Apoptosis during wound healing, fibrocontractive diseases and vascular wall injury

ht.

J. Eiochrm.

Copyright

Pergamon

Cell Bid

d’, 1997 Elsevier

PII: S1357-2725(96)00117-3

Vol. 29, No. I. pp. 19.-30. 1997 Somce Ltd. All rights reserved Printed in Great Britain 1357.2725/97

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REVIEW Apoptosis During Wound Healing, Fibrocontractive Diseases and Vascular Wall Injury ALEXIS DESMOULIkRE,‘” CHhRIF BADID,’ MARIE-LUCE BOCHATON-PIALLAT,’ GIULIO GABBIANI* ‘CNRS-URA 1459, Pathologie des Fibroses, Institut Pasteur de Lyon, Avenue Tony Garnier, 69365 L.von, cedex 07, France and ‘DPpartement de Pathologie, Centre Mt+dical Universitaire, 1, rue Michel-Servet, 1211 GenBve 4, Switzerland Following injury, tissue repair involves inflammation, granulation tissue formation and scar constitution. Granulation tissue develops from the connective tissue surrounding the damaged or missing area and contains mainly small vessels, inflammatory cells, fibroblasts and myofibroblasts. As the wound closes and evolves into a scar, there is a striking decrease in cellularity, including disappearance of typical myofibroblasts. The question arises as to what process is responsible for granulation tissue cell disappearance. Our results (in cutaneous wounds) and results of other laboratories (particularly in lungs and kidney) suggest that apoptosis is the mechanism responsible for the evolution of granulation tissue into a scar. During excessive scarring (hypertrophic scar or fibrosis), it is conceivable that the process of apoptosis cannot take place. After experimental endothelial injury in an artery, accumulation of smooth muscle cells participates in the formation of intimal thickening. Apoptotic features have been observed in cells of intimal thickening and also within human atherosclerotic plaques. In the case of atherosclerosis, apoptosis could be detrimental: since smooth muscle cells participate in plaque stability, apoptosis could lead to weakening and rupture of the plaque. These results underline the fact that both increased cell survival or excessive cell death can be associated with pathological disorders. Specific therapies devised to enhance or decrease the susceptibility of individual cell types to apoptosis development could modify the evolution of a variety of human diseases. 0 1997 Elsevier Science Ltd. All rights reserved Keywords: Int. J. Bioclwm.

Fibroblast

Smooth muscle cell

Cdl Biol. (1997)

29,

Myofibroblast

Actin

19-30

an atheromatous plaque). During wound healing and fibrocontractive diseases, fibroblasts acquire some morphological and biochemical features of smooth muscle cells (for review, see Schiirch et al., 1992). These modified fibroblasts called myofibroblasts contain cytoplasmic bundles of microfilaments or stress fibers, which, as in smooth muscle cells, play a role in contraction. Myofibroblasts are interconnected by gap junctions and are connected to the extracellular matrix by the fibronexus, a transmembrane complex involving intracellular microfilaments in apparent continuity with extracellular fibronectin fibers (Singer et al.,

INTRODUCTION

In normal adult individuals, the main function of fibroblasts is the secretion of extracellular matrix components while smooth muscle cells exert contractile activities (Desmouliere and Gabbiani, 1995). However, during pathological situations, fibroblasts may develop contractility (e.g. during wound healing) whereas smooth muscle cells may secrete important amounts of collagen (e.g. during the formation of ______ *To whom all correspondence should be addressed. Received 6 June 1996; accepted 3 September 1996. 19

Alexis Desmoulike

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1984). The nucleus of myofibroblasts shows indentations (Schiirch et al., 1992) an ultrastructural feature that has been correlated with cellular contraction in several systems (Franke and Schinko, 1969; Majno et ~1.. 1969). Myofibroblasts are partly enveloped by a basal lamina similar to that of smooth muscle cells. Cytoskeletal proteins, which are known to play a key role during the process of cell contraction, have been used to characterize different myofibroblast phenotypes that may co-express in addition to cytoplasmic actin isoforms: (a) vimentin, (b) vimentin and desmin, (c) vimentin and a-smooth muscle actin, (d) vimentin, desmin and x-smooth muscle actin. (e) vimentin, cc-smooth muscle actin and smooth muscle myosin heavy chains, and (f) vimentin, cr-smooth muscle actin. desmin and smooth muscle myosin heavy chains (Desmoulitre and Gabbiani, 1996). r-Smooth muscle actin, which is the actin isoform typical of contractile vascular smooth muscle cells, is expressed by practically all myofibroblastic populations in r.iz:o (Schmitt-G&I et al., 1994). In the arterial wall, subpopulations of smooth muscle cells, which express (at least in t.irro) different proportions of a-smooth muscle actin, desmin and smooth muscle myosin heavy chains, have been characterized (Bochaton-Piallat et al., 1992). During the atheromatous process. the expression of these markers of differentiation decreases and smooth muscle cells acquire a dedifferentiated phenotype similar to that of fetal smooth muscle cells and that resembles the fibroblast phenotype (Desmouliere and Gabbiani. 1992). In fact, during tissue aggression, fibroblasts and smooth muscle cells, which are both mesenchymal cells. adopt similar features: they migrate and proliferate to replace the defect of injured tissue. In pathological the proliferation and migration situations, of fibroblasts or smooth muscle cells can induce the development of organ fibrosis or the constitution of an atheromatous plaque, respectively. In some situations, the proliferation can be counterbalanced by cell loss and apoptosis could be the process through which cells are eliminated. GENERAL

COMMENTS PROCESS

ON

APOPTOTIC

Apoptosis has been recognized as a morphologically and biochemical distinct process of cell death. The morphological characteristics of

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this process have been firstly described by Kerr et al. (1972) using electron microscopy. It is characterized by the internal production of autodestructive substances without development of an inflammatory reaction. Apoptosis is involved in many processes such as embryonic development, normal cell turnover, maturation of the immune system and negative selection of immunocompetent T cells (Kerr et al., 1994). Moreover, in some cases, this form of cell death can be involved in non-physiological processes and play a role in degenerative situations such as Alzheimer’s disease or the acquired immunodeficiency syndrome (Thompson, 1995). By electron microscopy, cell shrinkage with chromatin condensation and ,formation of crescent shaped deposits along nuclear envelop are observed during the apoptotic process. The plasma membrane remains intact and the cytoplasmic organelles (Golgi and mitochondria) keep their integrity. However. modifications of electrolyte balance induce decrease of the cell size and formation of protuberances. Thereafter, the cell fragments into membrane bound apoptotic bodies that are phagocytosed by macrophages and neighboring cells. The cellular content is sealed within these bodies until phagocytosis, and no inflammatory responses are seen around apoptotic cells. The apoptotic process concerns generally isolated cells and is a very rapid phenomenon. Once chromatin condensation and other morphological changes typical of apoptotis occur, the uptake and degradation of apoptotic cells by phagocytosis is very fast; only 0.5-2.0 hr (clearance time) elapse before such cells are no longer detectable by conventional histology. Biochemically, apoptosis is characterized by activation of endogenous calcium dependent endonucleases resulting in the cleavage of chromatin into oligonucleosome DNA fragments detected on gel electrophoresis of extracted DNA (Kerr et al.. 1994). On histological sections, fragmented DNA can be detected by in .xitu tailing or nick translation techniques (Gold et ul., 1994). Endonuclease activation is a sign of irreversible cell death. Furthermore, the involvement of proteases in the mechanisms of apoptosis has been shown (Martin and Green, 1995). The role and the substrate of these proteases remain unknown but they seem to be involved in the chromatin changes and in the morphological modifications of the cell. The interleukin-lpconverting enzyme (ICE). a cysteine protease

Apoptosis during wound healing, fibrocontractive

that cleaves the 33 kD pro-interleukin-l/I into the 17.5 kD biologically active interleukin-l/?, is the mammalian homologue of the Caenorhabditis elegans cell death gene ted-3 and has been shown to induce apoptosis (Miura et al., 1993). Recently, Kayalar et al. (1996) have demonstrated that actin is a substrate for ICE. It is known that globular actin inhibits the endonucleolytic activity of DNase I, the major endonuclease associated with the internucleosoma1 fragmentation of DNA during apoptosis. ICE is able to cleave globular actin mainly at two sites (between Asp” and Asn” and between Asp“‘” and G~Y’~~)resulting in a decreased ability of globular actin to polymerize and to inhibit the endonuclease activity of DNase I. These reports underline that important modifications of cytoskeletal proteins occur during apoptosis allowing the formation of plasma membrane blebs and the retraction of cell processes. The tissue-type transglutaminase, an enzyme involved in the cross-linking of intracellular proteins, is increased in cells undergoing apoptosis (Fesus et al., 1991). The transglutaminase activity may serve to trap cytoplasmic components within the apoptotic body, preventing its leakage into the extracellular space where it might result in inflammation. Many genes have been implicated in the apoptosis induction or inhibition. For example, bc12 is a protooncogene involved in the negative regulation of apoptosis (Reed, 1994); in contrast, Fas transduces a signal for apoptosis after binding by specific antibodies or following contact with natural Fas ligand (Ogasawara et al., 1993). The expression of c-rv2~~ proto-oncogene has been implicated both in cell proliferation and apoptotic process (Evan et al., 1992; Bennett et al., 1993). The molecular mechanisms mediating these opposite functions of c-myc remain unclear but these observations support the possibilities that the expression of different genes, playing a role in apoptosis, depends on the balance of extracellular signals that induce cell proliferation, differentiation, quiescence, or death. It is noteworthy that a number of studies show that germinal center B cells, peritoneal B cells, thymocytes, and T cells can be protected from rapid apoptosis by stromal cells and/or fibroblasts (Merville et al., 1996). Apoptotic signals may be: loss of communications with the microenvironment, growth factor withdrawal, cytokine action or, virus infection. Interestingly, cells can undergo apoptosis when their anchorage is modified; this process implies integrin-

diseases and vascular wall injury

21

mediated signaling as the controlling factor (Ruoslahti and Reed, 1994). Up to now, this type of apoptotic response appears to be limited to endothelial and epithelial cells (Meredith et al., 1993; Boudreau et al., 1995) but we can suppose that a similar mechanism is implicated in fibroblastic cell apoptosis. APOPTOSIS

DURING WOUND

HEALING

Following tissue injury and after clot formation, inflammatory cells invade the lesion; then, fibroblasts migrate, proliferate, and synthetize extracellular matrix components, participating in the formation of granulation tissue. Granulation tissue formation and contraction is an important step of second intention wound healing (Grinnell, 1994). Granulation tissue develops from the connective tissue surrounding the damaged or missing area and its cellular components are mainly small vessels and inflammatory cells as well as fibroblasts and myofibroblasts surrounded by an important deposit of extracellular matrix components. As the wound closes and evolves into a scar, there is an important decrease in cellularity and in particular, myofibroblasts disappear (Clark, 1993). The question arises as to which process is responsible for this cellular loss. It has been shown that, during wound healing, myofibroblasts develop gradually and express temporarily E-smooth muscle actin (Darby et al., 1990). Furthermore, in late phases of wound healing, many myofibroblasts show changes compatible with apoptosis and it has been suggested that this type of cell death could be responsible for the disappearance of myofibroblasts. This hypothesis has been tested by means of morphometry at the electron microscopic level and by in situ labeling of fragmented DNA (Desmouliere et ul., 1995). Our results indicate that the number of myofibroblastic and vascular cells undergoing apoptosis increases importantly as the wound closes. In our rat model of wound healing (Darby et al., 1990) the wound is practically closed by 15 days. In the wall of granulation tissue vessels, isolated apoptotic cells appeared at 8 days and a maximum of labeled cells was observed between 16 and 25 days (Table 1). Apoptotic changes were seen in both pericytes and endothelial cells. Positive apoptotic staining in myofibroblasts appeared, albeit exceptionally, at 12 days when x-smooth muscle actin expression was maximal (Table I). The frequency of apoptotic myofibroblasts

22

Alexis

Table

1. Evaluation

of cells fragmented

showing in siru DNA a.h Granulation

Days

after

wounding 7 8 IO 12 16 20 25 30 60

Fibrobiasts f ++ + ++ ++ AI -

Desmouhere

labeling

of

tissue Vascular

cells --

tf + ++ ++ ++ + -

“The

staining was observed blindly and independently by two researchers and classified as: -, no staining; k, staining in less than 3% of cells; +, staining in 336% of cells; + +, staining in 669% of cells; + + f. staining in 9-12% of cells. hFrom Desmouliere et al. (Am. J. Pathoi. 1995, 146, 56-66). with permission.

measured as a percentage of labeled cells increased greatly by 16 days with a maximum at 20 days. We then observed a progressive decrease of labeled cells and at 60 days, labeled cells were absent. By electron microscopy, apoptotic fibroblasts and vascular cells showed condensation and margination of chromatin, cytoplasmic vacuoles, and convoluted cell surface (Fig. la, b and c). Macrophages containing phagolysosomes were seen near fibroblastic cells, suggesting that this is the major route of removal of apoptotic bodies (Fig. Id). Our results support the assumption that apoptosis is involved in the evolution of granulation tissue into scar. The regulation of apoptotic phenomena during wound healing may be important in scar establishment and development of pathological scarring. Indeed, when granulation tissue cells are not eliminated, there is development of hypertrophic scar or keloid, both characterized by a high degree of cellularity (Rockwell et al.. 1989). Only hypertrophic scars, which, contrary to keloids, contract, contain typical myofibroblasts expressing a-smooth muscle actin (Ehrlich et al., 1994). In both lesions, whatever the fibroblastic phenotype, cell proliferation allowing granulation tissue formation continues even after wound closure and re-epithelialization. During normal wound healing, apoptosis affects target cells consecutively rather than producing a single wave of cell disappearance. These observations are in line with the gradual resorption of granulation tissue after wound closure. In a recent work (Garbin et al., 1996), we have observed that covering granulation tissue with a skin flap results in a massive apoptotic process (Fig. 2). Thus, a skin flap

et u/

induces, in an accelerated way, the same phenomena that develop gradually during normal evolution of a wound into a scar. Total skin flaps were the most efficient to induce the apoptotic process compared to dermoepidermic flaps, which in turn were more efficient when compared to controls. Apoptosis took place only when surviving flaps were applied and did not take place in grafts not surviving. This suggests that the survival of the grafted tissue through vascularization is essential in order to stimulate apoptosis in granulation tissue cells. The nature of the stimuli supplied by the skin flap and causing selectively the death of granulation tissue fibroblastic and vascular cells is not defined. Jiirgensmeier et nl. (1994) have shown that the supernatant of transforming growth factorfl(TGF-/I)-treated fibroblasts induces apoptosis in transformed fibroblasts. TGF-o-induced elimination of transformed fibroblasts by their untransformed counterparts could represent a potential mechanism present during granulation tissue fibroblastic cell disappearance. APOPTOSIS

AND

ORGAN

FIBROSIS

In many organs, the repair process implicates the formation of a granulation tissue with cells acquiring myofibroblastic differentiation, i.e. expression of cr-smooth muscle actin. and finally scar formation (Schiirch et al., 1992). When the tissue injury is sufficiently limited in time, and when tissue loss does not destroy too many specialized cells, a scar is formed, which does not impair function. Alternatively. the damaging stimuli inducing granulation tissue formation can result in organ fibrosis. Apoptosis has been suggested to be one of the central mechanisms by which fibrosis is modulated. Apoptosis in lung there is a During acute lung injury. recruitment of many inflammatory cells in the alveolar wall and granulation tissue may develop in alveolar septa. Grigg et nl. (1991) have suggested that neutrophil apoptosis represents a mechanism by which tissue injury is reduced during the resolution of respiratory distress syndrome. They have studied bronchoalveolar fluid obtained from eight newborn babies (aged between 3 and 5 days) who presented this disease. In resolving conditions, electron microscopy showed typical apoptotic neutrophils, which were phagocytosed by

Apoptosis

during

wound

healing,

fibrocontractive

diseases

and vascular

wall

injury

Fig. I. Transmission electron micrographs showing different features of (a. b) apoptotic fibroblasts. (c) vascular structures containing apoptotic cells and (d) a macrophage. In fibroblasts undergoing apoptosis (a, b), variable degrees of chromatin condensation are visible. In (b), the nucleus appears m part extruded from the cytoplasm. In (c), the capillary is obstructed and cells with chromatin condensation are observed. The macrophage (d) contains apoptotic bodies and numerous phagolysosomes. (a. b): original magnification x 10 000: (c): original magnification x 5000; (d): original magnification x 8000. (d): From Garbin et rrl. (Wound Rep. Reg. 1996, 4, 244251). with permission.

24

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cl w l

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151

Desmouli&e

myofibrobiasts vessel well cells

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cells within the airspace granulation tissue. This study indicates that signal(s) inducing granulation tissue cell apoptosis is (are) present at the air lung interface in patients recovering from acute lung injury.

Apoptosis in kidney

6 Time

(hours)

24 after

48 flap

72

application

Fig. 2. Quantification of the different apoptotic cell populations in the granulation tissue underlying skin flap. The cell number/unit area was evaluated blindly and independently by two researchers. SEW were always lower than 5”/u of the values. From Garbin ef ul. (Wound Rep. Reg. 1996. 4, 244-251), with permission.

macrophages. Neutrophil infiltration persisted in infants who developed chronic bronchopulmonary dysplasia in which inflammation leads to the formation of fibrosis. Moreover, the chronic eosinophilic inflammation often observed in the lungs of asthmatic individuals in which a fibrotic process implicating myofibroblasts develops (Brewster et (11.. 1990) could be at least in part explained by defects of Fus-mediated resolution of eosinophils by apoptosis (Tsuyuki et al., 1995). After bleomycin-induced lung injury. a fibroproliferative response ensues, leading to extensive alveolar septum fibrosis. The expression of profibrotic factors including TGF-/I and tumor necrosis factor-cc has been shown to precede collagen deposition (Kapanci et al., 1995). Polunovsky et al. (1993) have studied the role of mesenchymal cell death in lung remodeling after acute injury (adult respiratory distress syndrome); when human lung fibroblasts and endothelial cells were cultivated with bronchoalveolar lavage fluid obtained from patients undergoing the inflammatory phase of the illness, the number of dead cells increased compared to the number observed in response to lavage fluid from patients in an earlier phase of the disease or control patients. Endothelial cells died by apoptosis while fibroblast death was morphologically distinct from necrosis and differed from typical apoptosis. Moreover, histological examination of lung tissue from patients after lung injury revealed the presence of apoptotic

Post-inflammatory scarring is an important cause of kidney disease, which can result in a chronic renal failure requiring, in many cases. a renal transplantation. Proliferative glomerulonephritis (GN) is characterized by hypercellularity with mesangial proliferation and an expansion of extracellular matrix. In some cases, one can observe a resolution of the disease (e.g. in post-streptococcal GN), but little is known about the repairing process of GN allowing glomerular structure and function to recover. Polymorphonuclear granulocytes are a prominent component of the inflammatory infiltrate in many types of human GN. Although essential in defense against invading micro-organisms, it is clear that neutrophils mediate injury to host tissue in a number of inflammatory processes, including GN. Savill et al. (1992) have studied the clearance of neutrophils 2 days after the induction in rats of experimental GN by means of anti-rat kidney serum. They show that not only macrophages but also glomerular cells participate in the clearance of apoptotic neutrophils. Because neutrophil apoptosis leads to macrophage and mesangial cell ingestion of apoptotic bodies which retain their toxic contents, it appears that this process represents a potentially ‘injurylimiting’ clearance mechanism (Savill, 1992). An experimental model of proliferative GN induced by an antibody to Thy1 .l antigen present on rat mesangial cells is well established and is characterized by acute mesangial proliferation, hypercellularity and expansion of the mesangial matrix resembling the morphological features of human proliferative GN. This lesion is transient and cell number spontaneously returns to normal with a decrease of extracellular matrix deposition. Baker c/ trl. (1994) and Shimizu et rrl. (1995) have demonstrated that the resolution of glomerular hypercellularity in experimental GN is mediated by mesangial cell apoptosis. In cultured mesangial cells, apoptosis is induced by growth factor deprivation or exposure to cycloheximide. stimuli known to increase apoptosis in other

Apoptosis

during

wound

healing,

fibrocontractive

cell types (Baker et al., 1994). Furthermore, Sato et al. (1996) have shown that in vitro, anti-Thy-l antibodies are able to induce apoptosis of glomerular mesangial cells. Failure to clear excess glomerular cells by apoptosis, perhaps because of local conditions promoting survival, could be a hitherto unrecognized condition in the pathogenesis of persistent mesangial cell hyperplasia and thus of glomerular scarring. Furthermore, it has been shown that apoptosis plays an important role in the pathogenesis of renal tubular atrophy associated with hydronephrosis induced by ureteral obstruction (Gobe and Axelsen, 1987). Interstitial fibrosis is a common outcome of long-term ureteral obstruction (Diamond et al., 1995; Wright et al., 1996); an increase of infiltrating cortical interstitial macrophages is observed with the proliferation of myofibroblasts and the accumulation of extracellular matrix. Before renal atrophy, a reduction in renal blood flow appears. Mild ischemia has been implicated as the cause of cell deletion by apoptosis in several other studies of pathological tissue atrophy (Wyllie et al., 1980). Gobe and Axelsen (1987) have suggested that contraction of interstitial myofibroblasts in the cortex with effects on the intertubular capillary circulation participate in the renal blood flow reduction. Apoptosis in liter Numerous works have described conditions in which hepatocyte apoptosis occurs (for review, see Pate1 and Gores, 1995). Particularly, the role of TGF-/j’ in the induction of hepatocyte apoptosis in cultured hepatocytes and in regressing liver has been discussed (Oberhammer et al., 1992). However, although non-parenchymal cells (e.g. Kupffer cells, endothelial cells, stellate cells) represent up to a third of all liver cells, the specific role of apoptosis in their maintenance and function has been poorly investigated. Regression of bile duct hyperplasia following elimination of the proliferative stimulus is widely recognized, but the mode of elimination of the excess biliary epithelial cells is not understood. Bhathal and Gall (1985) have performed a time course histological study following removal of the proliferative stimulus in two rat models of biliary epithelial cells hyperplasia. They show that decrease of biliary epithelial cells during regression of hyperplasia occurs principally by apoptosis. After common

diseases

and vascular

wall

injury

25

bile duct ligation in the rat, the formation of periportal fibrosis with slight inflammation and necrosis occurs together with the extensive proliferation of bile duct epithelial cells (Tuchweber et al., 1996). Abdel-Aziz et ul. (1990) have shown that hepatic fibrosis induced by experimental extrahepatic cholestasis in rat disappears in less than 3 weeks after relief of bile duct obstruction. They suggest that an active degradation of matrix proteins occurs; furthermore, an apoptotic process could be implicated in the elimination of myofibroblasts participating in the fibrogenesis. This hypothesis is currently under investigations in our laboratory. Apoptosis in other organs Ishizaki et al. (1994) have studied the evolution of granulation tissue in injured rabbit corneas. Cornea1 wound healing leads to the formation of opaque scars consisting of disorganized collagenous matrix. The fibroblastic cells in alkali-burned and lacerated corneas have the characteristics of myofibroblasts. In their study, the number of myofibroblasts in granulation tissue increased reaching a peak 3 weeks after injury and then declined. By electron microscopy and in situ labeling of fragmented DNA, the authors have shown that the regulation of the cellularity during cornea1 wound healing implicated the process of apoptosis. Itoh et al. (1995) have demonstrated that the infarcted myocardial cells that are located among normal myocardial cells or along boundaries between ischemic and non-ischemic areas show signs of apoptosis: these cells may suffer mild hypoxia and have the capacity to undergo a discreet elimination. APOPTOSIS

IN

VASCULAR

WALL

INJURY

Arterial intimal thickening induced in the rat after endothelial denudation by means of a balloon catheter is characterized by smooth muscle cell accumulation into the intima. It represents the most used experimental model for the development of the atheromatous plaque (for review, see Desmouliere and Gabbiani, 1992; Raines and Ross, 1996) restenosis after coronary angioplasty or stenosis after coronary bypass (for review, see Liischer et al., 1993). The endothelial lesion induces migration and proliferation of media smooth muscle cells into the intima (Clowes et al.. 1983). After a few

weeks, replication disappears and endothelial regeneration is achieved (Clowes et rrl.. 1983); moreover, a remodeling of the intimal thickening with an important decrease of smooth muscle cell number takes place (Kocher et ~1.. 1984). The mechanism of this phenomenon has not been clearly explained. By electron microscopy and in situ labeling of fragmented DNA. we have shown that, after an endothelial lesion of the rat aorta, apoptotic smooth muscle cells are observed within the intimal thickening, The number of apoptotic cells becomes important at 15 days after de-endothelialization and reaches a maximum at 20 days; at 45 days, the intimal thickening is re-endothelialized and no more apoptotic smooth muscle cells are detected (Bochaton-Piallat et al.. 1995). It is noteworthy that smooth muscle cell apoptosis appears latet than the mitotic events. Moreover. it mainly takes place in the cells close to the lumen similar to what has been described for mitotic changes (Clowes cl al., 1983), suggesting that highly replicative smooth muscle cells undergo apoptosis. Similar results were obtained in intimal thickening of rat iliac artery induced after endothelial injury, although, in this experiment, apoptotic and mitotic changes were temporally overlapping (Han et al.. 1995). These results indicate that apoptosis is an important mechanism in the regulation of intimal thickening evolution. Several years ago, Takebayashi et rrl. (1972) reported the presence of cells with typical features of apoptosis in human atherosclerosis. Recent works demonstrate the presence of apoptotic cells in primary atherosclerotic lesions (Han et ~zl., 1995; Isner et [I/., 1995), in restenosis after percutaneous atherecthomy (Isner et al., 1995) and in occluded human saphenous vein aorto-coronary grafts obtained during re-intervention bypass grafting (Kockx et ~1.. 1994). In these different situations. smooth muscle cells as well as macrophages undergo apoptosis (Kockx et al.. 1994; Geng and Libby, 1995; Isner et (II., 1995), which could be responsible for plaque rupture (Weissberg rz ~1.. 1996). In a healthy wall artery, apoptosis could regulate cell proliferation and remodeling. Interestingly, Cho et ul. (1995) have demonstrated that apoptosis is involved in the remodeling of the lamb abdominal aorta only after birth. By means of in vitro studies, several groups have attempted to define mechanisms of apoptosis induction in vascular smooth muscle

cells. Bennett et al. (1995a) have demonstrated that smooth muscle cells derived from the media of normal human coronary arteries and aorta undergo apoptosis only when they are cultured in absence of serum: insulin-like growth factor-l and platelet-derived growth factor were identified as serum survival factors. In contrast, smooth muscle cells isolated from coronary plaques (primary lesions) spontaneously die by apoptosis even when they are cultured in high serum concentrations. Treatment with combination of different cytokines secreted by immune cells present in atheromatous plaque (such as interferon-;,. tumor necrosis factor-r and interleukin- 1/i) promotes apoptosis of rat and human smooth muscle cells via nitric oxide-dependent and -independent mechanisms (Geng L’I rrl.. 1996a). Interestingly. rat smooth muscle cells treated with interferon-;! or tumor necrosis factor-r induce apoptosis of leukemic cells by production of nitric oxide (Geng et r/l., 1996b). Genes that regulate apoptosis in vascular smooth muscle cells are now being discovered. Constitutive expression of (‘+ZJY by rat aortic smooth muscle cells induces continuous cell proliferation as well as apoptosis (Bennett et nl., 1993). Although ~-I?~JYinduces apoptosis by increasing ~53 expression. transfection of hc.12 protects cells from death independently of pS3 (Bennett c/ (I/.. 1995b). Expression of hc.12 is also altered by inhibition of protein kinase C‘ (Leszczynski rl al., 1994). Taken together, irl l.itro results show that regulation of proliferation and apoptosis are mediated by closely linked mechanisms and it appears that smooth muscle cell apoptosis involved different pathways (Schwartz and Bennett. 1995). DIRECTIONS

FOR

FUTURE

RESEARCH

Apoptosis plays an opposite and complementary role to mitosis in the maintenance of cellular and tissue homeostasis. Recent evidence suggests that alterations in cell survival contribute to the pathogenesis of a number of human diseases (for review. see Thompson. 1995). The failure of this apoptotic process leads to imbalanced tissue homeostasis and is recognized as a mechanism of carcinogenesis. Alterations in the susceptibility of lymphocytes to die by apoptosis has been reported in several autoimmune diseases (Thompson, 1995). The suicide of an infected cell may be viewed as a defense

Apoptosis

during

wound

healing,

fibrocontractive

process to prevent viral propagation. To circumvent these host defenses, a number of viruses have developed mechanisms to disrupt the regulation of apoptosis within the infected cells. In contrast, some disorders are associated with excess of cell death. In acquired immunodeficiency syndrome, the viral infection is correlated with the depletion of CD4’ T cells. In neurodegenerative disorders (e.g. Alzheimer’s disease, Parkinson’s disease), the cell loss does not induce an inflammatory response and apoptosis appears to be the mechanism of cell death. Disorders of blood cell production, such as myelodysplastic syndrome and some forms of aplastic anemia, are associated with increased apoptotic cell death within the bone marrow. In polycystic kidney diseases (Woo, 1995) and in glomerular sclerosis (Sugiyama et al., 1996), the increase of apoptotic cell number correlated with deterioration of renal function. In the heart, apoptosis is implicated in some normal phenomena, particularly during post-natal morphogenesis, however, it has been suggested that apoptosis may be one of the mechanisms responsible for decrease of cardiac performance (Bennett and Evan, 1994). Apoptosis may be also a mechanism for the development of heart failure during chronic pressure overload (Bing, 1994). In atherosclerotic plaque-derived smooth muscle cells, a high rate of apoptosis is observed, which might lead to further weakening of the plaque (Weissberg et al., 1996). As underlined by James (1994), apoptosis is not always a normal process in the sense of being unfailingly beneficial but it may be involved in the pathogenesis of disease. During the repair of injured tissues, the mass of granulation tissue must be controlled and limited to prevent an anarchic remodeling and the development of fibrosis. Apoptosis has been suggested to be a central mechanism through which repair responses are completed. Tissue injury associated with inflammation is followed by healing if the injurious agent disappears or is inactivated by the host response. The process of healing depends on the type of inflammation, the extent of tissue damage, the cells involved, and the regenerative ability of parenchyma cells. It appears that, when injury is limited, tissue repair occurs with the development of granulation tissue followed by a discreet remodeling implicating an apoptotic process. Moreover, apoptosis is implicated in the resolution of inflammatory cells.

diseases

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27

The question that remains to be answered is, what are the stimuli leading to granulation tissue cell apoptosis during tissue repair and scar formation? Some cytokines and extracellular matrix components are known to modify the fibroblast phenotype. Our in vitro and in vivo results suggest that under normal conditions, the process of myofibroblast differentiation ends with the death of these cells. Thus, myofibroblasts could be considered to be terminally differentiated cells. A possible mechanism for apoptosis induction could be via direct action and/or withdrawal of cytokines. However, cytokines such as tumor necrosis factor-u (Laster et al., 1988) can induce both apoptotic and necrotic forms of cell lysis. Then, it is probable that subtle modifications of the microenvironment, which influence the balance of signals reaching the cell, induce the development of an apoptotic process. The extracellular matrix, which represents a reservoir of factors and which can modulate the activity of signals reaching the cell, plays an important role during apoptosis (Ruoslahti and Reed, 1994). In conclusion, homeostasis is maintained through a balance between cell proliferation and cell death. As in many biological processes, a wide variety of regulatory stimuli influence the decision of a cell to undergo proliferation, quiescence or apoptosis. It is well recognized that apoptosis plays a major role in physiological regulations and recent work suggests that apoptosis contributes to the pathogenesis of a number of human diseases. Further investigations will better define mechanisms inducing apoptosis and will lead to treatments specifically changing the behavior of cells during apoptotic processes in physiological or pathological situations. A~knoIr’ledgemmts~This work was supported by the Centre National de la Recherche Scientifique. the Swiss National Science Foundation, grant No. 31-40372.94, and the Association pour la Recherche sur le Cancer. We thank Mrs I. Berger for photographic work and Mrs M. Vitali for typing the manuscript. REFERENCES Abdel-Aziz G.. Lebeau G., Rescan P.-Y., Clement B., Rissel M., Deugnier Y., Campion J. P. and Guillouzo A. (1990) Reversibility of hepatic fibrosis in experimentally induced cholestasis in rat. Am. J. Pathol. 137, 1333-1342. Baker A. J., Mooney A., Hughes J., Lombardi D., Johnson R. J. and Savill J. (1994) Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. J. Clin. Inresr. 94, 210552116.

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Desmounliere

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