.D., and Martti Mosorin, M.D., for their contribution to this research. I am verygrateful to my co-author, Jari Satta, M.D., Ph.D., for his never-ending optimism,encouragement and valuable contribution to this research. Special thanks go to SailaKauppila, M.D., Ph.D., from whom I have learned so much and who has always been arole model for me, like a big sister in the scientific world.

Aimo Heinämäki, Ph.D., Päivi Annala, Kristiina Apajalahti, Tiina Holappa, UllaPohjoisaho and Sanna Sääskilahti are acknowledged for their expert technical assistance.I would also like to thank Anne Ollila for secretarial help. The photography laboratoryand the library of the Medical Faculty are greatly appreciated. I thank Sirkka-LiisaLeinonen, Lic. Phil., for the careful revision of the language of this thesis.

I wish to thank the whole staff of our laboratory. I am especially grateful to mycolleague Heidi Eriksen for her friendship and support during these years. Without her,the long and occasionally difficult days at work would have been unbearable.

Finally, I owe my deepest gratitude to my fiancé Joni Mäki, whose love,encouragement and confidence in me have been the thriving forces in my life since theday we first met at the freeze-dryer at the Department of Medical Biochemistry. I am alsoindebted to my mother Kaarina, and my grandparents Laila and Pentti, for their endless

love, support and care during my whole life. I hope that when I have children of my own,I will be able to provide them as good a home as I have had myself.

This work was financially supported by the Oulu University Hospital, SuomenLaboratoriolääketieteen Edistämissäätiö and my mother.

Oulu, February 2000 Michaela Bode

Abbreviations

AAA abdominal aortic aneurysmACP aldol condensation productAOD aortoiliac occlusive diseaseC- carboxy-CI confidence interval (95 %)CNBr cyanogen bromideCol 1 the most aminoterminal globular domain of aminoterminal propeptide

of a procollagenCol 2 carboxyterminal end of aminoterminal propeptideCol 3 collagenous helix in the middle of aminoterminal propeptideCTGF connective tissue growth factorDEAE diethylaminoethyldeH-DHLNL dehydrodihydroxylysinonorleucinedeH-HLNL dehydrohydroxylysinonorleucinedeH-LHNL dehydrolysinohydroxynorleucinedeH-LNL dehydrolysinonorleucineECM extracellular matrixEDS Ehlers-Danlos SyndromeEDTA ethylenediamine tetra-acetateHHL histidinohydroxylysinonorleucineHHMD histidinohydroxymerodesmosineHIS histidineHL-Pyridinoline hydroxylysylpyridinolineHL-Pyrrole hydroxylysylpyrroleHPLC high performance liquid chromatographyHSP47 heat shock protein 47 (a collagen specific chaperone)HYL hydroxylysineHYLALD hydroxylysylaldehydeICTP cross-linked carboxyterminal telopeptide of type I collagenIFN-γ interferon gammaIIICTP carboxyterminal telopeptide of type III collagenIIINTP cross-linked aminoterminal telopeptide of type III collagen

IL-1 interleukin-1LDL low density lipoproteinLO lysyl oxidaseLOL lysyl oxidase-like proteinLOR lysyl oxidase-related proteinL-Pyridinoline lysylpyridinolineLYS lysineLYSALD lysylaldehydeMMP matrix metalloproteinaseMT-MMP membrane type matrix metalloproteinaseN- amino-NTx aminoterminal telopeptide of type I collagenPAGE polyacrylamide gel electrophoresisPBS phosphate-buffered salinePDGF platelet derived growth factorPDI protein disulfide isomerasePICP carboxyterminal propeptide of type I procollagenPIIICP carboxyterminal propeptide of type III procollagenPIIINP aminoterminal propeptide of type III procollagenPINP aminoterminal propeptide of type I procollagenpN-collagen collagen molecule with retained aminoterminal propeptideRER rough endoplasmic reticulumSDS sodium dodecyl sulfateSMC smooth muscle cellSP4 linear synthetic peptide (SAGFDFSFLPQPPQEKY, amino acid

residues nos. 1193-1208 + tyrosine residue) corresponding to thecarboxyterminal telopeptide of the α1-chain of type I collagen

SP6 linear synthetic peptide (DVKSGVAVGGLAGY, amino acid residuesnos. 159-162) corresponding to the aminoterminal telopeptide of theα1-chain of type III collagen

TGF-β transforming growth factor betaTIMP tissue inhibitor of metalloproteinaseTPCK N-tosyl-L-phenylalanine chloromethyl ketone (enzyme inactivator)

List of original articles

This thesis is based on the following articles, which are referred to in the text by theirRoman numerals:

I Bode MK, Mosorin M, Satta J, Risteli L, Juvonen T & Risteli J (1999) Completeprocessing of type III collagen in atherosclerotic plaques. Arterioscler Thromb VascBiol 19: 1506-1511.

II Bode MK, Mosorin M, Satta J, Risteli L, Juvonen T & Risteli J (2000) Increasedamount of type III pN-collagen in abdominal aortic aneurysms. Submitted as arevised version.

III Bode MK, Karttunen TJ, Mäkelä J, Risteli L & Risteli J (2000) Type I and IIIcollagens in human colon cancer and diverticulosis. Scand J Gastroenterol. In press.

IV Kauppila S, Bode MK, Stenbäck F, Risteli L & Risteli J (1999) Cross-linkedtelopeptides of type I and III collagens in malignant ovarian tumours in vivo. Br JCancer 81: 654-661.

In addition to papers I, II, III and IV, some new results on abdominal aortic aneurysmsare included.

Contents

AbstractAcknowledgementsAbbreviationsList of original articlesContents1. Introduction...................................................................................................................132. Review of the literature.................................................................................................14

2.1. Structure and biosynthesis of collagens .................................................................142.2. Fibril-forming collagens ........................................................................................17

2.2.1. Type III collagen ..........................................................................................172.2.1.1. General features and assembly of type III collagen..........................172.2.1.2. Aminoterminal propeptide of type III procollagen, PIIINP .............182.2.1.3. Aminoterminal telopeptide of type III collagen, IIINTP..................202.2.1.4. Type III pN-collagen........................................................................212.2.1.5. Carboxyterminal telopeptide (IIICTP) and

propeptide (PIIICP) of type III collagen ...........................................222.2.1.6. Distribution of type III collagen in tissues .......................................232.2.1.7. Type III collagen mutations .............................................................23

2.2.2. Type I collagen and its metabolites ..............................................................242.2.3. Enzymatic cross-linking of type I and III collagens.....................................25

2.2.3.1. Lysyl oxidase....................................................................................252.2.3.2. Cross-linking pathways ....................................................................26

2.2.4. Degradation of type I and III collagens ........................................................282.3. Type I and III collagens in atherosclerosis and abdominal aortic aneurysms ........30

2.3.1. General features of atherosclerosis...............................................................302.3.2. Type I and III collagens in normal and atherosclerotic aorta .......................302.3.3. Synthesis and degradation of type I and III collagens

in atherosclerotic plaques .............................................................................322.3.4. Other collagen-related events in atherosclerotic plaques .............................332.3.5. Type I and III collagens in abdominal aortic aneurysms (AAA)..................34

2.4. Type I and III collagens in cancer of the ovary and colon .....................................352.5. Type I and III collagens in colon diverticulosis .....................................................36

3. Outlines of the present study.........................................................................................384. Materials and methods ..................................................................................................39

4.1. Tissue samples .......................................................................................................394.2. Purification of the aminoterminal telopeptide of type III collagen ........................404.3. Production of antiserum.........................................................................................404.4. Radioimmunoassay for IIINTP..............................................................................414.5. Other immunoassays for type I and III collagen domains......................................414.6. Immunohistochemical staining ..............................................................................414.7. Chromatographic analyses of type I and III collagen

antigens in tissue samples ......................................................................................424.8. Hydroxyproline measurements ..............................................................................424.9. Investigation of the proteolytic fragments of PIIINP.............................................434.10. Statistics ...............................................................................................................434.11. Ethical considerations ..........................................................................................43

5. Results...........................................................................................................................445.1. Purification of the human cross-linked aminoterminal

telopeptide of type III collagen (IIINTP)...............................................................445.2. New radioimmunoassays for type III collagen.......................................................445.3. Type I and III collagens in atherosclerotic plaques (I)...........................................455.4. Type I and type III collagens in human abdominal aortic aneurysms (II)..............465.5. Type I and III collagens in colon diverticulosis and colon cancer (III) .................485.6. Type I and III collagens in ovarian tumors (IV) ....................................................49

6. Discussion.....................................................................................................................516.1. Insoluble vessel wall collagens – methodological and structural aspects ..............516.2. Type I and III collagens – soil for atherosclerosis .................................................526.3. Abdominal aortic aneurysms and atherosclerosis ..................................................546.4. Type I and III collagens in AAA and diverticular disease of colon .......................566.5. Maturation and processing of type I and III collagens in ovarian and colon cancer........................................................................................57

7. Conclusions...................................................................................................................598. References.....................................................................................................................61

1. Introduction

Insoluble fibers, microfibrils, soluble proteins and glycoproteins are the components ofthe extracellular matrix (ECM). The ECM has an important role not only in providingtissues with their individual mechanical and physiological properties, but also ininfluencing cell attachment and cell migration.

Collagens are the most abundant proteins of the ECM. Each of the collagen typesshows a characteristic tissue distribution and thus has different functions and properties.Type I collagen is the major collagen type present in most tissues. Type III collagen is thesecond most abundant collagen in human tissues and occurs particularly in tissuesexhibiting elastic properties, such as skin, blood vessels and various internal organs.These collagen types are synthesized as large precursor molecules containing propeptidesat both ends. During extracellular processing, these propeptides are cleaved off and thecollagen molecule participates in the intermolecular cross-linking responsible for tensilestrength. Occasionally, the aminoterminal propeptides of type I and III collagens areretained, resulting in pN-collagen, which can only be found on the surface of collagenfibrils.

The present study examines the characteristics of type I and especially type IIIcollagens in the pathogenesis of several human diseases, in which these collagen typeshave been suggested to play a role. Type III collagen plays an important structural role inhollow organs, such as the aorta and bowel. Furthermore, type III collagen, together withtype I collagen, takes part in the invasion of malignant tumors. For these reasons,atherosclerosis, abdominal aortic aneurysms, diverticulosis, and colon and ovarian cancerwere studied in tissue samples by using specific immunoassays for several type I and IIIcollagen metabolites and corresponding purified antibodies for immunohistochemicalstudies. In addition, new methods for quantifying type III collagen in tissue digests weredeveloped. The immunomethods used here detect newly synthesized procollagens on theone hand and cross-linked, fibrillar type I and III collagens on the other, thus giving agood view into the metabolic state and matrix organization of tissues under investigation.

2. Review of the literature

2.1. Structure and biosynthesis of collagens

Collagens are one of the most important structural proteins in vertebrates. Thecharacteristic features of collagens are the presence of at least one triple-helical domainconsisting of polypeptide chains with a repeated Gly-X-Y sequence and the ability toform supramolecular aggregates with a structural function in the extracellular matrix(ECM) (see Kielty et al. 1993, Prockop & Kivirikko 1995, Bateman et al. 1996 forreviews). In addition to collagens, several other proteins, such as the complementcomponent C1q, acetylcholine esterase and macrophage scavenger receptors, containcollagenous sequences, but since these proteins do not have any structural functions inthe ECM, they are not classified as collagens (see Kielty et al. 1993).

Collagens consist of three identical or different polypeptide chains, called α-chains,each of which is coiled into a left-handed helix, the three together being twisted aroundone another to form a right-handed super-helix. Glysine in every third position,frequently hydroxylated proline and lysine residues in the X and Y positions, and exactregistration of the three α-chains are required for the zipper-like folding of the proteinand its consequent triple-helical conformation (for review, see Engel & Prockop 1991).

Collagens have traditionally been divided into two subgroups, fibril-forming and non-fibril-forming collagens, according to their structural features. The latter group may befurther divided into subfamilies: network-forming collagens, a beaded filament-formingcollagen, a collagen which forms anchoring fibrils, FACIT collagens (fibril-associatedcollagens with interrupted triple helices), collagens with a transmembrane domain, andMULTIPLEXINs (proteins with multiple triple helix domains and interruptions). To date,nineteen genetically distinct collagen types with specialized functions and characteristictissue distributions have been identified (Table 1) (see Chu & Prockop 1993, Kielty et al.1993, Prockop & Kivirikko 1995, Bateman et al. 1996, Adachi et al. 1997 for reviews).

After transcription of the procollagen genes and processing of the pre-mRNAs, the α-chains are synthesized on the ribosomes of the endoplasmic reticulum. The signalpeptides at the aminoterminal ends of the chains are removed by a signal peptidase aftertranslocation across the membrane of the rough endoplasmic reticulum (RER). A largenumber of post-translational modifications are involved in collagen biosynthesis, for

15which several specific enzymes are required. Proline and lysine residues in the Y-positionare hydroxylated to 4-hydroxyproline and hydroxylysine, respectively, and some of theprolines in the X-position are hydroxylated to 3-hydroxyproline. Galactosyl moieties canbe attached to some hydroxylysine residues by hydroxylysyl galactosyltransferase, andglucose can be attached to some of the galactosyl hydroxylysine residues by galactocylhydroxylysyl glucosyltransferase. In addition to the lysines in the triple-helical region,the lysines in the short telopeptides can also be glycosylated (Cannon & Davison 1978).Furthermore, the carboxyterminal propeptides of at least type I and III procollagenscontain asparagine-linked high-mannose type oligosaccharide side chains. Intra- andinterchain disulfide bonds are formed between cysteine residues by protein disulfideisomerase (PDI) (Koivu & Myllylä 1987), which is a subunit of prolyl-4-hydroxylase.Procollagen folding and association into a triple helix requires the involvement ofmolecular chaperones, such as HSP47 (Nakai et al. 1992, Clarke et al. 1993). HSP47 isneeded in collagen assembly and also during the transport of collagen from theendoplasmic reticulum to Golgi (see Nagata 1998). Increased HSP47 in the endoplasmicreticulum has been demonstrated in osteogenesis imperfecta fibroblasts, where thestructure of type I collagen can be abnormal (Kojima et al. 1998). In addition to HSP47,PDI has been shown to have chaperone functions as well (Wilson et al. 1998).

After the folding of the three polypeptide chains, the procollagen molecule istransported to the Golgi complex, where phosphorylation of some serine residues andsulfation of tyrosine residues in the propeptides of type I and III collagens take place,respectively. Finally, the procollagens are secreted out of the cell, where the extracellularprocessing takes place. Propeptides of the fibrillar collagens are cleaved off by specificN- and C- proteinases, the collagen is assembled into fibrils, and cross-links are formedbetween certain lysine and hydroxylysine residues after their conversion into aldehydederivatives by lysyl oxidase. For further reading and reviews on the biosynthesis ofcollagen, see Kielty et al. (1993), Prockop & Kivirikko (1995) and Bateman et al. (1996).

Table 1. Collagen types, their genes, molecular forms and distributions in human tissues.Type Subgroup Gene Molecular forms DistributionI Fibrillar COL1A1 [α1(I)]2α2(I) Most tissues

COL1A2 [α1(I)]3

II Fibrillar COL2A1 [α1(II)]3 Cartilage, cornea,vitreous humor,intervertebral disc

III Fibrillar COL3A1 [α1(III)]3 Soft tissues, with type Icollagen

IV Network-forming COL4A1 [α1(IV)]2α2(IV) Basement membranesCOL4A2 [α3(IV)]2α4(IV)COL4A3 other formsCOL4A4COL4A5COL4A6

16Table 1. continued.

Type Subgroup Gene Molecular forms DistributionV Fibrillar COL5A1 [α1(V)]3 Minor amounts in most

COL5A2 α1(V)α2(V)α3(V) tissues with type I collagenCOL5A3 other forms

VI Beaded filament- COL6A1 α1(VI)α2(VI)α3(VI) Minor amounts in mostforming COL6A2 tissues

COL6A3

VII Anchoring COL7A1 [α1(VII)]3 Skin, cervix, oral mucosafibril-forming

VIII Network-forming COL8A1 [α1(VIII)]2α2(VIII) Many tissuesCOL8A2

IX FACIT COL9A1 α1(IX)α2(IX)α3(IX) With type II collagen,COL9A2 e.g. cartilageCOL9A3

X Network-forming COL10A1 [α1(X)]3 Hypertrophic cartilage

XI Fibrillar COL11A1 α1(XI)α2(XI)α1(II) With type II collagenCOL11A2 other forms e.g. cartilageCOL2A1

XII FACIT COL12A1 [α1(XII)]3 Many tissues with type Icollagen

XIII Transmembrane COL13A1 Unknown Minor amounts in manydomain tissues

XIV FACIT COL14A1 [α1(XIV)]3 Many tissues with type Icollagen

XV MULTIPLEXINs COL15A1 Unknown Many tissues

XVI FACIT COL16A1 [α1(XVI)]3 Many tissues

XVII Transmembrane COL17A1 [α1(XVII)]3 Hemidesmosomes ofdomain stratified squamous

epithelia

XVIII MULTIPLEXINs COL18A1 Unknown Liver, kidney, placenta, etc.

XIX FACIT COL19A1 Unknown Several tissuesSee text for references.FACIT = fibril-associated collagens with interrupted triple helicesMULTIPLEXINs = proteins with multiple triple helix domains and interruptions

172.2. Fibril-forming collagens

The type I, II, III, V and XI collagens make up the group of fibrillar collagens. Byforming highly organized fibers, these collagens provide the structural support for thebody in the skeleton, skin, blood vessels, nerves, intestines, and the fibrous capsules ofthe organs. The characteristic features of fibrillar collagens include triple-helical domainsconsisting of about 1000 amino acids per chain, propeptides at both ends of the molecularprecursor, and finally, highly complex fibrillogenesis. Collagen fibril formation isintrinsically a self-assembly process, but considerable cellular control is also involved.For references and further reading, see Prockop & Kivirikko (1995) and Kadler et al.(1996).

Type I collagen is the most abundant collagen type, being present in most tissues, butmainly in bone, tendon and skin. Type III collagen is often found in association with typeI collagen in all soft tissues, but not in mineralized bone. In addition, hybrid fibrils oftype I and III collagens are found in at least skin, tendon and amnion (Keene et al. 1987).Type II collagen is often found in cartilage and vitreous humor in association with typeXI collagen (Sandberg et al. 1993), whereas type V usually occurs as a minor componentwith type I collagen fibers (Birk et al. 1988). In mammals, type I and III collagensaccount for approximately 70 % and 5 to 20 % of total collagen, respectively (see Adachiet al. 1997).

2.2.1. Type III collagen

2.2.1.1. General features and assembly of type III collagen

Type III collagen is a homotrimer of three α1(III) chains encoded by a single gene foundon human chromosome 2 at location q24.3-q31 (Emanuel et al. 1985). This gene, whichis called the COL3A1 gene, is about 44 kb in length and contains 52 exons (see Chu &Prockop 1993).

The isolation of collagen molecules with the chain composition of [α1 (III)]3 was firstreported almost 30 years ago (Miller et al. 1971). One α1(III) chain consists of anuninterrupted triple-helical domain about 1000 amino acids and 300 nm in length.Similarly to the other fibrillar collagens, type III collagen is also synthesized as a largeprecursor molecule containing propeptides at both ends. These propeptides are thenenzymatically cleaved off during the extracellular processing of type III collagen.Between these propeptides and the main triple helix, there are short, non-triple-helicaltelopeptides containing the sites essential for intermolecular cross-linking (see Yamauchi& Mechanic 1988, Kielty et al. 1993, Prockop & Kivirikko 1995 for reviews).

The folding of collagen requires association of the α-chains through the globular C-terminal domains, formation of a nucleus of triple-helical conformation at the C-terminus, and finally, propagation of the nucleus from the C-terminus to the N-terminaldomain with a zipper-like mechanism (see Engel & Prockop 1991, Baum & Brodsky1999 for reviews). Intra- and interchain disulfide bonds are formed within C-propeptide

18and C-telopeptide, and when the triple helix is forming, disulfide bonds are also formedwithin the N-propeptide (Bruckner et al. 1978).

There are a number of peptide bonds, to which proline contributes the nitrogen, in thecollagen polypeptide chains (see Engel & Prockop 1991). Since the peptide bonds mustbe in the trans configuration in the native triple helix, the cis-trans isomerization limitsthe rate of collagen folding. Type III collagen folds in two phases. In the fast phase, about25 tripeptide units fold between the disulfide bonds at the C-terminal region and the firstcis peptide bond. During the second, much slower phase, which is determined by theslow rate of cis-trans isomerization of the cis peptide bonds, the triple helix thenpropagates in a zipper-like fashion (Bächinger et al. 1978, Bächinger et al. 1980,Bächinger 1987, see Engel & Prockop 1991).

The currently available data indicates that the native type III collagen molecule isunusually susceptible to proteolysis by trypsin when compared to type I collagen, whichis highly resistant to this proteolytic activity. This phenomenon has been attributed to thelack of helicity in the trypsin-susceptible region of the type III collagen molecule (Milleret al. 1976). Furthermore, human and various animal type III collagens are differentiallysusceptible to hydrolysis by human collagenase 1 (MMP-1), probably due to variation inhelix stability (Welgus et al. 1985).

2.2.1.2. Aminoterminal propeptide of type III procollagen, PIIINP

The aminoterminal propeptide of type III procollagen, PIIINP, is cleaved off by a specificN-proteinase during the extracellular processing of type III procollagen molecules (Halila& Peltonen 1986). The PIIINP molecule has a molecular weight of approximately 42 000and consists of a cysteine-rich globular domain (Col 1), a triple-helical region (Col 3),and another non-collagenous domain (Col 2) ending in the N-telopeptide. The Col 1domain contains 5 intrachain disulfide bridges along with 79 amino acids. The Col 2domain is an area 12 amino acids long and located in the carboxyterminal region of thepropeptide containing three interchain disulfide bonds. Finally, the Col 3 domain with 39amino acids form a triple-helical structure between the Col 1 and Col 2 domains(Bruckner et al. 1978) (Fig. 1). The aminoterminus, known as Col 1, is the mostimmunogenic region in the molecule (Rohde et al. 1983). The Col 1 region has asubstrate site for transglutaminase (Bowness et al. 1987) (Fig. 1), which appears to playan important role in, for example, wound healing (Bowness et al. 1988) and the bindingof other molecules, such as fibrinogen and LDL, to procollagen (Bowness et al. 1989).The PIIINP molecule is stabilized by three interchain and five intrachain disulfidebridges. The melting temperature of the collagenous domain is 53°C, and refolding of thehelix from denatured peptides takes place extremely fast, probably due to the disulfidebonds that already keep the three chains together (Bruckner et al. 1978).

Tyrosine sulfation is a widespread post-translational modification (Huttner 1982). Thepresence of acidic amino acids, most frequently an aspartic or glutamic acid, is the keyfeature of tyrosine sulfation sites (Lee & Huttner 1983). Sulfation is a generalcharacteristic of type III procollagen, into which the anion is incorporated in the form oftyrosine-O-sulfate (Jukkola et al. 1986) (Fig. 1). Interestingly, inhibition of tyrosine

19sulfation does not seem to affect the secretion or processing of type III procollagen in invitro experiments (Jukkola et al. 1990). Since it has been suggested that charged andhydrophobic amino acids could contribute to the prevention of premature fibril formationand the regulation of collagen fiber diameter (see Fleischmajer & Perlish 1986), onepossible function of tyrosine sulfation lies in collagen fibrillogenesis. Furthermore, thesesulfate residues, due to their negative charge, could influence the interactions of type IIIprocollagen with other extracellular matrix components.

Intact circulating PIIINP is cleared by scavenger receptors located on the endothelialcells of the liver (Smedsrød et al. 1988, Melkko et al. 1994). The highly acidic nature ofthe PIIINP molecule due to sulfation is believed to play a role in this recognition. Themost aminoterminal part, Col 1, is present as monomers, which are eventually removedvia the kidneys. Because the metabolism of type III collagen is altered in various clinicalsituations, e.g. fibrosis and malignant processes, the measurement of PIIINP in humanserum by radioimmunoassay has established its status in clinical practice (Niemelä et al.1985, Risteli et al. 1988a, Risteli & Risteli 1995). Elevated levels of circulating PIIINPhave been observed in hormonally induced growth (Trivedi et al. 1989) and in diseasespresenting with fibrosis (see Risteli & Risteli 1990 for a review). Decreased PIIINPlevels have been observed in Ehlers-Danlos syndrome type IV (Steinmann et al. 1989).Furthermore, PIIINP assay may also be useful in monitoring dilated cardiomyopathy(Klappacher et al. 1995) or vascular sclerosis in hypertension (see Weber 1998). Inaddition, measurement of PIIINP concentrations in cerebrospinal fluid may be of value inthe clinical diagnosis of meningeal fibrosis (Sajanti & Majamaa 1999).

Fig. 1. Schematic illustration of the aminoterminal propeptide of type III procollagen. Thearrow represents the N-proteinase cleavage site. Possible tyrosine sulfation sites are indicatedby asterisks. The transglutamination site is marked by a circle. For clarity, thesemodifications are only indicated in one of the three chains. The bars indicate the location ofthe alleged disulfide bonds between cysteine residues (modified from Niemelä 1985, Risteli &Risteli 1990).

202.2.1.3. Aminoterminal telopeptide of type III collagen, IIINTP

A non-helical segment of 14 amino acid residues in type III collagen, the amino-terminaltelopeptide, has been recognized as a fundamental element for proper functioning of theprotein (Becker et al. 1976, Glanville & Fietzek 1976), since it contains intra- andintermolecular cross-linking sites. In addition, the tyrosine residues in IIINTP could besulfated, as described in the previous chapter concerning PIIINP. After the cleavage ofthe propeptides, the collagen molecules self-assemble into fibrils, which are thenstabilized by cross-links. In order to form these bonds, the collagen molecules are alignedin a staggered manner, so that the molecules are laterally displaced by about one-quarterof their length relative to the previous molecule along the axis of the fibril. Thus, theindividual 300-nm collagen molecules overlap each other by a distance of 67 nm, leavinga gap of 40 nm between the ends of the non-overlapping molecules. For further readingand references, see Kielty et al. (1993), Prockop & Kivirikko (1995) and Kadler et al.(1996).

Four homologous loci of cross-linking are present in the molecules of type IIIcollagen. Two are aldehyde sites, one in each telopeptide region. The other two sites arehydroxylysines located in the helical region at about 90 residues from each end of themolecule (see Eyre et al. 1984 for a review). During extracellular processing, a trivalentpyridinoline cross-link structure is formed between the lysine/hydroxylysine residues intwo aminoterminal telopeptides from the same collagen molecule and one in thecarboxyterminal triple-helical region of the neighboring collagen molecule (Fig. 2). Thereis also data to support the existence of divalent cross-links joining only one N-telopeptideto the carboxyterminal helical region (Henkel et al. 1979).

Fig. 2. Structure of the cross-linked aminoterminal telopeptide of type III collagen, IIINTP.The actual non-triple-helical telopeptide domain is underlined. The arrows represent the N-proteinase and trypsin cleavage sites. The asterisks indicate tyrosine residues, which canpossibly be sulfated. The pyridinoline cross-link connects two aminoterminal telopeptides ofone molecule with the carboxyterminal helical region of the adjacent collagen molecule.

212.2.1.4. Type III pN-collagen

The aminoterminal propeptides of both type I and III collagens have been suggested tohave a regulatory role in collagen fibril formation (see Fleischmajer & Perlish 1986). Theaminopropeptide of type III collagen is usually released more slowly than thecarboxyterminal propeptide (Fessler et al. 1981). Occasionally, the type III collagenmolecule retains its aminoterminal propeptide domain (PIIINP), resulting in type III pN-collagen. Fleischmajer et al. (1981) found type III pN-collagen along thin collagen fibrilsin human skin. Hedman et al. (1982) cultured amniotic epithelial cells, which onlysynthesize type III collagen, and demonstrated the incorporation of pN-type III collagenin early fibrillogenesis. Studies on human adult skin indicate that when the fibril reachesthe diameter of 35 – 55 nm, the aminoterminal propeptide of type III procollagen isretained at the surface of the fibril, possibly resulting in the prevention of further fibrilgrowth (Fleischmajer et al. 1985) (Fig. 3). This differs from type I collagen, where theaminoterminal propeptide is cleaved when the fibrils reach a diameter of 35-45 nm, thussuggesting that the aminopropeptide has only a transient role in fibrillogenesis(Fleischmajer et al. 1985). The same study also suggested that type I pN-collagen wasonly present on very thin collagen fibrils, which are typically seen in fetal skin, whereastype III pN-collagen was evident in both adult and fetal skin on fibrils ranging from 25 to55 nm in diameter. This observation has also been previously reported in studies onchicken embryo skin (Fleischmajer et al. 1983).

Fig. 3. Metabolism of type III collagen fibrils. When the procollagen molecule has beensecreted out of the fibroblast, the carboxyterminal propeptide (PIIICP) is cleaved. Theremaining type III pN-collagen, which still retains the aminoterminal propeptide domain, isattached to the surface of the fibril. Depending on the thickness of the existing fibril, thePIIINP molecule is cleaved off and released into the circulation. However, if the whole pN-collagen is degraded, the collagenous domain of PIIINP can also be further cleaved, so thatthe most aminoterminal region of the molecule, known as Col 1, is released (modified fromJensen & Høst, 1997).

22Evidence is also available to indicate that type III collagen remains associated with the

type I-containing collagen fibrils after removal of the amino- and carboxyterminalpropeptides, and that several tissues contain copolymers of at least type I and IIIcollagens (Keene et al. 1987, Fleischmajer et al. 1990). Furthermore, there are severalobservations to support the hypothesis that type III pN-collagen also regulates thediameter of type I collagen fibrils by coating the surface of the fibrils and therebyallowing tip growth but no lateral growth of the fibrils, leaving them thinner(Fleischmajer et al. 1990, Romanic et al. 1991).

Type III pN-collagen is suggested to be an important component of reticular fibers inlymph nodes, both under normal conditions and in Hodgkin’s disease (Karttunen et al.1988). The distribution of type III pN-collagen in the reticular fibers of the spleen isdifferent in developing and adult tissues, suggesting a possible role of type III collagen inthe development and function of normal spleen (Liakka et al. 1995). Interestingly, typeIII pN-collagen is reduced in severely photodamaged skin, possibly resulting in alteredorganization of fibrillar collagen and thus contributing to the wrinkled appearance ofinjured skin (Talwar et al. 1995).

2.2.1.5. Carboxyterminal telopeptide (IIICTP) and propeptide(PIIICP) of type III collagen

The C-propeptide (amino acid residues 1206-1466 of the pro-α1(III)-chain) plays a rolein the early stages of procollagen assembly (see McLaughlin & Bulleid 1998). The C-propeptide has a recognition signal, which directs the type-specific assembly ofindividual procollagen chains (Lees et al. 1997). By exchanging this sequence betweendifferent procollagen chains, it is possible to direct chain association and, potentially,assemble molecules with defined chain compositions (Lees et al. 1997). Bulleid et al.(1997) showed that the triple helix nucleation took place even when the C-propeptide wassubstituted with a transmembrane domain. Thus, it was concluded that the C-propeptideis required only to ensure association of the monomeric chains. Furthermore, the samestudy demonstrated that triple helix formation is directed by the most carboxyterminalGly-X-Y triplet of the triple helix and not by the propeptide.

The C-propeptide of the α1(III) chain contains 8 cysteine residues, which are essentialfor the formation of intra- and interchain disulfide bridges. Disulfide bonds are thought toform between the C-propeptide chains prior to the triple helix formation (Lukens 1976).However, mutant chains lacking the ability to form interchain disulfide bonds are stillable to assemble, indicating that interchain disulfide bonding within the C-propeptide orC-telopeptide is not required for triple helix formation (Bulleid et al. 1996).

The carboxyterminal propeptides of the secreted type III procollagen molecules arerapidly cleaved off by the same C-proteinase that is also known to cleave type I and IIcollagens (Kadler & Watson 1995). The C-telopeptide of the α1(III) chain contains aconserved partial sequence Glu-Lys-Ala involved in enzymatic cross-linking. Thissequence has a polar region where nuclear magnetic resonance studies have suggestedexistence of a β-turn (Liu et al. 1993, Henkel 1996). Apart from the interchain disulfide

23bridges within the C-telopeptide, there is also evidence of disulfide cross-links betweenadjacent collagen molecules (Cheung et al. 1983, Henkel 1996).

2.2.1.6. Distribution of type III collagen in tissues

Type III collagen is the second most abundant collagen in human tissues and occursparticularly in tissues exhibiting elastic properties, such as skin, blood vessels and variousinternal organs. Several studies concerning the relative amounts of type I and III collagenhave been published, all giving more or less different results, depending mainly on thetissue extraction methods used to solubilize the insoluble collagen (see Rauterberg et al.1993). For example, the initial work on arterial collagens suggested a predominance oftype III collagen over type I, but later results have suggested that type I collagen accountsfor 55 – 88 % of the total amount of these two (see Barnes 1985 for a review). Hanson &Bentley (1983) reported in one study that the vena cava contained 21 % of type IIIcollagen, whereas achilles tendon exhibited only type I collagen. The type III collagencontent of bone in normal subjects is about 4 % (Bailey et al. 1993). Type III collagenaccounts for 11 % and 20 % of the total collagen protein in normal myocardium andvalve leaflets, respectively (see Weber 1989). Dilated cardiomyopathy has recently beenfound to be associated with significant changes in the collagen type I/III ratio(Pauschinger et al. 1999). Liver has been reported to have as much as 45 % and humanlung 21 % of type III collagen (Kelley et al. 1984, van Kuppevelt et al. 1995). Theproportion of type III collagen out of the total collagen content is about 20 % and 50 % inadult human skin and embryonic dermis, respectively (Epstein & Munderloch 1975,Cheung et al. 1990).

There are also age-related changes in the relative amounts of type I and III collagens.In studies on rats, type III collagen represented about one-third of the sum of types I andIII in all tissues at 2 weeks of age. After this, the proportion of type III collagen increasedfor up to one year in both heart (54 %) and lung (48%), whereas it decreased in skin (19%) (Mays et al. 1988). Similar results on human skin collagen had also been obtainedpreviously (Chan & Cole 1984). Tagasako et al. (1992) reported the total quantitity oftype III collagen to decrease upon ageing in all tissues.

2.2.1.7. Type III collagen mutations

The whole nucleotide and amino acid sequences of the human type III procollagen chainwere presented in 1989 (Ala-Kokko et al. 1989). Since then, several disease-causingmutations in the COL3A1 have been found (see Kuivaniemi et al. 1997 for a review,Schwartze et al. 1997, Bateman et al. 1998, Gilchrist et al. 1999). Most of the mutationshave been seen in patients with Ehlers-Danlos syndrome IV, the most severe form ofEDS, which may cause sudden death due to a rupture of large arteries and hollow organs,in addition to causing skin and joint changes. The mutations include various single-basesubstitutions that mostly convert a codon of glycine to a codon of some other amino acid,

24in combination with deletions and mRNA splicing defects (see Kuivaniemi et al. 1997).EDS III is characterized by general laxity of joints and extensibility of skin, and aglycine-to-serine mutation in the α1(III) chain has also been reported in this disease(Narcisi et al. 1994). The different mutations in the COL3A1 gene have effects on thesecretion, folding, fibrillogenesis, and thermal stability of collagen as well as the tissuearchitecture (Anderson et al. 1997, Smith et al. 1997).

In order to study the role of COL3A1 in the body, the murine COL3A1 gene wasinactivated in embryonic stem cells (Liu et al. 1997). About 10 % of the homozygousmutant animals survived, but had a shortened lifespan. The major cause of death in themutant knock-out mice was rupture of the major blood vessels. Furthermore, it wasshown that type III collagen is essential for normal fibrillogenesis of type I collagen inthe cardiovascular system (Liu et al. 1997).

Mutations in the type III collagen gene are also associated with the pathogenesis ofabdominal aortic aneurysms without other features of EDS IV (Kontusaari et al. 1990,Anderson et al. 1996). Spontaneous cervical arterial dissections have also been studiedfor mutations, but even though some of the patients have a low ratio of type III to type Icollagen, mutations in the type III collagen gene are not considered responsible forcervical arterial dissections (van den Berg et al. 1998). The structural unstability of typeIII procollagen observed in patients with cerebral aneurysms (Majamaa et al. 1992) mightsuggest mutations in the COL3A1 gene. Such mutations are, however, an infrequentcause of these or any other familial aneurysms without other signs of connective tissuemanifestations (see Kuivaniemi et al. 1997).

2.2.2. Type I collagen and its metabolites

Type I collagen is the major collagen type in most tissues. For example, most of theorganic matrix of bone consists of type I collagen. The main cells synthesizing type Icollagen in soft tissues are fibroblasts, whereas bone collagen is produced by osteoblasts.The most abundant molecular form of type I collagen is a heterotrimer of two α1(I)chains and one α2(I) chain. In addition to this classical type I collagen, there is evidenceof a type I α1-homotrimer collagen consisting of three α1(I) chains (Jimenez et al. 1977,Wohllebe & Carmichael 1978). A third variant, the laminin-binding collagen found incolon and breast cancer tissue, has been suggested to consist of one α1(I) chain, oneα1(III) chain, and a unique acidic chain (Pucci-Minafra et al. 1993).

Similarly to other fibrillar collagens, type I collagen is synthesized as large precursormolecules containing extra propeptide domains at both ends. The aminoterminalpropeptide (PINP) and the carboxyterminal propeptide (PICP) of type I collagen arecleaved off enzymatically by specific endoproteinases, after the procollagen molecule hasentered the extracellular space (see Risteli & Risteli 1999). Cleavage of thecarboxyterminal propeptide is required for the initiation of fibril formation (Kadler et al.1987). The molecules can retain their aminoterminal propeptide for a while, resulting intype I pN-collagen, which has been suggested to regulate the lateral growth of type Icollagen fibers (Fleischmajer et al. 1985). The PINP molecule is similar to PIIINP andconsists of three structural subdomains: Col 1 (the globular part in the most

25aminoterminal region containing intrachain disulfide bridges, which is alsophosphorylated), Col 2 (the short non-helical part) and Col 3 (the central collagenousdomain) (see Risteli & Risteli 1999 for a review). The molecular mass of the PINPmolecule is 35 000, and it is cleared from the blood via scavenger receptors of liverendothelial cells (Melkko et al. 1994). The PICP molecule, in turn, is cleared via themannose receptor of the same liver endothelial cells (Smedsrød et al. 1990) and has amolecular mass of about 100 000. The PICP molecule is a glycoprotein containing high-mannose type oligosaccharides (Clark 1979).

The concentrations of PINP and PICP antigens in serum have been shown to reflectthe synthesis of type I collagen in both physiological and pathological situations (seeRisteli & Risteli 1999). Radioimmunossays for these two metabolites have beendeveloped for clinical use (Melkko et al. 1990, Melkko et al. 1996). Especially PINP hasproved useful in detecting changes in the rate of bone collagen metabolism, such as thoseseen during estrogen treatment in postmenopausal women (Suvanto-Luukkonen et al.1997), Paget’s disease of bone (Sharp et al. 1996), and in patients with aggressive breastcancer (Jukkola et al. 1997).

Between the propeptides and the helical collagen molecule, there are short, non-triple-helical telopeptide domains containing sites for intra- and intermolecular cross-linking.The lysine or hydroxylysine residue of the aminoterminal telopeptide of type I collagencan be oxidized into aldehyde, which can further mature into trivalent pyridinoline orpyrrole cross-links (see next chapter). The carboxyterminal telopeptide forms a divalentcross-link by binding to a helical region of another collagen molecule, which, in turn, canmature into pyridinoline or other mature structures (see next chapter) (see Bailey et al.1998, Risteli & Risteli 1999). An interesting feature in the C-terminal telopeptide is theβ-isomerization of the aspartic acid as a result of the normal ageing process (Fledelius etal. 1997), the biological effects of which are yet to be elucidated.

As for the propeptides of type I collagen, namely PINP and PICP, specific assays havealso been developed for the telopeptide regions in order to monitor the degradation oftype I collagen in serum and urine. The NTx assay detects the trivalently cross-linkedaminoterminal telopeptide structure of type I collagen (Hanson et al. 1992). The originalCrossLaps assay was developed for a synthetic peptide, the amino acid sequence(EKAHDGGR) of which was derived from the carboxyterminal telopeptide (Bonde et al.1994). The ICTP assay, which was also used in the current studies, detects degradationproducts of mature type I collagen with a trivalent cross-link (Risteli et al. 1993, Sassi etal. 2000).

2.2.3. Enzymatic cross-linking of type I and III collagens

2.2.3.1. Lysyl oxidase

Lysyl oxidase is a copper-dependent extracellular enzyme, which is responsible for theoxidation of certain lysine and hydroxylysine residues in collagen and lysine residues inelastin to form α-aminoadipic-δ-semialdehyde, which, in turn, can react with the

26neighboring molecules to form covalent cross-links (see Smith-Mungo & Kagan 1998 fora review). The cross-linking process goes on continuously on the surface of the growingfibril and therefore occurs at a very early stage of fibril formation (Cronlund et al. 1985).Up to date, lysyl oxidase, LO (Hämäläinen et al. 1991), has two identified isoforms: lysyloxidase-like protein, LOL (Kenyon et al. 1993, Kim et al. 1995) and lysyl oxidase-related protein, LOR (Saito et al. 1997). The existence of these highly related genesimplies the presence of a lysyl oxidase gene family, additional members of which mayyet remain to be identified (see Smith-Mungo & Kagan 1998).

The expression of lysyl oxidase responds to many physiological conditions, such asgrowth, development, ageing and wound healing. A number of genetic diseases affectingcopper metabolism and the function of lysyl oxidase, such as cutis laxa and Menkessyndrome, have been identified (see Smith-Mungo & Kagan 1998). LO expression isincreased in fibrotic conditions (see Kagan 1994). An opposite situation prevails inmalignant cell line cultures, where LO expression is down-regulated at the transcriptionallevel (Hämäläinen et al. 1995). In addition, there is evidence of a lack of LO in thestroma accompanying invading tumors, consistent with the deficiency of properly cross-linked fibers (Peyrol et al. 1997, see Smith-Mungo & Kagan 1998). Lysyl oxidase is alsoa candidate gene for a tumor suppressor (Contente et al. 1990).

2.2.3.2. Cross-linking pathways

Collagen cross-linking is essential for the tensile strength of tissue, since it increases theresistance of collagen fibers against proteolysis (Vater et al. 1979). Two pathways ofenzymatic cross-linking have been described for fibrillar collagens: one based on lysinealdehydes, and another based on hydroxylysine aldehydes. The cross-linking of type IIIcollagen has not been so widely studied as that of type I collagen, but the cross-linkingpathways have been thought to be similar in these two collagen types. The specifichydroxylation of lysine residues in the telopeptides is responsible for the nature of thecross-link and thus for the biomechanical properties of the tissue. The lysine aldehydepathway predominates at least in adult skin, cornea, sclera and rat tail tendon, whereas thehydroxylysine aldehyde pathway predominates in bone, cartilage, ligaments, tendons andmost internal organs, being also present in embryonic skin (see Eyre 1987, Yamauchi &Mechanic 1988, Bailey et al. 1998 for reviews). There is recent evidence of a bone-specific telopeptide lysyl hydroxylase, which could be partially responsible for the tissue-specific collagen cross-linking (Bank et al. 1999, Uzawa et al. 1999). Cross-linking doesnot occur only between the chains of one collagen type: type I collagen, for example, maybe co-polymerized with type III collagen (Henkel & Glanville 1982).

Enzymatic cross-linking of collagen is illustrated in Figure 4. The telopeptide lysyland hydroxylysyl aldehydes are capable to react with the unmodified lysine orhydroxylysine residues of another collagen molecule to form immature reducible divalentcross-links. In addition, two lysyl aldehydes from the same molecule can react with eachother to form an intramolecular aldol condensation product (ACP). In the hydroxylysylaldehyde pathway, the divalent cross-links spontaneously undergo an Amadorirearrangement, resulting in acid and heat-stable keto-imine cross-links, which then

27mature further into trivalent pyridinoline and pyrrole cross-links. Divalent cross-linksderived from the lysyl aldehyde pathway can react with hydroxylysine and histidineresidues resulting in trivalent histidinohydroxylysinonorleucine (HHL) and tetravalenthistidinohydroxymerodesmosine (HHMD) cross-links. However, the in vivo existence ofthe latter cross-link along with the pyrroles is controversial. Of the trifunctional cross-links, histidinohydroxylysinonorleucine (HHL) is found at least in skin and cornea,whereas hydroxylysylpyridinoline (HL-Pyridinoline) is very typical of type II collagen incartilage, and lysylpyridinoline (L-Pyridinoline) in calcified tissue. HL-Pyridinoline hasbeen reported to be located equally at both the N- and the C-terminus, whereas L-Pyridinoline and the pyrroles are preferentially located at the N- terminus of the molecule(Hanson & Eyre 1996). The other mechanism of intermolecular cross-linking of collagenis the non-enzymatic reaction with glucose. For references and more thorough reviews onthe cross-linking of collagen, see Eyre et al. (1984), Eyre (1987), Yamauchi & Mechanic(1988) and Bailey et al. (1998).

Fig. 4. Schematic representation of enzymatic cross-link formation and maturation in type Iand III collagens (modified from Yamauchi & Mechanic 1988, Bailey et al. 1998). Thepossible maturation product of deH-LNL is currently unknown (marked by a questionmark). For abbreviations, see the list of abbreviations at the beginning of this book.

The cross-link profiles are, to some extent, tissue-specific and may change with age.For example, the content of reducible cross-links in bone collagen decreases markedlybetween birth and 25 years (Eyre et al. 1988), followed by a suggested maturation mainly

28into pyrrolic trivalent structures, whereas the amount of pyridinolines may remain almostthe same (Knott et al. 1995). The age-related changes causing a functional deficiency ofcollagenous tissues are possibly due to increased intermolecular cross-linking (Bailey etal. 1998). In addition to normal ageing, changes in collagen cross-linking may beinvolved in the pathogenesis of many diseases, including liver fibrosis (Ricard-Blum etal. 1996), dilated cardiomyopathy (Gunja-Smith et al. 1996) and diabetes (Reiser 1991).

Cannon & Davison (1978) reported that type III collagen isolated from bovine amniontissue contained large amounts of immature lysine-derived cross-links, particularlydihydroxylysinonorleucine. In addition, almost 80 % of these cross-links wereglycosylated. Divalent hydroxylysinonorleucine cross-links containing only one N-telopeptide plus the carboxyterminal helical region and trivalent mature cross-linkscontaining two N-telopeptides in addition to the helical region have been found in humanuterine leiomyoma (Henkel et al. 1979). The cross-link analysis of the C-terminus of typeIII collagen from calf aorta revealed covalent cross-linking between the C-terminaltelopeptide of type III molecule and the N-terminal helical cross-linking region ofanother. This structure contained mainly dihydroxylysinonorleucine with a small amountof hydroxylysinonorleucine, indicating that the lysine residues involved in cross-linkingare both hydroxylated (Henkel 1996). In addition to the intermolecular aldehyde-derivedcross-links, type III collagen also has disulfide cross-links (Cheung et al. 1983). Forexample, Henkel (1996) suggested a presence of intermolecular disulfide cross-linksbetween the cysteine residues of adjacent collagen molecules in the C-terminus.

2.2.4. Degradation of type I and III collagens

Degradation of collagens can occur via intracellular or extracellular routes. Matrixmetalloproteinases (MMPs), which are zinc-dependent endopeptidases, play a crucial rolein the extracellular degradation of collagens. Breakdown of the ECM is needed in manyphysiological situations, such as development, tissue repair and angiogenesis. Severalpathological conditions involve changes in the synthesis and activity of the matrixmetalloproteinases. MMPs are grouped into collagenases, gelatinases, stromelysins andstromelysin-like MMPs, membrane type-MMPs (MT-MMPs), and novel MMPs,depending on their substrate specificity and primary structure. MMP activity is regulatedby gene transcription, activation of the inactive proenzyme forms and the action ofspecific inhibitors, the major ones being the group of tissue inhibitors of MMPs (TIMPs).For references and further reading, see Kähäri & Saarialho-Kere (1997) and Kähäri &Saarialho-Kere (1999).

Collagenase-1 (fibroblast collagenase, MMP-1), collagenase-2 (neutrophilcollagenase, MMP-8) and collagenase-3 (MMP-13) (Freije et al. 1994) can cleavefibrillar collagens at about three fourths of the way from the aminoterminal end, resultingin two separate fragments susceptible to denaturation and further proteolysis (see Kähäri& Saarialho-Kere 1997). MMP-8 preferentially degrades type I collagen, whereas MMP-1 cleaves type III and MMP-13 type II collagen (Knäuper et al. 1996a, see Kähäri &Saarialho-Kere 1997). In addition, human MMP-13 cleaves type I collagen in theaminoterminal non-helical telopeptide (Krane et al. 1996).

29Gelatinases are very important in the final degradation of collagens after cleavage by

collagenases. At least 72-kDa gelatinase (gelatinase A, MMP-2) has been shown tocleave native type I collagen into fragments identical to those generated by collagenases(Aimes & Quigley 1995). MMP-2 has also been shown to activate at least MMP-13(Knäuper et al. 1996b). Type I collagen, similarly to the types II and V, is digested in theN-terminal non-helical telopeptide by 92-kDa gelatinase (gelatinase B, MMP-9) (Okadaet al. 1995). The stromelysins and stromelysin-like MMPs are able to degrade a widerange of substrates in the extracellular matrix, including some of the collagens, but nottype I and III collagens in their native form. Of the membrane-type MMPs, MT1-MMP(MMP-14) degrades the native collagens I, II and III (D’Ortho et al. 1997, Ohuchi et al.1997) and activates gelatinases and MMP-13 (see Murphy et al. 1999). MT3-MMP hasbeen shown to cleave type III collagen (Matsumoto et al. 1997). The substratespecificities of the newer MMPs are yet to be established.

In addition to extracellular collagen degradation by specific endoproteinases, such asMMPs, fragments of collagen fibers can be endocytosed and degraded within thelysosomes. Furthermore, part of the newly synthesized collagen can be degraded before ithas even been secreted out of the cell (see Everts 1996 et al. for a review). Intracellulardegradation plays an important role particularly when the synthesized collagen isabnormal due, for example, to insufficient post-translational modifications or mutations(Berg et al. 1980, Bateman et al. 1984).

In addition to the degradation of type IV collagen in basement membranes, destructionof the stromal type I and III collagens is an essential step in the invasive and metastaticbehavior of tumor cells (see Kähäri & Saarialho-Kere 1999 for a review). TIMPs play akey role in malignant tissue, not only by controlling the activity of MMPs, but also byaffecting cell proliferation and survival (see Blavier et al. 1999). In addition to malignantprocesses, MMPs or TIMPs have been associated with at least atherosclerosis (Sukhovaet al. 1999, Lindstedt et al. 1999), abdominal aortic aneurysms (see Grange et al. 1997),congestive heart failure (Spinale et al. 1999), dilated cardiomyopathy (Thomas et al.1998), rheumatoid arthritis (Maeda et al. 1995) and fibrotic conditions, such asscleroderma of the skin (see Kähäri & Saarialho-Kere 1997). Unrevealing the detailedstructures of MMPs and TIMPs and their role in the pathogenesis of diseases haveprovided a basis for the development of drugs aimed at modulation of MMP activity (seeSkotnicki et al. 1999). Measurement of plasma or serum MMP and TIMP levels mayprovide important data for selecting and following patients considered for treatment withthese drugs (see Zucker et al. 1999 for a review).

In addition to MMPs, there are lysosomal cysteine proteinases, such as cathepsins,along with different serine and aspartic proteinases, all capable of degrading proteins ofthe extracellular matrix. Cathepsin K is abundant in at least human osteoclasts (Drake etal. 1996) and macrophages (Sukhova et al. 1998). Cathepsin K cleaves native type Icollagen at the N-terminal end of the triple helix and at multiple sites within the helicalregion (Garnero et al. 1998, Kafienah et al. 1998). Furthermore, this enzyme cleaves thetrivalently cross-linked C-terminal fragment of type I collagen (Sassi et al. 2000). As forthe other cathepsins, cathepsin B has been reported to raise the activity of the otherMMPs and to stimulate angiogenesis in malignancies, which together may promote tumorinvasion (Kostoulas et al. 1999).

302.3. Type I and III collagens in atherosclerosis and abdominal

aortic aneurysms

2.3.1. General features of atherosclerosis

Atherosclerosis is a response to injury to the endothelium, characterized by afibroproliferative inflammatory reaction, degenerative changes and accumulation ofextracellular matrix and lipids. One of the parameters associated with the endothelial celldysfunction that results from exposure to oxidized LDL is increased adherence ofmacrophages and other inflammatory cells. Macrophages are known to localizesubendothelially, where they scavenge lipids and turn into large foam cells. Proliferationand migration of vascular smooth muscle cells (SMCs) together with their transition fromthe “contractile” type into the “synthetic” phenotype are key features in the developmentof atherosclerosis. SMCs, T-cells and foam cells together form a fatty streak, which inturn may develop into an intermediate, fibrofatty lesion and, finally, into a fibrous plaquewith calcification. The whole process is extremely complex, involving growth factors,cytokines, hormones, hemostatic agents and changes in collagen synthesis and smoothmuscle cells (see Ross 1993, Rekhter 1999 for reviews).

2.3.2. Type I and III collagens in normal and atherosclerotic aorta

As in most connective tissues, the quantitatively most abundant group of arterialcollagens are the fibril-forming type I and III collagens (see Barnes 1985 for a review).These collagen types together are responsible for tensile strength, but also contribute,together with elastin, somewhat to the extensile properties of the aorta (Dobrin et al.1984, Dobrin & Mrkwicka 1994). The quantitative ratios of type I and III collagens innormal and atherosclerotic aortas have usually been determined after extraction of thecollagens or collagen fragments from tissue specimens and quantification, afterseparation, into different types (see Barnes 1985, Rauterberg et al. 1993 for reviews).

The increased proportion of type I collagen is thought to be characteristic of theatherosclerotic intimal plaque. In spite of extensive research, our knowledge of thequantitative ratios of type I and III collagens in normal and atherosclerotic aortic wall isincomplete and controversial (see Barnes 1985, Rauterberg et al. 1993). The currentlyexisting data on type I and type III collagen ratios in atherosclerosis are shownsummarized in Table 2.

Data from immunohistological studies indicate only small differences in the normalaorta in the distribution of type I and III collagens (see Rauterberg et al. 1993). Boththese collagen types are present in the normal fibrillar structures around smooth musclecells and elastic membranes (McCullagh et al. 1980, Shekhonin et al. 1985, Katsuda etal. 1992). Furthermore, they co-localize with fibronectin (Shekhonin et al. 1987) andstain more intensively in the adventitia than in the media (Katsuda et al. 1992). The type Iand III collagens appear together in the thickened intimas of atherosclerotic lesions,

31where their staining intensity is also greater than in healthy aorta (Katsuda et al. 1992,see Rauterberg et al. 1993).

Table 2. Summary of data on type I and III collagen contents in normal andatherosclerotic human aorta.Method Finding ReferencePepsin digestion, 65 % type I in atherosclerosis McCullagh &SDS-PAGE 30 % type I in normal aorta Balian 1975

Pepsin digestion, 66 % type I in atherosclerosis Ooshima 1981SDS-PAGE

Pepsin / 76 % type I in atherosclerosis Morton &CNBr cleavage, No major shift when compared to Barnes 1982SDS-PAGE healthy aorta

CNBr cleavage, 58 % type I in normal aorta Hanson &CMC chromatography, 90 % type I in atherosclerosis Bentley 1983SDS-PAGE

Pepsin, 38 % type I in normal aorta Hill & HarperDEAE-cellulose 1984chromatography,CNBr, SDS-PAGE

CNBr, SDS-PAGE 70 % type I in normal aorta Halme et al. 1986

Pepsin, 70 % type I in atherosclerosis Leuschner &CMC chromatography No major shift when compared to Haust 1986HPLC, CNBr, normal aortaSDS-PAGE

Pepsin, CNBr, 70 % type I collagen, type III ↓ Murata et al.SDS-PAGE with advancing atherosclerosis 1986

Pepsin, CNBr, 62 % type I in normal thoracic aorta Miller et al. 1991HPLC, UV absorbance 47 % type I in normal abdominal aorta

CNBr, HPLC, 59 % type I in normal thoracic aorta Deyl et al. 1997SDS-PAGE, capillary 52 % type I in normal abdominal aortaelectrophoresisCMC = carboxymethyl-cellulose; CNBr = cyanogen bromide; DEAE =diethylaminoethyl; HPLC = high-performance liquid chromatography; SDS-PAGE =sodium dodecyl sulfate polyacrylamide gel electrophoresis

322.3.3. Synthesis and degradation of type I and III collagens in

atherosclerotic plaques

The increased amount of type I collagen-producing cells has been established in all typesof human atherosclerotic lesions (Andreeva et al. 1997). In animal models, the collagencontent in atherosclerotic lesions has been shown to increase consistently over time(Kratky et al. 1999). The plaque collagen is mainly produced by smooth muscle cells(SMCs), but endothelial cells are also able to synthesize collagen (Canfield et al. 1992).Furthermore, cells different from typical SMCs produce type I collagen in humanatherosclerotic lesions (Rekhter et al. 1996, Andreeva et al. 1997, Tintut et al. 1998).SMC phenotype, proliferation, migration and collagen production play an important rolein the pathophysiology of atherosclerosis (see Rekhter 1999). There is evidence toindicate that ageing is associated with dysregulation of the proliferative responses ofSMCs from old aortas, possibly predisposing to atherosclerotic changes (McCaffrey et al.1988). In addition, DNA alterations have been found in SMCs of human atheroscleroticlesions, suggesting that mutational events may be part of the atherosclerotic process (DeFlora et al. 1997).

At least type I collagen synthesis has been clearly shown to be upregulated inatherosclerotic plaques compared with the media and normal arteries (Rekhter et al.1993). Recently published data suggest that genes consistent with increased proliferationand collagen production are upregulated in SMCs during atherogenesis (Laury-Kleintopet al. 1999). A number of cytokines and growth factors produced and secreted bymacrophages and other inflammatory cells have been described as being involved in theprocess of atherosclerotic plaque formation, in part by altering the rate of collagensynthesis (see Rekhter 1999 for a review). Transforming growth factor beta (TGF-β) isthe most potent and consistent stimulator of collagen synthesis in vascular SMCs(Amento et al. 1991). There is evidence of genomic instability of the type II TGF-β1receptor gene in human atherosclerotic and restenotic vascular cells, probably causingresistance to the antiproliferative effects mediated by this particular receptor type(McGaffrey et al. 1997). Platelet-derived growth factor (PDGF) (Amento et al. 1991),interleukin-1 (IL-1) (Amento et al. 1991), angiotensin-II (Kato et al. 1991),homocysteine (Majors et al. 1997), endothelin-1 (Rizvi et al. 1996) and oxidized LDL(Bachem et al. 1999) have also been reported to stimulate collagen synthesis in SMCculture. Human connective tissue growth factor (CTGF) is undetectable in normal bloodvessels, but overexpressed in atherosclerotic lesions, suggesting that CTGF plays a role inatherogenesis (see Oemar & Lüscher 1997). Furthermore, mechanical stress has beenshown to have a stimulatory effect on collagen synthesis (Kolpakov et al. 1995a). Incontrast, fibroblast growth factor (Majors & Ehrhart 1993, Pickering et al. 1997), nitricoxide (Kolpakov et al. 1995b, Myers & Tanner 1998), interferon gamma (IFN-γ)(Amento et al. 1991) and prostaglandins (Fitzsimmons et al. 1999) all inhibit collagenproduction (see Rekhter 1999). In addition, SMC may exhibit different biosyntheticactivity depending on the matrix environment, and the extracellular matrix itself is thusable to control collagen synthesis in SMC culture (Thie et al. 1991).

Plaque rupture is responsible for the acute manifestations of atherosclerosis. Ruptureprone plaques are characterized by a large lipid pool, a thin fibrous cap, an increasedamount of inflammatory cells and a reduced collagen and SMC content (see Lee & Libby

331997, van der Wal & Becker 1999 for thorough reviews). Previous studies (Henney et al.1991, Galis et al. 1994, Brown et al. 1995, Nikkari et al. 1995, Shah et al. 1995, Li et al.1996) have implicated matrix metalloproteinases, mostly MMP-1, gelatinases andstromelysins, together with certain tissue inhibitors of metalloproteinases, in thepathogenesis, destabilization and rupture of atherosclerotic lesions. Recently, Sukhova etal. (1999) demonstrated increased levels of MMP-1 and MMP-13 in atheromatous versusfibrous carotid atherosclerotic plaques. A large portion of matrix-degrading enzymes isproduced by inflammatory cells, especially macrophages. The numbers of mast cells, Tlymphocytes and MMP-9-containing macrophages increase as the atherosclerotic lesionsbecome more severe (Kaartinen et al. 1998). In addition to the MMPs, macrophages inhuman atheroma have been reported to contain abundant amounts of cathepsins K and S(Sukhova et al. 1998). Certain infections, such as Chlamydia pneumoniae (see Movahed1999 for a review), have been suggested to play a role in the pathogenesis ofatherosclerotic lesions, possibly by inducing the inflammatory process and thus theproduction of MMPs.

2.3.4. Other collagen-related events in atherosclerotic plaques

It has been suggested that oxidized low-density lipoprotein (LDL) is a key component inthe endothelial injury in atherosclerotic lesions. The type I collagen content in the aortasof cholesterol-fed birds has been shown to be increased compared to controls (Jarrold etal. 1999). LDL may directly injure the endothelium or play a role in the adherence andmigration of inflammatory cells (see Ross 1993). In addition, several collagen types areknown to bind oxidized lipoproteins, and the accumulation of collagen in the intima maythus promote lipoprotein accumulation (Greilberger et al. 1997). Furthermore, oxidizedLDL-containing immunocomplexes have been shown to play an important role inmacrophage activation, which, in turn, may result in the secretion of cytokines capable ofinducing collagen synthesis (Virella et al. 1995).

The aminoterminal propeptide of type III procollagen, PIIINP, has been found to beelevated in the blood of patients with coronary artery disease (Bonnet et al. 1988). Inaddition, thrombolytic treatment of patients with acute myocardial infarction results incollagen breakdown (Peuhkurinen et al. 1991, Peuhkurinen et al. 1996). Furthermore,thrombin, a very important peptide in blood coagulation and platelet activation,stimulates procollagen production in cultured SMCs and thus in the arterial wall(Dabbagh et al. 1998).

One of the most common features of advanced atherosclerosis is vascular calcification.Type I collagen has been shown to promote calcification of vascular cells in in vitroconditions (Watson et al. 1998). Type I collagen may also be important in plaqueneoangiogenesis (Jackson & Jenkins 1991) and the organization of thrombus (Rekhter etal. 1996). Furthermore, human coronary restenosis involves rapid accumulation ofcollagen fibers (Pickering et al. 1996). Collagen accumulation has also been found tocorrelate with the severity of restenosis after angioplasty in an animal model (Lafont etal. 1999). Not only synthesis but also degradation of collagen has an important role incollagen turnover after balloon angioplasty (see Rekhter 1999).

342.3.5. Type I and III collagens in abdominal aortic aneurysms (AAA)

Atherosclerotic plaques are present in aneurysmal walls, and atherosclerosis is thought tobe, at minimum, a permissive factor in aneurysm development (see Grange et al. 1997 fora review). There is evidence to suggest that the ratio of type III to type I collagen is notaltered in the aneurysmal aorta (Rizzo et al. 1989). However, occasional lower amountsof type III collagen at the tissue level have been described in patients with a familyhistory of aneurysms (Menashi et al. 1987, Powell & Greenhalg 1989). Kuga et al.(1998) suggested that the amount of type III collagen is significantly decreased inatherosclerotic AAAs compared with atherosclerotic aortas without aneurysmal changes.In addition, he found abnormal fragments of type III collagen in AAA tissue. Deak et al.(1992) and van Keulen et al. (1999) reported decreased type III procollagen secretion infibroblasts in a small group of patients with familial AAA. The expression of type I andIII collagens is increased in human abdominal aortic aneurysms (McGee et al. 1991,Mesh et al. 1992, Minion et al. 1993). The increased expression of collagen is located inadventitial fibroblasts, medial smooth muscle cells near the inflammatory infiltrates, andmyofibroblasts inside the plaque (Hunter et al. 1996, see Grange et al. 1997). Theexisting data on the amount of collagen are controversial: there is evidence of increased(Menashi et al. 1987, Rizzo et al. 1989, Baxter et al. 1994, He & Roach 1994),unchanged (McGee et al. 1991) or even decreased (Sumner et al. 1970) proportion oftotal collagen in AAAs.

Aneurysm formation is a complex remodeling process that involves both synthesis anddegradation of matrix proteins (see Ghorpade & Baxter 1996 for a review). Theidentification of at least the MMPs 1, 2, 3, 9 and, recently, 13 in AAA tissue has beenreported by several investigators (Irizarry et al. 1993, Newman et al. 1994, McMillan etal. 1995, Thompson et al. 1995, Sakalihasan et al. 1996, Tamarina et al. 1997, Mao et al.1999). Freestone et al. (1995) reported more MMP-2 activity in small aneurysms,whereas MMP-9 was significantly increased in large aneurysms. On the other hand,MMP-9 expression has also been reported to be significantly higher in medium-sizedAAAs than in either small or large aneurysms (McMillan et al. 1997). In addition, theactivity of several cathepsins has been found to be increased in aortic aneurysms (Gacko& Chyczewski 1997). Clinical methods of MMP inhibition are under development. Forexample, preoperative treatment with doxycycline has been associated with reduction inthe aortic wall expression of MMP-2 and MMP-9 (Thompson & Baxter 1999), andmarimastat (an MMP inhibitor) has been shown to significantly inhibit matrixdegradation and active MMP-2 production in a porcine model of aneurysm disease(Treharne et al. 1999). MMP-9, which is increased in the plasma of AAA patients, couldbe a potential diagnostic marker for clinical purposes (McMillan & Pearce 1999). Otherdiagnostic approaches have been studied as well: the aminoterminal propeptide of type IIIcollagen, PIIINP, has previously been found to be clinically useful in the follow-up ofpatients with abdominal aortic aneurysms (Satta et al. 1997).

Ehlers-Danlos syndrome IV (EDS IV), which is characterized by multiple aneurysmsand other connective tissue manifestations, is caused by mutations in the gene encodingthe α1-chain of type III collagen (see Kuivaniemi et al. 1997). In addition, there are manystudies reporting a strong familial tendency to aneurysms not associated with EDS IV(see Verloes et al. 1996 for a review). In these studies, the proportion of AAA probands

35with a positive family history has ranged from 12 to 20 %, and the relative risk forsiblings has been 10 - 28. Van Keulen et al. (1999) recently reported that 29 % of 56consecutive AAA patients had a positive family history of aneurysms. The characteristicfeatures of familial aneurysms are an early age of diagnosis and a higher risk of rupture.Furthermore, even though both sporadic and familial AAAs are more common in men,the patients with familial AAA are more likely to be women (see Verloes et al. 1996).Type III collagen gene mutations have been suggested to contribute to the developmentof AAAs (Kontusaari et al. 1990, Anderson et al. 1996), and other genes, such as thosefor collagenases and elastin, have also been mapped for abdominal aneurysms (seeVerloes et al. 1996). Some of the patients with familial aneurysms show decreased typeIII collagen production in cultured skin fibroblasts (Deak et al. 1992, van Keulen et al.1999), or even lack type III collagen in skin and aortic wall (Hamano et al. 1998).

2.4. Type I and III collagens in cancer of the ovary and colon

Altered synthesis and increased breakdown of extracellular proteins have been shown toplay very important roles in the invasion and growth of malignant ovarian tumors (Autio-Harmainen et al. 1993, Moser et al. 1994, Afzal et al. 1996, Wilson et al. 1996, Santalaet al. 1998). Probably due to the increased collagenolytic activity, the type I and IIIcollagen fibers are poorly organized and disintegrated in ovarian tumor tissue (Zhu et al.1993a, Zhu et al. 1995). The synthesis of type I and III procollagens is normally veryslow in ovarian tissue, and an increase of the production of these collagen types is relatedto the degree of malignancy (Zhu et al. 1993b, Kauppila et al. 1996). Some of themalignant cells even seem to have a capacity to produce type I collagen themselves (Zhuet al. 1995). Type I collagen is crucial for the invasion and metastasis of ovarian cancer,because it promotes the migration of carcinoma cells through an integrin-mediatedmechanism and induces the activation of at least MMP-2 (Fishman et al. 1998, Boyd &Balkwill 1999). Insertion polymorphism in the promoter region of the MMP-1 generesulting in greater transcriptional activity of the gene has been suggested to beassociated with at least ovarian cancer (Kanamori et al. 1999).

The increased synthesis and degradation of type I and III collagens in ovarianmalignancy can be monitored by measuring the type I and III collagen metabolites inserum, cyst fluid or ascitic fluid (Santala et al. 1998). Measurement of the circulatingaminoterminal propeptide of type III procollagen, PIIINP, has turned out valuable in theassessment of the prognosis and clinical course of the disease (Risteli et al. 1988b,Kauppila et al. 1989, Tomás et al. 1990). Elevated serum concentrations of the cross-linked carboxyterminal telopeptide of type I collagen, ICTP, reflecting increaseddegradation of type I collagen, are strongly related to impaired prognosis in patients withovarian cancer (Santala et al. 1995, Santala et al. 1999).

Altered synthesis and degradation of the extracellular matrix are also seen in humangastrointestinal malignancies, where the increased collagen synthesis takes place instromal fibroblasts (Ohtani et al. 1992). Even though the expression of type I and IIIprocollagens is increased in gastrointestinal carcinoma, the overall collagen content isdecreased (Turnay et al. 1989). Retained secretion of collagen molecules together with

36altered type I to type III procollagen ratios have been described in tumor-associatedfibroblast-like cells, which are responsible for the production of collagen in colonadenocarcinoma (Turnay et al. 1989, Turnay et al. 1991). Collagenolytic activity isincreased in colorectal malignant tissue and correlates with tumor differentiation (van derStappen et al. 1990). In various gastrointestinal cancers, the gene expression of MMP-1,which degrades particularly type III collagen, is very abundant in stromal cells, especiallyat the margin of tumor invasion (Otani et al. 1994). On the other hand, the invasive areasof human colon cancer display signs of active fibrosis and tissue contraction, which hasbeen suggested to be responsible for obstructing type colonic carcinomas (Miura et al.1993).

There is also evidence of a variant of type I and III collagens in both colon and breastcarcinoma tissue. This onco-fetal/laminin-binding collagen is a heterotrimer of α1(III)-chain, α1(I)-chain and a unique acidic chain, being completely absent in normal tissue,which makes it a possible marker of malignancy (Pucci-Minafra et al. 1993).Furthermore, α2(I) chains have been suggested to be either overmodified or completelyabsent in colon cancer tissue (Pucci-Minafra et al. 1998). The occurrence of these matrixirregularities in malignant tissue may promote tumor invasion by affecting thesupramolecular assembly of the collagenous matrix (Pucci-Minafra et al. 1998).

2.5. Type I and III collagens in colon diverticulosis

Diverticulosis of the colon is characterized by thickening of the muscle layers. The bowellumen is encroached by thickened bars of circular muscle, which divide it into a series ofsmall chambers (for reviews, see Smith 1986, Ming 1998). Surprisingly, the muscle cellsin diverticular disease appear to be normal, with no evidence of hyperplasia orhypertrophy (Whiteway & Morson 1985). There is, however, evidence of a threefoldincrease in the elastin content of the taeniae coli when compared with the controls(Whiteway & Morson 1985). Diverticular disease is also associated with increasedintraluminal pressure of the colon (Painter et al. 1965) and a deficiency of dietary fiberconsumption (Painter et al. 1971). Genetic factors are also involved, since diverticulosishas been reported in Marfan’s syndrome and Ehlers-Danlos syndrome IV (Beighton et al.1969, Suster et al. 1984), which involve mutations in fibrillin and type III collagen genes,respectively.

Type I and III collagens are the most abundant collagen types in colon tissue, beinginterspersed in the muscle layer and thus providing the tissue with tensile strength. Thereis a link between diverticular disease, the ageing process, and the decreased tensilestrength of the colonic wall. There is also evidence of different kinds of diverticulardisease, one being mainly associated with muscle abnormalities, whereas another, a non-muscle form, is possibly caused by a failure of collagen support (see Smith 1986, Ming1998). Collagen content has been suggested to decrease progressively or to be alteredupon ageing either by rearrangement of the matrix or by increasing weakness of theindividual fibers (see Smith 1986). Collagen fibrils in the left colon have been shown tobecome smaller and more tightly packed over increasing age (Thomson et al. 1987).Furthermore, the cross-linking of colonic collagen appears to increase with age and thus

37in diverticulosis (Wess et al. 1995). A high-fiber diet has been found to be associatedwith a decrease in collagen cross-linking in an animal model (Wess et al. 1996a).Furthermore, the offspring of rats on a high-fiber diet presented no colonic diverticulosis,suggesting that the maternal diet plays an important role in the development ofdiverticular disease (Wess et al. 1996b).

3. Outlines of the present study

Fibrillar collagens are the major proteins of the extracellular matrix (ECM) and providethe tissue with tensile strength. Furthermore, collagens play an important role inpathological remodeling of tissues, such as invasion of malignant tumors. At the time thepresent work was initiated, a lot of information was already available on the metabolismof type I and III collagens in various diseases. This information had mostly been gainedby measuring collagen metabolites in patient serum or other biological fluids, such asascites, pleural effusion and synovial fluid. However, the situation at tissue level hadusually only been studied with immunohistochemical techniques or hydroxyprolineanalyses, which were not able to shed light on the absolute amounts of the differentcollagen types or the state of maturation of their cross-links. The purpose of this studywas to develop new immunological methods that would allow us to investigate the cross-linked type I and III collagens in tissues and, eventually, to apply these methods to studyclinical conditions in which type III collagen, in particular, is known to play a role.

During the course of the work, atherosclerosis, abdominal aortic aneurysms, colon andovarian malignancies, and colon diverticulosis were chosen for further analysis. Thepresent results not only represent the changes in collagen metabolism and processing inselected clinical situations, but also increase our understanding of the complex nature ofcollagen metabolism in pathological conditions in general.

The specific aims of this study were as follows:

1. to characterize the cross-linking of type I and III collagens and the processing of typeIII pN-collagen in human atherosclerotic plaques

2. to examine the possible role of fibrillar collagens, particularly type III collagen andpN-collagen, in the pathogenesis of human abdominal aneurysms

3. to study the cross-linking and processing of type I and III collagens in malignantprocesses, such as cancer of the ovary and colon

4. to study cross-linking of type I and III collagens in diverticulosis of the human colon

4. Materials and methods

4.1. Tissue samples

The human leiomyomas for the purification of type III collagen telopeptide were obtainedfrom the Department of Obstetrics and Gynecology, Oulu University Hospital.

Altogether 17 atherosclerotic plaques from the carotid (n = 15) and femoral (n = 2)arteries and 8 pieces of atherosclerotic aorta (I) were obtained from routine surgery at theDepartment of Surgery, Oulu University Hospital. One piece of healthy aorta, obtainedfrom the Department of Pathology, Oulu University Hospital, was added to the study tovalidate the method. All the samples were stored at -20°C until processed.

Nine pieces of abdominal aortic aneurysms (II) were collected during operations at theDepartment of Surgery, Oulu University Hospital. The control material for this studyconsisted of ten pieces of extremely atherosclerotic abdominal aorta from autopsies at theDepartment of Forensic Medicine, Oulu University Hospital. All the samples were storedat -20°C until processed.

Altogether 24 pieces of colonic diverticulosis, six tissue samples of cancer of the colonand nine controls (III) were obtained from routine operations at the Department ofSurgery, Oulu University Hospital. The histology of the samples was confirmed by apathologist (TJK, see paper III) at the Department of Pathology, Oulu UniversityHospital. All the samples were stored at -20°C until processed.

The ovarian cancer material (IV) obtained from the Department of Obstetrics andGynecology, Oulu University Hospital, consisted of 18 samples of benign serouscystadenomas and malignant adenocarcinomas. The diagnoses were confirmed by routinehistopathological analysis at the Department of Pathology, and the samples were stored at-20°C. Parts of the tissue were used for immunohistochemical analysis and thereforefixed in 10 % buffered formalin, embedded in paraffin and cut into 5 µm sections.

404.2. Purification of the aminoterminal telopeptide of type III collagen

The aminoterminal telopeptide of type III collagen, IIINTP, was originally purified fromhuman uterine leiomyoma, which was first cut into small pieces, homogenized, andtreated with 6 mol/L urea in 0.05 mol/L Tris/HCl buffer (pH 7.4) at +4°C for 24 hours.The residue was then washed with distilled water and reduced with NaBH4 in 0.1 mol/Lsodium phosphate buffer, pH 7.4 (see Eyre 1987). The fat was extracted withacetone/methanol, and the residue washed briefly with distilled water and 0.2 mol/Lammonium bicarbonate (NH4HCO3), and freeze-dried.

The insoluble pellet was suspended in 500 ml of 0.2 mol/L NH4HCO3, denatured at+70°C for 1 hour, and treated with 1 mg of trypsin (Worthington; TPCK-treated) per 100mg of tissue at +37°C for 4 hours. After this, the residue was re-denatured, homogenizedwith an Ultra-Turrax homogenizer and digested with the same amount of trypsinovernight. The residual trypsin activity was destroyed at +70°C for 30 minutes, and thematerial was centrifuged at 10 000 g for 30 minutes. The supernatant was collected andfreeze-dried.

IIINTP was purified using Sephacryl S-100 (Pharmacia) gel filtration (equilibrated in0.2 mol/L NH4HCO3 at room temperature), chromatography on a reverse-phase C8column (Vydac) (0.4 % ammonium acetate + 0 → 60 % acetonitrile, pH 7.4), and finally,Sephacryl S-300 (Pharmacia) gel filtration (equilibrated in 0.2 mol/L NH4HCO3 at roomtemperature). Polyacrylamide gel electrophoresis was performed on 18 % gels withsodium dodecyl sulfate (SDS) to determine the purity and size of the purified peptide.The exact concentration was determined by amino acid analysis after acid hydrolysis.IIINTP was deblocked with pyroglutamate aminopeptidase (Sigma) (Podell & Abraham1978, Hörlein et al 1979) and sequenced.

4.3. Production of antiserum

Antibodies against the aminoterminal telopeptide region of type III collagen were raisedin New Zealand White rabbits by giving them purified or synthetic peptides conjugatedwith bovine thyroglobulin and mixed with equal amounts of 0.9 % NaCl and Freund’sincomplete adjuvant (Sigma) as intradermal injections at three- to four-week intervals.The conjugate was prepared by dissolving 2.5 mg of antigen and 10 mg of bovinethyroglobulin to 500 µl of 10 mM sodium phosphate buffer, to which 100 mg carbodi-imide (Sigma) diluted to 1 ml of sodium phosphate buffer was added. The mixture wasallowed to stay on ice in a mixer for two hours and dialyzed overnight against PBS at pH7.2. Finally, 100 to 200 µl of this conjugate-antigen mixture per one animal was used forimmunization.

414.4. Radioimmunoassay for IIINTP

The purified IIINTP was labelled with 125I by the Chloramine-T method, and free iodinewas separated from the iodinated antigen by disposable Sep pak C18 cartridges (Waters)(see Risteli & Risteli 1987). 100-µl aliquots of a properly diluted sample were incubatedwith 200 µl of the antiserum dilution and 200 µl of 125I-IIINTP solution for 2 h at 37°C.After that, 0.5 ml of the second antibody in 10 % polyethylene glycol (M.W. 6 000) wasadded, and the tubes were incubated at 4°C for 30 min. The samples were centrifuged at2000 x g for 30 min at 4°C and the radioactivity in the precipitates was counted.

4.5. Other immunoassays for type I and III collagen domains

Radioimmunoassays for the aminoterminal propeptide of type I procollagen (PINP)(Melkko et al. 1996), the carboxyterminal propeptide of type I procollagen (PICP)(Melkko et al. 1990), the carboxyterminal telopeptide of type I collagen (ICTP) (Risteliet al. 1993), and the aminoterminal propeptide of type III collagen (PIIINP) (Risteli et al.1988a) were used to determine their concentrations, when required. In addition, two in-house methods were used for analyses: an immunoassay for the synthetic peptide SP 6having the amino acid sequence DVKSGVAVGGLAGY (Neosystem Laboratories) fromthe aminoterminal telopeptide of the α1-chain of type III collagen (amino acid residuesnos. 159 – 162) and an immunoassay for the synthetic peptide SP 4 having the aminoacid sequence SAGFDFSFLPQPPQEKY (Neosystem), derived from the carboxyterminaltelopeptide of the α1-chain of type I collagen (amino acid residues nos. 1193 – 1208 +tyrosine residue). Samples in duplicate were diluted appropriately, and the analyses wereperformed as described above.

4.6. Immunohistochemical staining

The anti-aminoterminal propeptide of type I procollagen (anti-PINP), the anti-carboxyterminal telopeptide of type I collagen (anti-ICTP), and the anti-aminoterminalpropeptide of type III procollagen (anti-PIIINP) were used for immunohistochemicalanalysis. These antibodies were purified by cross-adsorption with several extracellularmatrix proteins (7S domain of human type IV collagen, P1 fragment of human laminin,different type I and III collagen domains) and with the specific antigen in questioncoupled to CNBr-activated Sepharose 4B.

The immunohistochemical stainings for type I and III collagens were carried out usingthe avidin-biotin-immunoperoxidase technique (Zhu et al. 1993a, Zhu et al. 1995).Deparaffinized, rehydrated and PBS-washed sections were treated with 0.4 % pepsin(Sigma) in 0.01 mol/L HCl, pH 2, at 37°C for 30 minutes to 2 hours, washed with PBSand incubated in 0.3 % H2O2 in 100 % methanol for 30 minutes. Afterwards, the sectionswere washed with distilled water and incubated with 2 % goat serum for 30 minutes and,finally, with primary antibody diluted 1:25 (anti-ICTP) and 1:100 (anti-PINP and anti-

42PIIINP) in PBS in a moist chamber for 2 hours at room temperature. Then the specimenswere incubated for 30 minutes with biotinylated anti-rabbit immunoglobulin diluted in1:400 in PBS and for 30 minutes with an avidin-peroxidase complex, washed with PBSand treated with 3,3-diaminobenzidine tetrahydrochloride and H2O2 in Tris buffer, pH7.4. The samples were then washed with distilled water and stained with hematoxylin.

4.7. Chromatographic analyses of type I and III collagen antigens intissue samples

Sephacryl S-300 gel filtration column was used to analyze the soluble tissue extracts. 2ml of sample (100 mg/ml) was put to a column equilibrated in PBS-Tween at roomtemperature, and fractions of 2 ml were collected at 20-minute intervals. These fractionswere used for PIIINP, PINP and PICP analyses after suitable dilutions.

The molecular sizes of telopeptides of insoluble cross-linked collagens, together withthe aminoterminal propeptide antigens of type III pN-collagen, were characterized on aSephacryl S-100 gel filtration column equilibrated in 0.2 mol/L NH4HCO3 at roomtemperature. 1 ml of sample (10 mg/ml) was analyzed, and fractions of 2 ml werecollected at 20-minute interval. ICTP, IIINTP, PIIINP, SP4 and SP6 were assayed inproperly diluted fractions (see chapters 4.4. and 4.5.).

4.8. Hydroxyproline measurements

The content of hydroxyproline was measured as described previously (Kivirikko et al.1967). The samples were hydrolyzed overnight in 6 mol/L HCl at 120°C. The hydrolyteswere then evaporated to dryness and diluted in 2 ml of distilled water. pH was adjusted toneutral by using 0.005 mol/L KOH. 1.5 g of KCl was used to saturate the samples, and250 µl of 10 % L-alanine and 500 µl of potassiumborate buffer (1 mol/L H3BO3, 3 mol/LKCl, pH 8.7) were added. The samples were stirred and incubated at room temperaturefor 20-30 minutes. Then, 500 µl of 0.2 mol/L chloramin-T (Riedel-deHaën) (2.82 g/ 50ml 1,3-propandiol) was added and the solution was allowed to incubate for 25 minutes.The reaction was stopped by adding 1.5 ml of 3.6 mol/L Na2S2O3. After this, 2.5 ml oftoluene was added, the samples were stirred and centrifuged at 2000 x g, and the upperphase was removed. The samples were then incubated in boiling water for 30 minutes,after which toluene was added and the samples were re-centrifuged as described above.After centrifugation, 2 ml of the upper phase was pipetted and mixed with 0.8 ml ofEhrlich´s reagent (Sigma) (120 g of p-dimethylaminobentsaldehyde, 400 ml of ethanoland 27.4 ml of H2SO4), and incubated for 30 minutes. Absorbances were measured at 560nm, and the amount of hydroxyproline was estimated from the standard curve (standards1 µg, 2.5 µg, 5 µg and 10 µg of L-hydroxyproline (S.A.F. Hoffmann – La Roche & Co.LTD.) in 2 ml of distilled water (standards prepared as the samples except forhydrolysis).

434.9. Investigation of the proteolytic fragments of PIIINP

PIIINP was purified from human ascitic fluid as described earlier (Niemelä et al. 1985,Risteli et al. 1988a) and digested with trypsin at 37°C after heat denaturation. A smallertrypsin-generated fragment of PIIINP, probably corresponding to the most aminoterminalpart of the monomeric PIIINP, was purified from the human uterine leiomyoma withSephacryl S-100 gel filtration, chromatographies on a reverse-phase C8 column (0.4 %ammonium acetate + 0 → 75 % acetonitrile, pH 7.4), DEAE (50 mmol/L ammoniumacetate + 0 → 0.3 mol/L NaCl, pH 5), and finally, a reverse-phase C18 column (0.1 %TFA + 0 → 70 % 2-propanol). The effect of trypsin treatment on the antigenic activity ofPIIINP was studied further in radioimmunoinhibition assay. The molecular sizes of thesetrypsin-generated peptides were characterized by polyacrylamide gel electrophoresis andSephacryl S-100 gel filtration.

4.10. Statistics

Statistical analyses were carried out using the SPSS analysis tool and CIA confidenceinterval analyses. Spearman’s rank correlation was used for correlation analysis. Meansand 95 % confidence intervals were calculated. Non-parametrical Mann-Whitney U testand one-way ANOVA with corrected LSD (Bonferroni) test were used for estimatingstatistical significances when appropriate.

4.11. Ethical considerations

Approvals of the local ethics committee for the collection of tissue samples wereobtained by our clinical collaborators for the studies I, II and IV and by our own researchgroup for study III. The tissue samples were obtained during routine surgical operations,and nothing else was removed from the patients. In the case of cadavers, only the piecesof tissue removed for diagnostic purposes were used.

5. Results

5.1. Purification of the human cross-linked aminoterminal telopeptideof type III collagen (IIINTP)

The cross-linked aminoterminal telopeptide of type III collagen, IIINTP, was purifiedfrom human uterine leiomyoma after extraction with urea, reduction with NaBH4, anddigestion with TPCK-treated trypsin, followed by a series of Sephacryl S-100 and S-300gel permeations and C8 reverse-phase chromatography on HPLC. The purity and size ofthe peptide were determined by SDS-polyacrylamide gel electrophoresis. Aminoterminalamino acid sequencing yielded a sequence equivalent to the aminoterminal telopeptide ofhuman type III collagen (QYDSYDV). Without deblocking with pyroglutamicaminopeptidase, the obtained amino acid sequence GAAGI corresponded to thecarboxyterminal triple-helical area of α1(III) containing a lysine residue involved inintermolecular cross-linking. Cross-link analysis of the IIINTP antigen indicated that atleast 80 % of the cross-links were hydroxylysylpyridinoline (unpublished data, courtesyof Jason Mansell, Bristol University). With the purified IIINTP antigen, aradioimmunoassay for the cross-linked aminoterminal telopeptide of type III collagenwas established.

5.2. New radioimmunoassays for type III collagen

A radioimmunoassay for IIINTP from human uterine leiomyoma was developed based onpolyclonal antibodies and iodine-labeled IIINTP antigen. A 1:400 dilution of theantibodies was chosen, giving 53 % binding. In paper IV, another antiserum with adifferent dilution (1:200) but similar immunological properties was used. Serum samplesdid not give inhibition in the assay, indicating that the physiological degradation productof type III collagen likely to exist in the circulation differs from the trypsin-generatedtelopeptide antigen. The intra- and interassay coefficients of variation were tested byusing 100 µl trypsin-digested human colon tissue diluted 1:1000. The intra- andinterassay coefficients of variance were 6 % and 10 %, respectively.

45An immunoassay for the synthetic peptide SP 6, having the sequence

DVKSGVAVGGLAGY from the aminoterminal telopeptide of the α1-chain of type IIIcollagen, was also established in the same way as the IIINTP assay. A 1:2000 dilution ofthe antibodies was chosen, giving 45 % binding in the assay. In this assay, the serumsamples gave linear inhibition, and 1:2 dilution of serum (about 50 % inhibition) wasused to determine the intra- and interassay variations, which were 4.3 and 11.5 %,respectively.

5.3 Type I and III collagens in atherosclerotic plaques (I)

Trypsin digestion was found to release effectively the cross-linked type I (ICTP) and typeIII (IIINTP) collagen telopeptide antigens from atherosclerotic plaques into analysis (seeFigure 1 in paper I). The ICTP and IIINTP antigens were partially purified from thetrypsin-digested plaque material, and both these cross-linked telopeptides were found tobe similar to those of the corresponding standard antigens, indicating mature cross-linking of these collagen types (see Figure 2 in paper I). In addition, the samples gave alinear inhibition in the ICTP and IIINTP assays. Furthermore, the IIINTP and ICTPantigens in the atherosclerotic plaques corresponded in size to the trivalent, fully cross-linked peptide standards, when studied by gel filtration (see Figure 5 in paper I).

The amount of type III collagen (calculated as a percentage of the sum of types I andIII, respectively), was 61 % (CI 58 – 65) in human atherosclerotic plaques (n = 17) and56 % (CI 44 – 68) in atherosclerotic aortic specimens (n = 8). The one healthy youngaorta contained 72 % of type III collagen. For comparison, in human leiomyoma andplacenta, the proportions of type III collagen calculated in the same way were 35 % and33%, respectively. In the calcified fraction of the atherosclerotic plaques, the meanproportion of type III collagen was 54 %.

The amount of type III pN-collagen was calculated from the molar amounts of PIIINPand IIINTP. To validate the results obtained, the effect of trypsin digestion on theantigenicity and size of the PIIINP antigen was studied. After trypsin digestion, atruncated form of the monomeric propeptide chain appeared, indicating partial cleavageof the propeptide leading to a 50 % decrease in the immunoreactivity of this peptide (seeFigures 3 and 4 in paper I). Thus, the trypsin loss of PIIINP was corrected by multiplyingthe obtained PIIINP concentrations by two. The amount of type III pN-collagen in theinsoluble matrix of the plaques was 0.0081 % (CI 0.0042-0.0121) (see Table 2 in paper I).

In the soluble tissue extracts, type III pN-collagen accounted for 42 % of the total typeIII collagen content. The mean concentrations of type I procollagen propeptides werevery low (PINP 0.13 µg/g and PICP 1.18 µg/g) in the soluble tissue extracts, indicating alow rate of type I procollagen synthesis. The mean PIIINP concentration was 0.31 µg/g.Gel filtration analysis of the soluble tissue extracts revealed that the type III collagen wasin the form of pN-collagen, whereas PINP was always present as a free propeptide. ForPICP, in turn, nearly equal amounts of authentically cleaved propeptide and type I pC-collagen were seen (see Figure 6 in paper I).

465.4. Type I and type III collagens in human abdominal

aortic aneurysms (II)

The average amounts of type I and type III collagens expressed per total hydroxyprolinewere 5.3 and 3.4 times higher in the abdominal aortic aneurysms (AAA) than in theatherosclerotic aortic samples (AOD), respectively (see upper panel of Figure 2 in paperII). However, the mean hydroxyproline concentrations were practically the same in bothAAAs (7.8 ± 1.4 mg/g) and AODs (7.8 ± 1.2 mg/g). The relative amount of type IIIcollagen was 44 % (CI 38-41) in AAAs (n = 9) and 60% (CI 53-67) in AODs (n = 10) (p< 0.001), indicating a significantly smaller relative amount of cross-linked type IIIcollagen in aneurysm tissue (see lower panel of Figure 2 in paper II). The concentrationsof ICTP and IIINTP antigens correlated significantly in both AAAs (r = 0.867; p < 0.002)and AODs (r = 0.648; p < 0.043). In both groups, most of the IIINTP antigen eluted asone peak corresponding to the elution position of the trivalently cross-linkedaminoterminal telopeptide antigen of type III collagen (see Figure 3 in paper II).

The mean absolute amounts of PIIINP in the insoluble fractions of AAA and AODsamples were 14.9 µg/g and 0.27 µg/g, respectively (see Figure 7A in paper II). Theaverage relative amount of type III pN-collagen in the insoluble collagenous matrix wasover 12 times higher in AAAs (0.74 %) than in AODs (0.06%) (see Table 1 in paper II).This difference was also evident in the gel filtration analysis, where the PIIINPantigenicity eluted mostly in one major peak in AAAs, whereas no propeptide derivedantigens were seen in AODs (see Figure 3 in paper II). The major peak of PIIINPantigenicity in insoluble AAA tissue shown in Figure 3 in paper II corresponded in sizeto the partially cleaved PIIINP molecule (see chapter 5.3. and Figure 3 in paper I). Thus,we were able to use the same correction factor 2 as in paper I. However, some PIIINPantigenicity, clearly smaller in size, was found in the gel-filtrated samples (see Figure 3in paper II). When purified from human uterine leiomyoma, this smaller peptide wasfound to be a single monomeric chain corresponding to the Col 1 part of theaminoterminal propeptide (see Figure 5 in paper II). This smaller form of digested PIIINPreacted about 30 times less intensely in the radioimmunoinhibition assay for the intactaminoterminal propeptide of type III collagen than did the larger trypsin-generatedPIIINP (see Figure 6 in paper II).

The mean absolute amounts of PIIINP in the soluble tissue extracts of AAA and AODwere 0.68 µg/g and 1.15 µg/g (see Figure 7B in paper II). As much as 78 % of PIIINPwas found to be soluble in AODs, whereas the corresponding percentage in AAAs wasonly 6 %. In the soluble tissue extracts, the relative amount of type III pN-collagenaccounted for 69 % and 41 % in AAAs and AODs, respectively (see Table I in paper II).The solubilized PIIINP antigens were studied on Sephacryl S-300 gel exclusionchromatography; most of the PIIINP antigenicity in AAAs eluted as type III pN-collagen,whereas AODs also appeared to contain free propeptide (see Figure 4 in paper II).

Immunohistochemical studies (M.K. Bode, Y. Soini, J. Melkko, J. Satta, L. Risteli, J.Risteli, unpublished data) indicated abundant PIIINP staining in the media of abdominalaortic aneurysms, whereas type I pN-collagen localized in the intima in both AAAs andAODs (Fig. 5). The healthy aorta, in turn, showed no immunoreactivity for either PIIINPor PINP (not shown).

47

Fig. 5. Immunohistochemical type I (A) and type III (B) pN-collagen staining in abdominalaortic aneurysms. Type I (C) and type III (D) pN-collagen staining in atheroscleroticabdominal aorta. Hematoxylin counterstain, original magnification, x 10, i = intima, m =media, a = adventitia.

485.5. Type I and III collagens in colon diverticulosis and

colon cancer (III)

The total collagen content was measured in trypsin-digested colon tissue byhydroxyproline analysis, which showed about 50 % less collagen in malignant tissue thancontrols (see Figure 1A in paper III). The amount of fibrillar type III collagen measuredby IIINTP presented a similar pattern: the cancer samples showed less cross-linked typeIII collagen than did the controls (0.14 mg/g and 0.36 mg/g, respectively). For type Icollagen, no such significant difference was found (see Table I in paper III). Theproportion of type III collagen (as a percentage of the sum of type I and III collagens)was significantly decreased in malignant samples (see Table I in paper III). Thecorrelations were 0.884 between hydroxyproline and ICTP, 0.940 betweenhydroxyproline and IIINTP, and 0.877 between ICTP and IIINTP. The amount of fibrillartype III pN-collagen was also assessed (calculated from the molar amounts of PIIINP andIIINTP). The mean amounts of type III pN-collagen were 0.48 % (range 0.00-2.97), 1.71% (range 0.15-5.96) and 0.40 % (range 0.05-1.38) in diverticulosis, cancer samples, andcontrols, respectively. However, due to the obvious large variation, there were nostatistically significant differences between the groups analyzed.

The trypsin-digested type I and III collagen antigens were characterized by gelfiltration analysis on Sephacryl S-100, to further analyze their molecular sizes and thestate of cross-linking. In order to do that, immunoassays for ICTP, IIINTP, SP4 and SP6were applied. In the samples of malignancy, the ICTP antigen eluted in one peak,corresponding to the trivalently cross-linked mature type I collagen. SP4, however, elutedin two peaks, the latter representing immaturely cross-linked type I collagen. In thediverticulosis and control samples, no such tendency was evident (see Figure 2 in paperIII). In all the samples, type III collagen eluted in one peak, representing the maturelycross-linked type III collagen, IIINTP (see Figure 2 in paper III).

The aminoterminal propeptide domains of type I and III procollagens, PINP andPIIINP, respectively, were quantified in the soluble fraction, where the molecules are intransit from biosynthesis to collagen fibers. The mean PINP concentrations weresignificantly higher in the colon cancer samples than in the controls (1.57 µg/g and 0.24µg/g, respectively). The mean PIIINP values, in turn, were significantly higher indiverticulosis than in the controls (1.73 µg/g and 0.83 µg/g, respectively). The proportionof type III collagen (as a percentage of the sum of type I and III collagen) wassignificantly decreased in the tumor samples (see Table II in paper III).

The sizes of the soluble PINP and PIIINP antigens were assessed by gel filtrationanalysis on Sephacryl S-300. The major PINP and PIIINP antigenicities in the tumorsamples eluted as free propeptides related to the ongoing synthesis. In diverticulosis,PINP eluted as a free propeptide, whereas the profile for PIIINP showed practicallyidentical amounts of type III pN-collagen and free aminoterminal propeptide. In thecontrol samples, most of the PINP and PIIINP antigenicities eluted in their pN-form (seeFigure 3 in paper III).

495.6. Type I and III collagens in ovarian tumors (IV)

The tissue contents of both ICTP and IIINTP were lower in the malignant than in thebenign tissues. For type I collagen, this tendency was even more obvious: the amount ofICTP in the benign cystadenomas was about 8 times higher than that in adenocarcinomas(mean 0.57 mg/g and 0.07 mg/g, respectively, see Table 2 in paper IV). Furthermore, theamount of total collagen measured by hydroxyproline analysis showed a nearly five-folddecrease in the malignant samples compared to the benign ones (see Table 2 in paper IV).There was a strong correlation between hydroxyproline and IIINTP (0.96),hydroxyproline and ICTP (0.98), and IIINTP and ICTP (0.96).

The amount of type III collagen (calculated as a percentage of the sum of type I and IIIcollagens) was 35 % (CI 28-42 %) in the benign ovarian tumor tissue. In the insolubletissue, the PIIINP/IIINTP ratio was used to estimate the amount of type III pN-collagen,which was clearly elevated in the malignant samples (3.20 % in adenocarcinomas and0.70 % in cystadenomas, see Table 3 in paper IV). There was no statistically significantdifference, however, in the total amount of PIIINP in the trypsin-digested ovarian tumorsbetween the two groups (see Table 3 in paper IV). In the soluble extracts, the mean PINPconcentration was higher in the carcinoma samples than in the benign tumors (2.45 µg/gand 1.00 µg/g, respectively). No such tendency was evident for PIIINP (2.04 µg/g formalignant and 1.44 µg/g for benign samples).

The sizes of the type I and III collagen telopeptide fragments were analyzed on gelexclusion chromatography. In the benign samples, most of the ICTP and IIINTP antigenseluted in one major peak, corresponding to the trivalently cross-linked telopeptideantigens (see Figures 1A-C and 2A in paper IV). In the malignant samples, however,there were also smaller forms present, indicating immaturely cross-linked type I and IIIcollagens (see Figures 1D-F and 2B in paper IV).

The soluble PINP and PIINP antigens were also characterized by gel filtration. In boththe benign and the malignant samples, the major PINP antigenicity eluted as a freepropeptide, whereas PIIINP was present in its pN-form (see Figure 3 in paper IV).

In the benign samples, PINP staining showed type I pN-collagen mainly at theepithelial-stromal junction, whereas in poorly differentiated tumors PINP was seen nearthe malignant cells. Mature cross-linked type I collagen (ICTP staining) was weaker inthe malignant samples compared to the benign ones. Type III pN-collagen stainingresembled that seen for type I pN-collagen. Immunostainings also revealed disorganizedtype I and type III collagen fibers in malignant tumors (see Figure 4 in paper III).

50Table 3. Summary of the quantitative and qualitative findings of type I and III collagensin the human tissues studied.

Type III/ Type III pN/ Immaturely cross-linked(Type III +I) % Total Type III % forms of

________________________________________________________

type I type III

Atheroscleroticplaques (n = 17) 61 0.01 -/+ -/+

AortaAOD (n = 10) 60 0.06 -/+ -/+AAA (n = 9) 44 0.74 -/+ -/+

Ovarian tumorBenign (n = 8) 35 0.70 -/+ -/+Malignant (n = 10) 42 3.20 +++ ++

ColonCancer (n = 6) 53 1.71 +++ -/+Diverticulosis (n = 24) 62 0.48 -/+ -/+Control (n = 9) 65 0.40 -/+ -/+

Placenta (n = 1) 33 8.50 ND NDLeiomyoma (n = 1) 35 1.41 ++ ++

ND = not determined-/+ = no or minor amounts ; ++ = moderate, and +++ = large amounts of immature forms,determined by gel exclusion chromatography by antigen size (qualitative analysis)

6. Discussion

6.1. Insoluble vessel wall collagens – methodological andstructural aspects

One of the most striking events of atherosclerosis is the accumulation of fibrillarcollagens, which is why the quantities of especially type I and III collagens inatherosclerotic lesions have been extensively investigated (see Barnes 1985, Rauterberget al. 1993 for reviews). However, the evidence has remained controversial. The initialwork by McCullagh & Balian (1975) suggested type III collagen to predominate innormal arterial wall. Since then, research results have indicated type I collagen to be themajor collagen type in both normal and atherosclerotic arteries (see Barnes 1985). Ourstudy (paper I), in turn, suggested that type III collagen predominates in atheroscleroticlesions.

The amounts of collagen types found in arterial wall may depend on the method ofextraction, and type III collagen seems to be more easily extractable than type I collagen(Szymanowicz et al. 1982). Pepsin, for example, cleaves collagen molecules from thetelopeptide region, which contains the sites for intermolecular cross-linking. Thus, pepsindigestion may result in different degrees of solubilization due to the different extents ofintermolecular cross-linking (see Rauterberg et al. 1993). Most previous studies on thequantitative ratios of type I and III collagens have been made by using cyanogen bromide(CNBr), which cleaves after the methionine residue (see Rauterberg et al. 1993). Thismethod has several advantages compared to pepsin digestion, one of the most importantbeing the high yield of soluble collagenous peptides. However, the cleavage of thecollagen chains may be incomplete due to methionine oxidation. In addition, the CNBrmethod is very often based on simple staining of the cleaved peptide fragments on SDS-PAGE, which is not ideal for collagenous sequences. Recently, improved methods basedon the quantitation of collagens by capillary electrophoresis after CNBr digestion havebeen developed (Deyl et al. 1997). Trypsin is known to cleave after the amino acidslysine and arginine, liberating the cross-linked telopeptides into analysis. Furthermore,previous studies have shown that trypsin solubilizes 99 % of cross-linked type I collagen(Kuboki et al. 1981). Due to these reasons, in addition to the easy availability and lowprice of the enzyme, trypsin was selected for these studies.

52The presence of differently organized type I and III collagen fibrils could theoretically

influence the solubility of collagens and the interpretation of the data. Two populations offibrils, the smaller ones about 28 nm and the larger ones about 42 nm in diameter, havebeen described in the aortic wall by Shiraishi et al. (1980). In contrast, Raspanti et al.(1989) detected only one kind of fibril population in the aortic media, but found evidenceof two morphologically different fibril types in other tissues, one arranged intounidirectionally packed fibrils and the other with a helical conformation. Raspanti et al.(1989) further suggested that unidirectional packing would be characteristic of skeletaltissues, whereas helical fibrils might be present in, for example, skin and vessel walls.There is evidence to suggest that helically arranged fibrils are present in tissues rich intype III collagen, but there are also exceptions to this rule (see Rauterberg et al. 1993).

Henkel (1996) previously investigated the supramolecular assembly and cross-linkingof the carboxyterminal telopeptide of type III collagen. He suggested that several dimerscontaining two quarter-staggered molecules with hydroxylysine or lysine-derived cross-links can be thought as being joined by disulfide bonds, resulting in a tetrameric structurestabilized by lysine derived cross-links connecting the N-telopeptides of different type IIIcollagen molecules (Henkel 1996). The different ways of organization and cross-linkingof fibrillar collagens are likely to influence the mechanical properties of tissues and thustheir functions. They may also have an effect on the results and their interpretations instudies aiming to quantify collagens in tissue digests.

The total collagen contents of tissue samples have been traditionally measured byhydroxyproline analysis (Kivirikko et al. 1967). Many different types of collagens havebeen described in vessel walls (see Barnes 1985, Rauterberg et al. 1993), and since theamount of one collagen type may increase at the same time as that of another decreases,hydroxyproline content cannot give any easily interpretable information on specificcollagen types. In paper II, the total amount of hydroxyproline was not significantlydifferent between AAAs and AODs, similarly to the finding published by Minion et al.(1994). However, type I and III collagen specific analyses showed the amounts of the twofibrillar collagen types to be larger in AAAs than in undilated atherosclerotic aortas.Thickening of the intima due to atherosclerosis may increase the amounts of at least typeV, VI (Katsuda et al. 1992) and VIII collagens (Plenz et al. 1999), which may explain thelower amounts of type I and III collagens per hydroxyproline in AODs. Furthermore,hydroxyproline is not collagen specific, since several other proteins, such as elastin, theC1q component of complement, scavenger and some other cell surface receptors, andacetylcholinesterase, contain hydroxyproline (see Kielty et al. 1993).

6.2. Type I and III collagens – soil for atherosclerosis

Fibrillar type I and III collagens constitute a major part of the total plaque protein (seeBarnes 1985 for a review) and occupy about 80 % of the section area in samples ofhuman coronary restenotic lesions (Pickering et al. 1996). Thus, these collagen types playan important role in plaque growth and arterial lumen narrowing. In addition, collagenstimulates atherogenesis by serving as a depot for proatherogenic peptides and molecules,such as lipoproteins, growth factors and cytokines, and modulates the responses of

53inflammatory cells and SMC proliferation (see Rekhter 1999). Furthermore, injury to theendothelium leads to activation of the coagulation system, since the thrombogenicmaterial of the plaque - lipids and collagens in particular - is exposed, resulting inthrombus formation.

A typical feature of advanced atherosclerosis is calcification, which shares manyhistological features with bone and involves many bone-related proteins, such asosteopontin, osteonectin and osteocalcin (Hirota et al. 1993, Bini et al. 1999). These bonematrix proteins, apolipoproteins, fibrinogen, and proteases, are suggested to interact inthe formation of calcification and thus the progression of the atherosclerotic lesions (Biniet al. 1999). Furthermore, intraplaque thrombus is suggested to contribute to arterialcalcification as a source of osteocalcin (Bini et al. 1999). Osteonectin promotes bindingof calcium to collagen (Termine et al. 1981), and vascular calcification is, in turn,promoted by type I collagen and fibronectin in in vitro conditions (Watson et al. 1998).In the current quantitative analyses of type I and III collagens in atherosclerotic plaques,no marked differences were seen between the non-calcified and calcified insoluble type Iand III collagens. Thus, despite the reported similarities in bone mineralization andvascular calcification, the presence of type III collagen in mineralized aortic tissue doesnot suggest the calcification process to be identical with that in bone.

The present study demonstrated that the amount of partially processed type IIIprocollagen molecules with a retained aminoterminal propeptide (type III pN-collagen) isnegligible in atherosclerotic plaques. In addition, the synthetic activity of these collagentypes was found to be very low, as suggested by the small concentrations of freepropeptides in the soluble fraction, where collagen molecules are in transit frombiosynthesis to collagen fibers. Thus, the present data supports the idea of metabolicallyinert, silent plaques (Kockx et al. 1998). Type I and III collagens appear to be fully cross-linked in atherosclerotic plaques. This is consistent with the previously reported increasedactivity of lysyl oxidase, an enzyme responsible for the cross-linking of collagen in therabbit aorta with diet-induced atherosclerosis (Kagan et al. 1981). Thus, mature cross-linking reflects the fibrotic process seen in atherosclerosis.

Several previous studies have implicated matrix metalloproteinases (MMPs), whichare enzymes responsible for collagen degradation, in the destabilization and rupture ofatherosclerotic lesions (Henney et al. 1991, Galis et al. 1994, Brown et al. 1995, Nikkariet al. 1995, Shah et al. 1995, Li et al. 1996, Sukhova et al. 1999). It can be suggested thatthe synthesis and degradation of collagens in the plaque are relatively balanced processes,but over time the degradation of collagens may overcome the synthesis, resulting inplaque rupture. On the other hand, if collagen synthesis decreases due, for example, to thereported inhibitory effect of monocyte prostaglandins (Fitzsimmons et al. 1999) and T-lymphocyte-derived IFN-γ (Amento et al. 1991) on collagen production, or apoptosis ofSMCs (Bauriedel et al. 1999), an acute lack of plaque-stabilizing collagenous matrixmight develop and result in complications (see Lee & Libby 1997, Rekhter 1999). This isconsistent with the finding that macrophages, T-lymphocytes and activated monocytesare particularly abundant at the site of plaque rupture, whereas the number of SMCs isdecreased (van der Wal et al. 1994, Kovanen et al. 1995). Many reports have suggestedfibrous plaques to be clinically more stable than those containing large pools of lipids(see van der Wal & Becker 1999). Feeley et al. (1991) reported that even thoughasymptomatic carotid plaques contain more fibrous material (88 %) than symptomatic

54plaques (66 %), no relationship between plaque composition and the number of ischemicevents was evident.

The development of atherosclerosis and restenosis is a very complex process involvingmany different but interrelated factors. Therefore, further studies on the regulation ofcollagen synthesis, processing and degradation are necessary to reveal the underlyingmechanisms.

6.3. Abdominal aortic aneurysms and atherosclerosis

Because atherosclerosis is an almost universal finding in the walls of aneurysms, AAAshave been thought to be caused by atherosclerosis. In addition, the risk factors for thesetwo clinical conditions – high blood pressure, high serum cholesterol, and cigarettesmoking – are the same (Reed et al. 1992).

Proliferation of SMCs is a typical feature in atherosclerosis (see Ross 1993), whereasthe density of these cells in aneurysmal wall is low due to apoptosis (López-Candales etal. 1997, Henderson et al. 1999). SMCs together with adventitial fibroblasts are thesource of collagens in the aortic wall. Atherosclerosis is characterized by accumulation ofthe extracellular matrix. Most of the existing data on AAAs suggest increasedprocollagen expression (McGee et al. 1991, Mesh et al. 1992, Minion et al. 1993). Theproportion of collagen has been reported to be increased (Menashi et al. 1987, Rizzo etal. 1989, Baxter et al. 1994, He & Roach 1994), unchanged (McGee et al. 1991) or evendecreased (Sumner et al. 1970) in AAAs compared to undilated aortas. In our study, nosuch comparison was possible due to the different extents of calcification between theAOD and AAA samples. However, since the amounts of aminoterminal propeptide oftype III procollagen, PIIINP, in the soluble tissue extracts of AAAs and AODs did notdiffer, the synthesis of type III procollagen does not seem to be markedly increased indilated aorta.

Type III collagen molecule with a retained aminoterminal propeptide is often found onthe surface of collagen fibrils. When the fibril has reached its final diameter, theaminoterminal propeptide is retained, thus preventing the attachment of other collagenmolecules to the fibril surface (Fleischmajer et al. 1985). Satta et al. (1995) found higherserum PIIINP concentrations below than above the aneurysmal region, which alsoprovided a rationale for us to investigate type III collagen at tissue level. Indeed, thecurrent study (paper II) indicated that the amount of type III pN-collagen is increased inabdominal aortic aneurysms, thus suggesting the presence of thinner than usual type IIIcollagen fibers. This might result in increased susceptibility of collagen to proteases,which are clearly increased in abdominal aortic aneurysms. There are studies indicatingthe presence of MMP-1, the main substrate of which is native type III collagen, in AAAtissue (Irizarry et al. 1991, Newman et al. 1994). Most studies have concentrated ongelatinases (MMP-2 and –9), the activities of which have usually been reported to behigher in AAAs compared to AODs, with a few exceptions, where no differencesbetween these two clinical conditions were seen (see Grange et al. 1997 for a review).More recent findings have suggested that MMP-9 is elevated in both AAA and AODtissue, whereas the MMP-2 mRNA and protein levels are only significantly higher in

55AAA tissue samples (Davis et al. 1998). At the same time, however, Elmore et al. (1998)reported completely opposite results, suggesting that MMP-9, but not MMP-2, is animportant factor in the etiology of AAAs. The role of these MMPs in aneurysmpathogenesis has been mainly attributed to their elastolytic properties. However, nativetype I and denatured collagen have also been reported as substrates for gelatinases (seeKähäri & Saarialho-Kere 1997).

Fragmentation and decreased concentrations of elastin have been described as one themost striking histologic features in aneurysm tissue (Rizzo et al. 1989, Baxter et al.1992). Changes in elastin content and architecture are not, however, exclusive to AAAs,but have also been described in AOD tissue (Campa et al. 1987). Degradation of elastinleads to aortic dilatation, whereas degradation of collagen results in both dilatation and,above all, rupture (Dobrin & Mrkvicka 1994). Thus, an increased amount of type III pN-collagen might be responsible for at least part of the clinical manifestations of aneurysms,making the collagen molecules more susceptible to degradation.

Infiltration of inflammatory cells is a characteristic feature of aortic aneurysms andatherosclerosis. Since some of the matrix-regulating peptides together with proteolyticenzymes are produced by inflammatory cells, this has an important role in both collagenand elastin metabolism (see Ghorpade & Baxter 1996, Grange et al. 1997). Furthermore,both atherosclerosis (see Movahed 1999) and abdominal aortic aneurysms (Juvonen et al.1997) have also been previously suggested to be associated with Chlamydia pneumoniaeinfection.

Our recent immunohistochemical studies have shown abundant type III pN-collagenstaining in the media of aneurysms (see Fig. 5 in Results-section). Type I pN-collagenstaining, in contrast, was seen in the intima of both AAA and AOD specimens. Thus, itseems that the atherosclerotic injury in the intima causes fibroproliferative reactionscharacterized by enhanced synthesis of type I procollagen, whereas the pathogenesis ofabdominal aortic aneurysms includes changes in type III collagen metabolism in themedia layer. Recent data suggests that the MMPs 1, 2 and 9 are differently expressed inthe aortic wall of AAAs and AODs (Palombo et al. 1999). Therefore, type III collagen inthe media of aneurysms might be under a more severe proteolytic attack than the wall ofa corresponding atherosclerotic aorta without dilatation.

In our biochemical studies (paper II), the relative amount of type III collagen wassignificantly lower in aneurysms than in AODs and atherosclerotic plaques. Theseobservations together with the previously reported observations on decreased type IIIcollagen in AAAs (Kuga et al. 1998, van Keulen et al. 1999) and cerebral aneurysms(Pope et al. 1981, van den Berg et al. 1997) suggest a very important role of type IIIcollagen in aneurysm formation. In addition, Pauschinger et al. (1999) reported adecreased relative amount of type III collagen in dilated cardiomyopathy, a disordersimilar to AAAs. The disorder in the processing of type III collagen might predisposeaneurysmal tissue to atherosclerosis, because the PIIINP molecule contains atransglutamination site available to LDL binding, for example (Bowness et al. 1989). Onthe other hand, this was not the case in atherosclerotic plaques in paper I, since theamount of type III pN-collagen was negligible.

A clear familial tendency of AAAs has been demonstrated on several occasions (seeVerloes et al. 1996). Ehlers-Danlos syndrome type IV is caused by several mutations inthe type III procollagen gene (see Kuivaniemi et al. 1997 for a review), and there is also

56evidence of type III procollagen gene mutations in familial aneurysms (Kontusaari et al.1990, Anderson et al. 1996). In the present study, all the AAAs were atherosclerotic andwithout a family history. Yet, the relative proportion of type III collagen in AAA tissuewas decreased. Therefore, one might question if there is a genetic factor involved, whichonly becomes apparent after the damaging effect of atherosclerosis on the aortic wall.

6.4. Type I and III collagens in AAA and diverticular disease of colon

The macroscopic appearance of both colon diverticula and abdominal aortic aneurysmssuggests a loss of tensile strength of the tissue. Abnormalities in the architecture ofelastin can be seen in both AAAs (Rizzo et al. 1989) and colon diverticulosis (Whiteway& Morson 1985). In addition, both of these clinical conditions may be associated with atleast genetic factors and increased intraluminal pressure. The collagen content has beenreported to be increased in AAAs (Menashi et al. 1987, Rizzo et al. 1989, Baxter et al.1994, He & Roach 1994). In colon diverticulosis, the total amount of collagen is notaltered, but an increase in the proportion of insoluble collagen has been suggested (Wesset al. 1995).

The present data indicates that type I and III collagens are maturely cross-linked inboth AAAs and colon diverticulosis. However, the increase in type III pN-collagen wasonly seen in aneurysm tissue. In contrast to Wess et al. (1995), there was no sign ofincreased cross-linking of collagen in colon diverticulosis. However, the result of Wess etal. was only based on hydroxyproline analysis and observations on collagen solubility inweak acids, which makes the obtained results susceptible to several errors. Maturecollagen cross-linking is thought to be a general feature of fibrotic conditions (Ricard-Blum et al. 1993), which is supported by the abundant ICTP staining in liver fibrosis(Ricard-Blum et al. 1996). Because the amount of maturely cross-linked telopeptides(ICTP and IIINTP) and hydroxyproline was not increased in colon diverticulosis, ourresults did not indicate a marked fibrotic process in this clinical situation.

In AAAs, mature cross-linking of type III collagen was found. There is evidence of alower total amount of cross-links in ruptured intracranial aneurysms than in unrupturedaneurysms or healthy controls (Gaetani et al. 1998). Furthermore, the involvement ofproteases in the pathogenesis of aneurysms has been established on several occasions.Thus, it seems that the whole situation may depend on the presence or absence of balancebetween the synthesis, cross-linking and degradation of collagen. On the other hand, wewere unable to find any signs of decreased cross-linking in the two AAA specimens,which had ruptured. The relative amount of type III collagen was decreased in AAAtissue but not in colon diverticulosis, suggesting that type III collagen has a differentfunction in the wall of the colon compared to the aorta. However, the rate of type IIIprocollagen synthesis seemed quite active in colon diverticulosis, where the amount ofPIIINP in the soluble tissue extract was significantly increased compared to normalcontrols. In addition, soluble PIIINP eluted partly in its cleaved form in gel filtrationanalysis, suggesting ongoing synthesis. In AAA tissue, in turn, the synthesis of type IIIprocollagen did not seem to be particularly increased when analyzed quantitatively, thus

57suggesting that the delayed maturation of type III collagen might be the reason forincreased type III pN-collagen.

6.5. Maturation and processing of type I and III collagens in ovarianand colon cancer

The previously reported increased expression of type I and III procollagens in ovarian(Kauppila et al. 1996) and type I collagen in gastrointestinal carcinomas (Ohtani et al.1992) was supported by the increased synthesis of type I collagen measured by PINPassay in the soluble tumor extracts. However, the amounts of cross-linked type I and IIIcollagen antigens and hydroxyproline were lower in the enzyme-digested malignanttissues than in the benign or normal controls. In addition, the cross-linking of especiallytype I collagen was found to be defective in tumor tissue. This is in agreement with theprevious findings indicating that the expression of lysyl oxidase, a key enzyme in cross-linking process of fibrillar collagens, is decreased in malignancies (Hämäläinen et al.1995, Peyrol et al. 1997). Thus, the malignant process seems to be very different frommany other situations, e.g. wound healing, where the expression of lysyl oxidaseincreases together with increasing collagen expression (Fushida-Takemura et al. 1996).This might suggest that lysyl oxidase, in addition to having a role in collagen cross-linking, also has many other important regulatory functions in malignant tissues. Indeed,it has been suggested that lysyl oxidase might have a function as a tumor suppressor(Contente et al. 1990).

In paper IV, the immunohistochemical studies on malignant ovarian tumors showedirregular arrangement of collagen fibers, as also described previously in our laboratory(Zhu et al. 1993a, Zhu et al. 1995). This is most probably due to the increased proteolyticactivity in malignant tissue. Increased levels of MMP-2 have been reported in bothovarian and gastrointestinal cancer (Levy et al. 1991, Autio-Harmainen et al. 1993,Moser et al. 1994, Afzal et al. 1996). In addition to MMP-2, MMP-1 has also beenshown to be associated with ovarian and colorectal cancer (van der Stappen et al. 1990,Kanamori et al. 1999). MMP-1 is able to cleave fibrillar type I and III collagens, whereasMMP-2 has been shown to degrade native type I collagen in addition to type IV collagen(Aimes & Quigley 1995). Indeed, MMP-1 produced by breast tumor cells has beenreported to effectively mediate invasion by degrading type I collagen (Benbow et al.1999). Interestingly, there is new data to suggest that MMP-1 is a target gene for tumorsuppressor protein p53 (Sun et al. 1999). Thus, the ECM degradation seen inmalignancies due to high levels of MMPs might be related to inactivation of p53. Manyother matrix metalloproteinases, e.g. MMP-7 in gastrointestinal cancer (Adachi et al.1999), have been associated with tumor progression and invasion as well (see Kähäri &Saarialho-Kere 1999), but these matrix metalloproteinases do not necessarily cleavefibrillar type I and III collagens.

Even though the type I collagen synthesis detected by PINP was increased inmalignant samples, no such tendency was seen in the case of type III collagen measuredby PIIINP. The amount of type III collagen with the retained aminoterminal propeptide,called type III pN-collagen, was significantly increased in the insoluble ovarian

58carcinoma tissue, suggesting enhanced type III collagen metabolism. This was not thecase in colon cancer. The increased amounts of type III pN-collagen might contribute totumor invasion by leaving the fibers thinner (Fleischmajer et al. 1985) and thus moresusceptible to proteolysis by MMPs. An interesting observation in both ovarian and coloncancers was that type I collagen was more poorly cross-linked than type III collagen. Thisphenomenon might suggest that type III collagen undergoes mature cross-linking morerapidly than type I collagen, being therefore more resistant to proteolysis. On the otherhand, firmly cross-linked collagen may have decreased endurance for tension (Allain etal. 1978), and hence be mechanically weak and susceptible to degradation. The abovedifferences between type I and III collagen synthesis and processing might be related to arecent observation by Boyd & Balkwill (1999) suggesting that type I collagen inducesMMP-2 production in ovarian carcinoma. Thus, type I collagen may be more importantin the regulation and function of tumor matrix proteins and malignant cells than type IIIcollagen.

7. Conclusions

The aim of the present study was to biochemically characterize the tissue contents of typeI and III collagens in various clinical conditions.

1. In contrast to the current opinion, human atherosclerotic plaques contained a lot oftype III collagen. In addition, the type III collagen was found to be completelyprocessed, since the amount of type III pN-collagen was low. Both type I and IIIcollagens were maturely cross-linked. This together with the low levels of type I andIII procollagen propeptides in soluble tissue extracts warrants the conclusion thatmetabolic inertia prevails in atherosclerotic plaques. Thus, the possiblethrombogenicity of type III collagen must be based on completely processed andcross-linked collagen.

2. In abdominal aortic aneurysms (AAAs), the relative amount of type III collagen wasdecreased, whereas the amount of incompletely processed type III collagen with theretained aminoterminal propeptide was increased. Because type III pN-collagen hasbeen reported to regulate the fibril diameter, it is suggested that the collagen fibers inAAAs are thinner, possibly resulting in susceptibility to proteolysis and decreasedtensile strength. Furthermore, the retained aminopropeptide might interfere with thecross-linking process.

3. Collagen synthesis was enhanced in ovarian and colon cancer, whereas the totalcollagen content in tissue was decreased. The cross-linking of especially type Icollagen was defective in both these clinical conditions. These findings suggest anincreased turnover of collagen in ovarian and colon cancer. The decreased cross-linking might be a general feature in malignant tissue, making collagen fibers moresusceptible to proteolysis and thereby promoting tumor invasion and growth.

4. Type I and III collagens were very similar in normal colon tissue and in colon withdiverticular disease. The only difference was the increased amount of freeaminoterminal propeptide of type III collagen, related to ongoing type III collagensynthesis. There was no evidence of the previously reported increased cross-linking ofcollagen.

60

Taken together, the current results suggest that disturbances in the processing, cross-linking and fibril formation of type I and III collagens may lead or contribute tonumerous pathological conditions. For example, the increased proteolysis in AAAs andmalignant processes reported on several occasions might require defective processing ofcollagen molecules in order to take place in significant amounts. New biochemicalmarkers for follow-up or even diagnostic purposes may arise when we learn about thepathogenesis of these conditions. Knowledge of the pathogenesis of these clinicalconditions may also facilitate the development of new therapeutic applications.

8. ReferencesAdachi E, Hopkinson I & Hayashi T (1997) Basement-membrane stromal relationships:

Interactions between collagen fibrils and the lamina densa. Int Rev Cytol 173: 73-156.Adachi Y, Yamamoto H, Itoh F, Hinoda Y, Okada Y & Imai K (1999) Contribution of matrilysin

(MMP-7) to the metastatic pathway of human colorectal cancers. Gut 45: 252-258.Afzal S, Lalani E-N, Foulkes WD, Boyce B, Tickle S, Cardillo MR, Baker T, Pignatelli M &

Stamp GWH (1996) Matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2expression and synthetic matrix metalloproteinase-2 inhibitor binding in ovarian carcinomas andtumor cell lines. Lab Invest 74: 406-421.

Aimes RT & Quigley JP (1995) Matrix metalloproteinase-2 is an interstitial collagenase. J BiolChem 270: 5872-5876.

Ala-Kokko L, Kontusaari S, Baldwin CT, Kuivaniemi H & Prockop DJ (1989) Structure of cDNAclones coding for the entire preproα1(III) chain of human type III procollagen. J Biol Chem260: 509-516.

Allain JC, Le Lous M, Bazin S, Bailey AJ & Delaunay A (1978) Isometric tension developedduring heating of collagenous tissues. Biochim Biophys Acta 533: 147-155.

Amento EP, Ehsani N, Palmer H & Libby P (1991) Cytokines and growth factors positively andnegatively regulate interstitial collagen gene expression in human vascular smooth muscle cells.Arterioscler Thromb 11: 1223-1230.

Anderson DW, Edwards TK, Ricketts MH, Kuivaniemi H, Tromp G, Stolle CA, Deak SB & BoydCD (1996) Multiple defects in type III collagen synthesis are associated with the pathogenesisof abdominal aortic aneurysms. Ann NY Acad Sci 800: 216-228.

Anderson DW, Thakker-Varia S, Tromp G, Kuivaniemi H & Stolle CA (1997) A glycine (415)-to-serine substitution results in impaired secretion and decreased thermal stability of type IIIprocollagen in a patient with Ehlers-Danlos syndrome type IV. Hum Mutat 9: 62-63.

Andreeva ER, Pugach IM & Orekhov AN (1997) Collagen-synthesizing cells in initial andadvanced atherosclerotic lesions of human aorta. Atherosclerosis 130: 133-142.

Autio-Harmainen H, Karttunen T, Hurskainen T, Höyhtyä M, Kauppila A & Tryggvason K (1993)Expression of 72 kilodalton type IV collagenase (gelatinase A) in benign and malignant ovariantumors. Lab Invest 69: 312-321.

Bachem MG, Wendelin D, Schneiderhan W, Haug C, Zorn U, Gross HJ, Schmid-Kotsas A &Grunert A (1999) Depending on their concentration oxidized low density lipoproteins stimulateextracellular matrix synthesis or induce apoptosis in human coronary artery smooth musclecells. Clin Chem Lab Med 37: 319-326.

Bailey AJ, Paul RG & Knott L (1998) Mechanisms of maturation and ageing of collagen. MechAgeing Dev 106: 1-56.

Bailey AJ, Wotton SF, Sims TJ & Thompson PW (1993) Biochemical changes in the collagen ofhuman osteoporotic bone matrix. Connect Tissue Res 29: 119-132.

62Bank RA, Robins SP, Wijmenga C, Breslau-Siderius LJ, Bardoel AFJ, Van der Sluijs HA, Pruijs

HEH & TeKoppele JM (1999) Defective collagen crosslinking in bone, but not in ligament orcartilage, in Bruck syndrome: indications for a bone-specific telopeptide lysyl hydroxylase onchromosome 17. Proc Natl Acad Sci USA 96:1054-1058.

Barnes MJ (1985) Collagens in atherosclerosis. Collagen Rel Res 5: 65-97.Bateman JF, Chiodo AA, Weng YM, Chan D & Haan E (1998) A type III collagen gly559 to arg

helix mutation in Ehlers-Danlos syndrome type IV. Hum Mutat Suppl 1: 257-259.Bateman JF, Lamandé SR & Ramshaw JAM (1996) Collagen superfamily. In: Comper WD (ed)

Extracellular matrix: Molecular components and interactions. Harwood Academic Publishers,Amsterdam, p 22-67.

Bateman JF, Mascara T, Chan D & Cole WG (1984) Abnormal type I collagen metabolism bycultured fibroblasts in lethal perinatal osteogenesis imperfecta. Biochem J 217: 103-115.

Baum J & Brodsky B (1999) Folding of peptide models of collagen and misfolding in disease. CurrOpin Struct Biol 9: 122-128.

Bauriedel G, Hutter R, Welsch U, Bach R, Sievert H & Luderitz B (1999) Role of smooth musclecell death in advanced coronary primary lesions: implications for plaque instability. CardiovascRes 41: 480-488.

Baxter BT, Davis VA, Minion DJ, Wang YP, Lynch TG & McManus BM (1994) Abdominal aorticaneurysms are associated with altered matrix proteins of the nonaneurysmal aortic segments. JVasc Surg 19: 797-803.

Baxter BT, McGee GS, Shively VP, Drummond IAS, Dixit SN, Yamauchi M & Pearce WH(1992). Elastin content, cross-links, and mRNA in normal and aneurysmal human aorta. J VascSurg 16: 192-200.

Becker U, Fietzek PP, Nowack H & Timpl R (1976) Isolation and structure of the amino-terminalcross-linking region in insoluble type III collagen. Hoppe-Seyler’s Z. Physiol Chem 357: 1409-1415.

Beighton PH, Murdoch JL & Votteler T (1969) Gastrointestinal complications of Ehlers-Danlossyndrome. Gut 10: 1004-1008.

Benbow U, Schoenermark MP, Orndorff KA, Givan AL & Brinckerhoff CE (1999) Human breastcancer cells activate procollagenase-1 and invade type I collagen: invasion is inhibited by all-trans retinoic acid. Clin Exp Metastasis 17: 231-238.

Berg RA, Schwartz ML & Crystal RG (1980) Regulation of the production of secretory proteins:intracellular degradation of newly synthesized “defective” collagen. Proc Natl Acad Sci USA77: 4746-4750.

Bini A, Mann KG, Kudryk BJ & Schoen FJ (1999) Noncollagenous bone matrix proteins,calcification and thrombosis in carotid artery atherosclerosis. Arterioscler Thromb Vasc Biol 19:1852-1861.

Birk DE, Fitch JM, Babiarz JP & Linsenmayer TF (1988) Collagen type I and type V are present inthe same fibril in the avian corneal stroma. J Cell Biol 106: 999-1008.

Blavier L, Henriet P, Imren S & Declerck YA (1999) Tissue inhibitors of matrix metalloproteinasesin cancer. Ann NY Acad Sci 878: 108-119.

Bonde M, Qvist P, Fledelius C, Riis BJ & Christiansen C (1994) Immunoassay for quantifying typeI collagen degradation products in urine. Clin Chem 40: 2022-2025.

Bonnet J, Garderes PE, Aumailley M, Moreau C, Gouverneur G, Benchimol D, Crockett R, LarrueJ & Bricaud H (1988) Serum type III procollagen peptide levels in coronary artery disease (amarker of atherosclerosis). Eur J Clin Invest 18: 18-21.

Bowness JM, Folk JE & Timpl R (1987) Identification of a substrate site for liver transglutaminaseon the aminopropeptide of type III collagen. J Biol Chem 262: 1022-1024.

Bowness JM, Tarr AH & Wiebe RI (1989) Transglutaminase-catalysed cross-linking: a potentialmechanism for the interaction of fibrinogen, low density lipoprotein and arterial type IIIprocollagen. Thromb Res 54: 357-367.

Bowness JM, Tarr AH & Wong T (1988) Increased transglutaminase activity during skin woundhealing in rats. Biochim Biophys Acta 967: 234-240.

Boyd RS & Balkwill FR (1999) MMP-2 release and activation in ovarian carcinoma: the role offibroblasts. Br J Cancer 80: 315-321.

63Brown DL, Hibbs MS, Kearney M, Loushin C & Isner JM (1995) Identification of 92-kD

gelatinase in human coronary atherosclerotic lesions: Association of active enzyme synthesiswith unstable angina. Circulation 91: 2125-2131.

Bruckner P, Bächinger HP, Timpl R & Engel J (1978) Three conformationally distinct domains inthe amino-terminal segment of type III procollagen and its rapid triple helix coil transition. Eur JBiochem 90: 595-603.

Bulleid NJ, Dalley JA & Lees JF (1997) The C-propeptide domain of procollagen can be replacedwith a transmembrane domain without affecting trimer formation or collagen triple-helix foldingduring biosynthesis. EMBO J 16: 6694-6701.

Bulleid NJ, Wilson R & Lees JF (1996) Type-III procollagen assembly in semi-intact cells: chainassociation, nucleation and triple-helix folding do not require formation of inter-chaindisulphide bonds but triple-helix nucleation does require hydroxylation. Biochem J 317: 195-202.

Bächinger HP (1987) The influence of peptidyl-prolyl cis-trans isomerase on the in vitro folding oftype III collagen. J Biol Chem 262: 17144-17148.

Bächinger HP, Bruckner P, Timpl R & Engel J (1978) The role of Cis-Trans isomerization ofpeptide bonds in the coil triple helix conversion of collagen. Eur J Biochem 90: 605-613.

Bächinger HP, Bruckner P, Timpl R, Prockop DJ & Engel J (1980) Folding mechanism of the triplehelix in type-III collagen and type-III pN-collagen. Eur J Biochem 106: 619-632.

Campa JS, Greenhalg RM & Powell JT (1987) Elastin degradation in abdominal aortic aneurysms.Atherosclerosis 65: 13-21.

Canfield AE, Wren FE, Schor SL, Grant ME & Schor AM (1992) Aortic endothelial cellheterogeneity in vitro. Lack of association between morphological phenotype and collagenbiosynthesis. J Cell Sci 102: 807-814.

Cannon DJ & Davison PF (1978) Crosslinking in type III collagen of fetal tissue. Biochem BiophysRes Commun 85: 1373-1378.

Chan D & Cole W (1984) Quantitation of type I and III collagens using electrophoresis of alphachains and cyanogen bromide peptides. Anal Biochem 139: 322-328.

Cheung DT, Benya PD, Perelman N, Dicesare PE & Nimni ME (1990) A highly specific andquantitative method for determining type III /I collagen ratios in tissues. Matrix 10: 164-171.

Cheung DT, DiCesare P, Benya PD, Libaw E & Nimni ME (1983) The presence of intermoleculardisulfide cross-links in type III collagen. J Biol Chem 258: 7774-7778.

Chu M-L & Prockop DJ (1993) Collagen: Gene structure. Royce PM & Steinmann B (eds)Connective tissue and its heritable disorders. Molecular, genetic and medical aspects. Wiley-Liss Inc., New York, p 149-165.

Clark CC (1979) The distributional initial characterization of oligosaccharide units on the COOH-terminal propeptide extensions of the pro-α2 chains of type I procollagen. J Biol Chem 254:10798-10802.

Clarke EP, Jain N, Brickenden A, Lorimer IA & Sanwal BD (1993) Parallel regulation ofprocollagen I and colligin, a collagen-binding protein and a member of the serine proteaseinhibitor family. J Cell Biol 121: 193-199.

Contente S, Kenyon K, Rimoldi D & Friedman RM (1990) Expression of gene rrg is associatedwith reversion of NIH 3T3 transformed by LTR-c-H-ras. Science 249: 796-798.

Cronlund AL, Smith BD & Kagan HM (1985) Binding of lysyl oxidase to fibrils of type I collagen.Connect Tissue Res 14: 109-119.

D’Ortho MP, Will H, Atkinson S, Butler G, Messent A, Gavrilovic J, Smith B, Timpl R, Zardi L &Murphy G (1997) Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrumproteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem 250: 751-757.

Dabbagh K, Laurent GJ, McAnulty RJ & Chambers RC (1998) Thrombin stimulates smoothmuscle cell procollagen synthesis and mRNA levels via PAR-1 mediated mechanism. ThrombHaemost 79: 405-409.

Davis V, Persidskaia R, Baca-Regen L, Itoh Y, Nagase H, Persidsky Y, Ghorpade A & Baxter BT(1998) Matrix metalloproteinase–2 production and its binding to the matrix are increased inabdominal aortic aneurysms. Arterioscler Thromb Vasc Biol 18: 1625-1633.

64De Flora S, Izzotti A, Walsh D, Degan P, Petrilli GL & Lewtas J (1997) Molecular epidemiology

of atherosclerosis. FASEB J 11: 1021-1031.Deak S, Ricotta JJ, Mariani TJ, Deak ST, Zatina MA, MacKenzie JW & Boyd CD (1992)

Abnormalities in the biosynthesis of type III procollagen in cultured skin fibroblasts from twopatients with multiple aneurysms. Matrix 12: 92-100.

Deyl Z, Novotná J, Mikšík I, Jelínková D, Uhrová M & Suchánek M (1997) Quantitation ofcollagen types I, III and V in tissue slices by capillary electrophoresis after cyanogen bromidesolubilization. J Chromatogr B 689: 181-194.

Dobrin PB, Baker WH & Gley WC (1984) Elastolytic and collagenolytic studies on arteries. ArchSurg 119: 405-409.

Dobrin PB & Mrkvicka R (1994) Failure of elastin or collagen as possible critical connective tissuealterations underlying aneurysmal dilatation. Cardiovasc Surg 2: 484-488.

Drake FH, Dodds RA, James IE, Connor JR, Debouk C, Richardson S, Lee-Rykaczewski E,Coleman L, Rieman D, Barthlow R, Hastings G & Gowen M (1996) Cathepsin K, but notcathepsins B, L, or S, is abundantly expressed in human osteoclasts. J Biol Chem 271: 12511-12516.

Elmore JR, Keister BF, Franklin DP, Youkey JR & Carey DJ (1998) Expression of matrixmetalloproteinases and TIMPs in human abdominal aneurysms. Ann Vasc Surg 12: 221-228.

Emanuel BS, Cannizarro LA, Seyer JM & Myers JC (1985) Human α1(III) and α2(V) procollagengenes are located on the long arm of chromosome 2. Proc Natl Acad Sci USA 82: 3385-3389.

Engel J & Prockop DJ (1991) The zipper-like folding of collagen triple helices and the effects ofmutations that disrupt the zipper. Annu Rev Biophys Biophys Chem 20: 137-152.

Epstein EH & Munderloch NH (1975) Isolation and characterization of CNBr peptides of human[α1(III)]3 collagen and tissue distribution of [α1(I)]2α2 and [α1(III)]3 collagens. J Biol Chem25: 9304-9312.

Everts V, van der Zee E, Creemers L and Beertsen W (1996) Phagocytosis and intracellulardigestion of collagen, its role in turnover and remodelling. Histochemical J 28: 229-245.

Eyre DR (1987) Collagen cross-linking amino acids. Methods Enzymol 144: 115-139.Eyre DR, Dickson IR & van Ness K (1988) Collagen cross-linking in human bone and articular

cartilage. Biochem J 252: 495-500.Eyre DR, Paz MA & Gallop PM (1984) Cross-linking in collagen and elastin. Annu Rev Biochem

53: 717-748.Feeley TM, Leen EJ, Colgan M-P, Moore DJ, Hourihane DO & Shanik GD (1991) Histologic

characteristics of carotid artery plaque. J Vasc Surg 13: 719-724.Fessler LI, Timpl R & Fessler JH (1981) Assembly and processing of procollagen type III in chick

embryo blood vessels. J Biol Chem 256: 2531-2537.Fishman DA, Kearns A, Chilukuri K, Bafetti LM, O’Toole EA, Georgacopoulos J, Ravosa MJ &

Stack MS (1998) Metastatic dissemination of human ovarian epithelial carcinoma is promotedby alpha2beta1-integrin-mediated interaction with type I collagen. Invasion Metastasis 18: 15-26.

Fitzsimmons C, Proudfoot D & Bowyer DE (1999) Monocyte prostaglandins inhibit procollagensecretion by human vascular smooth muscle cells: implications for plaque stability.Atherosclerosis 142: 287-293.

Fledelius C, Johnsen AH, Cloos PAC, Bonde M & Qvist P (1997) Characterization of urinarydegradation products derived from type I collagen. Identification of a β-isomerized Asp-Glysequence within the C-terminal telopeptide (α1) region. J Biol Chem 272: 9755-9763.

Fleischmajer R, Olsen BR, Timpl R, Perlish JS & Lovelace O (1983) Collagen fibril formationduring embryogenesis. Proc Natl Acad Sci USA 80: 3354-3358.

Fleischmajer R & Perlish JS (1986) The role of aminopropeptide in collagen fibrillogenesis.Rheumatology 10: 91-103.

Fleischmajer R, Perlish JS, Burgeson RE, Shaikh-Bahai F & Timpl R (1990) Type I and IIIcollagen interactions during fibrillogenesis. Ann NY Acad Sci 580: 161-175.

Fleischmajer R, Perlish JS & Timpl R (1985) Collagen fibrillogenesis in human skin. Ann NYAcad Sci 460: 246-257.

65Fleischmajer R, Timpl R, Tuderman L, Raisher L, Wiestner M, Perlish JS & Graves PN (1981)

Ultrastructural identification of extension aminopropeptides of type I and III collagens in humanskin. Proc Natl Acad Sci USA 78: 7360-7364.

Freestone T, Turner RJ, Coady A, Higman DJ, Greenhalg RM & Powell JT (1995) Inflammationand matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arterioscler ThrombVasc Biol 15: 1145-1151.

Freije JMP, Díetz-Itza I, Balbín M, Sánchez LM & Blasco R (1994) Molecular cloning andexpression of collagenase-3, a novel human matrix metalloproteinase produced by breastcarcinomas. J Biol Chem 269: 16766-16773.

Fushida-Takemura H, Fukuda M, Maekawa N, Chanoki M, Kobayashi H, Yashiro N, Ishii M,Hamada T, Otani S & Ooshima A (1996) Detection of lysyl oxidase gene expression in rat skinduring wound healing. Arch Dermatol Res 288: 7-10.

Gacko M & Chyczewski L (1997) Activity and localization of cathepsin B, D and G in aorticaneurysm. Int Surg 82: 398-402.

Gaetani P, Tartara F, Grazioli V, Tancioni F, Infuso L & Rodriguez y Baena R (1998) Collagencross-linkage, elastolytic and collagenolytic activities in cerebral aneurysms: a preliminaryinvestigation. Life Sci 63: 285-92.

Galis ZS, Sukhova GK, Lark MW & Libby P (1994) Increased expression of matrixmetalloproteinases and matrix degrading activity in vulnerable regions of human atheroscleroticplaques. J Clin Invest 94: 2493-2503.

Garnero P, Borel O, Byrjalsen I, Ferreras M, Drake FH, McQueney MS, Foged NT, Delmas PD &Delaissé J-M (1998) The collagenolytic activity of cathepsin K is unique among mammalianproteinases. J Biol Chem 273: 32347-32352.

Ghorpade A & Baxter BT (1996) Biochemistry and molecular regulation of matrix macromoleculesin abdominal aortic aneurysms. Ann NY Acad Sci 800: 138-150.

Gilchrist D, Schwartze U, Shields K, MacLaren L, Bridge PJ & Byers PH (1999) Large kindredwith Ehlers-Danlos syndrome type IV due to a point mutation (G571S) in the COL3A1 gene oftype III procollagen: low risk of pregnancy complications and unexpected longevity in someaffected relatives. Am J Med Genet 82: 305-311.

Glanville RW & Fietzek PP (1976) Amino acid sequence of the N-terminal non-triple-helical cross-link region of type III collagen. FEBS letters 71: 99-102.

Grange JJ, Davis V & Baxter BT (1997) Pathogenesis of abdominal aortic aneurysm: an update andlook toward the future. Cardiovasc Surg 5:256-265.

Greilberger J, Schmut O & Jürgens G (1997) In vitro interactions of oxidatively modified LDLwith type I, II, III, IV, and V collagen, laminin, fibronectin, and poly-D-lysine. ArteriosclerThromb Vasc Biol 17: 2721-2728.

Gunja-Smith Z, Morales AR, Romanelli R & Woessner JF Jr (1996) Remodeling of humanmyocardial collagen in idiopathic dilated cardiomyopathy. Role of metalloproteinases andpyridinoline cross-links. Am J Pathol 148: 1639-1648.

Halila R & Peltonen L (1986) Purification of human procollagen type III N-proteinase fromplacenta and preparation of antiserum. Biochem J 239: 47-52.

Halme T, Peltonen J, Sims TJ, Vihersaari T & Penttinen R (1986) Collagen in human aorta.Changes in the type III/I ratio and concentration of the reducible crosslink,dehydrohydroxylysyninorleucine in ascending aorta from healthy subjects of different age andpatients with annulo-aortic ectasia. Biochim Biophys Acta 881: 222-228.

Hamano K, Kuga T, Takahashi M, Fujioka K, Katoh T, Zempo N, Fujimura Y & Esato K (1998)The lack of type III collagen in a patient with aneurysms and an aortic dissection. J Vasc Surg28: 1104-1106.

Hanson AN & Bentley JP (1983) Quantitation of type I to type III collagen ratios in small samplesof human tendon, vessels and atherosclerotic plaques. Anal Biochem 130:32-40.

Hanson DA & Eyre DR (1996) Molecular site specificity of pyridinoline and pyrrole cross-linkingin type I collagen of human bone. J Biol Chem 271: 26508-26516.

Hanson DA, Weis MAE, Bollen A-M, Maslan SL, Singer FR & Eyre DR (1992) A specificimmunoassay for monitoring human bone resorption: Quantitation of type I collagen cross-linked N-telopeptides in urine. J Bone Miner Res 7: 1251-1258.

66He CM & Roach MR (1994) The composition and mechanical properties of abdominal aortic

aneurysms. J Vasc Surg 20: 6-13.Hedman K, Alitalo K, Lehtinen S, Timpl R & Vaheri A (1982) Deposition of an intermediate form

of procollagen type III (pN-collagen) into fibrils in the matrix of amniotic epithelial cells.EMBO J 1: 47-52.

Henderson EL, Geng Y-J, Sukhova GK, Whittemore AD, Knox J & Libby P (1999) Death ofsmooth muscle cells and expression of mediators of apoptosis by T lymphocytes in humanabdominal aortic aneurysms. Circulation 99: 96-104.

Henkel W (1996) Cross-link analysis of the C-telopeptide domain from type III collagen. BiochemJ 318: 497-503.

Henkel W & Glanville RW (1982) Covalent crosslinking between molecules of type I and type IIIcollagen. Eur J Biochem 122: 205-213.

Henkel W, Rauterberg J & Glanville RW (1979) Isolation of crosslinked peptides from insolublehuman leiomyoma. Eur J Biochem 96: 249-256.

Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R, Murphy G, Humphries S (1991)localization of stromelysin gene expression in atherosclerotic plaques by in situ hybridization.Proc Natl Acad Sci USA 88: 8154-8158.

Hill RJ & Harper E (1984) Quantitation of types I and III collagens in human tissue samples andcell culture by cyanogen bromide analysis. Anal Biochem 141: 83-93.

Hirota S, Imakita M, Kohri K, Ito A, Morii E, Adachi S, Kim H-M, Kitamura Y, Yutani C &Nomura S (1993) Expression of osteopontin messenger RNA by macrophages in atheroscleroticplaques: a possible association with calcification. Am J Pathol 143: 1003-1008.

Hunter GC, Smyth SH, Aguirre ML, Baxter BT, King DD, Wang YP, Hall KA & Putnam CW(1996) Incidence and histologic characteristics of blebs in patients with abdominal aorticaneurysms. J Vasc Surg 24: 93-101.

Huttner WB (1982) Sulphation of tyrosine residues is a widespread modification for proteins.Nature 299: 273-276.

Hämäläinen E-R, Jones TA, Sheer D, Taskinen K, Pihlajaniemi T & Kivirikko KI (1991)Molecular cloning of human lysyl oxidase and assignment of the gene to chromosome 5Q23.3-31.2. Genomics 11: 508-516.

Hämäläinen E-R, Kemppainen R, Kuivaniemi H, Tromp G, Vaheri A, Pihlajaniemi T & KivirikkoKI (1995) Quantitative polymerase chain reaction of lysyl oxidase mRNA in malignantlytransformed human cell lines demonstrates that their low lysyl oxidase activity is due to lowquantities of its mRNA and low levels of transcription of the respective gene. J Biol Chem 270:21590-21593.

Hörlein D, Fietzek PP, Wachter E, Lapière CM & Kühn K (1979) Amino acid sequence of theaminoterminal segment of dermatosparactic calf-skin procollagen type I. Eur J Biochem 99: 31-38.

Irizarry E, Newman KM, Gandhi RH, Nackman GB, Halpern V, Wishner S, Scholes JV & TilsonMD (1993) Demonstration of interstitial collagenase in abdominal aortic aneurysm disease. JSurg Res 54: 571-574.

Jackson CJ & Jenkins KL (1991) Type I collagen fibrils promote rapid vascular tube formationupon contact with the apical side of cultured endothelium. Exp Cell Res 192: 319-323.

Jarrold BB, Bacon WL & Velleman SG (1999) Expression and localization of the proteoglycandecorin during the progression of cholesterol induced atherosclerosis in Japanese quail:implication for interaction with collagen I and lipoproteins. Atherosclerosis 146: 299-308.

Jensen LT & Høst NB (1997) Collagen: scaffold for repair or execution. Cardiovasc Res 33: 535-539.

Jimenez SA, Bashey RI, Benditt M & Yankowski R (1977) Identification of collagen α1(I) trimerin embryonic chick tendons and calvaria. Biochem Biophys Res Commun 78: 1354-1361.

Jukkola A, Risteli J, Niemelä O & Risteli L (1986) Incorporation of sulphate into type IIIprocollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate. Eur J Biochem154: 219-224.

67Jukkola A, Risteli J & Risteli L (1990) Chlorate inhibits tyrosine sulfation of human type III

procollagen without affecting its secretion or processing. Biochem Biophys Res Commun 170:264-269.

Jukkola A, Tähtelä R, Thölix E, Vuorinen K, Blanco G, Risteli L & Risteli J (1997) Aggressivebreast cancer leads to discrepant serum levels of the type I procollagen propeptides PINP andPICP. Cancer Res 57: 5517-5520.

Juvonen J, Juvonen T, Laurila A, Alakärppä H, Lounatmaa K, Surcel H-M, Leinonen M,Kairaluoma MI & Saikku P (1997) Demonstration of Chlamydia pneumoniae in the walls ofabdominal aortic aneurysms. J Vasc Surg 25: 499-505.

Kaartinen M, van der Wal AC, van der Loos CM, Piek JJ, Koch KT, Becker AE & Kovanen PT(1998) Mast cell infiltration in acute coronary syndromes: implications for plaque rupture. J AmColl Cardiol 32: 606-12.

Kadler KE, Hojima & Prockop DJ (1987) Assembly of collagen fibrils de Novo by cleavage of thetype I pC-collagen with procollagen C-proteinase. J Biol Chem 260: 15696-15701.

Kadler KE, Holmes DF, Trotter JA & Chapman JA (1996) Collagen fibril formation. Biochem J316: 1-11.

Kadler KE & Watson RB (1995) Procollagen C-peptidase: procollagen C-proteinase. MethodsEnzymol 248: 771-781.

Kafienah W, Brömme D, Buttle DJ, Croucher LJ & Hollander AP (1998) Human cathepsin Kcleaves native type I and II collagens at the N-terminal end of the triple helix. Biochem J 331:727-732.

Kagan HM (1994) Lysyl oxidase: mechanism, regulation and relationship to liver fibrosis. Path ResPract 190: 910-919.

Kagan HM, Raghavan J & Hollander W (1981) Changes in aortic lysyl oxidase activity in diet-induced atherosclerosis in the rabbit. Arteriosclerosis 1: 287-291.

Kanamori Y, Matsushima M, Minaguchi T, Kobayashi K, Sagae S, Kudo R, Terakawa N &Nakamura Y (1999) Correlation between expression of the matrix metalloproteinase-1 gene inovarian cancers and an insertion/deletion polymorphism in its promoter region. Cancer Res 59:4225-4227.

Karttunen T, Alavaikko A, Apaja-Sarkkinen M & Autio-Harmainen H (1988) Animmunohistochemical study of laminin, type IV collagen and type-III pN-collagen with relationto reticular fibres in Hodgkin´s disease. Int J Cancer 41: 52-58.

Kato H, Suzuki H, Tajima S, Ogata Y, Tominaga T, Sato A & Saruta T (1991) Angiotensin IIstimulates collagen synthesis in cultured vascular smooth muscle cells. J Hypertens 9: 17-22.

Katsuda S, Okada Y, Minamoto T, Oda Y, Matsui Y & Nakanishi I (1992) Collagens in humanatherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies.Arterioscler Thromb Vasc Biol 12: 494-502.

Kauppila A, Puistola U, Risteli J & Risteli L (1989) Amino-terminal propeptide of type IIIprocollagen: A new prognosis indicator in human ovarian cancer. Cancer Res 49: 1885-1889.

Kauppila S, Saarela J, Stenbäck F, Risteli J, Kauppila A & Risteli L (1996) Expression for mRNAsfor type I and III procollagens in serous ovarian cystadenomas and cystadenocarcinomas. Am JPathol 148: 539-548.

Keene DR, Sakai LY, Bächinger HP & Burgeson RE (1987) Type III collagen can be present onbanded collagen fibrils regardless of fibril diameter. J Cell Biol 105: 2393-2402.

Kelley J, Chrin L & Evans JN (1984) Microquantitation of insoluble tissue collagen (types I andIII) by radiodilution assay. Anal Biochem 139: 115-125.

Kenyon K, Modi WS, Contente S & Friedman RM (1993) A novel human cDNA with a predictedprotein similar to lysyl oxidase maps to chromosome 15q24-q25. J Biol Chem 268: 18435-18437

Kielty CM, Hopkinson I & Grant ME (1993) Collagen: the collagen family: structure, assembly,and organization in the extracellular matrix. In: Royce PM & Steinmann B (eds) Connectivetissue and its heritable disorders. Molecular, genetic and medical aspects. Wiley-Liss Inc., NewYork, p 103-147.

Kim Y, Boyd CD & Csiszar K (1995) A new gene with sequence and structural similarity to thegene encoding human lysyl oxidase. J Biol Chem 270: 7176-7182.

68Kivirikko KI, Laitinen O, Prockop DJ (1967) Modifications of a specific assay for hydroxyproline

in urine. Anal Biochem 19: 249-255.Klappacher G, Franzen P, Haab D, Mehrabi M, Binder M, Plesch K, Pacher R, Grimm M, Pribill I,

Eichler HG & Glogar HD (1995) Measuring extracellular matrix turnover in the serum ofpatients with idiopathic or ischemic dilated cardiomyopathy and impact on diagnosis andprognosis. Am J Cardiol 75: 913-918.

Knott L, Whitehead CC, Fleming RH & Bailey AJ (1995) Biochemical changes in the collagenousmatrix of osteoporotic avian bone. Biochem J 310: 1045-1051.

Knäuper V, López-Otin C, Smith B, Knight G & Murphy G (1996a) Biochemical characterizationof human collagenase-3. J Biol Chem 271: 1544-1550.

Knäuper V, Will H, López-Otin C, Smith B, Atkinson SJ, Stanton H, Hembry RM & Murphy G(1996b) Cellular mechanisms for human procollagenase-3 (MMP-13) activation. J Biol Chem271: 17124-17131.

Kockx MM, De Meyer GR, Buyssens N, Knaapen MW, Bult H & Herman AG (1998) Cellcomposition, replication, and apoptosis in atherosclerotic plaques after 6 months of cholesterolwithdrawal. Circ Res 83: 378-387.

Koivu J & Myllylä R (1987) Interchain disulfide bond formation in types I and II procollagen. JBiol Chem 262: 6159-6164.

Kojima T, Miyaishi O, Saga S, Ishiguro N, Tsutsui Y & Iwata H (1998) The retention of abnormaltype I procollagen and correlated expression of HSP 47 in fibroblasts from a patient with lethalosteogenesis imperfecta. J Pathol 184: 212-218.

Kolpakov V, Gordon D & Kulik TJ (1995b) Nitric oxide-generating compounds inhibit totalprotein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res 76: 305-309.

Kolpakov V, Rekhter MD, Gordon D, Wang WH & Kulik TJ (1995a) Effect of mechanical forceson growth and matrix protein synthesis in the in vitro pulmonary artery. Analysis of the role ofindividual cell types. Circ Res 77: 823-831.

Kontusaari S, Tromp G, Kuivaniemi H, Romanic AM & Prockop DJ (1990) A mutation in the genefor type III procollagen (COL3A1) in a family with aortic aneurysms. J Clin Invest 86: 1465-1473.

Kostoulas G, Lang A, Nagase H & Baici A (1999) Stimulation of angiogenesis through cathepsin Binactivation of the tissue inhibitors of matrix metalloproteinases. FEBS Lett 455: 286-290.

Kovanen PT, Kaartinen M & Paavonen T (1995) Infiltrates of activated mast cells at the site ofcoronary atheromatous erosion or rupture in myocardial infarction. Circulation 92: 1084-1088.

Krane SM, Byrne MH, Lemaître V, Henriet P, Jeffrey JJ, Witter JP, Liu X, Wu H, Jaenisch R &Eeckhout Y (1996) Different collagenase gene products have different roles in degradation oftype I collagen. J Biol Chem 271: 28509-28515.

Kratky RG, Ivey J & Roach MR (1999) Local changes in collagen content in rabbit aorticatherosclerotic lesions with time. Atherosclerosis 143: 7-14.

Kuboki Y, Tsuzaki M, Sasaki S, Liu CF & Mechanic GL (1981) Location of the intermolecularcross-links in bovine dentin collagen, solubilization with trypsin and isolation of cross-linkpeptide containing dihydroxylysinonorleucine and pyridinoline. Biochem Biophys ResCommun 102: 119-126.

Kuga T, Esato K, Zempo N, Fujioka K & Nakamura K (1998) Detection of type III collagenfragments in specimens of abdominal aortic aneurysms. Surg Today 28: 385-390.

Kuivaniemi H, Tromp G & Prockop DJ (1997) Mutations in fibrillar collagens (types I, II, III, andXI), fibril-associated collagen (type IX), and network-forming collagen (type X) cause aspectrum of diseases of bone, cartilage, and blood vessels. Hum Mutat 9: 300-315.

Kähäri V-M & Saarialho-Kere U (1997) Matrix metalloproteinases in skin. Exp Dermatol 6: 199-213.

Kähäri V-M & Saarialho-Kere U (1999) Matrix metalloproteinases and their inhibitors in tumourgrowth and invasion. Ann Med 31: 34-45.

Lafont A, Durand E, Samuel JL, Besse B, Addad F, Lévy BI, Desnos M, Guérot C & BoulangerCM (1999) Endothelial dysfunction and collagen accumulation. Two independent factors forrestenosis and constrictive remodeling after experimental angioplasty. Circulation. 100: 1109-1115.

69Laury-Kleintop LD, Gleason M & Tulenko TN (1999) Expression of the heterogenous nuclear

ribonucleoprotein complex K protein and the prolyl-4-hydroxylase α-subunit in atheroscleroticarterial smooth muscle cells. Biochem Biophys Res Commun 260: 382-389.

Lee RT & Libby P (1997) The unstable atheroma. Arterioscler Thromb Vasc Biol 17: 1859-1867.Lee RWH & Huttner WB (1983) Tyrosine-O-sulfated proteins of PC 12 pheochromocytoma cells

and their sulfation by tyrosylprotein sulfotransferase. J Biol Chem 258: 11326-11334.Lees JF, Tasab M & Bulleid NJ (1997) Identification of the molecular recognition sequence which

determines the type-specific assembly of procollagen. EMBO J 16: 908-916.Leuschner JRA & Haust MD (1986) Interstitial collagens in fibrous atherosclerotic lesions of

human aorta. Path Biol 34: 14-18.Levy AT, Cioce V, Sobel ME, Garbisa S, Grigioni WF & Liotta LA (1991) Increased expression of

the Mr 72,000 type IV collagenase in human colonic adenocarcinoma. Cancer Res 51: 439-444.Li Z, Li L, Zielke R, Chenng L, Xiao R, Crow MT & Stetler-Stevenson WG (1996) Increased

expression of 72-kd type IV collagenase (MMP-2) in human aortic atherosclerotic lesions. Am JPathol 148: 121-128.

Liakka A, Karjalainen H, Virtanen I & Autio-Harmainen H (1995) Immuno-electron-microscopiclocalization of types III pN-collagen and IV collagen, laminin and tenascin in developing andadult human spleen. Cell Tissue Res 282: 117-127.

Lindstedt L, Saarinen J, Kalkkinen N, Welgus H & Kovanen PT (1999) Matrix metalloproteinases-3, -7, and –12, but not –9, reduce high density lipoprotein-induced cholesterol efflux fromhuman macrophage foam cells by truncation of the carboxyl terminus of apolipoprotein A-I.Parallel losses of pre-beta particles and the high affinity component of efflux. J Biol Chem 274:22627-22634.

Liu X, Otter A, Scott PG, Cann JR & Kotovych G (1993) Conformational analysis of the type IIand type III collagen α-1 chain C-telopeptides by 1H NMR and circular dichroism spectroscopy.J Biomol Struct Dyn 11: 541-555.

Liu X, Wu H, Byrne M, Krane S & Jaenisch R (1997) Type III collagen is crucial for collagenfibrillogenesis and for normal cardiovascular development. Proc Natl Acad Sci 94: 1852-1856.

López-Candales A, Holmes DR, Liao S, Scott MJ, Wickline SA & Thompson RW (1997)Decreased vascular smooth muscle cell density in medial degeneration of human abdominalaortic aneurysms. Am J Pathol 150: 993-1007.

Lukens LN (1976) Time of occurrence of disulfide linking between procollagen chains. J BiolChem 251: 3530-3538.

Maeda S, Sawai T, Uzuki M, Takahashi Y, Omoto H, Seki M & Sakurai M (1995) Determinationof interstitial collagenase (MMP-1) in patients with rheumatoid arthritis. Ann Rheum Dis 54:970-975.

Majamaa K, Savolainen ER & Myllylä VV (1992) Synthesis of structurally unstable type IIIprocollagen in patients with cerebral artery aneurysm. Biochim Biophys Acta 1138: 191-196.

Majors A & Ehrhart LA (1993) Basic fibroblast growth factor in the extracellular matrix suppressescollagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cell cultures.Arterioscler Thromb 13: 680-686.

Majors A, Ehrhart A, Pezacka EH (1997) Homocysteine is a risk factor for vascular disease –enhanced collagen production and accumulation by smooth muscle cells. Arterioscler ThrombVasc Biol 17: 2074-2081

Mao D, Lee JK, VanVickle SJ, & Thompson RW (1999) Expression of collagenase-3 (MMP-13) inhuman abdominal aortic aneurysms and vascular smooth muscle cells in culture. BiochemBiophys Res Commun 261: 904-910.

Matsumoto S, Katoh M, Saito S, Watanabe T & Masuho Y (1997) Identification of soluble type ofmembrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA. BiochimBiophys Acta 1354: 159-170.

Mays PK, Bishop JE & Laurent GJ (1988) Age-related changes in the proportion of types I and IIIcollagen. Mech Ageing Dev 45: 203-212.

McCaffrey TA, Du B, Consigli S, Szabo P, Bray PJ, Hartner L, Weksler BB, Sanborn TA,Bergman G & Bush HL, jr. (1997) Genomic instability in the type II TGF-β1 receptor gene inatherosclerotic and restenotic vascular cells. J Clin Invest 100: 2182-2188.

70McCaffrey TA, Nicholson AC, Szabo PE, Weksler ME & Weksler BB (1988) Aging and

arteriosclerosis. The increased proliferation of arterial smooth muscle cells isolated from old ratsassociated with increased platelet-derived growth factor-like activity. J Exp Med 167: 163-174.

McCullagh KA & Balian G (1975) Collagen characterization and cell transformation in humanatherosclerosis. Nature 258: 73-75.

McCullagh KG, Duance VC & Bishop KA (1980) The distribution of collagen types I, III and V(AB) in normal and atherosclerotic human aorta. J Pathol 130: 45-52.

McGee GS, Baxter BT, Shively VP, Chisholm R, McCarthy WJ, Flinn WR, Yao JST & Pearce WH(1991) Aneurysm or occlusive disease – factors determining the clinical course ofatherosclerosis of the infrarenal aorta. Surgery 110: 370-376.

McLaughlin SH & Bulleid NJ (1998) Molecular recognition in procollagen chain assembly. MatrixBiol 16: 369-377.

McMillan WD, Patterson BK, Keen RR & Pearce WH (1995) In situ localization and quantificationof seventy-two-kilodalton type IV collagenase in aneurysmal, occlusive, and normal aorta. JVasc Surg 22: 295-305.

McMillan WD & Pearce WH (1999) Increased plasma levels of metalloproteinase-9 are associatedwith abdominal aortic aneurysms. J Vasc Surg 29: 122-129.

McMillan WD, Tamarina NT, Cipollone M, Johnson DA, Parker MA & Pearce WH (1997) Sizematters: the relationship between MMP-9 expression and aortic diameter. Circulation 96: 2228-2232.

Melkko J, Hellevik T, Risteli L, Risteli J & Smedsrød B (1994) Clearance of NH2-terminalpropeptides of types I and III procollagen is a physiological function of the scavenger receptorin liver endothelial cells. J Exp Med 179: 405-412.

Melkko J, Kauppila S, Risteli L, Niemi S, Haukipuro K, Jukkola A & Risteli J (1996)Immunoassay for the intact amino-terminal propeptide of human type I procollagen. Clin Chem42: 947-954.

Melkko J, Niemi S, Risteli L & Risteli J (1990) Radioimmunoassay of the carboxy-terminalpropeptide of human type I procollagen. Clin Chem 36: 1328-1332.

Menashi S, Campa JS, Greenhalg RM & Powell JT (1987) Collagen in abdominal aneurysm:Typing, content, and degradation. J Vasc Surg 6: 578-582.

Mesh CL, Baxter BT, Pearce WH, Chisholm RL, McGee GS & Yao JST (1992) Collagen andelastin gene expression in aortic aneurysms. Surgery 112: 256-262.

Miller EJ, Epstein EH Jr. & Piez KA (1971) Identification of three genetically distinct collagens bycyanogen bromide cleavage of insoluble human skin and cartilage collagen. Biochem BiophysRes Commun 42: 1024-1029.

Miller EJ, Finch JE jr., Chung E & Butler WT (1976) Specific cleavage of the native type IIIcollagen molecule with trypsin. Arch Biochem Biophys 173: 631-637.

Miller EJ, Furuto DK & Narkates AJ (1991) Quantitation of type I, III and V collagens in humantissue samples by high-performance liquid chromatography of a selected cyanogen bromidepeptides. Anal Biochem 196: 54-60.

Ming SC (1998) Diverticular diseases of the colon. In: Ming SC & Goldman H (eds) Pathology ofthe gastrointestinal tract. Williams & Wilkins, Baltimore, Maryland, p 801-818.

Minion DJ, Davis VA, Nejezchleb BS, Wang Y, McManus BM & Baxter BT (1994) Elastin isincreased in abdominal aortic aneurysms. J Surg Res 57: 443-446.

Minion DJ, Wang Y, Lynch TG, Fox IJ, Prorok GD & Baxter BT (1993) Soluble factors modulatechanges in collagen gene expression in abdominal aortic aneurysms. Surgery 114:252-257.

Miura S, Kodaira S & Hosoda Y (1993) Immunohistologic analysis of the extracellular matrixcomponents of the fibrous stroma of human colon cancer. J Surg Oncol 53: 36-42.

Morton LF & Barnes MJ (1982) Collagen polymorphism in the normal and diseased blood vesselwall. Investigation of collagens types I, III and V. Atherosclerosis 42: 41-51.

Moser TL, Young TN, Rodriguez GC, Pizzo SV, Bast RTC Jr & Stack MS (1994) Secretion ofextracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma. Int JCancer 56: 552-559.

Movahed MR (1999) Infection with Chlamydia pneumoniae and atherosclerosis: a review. JSCMed Assoc 95: 303-308.

71Murata K, Motayama T & Kotake C (1986) Collagen types in various layers of the human aorta

and their changes with the atherosclerotic process. Atherosclerosis 60: 251-262.Murphy G, Knäuper V, Cowell S, Hembry R, Stanton H, Butler G, Freije J, Pendás AM & López-

Otín C (1999) Evaluation of some newer matrix metalloproteinases. Ann NY Acad Sci 878: 25-39.

Myers PR & Tanner MA (1998) Vascular endothelial cell regulation of extracellular matrixcollagen: role of nitric oxide. Arterioscler Thromb Vasc Biol 18:717-722.

Nagata K (1998) Expression and function of heat shock protein 47: a collagen-specific molecularchaperone in the endoplasmic reticulum. Matrix Biol 16: 379-386.

Nakai A, Satoh M, Hirayoshi K & Nagata K (1992) Involvement of the stress protein HSP47 inprocollagen processing in the endoplasmic reticulum. J Cell Biol 117: 903-914.

Narcisi P, Richards AJ, Ferguson SD & Pope FM (1994) A family with Ehlers-Danlos syndrometype III/articular hypermotility syndrome has a glycine 637-to-serine substitution in type IIIcollagen. Hum Molec Genet 3: 1617-1620.

Newman KM, Malon AM, Shin RD, Scholes JV, Ramey WG & Tilson MD (1994) Matrixmetalloproteinases in abdominal aortic aneurysm: characterization, purification, and theirpossible sources. Connect Tissue Res 30: 265-276.

Niemelä O (1985) Radioimmunoassays for type III procollagen amino-terminal peptides inhumans. Clin Chem 31: 1301-1304.

Niemelä O, Risteli L, Parkkinen J & Risteli J (1985) Purification and characterization of the N-terminal propeptide of human type III procollagen. Biochem J 232: 145-150.

Nikkari ST, O’Brien KD, Ferguson M, Hatsukami T, Welgus HG, Alpers CE & Clowes AW(1995) Interstitial collagenase (MMP-1) expression in human carotid athesclerosis. Circulation92: 1393-1398.

Oemar BS & Lüscher TF (1997) Connective tissue growth factor. Friend or foe? ArteriosclerThromb Vasc Biol 17: 1483-1489.

Ohtani H, Kuroiwa A, Obinata M, Ooshima A & Nagura H (1992) Identification of type I collagen-producing cells in human gastrointestinal carcinomas by non-radioactive in situ hybridizationand immunoelectron microscopy. J Histochem Cytochem 40: 1139-1146.

Ohuchi E, Imai K, Fujii Y, Sato H, seiki M & Okada Y (1997) Membrane type I matrixmetalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. JBiol Chem. 272: 2446-2451.

Okada Y, Naka K, Kawamura K, Matsumoto T, Nakanishi I, Fujimoto N, Sato H & Seiki M (1995)Localization of matrix metalloproteinase-9 (92 kDa gelatinase/type IV collagenase=gelatinaseB) in osteoclasts; implications for bone resorption. Lab Invest 72: 311-322.

Ooshima A (1981) Collagen αB chain: Increased proportion in human atherosclerosis. Science 213:666-668.

Otani Y, Okazaki I, Arai M, Kameyama K, Wada N, Maruyama K, Yoshino K, Kitajima M,Hosoda Y & Tsuchiya M (1994) Gene expression of interstitial collagenase (matrixmetalloproteinase 1) in gastrointestinal tract cancers. J Gastroenterol 29: 391-397.

Painter NS & Burkitt DP (1971) Diverticular disease of the colon: a deficiency disease of westerncivilization. Br Med J 2: 450-454.

Painter NS, Truelove SC, Ardran GM & Tuckey M (1965) Segmentation and the localization ofintraluminal pressures in the human colon, with special reference to the pathogenesis of colonicdiverticula. Gastroenterology 49: 169-177.

Palombo D, Maione M, Cifiello BI, Udini M, Maggio D & Lupo M (1999) Matrixmetalloproteinases. Their role in degenerative chronic diseases of abdominal aorta. J CardiovascSurg 40: 257-260.

Pauschinger M, Knopf D, Petschauer S, Doerner A, Poller W, Schwimmbeck PL, Kuhl U &Schultheiss HP (1999) Dilated cardiomyopathy is associated with significant changes incollagen type I/III ratio. Circulation 99: 2750-2760.

Peuhkurinen K, Risteli L, Jounela A & Risteli J (1996) Changes in interstitial collagen metabolismduring acute myocardial infarction treated with streptokinase or tissue plasminogen activator.Am Heart J 131: 7-13.

72Peuhkurinen KJ, Risteli L, Melkko J, Linnaluoto M, Jounela A & Risteli J (1991) Thrombolytic

therapy with streptokinase stimulates collagen breakdown. Circulation 83: 1969-1975.Peyrol S, Raccurt M, Gerard F, Gleyzal C, Grimaud JA & Sommer P (1997) Lysyl oxidase gene

expression in the stromal reaction to in situ and invasive ductal breast carcinoma. Am J Pathol150: 497-507.

Pickering JG, Ford CM & Chow LH (1996) Evidence for rapid accumulation and persistentlydisordered architecture of fibrillar collagen in human coronary restenosis lesions. Am J Cardiol78: 633-637.

Pickering JG, Ford CM, Tang B & Chow LH (1997) Coordinated effects of fibroblast growthfactor-2 on expression of fibrillar collagens, matrix metalloproteinases and tissue inhibitors ofmatrix metalloproteinases by human vascular smooth muscle cells. Evidence for repressedcollagen production and activated degradative capacity. Arterioscler Thromb Vasc Biol 17: 475-482.

Plenz G, Dorszewski A, Völker W, Ko YS, Severs NJ, Breithardt G & Robenek H (1999)Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteriesof rabbit. Arterioscler Thromb Vasc Biol 19: 2395-2404.

Podell DN & Abraham GN (1978) A technique for the removal of pyroglutamic acid from theamino terminus of proteins using calf liver pyroglutamate amino peptidase. Biochem BiophysRes Commun 81: 176-185.

Pope FM, Nicholls AC, Narcisi P, Bartlett J, Neil-Dwyer G & Doshi B (1981) Some patients withcerebral aneurysms are deficient in type III collagen. Lancet 1:973-975.

Powell J & Greenhalgh RM (1989) Cellular, enzymatic, and genetic factors in the pathogenesis ofabdominal aortic aneurysms. J Vasc Surg 9: 297-304.

Prockop DJ & Kivirikko KI (1995) Collagens: Molecular biology, diseases, and potentials fortherapy. Annu Rev Biochem 64: 403-434.

Pucci-Minafra I, Andriolo M, Basiricó L, Luparello C, Buccellato C, Garbelli R & Minafra S(1998) Absence of regular α2(I) collagen chains in colon carcinoma biopsy fragments.Carcinogenesis 19: 575-584.

Pucci-Minafra I, Luparello C, Andriolo M, Basiricó L, Aquino A & Minafra S (1993) A new formof tumor and fetal collagen that binds laminin. Biochemistry 32: 7421-7427.

Raspanti M, Ottani V & Ruggeri A (1989) Different architectures of the collagen fibril:morphological aspects and functional implications. Int J Macromol 11: 367-371.

Rauterberg J, Jaeger E & Althaus M (1993) Collagens in atherosclerotic vessel wall lesions. In:Roessner A, Vollmer E (eds) Current Topics in Pathology. Springer-Verlag, Berlin, p 163-192.

Reed D, Reed C, Stemmermann G & Hayashi T (1992) Are aortic aneurysms caused byatherosclerosis? Circulation 85: 205-211.

Reiser KM (1991) Nonenzymatic glycation of collagen in aging and diabetes. Proc Soc Exp BiolMed 196: 17-29.

Rekhter MD (1999) Collagen synthesis in atherosclerosis: too much and not enough. CardiovascRes 41: 376-384.

Rekhter MD, O’Brien E, Shah N, Schwartz SM, Simpson JB & Gordon D (1996) The importanceof thrombus organization and stellate cell phenotype in collagen I gene expression in human,coronary atherosclerotic and restenotic lesions. Cardiovasc Res 32: 496-502.

Rekhter MD, Zhang K, Narayanan AS, Phan S, Schork MA & Gordon D (1993) Type I collagengene expression in human atherosclerosis. Localization to specific plaque regions. Am J Pathol143: 1634-1648.

Ricard-Blum S, Bresson-Hadni S, Guerret S, Grenard P, Volle PJ, Risteli L, Grimaud JA & VuttonDA (1996) Mechanism of collagen network stabilization in human irreversible granulomatousliver fibrosis. Gastroenterology 111: 172:182.

Ricard-Blum S, Esterre P & Grimaud JA (1993) Collagen cross-linking by pyridinoline occurs innon-reversible skin fibrosis. Cell Mol Biol 39: 723-727.

Risteli J, Elomaa I, Niemi S, Novamo A & Risteli L (1993) Radioimmunoassay for the pyridinolinecross-linked carboxy-terminal telopeptide of type I collagen: a new serum marker of bonecollagen degradation. Clin Chem 39: 635-640.

73Risteli J, Niemi S, Trivedi P, Mäentausta O, Mowat AP & Risteli L (1988a) Rapid equilibrium

radioimmunoassay for the amino-terminal propeptide of human type III procollagen. Clin Chem34: 715-718.

Risteli J & Risteli L (1995) Analysing connective tissue metabolites in human serum. Biochemical,physiological and methodological aspects. J Hepatol 22(Suppl. 2): 77-81.

Risteli J & Risteli L (1999) Products of bone collagen metabolism. In: Dynamics of bone andcartilage metabolism: principles and clinical applications. Seibel MJ, Robins SP, Bilezikian JP(eds) Academic Press, London, p 275-287.

Risteli L, Kauppila A, Mäkilä UM & Risteli J (1988b) Aminoterminal propeptide of type IIIprocollagen in serum – an indicator of clinical behavior of advanced ovarian carcinoma? Int JCancer 41: 409-414.

Risteli L & Risteli J (1987) Analysis of extracellular matrix proteins in biological fluids. MethodsEnzymol 145: 391-411.

Risteli L & Risteli J (1990) Noninvasive methods for detection of organ fibrosis. In: Connectivetissue in health and disease. Rojkind M (ed) CRC press, Boca Raton, FL, p 61-98.

Rizvi MAD, Katwa L, Spadone DP & Myers PR (1996) The effects of endothelin-1 on collagentype I and type III synthesis in cultured porcine coronary artery vascular smooth muscle cells. JMol Cell Cardiol 28: 243-252.

Rizzo RJ, McCarthy WJ, Dixit SN, Lilly MP, Shively VP, Flinn WR & Yao JST (1989) Collagentypes and matrix protein content in human abdominal aneurysms. J Vasc Surg 10: 365-373.

Rohde H, Bruckner P & Timpl R (1983) Immunochemical properties of the aminopropeptide ofprocollagen type III. Eur J Biochem 135: 197-202.

Romanic AM, Adachi E, Kadler KE, Hojima Y & Prockop DJ (1991) Copolymerization ofpNcollagen III and collagen I. J Biol Chem 266: 12703-12709.

Ross R (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801-809.

Saito H, Papaconstantinou J, Sato H & Goldstein S (1997) Regulation of a novel gene encoding alysyl oxidase-related protein in cellular adhesion and senescence. J Biol Chem 272: 8157-8160.

Sajanti J & Majamaa K (1999) Detection of meningeal fibrosis after subarachnoid haemorrhage byassaying procollagen propeptides in cerebrospinal fluid. J Neurol Neurosurg Psychiatry 67: 185-188.

Sakalihasan N, Delvenne P, Nusgens BV, Limet R & Lapiere CM (1996) Activated forms ofMMP2 and MMP9 in abdominal aortic aneurysms. J Vasc Surg 24: 127-133.

Sandberg MM, Hirvonen HE, Elima KJ & Vuorio E (1993) Co-expression of collagens II and XIand alternative splicing of exon 2 of collagen II in several developing human tissues. Biochem J294: 595-602.

Santala M, Risteli J, Risteli L, Puistola U, Kacinski BM, Stanley ER & Kauppila A (1998)Synthesis and breakdown of fibrillar collagens: concomitant phenomena in ovarian cancer. Br JCancer 77: 1825-1831.

Santala M, Risteli L, Puistola U, Risteli J & Kauppila A (1995) Elevated serum ICTPconcentrations reflect poor prognosis in patients with ovarian carcinoma. Ann Med 27: 57-61.

Santala M, Simojoki M, Risteli J, Risteli L & Kauppila A (1999) Type I and III collagenmetabolites as predictors of clinical outcome in epithelial ovarian cancer. Clin Cancer Res 5:4091-4096.

Sassi M-L, Eriksen H, Risteli L, Niemi S, Mansell J, Gowen M & Risteli J (2000)Immunochemical characterization of the assay for the carboxyterminal telopeptide of humantype I collagen (ICTP). Loss of antigenicity by treatment with cathepsin K. Bone, in press.

Satta J, Haukipuro K, Kairaluoma MI & Juvonen T (1997) Aminoterminal propeptide of type IIIprocollagen in the follow-up of patients with abdominal aortic aneurysms. J Vasc Surg 25: 909-915.

Satta J, Juvonen T, Haukipuro K, Juvonen M & Kairaluoma MI (1995) Increased turnover ofcollagen in abdominal aortic aneurysms, demonstrated by measuring the concentration of theaminoterminal propeptide of type III procollagen in peripheral and aortal blood samples. J VascSurg 22: 155-160.

74Schwartze U, Goldstein JA & Byers PH (1997) Splicing defects in the COL3A1 gene: marked

preference for 5’ (donor) spice-site mutations in patients with exon-skipping mutations andEhlers-Danlos syndrome type IV. Am J Hum Genet 61:1276-1286.

Shah PK, Falk E, Badimon JJ, Fernandez-Ortiz A, Mailhac A, Villareal-Levy G, Fallon JT,Regnstrom J & Fuster V (1995) Human monocyte-derived macrophages induce collagenbreakdown in fibrous caps of atherosclerotic plaques. Potential role of matrix-degradingmetalloproteinases and implications for plaque rupture. Circulation 92: 1565-1569.

Sharp CA, Davie MWJ, Worsfold M, Risteli L & Risteli J (1996) Discrepant blood concentrationsof type I procollagen propeptides in active Paget’s disease of bone. Clin Chem 42: 1121-1122.

Shekhonin BV, Domogatsky SP, Idelson GL, Koteliansky VE & Rukosuev VS (1987) Relativedistribution of fibronectin and type I, III, IV, V collagens in normal and atherosclerotic intimaof human arteries. Atherosclerosis 67: 9-16.

Shekhonin BV, Domogatsky SP, Muzykantov VR, Idelson GL & Rukosuev VS (1985) Distributionof type I, III, IV and V collagen in normal and atherosclerotic human arterial wall:Immunomorphological characteristics. Collagen Rel Res 5: 355-368.

Shiraishi M, Segawa M & Takebayashi S (1980) An electron microscopic study on aging ofcollagenous fibrils in the large blood vessels of humans. J Electron Microsc 29: 139-43.

Skotnicki JS, Zask A, Nelson FC, Albright JD & Levin JI (1999) Design and syntheticconsiderations of matrix metalloproteinase inhibitors. Ann NY Acad Sci 878: 61-72.

Smedsrød B (1988) Aminoterminal propeptide of type III procollagen is cleared from thecirculation by receptor-mediated endocytosis in liver endothelial cells. Coll Rel Res 8: 375-388.

Smedsrød B, Melkko J, Risteli L & Risteli J (1990) Circulating C-terminal propeptide of type Iprocollagen is cleared mainly via the mannose receptor in liver endothelial cells. Biochem J271: 345-350.

Smith AN (1986) Colonic muscle in diverticular disease. Clin Gastroenterol 15:917-935.Smith LT, Schwartze U, Goldstein J & Byers PH (1997) Mutations in the COL3A1 gene result in

the Ehlers-Danlos syndrome type IV and alterations in the size and distribution of the majorcollagen fibrils of the dermis. J Invest Dermatol 108: 241-217.

Smith-Mungo LI & Kagan HM (1998) Lysyl Oxidase: properties, regulation and multiple functionsin biology. Matrix Biol 16: 387-398.

Spinale FG, Coker ML, Krombach SR, Mukherjee R, Hallak H, Houck WV, Clair MJ, Kribbs SB,Johnson LL, Peterson JT & Zile MR (1999) Matrix metalloproteinase inhibition during thedevelopment of congestive heart failure. Effects on left ventricular dimensions and function.Circulation Res 85: 364-376.

Steinmann B, Superti-Furga A, Joller-Jemelka H, Cetta G & Byers PH (1989) Ehlers-Danlossyndrome type IV: A subset of patients distinguished by low serum levels of the amino terminalpropeptide of type III procollagen. Am J Med Genet 34: 68:71.

Sukhova GK, Schönbeck U, Rabkin E, Schoen FJ, Poole R, Billinghurst RC & Lippy P (1999)Evidence for increased collagenolysis by interstitial collagenases-1 and –3 in vulnerable humanatheromatous plaques. Circulation 99: 2503-2509.

Sukhova GK, Shi GP, Simon DI, Chapman HA & Libby P (1998) Expression of the elastolyticcathepsins S and K in human atheroma and regulation of their production in smooth musclecells. J Clin Invest 102: 576-583.

Sumner DS, Hokanson DE & Strandness DE Jr. (1970) Stress-strain characteristics and collagen-elastin content of abdominal aortic aneurysms. Surg Gynecol Obstet 130: 459-466.

Sun Y, Sun Y, Wenger L, Rutter JL, Brinckerhoff CE & Cheung HS (1999) Humanmetalloproteinase-1 (collagenase-1) is a tumor suppressor protein p53 target gene. Ann NYAcad Sci 878: 638-641.

Suster SM, Ronnen M & Bubis JJ (1984) Diverticulosis coli in association with Marfan´ssyndrome. Arch Intern Med 144: 203.

Suvanto-Luukkonen E, Risteli L, Sundström H, Penttinen J, Kauppila A & Risteli J (1997)Comparison of three serum assays for bone collagen formation during postmenopausalestrogen-progestin therapy. Clin Chim Acta 266: 105-116.

75Szymanowicz AG, Bellon G, Laurain-Guillaume G, Delvincourt T, Randoux A, Caulet T & Borel

JP (1982) An evaluation by sequential extraction of the proportions of collagen types frommedium sized arteries. Artery 10: 250-265.

Tagasako T, Nakamura K, Kashiwagi S, Inoue S-H, Oto H & Takeo K (1992) Analysis of collagentype III by uninterrupted sodium dodecyl sulfate-polyacrylamide gel electrophoresis andimmunoblotting: Changes in collagen type III polymorphism in aging rats. Eletrophoresis 13:373-378.

Talwar HS, Griffiths CEM, Fisher GJ, Hamilton TA & Voorhees JJ (1995) Reduced type I and typeIII procollagens in photodamaged adult human skin. J Invest Dermatol 105: 285-290.

Tamarina NA, McMillan WD, Shively VP & Pearce WH (1997) Expression of matrixmetalloproteinases and their inhibitors in aneurysms and normal aorta. Surgery 122: 264-271.

Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML & Martin GR (1981)Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26: 99-105.

Thie M, Schlumberger W, Semich R, Rauterberg J & Robenek H (1991) Aortic smooth musclecells in collagen lattice culture: effects on ultrastructure, proliferation and collagen synthesis.Eur J Cell Biol 55: 295-304.

Thomas CV, Coker ML, Zellner JL, Handy JR, Crumbley AJ & Spinale FG (1998) Increasedmatrix metalloproteinase activity and selective upregulation in LV myocardium from patientswith end-stage dilated cardiomyopathy. Circulation 97: 1708-1715.

Thompson RW & Baxter BT (1999) MMP inhibition in abdominal aortic aneurysms. Rationale fora prospective randomized clinical trial. Ann NY Acad Sci 878: 159-178.

Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG & ParksWC (1995) Production and localization of 92-kilodalton gelatinase in abdominal aorticaneurysms. J Clin Invest 96: 318-326.

Thomson HJ, Busuttil A, Eastwood MA, Smith AN & Elton RA (1987) Submucosal collagenchanges in the normal colon and in diverticular disease. Int J Colorect Dis 2: 208-213.

Tintut Y, Parhami F, Boström K, Jackson SM & Demer LL (1998) cAMP stimulates osteoblast-likedifferentiation of calcifying vascular cells. Potential signaling pathway for vascularcalcification. J Biol Chem 273: 7547-7553.

Tomás C, Penttinen J, Risteli J, Risteli L & Kauppila A (1990) Simultaneous evaluation ofepithelial cell function by CA 125 and stromal cell activity by aminoterminal propeptide of typeIII procollagen (PIIINP) in ovarian carcinoma. Ann Med 22: 115-121.

Treharne GD, Boyle JR, Goodall S, Loftus IM, Bell PR & Thompson MM (1999) Marimastatinhibits elastin degradation and matrix metalloproteinase 2 activity in a model of aneurysmdisease. Br J Surg 86: 1053-1058.

Trivedi P, Hindmarsh P, Risteli J, Risteli L, Mowat AP & Brook CGD (1989) Growth velocity,growth hormone therapy, and serum concentrations of the amino-terminal propeptide of type IIIprocollagen. J Pediatr 114: 225-230.

Turnay J, Olmo N, Gavilanes JG & Lizarbe MA (1989) Collagen metabolism in human colonadenocarcinoma. Connect Tissue Res 23: 251-260.

Turnay J, Olmo N, Gavilanes JG & Lizarbe MA (1991) Fibroblastlike primary cells from humancolon adenocarcinoma explants: collagen biosynthesis. In Vitro Cell Dev Biol 27A: 447-452.

Uzawa K, Grzesik WJ, Nishiura T, Kuznetsov SA, Robey PG, Brenner DA & Yamauchi M (1999)Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro. J Bone Mineral Res 14:1272-1280.

van den Berg JS, Limburg M, Kappelle LJ, Pals G, Arwert F & Westerveld A (1998) The role oftype III collagen in spontaneous cervical arterial dissections. Ann Neurol 43: 494-498.

van den Berg JS, Limburg M, Pals G, Arwert F, Westerveld A, Hennekam RC, Albrecht KW(1997) Some patients with intracranial aneurysms have a reduced type III/type I collagen ratio.A case-control study. Neurology 49: 1546-1551.

van der Stappen WJ, Hendriks T & Wobbes T (1990) Correlation between collagenolytic activityand grade of histological differentiation in colorectal tumors. Int J Cancer 45: 1071-1078.

van der Wal AC & Becker AE (1999) Atherosclerotic plaque rupture – pathologic basis of plaquestability and instability. Cardiovasc Res 41: 334-344.

76van der Wal AC, Becker AE, van der Loos CM & Das PK (1994) Site of intimal rupture or erosion

of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory processirrespective of the dominant plaque morphology. Circulation 89: 36-44.

van Keulen CJ, van de Akker E, Pals G & Rauwerda JA (1999) The role of type III collagen in thedevelopment of familial abdominal aortic aneurysms. Eur J Endovasc Surg 18: 65-70.

van Kuppevelt TH, Veerkamp JH & Timmermans JAH (1995) Immunoquantification of type I, III,IV and V collagen in small samples of human lung parenchyma. Int J Biochem Cell Biol 27:775-782.

Vater CA, Harris ED & Siegel RC (1979) Native cross-links in collagen fibrils induce resistance tohuman synovial collagenase. Biochem J 181: 639-645.

Verloes A, Sakalihasan N, Limet R & Koulischer L (1996) Genetic aspects of abdominal aorticaneurysm. Ann NY Acad Sci 800: 44-55.

Virella G, Munoz JF, Galbraith GM, Gissinger C, Chassereau CH & Lopes-Virella MF (1995)Activation of human monocyte-derived macrophages by immune complexes containing lowdensity lipoprotein. Clin Immunol Immunopathol 75: 179-189.

Watson KE, Parhami F, Shin V & Demer LL (1998) Fibronectin and collagen I matrices promotecalcification of vascular cells in vitro, whereas collagen IV matrix is inhibitory. ArteriosclerThromb Vasc Biol 18: 1964-1971.

Weber KT (1989) Cardiac interstitium in health and disease: The fibrillar collagen network. J AmColl Cardiol 13: 1637-1652.

Weber KT (1998) Monitoring vascular sclerosis in hypertension. A new window of opportunity.Circulation 98: 498-500.

Welgus HG, Burgeson RE, Wootton JAM, Minor RR, Fliszar C & Jeffrey JJ (1985) Degradation ofmonomeric and fibrillar type III collagens by human skin collagenase. J Biol Chem 260: 1052-1059.

Wess L, Eastwood M, Busuttil A, Edwards C & Miller A (1996b) An association between maternaldiet and colonic diverticulosis in an animal model. Gut 39: 423-427.

Wess L, Eastwood MA, Edwards CA Busuttil A, Miller A (1996a) Collagen alteration in an animalmodel of colonic diverticulosis. Gut 38: 701-706.

Wess L, Eastwood MA, Wess TJ, Busuttil A & Miller A (1995) Cross linking of collagen isincreased in colonic diverticulosis. Gut 37: 91-94.

Whiteway J & Morson BC (1985) Elastosis in diverticular disease of the sigmoid colon. Gut 26:258-266.

Wilson KE, Langdon ASP, Lessells AM & Miller WR (1996) Expression of the extracellularproteins tenascin in malignant and benign ovarian tumours. Br J Cancer 74: 999-1004.

Wilson R, Lees JF & Bulleid NJ (1998) Protein disulfide isomerase acts as a molecular chaperoneduring the assembly of procollagen. J Biol Chem 273: 9637-9643.

Wohllebe M & Carmichael DJ (1978) Type-I trimer and type-I collagen in neutral-salt-solublelathyritic-rat dentine. Eur J Biochem 92: 183-188.

Yamauchi M & Mechanic GL (1988) Cross-linking of collagen. In: Nimni ME (ed) Collagen. CRCpress, Boca Raton, FL, p 157-172.

Zhu GG, Risteli J, Puistola U, Kauppila A & Risteli L (1993b) Progressive ovarian carcinomainduces synthesis of type I and type III procollagens in the tumor tissue and peritoneal cavity.Cancer Res 53: 5028-5032.

Zhu GG, Risteli L, Mäkinen M, Risteli J, Kauppila A & Stenbäck F (1995) Immunohistochemicalstudy of type I collagen and type I pN-collagen in benign and malignant ovarian neoplasms.Cancer 75: 1010-1017.

Zhu GG, Stenbäck F, Risteli L, Risteli J & Kauppila A (1993a) Organization of type III collagen inbenign and malignant ovarian tumors. An immunohistochemical study. Cancer 72: 1679-1684.

Zucker S, Hymowitz M, Conner C, Zarrabi HM, Hurewitz AN, Matrisian L, Boyd S, Nicolson G &Montana S (1999) Measurement of matrix metalloproteinases and tissue inhibitors ofmetalloproteinases in blood and tissues. Clinical and experimental applications. Ann NY AcadSci 878: 212-227.