Zeitschrift ffir Zellforschung 63, 759--766 (1964)

From the Department of Pathology, Yale University Medical School, New Haven, Connecticut (U.S.A.)

F I B R I L L A R S T R U C T U R E S R E S E M B L I N G L E I O M Y O F I B R I L S I N E N D O T H E L I A L CELLS OF MAMMALIAN P U L M O N A R Y

BLOOD V E S S E L S *

By

K. G. BENSOH, G . B . GORDON and L. MILLER

With 6 Figures in the Text

(Received April 30, 1964)

Electronmicroscopic studies of normal and diseased lungs of mammals lead to the incidental observat ion of bundled f i lamentous structures wi thin endothelial cells of arteries and arterioles. Contained hi this publ icat ion are the description and i l lus t ra t ion of these structures as found in the lungs of heal thy guinea pigs and the normal pu lmona ry parenchyma of lobes of h u m a n lung which were surgically resected for benign in t rabronchia l tumors.

Perusal of the l i terature did no t reveal an y publicat ions describing electron- microscopic observations of identical or similar s tructures in endothelial cells of lung vessels.

Material and Methods

Immediately after death, the lungs of normal healthy young ~dult guinea pigs were in- flated with a cold 5% glutaraldehyde solution buffered with 0.1 M Cacodylate to a pH 7.2 and immersed thereafter in the same fixative for two days at refrigerator temperature (SABATINI, BENSCl~[ and BARRNETT), (SCHAEFER, AVERY and BENSCH). The lungs were then transferred into containers with a wash solution consisting of 0.07 M Cacodylate buffer pH 7.2 adjusted with Sucrose to 290 m Os. After storage at 4~ 1 mm 3 blocks of lung tissue were immersed overnight in 12 ce. glass containers with the above wash solution and then postfixed in 1% Osmium tetroxide (PALADE, 1952). The human pulmonary tissue which consisted of the healthy parts of the surgically removed lobes of lung was treated in identical fashion.

The osmieated tissue was embedded in Maraglas (SPURLOCK, KATTINE and FREEMAN), sectioned with an LKB Microtome and stained with lead hydroxide either by the Method of KARNOVSKu or REYNOLDS before examination with an Elmiskop 1.

Results

The guinea pigs' pu lmonary arteries conta in endothelial cells with bundled filaments. These animals have very characteristic beaded pu lmonary arteries because of the periodic var ia t ion in the thickness of the vascular smooth muscle coat. I n the h u m a n tissue, the vessels with fibrils in the endothelial cells were those providing the blood supply for the walls of lobar bronchi. The la t ter vessels were arterioles measuring as little as 20 # in diameter. The type of vessel was identif ied in 1/~ thick Toluidin blue s tained sections.

On electron microscopic examinat ion , the endothelial cells of these vessels contained bundles of fibrillar structures. These bundles measured up to 4.5 #

* Supported by U.S.F.H.S. Grants A 5514-03, GM-K3-14834, 18573-R2 and CA-11, 77-01A1.

760 K.G. BENSCtt, G. B. GORDON, and L. MILLER:

Fig. 1. P a r t of a c o n t r a c t e d p u l m a n a r y a r t e r y of a g u i n e a p ig . A l l ce l lu la r e l e m e n t s are e n d o t h e l i a l cel ls e x c e p t for two c ross - sec t ioned s m o o t h musc le cel ls ( r i g h t lower corner) . The e n d o t h e l i a l ce l ls b u l g e i n t o t h e v a s c u l a r l u m e n ( top ha l f of i l l u s t r a t i o n ) . The t h i n a r rows p o i n t a t some of t he n u m e r o u s b u n d l e s of f i l a m e n t o u s m a t e r i a l in t he e n d o t h e l i a l cells wh i l e t he t h i c k a r rows i n d i c a t e t he ends of

one of t he se f i b r i l l a r s t r u c t u r e s . M a g n i f i c a t i o n : 8000 •

in length, 0.2--0.3/~ (seldom more) in thickness and consisted of as much as 20--30 parallel filaments (Figs. 1--3). Individual filaments measured up to 1/~ in length before leaving the plane of a section. The diameter of individual fila-

Fibrils in endothelial cells 761

Fig. 2. Same as Fig . 1, a t h igher magn i f i ca t ion . The endothel ia l cell in the r i g h t uppe r corner conta ins also f i l amentous s t ruc tu re s (arrows). Magni f i ca t ion : 34000 •

ments was in the neighborhood of 75--80 A on cross section (Fig. 4). The distance between the filaments was more than their diameter although cross sections showed occasional filaments in closer approximation.

The fibrils run roughly parallel to the cell surface (Figs. 2 4). The long axis of the bundled filaments generally appeared to be at right angles to the long

762 K .G . BENSC~, G. B. GORDON, and L. MILLER: Fibrils in endothelial cells

Fig . 3. Same as F ig . 1. B u n d l e d f i l a m e n t s in t he p a r t of an e n d o t h e l i a l cell w h i c h p r o t r u d e s i n t o the v a s c u l a r l u m e n . M a g n i f i c a t i o n : 43000 x

axis of the vessel. Sections conta in ing more t han one endothel ia l cell in the same vessel d id usua l ly show the fibrils of the different cells a r ranged in paral le l fashion to one another . Excep t ion to this was no t in f requen t ly observed and occasional ly the ind iv idua l bundles of f i laments within the same cell would follow different courses.

F i g . 4. S a m e a s F i g . 1. P a r t s of t w o a d j a c e n t e n d o t h e l i a l cel ls w i t h c r o s s - s e c t i o n e d f i l a m e n t s i n one of t h e s e ( a r rows) . T h e cel l o c c u p y i n g t h e u p p e r h a l f of t h e i l l u s t r a t i o n c o n t a i n s a l o n g i t u d i n a l l y

s e c t i o n e d f i b r i l l a r s t r u c t u r e . M a g n i f i c a t i o n : 70000 •

F i g . 5. T h e c e n t r a l one t h i r d of t h e a t t e n u a t e d p o r t i o n of a n e n d o t h e l i a l cel l c o n s i s t s of t a n g e n t i a l l y s e c t i o n e d f i l a m e n t o u s m a t e r i a l N o t e t h e i n t e r c e l l u l a r a t t a c h m e n t p l a t e ( d e s m o s o m e ) (a r row) . T h e l o w e r one h a l f of t h e i l l u s t r a t i o n s h o w s p a r t s of s m o o t h m u s c l e cells o5 t h e v a s c u l a r coa t . ( g u i n e a p ig) .

Ma~o*nifieation: 30000 •

764 K.G. BENSC~, G. B. GORDON, and L. MILLER:

F i g . 6. P a r t of a n e n d o t h e l i a l cel l of a p r e c a p i l l a r y of h u m a n lung . T h e l u m e n of t h e v e s s e l i s s e e n in t h e u p p e r p a r t of t h e i l l u s t r a t i o n , T h e c y t o p l a s m of t h e e n d o t h e l i a l cel l is f i l l ed w i t h f i l a m e n t s w h i c h a re b u n d l e d i n t h e l e f t h a l f of t h e p i c t u r e . S e v e r a l c i s t e r n ~ e of t h e r o u g h e n d o p l a s m i c r c t i c u l u m

a r c d i l a t e d . M a g n i f i c a t i o n : 28000 •

The amount of cytoplasm occupied by the bundles varied greatly. In some sections of the attenuated parts of the endothelial cells, the central 3/5 to 5/7 of a 0.3/~ thick endothelial cell were filled with bundled filamentous material (Figs. 5 and 6).

Attachment of the fibrils to the cell wall or attachment plates of the cells (desmosomes) was never observed although particular attention was given to the latter (Fig. 5).

High magnification micrographs revealed a similarity between the filamentous structures in endothelial cells and the adjacent vascular smooth muscle. Glycogen was identified only in the smooth muscle cells of the vessel wall and attachment plates (desmosomes) only between endothelial elements. The pinocytotic activity was about equal in both cell types and pronounced.

Neural elements were not found to abutt on endothelial cells. A periodicity of the individual filaments or bundles was not observed, nor crosslinks between individual filaments.

Discussion

Physiologists are still involved in a discussion of long standing as to whether capillaries possess constrictive properties and whether these are provided by endothelial cells, pericytes (Rouget cells) or other means. Microscopic evidence of fibrillar structures in endothelial cells was first provided by Rio HORTEGA in 1917. A few publications of electronmicroscopie observations of filamentous elements in endothelial cells of capillaries, arterioles and arteries appeared in recent years. PALADE reported in ] 953 on the existence of fibrils in endothelial

Fibrils in endothelial cells 765

cells. FAWCETT confirmed this observation in 1959. He described, but did not illustrate, fibrous tracts in endothelial cells and found tha t the individual filaments were less than 100 A thick instead of 240 A as reported by PALADE. RHODIN described briefly similar structures in endothelial cells of arteries and arterioles of mouse intestine. He found their diameter to be less than 70 A and their length about 1/3 of one/~. More extensive studies of endothelial cells with fibrous components were reported by L. 0. and H. L. ZWILLENBERG and L. WEISS. These authors observed abundant filamentous elements in the well-known spe- cialized endothelial cells of the sheathed segment of the penicilliary arteries of the spleen of the rabbit, dog, cat and horse. The former of these two groups of authors found also a close association of neural elements with the fibril con- taining endothelial cells. A report by S. MAN and J. AVERY described filamentous structures (diameter 60--70 A) in endothelial cells of vessels of the hamster dental pulp, which in contrast to all previous and our findings, were at tached to the cell wall at points of intercellular junction (adhesion plates), similar to the tonofilaments of the epidermis. Only two publications on the presence of fibrillar structures in endothelial cells of non-mammalian species were found. HANA described filamentous elements of 60--80 A diameter in cells of amphibian capil- laries and some of the vessels of the earthworm. The latter was long known to have contractile capillaries (FEDERIGHI). HANA emphasizes the microscopic absence of fibrous tracts in endothelial cells of mammalian blood vessels. Other authors also failed to find them (MooRE and RUSKA), (PoLICARD, COLLET and PREGERMAIN), (BENNETT, LUFT and HAMPTON).

Our studies clearly demonstrated bundled filaments in endothelial cells of pulmonary arteries, arterioles, and so-ealled preeapillaries (metarterioles) (dia- meter 18--30 #), (BAEz), (HAYEK). The chemical composition and physiologic properties of these fibrils are still in question. Their resemblance to smooth muscle, lack of a t tachment to the cell wall, parallel grouping in bundles of often considerable length, presence in vessels without muscular coat (precapillaries) and their similarity to filamentous structures of endothelial cells of annelids and the splenic penicilliary arteries suggests a function similar to tha t of muscle. The presence of contractile elements in the pulmonary precapillaries would help explain the findings of in vivo studies on the mierocirculation of the alveolus. I t was found in experiments by WEAI~N, BARR, and GERMAN that certain capil- laries of an alveolus always were filled with blood (flow or resting capillaries), other only intermittently (working capillaries). Yet, it should be mentioned at this point that the individual filaments in endothelial cells resemble structurally also gliofilaments, neurofilaments and tonofilaments.

The variation in the diameter of the endothelial filaments is probably due to species differences. This may be analogous to the variation in diameter of the contractile elements of smooth muscle of different species (RHoDIN), (KABRER).

Fibrillar structures in endothelial cells of pulmonary capillaries (diameter 6--12 ~t.) were never observed.

Summary

Endothelial cells of mammalian pulmonary arteries, arterioles and pro- capillaries (metarterioles) posses bundled filamentous structures which resemble leiomyofibrils.

766 K . G . BENSCt~, G. B. GORDON, and L. MILLER: Fibrils in endothelial cells

B i b l i o g r a p h y

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Drs. KLAUS BENSCIt, GERALD GORDON, and LEONARD MILLER: Department of Pathology, Yale University School of Medicine, New Haven,

Connecticut (U.S.A.)