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Plasminogen in periodontitis and wound repair
Rima Šulniūtė
Department of Medical Biochemistry and Biophysics Umeå 2013
Responsible publisher under Swedish law: the Dean of the Medical FacultyThis work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7459-547-5 ISSN: 0346-6612 Ev. info om Omslag/sättning/omslagsbild: Elektronisk version tillgänglig på http://umu.diva-portal.org/ Tryck/Printed by: Servicecenter KBC Umeå, Sweden 2013
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Table of contents Table of contents 3
Abstract 5
List of papers 7
Abbreviations 8
Introduction 9
1. The Plasminogen Activator System 10
1.1. Plasminogen/plasmin 10
1.2. Plasminogen activators 12
1.2.1. Tissue-type plasminogen activator 12
1.2.2. Urokinase-type plasminogen activator 12
1.3. Inhibitors of the plasminogen activator system 13
1.3.1. Plasmin inhibitors: α2-antiplasmin and α2-macroglobulin 13
1.3.2. Plasminogen activator inhibitors 14
1.4. Receptors of the plasminogen activator system 14
1.4.1. Plasminogen receptors 15
1.4.2. The urokinase receptor 17
1.5. Plasminogen in cell signaling 17
1.6. The plasminogen activator system in tissue remodeling 18
1.6.1. Fibrinolysis 18
1.6.2. Bone remodeling 19
1.6.3. Other tissue remodeling processes requiring the plasminogen activator system 19
1.7. The plasminogen activator system in infection 20
1.8. The plasminogen activator system in inflammation 21
2. Periodontitis 22
2.1. The periodontium in health and disease 22
2.1.1. Gingiva 23
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2.1.2. Periodontal ligament and root cementum 24
2.1.3. Alveolar bone in health and disease 25
2.2. Pathogenesis of periodontitis 26
2.3. The plasminogen activator system in periodontitis 27
2.4. Animal models in periodontal research 28
3. Wound Healing 30
3.1. Phases of wound repair 30
3.1.1. The inflammatory phase 30
3.1.2. The proliferative phase 31
3.1.3. The remodeling phase 32
3.2. The plasminogen activator system in wound healing 32
3.3. Cutaneous wound models in mice 33
Results and Discussion 35
Plasmin is essential in preventing periodontitis in mice (Paper I) 35
Plasminogen is a critical regulator of cutaneous wound healing (Paper II) 36
Plasminogen is a key pro-inflammatory regulator that accelerates the healing of acute and diabetic
wounds (Paper III) 37
Concluding Remarks 39
Acknowledgements 40
References 42
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Abstract
The plasminogen activator (PA) system plays a critical role in many physiological and
pathological processes, such as fibrinolysis, extracellular matrix (ECM) degradation, wound healing,
inflammation, and cancer. The key component of the PA system is plasmin, a broad-spectrum serine
protease that is derived from its inactive form, plasminogen. The first aim of this thesis research was to
determine the role of plasminogen in periodontitis, an inflammatory oral disease. The second aim was to
explore the molecular mechanism by which plasminogen contributes to wound healing in the skin. Finally,
the third aim was to investigate the possibility of using plasminogen as a treatment for skin wounds,
especially for chronic wounds, such as diabetic wounds.
Periodontitis is an oral disease that involves a bacterial infection, the inflammation of the
periodontium, and the degradation of gum tissue and alveolar bone. This disease is irreversible and, in
severe cases, can lead to loss of teeth due to the degradation of the periodontal ligament and alveolar
bone. To study the effects of the PA system on oral health, we monitored the development of periodontitis
in plasminogen-deficient mice and plasminogen activator-deficient mice. In control wild-type mice,
periodontitis did not occur. However, in plasminogen-deficient mice, periodontitis developed rapidly
within 20 weeks after birth. The morphological studies of plasminogen-deficient mice showed the
detachment of gingival tissues, resorption of the cementum layer, formation of necrotic tissue, and severe
alveolar bone degradation. Immunohistochemical staining showed the massive infiltration of neutrophils
into the periodontal tissues. Interestingly, doubly deficient mice lacking both tissue-type plasminogen
activator (tPA) and urokinase-type PA (uPA) developed periodontitis at a similar rate as the plasminogen-
deficient mice, but mice lacking only tPA or uPA remained healthy. The intravenous injection of human
plasminogen for 10 days into plasminogen-deficient mice led to the absorption of necrotic tissue, the
diminution of inflammation, and the full regeneration of gum tissues. Notably, there was also partial re-
growth of degraded alveolar bone.
The wound healing process consists of three overlapping phases: inflammatory,
proliferative, and remodeling. It has been postulated that the PA system plays an integral role in this
process, and a lack of plasminogen leads to delayed wound healing in mice. To study the role of the PA
system in wound healing, we monitored the responses of wild-type, plasminogen-deficient and diabetic
mice to incision and burn wounds. We found that in addition to being delayed, the wound healing process
in plasminogen-deficient mice was only superficial in nature. The plasminogen-deficient mice were unable
to clear the provisional matrix after the formation of granulation tissue, and an extensive fibrin
deposition. In addition, persistent inflammation was still present subcutaneously in these mice 60 days
after introduction of the wound.
The essential role of plasminogen in burn and incision wounds healing was further
confirmed by reconstitution experiments. Both intravenous and subcutaneous administrations of human
plasminogen to plasminogen-deficient mice led to a restored healing rate and wound maturation that was
comparable to those of wild-type mice. We also demonstrated that plasminogen supplementation of
plasminogen to wild-type and diabetic mice significantly improved the healing of cutaneous wounds.
Plasminogen levels were not only temporally increased during the inflammation phase but also spatially
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concentrated at the site of the wound. The wound-specific accumulation of plasminogen after systemic
supplementation is mainly due to the transportation of plasminogen by neutrophils and macrophages.
Furthermore, the increased expression of interleukin 6 and the enhanced phosphorylation of STAT3 were
observed in the wound after plasminogen treatment. These data indicate that plasminogen acts as a key
pro-inflammatory regulator. It enhances pro-inflammatory cytokines and activates intracellular signaling
events during wound healing.
Taken together, the data obtained during the course of this project indicate that
plasminogen is crucial for oral health in mice. We also demonstrate that supplementation of plasminogen
to mice with periodontitis results in healing of gum tissues and significant re-growth of alveolar bone.
Therefore, plasminogen may be a new drug that will be competitive to currently used oral health-related
procedures, such as implantations and surgeries. Furthermore, we demonstrate for the first time that, in
addition to its role in extracellular matrix degradation, plasminogen is a key pro-inflammatory factor that
accumulates at the wound and potentiates the early inflammatory response during wound healing. Based
on our findings, we propose the administration of plasminogen as a novel therapeutic strategy for the
treatment of different types of wounds, including chronic diabetic wounds.
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List of papers
I Sulniute R., Lindh T., Wilczynska M., Li J., and Ny T. Plasmin is essential in
preventing periodontitis in mice. Am. J. Path. 2011 179(2): 819-28.
II Sulniute R., Shen Y., Ahlskog N., Guo Y-Z., Li J., Wilczynska M, and Ny T.
Plasminogen is a critical regulator of cutaneous wound healing. Manuscript
III Shen Y., Guo Y-Z., Mikus P., Sulniute R., Wilczynska M., Ny T*, and Li J*.
Plasminogen is a key pro-inflammatory regulator that accelerates the healing of acute and diabetic
wounds. Blood. 2012 119(24): 5879-87.
* Corresponding authors
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Abbreviations
α2-AP α2-antiplasmin
α2-M α2-macroglobulin
aa amino acid
CEJ cemento-enamel junction
ECM extracellular matrix
EGF epidermal growth factor
FBN fibrinogen
K1-K5 kringle 1 to kringle 5
kDa kilodalton
MMP matrix metalloproteinase
PA plasminogen activator
PAI-1 plasminogen activator inhibitor type 1
PAI-2 plasminogen activator inhibitor type 2
PAP plasminogen N-terminal activation peptide
Plg-R plasminogen receptor
PMN polymorphonuclear leukocyte
PN-1 protease nexin-1
tPA tissue-type plasminogen activator
uPA urokinase-type plasminogen activator
uPAR urokinase-type plasminogen activator receptor
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Introduction
The key component of the plasminogen activator (PA) system is the broad-spectrum serine
protease plasmin, which is formed from its inactive form, plasminogen. Plasminogen is activated by either
of the two plasminogen activators (PAs), tissue-type PA (tPA) or urokinase-type PA (uPA). These
proteases are regulated by several specific inhibitors against both PAs and plasmin. The PA system plays a
key role in many physiological and pathological processes, such as fibrinolysis, extracellular matrix (ECM)
degradation, tissue remodeling, inflammation, and cell migration. These processes contribute to
development of periodontitis and occur during wound healing.
Periodontitis is an oral disease that involves a bacterial infection, the inflammation of the
periodontium, and the degradation of gum tissue and alveolar bone. It normally starts with formation of
gingivitis that is a mild, reversible inflammation of the gums caused by bacterial plaque that accumulates
on the teeth at the gingival margin. In some patients, gingivitis develops into periodontitis. Periodontitis is
an irreversible disease and, in severe cases, can lead to tooth loss due to degradation of the periodontal
ligament and the alveolar bone.
The wound healing process consists of three overlapping phases: inflammation,
proliferation, and remodeling. The PA system is involved in all phases of the healing process beginning
with clot formation and ending with the remodeling of granulation tissue. The improper or delayed
healing of wounds caused by either trauma or surgical procedures may lead to chronic infections and
severe scarring.
The aims of thesis were three-fold:
1) to study the role of plasminogen in oral health by monitoring the development of periodontitis in wild-
type mice, plasminogen-deficient mice, and mice lacking the plasminogen activators tPA and uPA;
2) to study plasminogen’s contribution to, and acceleration of, the healing of cutaneous wounds in
plasminogen-deficient and diabetic mice;
3) to investigate the molecular mechanism by which supplemental plasminogen improves the healing of
cutaneous wounds in diabetic mice.
Conclusion: The studies included in this thesis suggest that plasminogen has a potential as a new drug
to treat periodontal disease and to be an alternative for presently used oral health-related procedures,
such as implantations and surgeries. These studies also indicate that plasminogen is an important
regulator of wound healing, and it enhances the rate and quality of healing of acute and chronic cutaneous
wounds.
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1. The Plasminogen Activator System
The PA system, traditionally called the fibrinolytic system, is a well-balanced enzymatic
cascade. The central molecule of this system, plasmin, is formed by proteolytic activation of the
proenzyme, plasminogen, by tPA or uPA [1]. The activity of this system is regulated at many levels, which
includes inhibition by plasmin inhibitors such as α2-AP and α2-macroglobulin (α2-M) and PA inhibitors
(PAIs) such as PAI-1 and PAI-2 (Figure 1).
Plasmin is a serine protease that degrades a large number of ECM proteins, including
fibrin, gelatin, fibronectin, and proteoglycans [2]. The PA system has been implicated in important
physiological events such as embryogenesis, wound healing, and angiogenesis, as well as in a variety of
pathological conditions such as infection, tumor growth, and metastasis [3-7]. The development and
characterization of mice deficient in plasminogen and other members of the PA system has provided a
number of animal models that mimic human conditions and contribute to an understanding of the
mechanisms associated with many pathological events. Such animal models have been utilized in this
thesis to determine novel roles for plasminogen in the development of periodontitis and in healing of
cutaneous wounds.
Figure 1. A schematic representation of the PA system. The proenzyme plasminogen is converted to the
active enzyme plasmin by tPA or uPA. Plasmin degrades fibrin and can convert MMPs and TGF-β into
their active forms. Inhibition of plasmin can occur at the PA level by PAIs and at the plasmin level by α2-
AP or α2-M. Adapted from C. Moali and D.J. Hulmes [7].
1.1. Plasminogen/plasmin
Human plasminogen is synthesized as an 810 amino acid (aa) single polypeptide protein
that is cleaved during secretion from the liver to form a 791 aa protein with a molecular mass of 92 kDa
[8]. The concentration of plasminogen in blood plasma is approximately 2 µM (200 µg/ml), and its half-
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life is 2.2 days [9-11]. Plasminogen is composed of an amino-terminal domain containing five kringle
structures that are stabilized by three intra-chain disulfide bridges (the heavy chain) and a carboxyl-
terminal trypsin-like serine protease domain that contains the catalytic triad His603, Asp646, and Ser741 (the
light chain) (Figure 2) [12]. The five kringle domains (K1-K5) on the heavy chain contain lysine binding
sites, and K1 has the highest affinity for lysine [13]. The lysine binding sites are crucial for the specific
recognition of fibrin, cell surface receptors, and α2-AP [13].
Plasminogen exists in two different molecular forms: Glu-plasminogen and Lys-
plasminogen. Glu-plasminogen is the native, uncleaved plasminogen with an amino-terminal glutamic
acid residue. Upon cleavage of the Lys76-Lys77 bond, Lys-plasminogen is formed, which is 76-aa shorter
than Glu-plasminogen and has an amino-terminal lysine residue [14, 15]. Lys-plasminogen has a higher
binding affinity to fibrin but has a shorter half-life (0.8 days) compared to Glu-plasminogen (2.2 days) [13,
16]. Both forms of plasminogen can be activated by the physiological PAs [17]. When the Arg560-Val561
bond of the single-chain plasminogen is cleaved by tPA or uPA, a two-chain disulfide-linked active serine
protease is formed [18]. Plasminogen is primarily synthesized in the liver [19, 20], but several other
organs, including the adrenal gland, kidney, brain, testis, heart, lung, uterus, spleen, thymus, and gut,
have been identified as possible sources of plasminogen [21].
Figure 2. The structural features of plasminogen and PAs. Adapted from C. Moali and D.J. Hulmes [7].
Plasmin is responsible for the degradation of ECM components, which include fibrin,
fibronectin, and laminin, into soluble fragments [4, 5]. It can also indirectly degrade additional
components of the ECM by activating the precursors of the matrix metalloproteinases (MMPs) [6, 22].
Both the degradation and cell surface binding abilities of plasmin play a crucial role in cell migration
during angiogenesis, inflammation, wound healing and cancer. The existence of protease-activated
receptors (PARs) and the newly discovered plasminogen receptors (Plg-Rs) suggest an even broader
spectrum of action for plasminogen through cell signaling pathways [23-25].
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1.2. Plasminogen activators
Similar to plasminogen, the two main physiological PAs, tPA and uPA, are multidomain
serine proteases [26]. Although tPA and uPA catalyze the same reaction and share similar overall
structures in terms of kringle and epidermal growth factor-like (EGF) domains (Figure 2), tPA and uPA
have different biological roles. Traditionally, tPA is thought to activate plasminogen during fibrinolysis,
whereas uPA activates cell-associated plasminogen during cell migration [4]. However, functional studies
in PA-deficient mice have shown that tPA and uPA are involved in many other physiological reactions and
may also have partially overlapping physiological functions [27, 28].
1.2.1. Tissue-type plasminogen activator
Human tPA is secreted as a 527 aa single-chain, multidomain glycoprotein with a molecular
mass of approximately 70 kDa [29, 30]. The normal plasma concentration of tPA is 5-10 ng/ml. The non-
catalytic amino-terminal domain (the heavy chain) of tPA contains the fibrin-binding finger domain, the
EGF domain, and two kringle domains, where the second kringle domain has a high binding affinity for
lysine [1]. The carboxyl-terminal region (the light chain) contains the serine protease domain. The single-
chain form of human tPA is converted to the two-chain form upon cleavage of the Arg275 – Ile276 peptide
bond by plasmin or kallikrein [31]. Interestingly, both the single-chain and two-chain forms are active,
which makes tPA unique among serine proteases [32, 33].
tPA is involved in biological functions that are not related to the activation of plasminogen,
such as long-term potentiation in the hippocampus and excitotoxic damage [34]. Several tPA-binding sites
on different cell types have been described, but a specific receptor for tPA has not yet been identified [35-
37]. tPA is mainly produced by endothelial cells, but it is also produced by keratinocytes, neurons, and
melanocytes [38-40]. Additionally, tPA is broadly distributed in the brain and plays important roles
during physiological and pathological conditions [41]. Elevated levels of tPA lead to hyperfibrinolysis and
excessive bleeding disorders [42].
1.2.2. Urokinase-type plasminogen activator
Human uPA is synthesized as an inactive, single-chain, multidomain glycoprotein of 411 aa
(53 kDa). The concentration of uPA in plasma varies between 2 and 20 ng/ml [43, 44]. The single-chain
uPA is an inactive proenzyme that is converted to the active two-chain form of uPA upon cleavage of the
Lys158-Ile159 bond by proteases such as plasmin, kallikrein, coagulation factor XIIa, or cathepsin B [28, 45].
The amino-terminal region (the heavy chain) contains the EGF domain and the single kringle domain
(Figure 2), and the carboxyl-terminal region (the light chain) contains the active site with the classical
serine protease catalytic triad – His204, Asp255, and Ser356 [1, 45, 46]. In contrast to tPA, uPA has no fibrin-
binding capability. However, uPA does have a well-characterized receptor, the uPA receptor (uPAR), that
is expressed on the surface of many cell types. This receptor enables cell-localized accumulation of uPA
and the high local activation of plasminogen [3]. In vitro studies in cultured pre- and postnatal human
13
tissues and cells have shown that uPA is expressed in lung and kidney cells, as well as in keratinocytes and
endothelial cells [47, 48]. Increased levels of expression of uPA and/or uPAR are seen in the cancer cells
from tumors of the breast, colon, ovary, stomach, kidney, brain, and other organs [49, 50].
1.3. Inhibitors of the plasminogen activator system
Tight regulation of proteolytic activity is fundamental in many biological processes. The
three major serine protease inhibitor (serpin) molecules, α2-AP, PAI-1, and PAI-2, regulate the activity of
plasmin and PAs. The native state of serpins, the stressed (S)-state, is metastable, and the proteolytic
cleavage of the reactive center loop (RCL) results in a dramatic conformational change to the relaxed (R)-
state along with an increase in the thermal stability of the serpin molecule. This family of inhibitors is
known as suicide inhibitors – the serpin-protease complex, in which inhibition of the protease is
maintained by covalent linkage to the serpin, is irreversible [51-53]. Serpins regulate very diverse
physiological processes that require proteolytic activity, including coagulation and fibrinolysis,
complement activation, inflammation, angiogenesis, apoptosis, neoplasia, and viral pathogenesis [54].
1.3.1. Plasmin inhibitors: α2-antiplasmin and α2-macroglobulin
α2-AP, also known as α2-plasmin inhibitor (A2PI), is the major physiological inhibitor of
plasmin, but it can also inhibit chymotrypsin and trypsin. Human α2-AP is a 452 aa single-chain
glycoprotein with a molecular mass of approximately 70 kDa. The concentration in plasma is 1 µM (70
µg/ml), and the half-life is 2.6 days [55, 56]. α2-AP rapidly binds to the active center and the lysine-
binding sites of plasmin, thereby inhibiting both its enzymatic activity and its lysine-binding capability.
Both of these sites must be free for α2-AP to inhibit plasmin, which means that plasmin bound to fibrin is
protected from inhibition by α2-AP [57-59]. An α2-AP deficiency in humans increases the chances of
excessive bleeding, especially during trauma, surgery or dental extraction [60]. In contrast, the lack of α2-
AP in mice increases platelet microaggregation, resulting in thrombus formation [61]. α2-AP is mainly
synthesized in the liver, but is also produced to some extent in the kidneys, intestines, and brain [62, 63].
Human α2-M is a disulfide-linked homotetramer of 1451 aa (725 kDa) with a relatively high
plasma concentration of 2.5 mg/ml [64-67]. It is a member of the I39 family of protease inhibitors, and it
inhibits proteases, regardless of their catalytic type, through a unique mechanism often referred to as the
‘trap mechanism’ [68]. Specific cleavage by the active protease in the ‘bait’ region of α2-M induces a large
conformational change that results in the trapping of the protease within the large central cavity of the
inhibitor. In this way, α2-M bound to a protease prevents the protease from reacting with high molecular
weight substrates such as fibrin and collagen, but leaves it free to interact with low molecular weight
substrates. The liver is the main producer of this inhibitor, but other organs may also produce it. A lack of
α2-M is associated with Alzheimer disease [69].
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1.3.2. Plasminogen activator inhibitors
PA inhibitor type 1 (PAI-1) is a single-chain glycoprotein of 379 aa (45 kDa) and has a
plasma concentration of approximately 20 ng/ml [70]. PAI-1 is in its active form when secreted but
spontaneously converts to its latent form [71, 72]. This serpin rapidly inhibits single-chain tPA, two-chain
tPA, and two-chain uPA, but not single-chain uPA [70, 73], by forming covalently bound one-to-one
complexes [53, 74, 75]. PAI-1 is produced at low levels by several different cell types in response to growth
factors, cytokines, phorbol esters, endotoxins, and hormones [76]. A primary function of PAI-1 in vivo is
to balance the proteolytic activity of the PAs during fibrinolysis [77]. Beside its role in regulating
hemostasis, PAI-1 is important for the regulation of cell adhesion, cell migration, and PA- or plasmin-
dependent tumor invasion [78, 79]. Studies in transgenic mice have also revealed a functional role for
PAI-1 in wound healing, atherosclerosis, metabolic disturbances such as obesity and insulin resistance,
tumor angiogenesis, chronic stress, bone remodeling, asthma, rheumatoid arthritis, fibrosis,
glomerulonephritis, and sepsis [80].
PA inhibitor type 2 (PAI-2) is a single-chain, 425 aa serpin that is detectable in plasma
only during pregnancy and reaches concentrations from 5 ng/ml up to 250 ng/ml [81-83]. PAI-2 is not
efficiently secreted and, therefore, accumulates intracellularly as a non-glycosylated 47 kDa protein [84].
A small portion of single-chain PAI-2 is secreted as a 60 kDa plasma glycoprotein [85]. PAI-2 can
spontaneously polymerize, and the CD-loop of PAI-2 functions as a redox-sensitive molecular switch that
converts PAI-2 between a stable, active, monomeric conformation and a polymeric conformation [86].
This serpin is produced by several cell types, including monocytes/macrophages, keratinocytes, and
epithelial cells. Extracellular PAI-2 inhibits both uPA and two-chain tPA, although PAI-2 is a poor
inhibitor of single-chain tPA, with up to 10,000-fold lower inhibition compared to uPA inhibition [87].
The extracellular form of PAI-2 is also considered to be a regulator of uPA activity in blood vessels and in
the ECM during the pregnancy, cancer formation, and inflammation [88, 89]. The role of intracellular
PAI-2 remains unclear, although several studies have suggested that it may play a role in protecting cells
from apoptosis [81, 90].
1.4. Receptors of the plasminogen activator system
Cell surface-associated proteolysis plays a crucial role in cell migration during
embryogenesis, angiogenesis, inflammation, healing, and invasion. There are receptors for uPA and
plasminogen, and co-localization of plasminogen and PAs on the cell surface ensures that sufficient local
proteolytic activity is available. Cell-bound plasmin is protected from its physiological inhibitors α2-AP
and α2-M [91]. The interaction between the cell surface and plasminogen is mediated by a heterogeneous
group of Plg-Rs. Many, but not all, Plg-Rs interact through their carboxy-terminal lysine with the lysine-
binding sites within the kringle domains of plasminogen [92, 93]. These receptors can be categorized into
two groups: tailless (those without a cytoplasmic tail) and tailed (those with a cytoplasmic tail) [94]. Most
Plg-Rs, including enolase-1, annexin 2, Pg-RKT, and histone H2B, belong to the tailless group, and the
tailed group contains protease-activated receptors and integrins (αMβ2, αIIbβ3, αvβ3, α4β1, and α5β1).
15
Although β2 integrins do not contain carboxyl-terminal lysines, they are still able to interact with the
lysine binding site of plasminogen. Both tailless and tailed Plg-Rs promote plasminogen activation and
pericellular proteolysis.
1.4.1. Plasminogen receptors
Plasminogen receptors (Plg-R) are a diverse group of proteins that are expressed on the
surface of many types of cells. Plasminogen binding has been observed on B cells, smooth muscle cells,
fibroblasts, keratinocytes, endothelial cells, renal epithelial cells, leukocytes, platelets, monocytes or
monocytoid cell lines, and metastatic colorectal and mammary carcinoma cells [91, 93, 95-99]. The tailless
Plg-Rs are responsible for concentrating the local proteolytic activity of plasminogen on the cell surface,
and this promotes sufficient cell mobility (Figure 3). The tailed receptors, on other hand, might be
involved not only in cell surface-associated proteolytic activity but also in cell signaling.
Figure 3. Plg-R activity during monocyte/macrophage recruitment. Monocyte activation by external
stimuli leads to expression of Plg-Rs at the cell surface. Plasminogen binds to the Plg-R and is converted
to plasmin by PAs. When monocytes differentiate into macrophages, additional Plg-Rs are exposed on the
cell surface to maintain further migration and activation of intracellular signaling at the inflammation site.
Adapted from E.F. Plow and R. Das [100].
Enolase-1, also called α-enolase, is a 48 kDa glycolytic enzyme that acts as a 2-phospho-
D-glycerate hydrolase in the cytoplasm of prokaryotic and eukaryotic cells [101]. Enolase-1 was first
described as a plasminogen receptor on the surface of monocytoid cells [92] and was later detected on the
surfaces of other cell types [102]. The plasminogen-enolase-1 interaction is mediated by the binding of
plasminogen’s kringle domains to the carboxyl-terminal lysine residue of enolase-1 [92, 103]. On the cell
16
surface, the interaction of plasminogen with enolase-1 is enhanced by tPA, and protects plasminogen from
inhibition by α2-antiplasmin [103]. Enolase-1 plays a crucial role in plasminogen-mediated recruitment of
monocytes to inflamed areas of the lung. Using a monocytoid cell line and isolated blood monocytes, cell-
surface expression of enolase-1 was also shown to increase dramatically upon lipopolysaccharide
stimulation [104].
Annexin 2 is a member of the annexin superfamily of calcium-dependent, phospholipid-
binding proteins. Annexin 2 is 38 kDa protein containing a large, conserved carboxyl-terminal ‘core’
domain (34.2 kDa) and a smaller (3.4 kDa) more variable amino-terminal ‘tail’ domain [105]. This
receptor does not contain a carboxyl-terminal lysine, but its binding partner, p11 (S100A10), does, and
this allows annexin 2 to bind plasminogen, tPA and plasmin [106]. The annexin 2 can form heterodimer,
which consists of two annexin 2 monomers and two p11 subunits, which have even greater plasmin
generation ability than monomeric annexin 2 [107]. Annexin 2 was first identified as a Plg-R on the
surface of endothelial cells, but it is also abundant on monocytoid, monocytes/macrophages, myeloid
cells, developing neuronal cells, and some tumor cells [108]. Mice lacking annexin 2 show an impaired
clearance of arterial thrombi, deposition of fibrin in the microvasculature, and angiogenic defects in a
variety of tissues. Interestingly, p11 is expressed at very low levels in the absence of annexin 2 [109, 110].
In rats, arterial thrombosis is diminished by intravenous treatment with recombinant annexin 2 [111].
Plg-RKT is the first transmembrane receptor for plasminogen that has been identified. This
membrane-integrated, 147 aa, and 17 kDa protein has a carboxyl-terminal lysine and is predicted to
contain two transmembrane helices. It is widely expressed on the surface of cells known to bind
plasminogen and is highly co-localized on the cell surface with urokinase receptor (uPAR) [25]. Plg-RKT
plays a key role in the plasminogen-dependent regulation of macrophage recruitment during an
inflammatory response [112]. This novel Plg-R is highly expressed in murine and human adrenal
medullary chromaffin cells, human pheochromocytoma tissue, and murine hippocampal cells [113]. The
expression of Plg-RKT in catecholaminergic tissues is responsible for the regulation of catecholamine
release through cell surface plasminogen activation [113].
Integrin αMβ2, a non-covalently linked heterodimer of αM and β2 subunits, belongs to a
large family of heterodimeric cell adhesion receptors that mediate a wide spectrum of biological functions
[114]. Integrin αMβ2 is a tailed Plg-Rs that does not contain a carboxyl-terminal lysine but still interacts
with the lysine binding sites in plasminogen’s kringle domains. This receptor binds both plasminogen and
uPA in a dose-dependent, saturable manner, and this binding leads to a 50-fold acceleration of plasmin
generation [115]. Integrin αMβ2 also forms a tight complex with uPAR that leads to enhanced integrin-
dependent leukocyte responses [116]. As a tailed Plg-R, αMβ2 takes part in cell signaling in which
plasminogen triggers activation of ERK1/2 and Akt through αMβ2 in neutrophils [117]. Integrin αMβ2 is
expressed in monocytes, granulocytes, macrophages, and natural killer cells, and it has been implicated in
the diverse functions of these cells, which include phagocytosis, cell-mediated killing, chemotaxis, and
cellular activation [118-120]. The characterization of αMβ2-deficient mice showed a variety compromised
leukocyte-dependent responses [118-120].
Protease-activated receptors (PARs) are G-protein-coupled receptors that are
activated by proteolytic cleavage of their extracellular amino-terminus. The cleaved part of the receptor
17
interacts with its extracellular loop and leads to a transmembrane signal. There are four known human
PARs – PAR1, PAR2, PAR3, and PAR4 [121] – and these receptors are found on the surfaces of platelets,
endothelial cells, neurons, and myocytes. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce
activation of platelets and vascular cells, whereas PAR2 is preferentially cleaved by trypsin. Alternatively,
plasmin can activate PAR1 and PAR4 [122, 123]. Plasmin stimulates an inflammatory response in human
dental pulp cells by activating PAR1 [124], and it also mediates platelet aggregation through proteolytic
cleavage of PAR4 [123]. PAR1-mediated signaling is involved in cell growth and division, which suggests
additional biological roles for plasmin [125, 126]. In fibroblasts, plasmin is able to induce p44/42
mitogen-activated protein kinase (MAPK) activation and to upregulate Cyr61 gene expression through
PAR1 [122]. This expression of a growth factor-like gene can potentially contribute to fibroblast
proliferation and migration, especially during the wound healing process.
1.4.2. The urokinase receptor
uPAR is a 335 aa polypeptide that includes a 22 residue amino-terminal signal peptide.
The mature uPAR protein (283 aa) is highly glycosylated and composed of three similarly sized
homologous domains [127]. uPAR is anchored to the plasma membrane through
glycosylphosphatidylinositol (GPI) linkages [128] and is the primary, specific cellular receptor for uPA
[129]. uPAR-bound uPA is active and susceptible to inhibition by PAI-1 or PAI-2. Furthermore, uPAR co-
localizes with integrins at the leading edge of migrating cells. This receptor not only facilitates uPA-
dependent proteolytic activity at the cell surface but can also initiate intracellular signaling [130].
The plasma levels of PAs are tightly regulated in vivo to maintain a balance between
fibrinolysis and thrombosis. One of the mechanisms responsible for this balance is the rapid clearance of
PAs via receptor-mediated endocytosis. uPAR can function as one such clearance receptor in cooperation
with low density lipoprotein receptor-related protein (LRP) [131]. PAIs inhibit uPAR-bound uPA and
cause this complex to be internalized and undergo degradation in the lysosomes of hepatic and
monocytoid cells.
uPAR is expressed on monocytes, macrophages, fibroblasts, endothelial cells, and a variety
of tumor cells [129], and its expression is increased in many pathological conditions, including cancer,
inflammation, and infection [132]. uPAR-deficient mice are essentially healthy, with normal fertility,
development, and hemostasis, even though macrophages from these mice showed reduced activation by
plasminogen [133]. The only obvious health issue these mice have is an increased sensitivity to induced
urinary tract infections. Neutrophil recruitment is similar in both uPAR-deficient and wild-type mice, but
neutrophils of uPAR-deficient mice have impaired bacterial clearance [134].
1.5. Plasminogen in cell signaling
Cell-bound plasminogen has traditionally been regarded to only be responsible for cell migration through
its proteolytic degradation of the ECM. However, in vivo studies have demonstrated that plasmin is able
to trigger functional changes in cells, which suggests that plasminogen can activate receptor-mediated
18
signaling pathways [23]. Several receptors in the PA system (annexin 2, integrins, PARs, and uPAR) also
induce intracellular signaling pathways that are critically important for the expression of many pro-
inflammatory mediators in various cells [135]. In monocytes, plasmin induces the expression of cytokines
(IL-1α, IL-β, and TNF-α) and tissue factor (TF) via the NF-κB pathway [136]. Plasmin also triggers pro-
inflammatory cytokine expression in macrophages via the annexin 2 receptor and is involved in the
activation of multiple signaling pathways, including the JAK/STAT, p38 MAPK, ERK1/2, and NF-κB
pathways [137]. Notably, the MAPK pathway is one of the major signaling pathways that control the
expression of different pro-inflammatory genes [138]. In Paper II, we also show that plasminogen
accumulates around wounds, induces the expression of IL-6, and activates STAT3. Additionally, plasmin
induces the expression of the angiogenic and wound-healing promoter Cyr61 in fibroblasts via the PAR1
receptor [122]. Furthermore, plasmin leads to a decrease in the expression of the pro-apoptotic protein
BimEL in primary hepatocytes via the ERK1/2 signaling pathway [139]. Taken together, these studies have
revealed a much broader range of functions for plasmin than was previously known.
1.6. The plasminogen activator system in tissue remodeling
The PA system has been implicated in several physiological and pathological processes that
require tissue remodeling. The most studied of these processes are fibrinolysis and the degradation of the
ECM. Plasmin has a proteolytic activity against various components of the ECM, which include laminin,
fibronectin, and collagens. Furthermore, plasmin may activate other ECM-degrading proteases, such as
MMPs, and in this way, it may promote additional degradation processes [4]. Several cell types can bind
plasminogen/plasmin and/or other components of the PA system on their surfaces to initiate cell
migration, tumor invasion, and angiogenesis [3, 140].
1.6.1. Fibrinolysis
The fibrinolytic activity of plasmin is crucial for the degradation of fibrin clots in the
vascular system. A localization of this proteolytic activity at the site of a developing thrombus, where
plasmin and PAs are protected from circulating inhibitors, is an essential process in regulating fibrinolysis
[1]. tPA is the main molecule responsible for activating fibrin-bound plasminogen because uPA lacks a
fibrin-binding domain. However, in the absence of tPA, uPA may take over the plasminogen-activating
function in fibrinolysis [27]. It is necessary to maintain a strict balance between plasminogen activation
and inhibition of the PAs. Patients with reduced concentrations of PA inhibitors suffer from excessive
bleeding, whereas elevated levels of PA inhibitors can lead to myocardial infarction, embolism, and
atherosclerosis [141-143].
The fibrinolytic activity of plasmin plays a critical role in tissue remodeling during wound
healing [144, 145]. In Paper II, we also have shown that fibrinogen deposition remained at the wound site
even after 60 days of healing in plasminogen-deficient mice. Bugge et al demonstrated that mice deficient
19
in both plasminogen and fibrinogen have restored rates of re-epithelialization comparable to wild-type
mice, but the quality of the healed wound may not be the same as that of wild-type mice [146].
1.6.2. Bone remodeling
Bone remodeling includes bone formation and bone resorption. These processes are
regulated by a number of genetic, hormonal, nutritional, growth and other factors and continues during
the lifetime of the bone. During the bone remodeling process, mineralized bone is removed by osteoclasts
followed by the formation of a new bone matrix by osteoblasts [147]. The balance between osteoclast-
osteoblast functions has to be tightly regulated, as excessive bone resorption can lead to diseases such as
osteoporosis.
Both bone-producing and bone-degrading cells express components of the PA system
including uPA, tPA, uPAR, and PAI-1, indicating that the PA system is involved and tightly regulated in
bone remodeling [148]. A number of in vitro studies have shown that the PA system mainly participates in
bone resorption. Plasmin can, to some extent, activate TGF-β and release insulin-like growth factor (IGF-
1), which may stimulate osteoblast proliferation and activity and lead to bone formation processes [149].
Recently, Kanno et al have demonstrated the direct involvement of plasminogen in bone metabolism by
showing that the pre-osteoclast cell population in plasminogen-deficient mice was higher than that in
wild-type. The absence of plasminogen caused reduced expression of osteoprotegerin, also known as
osteoclastogenesis inhibitory factor, and resulted in decreased proliferation of osteoblasts [150].
Interestingly, exogenous plasmin induced the expression of osteoprotegerin in plasminogen-deficient
mice. The bone mineral density (BMD) of the trabecular bone at four to six weeks of age was significantly
lower in the tibia of plasminogen-deficient mice compared to wild-type mice, but this difference
diminished over time. The cortical bone BMD was lower in plasminogen-deficient mice into adulthood (up
to 18 weeks old) [150].
Persistent inflammation of the periodontium induces osteoclast activation and resorption of alveolar
bone, which leads to flexibility of the teeth and eventual tooth loss. As we have shown, plasminogen-
deficient mice develop spontaneous periodontitis in which the alveolar bone is severely degraded.
Exogenous plasminogen was not only able to heal the periodontium in these mice, but it also induced
alveolar bone re-growth (Paper I, this thesis). Thus, a more thorough understanding of the role of the PA
system in bone remodeling might lead to novel therapeutic interventions for treating bone diseases.
1.6.3. Other tissue remodeling processes requiring the plasminogen activator system
The study of mice deficient in most members of the PA system has revealed the importance
of the PA system in many physiological and pathological events, including reproduction, angiogenesis, and
cancer. These are processes where tissue remodeling is crucial.
Reproduction involves complex biological processes, such as ovulation, fertilization,
embryo implantation, and embryogenesis, in which the PA system may play an important role. However,
20
studies of gonadotropin-induced and physiological ovulation in plasminogen-deficient mice have
demonstrated only slightly reduced ovulation efficiency [151]. Plasminogen-deficient mice treated with a
broad-spectrum MMP inhibitor showed only a 14% reduction in ovulation efficiency [152]. However,
because plasminogen plays a major role in wound healing and angiogenesis, plasminogen-deficient mice
might recover poorly after litter delivery, which in turn could affect subsequent reproduction processes.
Interestingly, the serine protease inhibitor protease nexin-1 (PN-1), which modulates the proteolytic
activity of plasminogen, PAs, thrombin, and trypsin, was shown to play a crucial role in male fertility. Mice
lacking PN-1 are infertile due to enhanced proteolytic activity in the seminal fluid [153].
Angiogenesis, the formation of new blood vessels, is important during several
physiological and pathological processes, such as placental development, embryonic development, corpus
luteum formation, and wound healing [154]. Pathological angiogenesis occurs during the progress of
several disease conditions, such as tumor growth and metastasis, rheumatoid arthritis, and
atherosclerosis [154]. During angiogenesis, the endothelial cells of existing blood vessels degrade the
basement membranes and make space for the formation of new vessels [155]. This degradation process
involves the PA system and other systems such as the MMPs [156, 157].
Tumor growth and metastasis requires degradation of the ECM of the basement
membrane. The PA and MMP systems provide the proteolytic activity that is required for this process. The
PA system appears to be involved not only in tumor cell migration and invasion but also in other processes
in tumors such as angiogenesis and desmoplasia (stimulation of fibroblast proliferation and synthesis of
ECM proteins) [158]. Mice lacking plasminogen or PAs generally show a reduced rate of tumor growth
[159-161]. In humans, high levels of uPA and its receptor uPAR are associated with poor prognosis in
many types of cancer [49].
1.7. The plasminogen activator system in infection
An infection is caused by the invasion of a pathogenic bacteria, virus or parasite into a host
organism and depends on successful colonization of the host, avoidance of host defenses, and replication.
Infectious and parasitic diseases currently cause approximately one-third of all human deaths in the
world, which is more than all forms of cancer combined [162].
Some bacterial pathogens use host plasmin-mediated proteolysis to enhance their invasion
abilities and evade the innate immune system [163]. However, the PA system also participates in the
host’s defense against infection through its important roles in the inflammatory response. Numerous
bacteria, both gram-negative and gram-positive, carry receptors for plasmin/plasminogen or PAs [164,
165], and this provides them with the ability to invade the host by using the proteolytic activity of the host-
derived PA system. Several pathogens also produce pathogenic PAs, such as streptokinase (from various
species of streptococci), staphylokinase (from Staphylococcus aureus), and the Pla protease (from
Yersinia pestis) [163, 166]. Streptokinase, produced by group A streptococci, is very specific to human
plasminogen and has very low or no activity against other species, including mice. In transgenic mice
expressing human plasminogen, streptokinase caused increased mortality following a skin infection [167].
21
The periodontal bacterium Fusobacterium nucleatum subsp. nucleatum binds
plasminogen in vitro, after which it becomes a proteolytic organism with the potential to invade
periodontal tissues [168]. Furthermore, the periodontal pathogen Porphyromonas gingivalis is able to
inactivate plasmin inhibitors α2-AP and α2-M [169, 170]. Although a number of bacterial species bind
plasminogen, the pathogenic properties of plasminogen activation have only been studied in a limited
group of tissue invasive bacteria. In vivo data that reveal the role of PA system in bacterial infections are
limited and have come from studies using plasminogen-deficient mice, even though plasminogen has been
shown to have a protective role in a Staphylococcus aureus-induced arthritis model [171]. Based on the
collective evidence, the PA system appears to play a dual role during infection.
1.8. The plasminogen activator system in inflammation
Inflammation is a protective process through which an organism removes injurious stimuli,
such as pathogens, damaged cells, or irritants, and initiates the healing process. Inflammation can be
classified as acute or chronic. Acute inflammation is the initial response to harmful stimuli in which
leukocytes migrate from the blood and into the injured tissues. A cascade of biochemical events leads to
the inflammatory response and involves the local vascular system, the immune system, and various cells
within the injured tissue. Chronic inflammation differs in the type of cells present at the site of
inflammation and is characterized by continuous destruction and healing of the tissue under the
stimulation of inflammatory processes [172, 173].
The PA system participates in all steps of the inflammation process, including
vasodilatation, exudation, release of pain mediators, and the clearance of the fibrinous exudates [174].
Plasmin is capable of inducing an inflammatory response by activating the complement system that
generates C3a and C5a. In turn, C3a and C5a induce mast cell degranulation, histamine-dependent
vasodilatation, and an increased permeability of the venules. uPAR-mediated generation of bradykinin, a
potent endothelium-dependent vasodilator, provides another functional role of the PA system in
vasodilation. Acute inflammation disturbs the equilibrium of fluid and protein exchange between the
microcirculation and tissues that provides cells and fluids that are needed at the site of injury. The PA
system plays a major role in leukocyte recruitment during inflammation. Plasmin triggers the aggregation
of neutrophils and the degranulation of platelets [175], and it also induces expression of cytokines and
tissue factor (TF) in human monocytes [136, 176, 177]. Plasmin binds to a variety of cell types, including
inflammatory cells, through its lysine binding sites. Such cell-surface binding leads to a full-scale pro-
inflammatory activation [136]. The lack of uPA is associated with an impaired influx of macrophages into
the injured area, which results in delayed or abolished cardiovascular wound healing [178, 179]. In vivo
studies also have shown a critical role for uPAR in the regulation of neutrophil recruitment to inflamed
sites [134].
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2. Periodontitis
The inherited or acquired disorders in the tissues surrounding and supporting the teeth are
assigned to eight major classes of periodontal disease: gingival diseases (I), chronic periodontitis (II),
aggressive periodontitis (III), periodontitis as a manifestation of systematic diseases (IV), necrotizing
periodontal diseases (V), abscesses of the periodontium (VI), periodontitis associated with endodontic
lesions (VII), and developmental and acquired deformities and conditions (VIII) [180, 181]. Most
periodontal diseases are inflammatory in nature and are caused by pathogenic oral microflora. Depending
on severity, periodontal diseases can be broadly divided into gingivitis and periodontitis. The mildest form
of periodontal disease, gingivitis is a highly prevalent inflammation of the gums caused by bacterial plaque
that accumulates on the teeth at the gingival margin. Gingivitis is usually reversible through effective
dental hygiene. In some patients gingivitis can develop into periodontitis – a severe and chronic
inflammatory disease affecting tissues that support and anchor teeth (the periodontium). Periodontitis
appears as progressive and irreversible loss of gingival tissue, the periodontal ligament, and alveolar bone
that eventually leads to loss of teeth [180]. Periodontal diseases are highly prevalent and affect up to 90%
of the worldwide population [182]. Until now, no treatment for periodontitis had been developed that
would be able to regenerate all of the damaged periodontal tissues, especially the alveolar bone.
Smoking and type 1 and type 2 diabetes are major risk factors for periodontitis, and
microorganisms such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and
Tannerella forsythia (formely Bacteroides forsythus) are considered to be main risk indicators [183].
Severe periodontal disease often occurs in individuals whose host response mechanisms or immune
systems have been impaired, for example, by defects in neutrophil function. Various systemic diseases
such as leukemia, thrombocytopenia, and leukocyte disorders are associated with the increased severity of
periodontal disease [180]. Periodontitis itself can be one of the risk factors for preterm delivery, diabetes,
cardiovascular disease, and pulmonary disease by spreading inflammatory factors as a result of persistent
inflammation in the oral cavity.
The PA system is closely associated to processes such as bacterial invasion, tissue
degradation, bone remodeling, and inflammation, all of which typically occur during the development of
periodontal disease. The PA system can also participate in the regeneration of periodontal tissues due to
its ability to activate growth factors and regulate tissue remodeling processes.
2.1. The periodontium in health and disease
The tissues surrounding the teeth, such as the gingiva, the periodontal ligament, the root
cementum, and the alveolar bone are called the periodontium (Figure 4). The main function of the
periodontium is to support and anchor teeth, but it also provides tissue integrity, defense against the
invasion of pathogens, and maintenance of local hemostasis; damage to any one of the periodontal tissues
can lead to periodontal disease. The protection against periodontal infection is provided by the extrinsic
and intrinsic defense systems. The extrinsic protection is derived from secretions of the salivary glands
that are rich in immunoglobulin A. The intrinsic protection is located within and beneath the oral gingival
epithelium. The gingivial epithelium contains a specific tissue architecture and leukocytes, which are the
most important for defense of periodontium [184].
23
Figure 4. The periodontium comprises the following tissues: the gingiva, the periodontal ligament, the
root cementum, and the alveolar bone. The tooth consists of the crown and the roots, where the crown is
covered with enamel and root surface with the cementum layer. Dental pulp, surrounded by dentin,
contains nerves and blood vessels. Modified from J. Lindhe et al [185].
2.1.1. Gingiva
The gingiva is the part of the oral mucosa that covers the alveolar bone and surrounds the
teeth. The gingival epithelium provides a physical barrier between infection agents from the environment
and the host tissues. The major population of cells in the gingival epithelium consists of keratinocytes, but
this layer also contains melanocytes, Langerhans cells, and Merkel cells. These epithelial cells are
important for innate host defense by responding to attempted bacterial invasions and by transmitting
signals to other parts of the host organism.
The junctional epithelium is firmly attached to the tooth surface and forms an epithelial
barrier against bacterial plaque. It plays an important role by providing access for gingival fluid,
inflammatory cells, and components of the immune defense to the gingival margin. Junctional epithelial
cells exhibit rapid turnover, which contributes to the host-bacteria equilibrium and to the rapid repair of
damaged tissue. In the case of periodontitis, the junctional epithelium migrates down to the pocket, and
this lowers the level of the cemento-enamel junction (CEJ).
The gingival connective tissue (lamina propria) is the predominant tissue of the gingiva.
This gingival connective tissue has a high turnover rate, a good healing and regenerative capacity, and
leaves little evidence of scarring after surgical procedures. The major components of the connective tissue
are collagen fibers (60% by volume), fibroblasts (5%), blood vessels, nerves and matrix (35%) [185]. The
gingival connective tissue fibers are produced mostly by fibroblasts and consist of collagen, reticular
fibers, and elastic fibers. The collagen fibers hold the marginal gingiva firmly against the tooth and
24
provide rigidity against the forces that develop during chewing. Collagen type I is most predominant in the
lamina propria and provides the tensile strength of the gingival tissue.
Different cell types, such as fibroblasts, mast cells, macrophages, and inflammatory cells,
are present in the connective tissue. The fibroblasts are the most predominant connective tissue cell type
(65% of the total cell population) and produce various types of fibers and ECM components. Fibroblasts
also participate in immune reactions and inflammation [186]. Cytokines released during inflammation
play a central role in the modulation of inflammatory systems. They are produced not only by
inflammatory/immune cells but also by epithelial, endothelial cells, and fibroblasts. Cells respond to
various inflammatory cytokines and growth factors and notably influence the balance between ECM
synthesis and degradation. Alterations in this balance can lead to pathological lesions, such as tissue
destruction in periodontitis.
2.1.2. Periodontal ligament and root cementum
The periodontal ligament is a highly vascularized connective tissue that surrounds the roots
of the teeth and joins the root cementum to the alveolar bone [185]. The functions of the periodontal
ligament can be grouped into physical functions, formative and remodeling functions, and nutritional and
sensory functions. The periodontal ligament anchors the tooth to the alveolar bone and distributes
masticatory forces. Its brush-like fibrils, mainly containing type I collagen, form a meshwork that
stretches out and connects the cementum of one tooth to that of the adjacent tooth (Figure 4) and to the
alveolar bone [187]. Type III collagen is present as mixed fibers, and type I collagen is present as Sharpey’s
fibers that insert from the periodontal ligament into the alveolar bone and provide a stable connection
with the tooth [188]. Type I, V and XII collagens are expressed by osteoblasts, and type III and some type
XII collagen fibers appear to be produced by fibroblasts during the formation of the periodontal ligament.
The collagen fibrils in the bone are stabilized by intermolecular cross-linking [189].
The periodontal ligament consists of fibroblasts, osteoblasts, cementoblasts, osteoclasts,
and epithelial and nerve cells. The periodontal ligament is also rich in mesenchymal stem cells, which are
important for regeneration. The fibroblasts are aligned along the principal fibers, the cementoblasts line
the surface of the cementum, and the osteoblasts line the alveolar bone surface. The periodontal ligament
undergoes constant remodeling in which old cells and fibers are broken down and replaced by new ones.
The cementum, bone, and gingiva are supplied with needed nutrients through a rich blood vessel network
that extends throughout the periodontal ligament.
The root cementum is a calcified tissue that covers the root of the tooth and provides an
attachment for the periodontal ligament fibers to the tooth (Figure 5). The cementum contains no blood or
lymph vessels, has no innervation, and does not undergo physiologic resorption or remodeling, but it is
continually deposited throughout the life of the organism. The cementum’s main function is to attach the
fibers of the periodontal ligament to the tooth, but the cementum also contributes to the process of repair
after damage to the root surface.
25
Figure 5. A histological cross-section illustrating the periodontal ligament between two molars. The
periodontal ligament, containing brush-like fibrils of collagen, anchors the tooth in the pocket by
connecting cementum layers between teeth and the alveolar bone. The Safranin O-stained lower jaw of a
wild type mouse, magnification 400×.
In the case of periodontitis, persistent inflammation leads to severe tissue degradation that
includes the degradation of the periodontal ligament. The cementum layer is gradually resorbed, and the
fibers of the periodontal ligament loosen. Eventually this leads to the flexibility of the tooth and, in a worst
case scenario, to the loss of teeth accompanied by severe alveolar bone degradation.
2.1.3. Alveolar bone in health and disease
The alveolar bone, also called the alveolar process, is a specialized part of the mandible
(lower jaw) and maxilla (upper jaw) that forms the primary support for the teeth. The alveolar bone
consists of two components: cortical bone (also called the alveolar proper), which lines the walls of tooth
sockets, and the cancellous (trabecular) bone, which fills up space between the sockets and between the
compact jaw bone walls. Compared to other bone tissues in the body, the alveolar bone undergoes more
rapid remodeling due to tooth eruption and masticatory demands. Osteoblasts and osteoclasts are
involved in bone remodeling and form and resorb the mineralized connective tissues of bone, respectively.
The balance between the resorption by osteoclasts and the formative properties of osteoblasts must be
tightly regulated. The formation of bone involves the proliferation of stem cells and their differentiation
into osteoblasts. This differentiation is regulated by various hormones, cytokines, and growth factors. The
organic matrix that is produced by osteoblasts consists predominantly of type I collagen and various other
non-collageneous bone proteins. Following maturation, osteoblasts may undergo apoptosis, become
encased in matrix as osteocytes, or remain on the bone surface as bone lining cells [190]. Resorption of
mineralized bone requires osteoclasts, which are produced by the monocyte/macrophage lineage of
hematopoietic cells derived from bone marrow [190]. Osteoclasts are large, multinucleated cells with a
26
ruffled border on one side of the cell. These cells are located in small cavities called Howship’s lacunae
that are formed during the digestion of the underlying bone. Resorption occurs through the release of
acidic substances that are able to dissolve the mineralized matrix of the bone. The remaining organic part
of the bone is eliminated by osteoclastic enzymes and phagocytosis [185].
Figure 6. The relationship between an inflammatory infiltration and periodontal bone loss. When
inflammatory infiltrate is at a distance from bone (left panel), it causes a mild, reversible form of oral
disease – gingivitis. If inflammation moves closer to the bone (right panel), osteoclastogenesis is induced
and irreversible bone resorption occurs that leads to a severe, irreversible form of oral disease –
periodontitis. Modified from D. T. Graves et al [191].
Inflammation and bone loss are the main symptoms of periodontitis (Figure 6). The
inflammation-mediated bone loss in periodontitis is enhanced by osteoclast activity without any
corresponding increase in bone formation [192]. Bone resorption during periodontitis depends on the
concentration of proinflammatory cytokines, such as IL-1, IL-6, IL-11, IL-17, and TNF-α, in gingival
tissues and how far these inflammatory mediators penetrate the alveolar bone [193, 194]. The combined
inhibition of IL-1 and TNF-α in an experimental model of periodontitis reduced alveolar bone loss by up
to 60% [195]. Receptor activator of nuclear factor kappa-B ligand (RANKL) is critical for bone metabolism
regulation because it is a key mediator in the process of osteoclast formation. RANKL is expressed mainly
on osteoblasts, and its increased expression can tip the balance towards osteoclastogenesis and an
increase in the bone loss observed during the development of periodontitis [192]. Thus, there is a great
need for a treatment to stimulate the formation of the alveolar bone that is lost during periodontal disease.
2.2. Pathogenesis of periodontitis
Bacteria and their pathogenic components are responsible for initiation of the tissue
destruction that occurs during the development of periodontitis. Pathogens that cause periodontitis have
classically been identified as gram-negative anaerobic bacteria that can survive in the gingival sulcus (the
27
space between the tooth and the adjacent gingival epithelium) [191, 196]. Over 700 species of bacteria
have been identified in the human oral cavity, and one individual usually has approximately 100–200
different species of periodontal bacteria. Porphyromonas gingivalis, Tannerella forsythia, and
Aggregatibacter actinomycetemcomitans have been identified as key pathogens [197]. Interestingly, the
most pathogenic complex, comprised of Porphyromonas gingivalis, Tannerella forsythia, and
Treponema denticola, was shown to be dependent on prior colonization of the tooth pocket by a complex
of less pathogenic organisms [198]. Dental plaque is a biofilm containing several species of colonizing
bacteria [199]. Bacteria induce tissue destruction indirectly by activating host defense cells, which in turn
produce and release inflammatory mediators that stimulate connective tissue destruction [196]. The
components released by bacterial plaque induce the infiltration of inflammatory cells including
lymphocytes, macrophages, and polymorphonuclear leukocytes (PMNs) [200]. Bacterial LPS activates the
macrophages to synthesize and secrete the proinflammatory cytokines IL-1 and TNF-α, prostaglandins
(especially PGE2), and hydrolytic enzymes. The cytokines IL-1 and TNF-α are the main proinflammatory
mediators that lead to tissue and alveolar bone degradation in periodontitis [195, 201]. IL-1 and TNF-α
influence endothelial cells to recruit neutrophils and monocytes to the inflammation site. Moreover, IL-1
stimulates the macrophages and gingival fibroblasts to produce PGE2, a vasodilator and a cofactor
involved in increased vascular permeability and bone demineralization. Periodontal research has
traditionally focused on the pathogenesis of periodontitis by analyzing bacterial infection and risk factors,
but there have been a number of recent studies that highlight the host response factors that actually drive
the development of periodontal disease.
2.3. The plasminogen activator system in periodontitis
The PA system plays a central role in the degradation of the ECM by generating plasmin
that can degrade non-collagenous ECM proteins such as laminin and fibronectin. Plasmin can also act
indirectly by activating proMMPs into proteases that are specific for other connective tissue proteins such
as elastin and collagen [202]. The role of MMPs in gingival/periodontal tissues has been extensively
studied, while the PA system, which has a central regulatory role, has received less attention [203].
All members of the PA system are localized to periodontal tissues. Plasmin has been found
in the gingival fluid of both healthy and diseased patients, and the level of plasmin activity has been
observed to increase along with the severity of periodontitis and is related to the bone resorption rate
[204]. The concentrations of tPA and PAI-2 are approximately 300- and 10-times higher, respectively, in
the gingival cervical fluid compared to their plasma levels [205], and uPA and PAI-1 have also been
detected in gingival cervical fluid at concentrations above normal plasma levels. These factors are thought
to be produced locally [205]. In vitro studies have shown that gingival fibroblasts can alter their
production of tPA and PAI-2 in response to exposure to LPS from the periodontal pathogens A.
actinomycetemcomitans, F. nucleatum, and P. gingivalis [206]. Furthermore, the expression of uPA and
uPAR by gingival fibroblasts increases following exposure to LPS from P. gingivalis [207]. Bacteria induce
tissue destruction indirectly by activating host defense cells, which in turn produce and release mediators
that stimulate the effector molecules of connective tissue breakdown [199].
The direct involvement of plasminogen in oral health is demonstrated by human
plasminogen deficiency. Clinical plasminogen mutations have been reported in patients with ligneous
28
gingivitis/periodontitis, which is a rare genetic periodontal disease with gingival enlargement and
periodontal breakdown [208-211]. In these patients, the disease re-occurs even after surgical treatments
or the application of heparin combined with antibiotics [212].
In Paper I of this thesis, we show that the lack of plasminogen in mice leads to
periodontitis. The intravenous restoration of plasminogen for 10 days in plasminogen-deficient mice
resolved the persistent inflammation of the gum tissue and induced alveolar bone regeneration.
Preventing or minimizing alveolar bone degradation is a major clinical aim in dentistry, and the
restoration of bone mass is extremely difficult. The regeneration of periodontal tissues involves complex
interactions between different cell types, the extracellular matrix, and local mediators such as growth
factors and cytokines. There are many conventional treatments that are currently used for periodontitis,
but few of them result in the complete regeneration of periodontal soft tissue [213]. The development of
therapeutic treatments to treat bone diseases are more focused on inhibition of the formation or activity of
osteoclasts, and far less attention has been paid to promoting bone formation [214]. Based on our data, we
suggest that plasminogen could be a candidate for the treatment of periodontitis by promoting the
clearance of persistent inflammation, stimulating the healing process, and, possibly, regenerating the
alveolar bone.
2.4. Animal models in periodontal research
Several animal models have been developed for studying the events associated with
inflammatory periodontal disease. Selection of the appropriate animal model depends on the similarity of
the periodontium and the nature of the disease to that of humans. In most mammalian species, the
inflammatory destruction of the periodontium can be spontaneous or experimentally induced.
Non-human primates have very similar dental formulas, root morphologies and
periodontium structure to humans, and they also have similarly occurring dental plaque, calculus, and
oral microbial pathogens (e.g., P. gingivalis). Late in life, these primates often acquire naturally occurring
periodontal disease. Although periodontitis in primates most closely resembles the human disease, the
expense and special requirements of these animals limit their use in periodontal studies [215]. In addition,
they are prone to infectious diseases such as tuberculosis, which makes them a less practical model for
studying periodontal diseases.
Mouse models make up the bulk of animal studies related to periodontal research [216].
The availability of different strains and transgenic mice allows for the study periodontal disease under
well-defined conditions [217, 218]. We have utilized mouse models in our studies, which has allowed us to
use mice deficient in plasminogen and PAs.
Rats and hamsters, like mice, have different dental formulas compared to humans, but
rats are often used as models of experimental periodontitis because the periodontal anatomy in the molar
region shares some similarities with that of humans. Furthermore, rats are easy to handle, can be obtained
with different genomes and microbial status, and are easily induced with infections. There is clear
evidence from the literature demonstrating horizontal bone loss in rats infected with A.
actinomycetemcomitans [219] and P. gingivalis [220].
29
Beagle dogs are also widely used in periodontal research. They have two sets of teeth,
primary and permanent, with similar root morphology as humans. Gingivitis and periodontitis lesions in
dogs are more closely related to humans than other non-primate laboratory animals [221].
Each animal model for periodontal disease has advantages and disadvantages; finding an
animal model of periodontal disease that can reproduce all the aspects of human disease is difficult.
30
3. Wound Healing
Wound healing is a very complex biological process that requires the interplay between
different tissue structures, a large number of resident and infiltrating cell types, and a variety of factors
released into the cellular milieu. The tissue damage must be repaired quickly to avoid complications, such
as blood loss and infection, and to reduce scarring. There are a number of wound types – incisions
(surgeries, accidents), excisions, burns, ulcers, and chronic wounds due to diabetes mellitus, infections, or
immunodeficiencies. The discovery of effective therapeutic treatments, that accelerate wound healing and
perhaps even result in non-scarring healing of the wound, would be of great benefit clinically. However,
due to the multiplicity and complexity of wound healing events, this is challenging.
3.1. Phases of wound repair
The healing of all wound types share the same ordered steps that can be divided into three
overlapping phases: the inflammatory phase, the proliferative phase (or the re-epithelialization phase),
and the remodeling phase [222]. Wound healing involves many cell types, as well as the ECM, and
chemical mediators. Each cell type has a specific function (or functions) in achieving normal repair of
damaged tissue, and disturbances of the healing process can lead to complications such as delayed and
improper healing, infection, and scarring.
3.1.1. The inflammatory phase
One of the first steps in wound healing is blood clotting, which is induced by platelets
exposed to collagen and the ECM at the site of the wound (Figure 7). The platelets release clotting factors,
growth factors, and cytokines [223], and this initiates hemostasis in the form of a fibrin clot and wound
contractions. The clot, formed from fibrin and fibronectin, also provides a matrix scaffold for the
recruitment of monocytes, fibroblasts, neutrophils, and endothelial cells to the injured site where the
inflammatory phase occurs [224].
Lymphocytes, monocytes and neutrophils are the main immune cells active in the
inflammatory process and subsequent tissue repair [225]. During the inflammatory phase, neutrophils are
first to infiltrate the wound site and play a key role in protecting the tissue against infection. These
neutrophils are responsible for phagocytosis, activation of the complement system, and producing
antibacterial oxygen radicals. However, the reactive oxygen species and enzymes (e.g., myeloperoxidase)
that are released by neutrophils may also cause damage to the host tissue. During the latter part of the
inflammatory phase, macrophages migrate into the wound site and phagocytize the neutrophils. At the
same time, the macrophages supply the wound with a continuous stream of cytokines and growth factors
that are necessary for the formation of new tissue in the wound [226].
31
Figure 7. The inflammatory phase of wound repair three days after injury. When a blood clot is formed,
the platelets release growth factors and cytokines. Neutrophils are the first to arrive at the site of wound to
phagocytize debris, pathogens, and dead cells. Macrophages then migrate to the wound to clear the
neutrophils and to release cytokines and growth factors. The keratinocytes at the wound edges start to
migrate towards each other to close the open wound. Modified from R.A.F. Clark [222].
3.1.2. The proliferative phase
Within a few hours after a wound has been made, the epithelial cells at the edge of the
wound undergo a phenotypic transition in response to growth factors, such as platelet-derived growth
factor, transforming growth factors α and β, and cytokines [226]. These epithelial cells loosen from the
basement membrane and, with help of integrin receptors, migrate over the wound separating the
hemostatic scab from the underlying viable granulation tissue [227, 228]. The degradation of the ECM
and collagen through the activation of plasminogen and MMPs allows migrating epidermal cells to dissect
their way between the collagenous dermis and the fibrin clot [229]. After the first or second day post-
injury, epidermal cell proliferation is induced behind the actively migrating keratinocytes to maintain the
thickness of the epidermal cell layer [222, 226].
Fibroblasts that migrate to the wound site produce new ECM and collagen that are
deposited in the granulation tissue (Figure 8). The amount of collagen deposited during this phase is a
determining factor for the tensile strength of the tissue after healing. The collagen also serves as a scaffold
for the keratinocytes to migrate on and adhere to as soon as they separate the granulation tissue from the
fibrin clot [230]. The epidermal attachment to the basement membrane is re-established in a zipper-like
fashion [231, 232]. The formation of new blood vessels is a complex process, which relies on the ECM and
a number of growth factors [233]. Increased angiogenesis at the wound site is important in providing
essential nutrients during the wound repair process [234].
32
Figure 8. The proliferative phase of wound repair five days after injury. During this phase, the wounded
area is divided into the hemostatic scab and granulation tissue. The granulation tissue consists of collagen
and the ECM that has been newly produced by fibroblasts. Angiogenesis occurs, and the vascular network
is re-established in the wounded area. Modified from R.A.F. Clark [222].
3.1.3. The remodeling phase
The matrix formation and the remodeling phase follow the re-epithelialization and
granulation formation phase. During this remodeling phase, the collagen formed in the granulation tissue
is collected into larger bundles that are held together by an increasing number of cross-linkages [235]. The
remodeling of collagen is crucial for the strength of the healed tissue and scarring. The collagen
remodeling process is dependent on the continuous synthesis and degradation of collagen that is
controlled by several of the proteolytic enzymes released by macrophages, fibroblasts, and epidermal and
endothelial cells [222]. Unfortunately, the tensile strength of a mature scar never exceeds 70–80% that of
undamaged skin [236, 237]. Depending on the severity of injury, this remodeling phase can take over a
year.
3.2. The plasminogen activator system in wound healing
The PA system is intricately involved in all of the processes that are important for wound
healing, including fibrinolysis, ECM degradation, activation of proMMPs, regulation of cell migration,
release and activation of growth factors, angiogenesis, and collagen remodeling [146, 229, 238-240].
Using PA- and plasminogen-deficient mice, different groups have shown the importance of plasminogen
in wound healing [144, 241, 242][Paper II and paper III in this thesis]. Initially it was reported that the
lack of plasminogen in mice leads to impaired wound repair. However, even in plasminogen-deficient
mice, cutaneous wounds will eventually achieve complete epithelial closure (144). Treatment with the
MMP inhibitor galardin, in combination with plasminogen deficiency, arrested wound healing completely
33
in plasminogen-deficient mice [229]. Wound closure in mice that are doubly deficient in uPA and tPA is
not as severely impaired as it is in plasminogen-deficient mice [145]. Notably, treatment of doubly
deficient uPA and tPA mice with the MMP inhibitor galardin further delayed, but did not completely
block, wound closure, which occurred in plasminogen-deficient mice treated with galardin [145].
Furthermore, the impaired wound re-epithelization in plasminogen-deficient mice was fully restored by
an additional deficiency in fibrinogen [146]. In a different type of wound healing model – tympanic
membrane perforation – the healing process was shown to be completely arrested in the absence of
plasminogen [241]. This earlier study of tympanic membrane healing was the impetus for us to further
investigate the novel roles of plasminogen in wound repair. Our studies described in Paper II show that
burn and incision wounds successfully re-epithelialized with some delay in plasminogen-deficient mice.
However, neutrophils and fibrin deposits are present in the underlying tissues even after 60 days of
healing, and the healing is, therefore, not complete. In contrast, healing was restored in plasminogen-
deficient mice that were treated with human plasminogen, and the quality of healed burn wounds was
comparable to that of wild-type mice.
Plasminogen has been continuously observed in human wound biopsies analyzed at 10 days
after a wounding event in association with macrophages and fibroblasts. uPA was also detected in
macrophages and fibroblasts, whereas uPAR is only observed in macrophages of human wound
granulation tissue [243]. uPAR provides a mechanism to localize the production of the proteolytic activity
to the wounded area. The expression of uPA and uPAR, as well as PAI-1, is induced in the migrating
epithelial cell layer early during the re-epithelialization phase, when the proteolytic activity of plasmin
plays crucial role [144, 244, 245]. uPAR is also important for signal transduction in platelets and during
cell migration [246-249].
Increasing in vitro evidence also suggests that plasminogen is able to induce cell signaling
and cause the release and/or activation of growth factors, expanding the role the PA system in wound
repair [23, 250, 251]. Our data in Paper III demonstrate that plasminogen is a key regulatory molecule
with a pronounced pro-inflammatory effect that potentiates the early inflammatory response during
wound healing via the STAT3 pathway.
3.3. Cutaneous wound models in mice
Wound healing disorders are likely to increase, especially as the rates of diseases such as
diabetes, hypertension, and obesity increase. Animals have been used to experimentally determine the
molecular and cellular mechanisms underlying and controlling the healing process. A variety of
specialized wound models, such as those for incisions, excisions, burns, and chronic wounds, are needed
because each wound type has a unique healing pattern that depends on which tissues are wounded and
the manner in which they are wounded. The most common types of wounds established in mice models
are incision and excision wounds, burns and ulcers. The use of gene deficient or transgenic mice provides
an opportunity to study the role of specific genes during the wound healing processes.
The immediate reaction to an incisional or excisional injury is the formation of a fibrin-rich
hemostatic clot to stop the bleeding. Incision wounds are very abundant in a number of surgical
procedures and cause the least damage to epithelial and connective tissues. Incision wound models are the
most commonly used for studying the cellular mechanisms of wound healing with the aim of accelerating
34
the healing process and diminishing scaring. Like all other wound types, the healing of excisional wounds
follows the inflammatory, proliferative and remodeling phases of wound healing but requires greater
amounts of granulation tissue formation, re-epithelialization, and cell proliferation. The excision model is
often used to monitor the effect of treatments on the rate of wound healing and to dissect the molecular
mechanisms of wound healing.
Burns cause cauterization of the blood vessels, and the top layer of the skin becomes
necrotic. Morphologically, severe burn wounds are divided into three layers. The top layer of the wound
consists of necrotic tissue that forms a burn scab. The underlying zone of stasis is still viable but risks
ischemic damage due to the decreased perfusion of oxygen to the area. The hyperemia zone sustains the
least amount of cellular damage and responds to the injury with vasodilatation and increased blood flow
[252].
Diabetes is one of the major causes of impaired wound healing [253, 254]. Patients tend to
develop wounds and have difficulty healing simple wounds, which often become chronic and infected.
Therefore, mouse strains that develop insulin resistance, for example, obese (ob/ob) and diabetic (db/db)
mice, are widely used as a chronic wound model [255].
35
Results and Discussion
This chapter summarizes the three papers (Paper I-III) included in this thesis. The figures cited
correspond to those used in the original papers.
Plasmin is essential in preventing periodontitis in mice (Paper I)
Periodontitis involves infection, inflammation of the periodontium, degradation of gum
tissue, and alveolar bone resorption, all of which are processes in which the PA system is tightly involved.
We first monitored the oral health of plasminogen-deficient mice in comparison to wild-
type mice. Interestingly, all of the plasminogen-deficient mice developed periodontitis, while no
pathological changes were observed in the periodontal tissues of wild-type mice up to 20 weeks of age
(Figure 1). The alveolar bone in wild-type mice was intact (Figure 2A), but in plasminogen-deficient mice,
it was severely degraded (Figure 2B). The quantification of alveolar bone degradation by measuring the
distance between the CEJ and the alveolar bone crest revealed a significant age-associated increase in
bone degradation in plasminogen-deficient mice (Figure 2C). Furthermore, extensive neutrophil
accumulation was detected in the diseased gums of plasminogen-deficient mice, showing that recruitment
of inflammatory cells to the site of inflammation was not impaired (Figure 3D, F, and H). Fibrin deposits
were detected only in the damaged gum tissue (Figure 4D and 4F), suggesting that these deposits are a
consequence of disease, not the cause of it. Additionally, we demonstrated that mice doubly deficient in
uPA and tPA developed periodontitis in an identical manner to plasminogen-deficient mice (Figure 5),
leading to the conclusion that the proteolytic activity of plasmin is essential for normal oral health.
The supplementation of plasminogen-deficient mice with human plasminogen for 10 days
reestablished the oral health of two age groups of mice (8–12 and 17–20 weeks of age); the necrotic tissue
was significantly reduced (Figure 7A and 7B) and new epithelium and connective tissue fibers formed and
reattached (Figure 6C and 6D). Surprisingly, alveolar bone re-growth was observed along with soft gum
tissue regeneration (Figure 7C and 7D). The distance between the CEJ and alveolar bone crest in
plasminogen-treated plasminogen-deficient mice decreased by 17 to 25% and indicated a significant re-
growth of alveolar bone. Alkaline phosphatase staining confirmed osteoblast activity at the site of bone
formation (Figure 7E). As expected, there was no alkaline phosphatase activity detected at the alveolar
bone of PBS-treated plasminogen-deficient mice.
The data collected for this manuscript revealed the importance of plasmin for maintenance
of healthy periodontal tissues and that plasminogen has the potential for future development into a
therapeutic treatment for periodontal diseases.
36
Plasminogen is a critical regulator of cutaneous wound healing (Paper II)
In a previous study, the lack of plasminogen caused a delayed healing of cutaneous incision wounds in
mice, which was caused by slower fibrin degradation in the wound and subsequent impairment of
keratinocyte migration over the wound [144]. Full re-epithelialization in some plasminogen-deficient mice
was not reached until 20–45 days, while in wild-type mice, full re-epithelialization occurred within 10
days [144]. However, our data on the role of plasminogen in tympanic membrane healing had revealed
that plasminogen is essential in healing of tympanic membrane perforations [242]. We therefore extended
our studies by monitoring cutaneous wound healing for up to 60 days in plasminogen-deficient mice. Both
incisional and burn wounds were used. Incision and burn wounds differ in the type of tissue damage.
Incision wounds have minimal damage to the surrounding tissue and form a fibrin-rich hemostatic clot,
whereas burn wounds caused by heat give, in addition to a clot, a layer of necrotic tissue closest to the heat
source and demand more effort to re-epithelialize the broader wound area.
We have confirmed that incision wounds in plasminogen-deficient mice eventually achieve
wound closure. However, morphologic Masson-Trichrome staining revealed a lower density of collagen in
re-epithelialized wound areas in plasminogen-deficient mice compared to wild-type mice (Figure 1F and
1H). Immunohistochemical staining allowed us to detect the accumulation of neutrophils, fibrin, and
fibrinogen in re-epithelialized incision wounds of plasminogen-deficient mice, even after 30 and 60 days
post-wounding (Figure 2E-H). Neutrophils, fibrin, and fibrinogen were absent in re-epithelialized incision
wounds of wild-type mice after 14 days (Figure 2A and 2B). Using a burn wound model, we showed that
plasminogen deficient mice also had delayed healing compared to wild-type mice (Figure 3). Neutrophils,
fibrin, and fibrinogen accumulated under the re-epithelialized area of the burn wound in plasminogen-
deficient mice after 30 and 60 days post-wounding (Figure 4E-H), and none of these were detected in
wild-type mice after 14 days of healing (Figure 4A and 4B). The persistent accumulation of neutrophils
around the edge of the wound in plasminogen-deficient mice suggests a nonresponsive inflammatory
system in these mice, and there remains the possibility that the presence of fibrinogen might play a role as
a chemoattractant for neutrophils [224, 256].
We also performed plasminogen reconstitution experiments to determine if daily
intravenous or subcutaneous injections of purified human plasminogen could improve or even restore the
wound healing process in burn wounds of plasminogen-deficient mice. Intravenous injections were
started at the time of the wounding and continued for two weeks. After four to five days of treatment of
plasminogen-deficient mice with human plasminogen, the rate of wound healing was similar to the
control group. However, at 7 days post-wounding, the plasminogen-treated group demonstrated increased
speed in wound closure and the acceleration of healing continued for the duration of the treatment period
(Figure 5A). After two weeks, the wounds of the plasminogen-treated mice had completely healed (Figure
5C), while approximately 40% of the wound area was not re-epithelialized in the PBS-treated mice (Figure
5A and 5E). The morphology and time of healing of the plasminogen-deficient mice that were treated with
plasminogen mimicked the wild-type mouse burn wound healing pattern (Figure 3C and 5C).
Subcutaneous injections around burn wounds were started five days after administration of
the burn in plg-deficient mice. During the first five to seven days of treatment, there was no obvious
37
improvement in the healing of plasminogen-treated mice compared to PBS-treated plg-defient mice. By
two weeks after wounding, however, the mice treated locally with plasminogen showed an increased
wound healing rate compared to control mice, and only 25% of the wound area had yet to be re-
epithelialized (Figure 5A). Plasminogen-treated mice had completely healed wounds (Figure 5D), while
PBS-treated mice still had 10% of the open wound remaining on the last day of treatment (Figure 5B and
5F).
Improper and slow wound healing is a major concern in medical care. Delayed re-
epithelialization might lead to wound infection and, in the worst case scenario, to sepsis. In addition,
proper wound closure and maturation is very important to avoid scarring. Our results show that
plasminogen could be a possible therapeutic agent for improvement of wound healing by accelerating the
healing rate and resolving persistent inflammation around the wound area.
Plasminogen is a key pro-inflammatory regulator that accelerates the
healing of acute and diabetic wounds (Paper III)
The finding that plasminogen, in addition to its fibrinolytic properties, has the ability to
affect inflammatory processes and to induce cell signaling encouraged us to continue more detailed
studies on wound healing to dissect the molecular mechanisms behind plasminogen and to test whether
plasminogen can be used as a therapeutic agent. In the current study, we used a standard burn injury
mouse model to explore the healing potential of plasminogen and to reveal the molecular mechanism
underlying its effect in acute and diabetic wounds.
In the present study, we demonstrated that plasminogen supplementation improves and
speeds up skin wound healing in wild-type mice. Full-thickness standardized burn wounds were induced
in wild-type mice, and immediately after the injury, the mice were given 2 mg of plasminogen by
intravenous injection. The control group received 2 mg of bovine serum albumin (BSA) by intravenous
injection. Quantification of the wound area at different time points showed that from day 6 post-injury,
the healing rate of the plasminogen-treated group was significantly faster compared to that of the control
group (Figure 1A). A morphological analysis at 11 days post-injury revealed that the epithelium layer in the
control group remained open and was covered by a large scab (Figure 1C). However, in the plasminogen-
treated group, the re-epithelization was complete and only a small scab remained lightly attached above
the wound (Figure 1D). Furthermore, we also demonstrated that plasminogen levels specifically increase
at the inflammation site and in a limited area around the wound. As shown in Figure 2A, the plasminogen
levels were highest during the early inflammatory phase, became lower during the tissue formation phase,
and nearly returned to basal levels during the late tissue remodeling phase. Moreover, a spatial analysis
demonstrated that the accumulation of plasminogen was wound-specific (Figure 2B and 2C). The level of
plasminogen at the wound site was increased up to 20-fold when wild-type mice were supplemented
intravenously with plasminogen (Figure 2D). Taken together, these data suggest that the healing effect of
plasminogen is related to its wound-specific accumulation and that the extent of the plasminogen
accumulation can be further enhanced by supplementation with exogenous plasminogen.
38
Inflammatory cell depletion experiments revealed that the accumulation of plasminogen at
the wound site is mainly due to migrating neutrophils and macrophages. Plasminogen seems to be
transported to the inflammation site bound to surface receptors present on these cells. As shown in Figure
3A, there was no significant reduction in plasminogen accumulation in the wounds of fibrinogen-depleted
mice compared to the control group. This indicates that the binding of plasminogen to fibrin is not a
major mechanism for the local accumulation of plasminogen in the wound. However, the level of
plasminogen in the wound was reduced by 52% following macrophage depletion, compared with the no
depletion control group (Figure 3B). Similarly, the plasminogen accumulation was reduced by 23%
following neutrophil depletion (Figure 3C). The inflammatory phase-specific accumulation of
plasminogen at the wound site suggests that plasminogen has a regulatory role as a pro-inflammatory
regulator in wound healing. Thus, we measured the level of IL-6, which is an important mediator of
inflammatory host responses to tissue injury. As shown in Figure 4A, IL-6 levels in unwounded skin were
low irrespective of genotype and treatment. However, the level of IL-6 in the wounded skin of wild-type
mice was significantly higher than in the wounded skin of plasminogen-deficient mice. After plasminogen
treatment, the levels of IL-6 were enhanced in plasminogen-deficient mice and were further elevated in
the wild-type mice. Moreover, the level of phosphorylated STAT3 (pSTAT3), which is an intracellular
inflammatory mediator, was higher in wounded skin than in unwounded skin, and the pSTAT3 level was
further increased following plasminogen treatment (Figure 4B). These data indicate that during wound
healing, plasminogen treatment enhances the expression of pro-inflammatory cytokines and activates
intracellular signaling events.
An imbalanced inflammatory response is one reason for wound healing defects in diabetic
patients. To explore the healing potential of plasminogen in diabetic wounds, we utilized db/db mice,
which suffer from a severe wound healing impairment. Immediately after the burn injury, the mice were
intravenously treated with plasminogen, whereas in the control group, the mice received BSA. As shown in
Figure 5, the healing rate in the plasminogen-treated group was significantly faster than that of the control
group. Furthermore, we determined the plasminogen level in wounded and unwounded skin in db/db and
non-diabetic control mice. As shown in Figure 6A, the plasminogen levels in the unwounded skin were low
irrespective of genotype or treatment. After burn induction, the plasminogen levels in the wounds of the
db/db mice were only slightly enhanced compared with the control mice, where the plasminogen level
after wounding increased approximately 4-fold. However, after receiving systemic plasminogen treatment,
the plasminogen levels in the wounds of the db/db mice reached approximately the same level as in the
control mice and the IL-6 levels in the wounds of the db/db mice were clearly increased (Figure 6B). The
levels of pSTAT3 in the wound sites increased after wounding and further increased after plasminogen
treatment (Figure 6C). These data indicate that the impaired wound healing in diabetic db/db mice may,
in part, be due to the dysregulation of plasminogen. Plasminogen supplementation appears to rebalance
the early inflammatory response in these mice, which leads to improved wound healing.
Taken together, our data show that plasminogen is a key regulatory molecule with a
pronounced pro-inflammatory effect that potentiates the early inflammatory response during wound
healing. Administration of additional plasminogen not only accelerates the healing of acute wounds but
also initiates and improves the healing of chronic diabetic wounds. Plasminogen treatment may, therefore,
become a novel therapeutic strategy for the treatment of cutaneous wounds.
39
Concluding Remarks
Plasminogen-deficient mice spontaneously develop periodontitis, and the administration of plasminogen
to these mice leads to regeneration of the periodontium and partial re-growth of the alveolar bone.
Plasminogen accumulated at the area of wound and is essential for proper cutaneous wound healing.
In addition to its role in extracellular matrix degradation, plasminogen plays a pivotal role as a key pro-
inflammatory factor that potentiates the early inflammatory response during wound healing.
Delivery of plasminogen to the wounded area seems to be dysregulated in diabetic wound healing, and
plasminogen supplementation results in significantly improved healing of diabetic wounds.
40
Acknowledgements
I am lucky to meet many good helpful people and I appreciate each of them for supporting and making me
happy during all those years here in Umeå.
For my PhD time I would like to thank my supervisor Tor Ny. Thank you for sharing with me your
experience in research and teaching me how to become well organized and thoughtful. Best wishes and
huge thanks to previous and current group members – Guo for fun conversations, your smile and help;
Shen for knowing and sharing all lab news; Peter for your calm attitude, Elin – for ‘surviving’ with me
introduction to the lab; Patrik for conceding your lab-bench for me – it was very inspirational; Nina for
making our lab so organized and all the fun we had sharing an office; Chun – it was great to have you
around to talk and laugh; Jinan for periodontitis project. Kui Liu and his group, especially Reddy,
Wenjing and Krishna. Malgorzata for friendship and laughs, chance to share all, work related and
personal – without you I wouldn’t make it. Nelson, I still can’t decide to curse you or to thank you, in any
case I am very happy I got a chance to meet you. Tomas Lindh for your endless enthusiasm and good
mood. Thomas Borén, I extremely your knowledge, advices and generosity. Anna Arnqvist for
Christmas gatherings at the most welcoming lab. Richard Lundmark for helping me out by involving a
bit in your projects. Erik Lundgren and Kristina Ruuth for introducing me to research world at Umeå
University. Ulf Ahlgren for my happiest period in Umeå – you have a golden heart! Ingrid, the most
elegant and gorgeous woman I have ever met, for the friendship, advices and warmth. Kenneth for your
irreplaceable personality and immediate help in any situation. Elisabeth for your smile and always good
mood. Animal house staff, especially Lena for your hard work and enthusiasm. Cecilia Elofsson, my
sunshine especially when it comes to paper work/applications-your good mood was very supportive.
My colleagues/friends: Katja – for supporting me and always overpricing me, for best ‘caipirinha’ talks –
I miss you a lot. Annelie – you are my star, your goodness and lack of selfishness are just endless! Love
you very much. Sara – I love you for being so honest, smart, and fun. I always have a great time with you.
Anna S, Zhanna, Olena, Melissa, and Jenny – you are all so charming in so different ways and I
always enjoy your lovely company. Pär, Miriam, Mantas, Anna E, Isabelle, Louise, Göran L,
Erika, Emma, Peter, Karin, Linus, Lisette, Göran B, Anna G, Björn, Verena and Sarantis for
sharing and enjoying ‘coffee tea vodka’ time with me. Malin for finally making teaching enjoyable.
Nasim and Nils – it’s so simple to love you for being so cute and friendly. Clas – for so many things…
caring so much and making me smile all the time.
My new colleagues at Molecular Periodontology – Ingrid, Ulf, Lillemor, Inger, Anita, Chrissie,
Anders, Cecilia, Ali and Fredrik – you are very welcoming and caring – I am very lucky to work with
you. Special thanks to my gorgeous boss Pernilla – your smile can move mountains.
Lelle (Lennart Carlsson), you do have one of the biggest hearts – thank you for helping me! Amir and
Tomas for all that fun we had working together – thank you for being there for me in good and hard
times.
41
Patrycja – you are so brave and gorgeous friend of mine – I have missed you so badly. Tomek for
hangouts together with nice food and great mood. Lijana and Fredrik – už jūsų neeilinį svetingumą.
Jūratei ir Dariui už jaukius jūsų namus, kur iškart pasijunti savas, aukšto pilotažo kulinariją ir krepšinio
transliacijas. Arūnui ir Ramunei už tai, kad jūs tiesiog nepaprastai smagūs ir svetingi – visada smagu
jus aplankyt. Eglei ir Ernestui – už džiazą ir skanėstus. Kotryna, ačiū, kad priverti mane pasitempti bet
kuriuo klausimu and Andreas for making our queen of dresses so happy. Milžiniškas dėkui draugams ir
šeimai Lietuvoj. Rokai, Rasele, Loreta, Dalyte, Arūnai ir Gintai – ačiū, kad visada jaučiuos laukiama
ir atvykus jaučiuos lyg niekad nebuvau išvykus. Jūs man be galo brangūs! Evelina, mano ištikimiausia
vaikystės drauge, tu mane įkvėpi savo kūrybingumu ir paprastumu. Jovita ir Evaldai, nuo studijų laikų
mes kartu ir nesvarbu, kad mus dabar skiria jūra. Ačiū jum už neišsenkantį laukimą.
Mamyte, už man įdiegtą optimizmą ir paveldėtą smagų charakterį, dar tik tavo stiprybės pritrūkau.
Sesutei Elytei, už nepakeičiamumą – visada buvimą su manimi, kur aš bebūčiau. Algimantui didžiulis
ačiū už visus reikalų tvarkymus ir pagalbą.
Rimui už kavą ir smagius pasakojimus, o Juzefai už nepamainomą pagalbą prižiūrint mažylius tiek
rašant šias tezes, tiek apsiginant.
Karoliui, Gretai ir Ariui už tą neapsakomą laimės ir džiaugsmo jausmą patiriamą kiekvieną dieną.
Paskutinės eilutės skiriamos labai ypatingam žmogui, kurio jau deja nėra tarp mūsų... Mylimam Tėčiui.
Žinau, kad labai džiaugtumeis ir didžiuotumeis manim. Prisiminimai apie Tave visada priverčia
nusišypsoti.
42
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