EXPERIMENTAL

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Horseradish

PATHOLOGY

53,81-97 (1%))

Peroxidase as a Permeability Marker in Injured Rat Caudal and Iliac Arteries

GABIUELLA ELEMERAND INSERM

MARY J. OSBORNE-PELLEGRIN'

U7, Hdpital Necker, 161 rue de Shres, 75015 Paris, France

Received February 2, 1990, and in revised form June 6, 1990 The permeability to Horseradish Peroxidase (HRP) of the rat caudal artery at the level of spontaneous lesions was evaluated by electron microscopy and compared with that of lesions experimentally induced by pinching or internal scraping of the caudal and iliac arteries. No HRP reaction product is observed in the extracellular space of the arterial wall when (i) the internal elastic lamina (IEL) and the endothelium are absent, (ii) the IEL is maintained and the endothelium is absent and (iii) the TEL is absent and the endothelium has regenerated. That HRP does enter the arterial wall in cases of gross endothelial damage is shown by its selective retention in damaged smooth muscle cells in such cases. In contrast, HRP reaction product is detected in the subendothelial space when the IEL is maintained and is covered by a regenerating or recently regenerated endothelium. Furthermore, the amount of tracer visualized under the same experimental conditions is greater in the iliac than in the caudal artery. We conclude that the detection of HRP in the subendothelial space of the artery wall requires the presence of regenerating or recently regenerated endothelial cells lying on an intact IEL. It is thus not simply related to endothelial permeability but depends also upon the retention of HRP by extracellular substances. In addition, the quantity of marker retained varies between different sites in the arterial tree. 0 1990 Academic Press, Inc.

INTRODUCTION Increased permeability of the arterial wall has long been considered to be one of the factors involved in the pathogenesis of atherosclerosis. The endothelium is thought to act as a permeability barrier preventing the influx of plasma constituents into the arterial wall. The access of 12?-albumin to the rabbit aortic media has been shown to be greatly increased shortly after experimental deendothelialization (Ramirez et al., 1984). When the artery is experimentally denuded of its endothelial lining, the passage of plasma constituents into the media may contribute to the proliferation of smooth muscle cells and the accumulation of lipids which follow (Bjorkerud and Bondjers, 1971; Schwartz et al., 1975; Fishman et al., 1975; Walton and Morris, 1977; Ross 1986). Horseradish peroxidase (HRP) has often been used as a permeability marker in various experimental conditions, such as hypertension (Giacomelli et al., 1970; Huttner et al., 1973b; Gabbiani et al., 1979; Limas et al., 1980), hypercholesterolemia (Stemerman, 1981) and mechanical injury to the arterial wall (Webster et al., 1974; Clowes et al., 1978). The accumulation of this protein tracer (molecular weight 40,000, diameter about 30 A) in the subendothelial space and in the media has been interpreted as a result of an altered dysfunctional endothelial cell layer with loss of macromolecular barrier function (Gimbrone, 1979; Stemerman, 1981). ’ To whom correspondence and reprint requests should be addressed. 81 0014-4800/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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In this study, we have turned our attention to the use of HRP as a permeability marker in a small muscular artery, the caudal artery, and compared it to a small elastic artery of the same size, the iliac artery. Lesions developing spontaneously with age in the rat caudal artery characterized by a break in the internal elastic lamina (IEL) and damage to the endothelium followed by rapid repair (OsbornePellegrin, 1979; Osborne-Pellegrin and Weill, 1983) appeared to us as a suitable in vivo model to evaluate the permeability role of the endothelium. However, in preliminary experiments we observed that in freshly formed lesions of the rat caudal artery, where gross endothelial disruption occurs on the lesion surface (Osborne-Pellegrin and Weill, 1983), no HRP was detectable within the artery wall. In view of this surprising result, we decided to study this phenomenon in detail, to determine under which conditions HRP may be visualized within the caudal artery wall. Since the spontaneous caudal artery lesions involve both breaks in the IEL and damage to endothelium and are present in any one rat in different stages of repair depending on the age of the lesion, to further our analysis we have compared them to lesions induced experimentally. By external pinching or internal scraping of the artery wall we have tried to induce situations with and without the endothelium and with and without the TEL in order to evaluate the role of these two components in the permeability to HRP. Similar lesions were also induced in the iliac artery for comparison. In addition, in all cases where deendothelialization was likely, another tracer, colloidal carbon, was used for comparison with HRP. MATERIALS

AND METHODS

A total of 220 male Wistar rats were used for this study. For the study of spontaneous lesions of the caudal artery, rats aged 7-8 weeks were used, as at this age many spontaneous lesions form (Osborne-Pellegrin, 1979). For the experimental induction of arterial lesions, rats aged 5 weeks (90-120 g body weight) were used, i.e., before the formation of spontaneous caudal artery lesions. Experimental

Induction

of Arterial

Lesions

(i) Pinching of the artery. After exposure of the artery (caudal or iliac) in rats anaesthetized with sodium methohexital (Brietal; Lilly, 30 mg/kg ip) the artery was pinched lightly for a few seconds with fine metallic tweezers and then released. Several such pinches were made at 5-mm intervals along the proximal 3 cm of the caudal artery or the proximal 1.5 cm of the iliac artery. (ii) Znternal scraping of the artery. After temporary interruption of flow by placing a clamp on the proximal part of the artery, a catheter (PElO, external diameter 0.61 mm) was introduced against the flow into the distal part of the caudal artery or into the external femoral artery and thus into the iliac artery. It was then pushed up inside the caudal or iliac artery and then pulled back and withdrawn. Histological controls showed us that this procedure was sufficient to damage the endothelial lining of the artery. After withdrawal of the catheter, the artery was ligated to prevent bleeding and flow was reestablished via branches. In all cases, after the intervention the wound was closed and the animal left to recover. These lesions were examined 2 h, 24 h, 48 h, 4 days, 8 days, and in some cases 15 days after their experimental induction.

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Markers

Horseradish Peroxidase (Type II, Sigma Chemical Co., USA) at a dose of 5 mg per 100 g body weight in 0.15 ml of saline, or colloidal carbon (Pelikan Cl 1/1431A, Gunther Wagner, Hanover, Germany), 0.1 ml per 100 g body weight was injected into the jugular vein under sodium pentobarbital anaesthesia (40 mg per kg ip) 5 min before fixation of arteries. Fixation

of Arteries

Caudal and iliac arteries were fixed by perfusion of 3% glutaraldehyde in cacodylate buffer (pH 7.4) via a catheter placed in the abdominal aorta in the direction of flow. The vena cava was cut at the onset of perfusion for outflow of fixative. After 5 min of perfusion, caudal or iliac arteries were dissected out and fixed by immersion in the same fixative for a further 5 hr. They were then cut into lengths of 0.5 cm and each piece cut lengthwise in two to permit access of reagents to the endothelium. Blocks were then washed overnight in cacodylate buffer at 4°C. The HRP reaction product was then developed using diaminobenzedine as previously described (Huttner et al., 1973a, b; Gabbiani et al., 1979). Postfixation was performed with 2% 0~0, in sym-collidine buffer (pH 7.4) and the blocks were treated with saturated aqueous uranyl acetate, dehydrated in alcohol and flatembedded in Epon. Sections of l-2 pm thickness were prepared and examined, either unstained or stained with 0.5% toluidine blue in 1% sodium borate, by light microscopy. Thin sections of selected blocks were cut on a Reichert OMU, ultramicrotome and examined, unstained, in Philips EM 200 and 300 microscopes. Zmmunojluorescence Another group of rats underwent the same operative procedures as described above (pinch and scrape). At the required time after the operation, caudal or iliac arteries were perfused with 1% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 5 min. Relevant parts of arteries were dissected out, rapidly frozen, sectioned longitudinally at 4 p,rn in a cryostat, and mounted on clean slides. Caudal arteries of nonoperated rats were processed identically to study spontaneous lesions. Slides were left to dry for 2 hr, washed with phosphate-buffered saline, incubated with rabbit antiserum to rat factor VIII (kindly donated by Drs. Roger E. Benson and W. Jean Dodds, Division of Laboratories and Research, New York State Department of Health, New York), diluted l:lO, for 30 min, rinsed in PBS, and then incubated with the fluorescein-conjugated IgG fraction of goat antiserum to rabbit IgG (Nordic Immunological Laboratories, Tilburg, Holland), diluted 1:20 for 30 min. Controls were performed with normal rabbit serum. Sections were rinsed in PBS and observed in a fluorescence microscope and photos were taken immediately. Scanning Electron Microscopy Spontaneous lesions and some experimental lesions induced by pinching were taken for examination by SEM. Arteries were fixed by perfusion with glutaraldehyde as described above and then cut into 0.5-cm lengths and washed overnight in cacodylate bulfer at 4°C. After postfixation in 0~0, they were dehydrated in acetone, critical point dried with a Polaron E 3000 apparatus (Polaron Equipment

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and examined

using a Scientific

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scanning

RESULTS Low power light photomicrographs illustrating caudal artery with a spontaneous lesion (a), early and late after pinching (b and c), and early after internal scraping (d) are shown in Fig. 1. They will be commented on below in relevant sections, along with the electron micrographs. Control Areas of Caudal and Iliac Arteries HRP reaction product was present only in some plasmalemmal vesicles of the endothelial cells in control areas of caudal and iliac arteries, i.e., outside spontaneous or experimentally induced lesions, where a normal flat endothelium lay on an intact IEL. Occasionally, HRP reaction product was present in naturally occurring intimal thickenings associated with branchpoints. In rats injected with colloidal carbon, no carbon particles were visible either within or on the surface of the arterial wall outside the lesioned area. Spontaneous

Lesions of the Caudal Artery

The morphology of these lesions has been described in detail elsewhere (Osborne-Pellegrin, 1979; Osborne-Pellegrin and Weill, 1983). Briefly, they consist

FIG. 1. Photomicrographs of longitudinal sections of caudal artery, stained with toluidine blue (x230). (a) Spontaneous lesion; note the interruption of the internal elastic lamina (IEL). (b, c, and d) Experimentally induced lesions; (b) 2 hr after pinching; note damaged medial smooth muscle cells on periphery of lesion containing HRP reaction product ( r ) and leukocytes and red blood cells infiltrated into the media in the center of the lesion; (c) 4 days after pinching, where damaged smooth muscle cells and infiltrated leukocytes are no longer visible; (d) 2 hr after internal scraping; note continuity of the IEL and absence of endothelium. L, lumen; M, media; A, adventitia.

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essentially of ruptures in the internal elastic lamina associated with deendothelialization and damage to some smooth muscle cells of the inner media, rapidly followed by repair. Despite some local neosynthesis of collagen and elastic fibers, the interruption of the IEL remains throughout life and thus marks the site of damage (Fig. la). In no case was HRP reaction product observed within the arterial wall at the site of these spontaneous lesions, i.e., where the IEL was absent. This was true whether the lesions were freshly formed (deendothelialized with the luminal surface covered with a layer of platelets and some polymorphonuclear leukocytes (PMNs), Fig. 2a) or repaired (covered with a layer of newly regenerated cells, Fig. 2b). In some repaired lesions traces of HRP reaction product were present at the edge of the lesion between the endothelium and the IEL at the site of rupture. In rats having received an injection of colloidal carbon, no carbon particles were observed in these lesions whether deendothelialized or repaired. Experimentally

Induced

Lesions

Caudal artery pinching. Early (2-24 hr) after pinching the lesion was characterized by a break in the IEL, an absence of endothelium with the luminal surface covered by a layer of platelets and associated PMNs, and various degrees of damage to the smooth muscle cells of the media at the level of the pinch (ranging from rupture of the plasma membrane for the innermost cells of the media to cell edema, mitochondrial swelling, and dilatation of the RER for those on the periphery of the lesion). Infiltration of red blood cells and leukocytes into the media was also observed, which, together with the extensive injury to the medial smooth muscle cells (Fig. lb), enabled us to distinguish these experimentally induced lesions from any spontaneous lesion which might be present. No HRP reaction product was observed in the extracellular space of the arterial wall at the site of the experimental lesion, 2-24 hr after the intervention, despite the lack of both the endothelium and the IEL (Fig. 3a). However, unlike the spontaneous lesion, many damaged smooth muscle cells were diffusely imbued with the HRP reaction product, especially on the periphery of the lesion (Fig. lb). In rats having received colloidal carbon, carbon particles appeared on the luminal surface associated with platelets but were never seen to enter the artery wall despite the absence of the endothelium and the IEL. Later (48 hr-4 days) after pinching, the majority of experimental lesions were repaired, with the luminal surface covered by a layer of enlarged, bulging cells. They contained an oval nucleus, many ribosomes, and abundant RER but few mitochondria. They exhibited prominent filament bundles in their basal cytoplasm, composed of filaments measuring 40-70 A in diameter with a few filaments 100-120 A in diameter scattered between them. The endothelial cells on the edge of the lesion showed the same morphology as the cells covering the lesion surface. Within the lesion where the IEL was absent, no trace of HRP reaction product was visible within the arterial wall (Fig. 3b). However, often on the edge of the lesion where the IEL was present, from 24 hr after the intervention HRP reaction product was visible in the subendothelial space (Fig. 3~). At 48 hr, the smooth muscle cells of the media no longer contained the tracer but the media still contained numerous leukocytes, which had disappeared by 4 days (Fig. lc). At 8 and 15 days after pinching, the whole arterial wall was negative for HRP reaction

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FIG. 2. Electron micrographs of longitudinal sections of spontaneous lesions of the caudal artery in rats having received HRP; (a) recently formed lesion where IEL and endothelium are absent and the luminal surface is lined with a layer of platelets (p) and associated polymorphonuclear leukocytes (PMN) (x 11,000); (b) repaired lesion where luminal surface is lined with regenerated cells (R), (~8000). Note absence of HRP reaction product in the arterial wall. SMC, smooth muscle cell; L, lumen.

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FIG. 3. Electron micrographs of caudal artery lesions induced experimentally by pinching (longitudinal sections) in rats having received HRP; (a) 2 hr after pinching, where endothelium and IEL are absent and the luminal surface is lined with a layer of platelets (p) and polymorphonuclear leukocytes (PMNs), (x7000); (b) 48 hr after pinching where the luminal surface is lined with regenerated cells (R), (X 11,000); (c) edge of lesion 24 hr after pinching, where endothelium (E) lies on an intact internal elastic lamina (IEL) (X 11,000). Note absence of HRP reaction product in the subendothelial space in (a) and (b) and its presence ( t ) in (c). SMC, smooth muscle cell; L, lumen; S, subcndothelial space.

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product. As for rats injected with colloidal carbon, in all cases where cells covered the luminal surface, no traces of carbon were observed in the arterial wall. Cuudal artery after internal scraping. Early (2-24 hr) after scraping, the endothelium was absent from the luminal surface and platelets and leukocytes (mainly PMNs) adhered to the exposed subendothelium. The IEL remained intact (Fig. Id). In some cases the luminal surface was covered with damaged endothelial cells which appeared to be detaching. Some smooth muscle cells of the media showed signs of injury (moderate dilatation of the RER, slight swelling of the mitochondria), most probably due to stretching during the passage of the catheter. In rats having received colloidal carbon, electron dense carbon particles were observed only on the luminal surface, associated with the platelets, and never crossed the IEL to enter the artery wall (Fig. 4a). Despite the absence of endothelial barrier, HRP reaction product was absent from the arterial wall (Fig. 4b) with the exception of some damaged endothelial cells where it was present in the cytoplasm. From 24 to 48 hr after scraping, surface covering cells began to appear and most of the luminal surface was relined with cells by 4 days. These newly regenerated cells were plumper than control endothelium and moderately rich in organelles, containing some ribosomes, RER, and microfilament bundles. Focal deposits of HRP reaction product were present in the subendothelial space (Fig. 4~). By 8 days, this focal presence of tracer in the subendothelial space became less frequent and had disappeared by 15 days, with the exception of a few cases where smooth muscle cell intimal thickening had occurred, in which case HRP reaction product was visible in the intima, in the subendothelial space, and between the intimal smooth muscle cells, In no case where the luminal surface was relined with cells were carbon particles visible in the artery wall in rats where they had been administered. Iliac artery after pinching. Early (2-24 hr) after pinching the lesion site could be recognized by the presence of many injured smooth muscle cells in the media which were diffusely imbued with HRP reaction product. The IEL remained intact but endothelial cells were either damaged and HRP positive or absent. No HRP reaction product was visible in the extracellular space, with the exception of the edges of some lesions 24 hr after injury where the endothelium was present and intense deposits were observed in the subendothelial space. In carboninjected rats, the results were the same as in the caudal artery, i.e., carbon particles did not enter the artery wall where the endothelium was absent but were visible only on the luminal surface between platelets or attached to the platelet surface. Later (48 hrd days), the whole luminal surface had been covered with new cells and intense focal deposits of HRP reaction product were present in the subendothelial space (Fig. 5a). The smooth muscle cells of the media were no longer HRP-positive. The subendothelial deposits of HRP reaction product were less intense at 8 days after injury and were absent 15 days after injury. In carboninjected rats, in all cases where the lesion was repaired, no carbon particles were visible within the artery wall. Iliac artery after scraping. Early (2 hr) after scraping, as in the caudal artery, the endothelium was either absent and replaced by a layer of platelets and associated PMNs or was damaged and detaching, the injured cells containing HRP reaction product. The IEL remained intact. Some smooth muscle cells of the media were injured and likewise imbued with HRP reaction product. As with the

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FIG. 4. Electron micrographs of longitudinal sections of lesions induced experimentally by internal scraping in the caudal artery; (a) 2 hr after scraping in a rat having received colloidal carbon, where endothelium is absent and replaced by a layer of platelets (p) on an intact internal elastic lamina (IEL). Note carbon particles on the huninal surface associated with platelets (p), (X 11,600). (b) Two hours after scraping in a rat having received HRP (X 11,000). Note absence of HRP reaction product in the arterial wall. (c) Twenty-four hours after scraping in a rat having received HRP, where regenerated cells (R) lie on an intact IEL, (X 10,000). Note the presence of small deposits of HRP reaction product in the subendothelial space ( t ). SMC, smooth muscle cell; PMN, polymorphonuclear leukocyte; L, lumen.

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FIG. 5. Electron micrographs of lesions induced experimentally in the iliac artery of rats having received HRP (longitudinal sections); (a) 4 days after pinching (x8fltXl); (b) 48 hr after internal scraping (~16,400). In both cases regenerated cells (R) lie on an intact internal elastic lamina (IEL). Note intense deposits of HRP reaction product in the widened subendothelial space ( t ). SMC, smooth muscle cell; L, lumen.

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caudal artery, in carbon-injected rats, carbon particles were only seen associated with platelets and never within the deendothelialized artery wall. From 24 to 48 hr after scraping, the majority of lesions had been covered by regenerated cells. The subendothelium appeared enlarged when compared to that of controls and contained intense HRP reaction product (Fig. 5b). Occasionally in the inner media HRP reaction product was present in the extracellular space around smooth muscle cells of the synthetic phenotype (containing welldeveloped RER and few myofilaments). Four to eight days after scraping, HRP reaction product was still present in the subendothelium and inner media but in lesser quantities than at 24-48 hr. No carbon particles were visible in repaired lesions after scraping. Zmmunojluorescence In cases where the luminal surface was covered with cells which we consider to have regenerated (late spontaneous lesions or late after pinching or scraping), the question arises as to the nature of these cells. Do they represent regenerated endothelium or luminal smooth muscle cells which have been described in certain cases of extensive experimental deendothelialization (Clowes et al., 1978)? For this reason we performed immunofluorescence studies with rabbit anti-serum to rat factor VIII, which has been previously reported to be present in endothelial cells and not in smooth muscle cells (Hoyer el al., 1973; Clowes ef al., 1978). In the majority of cases of repaired lesions (spontaneous, pinch, or scrape), with or without the presence of the IEL, linear staining was observed along the luminal surface showing that the luminal cells contained factor VIII, indicating that they are endothelial and not smooth muscle cells (Figs. 6a and 6b). No such staining was seen when normal rabbit serum was used (Fig. 6~).

FIG. 6. Immunofluorescent microphotographs of longitudinal frozen sections of caudal artery (x480); (a) at the level of a spontaneous lesion; (b) at the level of a repaired lesion induced by pinching. (a and b) Stained with antibody to rat factor VIII. Note linear fluorescence of cells at the luminal surface within the lesions ( t ). (c) Section stained with normal rabbit serum. Note absence of fluorescence at the luminal surface. L, lumen; M, media.

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Scanning Electron Microscopy

AND OSBORNE-PELLEGRIN

(SEMI

SEM of the luminal surface of spontaneous lesions showed several cases of bulging luminal cells with numerous surface microvilli (Fig. 7), which have been previously described to correspond to proliferating endothelial cells in vivo (De Chastonay et al., 1983) and in vitro (Ausprunk and Berman, 1978). Similar surface morphology was observed in repaired lesions early after pinching. The essential findings of these experiments are summarized in Table 1. It appears that in these two arteries, HRP reaction product is absent from the extracellular space of the artery wall in cases where (i) the IEL and the endothelium are absent (fresh spontaneous lesions or early after pinching in the caudal artery), (ii) the IEL is maintained and the endothelium absent (early after scraping), (iii) the TEL is absent and the endothelium regenerated (repaired spontaneous lesions or late after pinching in the caudal artery). By contrast, HRP reaction product is present in the subendothelial space when the IEL is maintained and is covered by a regenerating or recently regenerated endothelium (edge of spontaneous lesions and those induced by pinching, late after scraping). Furthermore, the amount of tracer visualized under the same experimental conditions is greater in the iliac than in the caudal artery. Colloidal carbon was only visible in cases of deendothelialization, in association with platelets adhering to the luminal surface, and was never seen within the artery wall. DISCUSSION The dose of HRP used in these experiments (5 mg per 100 g body weight) has been shown previously not to enter the subendothelial space in the aorta of the

FIG. 7. Scanning electron micrograph of the luminal surface of the rat caudal artery at the level of a spontaneous lesion. Note luminal cells rich in microvilli ( f ) (X 6000).

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TABLE I Localization of HRP Reaction Product in Caudal and Iliac Arteries as a Function of Presence and Absence of the Endothelium and IEL (Details Are Given in the Results Section) Experimental Caudal artery pinch

Iliac artery pinch Caudal artery scrape Iliac artery scrape Caudal artery spontaneous lesions

model Early (2-24 hr) Late (4 days) Center Edges Early (2-24 hr) Late (4 days) Early (2-24 hr) Late (4 days) Early (2-24 hr) Late (24 days) Early Late Center Edges

Endothelium

IEL

Absent

Absent

HRPin subendothelium -

Present Present Absent Present Absent Present Absent Present Absent

Absent Present Present Present Present Present Present Present Absent

+ ++ + ++ -

Present Present

Absent Present

+

control rat 5 min after the injection, suggesting that the endothelium does indeed, under normal conditions, constitute a relative barrier to the passage of this macromolecule into the arterial wall (Gabbiani et al., 1979). With the same amount of tracer, dense deposits of HRP reaction product have been shown to be present in the subendothelial space of the rat aorta in some types of experimental hypertension, suggesting an increase in endothelial permeability to HRP under these conditions (Huttner et al., 1973b; Gabbiani et al., 1979). In our preliminary experiments, we observed that in the case of freshly formed spontaneous lesions of the caudal artery, despite the absence of endothelium, no HRP reaction product is detectable within the arterial wall. This surprising result led us to investigate further this phenomenon by inducing other lesions experimentally in the caudal artery and also in the iliac artery for comparison. The results of our experiments show that in the caudal and iliac arteries of the rat 5 min after intravenous injection of HRP, HRP reaction product is present in the subendothelial space only in cases where a regenerating or recently regenerated endothelium lies on an intact IEL. Since the endothelium is considered to be a barrier to the passage of macromolecules into the arterial wall, one would expect an increase in permeability when this barrier is missing; i.e., in cases of deendothelialization. In our experiments, in the cases where the endothelium was absent, both in spontaneous and experimentally induced lesions, no HRP reaction product was observed in the extracellular space of the artery wall. The fact that the marker was observed in the cytoplasm of damaged smooth muscle cells nevertheless shows that HRP had penetrated into the arterial wall and that it had been injected in quantities suBicient to be visualized if present. It is well known that damaged cells, whose membrane permeability is increased, become diffusely impregnated with HRP and this fact has prompted some authors to use this as a criterion of cell injury (Geyer et al., 1979). Our inability to detect the marker in the extracellular space in cases of deendothelialization suggests that HRP, which is a rapidly diffusing tracer, is not retained at this site. The possibility that the enzyme has been inactivated

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cannot be ruled out but this is unlikely as HRP reaction product is visible within the adjacent damaged smooth muscle cells. Our previous results suggest that the repaired spontaneous lesions were covered by a regenerated endothelium (Osborne-Pellegrin and Weill, 1983). This conclusion was based on results of autoradiography with tritiated thymidine and on the morphological similarity of the regenerating cells with regenerating endothelium described by others (Schwartz et al., 1975; Fishman et al., 1975; Haudenschild and Schwartz, 1979; Reidy and Schwartz, 1981). Here we present results of immunofluorescence studies which show that these regenerated luminal cells do, in the majority of cases, represent endothelial cells, not only in the spontaneous lesions but also in lesions experimentally induced by pinching and scraping, since the presence of factor VIII distinguishes them from smooth muscle cells (Hoyer et al., 1973; Clowes et al., 1978). In view of the relatively small size of these lesions, it is nor surprising that the endothelium rapidly recovers the denuded surface. Indeed, in the carotid artery, where modified smooth muscle cells have been described as forming a nonthrombogenic luminal covering, much larger areas of endothelium had been damaged (Clowes et al., 1978). Our observations in scanning electron microscopy also support this view as the surface morphology of the regenerated cells resembles that described by Auspnmk and Berman (1978) for bovine endothelial cells in culture which are undergoing spreading and attachment after mitosis. In all cases where a regenerated endothelium was present in the absence of IEL, no trace of HRP reaction product was observed in the artery wall. In contrast, in cases where a regenerating or recently regenerated endothelium lay on an intact IEL, HRP reaction product was present in the subendothelial space, albeit focally and only in small quantities in the caudal artery. The fact that regenerating endothelial cells have been shown to possess poorly formed junctions (Schwartz et al., 1975; Spagnoli er al., 1982), which have been proposed to be leaky (Weinbaum et al., 1985), could explain the arrival of HRP reaction product in the subendothelial space. Indeed, Webster et al., (1974) have shown that newly regenerated endothelial cells show a temporary increase in permeability to HRP. It appears likely, however, from our results that the presence of the tracer is related not only to the existence of regenerating or recently regenerated endothelial cells but also to the simultaneous presence of an intact IEL. The IEL may present a barrier to the further diffusion of HRP into the media as previously suggested for other macromolecules (Smith and Staples, 1980) or the IEL may have some impact upon the metabolic activity or functioning of the overlying endothelial cells. We observed in our experiments, especially in the iliac artery, a widening of the subendothelial space associated with the accumulation of HRP reaction product. A similar observation was made by Gabbiani et al. (1979) in the aorta, where in all cases where HRP reaction product accumulated in the subendothelial space, the thickness of this space was markedly increased. This observation suggests that the endothelial cells might produce an extracellular substance which fills the subendothelial space and is responsible for its enlargement and that this same substance may bind or retain HRP, thus explaining the selective retention of the tracer. It has been shown that endothelial cells in culture can produce proteoglycans, whose principal glycosaminoglycan constituent is heparan sulfate (Oohira et al., 1983) and newly endothelialized arteries in vivo have been shown to contain increased amounts of glycosaminoglycans (Wight et al., 1983; Moore, 1979; Rich-

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ardson et al., 1980). Although these studies relate to longer times after injury, it is still probable that in our experiments the extracellular substance filling the subendothelial space contains glycosaminoglycans. Since glycosaminoglycans are negatively charged molecules (Alavi et al., 1989) and the HRP which we injected is slightly positively charged (PZ = 7.94-8.17) (Davies et al., 1981) electrostatic attraction could thus explain its retention in the subendothelial space. We thus investigated the permeability of the arterial wall to colloidal carbon under the same conditions, since this permeability marker is an uncharged molecule. It also has a much larger diameter than HRP. We observed that in cases of gross endothelial disruption the carbon particles remained on the luminal surface, associated with the platelets, and did not enter the artery wall. Thus, HRP appears to enter the deendothelialized artery wall more readily than colloidal carbon not only because of its smaller diameter compared to carbon but probably also because of its increased charge. It is then retained in certain areas, i.e., in injured cells or in the extracellular space of the intima, presumably by electrostatic attraction. The observation of HRP reaction product in the extracellular space of some naturally occurring, smooth muscle-containing, intimal thickenings at branch points could be related to endothelial dysfunction at sites of increased hemodynamic stress. However, as HRP reaction product was also observed around smooth muscle cells in intimal thickening late after scraping and occasionally in the inner media of the iliac artery after scraping around smooth muscle cells of the synthetic phenotype, it appears possible that in some conditions smooth muscle cells can also synthesize extracellular material which retains HRP. Indeed, it is known that smooth muscle cells can synthesize and release glycosaminoglycans, although not the same molecules as endothelial cells (Wight et al., 1983). In addition, it appears that regenerating endothelium can influence the production of GAGS by the underlying smooth muscle cells (Wight et al., 1983). It was evident from our experiments that HRP reaction product accumulated more readily in the reendothelialized iliac artery than in the reendothelialized caudal artery and that the intensity of HRP reaction product deposits paralleled the widening of the subendothelial space, which was greater in the iliac than in the caudal artery under the same conditions. Other authors have also noted variations in HRP retention in different parts of the arterial tree. In general, the nearer the artery is to the heart, the more HRP accumulates, suggesting that hemodynamic factors may be involved. Huttner et al. (1973a) have shown that the endothelium of the rat abdominal aorta and carotid and iliac arteries is less permeable to HRP than that of the thoracic aorta, and Okuda and Yamamoto (1983) have shown, under different experimental conditions (10 mg HRP/lOO g body weight), that HRP reaction product accumulates in the subendothelial space of the aorta and not in that of the basilar artery of the same animal. Limas et al. (1980) reported that in the SHR the widened subendothelial space associated with HRP deposits was confined to the aorta and disappeared within a few micrometers of the ostium in medium-sized arteries originating in the aorta. In addition, pressure has been shown to modify HRP accumulation. Acute high blood pressure has been shown to increase the passage of HRP across the endothelium in rat thoracic aorta (Huttner et al., 1973b) and other authors report that the perfusion pressure is important in determining the passage of HRP into the arterial wall both in vivo in dogs and post-mortem in human arteries (Goldman et al., 1987). It is thus possible

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that hemodynamic factors may influence both the passage of HRP across the endothelium and also the synthesis and accumulation of subendothelial extracellular material which is liable to retain it. In conclusion, we have shown that the presence of HRP reaction product in the arterial subendothelial space of perfusion-fixed arteries after injection of HRP into the circulation is not simply a question of endothelial permeability but depends also upon the presence of regenerating or recently regenerated endothelial cells lying on an intact IEL and may vary within the same animal according to the type of artery considered. The presence of HRP appears to be related to its retention by newly synthesizedextracellular material present in the widened subendothelial space, probably produced by regenerating, hyperactive endothelial cells. ACKNOWLEDGMENTS This work was supported by the Institut de la Sante et de la Recherche Medicale. We thank the Laboratoire de Biologie, Ministbre de l’Arm6e de IAir, Paris for the use of the scanning electron

REFERENCES ALAVI, M. Z., RICHARDSON, M. and MOORE, S. (1989). The “in vitro” interactions between serum lipoproteins and proteoglycans of the neointima of rabbit aorta after a single balloon catheter injury. Amer. .I. Pathol. 134, 287-294. AUSPRUNK, D. H., and BERMAN, H. J. (1978). Spreading of vascular endothelial cells in culture: Spatial reorganization of cytoplasmic fibers and organelles. Tissue Cell 10, 707-724. BJOKKERUD, S. and BONDJERS, G. (1971). Arterial repair and atherosclerosis after mechanical injury. 1. Permeability and light microscopic characteristics of endothelium in nonatherosclerotic and atherosclerotic lesions. Atherosclerosis 13, 355-363. CLOWES, A. W., COLLAZZO, R. E. and RAKNOVSKY, M. J. (1978). A morphologic and permeability study of luminal smooth muscle cells after arterial injury in the rat. Lab. Invest. 39, 141-150. DAVIES, P. F., RENNKE, H. G., and COTRAN, R. S. (1981). The infIuence of molecular charge upon the endocytosis and intracellular fate of peroxidase activity in cultured arterial endothelium. J. Cell Sci. 49, 69-86. DE CHASTONAY, C., GABBIANI, G., ELEMER, G., and HUTTNER, I. (1983). Remodeling of the rat aortic endothelial layer during experimental hypertension: Changes in replication rate, cell density and surface morphology. Lab. Invest. 48, 45-52. FISHMAN, J. A., RYAN, G. B., and KARNOVSKY, M. J. (1975). Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening. Lab. Invest. 32, 339-351. GABBIANI, G., ELEMER, G., GUELPA, C., VALLOTON, M. B., BADONNEL, M. C., and HUTTNER, I. (1979). Morphologic and functional changes of the aortic intima during experimental hypertension. Amer. J. Pathol. 96, 39p422. GEYER, G., SCHMIDT, H. P., and BIEDERMANN, M. (1979). Horseradish peroxidase as a label of injured cells. Histochemical J. 11, 337-344. GIACOMELLI, F., WEINER, J., and SPIRO, D. (1970). The cellular pathology of experimental hypertension. V. Increased permeability of cerebral arterial vessels. Amer. J. Pathol. 59, 133-160. GIMBRONE, M. A., JR. (1979). Endothelial dysfunction and the pathogenesis of atherosclerosis. In “Atherosclerosis V. Proceedings of the 5th International Symposium.” A. M. Gotto, L. C. Smith, and B. Allen, (Eds.), p. 415, Springer Verlag, New York/Heidelberg/Berlin. GOLDMAN, B., BLANKE, H. and WOLINSKY, H. (1987). Intluence of pressure on permeability of normal and diseased muscular arteries to horseradish peroxidase. Atherosclerosis 65, 215-225. HAUDENSCHILD, C. C., and SCHWARTZ, S. M. (1979). Endothelial regeneration. II. Restitution of endothelial continuity. Lab. Invest. 41, 407-418. HOYER, L. W., DE Los SANTOS, R., and HOYER, J. R. (1973). Antihemophilic factor antigen: Localization in endothelial cells by immunofluorescent microscopy. J Clin. Invest. 52, 2737-2744. HUTTNER, I., BOUTET, M. and MORE, R. H. (1973a). Studies on protein passage through arterial

HRP

IN INJURED

RAT

CAUDAL

AND

ILIAC

ARTERIES

97

endothelium. II. Regional differences in permeability to tine structural protein tracers in arterial endothelium. Lab. Znvesr. 28, 678-685. HU~NER, I., BOIJTET, M., RONA, G., and MORE, R. H. (1973b). Studies on protein passage through arterial endothelium. III. Effect of blood pressure levels on the passage of fine structural protein tracers in arterial endothelium of normotensive rat. Lab. Invest. 29, 536-546. LIMAS, C., WESTRUM, B., and LIMAS, C. J. (1980). The evolution of vascular changes in the spontaneously hypertensive rat. Amer. J. Puthol. 98, 357-384. Mooan, S. (1979). Endothelial injury and atherosclerosis. Exp. Mol. Pathol. 31, 182-190. OKUDA, T., and YAMAMOTO, T. (1983). The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase. Cell Tissue Res. 231, 117-128. OOHIRA, A., WIGHT, T. N., and BORNSTEIN, P. (1983). Sulfated proteoglycans synthesized by vascular endothelial cells in culture. J. Biol. Chem. 258, 2014-2021. OSBORNE-PELLEGRIN, M. J. (1979). “Spontaneous” lesions of the intima in the rat caudal artery. Principal morphologic characteristics and occurance as a function of age and sex. Lab. Invest. 40, 668-677.

OSBORNE-PELLEGRIN, M. J., and WEILL, D. (1983). “Spontaneous” endothelial injury in the rat caudal artery. Exp. Mol. Pathol. 39, 61-79. RAMIREZ, C. A., COLTON, C. K., SMITH, K. A., STEMERMAN, M. B., and LEES, R. S. (1984). Transport of 1Z51-albumin across normal and deendothelialized rabbit thoracic aorta in vivo. Arteriosclerosis 4, 283-29 1. REIDY, M. A., and SCHWARTZ, S. M. (1981). Endothelial regeneration. III. Time course of intimal changes after small defined injury to rat aortic endothelium. Lab. Invest. 44, 301-308. RICHARDSON, M., IHNATOWYCZ, I., and MOORE, S. (1980). Glycosaminoglycan distribution in rabbit aortic wall following balloon catheter de-endothelialization: An ultrastructural study. Lab. Invest. 43, 509-516.

Ross, R. (1986). The pathogenesis of atherosclerosis. N. Engl. J. Med. 314, 488-500. SCHWARTZ, S. M., STEMERMAN, M. B., and BENDITT, E. P. (1975). The aortic intima. II. Repair of aortic lining after mechanical denudation. Amer. J. Pathol. 81, 15-42. SMITH, E. B., and STAPLES, E. M. (1980). Distribution of plasma proteins across human aortic wall. Atherosclerosis

17, 579-590.

SPAGNOLI, L. G., PIETRA, G. G., VILLASCHI, S. and JOHNS, L. W. (1982). Morphometric analysis of gap junctions in regenerating arterial endothelium. Lab. Invest. 46, 139-148. STEMERMAN, M. B. (1981). Effects of moderate hypercholesterolemia on rabbit endothelium. Arteriosclerosis

1, 25-32.

WALTON, K. W., and MORRIS, C. J. (1977). Studies on the passage of plasma proteins across the arterial endothelium in relation to atherogenesis. Prog. Biochem. Pharmacol, 14, 138-152. WEBSTER, W. S., BISHOP, S. P., and GEER, J. C. (1974). Experimental aortic intimal thickening. II. Endothelialization and permeability. Amer. J. Pathol. 76, 265-284. WEINBAUM, S., TZEGHAI, G., GANATOS, P., PFEFFER, R., and CHIEN, S. (1985). Effect of cell turnover and leaky junctions on arterial macromolecular transport. Amer. J. Physiol. 248, H945H960. WIGHT, T. N., CURWEN, K. D., LITRENTA, M. M., ALONSO, D. R., ~~~MINICK, C. R. (1983). Effect of endothelium on glycosaminoglycan accumulation in injured rabbit aorta. Amer. J. Pathol. 113, 156-164.