On pericytes, particularly their existence on lung capillaries

MICROVASCULARRESEARCH,8,218-235

On Pericytes,

(1974)

Particularly Their on Lung Capillaries’ EWALD

R.

Existence

WEIBEL

University of Berne, Switzerland Received February 19,1974 The existence of pericytes on the capillaries of lung alveoli has long been questioned. A systematic electron microscopic study has shown them to exist in human lungs, as well as in lungs of dogs, guinea pigs, and rats, whereas they could not be found in the lungs of the smallest mammal, the Etruscan shrew. The study of systemic capillaries (myocardium, pancreas) shows the pericyte to be characterised by a close association with the capillary basement membrane within which it is ensheathed, by the formation of branched cytoplasmic processes which approach the endothelial cell more and more the finer they become, by the presence of a web of cytoplasmic filaments along the membrane close to the endothelium, and by the exclusive or predominant occurrence of pinocytosis along the outer cell membrane; lysosomal elements are a rare occurrence. These features are comparable to those characterising smooth muscle cell processes in venules. Several cells and numerous cell processes showing all these characteristics have been found associated with lung capillaries. It is concluded that pulmonary alveolar capillaries are also provided with pericytes, but that these appear to be rarer than on systemic capillaries and that they are less densely branched. Their frequency furthermore appears to be proportional to body and lung size.

It is now just over 100years sincethe French histologist C. Rouget discovered in 1873 the existence of highly ramified cells at the surface of capillaries. He gave a meticulous description of what is now called the pericyte and concluded that these cells constitute a contractile sleeve around the capillaries, so that all vessels,from the heart to the capillaries, had to be conceived as made up of an endothelial cell lining, an amorphous enveloping membrane, and a sleeveof contractile cells, i.e., muscle cells which would be reduced to a thin network of pericyte processesin the capillaries. These fundamental discoveries remained unnoticed until around the turn of the century when S. Mayer (1902) confirmed Rouget’s observation that “muscular” elements coated the capillary wall. But pericytes came into fashion only in the 1920’s when Zimmermann (1923) published his now classical paper “Der feinere Bau der Blutcapillaren,” and Vimtrup (1922, 1923) of the school of August Krogh (1929) devoted much work to the problem of capillary wall contractility. Since then pericytes are an acceptedentity, but the number of papersdealing with this intriguing cell are scanty (Majno, 1964), and there still is no consensuswhether they represent contractile or phagocytic cells, both functions having beenclaimed repeatedly. Zimmermann (1923) showed the existence of pericytes on capillaries from a wide variety of tissues in numerous species: mammals, amphibians, birds, and fishes. They appeared to occur everywhere with one important exception: the mammalian lung. Zimmermann (1923) did not find them there, although he found them in frog lungs; 1 Supported by grant 3.682.71 from the Swiss National Science Foundation. Copyright Q 1974by Academic Press,Inc. All rights of reproduction in any form reserved. Printed in Great Britain

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neither did Seemann(1931) or Pfuhl(1933), although this author specifically searched for them together with H. and E. Loeschke (quoted by Pfuhl). Most descriptions of lung structure leave pericytes unmentioned, but Krogh (1929), Bargmann (1936) and von Hayek (1953 and 1970)positively state that pericytes have not been discovered on lung capillaries. More recently, electron microscopic studies showed that pericytes are inserted within the split capillary basement membrane (Majno, 1964; Hogan and Feeney, 1963; Kuwabara and Cogan, 1963), a finding which Plenk (1927) had already obtained by silver impregnation techniques and light microscopy. In the numerous studies on lung ultrastructures no attention was, however, given the question whether pericytes existed or not; occasionally one will meet the label “pericyte” on a micrograph, but this is never pursued. This appeared an intriguing situation, mainly since one can often observe on electron micrographs cell processesintimately associated with the endothelial wall, which one would be tempted to call “pericytes” (Weibel, 1973).We have therefore systematically explored the question whether lung capillaries are provided with pericytes and have concluded that this is the case.In the following the evidencein support of this conclusion is presented. MATERIAL AND METHODS This study was done on electron microscope preparations of lungs of man, dogs, guinea pigs, rats, and shrews. They had all been fixed by our standard triple fixation with 1.5y< glutaralbhyde (instilled into the trachea at controlled pressure), osmium tetroxide, and uranyl acetate solutions all adjusted for pH and osmolarity (Weibel, 1970).The sections were contrasted with lead citrate, and examined in a Philips EM 300 operated at 60 kV. As controls, capillaries in tissue samples from heart muscle and pancreas, fixed by immersion with essentially the samesolutions, were examined. In addition we had the privilege to reexamine some of Prof. K. W. Zimmermann’s original silver-impregnated specimensof heart and tongue muscle and to confirm his observations. RESULTS Periqtes in SJvtemic Capillaries

In order to establish a basis of referencefor the searchfor pericytes in lung capillaries it is first necessary to survey the appearance of pericytes in capillaries where their existence is unambiguously established. Figure 1 shows a montage of light micrographs of capillary pericytes from heart FIGURES. Keyto symbols:A, alveolus; B, basement membrane;C, capillary lumen; cf, collagen fibrils; E, endothelium; ef, elastic fibers; EP, alveolar epithelium; er, endoplasmic reticulum; F, fibroblast; f, cytoplasmic filament; J, intercellular junction; L, lysosomal vesicles; M, muscle cell; N, nonmyelinated nerve; P, pericyte (cell body); PP, pericyte process; p. pinocytotic vesicles; T, thrombocyte; t, microtubules; V, venule. FIG. 1. Montage of three pericytes (P) from cardiac muscle recorded from original Golgi-Kopsch preparation of K. W. Zimmermann, together with model of capillaries (C) branching from arteriole (A) also prepared by Zimmermann. Note that pericytes with their long processes (PP) and multiple side branches appear to develop continuously from branched smooth muscle cells (M) of the arteriole. The model corresponds to Fig. 106 of Zimmennann’s treatise of 1923. Micrographs magnified 1400 x.

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muscle, recorded from one of the original preparations of K. W. Zimmermann’. Also shown is one of the models made by Zimmermann from his preparations. The fundamental features of pericytes are (i) an ellipsoidal nucleus lies along-side the capillary and protrudes slightly into the connective tissue, (ii) two long slender processes course longitudinally along the capillary, (iii) numerous finer processes extend laterally from these main stems and form a partial girdle of parallel semicircular bands around the capillary. In the electron micrograph of Fig. 2 a capillary from rat cardiac muscle is obliquely sectioned. A cell is observed to lie close to the endothelium, but to remain separated from it by a basement-membrane-like space. The nucleus is surrounded by a slim rim of cytoplasm from which fine processes extend which approach the endothelium. As shown at higher power in Fig. 3, these processes extend beneath the capillary basement membrane; they make close contact with the endothelial cell in a few points, but for the most the two cells remain separated by a gap of some 20-30 nm which contains a fine layer of basement-membrane-like material. In the vessel of Fig. 4 the cell body and a large pericyte process are seen to be ensheathed completely within the capillary basement membrane sleeve which splits up to send one thinner leaflet into the interstice between endothelium and pericyte. The pericyte processes are always located beneath or within the capillary basement membrane (Figs. 5 and 6). The larger processes are separated from the endothelium by a thick layer of basement membrane material; it is interesting that a less electron dense layer of the basement membrane is present both towards the endothelium and towards the pericyte (Figs. 4-6). The leaflet separating smaller processes from the endothelial cell is thinner and may disappear in part (Fig. 5). It is tempting to speculate that the thicker processes correspond to the longitudinal stems, the finer ones to the circumferential branches (Fig. l), but this would need to be established by further evidence. Figure 5 shows very clearly the difference between pericyte and fibroblast processes, the latter being free of a basement membrane sheath. The cytoplasm of pericyte processes shows a distinct structure: on the side adjacent to the endothelium it contains a dense meshwork of fine cytoplasmic filaments, whereas on the outer surface numerous pinocytotic vesicles are observed (Figs. 4-6); this has been observed by a number of authors (Majno, 1964; Mohamed et al., 1973; and others). Pinocytotic vesicles are usually missing in the finest processes. Occasionally, one may also observe lysosomal elements in larger pericyte processes. A comparison of Figs. 6 and 7 reveals the great similarity between pericyte processes and smooth muscle cells from a venule: the distribution of filaments and pinocytosis is identical; furthermore, the smooth muscle cell of venules, as that of arterioles, is separated by an incomplete basement membrane from the endothelium, so that muscle and endothelial cells are closely approximated in some places. In summary, then, the pericyte is characterised by its close association with the capillary basement membrane, by the formation of cytoplasmic processes which L The author is the third successor to Karl Wilhelm Zimmermann as Professor of Anatomy at the University of Berne. Some of Zimmermann’s original preparations, as well as the models he made from his observations, are still preserved in the archives of the Institute. A comparison of the preparations with the drawings published in his treatise of 1923 induce great respect for the extraordinary skill for observation and meticulous reproduction of this great histologist. A photograph can only partly render justice to the perfection of the preparations.

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FIG. 2. Electron micrograph of capillary (C) from rat cardiac muscle with pericyte (P). Note processes (arrows) approaching endothelium. Magnification: 12,000 x. FIG. 3. Higher magnification of pericyte in Fig. 2. Note basement laminae (B) around pericyte, and, partially, between pericyte and endothelium. Magnification: 20,500 x.

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FIG. 4. Pericyte (P) and large pericyte process (PP) are separated from endothelium (E) by basement membrane lamina (B), except in region of close approximation (arrows). Rat cardiac muscle. Magnification: 12,850 x.

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FIG. 5. Compare location of pericyte processes (arrows) within capillary basement membrane (B) with free interstitial location of fibroblasts (F). Rat cardiac muscle. Magnification: 18,700 x.

approach the endothelial cell more and more the finer they become, by the presence of cytoplasmic filaments along the membrane close to endothelium, and by the (exclusive) presence of pinocytotic vesicles along the outer cell membrane of larger processes.Lysosomal elements are a rare occurrence.

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FIG. 6. Higher magnification ofpericyte process (PP) enframed in Fig. 5 to show distribution of web ofcytoplasmic filaments (f) and pinocytotic vesicles(p). Note location within split basement membrane. Magnification: 52,670 x. FIG. 7. Smooth muscle cell on venule (V) shows identical distribution offilaments (f) and vesicles (p). Note interruption of intercalated basement membrane (B) where smooth muscle approaches endothelium (E) at arrows. Magnification: 38,300 x.

EWALD R. WEIBEL

FIGS. 8 and 9. Alveolar capillary (C) from human lung with pericyte (P) which is partially separated from endothelium by basement membrane lamina (B) and extends with processestowards endothelium (arrows). Magnifications: Fig. 8, 7,180 x; Fig. 9, 19,420 x.

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FIG. IO. Pericyte (P) on human lung capillary which approaches endothelium with two processes (PP) which lie beneath basement membrane (B). Magnification: 26,330 x.

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Pericytes in Pulmonary Alveolar Capillaries

In Fig. 8 an alveolar capillary from a human lung is longitudinally sectioned; a cell is observedto lie alongside the capillary from whose endothelium it is separated,for the greater part, by a basement membrane leaflet. This cell is completely enveloped by basement membrane (Fig. 9), but it forms processeswhich broadly approach the endothelial cell, the gap between the two cells being irregular. The cytoplasm does contain filamentous material, mainly in the processesapproaching the endothelium.

FIG. 11. Higher power of enframed area in Fig. 10 shows cytoplasmic filaments (f), microtubules (t), and pinocytotic vesicles (p) in typical location. Magnification: 89,770 x.

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A similar cell is shown in Fig. 10: its nucleus is surrounded by a thin shell ofcytoplasm, and two extensions are seen to come close to the endothelium, sending smaller processes beneath the basement membrane. The cytoplasm contains bundles of fine filaments (Fig. 11) and also some microtubules, but it should be noticed that filaments are abundant in the adjacent fibroblast processes too. The gap between this cell and the

FIGS. 12 and 13. Pericyte (P) connecting two capillaries (C) in septum between two alveoli (A) in human lung. Note that process(PP) lies beneath basement membrane. Magnifications: Fig. 12.9,300 x; Fig. 13, 19,400 x.

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FIGS. 14 and 15. Pericytes (P) on alveolar capillary (C) of guinea pig lung. Large processes (PP) are rich in organelles and separated from endothelium (E) by basement membrane, whereas small processes (arrows) contain mainly filamentous web and lie close to endothelium. Magnifications: Fig. 14, 18,600 x ; Fig. 15,3,6.50 x.

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endothelium is of irregular width, averaging about 30 nm; it appears mostly empty with some scanty light material occurring in spots. An interesting cell is finally seento “bridge” two adjacent capillary segments(Fig. 12); it is apposed to one of the segmentsand sends a long process to the other one. This process penetrates beneath the endothelial basement membrane and establishes close contact with the endothelial cell; its cytoplasm contains filaments (Fig. 13). All three cells fulfill the criteria defining pericytes: they are closely apposed to the capillary endothelium, are envoloped by its basementmembrane, but remain separated from the endothelium by basementmembrane leaflets,except in regions where processes approach the endothelial cell; and their processesshow a cytoplasmk differentiation similar to that of pericytes. We would, therefore, conclude that these cells represent pulmonary capillary pericytes. These cells were found in human lungs; but Figs. 14 and 15show that identical cells may also be found in guinea pig lungs. We have also observed some in rat lungs, but it appears that they are not as frequent there as in human, dog, or guinea pig lungs. We have not been able to find a pericyte in the lung of the Etruscan shrew, the smallest mammal. It is evidently quite rare to find entire pericytes sectioned; mostly the section will only cut one of the processes.A selection of those identified on our preparations is shown in Figs. 16-17. It is apparent that the larger of these processesare intercalated between leaflets of the split basement membrane, whereas the smaller processeslie between basement membrane and endothelial cell. Very often small and large processesare continuous (Figs. 14 and 15); the basement membrane leaflet intercalated between them and the endothelium is then discontinuous. This is identical to the situation in systemic capillaries (Figs. 3 and 4). The cytoplasmic differentiation is also similar to that in systemicpericytes in that fine filaments form a web near the inner cell membrane (Figs. 10, 11, 15-18), whereas pinocytosis occurs predominantly at the outer membrane (Figs. 10, 11, 14, 18). Here again, the distribution of filaments and pinocytotic vesiclesis identical to that in muscle cells in a pulmonary venule, as is evident in comparing Figs, 18 and 19. It should also be noted that both may contain microtubules. It should, however, be mentioned that densecytoplasmic filaments are not a unique feature of pericytes in the aveolar wall; fibroblasts may also contain many (Figs. 11, 13, 14, 17, 18). Furthermore, endothelial cells are rich in cytoplasmic filaments which are particularly concentrated in the cytoplasmic lip adjacent to intercellular junctions (Figs. 13and 16). DISCUSSION The evidence presented in this paper unambiguously supports the existence of pericytes on pulmonary alveolar capillaries. All the morphological features characterFIG. 16. 4 large pericyteprocess (PP) is ensheathed by basement membrane (B) in human lung capillaries. Smaller process (arrow) is beneath basement membrane. Cytoplasmic filaments (f) fill the smaller process, but occupy the region close to endothelium in the larger branch. Note filament web (Ef) in cytoplasmic lip of endothelial cell near intercellular junction (J). Magnification: 30,100 X. FIG. 17. Small pericyte branches (arrows) from alveolar capillary in guinea pig lung are beneath basement membrane, and are stuffed with filaments. Magnification: 20,500 x. 9

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FIGS. IS,19

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ising pericytes on capillaries of the systemic circulation are also demonstrated in pulmonary alveolar capillary pericytes : (i) the cell body is ensheathedby the endothelial basement membrane and protrudes above the capillary wall, (ii) larger processes (stems)remain separatedfrom the endothelium by an intercalated basementmembrane leaflet, (iii) finer processespenetrate beneath the basement membrane and establish close contact with the endothelial cell, (iv) the cytoplasm contains a filamentous web on the inner and pinocytotic vesicles on the outer surface, (v) rare lysosomal elements occur. The main difference to systemiccapillaries appearsto lie in the frequency of pericytes and, probably, also in the number of processesformed. Whereas it is very easy to find pericytes in cardiac muscle or pancreas capillaries, for example, one must search for them in the lung. The relatively rare small processesobserved also suggest that the cytoplasmic branches must be quite loosely arranged around the capillary, whereas in systemic capillaries they may form a densegirdle (Fig. 1). There are, furthermore, considerable speciesdifferences: whereaspericytes appear to be quite frequent in human and dog lungs, as well as in the guinea pig, they are rarer in rat lungs, and we have not beenable to find a pericyte processin the lung of the Etruscan shrew, the smallest mammal (body weight, 2 g), in which the pulmonary air-blood barrier is extraordinarily thin. It hence appears that the smaller the animal, the rarer pericytes. This may explain why pericytes have not yet been described for lung capillaries : most studies have been done on lungs of small rodents. A few remarks on the functional role of pericytes are in place. It has been claimed by some that they are contractile elements,whereasothers consider them to be phagocytic cells; they may be both (Majno, 1964). The few lysosomal elements observed in this study would not suggest an important level of phagocytotic activity in the normal tissue, but this may evidently be different in pathological states.The regular occurrence of a filamentous web in the cytoplasm is certainly suggestive of contractility of the pericyte processes,particularly since the systematic localisation of these webs at the inner cell surface is identical to the distribution of this cytoplasmic constituent in venular smooth muscle cells. It should be recalled that pericytes had been considered the main contractile component of the capillaries by the older investigators @tricker, 1876; Krogh, 1929; Vimtrup, 1923) who had observed contractility in the living capillary. Kapanci et al. (1974) have recently demonstrated that strips of lung tissue are contractile and that some cells of the alveolar wall bind anti-actin antibodies; one could speculate that this was due to pericytes. This would be a convenient hypothesis because Sobin et al. (1972) postulate the existence of a supportive system for the alveolar capillaries in their sheetflow model; they claim to have demonstrated a sheath of connective tissue around the capillaries with “posts” extending across the alveolar FIG. 18. Longitudinal section of large pericyte process (PP) which is partially separated by basement membrane fragments (B) from endothelium (E). Note location of filament web (f), microtubules (t), and pinocytotic vesicles (p). Guinea pig lung. Magnification: 24,600 x. FIG. 19. Smooth muscle cell (M) from pulmonary venule in the same specimen as Fig. 18 shows identical location of filaments (f), microtubules (t), and pinocytotic vesicles as in pericyte. It is mostly separated from endotheliurn (E) by broad basement membrane (B), but establishes close contact (arrow) in one spot. Guinea pig lung. Magnification: 60,200 x.

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septum (Rosenquist er al., 1973), but evidently a sleeveof contractile pericytes would be an attractive alternative. Such speculation must however be cautioned. Firstly, it is not yet demonstrated that these filamentous.webs are indeed contractile. Secondly, the pericytes are not the only cells to contain such filaments; they may not even contain a particularly great amount of them. Endothelial cells themselves(Majno.er al., 1969),as well as fibroblasts, may be particularly rich in this component, as was shown for the lung by Hung et al. (1972) and Kapanci ef al. (1974), as well as in this study. Whereasthis exploration has shown that pericytes may be found on the capillaries of the pulmonary alveolus, there still remain the questions, why thesecells are particularly sparse in this organ, and why smaller animals may have extremely few of them. Is this related to the lower blood pressureprevailing in the lessercirculation? And could their

greater frequency in larger lungs be due to hydrostatic effects, becausethe capillaries are suspended in air? The answer to these questions will have to come from further experimental studies. ACKNOWLEDGMENTS The author expresses his sincere gratitude for the skillful and devoted collaboration of Miss Gertrud Reber, Miss Helgard Claassen, Mrs. Rosmarie Bachmann, and Mr. Karl Babl. REFERENCES BARGMANN,W. (1936). Die Lungenalveole. In “Handbuch der Mikroskopischen Anatomie,” Vol. 5, pp. 799-859. Springer, Berlin. HAYEK, H. VON(1953). “Die Menschliche Lunge,” 1st edit. Springer, Berlin. HAYEK, H. VON(1970). “Die Menschliche Lunge,” 2nd edit. Springer, Berlin. HOGAN, M. J., AND FEENEY;L. (1963). The ultrastructure of the retinal vessels. II. The small vessels. J. Ultrastruct.

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HUNG, K.-S., HERTWECK,M. S., HARDY, J. D., AND LOOSLI,C. G. (1972). Filaments in fibroblast in pulmonary alveolar wall. In “30th Annual Proceedings, Electron Mic;oscopy Society of America” (C. I. Arceneaux, ed.), Los Angeles, Calif. KAPANCI, Y., ASSIMACOPOULOS, A., ZWAHLEN, A., IRLE, C., AND GABBIANI, G. (1974). “Contractile interstitial cells” in pulmonary alveolar septa. A possible regulator of ventilation/perfusion ratio? J. Cell Biol. 60, 375-392.

KROGH, A. (1929). “Anatomie und Physiologie der Capillaren.” Springer, Berlin. KUWABARA,T., AND COGAN, D. G. (1963). Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Arch. Ophthulmoi. 69, 492-502. MAJNO, G. (1964). Ultras’tructure of the vascular membrane. In “Handbook of Physiology,” Circulation Vol. III, pp. 2293-2375, Amer. Physiol. Sot., Washington, DC. MAJNO, G., Shea, ST. M., AND LEVENTHAL,M. (1969). Endothelial contraction induced by histaminetype mediators. J. Ceil Biol. 42, 647-672. MAYER, S. (1902). Die Muscularisierung der capillaren Blutgeftisse. Anut, Anzeiger 21, 442455. MOHAMED,A. H., WATERHOUSE, J. P., AND FRIEDERICI,H. H. R. (1973). The fine structure of gingival terminal vascular bed. Microuusc. Res. 6, 137- 152. PLENK, H. (1927). Ueber argyrophile Fasern (Gitterfasern) und ihre Bildungszellen. Ergeb. And. Entwicklungsgesch. 21, 302-412. PFUHL, W. (1933). Physiologische Anatomie der Blutkapillaren. Z. Zellforsch. u. Mikrosk. Anat. 20, 390416. ROSENQUIST, T. H., BERNICK,S., SOBIN,S. S., AND FUNG, Y. C. (1973). The structure of the pulmonary interalveolar microvascular sheet. Microvasc. Res. 5, 199-212. ROUGET,C. (1873). Memoire sur le dbveloppement, la structure et les propri&es physiologiques des capillaries sanguins et lymphatiques. Arch. Physiof. Norm. Pathol. 5,603-663.

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SEEMANN,G. (1931). “Histobiologie der Lungenalveole.” Jena. SOBIN,S. S., FUNG, Y. C., TREMER,H. M., AND R~SENQUIST, T. H. (1972). Elasticity of the pulmonary alveolar microvascular sheet in the cat. Cc. Res. 30,440-450. STRICKER,S. (1876). Untersuchungen iiber die Contractilitat der Capillaren. Sitzungsber. d. Akad. Wien, Marh. Naturw. Klasse III, Abt. 74. VIMTRUP, B. J. (1922). Beitrlge zur Anatomie der Kapillaren. I. Ueber kontraktile Elemente in der Gefasswand der Blutkapillaren. Z. Gesamte Anat. 65,156182. VIMTRUP, B. J. (1923). Beitrlge zur Anatomie der Kapillaren. II. Weieret Untersuchungen iiber kontraktile Elemente in der Gefasssand der Blutkapillaren. Z. Anat. Entwickluttgsgesch. 68,469-482. WEIBEL,E. R. (1970). Morphometric estimation of pulmonary diffusion capacity. I. Model and method. Resp. Physiol. 11, 54-75. WEIBEL,E. R. (1973). Morphological basis of alveolar-capillary gas exchange. Physior Reo. 53,419495. ZIMMERMANN,K. W. (1923). Der feinere Bau der Blutcapillaren. Z. Anat. Enfwicklungsgesch. 68,3-109.