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Role of the pericyte in wound healing
EXPERIMENTAL
AND
MOLECULAR
Role
13, 51-65
PATHOLOQY
of the
Pericyte
in Wound
An Ultrastructural DAN J. CROCKER, TARIQ Department
of Pathology,
The
M.
Ohio
Received
(1970)
Study’
MURAD, State
February
Healing
AND
University,
JACK
C. GEER
Columbus, O’aio 43210
18, i970
Mesenchymal cells present in earlywoundswere divided into two groups: primitive mesenchymal cells and macrophages. Primitive mesenchymal cells appeared to become incorporated in the thick basement membrane of recently formed capillaries, and they also appeared to differentiate into fibroblasts. After becoming encased in vascular basement membrane these cells, pericytes, featured areas of cytoplasmic contact with underlying endot.helium. It is proposed that the pericyte-endothelial “contacts” act as a regulatory mechanism for capillary proliferation.
Pericytes are perivascular cells that are associated with the capillaries of many tissues and organs. The ultrastructural morphology of these cells and their relation to small blood vessels have been previously described (Fawcett, 1959; Movat and Fernando, 1964; Murad and von Haam, 1968). The origin of pericytes and their function is not known and has been a subject for speculation (Clark and Clark, 1925; Cliff, 1963; Rhodin, 1968). In the healing wound the presenceof pericytes has been inferred to be associated with the maturation of proliferating vessels (Cliff, 1963). The present report is a sequential electron microscopic study of the healing wound in hamsters. The incorporation of pericytes within the basement membrane of proliferating capillaries is proposed as the mechanism for inhibition of capillary proliferation. MATERIALS
AND METHODS
Sixteen male hamsters ranging from 6 to 8 months of age and weighing about 100 gm each, were used for this study. The dorsal flanks of the animals were shaved and circular wounds were made with an S-mm dermal punch. The wounds extended to the depth of the superficial fascia. After wounding, animals were killed on the following days, 2-7, 14, and 21. The excised wounds from each animal were fixed in 5 % cold glutaraldehyde in phosphate buffer (Sabatini et al., 1963) for l-lx hours. After fixation, the edge of the wound was examined under a stereoscope and dissected away from adjacent tissue, then cut into small pieces (ca 1.0 mm). The small pieces of tissue were then placed in 1% osmium tetroxide in phosphate buffer at pH 7.3 (Millonig, 1961). The post-fixed tissue was embedded in DER (Lockwood, 1964). Sections 1~ thick were cut, mounted on slides, stained with toluidine blue (Trump et al., 1961) and 1 This investigation was supported in part by a General Research Support Grant from the National Institutes of Health and HE 11897 from the National Heart and Lung Institute of the National Institutes of Health. 51
52
CROCKER,
MURAD,
AND
GEER
examined with light microscopy. Selected areas were thin-sectioned, mounted on copper grids, and double-stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). An RCA EMU-3F electron microscope was used to examine the stained sections. RESULTS LIGHT
MICROSCOPY
Wounds on days 2, 3, and 4 of healing were characterized by the presence of inflammatory exudate composed of neutrophils and fibrin (Fig. 1). At the margin of the wound mononuclear cells were present. At day 4 newly formed capillaries were seen at the edge of the wound. Numerous newly formed capillaries were present by day 7. The intercapillary interstitial tissue at day 7 contained numerous cells of varying morphology that appeared to represent neutrophils, fibroblasts, and macrophages (Fig. 2). At days 14 and 21 of healing the wound was characterized by abundant collagen with fibrocytes, and the number of capillaries was markedly reduced from that seen at day 7. ELECTRON
MICROSCOPY
The inflammatory exudate of the wound at 2 and 3 days was composedprimarily of fibrin and numerous partially degranulated neutrophils (Fig. 3). In the fibrin network there were clusters of extracellular neutrophil granules and cells resembling blood monocytes in addition to the partially degranulated neutrophils. The extracellular neutrophil granules typically were surrounded by an electron-lucid zone (Fig. 4). Mononuclear cells in the wound at 3 and 4 days could be divided into two morphologic types based upon the number of electron-dense cytoplasmic inclusions. Those cells with numerous electron-dense, membrane-bound inclusions were designated macrophages (Fig. 5). The macrophage generally was larger than the second type mononuclear cell. The nucleus of the macrophage typically was ovoid or kidney-shaped with a narrow rim of heterochromatin along the inner nuclear membrane and contained a single nucleolus. The cytoplasm demonstrated the usual organelles including scattered parallel arrays of granular endoplasmic reticulum. The second type of mononuclear cell could not be distinguished from the macrophage on the basis of nuclear morphology or cytoplasmic organelles. In general, the second-type cell had a lesser cytoplasmic area than the macrophage, but the distinguishing feature was the absenceof electron-dense cytoplasmic inclusions (Fig. 6). We have designated this second type of cell as a primitive mesenchymal cell for purposes of this presentation. Primitive mesenchymal cells were more abundant than macrophages at days 3 and 4 and often were present in clusters about small blood vesselsas well as within the fibrin network of the exudate (Fig. 7). The perivascular mesenchymal cell was distinguishable from a pericyte by its lack of surrounding basement membrane. A prominent feature of the growing capillaries at the margin of the wound at day 4 was a thick basement membrane (Figs. 8 and 11). Mitotic figures were seen
PERICYTE
IN WOUND
HEALING
53
FIG. 1. Early wound filled with acute inflammatory exudate composed of fibrin mesh and leukocytes. X 200. FIG. 2. Seven-day-old wound featuring numerous blood vessels, polymorphonuclear leukocytes, and mononuclear cells. X 700.
54
FIG. FIG. rounded
CROCKER,
3. Partially degranulated neutrophils 4. Extracellular neutrophil granules by a clear zone. x 29,000.
MURAD,
AND
GEER
surrounded by serum protein. X 5200. entrapped in fibrin. Many of the granules
are sur-
PERICYTE
IN WOUND
HEALING
55
FIG. 5. Macrophage featuring numerous membrane bound cytoplasmic inclusions of varying electron density. X 13,000. FIG. 6. Primitive mesenchymal cells with a paucity of formed cytoplasmic elements. x 10,500.
56
CROCKER,
MURAD,
AND
GEER
FIG. 7. Primitive mesenchymal cells (PMC) are seen clustered near a venule. Note the similarity in the eytoplasmic profile of the pericyte (P) and the primitive mesenchymal cells. x 7c!oo. FIG. 8. Forming capillary with a thick basement membrane which contains a pericyte (P). The capillary exhibits a narrow slitlike lumen (arrow). X 13,ooO.
PERICYTE
IN WOUND
HEALING
57
in the endothelial cells (Fig. 9) and in the perivascular primitive mesenchymal cells (Fig. 10). By days 5 and 6 of healing many of the primitive mesenchymal cells were elongated and enclosed within the capillary basement membrane, thus having the location of pericytes. At days 5 and 6 of healing areas of the plasma membranes of “pericytes” and endothelium were seen closely approximated to one another (Fig. 11). At day 7 of healing the capillary basement membrane was the same thickness as in the early stages of healing but relative to the greater diameter of the capillary appeared reduced in thickness. The capillary endothelium was more attenuated than at days 4 to 6, though portions of the cytoplasm were still quite thick (Fig. 12). The pericytes partially enveloped the capillaries and showed a marked increase in cytoplasmic organelles, especially granular endoplasmic reticulum, over that seen in the perivascular primitive mesenchymal cell. The primitive mesenchyma1 cells in the interstitial tissue changed from a round shape to elongate cells, and the cytoplasm showed an increased number of profiles of granular endoplasmic reticulum (Fig. 13). The capillaries at the 14th day of healing showed numerous areas of close association of endothelial and pericyte plasma membranes (Fig. 14). The interstitial tissue was composed primarily of collagen fibers and elongated cells having within their cytoplasm numerous profiles of dilated granular endoplasmic reticulum (Fig. 15). These cells morphologically appeared to be fibroblasts. The capillaries at day 21 of healing were far fewer in number than in the earlier days. The capillaries were characterized by having an endothelial lining of rather uniform thickness in contrast to the irregular thickenings seen at 4-7 days. The pericytes partially enveloped the endothelium with narrow processes of electrondense cytoplasm (Fig. IS), and areas where these cells closely approximated one another were readily seen (Fig. 17). Few fibroblasts were present among the collagen fibers of the intercapillary tissue. DISCUSSION There are conflicting views regarding the origin and function of mesenchymal cells in the healing wound. The studies of Volkman and Gowans (1965a, 196513) indicate that most macrophages in the wound are derived from blood monocytes. The observations of Ebert and Florey (1939) also indicate that blood monocytes may become tissue macrophages. Ross and Benditt (1961) concluded from their observations that fibroblasts probably arise from cells of hematogenous origin, such as the blood monocyte, or a pleuripotential cell from the blood. There is evidence indicating that certain cells derived from blood appear to have a potential for the production of hydroxyproline (Allgijwer and Hullinger, 1960); however, autoradiographic studies of wound healing (Grillo, 1963; MacDonald, 1959) strongly indicate that the main source of fibroblasts in the healing wound is from the connective tissue at the wound edge. ClifI (1963) concluded from hi observations that there is no evidence for the presence of primitive, or pleuripotential mesenchymal cells in the wound. He believes that fibroblasts give rise to fibroblasts, monocytes give rise to macrophages, and that the endothelium of proliferating capillaries has as its source the endothelium of preexisting capillaries. His observations give no
58
x
CROCKER,
FIG. 9. Pericyte 17,000. FIG. 10. Capillary
(P) (C)
closely featuring
MURAD,
nestled
AND
on endothelial
a pericyte
undergoing
GEER
cell
(E) mitosis.
that
is undergoing
X 13,000.
mitosis.
FIN. 11. Cytoplasmic processes of a pericyte are seen in the thick basement membrane of a capillary. Areas of “contact” (arrows) are seen between the plasma membranes of endothelium and pericyte cytoplasmic extensions. X 10,u)o. FIG. 12. Capillary invested by a pericyte (P). Focally condensed bundles of filaments are seen along the cytoplasmic membrane of the pericyte suggestive of early hemidesmosome formation (arrows). X 10,200. 59
PERICYTE
IN WOUND
HEALING
61
FIG. 15. Fibroblasts with well developed granular cytoplasm situated next to venule. x 7060. FIG. 16. Thin cytoplasmic process of a pericyte extending around a capillary. An area of incomplete “contact” is readily seen between the pericyte and endothelium (arrow). X 21,000.
62
CROCKER,
MURAD,
AND
GEER
FIG. 17. High magnification of pericyte-endothelial “contact” of plasma membrane and the cytoplasmic fusion of the contacting lium features numerous pinocytotic vesicles. X 50,OCMl.
(arrow). Notice the absence cells (arrow). The endothe-
credence to the thought that there are transformations from one cell type to another. We have divided the mononuclear cells found in early wounds into two groups: macrophages and primitive mesenchymal cells. Those cells fitting the description of macrophages as described by Palde (1955) were designated as such. However, the second population of cells lacked certain features ascribed to the macrophage, namely, electron-dense bodies and phagocytosed material in their cytoplasm. Since we were unsure of the origin of this cell, and becauseits cytoplasmic profile was not indicative of a specific cell-type, we chose to designate this group as primitive mesenchymal cells. During the course of healing, cells of this type were seen nested around newly formed capillaries and appeared to become incorporated in membrane, thus becoming pericytes. It appeared that primitive vascular basement mesenchymal cells also differentiated into fibroblasts during the process of healing. That newly formed capillaries found in healing wounds come from the severed ends of preexisting capillaries has been demonstrated (Cliff, 1963; Ebert et al., 1939; Schoefl, 1963). The genesis of newly formed capillaries from the terminal portion of severed vesselshas been observed, and the endothelial cells of proliferating capillaries are seento be derived frqm proximal endothelial cells via the process of mitosis and subsequent migration distally. Schoefl (1963) also suggested the possibility that pericytes may contribute to the endothelial lining of regenerating capillaries.
PERICYTE
IN WOUND
HEALING
63
At the advancing tip of proliferating capillaries the observation has been made that the vascular basement membrane is devoid of a cellular constituent (Cliff, 1963; Schoefl, 1963). However, proximal to the advancing tip cells are seen incorporated in the vascular basement membrane. These cells are described as coming from the surrounding interstitium (Cliff, 1963) and being fibroblastlike in character. Kuwabara and Cogan (1963) have suggested that the absence of pericytes in vascular basement membrane permits endothelial proliferation. Cliff (1963) implies that there is vascular maturation with the envelopment of interstitial in vascular basement membrane. Observations of this study lead the authors to hypothesize that it is the incorporation of interstitial cells in the basement membrane of newly formed capillaries with the subsequent close association of areas of their cytoplasmic membranes that is responsible for inhibiting endothelial proliferation. And, according to our classification this interstitial cell is of primitive mesenchymal type. Studies of cell culture monolayers have demonstrated that once cells reach a critical cell density they cease to proliferate. This phenomenon has been called “contact inhibition“ of cell division (Todaro et al., 1965)) and it is thought to be dependent on direct physical contacts, not humoral factors (Levine et al., 1965; Stoker, 1964). Schutz and Mora (1968) have concluded from their studies of contact inhibition in cell cultures that it is necessary for cells to have close contact over large areas of their surface for contact inhibition to occur. The proposal has been made that the contacting of cells in culture initiates a sequence of intracellular events which activates the internal release of an inhibitor of macromolecular synthesis, which in turn, produces a cessation of replication (Fischer and Yeh, 1967; Yeh and Fischer, 1969). In their studies they have shown that this inhibitor acts specifically to stop RNA synthesis of contact inhibited cells, and that this inhibition is reversible. In this study, areas of close proximity between pericyte and endothelial plasma membranes made their appearance in the proliferative phase and reach their greatest number by day 14 of healing. For the purpose of simplicity the areas of close approximation of pericytes and endothelium are designated as “contacts” although no direct physical contacts between the cell membranes were observed. The increased number of “contacts” between pericytes and endothelium may act to inhibit endothelial proliferation through a mechanism similar to that ascribed to cell culture. Specifically, the increased number of “contacts” could correspond to the activation of a sequence of intracellular events leading to inhibition of macromolecular synthesis thereby stopping endothelial proliferation. ACKNOWLEDGMENT The authors thank Miss Theresa Krick for her technical son for typing the manuscript.
assistance and Mrs. Janice Garrett-
REFERENCES ALLGBWER, M., and HULLIGER, L. (1960). Origin of fibroblasts from mononuclear blood cells: A study of in vitro formation of the collagen precursor, hydroxyproline, in buffy coat cultures. Surgery 47, 663-610. CLARK, E. R., and CLARK, E. L. (1925). A. The development of adventitial (Rouget) cells on the blood capillaries of amphibian larvae. Amer. J. Anat. 35, 239-264.
64
CROCKER,
MURAD,
AND
GEER
CLARK, E. R., and CLARK, E. L. (1939). Microscopic observations on the growth of blood vessels in the living mammal. Amer. J. Anat. 64, 251-299. CLIFF, W. J. (1963). Observations on healing tissue: A combined light and electron microscopic investigation. Phil. Trans. Roy. Sot. London, Ser. B 246, 305-325. (1939). A modification of a SandisonEBERT, R. H., FLOREY, H. W., and PULLINGER, B. D. Clark chamber for observation of transparent tissue in the rabbit’s ear. J. Pathol. Bacterial. 48, 79-94. EBERT, R. H., and FLOREY, H. W. (1939). The extravascular development of the monocyte observed in vivo. Brit. J. Exp. Pathol. 20, 342-356. FAWCETT, D. W. (1959). The fine structure of capillaries, arterioles and small arteries. In “The Microcirculation” (S. R. M. Reynolds, and B. Zweifach, eds.), pp. l-27. Univ. of Illinois Press, Urbana, Illinois. FISHER, H. W., and YEH, J. (1967). Contact inhibition in colony formation. Science 155, 581-532. GRILLO, H. C. (1963). Origin of fibroblasts in wound healing: An autoradiographic study of inhibition of cellular proliferation by local x-irradiation. Ann. Surg. 157, 453467. KUWABARA, T., and COGAN, D. G. (1963). Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Arch. Ophthal. N.S. 69, 492-502. LEVINE, E. M., BECKER, Y., BOONE, G. W., and EAGLE, H. (1965). Contact inhibition, macromolecular synthesis, and polyribosomes in cultured human diploid fibroblasts. Proc. Nat. Acad. Sci. U.S.A. 53, 350-356. LOCKWOOD, W. R. (1964). A reliable and easily sectioned epoxy embedding medium. Anat. Rec. 150, 129-140. MACDONALD, R. A. (1959). Origin of fibroblasts in experimental healingwounds: Autoradiographic studies using tritiated thymidine. Surgery 46, 376-382. MILLONIG, G. J. (1961). Advantages of a phosphate buffer for 0~04 solution in fixation. J. Appt. Physics (Abstr.) 32, 1637. MOVAT, H. Z., and FERNANDO, N. V. P. (1964). The fine structure of the terminal vascular bed. IV. The venules and their perivascular cells (pericytes, adventitial cells). Exp. Mol. Pathol. 3, 98-114. MURAD, T. M., and VON HAAM, E. (1968). An ultrastructural study of pericytes. Proc. Elec. Micro. Sot. Amer., 26th Ann. Meet., pp. 6667. PALADE, G. E. (1955). Relations between the endoplasmic reticulum and the plasma membrane in macrophages. Anat. Rec. (Abstr.) 121, 445. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. CeZZ Biol. 17, 208-212. RHODIN, J. A. G. (1968). Ultrastructure of mammalian venous capillaries, venules, and small collecting veins. J. Ultrastruct. Res. 25, 452300. Ross, R., and BENDITT, E. P. (1961). Wound healing and collagen formation. I. Sequential changes in components of guinea pig skin wounds observed in the electron microscope. J. Biophys. Biochem. Cytol. 11, 677-700. SABATINI, D. D., BENSCH, K., and BARRNETT, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol. 17, 19-58. SCHOEFL, G. I. (1963). Studies on inflammation. III. Growing capillaries: Their structure and permeability. Virchows Arch. Pathol. Anat. 337, 97-141. SCHUTZ, L., and MORA, P. T. (1968). The need for direct cell contact in “contact” inhibition of cell division in culture. J. Cell Physiol. 71, l-6. STOKER, M. (1964). Regulation of growth and orientation in hamster cells transformed by polyoma virus. Virology 24, 165-174. TODARO, G. J., LAZAR, G. K., and GREEN, H. (1965). The initiation of cell division in a contact-inhibited mammalian cell line. J. Cell. Comp. Physiol. 66,325334. TRUMP, B. F., SMUCKLER, E. A., and BENDITT, E. P. (1961). A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5, 343-348.
PERICYTE
IN WOUND
HEALING
65
A., and GOWANS, J. L. (1965). The production of macrophages in the rat. Brit. J. Exp. Palhol. 46, 50-61. VOLKMAN, A., and GOWANS, J. L. (1965). The origin of macrophages from bone marrow in the rat. Brit. J. Exp. Pathol. 46, 62-70. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biocham. Cytol. 4, 475-478. YEH, J., and FISHER, H. W. (1969). A diffusible factor which sustains contact inhibition of replication. J. Cell Biol. 40, 382-388. VOLKMAN,
AND
MOLECULAR
Role
13, 51-65
PATHOLOQY
of the
Pericyte
in Wound
An Ultrastructural DAN J. CROCKER, TARIQ Department
of Pathology,
The
M.
Ohio
Received
(1970)
Study’
MURAD, State
February
Healing
AND
University,
JACK
C. GEER
Columbus, O’aio 43210
18, i970
Mesenchymal cells present in earlywoundswere divided into two groups: primitive mesenchymal cells and macrophages. Primitive mesenchymal cells appeared to become incorporated in the thick basement membrane of recently formed capillaries, and they also appeared to differentiate into fibroblasts. After becoming encased in vascular basement membrane these cells, pericytes, featured areas of cytoplasmic contact with underlying endot.helium. It is proposed that the pericyte-endothelial “contacts” act as a regulatory mechanism for capillary proliferation.
Pericytes are perivascular cells that are associated with the capillaries of many tissues and organs. The ultrastructural morphology of these cells and their relation to small blood vessels have been previously described (Fawcett, 1959; Movat and Fernando, 1964; Murad and von Haam, 1968). The origin of pericytes and their function is not known and has been a subject for speculation (Clark and Clark, 1925; Cliff, 1963; Rhodin, 1968). In the healing wound the presenceof pericytes has been inferred to be associated with the maturation of proliferating vessels (Cliff, 1963). The present report is a sequential electron microscopic study of the healing wound in hamsters. The incorporation of pericytes within the basement membrane of proliferating capillaries is proposed as the mechanism for inhibition of capillary proliferation. MATERIALS
AND METHODS
Sixteen male hamsters ranging from 6 to 8 months of age and weighing about 100 gm each, were used for this study. The dorsal flanks of the animals were shaved and circular wounds were made with an S-mm dermal punch. The wounds extended to the depth of the superficial fascia. After wounding, animals were killed on the following days, 2-7, 14, and 21. The excised wounds from each animal were fixed in 5 % cold glutaraldehyde in phosphate buffer (Sabatini et al., 1963) for l-lx hours. After fixation, the edge of the wound was examined under a stereoscope and dissected away from adjacent tissue, then cut into small pieces (ca 1.0 mm). The small pieces of tissue were then placed in 1% osmium tetroxide in phosphate buffer at pH 7.3 (Millonig, 1961). The post-fixed tissue was embedded in DER (Lockwood, 1964). Sections 1~ thick were cut, mounted on slides, stained with toluidine blue (Trump et al., 1961) and 1 This investigation was supported in part by a General Research Support Grant from the National Institutes of Health and HE 11897 from the National Heart and Lung Institute of the National Institutes of Health. 51
52
CROCKER,
MURAD,
AND
GEER
examined with light microscopy. Selected areas were thin-sectioned, mounted on copper grids, and double-stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). An RCA EMU-3F electron microscope was used to examine the stained sections. RESULTS LIGHT
MICROSCOPY
Wounds on days 2, 3, and 4 of healing were characterized by the presence of inflammatory exudate composed of neutrophils and fibrin (Fig. 1). At the margin of the wound mononuclear cells were present. At day 4 newly formed capillaries were seen at the edge of the wound. Numerous newly formed capillaries were present by day 7. The intercapillary interstitial tissue at day 7 contained numerous cells of varying morphology that appeared to represent neutrophils, fibroblasts, and macrophages (Fig. 2). At days 14 and 21 of healing the wound was characterized by abundant collagen with fibrocytes, and the number of capillaries was markedly reduced from that seen at day 7. ELECTRON
MICROSCOPY
The inflammatory exudate of the wound at 2 and 3 days was composedprimarily of fibrin and numerous partially degranulated neutrophils (Fig. 3). In the fibrin network there were clusters of extracellular neutrophil granules and cells resembling blood monocytes in addition to the partially degranulated neutrophils. The extracellular neutrophil granules typically were surrounded by an electron-lucid zone (Fig. 4). Mononuclear cells in the wound at 3 and 4 days could be divided into two morphologic types based upon the number of electron-dense cytoplasmic inclusions. Those cells with numerous electron-dense, membrane-bound inclusions were designated macrophages (Fig. 5). The macrophage generally was larger than the second type mononuclear cell. The nucleus of the macrophage typically was ovoid or kidney-shaped with a narrow rim of heterochromatin along the inner nuclear membrane and contained a single nucleolus. The cytoplasm demonstrated the usual organelles including scattered parallel arrays of granular endoplasmic reticulum. The second type of mononuclear cell could not be distinguished from the macrophage on the basis of nuclear morphology or cytoplasmic organelles. In general, the second-type cell had a lesser cytoplasmic area than the macrophage, but the distinguishing feature was the absenceof electron-dense cytoplasmic inclusions (Fig. 6). We have designated this second type of cell as a primitive mesenchymal cell for purposes of this presentation. Primitive mesenchymal cells were more abundant than macrophages at days 3 and 4 and often were present in clusters about small blood vesselsas well as within the fibrin network of the exudate (Fig. 7). The perivascular mesenchymal cell was distinguishable from a pericyte by its lack of surrounding basement membrane. A prominent feature of the growing capillaries at the margin of the wound at day 4 was a thick basement membrane (Figs. 8 and 11). Mitotic figures were seen
PERICYTE
IN WOUND
HEALING
53
FIG. 1. Early wound filled with acute inflammatory exudate composed of fibrin mesh and leukocytes. X 200. FIG. 2. Seven-day-old wound featuring numerous blood vessels, polymorphonuclear leukocytes, and mononuclear cells. X 700.
54
FIG. FIG. rounded
CROCKER,
3. Partially degranulated neutrophils 4. Extracellular neutrophil granules by a clear zone. x 29,000.
MURAD,
AND
GEER
surrounded by serum protein. X 5200. entrapped in fibrin. Many of the granules
are sur-
PERICYTE
IN WOUND
HEALING
55
FIG. 5. Macrophage featuring numerous membrane bound cytoplasmic inclusions of varying electron density. X 13,000. FIG. 6. Primitive mesenchymal cells with a paucity of formed cytoplasmic elements. x 10,500.
56
CROCKER,
MURAD,
AND
GEER
FIG. 7. Primitive mesenchymal cells (PMC) are seen clustered near a venule. Note the similarity in the eytoplasmic profile of the pericyte (P) and the primitive mesenchymal cells. x 7c!oo. FIG. 8. Forming capillary with a thick basement membrane which contains a pericyte (P). The capillary exhibits a narrow slitlike lumen (arrow). X 13,ooO.
PERICYTE
IN WOUND
HEALING
57
in the endothelial cells (Fig. 9) and in the perivascular primitive mesenchymal cells (Fig. 10). By days 5 and 6 of healing many of the primitive mesenchymal cells were elongated and enclosed within the capillary basement membrane, thus having the location of pericytes. At days 5 and 6 of healing areas of the plasma membranes of “pericytes” and endothelium were seen closely approximated to one another (Fig. 11). At day 7 of healing the capillary basement membrane was the same thickness as in the early stages of healing but relative to the greater diameter of the capillary appeared reduced in thickness. The capillary endothelium was more attenuated than at days 4 to 6, though portions of the cytoplasm were still quite thick (Fig. 12). The pericytes partially enveloped the capillaries and showed a marked increase in cytoplasmic organelles, especially granular endoplasmic reticulum, over that seen in the perivascular primitive mesenchymal cell. The primitive mesenchyma1 cells in the interstitial tissue changed from a round shape to elongate cells, and the cytoplasm showed an increased number of profiles of granular endoplasmic reticulum (Fig. 13). The capillaries at the 14th day of healing showed numerous areas of close association of endothelial and pericyte plasma membranes (Fig. 14). The interstitial tissue was composed primarily of collagen fibers and elongated cells having within their cytoplasm numerous profiles of dilated granular endoplasmic reticulum (Fig. 15). These cells morphologically appeared to be fibroblasts. The capillaries at day 21 of healing were far fewer in number than in the earlier days. The capillaries were characterized by having an endothelial lining of rather uniform thickness in contrast to the irregular thickenings seen at 4-7 days. The pericytes partially enveloped the endothelium with narrow processes of electrondense cytoplasm (Fig. IS), and areas where these cells closely approximated one another were readily seen (Fig. 17). Few fibroblasts were present among the collagen fibers of the intercapillary tissue. DISCUSSION There are conflicting views regarding the origin and function of mesenchymal cells in the healing wound. The studies of Volkman and Gowans (1965a, 196513) indicate that most macrophages in the wound are derived from blood monocytes. The observations of Ebert and Florey (1939) also indicate that blood monocytes may become tissue macrophages. Ross and Benditt (1961) concluded from their observations that fibroblasts probably arise from cells of hematogenous origin, such as the blood monocyte, or a pleuripotential cell from the blood. There is evidence indicating that certain cells derived from blood appear to have a potential for the production of hydroxyproline (Allgijwer and Hullinger, 1960); however, autoradiographic studies of wound healing (Grillo, 1963; MacDonald, 1959) strongly indicate that the main source of fibroblasts in the healing wound is from the connective tissue at the wound edge. ClifI (1963) concluded from hi observations that there is no evidence for the presence of primitive, or pleuripotential mesenchymal cells in the wound. He believes that fibroblasts give rise to fibroblasts, monocytes give rise to macrophages, and that the endothelium of proliferating capillaries has as its source the endothelium of preexisting capillaries. His observations give no
58
x
CROCKER,
FIG. 9. Pericyte 17,000. FIG. 10. Capillary
(P) (C)
closely featuring
MURAD,
nestled
AND
on endothelial
a pericyte
undergoing
GEER
cell
(E) mitosis.
that
is undergoing
X 13,000.
mitosis.
FIN. 11. Cytoplasmic processes of a pericyte are seen in the thick basement membrane of a capillary. Areas of “contact” (arrows) are seen between the plasma membranes of endothelium and pericyte cytoplasmic extensions. X 10,u)o. FIG. 12. Capillary invested by a pericyte (P). Focally condensed bundles of filaments are seen along the cytoplasmic membrane of the pericyte suggestive of early hemidesmosome formation (arrows). X 10,200. 59
PERICYTE
IN WOUND
HEALING
61
FIG. 15. Fibroblasts with well developed granular cytoplasm situated next to venule. x 7060. FIG. 16. Thin cytoplasmic process of a pericyte extending around a capillary. An area of incomplete “contact” is readily seen between the pericyte and endothelium (arrow). X 21,000.
62
CROCKER,
MURAD,
AND
GEER
FIG. 17. High magnification of pericyte-endothelial “contact” of plasma membrane and the cytoplasmic fusion of the contacting lium features numerous pinocytotic vesicles. X 50,OCMl.
(arrow). Notice the absence cells (arrow). The endothe-
credence to the thought that there are transformations from one cell type to another. We have divided the mononuclear cells found in early wounds into two groups: macrophages and primitive mesenchymal cells. Those cells fitting the description of macrophages as described by Palde (1955) were designated as such. However, the second population of cells lacked certain features ascribed to the macrophage, namely, electron-dense bodies and phagocytosed material in their cytoplasm. Since we were unsure of the origin of this cell, and becauseits cytoplasmic profile was not indicative of a specific cell-type, we chose to designate this group as primitive mesenchymal cells. During the course of healing, cells of this type were seen nested around newly formed capillaries and appeared to become incorporated in membrane, thus becoming pericytes. It appeared that primitive vascular basement mesenchymal cells also differentiated into fibroblasts during the process of healing. That newly formed capillaries found in healing wounds come from the severed ends of preexisting capillaries has been demonstrated (Cliff, 1963; Ebert et al., 1939; Schoefl, 1963). The genesis of newly formed capillaries from the terminal portion of severed vesselshas been observed, and the endothelial cells of proliferating capillaries are seento be derived frqm proximal endothelial cells via the process of mitosis and subsequent migration distally. Schoefl (1963) also suggested the possibility that pericytes may contribute to the endothelial lining of regenerating capillaries.
PERICYTE
IN WOUND
HEALING
63
At the advancing tip of proliferating capillaries the observation has been made that the vascular basement membrane is devoid of a cellular constituent (Cliff, 1963; Schoefl, 1963). However, proximal to the advancing tip cells are seen incorporated in the vascular basement membrane. These cells are described as coming from the surrounding interstitium (Cliff, 1963) and being fibroblastlike in character. Kuwabara and Cogan (1963) have suggested that the absence of pericytes in vascular basement membrane permits endothelial proliferation. Cliff (1963) implies that there is vascular maturation with the envelopment of interstitial in vascular basement membrane. Observations of this study lead the authors to hypothesize that it is the incorporation of interstitial cells in the basement membrane of newly formed capillaries with the subsequent close association of areas of their cytoplasmic membranes that is responsible for inhibiting endothelial proliferation. And, according to our classification this interstitial cell is of primitive mesenchymal type. Studies of cell culture monolayers have demonstrated that once cells reach a critical cell density they cease to proliferate. This phenomenon has been called “contact inhibition“ of cell division (Todaro et al., 1965)) and it is thought to be dependent on direct physical contacts, not humoral factors (Levine et al., 1965; Stoker, 1964). Schutz and Mora (1968) have concluded from their studies of contact inhibition in cell cultures that it is necessary for cells to have close contact over large areas of their surface for contact inhibition to occur. The proposal has been made that the contacting of cells in culture initiates a sequence of intracellular events which activates the internal release of an inhibitor of macromolecular synthesis, which in turn, produces a cessation of replication (Fischer and Yeh, 1967; Yeh and Fischer, 1969). In their studies they have shown that this inhibitor acts specifically to stop RNA synthesis of contact inhibited cells, and that this inhibition is reversible. In this study, areas of close proximity between pericyte and endothelial plasma membranes made their appearance in the proliferative phase and reach their greatest number by day 14 of healing. For the purpose of simplicity the areas of close approximation of pericytes and endothelium are designated as “contacts” although no direct physical contacts between the cell membranes were observed. The increased number of “contacts” between pericytes and endothelium may act to inhibit endothelial proliferation through a mechanism similar to that ascribed to cell culture. Specifically, the increased number of “contacts” could correspond to the activation of a sequence of intracellular events leading to inhibition of macromolecular synthesis thereby stopping endothelial proliferation. ACKNOWLEDGMENT The authors thank Miss Theresa Krick for her technical son for typing the manuscript.
assistance and Mrs. Janice Garrett-
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