Haemostasis S: 149-157 (1979)
In vivo and in vitro Studies on Endothelial Regeneration P. Clopath, K. Muller, W. Stäubliand R.R. Burk1 Research Department, Pharmaceuticals Division, Ciba-Geigy Ltd., Basle and Friedrich Miescher Institute, Basle
Key Words. Endothelium • Ballooning • Evans-blue • Glucocorticoids • Hyperlipemia • Migration • 3T3 • Rabbit • Minipig • Atherosclerosis Abstract. Endothelial regeneration was studied in rabbit aorta after intra-arterial balloon catheterization. Most of the regenerated endothelium originated from existing branches which was assessed by the Evans-blue uptake pattern and confirmed by trans mission and scanning electron microscopy. Glucocorticoid treatment enhanced re-endothelialization whereas hyperlipemic diet inhibited. Sera from minipigs fed an atherogenic diet consistently have less ability than sera from control pigs to stimulate in vitro the regeneration of wounded endothelium-like monolayers of 3T3-B cells. The deficiency is probably due to an inhibitor which appears and disappears with changes in the diet.
Introduction
Endothelial cells form the barrier between the circulating blood and the vessel wall of arteries and veins (1). These cells are of importance in physiologic hemostasis, in the control of the passage of blood constituents into the vessel wall, as well as in mediation of a variety of physiologic and pathologic stimuli (2) and in the non-thrombogenicity of the vessel wall. Thus the vital function of blood vessels is largely dependent upon the integrity of the endothelial cell layer ( 3) .
Injury to and subsequent regeneration of the endothelial cells are supposed to be important factors in diseases such as atherosclerosis and thrombosis (4).
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' We thank R. Bachmann, C. Briicher, M. Erard, K. Handloser, G. Steck and J. Suter for their excellent technical assistance.
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Normally, the injured endothelium regenerates quickly and seals off the blood from the subendothelial tissue (5). Atherosclerotic lesions are thought to devel op at sites of persistent loss of endothelium which may occur when the endothelium is repeatedly injured, or when the blood does not support replace ment of endothelium at a sufficient speed (6, 7). Under these conditions, the subendothelial connective tissue is continuously exposed to the blood elements, platelets adhere and release factors which are thought to stimulate migration of smooth muscle cells through the fenestrae of the internal elastic lamina and their proliferation, leading to fibromuscular intimal thickening, lipid accumulation, and, ultimately, atherosclerosis (8, 9). Re-endothelialization appears to limit the development of such intimal hyperplasia (10, 11). In this paper we wish to report on factors influencing endothelial regenera tion and endothelial permeability in vivo as well as on factors influencing cell migration in vitro. The in vivo studies were carried out in rabbits. Changes in endothelial permeability and the extent of endothelial regeneration after mech anical injury were quantitated by intravenous injection of Evans-blue, an azo-dye known to bind to serum albumin (12). This dye-protein complex actively stains areas of enhanced permeability as well as vessels devoid of endothelium (13). Quantification was achieved by spectrophotometry after extraction of the dye with formamide. With this experimental set-up, the effects of glucocorticoid treatment and hyperlipidemia upon intimal healing was studied. Re-endothelialized areas were also studied by transmission and scanning electron microscopy. For the in-vitro studies, we investigated cellular migration of Balb/c 3T3A31 cells, designated by us as 3T3-B, a mouse cell line reported to have endothelial properties (14, 15). Specifically, the repair of mechanically induced defects (‘wounds’) in quiescent monolayers 3T3-B cells was studied in the presence of sera from either normo- or hypercholesterolemic pigs.
In vivo Studies Male chinchilla rabbits were used in this study. They were about 6 months old and weighed approximately 3 kg. Endothelial regeneration was studied in the thoracic aortic segment after the endothelium was abraded by passage of a balloon catheter through the lumen of the aorta according to the method developed by Baumgartner and Studer ( 16). The intact aortic arch and the intact abdominal aorta segments were used to study endothelial permeability. In a first experiment, we examined the influence of hyperlipemia on endothelial regeneration and permeability. The thoracic segment of one group of animals was ballooned
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and the animals were kept on a normal diet. A second group of animals was identically injured but placed on a high cholesterol/high lipid diet, consisting of peanut oil 6%, cholesterol 0.8%, and commercial rabbit chow 93.2%. Re-endothelialization and endothelial permeability were studied 3 weeks’ post ballooning. In a second experiment, the effects of prednisolone treatment on endothelial regenera tion and permeability were studied, whereby the thoracic segment of 2 groups of animals was again denuded of endothelium. The rabbits of the control group had daily oral administration of 1.0 ml/kg of 35% alcohol, whereas the rabbits of the test group were pretreated for 4 days with 2 mg/kg of prednisolone in 1.0 ml/kg of 35% ethanol, then ballooned and treated thereafter again with 2 mg/kg of prednisolone for 3 weeks. All animals were placed on commercial rabbit chow. Quantification o f Endothelial Permeability and Regeneration 2 h prior to autopsy, the animals were injected intravenously with 20 mg/kg of Evans-blue (Fluka), given as a 20-mg/ml solution in sterile saline. The animals were sacrificed by injection of 20-ml air. The whole aorta was immediately removed and carefully freed from adhering fat and connective tissue. The aorta was opened longitudinally, washed in distilled water, separated into the segments: aortic arch, thoracic and abdominal part, weighed and photographed. Each segment was placed in 2 ml of formamide (Merck, reagent grade) and the dye was extracted at 80 °C for 2 h. The amount of Evans-blue taken up by the vessel was quantitated by spectrophotometry [ \ max (Evans-blue/formamide) 623 nm]. Tissue Preparation for Scanning and Transmission Electron Microscopy Single rabbits were sacrificed after the injection of Evans-blue by fixation of animals with 1.25% glutaraldehyde, 1.0% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) under conditions of physiologic pressure. After fixation in situ, the aortae were fixed by immer sion in double strength of the perfusion fixative and post-fixed in 1% OsO„ in 0.1 M cacodylate buffer (pH 7.4). Sections of aortas were dehydrated in graded acetone solutions. For scanning, electron microscopy specimens were critical point dried, mounted, and thereafter sputter-coated with gold. For transmission electron microscopy, the specimens were embedded in Spurr’s low-viscosity embedding medium.
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In vitro Studies Cellular migration of 3T3-B cells was assayed as previously described (17). To assess the effect of sera from either normo- or hyperlipemic pigs on migration activity, two groups of pigs were studied. One group of 12 animals was fed a commercial minipig chow and the second group of 13 animals was placed on an atherogenic diet, consisting of lard 25.6%, peanut oil 2.65%, cholesterol 2.65%, and commercial minipig chow 69.1% (18). After a feeding period of 4 weeks, blood was drawn from each minipig after a,n 18-hour fast from the orbital sinus under general anaesthesia and serum was prepared. In a second experiment, the effects of sera from two pigs on migration activity was determined whereby these animals were first placed on a normal diet for 4 weeks, thereafter on the atherogenic diet for 4 weeks, and then changed again to the control diet for an additional period of 8 weeks. The animals were weekly bled and each time serum was prepared.
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Table I. The effect of hyperlipemia or glucocorticoid treatment on Evans-blue uptake of rabbit aorta Treatment
(a) Control diet Atherogenic diet (3 weeks)
Aortic segment number of animals
aortic arch not ballooned
thoracic aorta ballooned
abdominal aorta not ballooned
4 4
14.9 ± 0.8 20.5 ± 3.6
125.6 ± 8.7 143.0 ± 5.4
18.4 ± 0.9 28.1 ± 1.7
p < 0.05
NS
p < 0.05
27.3 ± 3.6 25.9 ± 6.1
194.9 ± 13.1 129.9 ± 12.7
32.2 ± 3.0 31.7 ± 7.2
NS
p < 0.05
NS
Statistics (b) Placebo 5 Prednisolone 5 2 mg/kg p.o. 4 days’ pretreatment 3 weeks’ post treatment Statistics
Data expressed as uptake of pg Evans-blue/g tissue ± SEM.
Results
Hyperlipemia. Feeding the atherogenic diet for 3 weeks caused enhanced vascular permeability which was assessed by a 30—40% increase in Evans-blue uptake in the undamaged segments of the vessel wall. In addition, we found that the uptake of Evans-blue by the thoracic, ballooned segment was slightly lower on the normal diet than on the atherogenic diet 3 weeks after ballooning (table la). A SEM picture of the boundary between a blue and a white area in a rabbit on the atherogenic diet is shown in figure lb. The blue area at the right clearly lacks the endothelial cell layer (fig. lc) whereas the white area is covered by regenerated endothelium (fig. la).
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In vivo Studies Mechanical Injury. The data in table la for the rabbits on the control diet show that the thoracic segment, the endothelium of which had been mechanical ly removed, took up 7—8 times more Evans-blue than the undamaged arch and abdominal part.
Fig. 1. Surface of a thoracic aortic segment of a rabbit on the atherogenic diet, as visualized by scanning electron microscopy. There is a distinct regenerated endothelium in the white area (a- b), whereas the blue area still lacks endothelium (b-c). Magnification: a, c X 1,200; b X 240.
Effects o f Prednisolone o f Endothelial Permeability and Regeneration. Pred nisolone caused no alteration of the vascular permeability in the aortic arch or the abdominal aorta with intact endothelium. However, the amount of Evansblue taken up by the ballooned thoracic segment was markedly reduced by the steroid (table lb). We take this result as indication that treatment with predni solone enhanced endothelial regeneration after mechanical injury.
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In vitro Studies Influence o f Normo- and Hyperlipemic Pig Serum on Cell Migration. Feeding a cholesterol-rich/lipid-rich diet to minipigs for 4 weeks resulted in a 16-fold increase in total serum cholesterol concentration (1,022 ± 88 m g/100 ml versus 64.5 mg/100 ml). Migration of 3T3-B cells in the presence of 4% serum
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Fig. 2. Effect of changing diet on serum migration activity.
from the normolipemic pigs amounted to 42.3 ± 2.1 cells/mm length of wound, whereas migration activity in the presence of hypercholesterolemic serum was reduced to 17.4 ± 1.9 cells/mm. The effect of changes of the diet on migration activity was studied by taking serum weekly from 2 pigs, which were fed the normal diet for the first 4 weeks, then the atherogenic diet for 4 weeks and again the normal diet for 8 additional weeks. Figure 2 shows the time-course of the average migration activity and serum cholesterol concentration. During the initial period on the normal diet, migration amounted to 15 cells/mm length of wound. Placing the animal on the atherogenic diet reduced the amount of cells crossing the edge artificial wound to about 3 cells/mm. By feeding the animals again a normal diet, the migration activity of these sera returned to normal values. Figure 2 shows, in addition, that the cell migration activity changes concomitantly with the serum cholesterol concentration.
In vivo Studies Endothelial permeability and endothelial regeneration were quantitatively studied by the amount of Evans-blue taken up by the vessel wall. We used the protein-binding azo-dye in our studies because it has been shown that areas of
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pathologically-increased permeability do correspond with areas of enhanced Evans-blue uptake (19). In addition, it has been demonstrated that areas with intact endothelium remain macroscopically unstained, while denuded surfaces present with an intense blue colour (8).
Effects o f Glucocorticoid Treatment. Our results presented here suggest that re-endothelialization after ballooning was enhanced by prednisolone treatment. Manthorpe et al. (23) have published on the effects of glucocorticoids on vascular connective tissue during repair. These authors found that prednisolone inhibited in rabbits intimal thickening and the biosynthesis of non-dialysable [14C]-hydroxyproline. These results seem to fit very well in the hypothesis discussed by Haudenschild (personal commun.) who believes that after endo thelial injury in vivo there is competition between re-endothelialization and proliferation of smooth muscle cells. Glucocorticoid treatment would enhance re-endothelialization because the underlying smooth muscle cell proliferation and their connective tissue synthesizing capacity is delayed by prednisolone. The possibility of competition between smooth muscle cell proliferation and re-endo thelialization also allows an explanation of the resistance to experimental athero sclerosis in pigs with von Willebrand’s disease (24). It has been shown that the platelets of these pigs cannot effectively adhere to the subendothelial surfaces.
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Effects o f Hyperlipemia. Hyperlipemia resulted in increased endothelial permeability of mechanically undamaged aortic segments. This finding is in agreement with many reports in the literature, in particular with the results published by Stefanovitch and Gore (20) who found increased permeability of the aortic wall to 1311-labeled albumin in rabbits during dietary atherogenesis. In addition, Ross and Harker (21) published that chronic hyperlipemia in monkeys caused endothelial cell desquamation. Hyperlipemia might thus be considered as a condition by which the arterial endothelium is chemically injured. After injury of the endothelium with a balloon catheter, re-endothelialization takes place, starting from the branches of the intercostal arteries. We found that intimal healing occurs preferentially along the axis of the aorta. This observation was first described by Schwartz et al. (22) who demonstrated that endothelial cells regenerate faster in the direction parallel to the blood flow than perpendicular to it. The macroscopic picture of white and of blue areas in the thoracic segment (unpublished) together with the figures of the amount of Evans-blue taken up, might be interpreted as retarded re-endothelialization in hyperlipemic rabbits.
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Therefore, the amount of mitogenic factors released from platelets might be reduced, causing fewer smooth muscle cells to proliferate. Delayed smooth muscle cell proliferation would allow enhanced re-endothelialization, leading to less atherosclerosis. In vitro Studies It has been reported that 3T3-B cells in culture show many characteristics of genuine endothelial cells (14, 15). One may therefore assume that a monolayer of 3T3-B cells in a culture dish is a convenient model for intact endothelium. In that case, repopulation of artificial wounds would mimic re-endotheli alization occurring in vivo after in-arterial balloon catheterization. We showed that hyperlipemic serum reduced migration of these cells and also pig aorta endothelial cells (unpublished results) after wounding a confluent monolayer. Of particular interest seems to be the fact that we were able to follow the dietary status of our pigs. Changes in serum lipids were accompanied by changes of cell migration activity. Atherosclerotic lesions are thought to develop at sites of persisting endo thelial damage. If our migration assay in vitro gives a true measure of the ability of the blood to promote regeneration of damaged endothelium in vivo, then it may become possible to turn the migration assay into a diagnostic tool for one important factor causing atherosclerosis.
References 1 Jaffe, E.A.: Endothelial cells and the biology of factor VIII. New Engl. J. Med. 296: 377-383 (1977). 2 Schwartz, C.J.; Gerrity, R.G.; Lewis, L.J.; Chisholm, G.M., and Brotherton, K.N.: Arterial endothelial permeability to macromolecules; in Schettler, Goto, Hata and
4 5 6 7
Baumgartner, H.R.: Zur Pathogenese der Atherosklerose. Schweiz, med. Wschr. 107: 717-722 (1977). Ross, R. and Glomset, J.A.: The pathogenesis of atherosclerosis. N. Engl. J. Med. 295: 420-425 (1976). Harker, LA.; Ross, R., and Glomset, J.: Role of the platelet in atherogcnesis. Ann. N.Y. Acad. Sei. 275: 321-329 (1976). Ross, R.: Atherosclerosis: the role of endothelial injury, smooth muscle proliferation and platelet factors. Triangle 15: 45-51 (1975). Sholley, M.M.; Gimbrone, M.A., and Cotran, R.S.: Cellular migration and replication in endothelial regeneration. Lab. Invest. 36: 18-25 (1977).
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K lose, A th ero sclero sis V; pp. 1 - 1 1 (Springer, Berlin 1977).
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Minick, C.R.; Stemerman, M.B., and Insull, W.: Effect of regenerated endothelium on lipid accumulation in the arterial wall. Proc. natn. Acad. Sci. 74: 1724-1728 (1977). Clopath, P.: Arteriosclerotic and atherosclerotic lesions induced in swine by single and repeated endothelial cell injury. Artery 4: 275-288 (1978). Schwartz, S.M.; Stemerman, M.B., and Benditt, E.P.: The aortic intima. Repair of the aortic lining after mechanical denudation. Am. J. Pathol. 81: 15-42 (1975). Stemerman, M.B. and R,oss, R.: Experimental arteriosclerosis. I. Fibrous plaque formation in primates, an electron microscope study. J. exp. Med. 136: 769-789 (1972). Collatz Christensen, B.; Chemnitz, J.; Tkocz, I., and Blaabjerg, O.: Repair in arterial tissue. Acta path, microbiol. scand. Sect. A 85: 297-310 (1977). Caplan, B.A.; Gerrity, R.G., and Schwartz, C.J.: Endothelial cell morphology in focal areas of in vivo Evans-blue uptake in the young pig aorta. Expl molec. Path. 21: 102-117 (1974). Boone, C.W.; Takeichi, N.; Paranjpe, M., and Gilden, R.: Vasoformative sarcomas arising from BALB/3T3 cells attached to solid substrates. Cancer Res. 36: 1626-1633 (1976). Porter, K.R.;Todaro, G.J., and Fonte, V.A.: A scanning electron microscope study of surface features of viral and spontaneous transformants of mouse BALB/3T3 cells. J. Cell Biol. 59: 633 642 (1973). Baumgartner, H.R. and Studer, A.: Gezielte Oberdehnung der Aorta abdominalis am normo- und hypercholesterinamischen Kaninchen. Pathol. Microbiol. 26: 129-148 (1963). Burk, R.R.: A factor from a transformed cell line that affects cell migration. Proc. natn. Acad. Sci. USA 70: 36 9 - 3 72 (19 73). Clopath, P.: Rapid production of advanced atherosclerosis in swine. Artery 3: 429-438(1977). Gerrity, R.G.; Richardson, M.; Somer, J.B.; Bell, F.B., and Schwartz, C.J.: Endothelial cell morphology in areas of in vivo Evans-blue uptake in the aorta of young pigs. Am. J. Pathol. 89: 313-334 (1977). Stefanovitch, V. and Gore, I.: Cholesterol diet and permeability of rabbit aorta. Expl molec. Path. 14: 20-29(1971). Ross, R. and Harker, L.: Hyperlipidemia and atherosclerosis. Science, N.Y. 193: 1094-1100 (1976). Schwartz, S.M.; Haudenschild, C.C., and Eddy, E.M.: Endothelial regeneration. Lab. Invest. 38: 568-580 (1978). Manthorpe, R.; Garbarsch, C.; Kofod, B., and Lorenzen, I.: Glucocorticoid effects on vascular connective tissue during repair. Importance of dose level and pre- and post-injury treatment. Acta endocr., Copenh. 86: 437-448 (1977). Fuster, V.; Bowie, E.J.W.; Lewis, J.C.; Fass, D.N.; Owen, C.A., and Brown, A.L.: Resistance to arteriosclerosis in pigs with von Willebrand’s disease. J. clin. Invest. 61: 722-730 (1978).
P. Clopath, Ciba-Geigy Ltd., CH-4002 Basle (Switzerland) Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/17/2019 10:16:51 AM
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In vivo and in vitro Studies on Endothelial Regeneration P. Clopath, K. Muller, W. Stäubliand R.R. Burk1 Research Department, Pharmaceuticals Division, Ciba-Geigy Ltd., Basle and Friedrich Miescher Institute, Basle
Key Words. Endothelium • Ballooning • Evans-blue • Glucocorticoids • Hyperlipemia • Migration • 3T3 • Rabbit • Minipig • Atherosclerosis Abstract. Endothelial regeneration was studied in rabbit aorta after intra-arterial balloon catheterization. Most of the regenerated endothelium originated from existing branches which was assessed by the Evans-blue uptake pattern and confirmed by trans mission and scanning electron microscopy. Glucocorticoid treatment enhanced re-endothelialization whereas hyperlipemic diet inhibited. Sera from minipigs fed an atherogenic diet consistently have less ability than sera from control pigs to stimulate in vitro the regeneration of wounded endothelium-like monolayers of 3T3-B cells. The deficiency is probably due to an inhibitor which appears and disappears with changes in the diet.
Introduction
Endothelial cells form the barrier between the circulating blood and the vessel wall of arteries and veins (1). These cells are of importance in physiologic hemostasis, in the control of the passage of blood constituents into the vessel wall, as well as in mediation of a variety of physiologic and pathologic stimuli (2) and in the non-thrombogenicity of the vessel wall. Thus the vital function of blood vessels is largely dependent upon the integrity of the endothelial cell layer ( 3) .
Injury to and subsequent regeneration of the endothelial cells are supposed to be important factors in diseases such as atherosclerosis and thrombosis (4).
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' We thank R. Bachmann, C. Briicher, M. Erard, K. Handloser, G. Steck and J. Suter for their excellent technical assistance.
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Normally, the injured endothelium regenerates quickly and seals off the blood from the subendothelial tissue (5). Atherosclerotic lesions are thought to devel op at sites of persistent loss of endothelium which may occur when the endothelium is repeatedly injured, or when the blood does not support replace ment of endothelium at a sufficient speed (6, 7). Under these conditions, the subendothelial connective tissue is continuously exposed to the blood elements, platelets adhere and release factors which are thought to stimulate migration of smooth muscle cells through the fenestrae of the internal elastic lamina and their proliferation, leading to fibromuscular intimal thickening, lipid accumulation, and, ultimately, atherosclerosis (8, 9). Re-endothelialization appears to limit the development of such intimal hyperplasia (10, 11). In this paper we wish to report on factors influencing endothelial regenera tion and endothelial permeability in vivo as well as on factors influencing cell migration in vitro. The in vivo studies were carried out in rabbits. Changes in endothelial permeability and the extent of endothelial regeneration after mech anical injury were quantitated by intravenous injection of Evans-blue, an azo-dye known to bind to serum albumin (12). This dye-protein complex actively stains areas of enhanced permeability as well as vessels devoid of endothelium (13). Quantification was achieved by spectrophotometry after extraction of the dye with formamide. With this experimental set-up, the effects of glucocorticoid treatment and hyperlipidemia upon intimal healing was studied. Re-endothelialized areas were also studied by transmission and scanning electron microscopy. For the in-vitro studies, we investigated cellular migration of Balb/c 3T3A31 cells, designated by us as 3T3-B, a mouse cell line reported to have endothelial properties (14, 15). Specifically, the repair of mechanically induced defects (‘wounds’) in quiescent monolayers 3T3-B cells was studied in the presence of sera from either normo- or hypercholesterolemic pigs.
In vivo Studies Male chinchilla rabbits were used in this study. They were about 6 months old and weighed approximately 3 kg. Endothelial regeneration was studied in the thoracic aortic segment after the endothelium was abraded by passage of a balloon catheter through the lumen of the aorta according to the method developed by Baumgartner and Studer ( 16). The intact aortic arch and the intact abdominal aorta segments were used to study endothelial permeability. In a first experiment, we examined the influence of hyperlipemia on endothelial regeneration and permeability. The thoracic segment of one group of animals was ballooned
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Material and Methods
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and the animals were kept on a normal diet. A second group of animals was identically injured but placed on a high cholesterol/high lipid diet, consisting of peanut oil 6%, cholesterol 0.8%, and commercial rabbit chow 93.2%. Re-endothelialization and endothelial permeability were studied 3 weeks’ post ballooning. In a second experiment, the effects of prednisolone treatment on endothelial regenera tion and permeability were studied, whereby the thoracic segment of 2 groups of animals was again denuded of endothelium. The rabbits of the control group had daily oral administration of 1.0 ml/kg of 35% alcohol, whereas the rabbits of the test group were pretreated for 4 days with 2 mg/kg of prednisolone in 1.0 ml/kg of 35% ethanol, then ballooned and treated thereafter again with 2 mg/kg of prednisolone for 3 weeks. All animals were placed on commercial rabbit chow. Quantification o f Endothelial Permeability and Regeneration 2 h prior to autopsy, the animals were injected intravenously with 20 mg/kg of Evans-blue (Fluka), given as a 20-mg/ml solution in sterile saline. The animals were sacrificed by injection of 20-ml air. The whole aorta was immediately removed and carefully freed from adhering fat and connective tissue. The aorta was opened longitudinally, washed in distilled water, separated into the segments: aortic arch, thoracic and abdominal part, weighed and photographed. Each segment was placed in 2 ml of formamide (Merck, reagent grade) and the dye was extracted at 80 °C for 2 h. The amount of Evans-blue taken up by the vessel was quantitated by spectrophotometry [ \ max (Evans-blue/formamide) 623 nm]. Tissue Preparation for Scanning and Transmission Electron Microscopy Single rabbits were sacrificed after the injection of Evans-blue by fixation of animals with 1.25% glutaraldehyde, 1.0% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) under conditions of physiologic pressure. After fixation in situ, the aortae were fixed by immer sion in double strength of the perfusion fixative and post-fixed in 1% OsO„ in 0.1 M cacodylate buffer (pH 7.4). Sections of aortas were dehydrated in graded acetone solutions. For scanning, electron microscopy specimens were critical point dried, mounted, and thereafter sputter-coated with gold. For transmission electron microscopy, the specimens were embedded in Spurr’s low-viscosity embedding medium.
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In vitro Studies Cellular migration of 3T3-B cells was assayed as previously described (17). To assess the effect of sera from either normo- or hyperlipemic pigs on migration activity, two groups of pigs were studied. One group of 12 animals was fed a commercial minipig chow and the second group of 13 animals was placed on an atherogenic diet, consisting of lard 25.6%, peanut oil 2.65%, cholesterol 2.65%, and commercial minipig chow 69.1% (18). After a feeding period of 4 weeks, blood was drawn from each minipig after a,n 18-hour fast from the orbital sinus under general anaesthesia and serum was prepared. In a second experiment, the effects of sera from two pigs on migration activity was determined whereby these animals were first placed on a normal diet for 4 weeks, thereafter on the atherogenic diet for 4 weeks, and then changed again to the control diet for an additional period of 8 weeks. The animals were weekly bled and each time serum was prepared.
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Table I. The effect of hyperlipemia or glucocorticoid treatment on Evans-blue uptake of rabbit aorta Treatment
(a) Control diet Atherogenic diet (3 weeks)
Aortic segment number of animals
aortic arch not ballooned
thoracic aorta ballooned
abdominal aorta not ballooned
4 4
14.9 ± 0.8 20.5 ± 3.6
125.6 ± 8.7 143.0 ± 5.4
18.4 ± 0.9 28.1 ± 1.7
p < 0.05
NS
p < 0.05
27.3 ± 3.6 25.9 ± 6.1
194.9 ± 13.1 129.9 ± 12.7
32.2 ± 3.0 31.7 ± 7.2
NS
p < 0.05
NS
Statistics (b) Placebo 5 Prednisolone 5 2 mg/kg p.o. 4 days’ pretreatment 3 weeks’ post treatment Statistics
Data expressed as uptake of pg Evans-blue/g tissue ± SEM.
Results
Hyperlipemia. Feeding the atherogenic diet for 3 weeks caused enhanced vascular permeability which was assessed by a 30—40% increase in Evans-blue uptake in the undamaged segments of the vessel wall. In addition, we found that the uptake of Evans-blue by the thoracic, ballooned segment was slightly lower on the normal diet than on the atherogenic diet 3 weeks after ballooning (table la). A SEM picture of the boundary between a blue and a white area in a rabbit on the atherogenic diet is shown in figure lb. The blue area at the right clearly lacks the endothelial cell layer (fig. lc) whereas the white area is covered by regenerated endothelium (fig. la).
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In vivo Studies Mechanical Injury. The data in table la for the rabbits on the control diet show that the thoracic segment, the endothelium of which had been mechanical ly removed, took up 7—8 times more Evans-blue than the undamaged arch and abdominal part.
Fig. 1. Surface of a thoracic aortic segment of a rabbit on the atherogenic diet, as visualized by scanning electron microscopy. There is a distinct regenerated endothelium in the white area (a- b), whereas the blue area still lacks endothelium (b-c). Magnification: a, c X 1,200; b X 240.
Effects o f Prednisolone o f Endothelial Permeability and Regeneration. Pred nisolone caused no alteration of the vascular permeability in the aortic arch or the abdominal aorta with intact endothelium. However, the amount of Evansblue taken up by the ballooned thoracic segment was markedly reduced by the steroid (table lb). We take this result as indication that treatment with predni solone enhanced endothelial regeneration after mechanical injury.
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In vitro Studies Influence o f Normo- and Hyperlipemic Pig Serum on Cell Migration. Feeding a cholesterol-rich/lipid-rich diet to minipigs for 4 weeks resulted in a 16-fold increase in total serum cholesterol concentration (1,022 ± 88 m g/100 ml versus 64.5 mg/100 ml). Migration of 3T3-B cells in the presence of 4% serum
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Fig. 2. Effect of changing diet on serum migration activity.
from the normolipemic pigs amounted to 42.3 ± 2.1 cells/mm length of wound, whereas migration activity in the presence of hypercholesterolemic serum was reduced to 17.4 ± 1.9 cells/mm. The effect of changes of the diet on migration activity was studied by taking serum weekly from 2 pigs, which were fed the normal diet for the first 4 weeks, then the atherogenic diet for 4 weeks and again the normal diet for 8 additional weeks. Figure 2 shows the time-course of the average migration activity and serum cholesterol concentration. During the initial period on the normal diet, migration amounted to 15 cells/mm length of wound. Placing the animal on the atherogenic diet reduced the amount of cells crossing the edge artificial wound to about 3 cells/mm. By feeding the animals again a normal diet, the migration activity of these sera returned to normal values. Figure 2 shows, in addition, that the cell migration activity changes concomitantly with the serum cholesterol concentration.
In vivo Studies Endothelial permeability and endothelial regeneration were quantitatively studied by the amount of Evans-blue taken up by the vessel wall. We used the protein-binding azo-dye in our studies because it has been shown that areas of
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pathologically-increased permeability do correspond with areas of enhanced Evans-blue uptake (19). In addition, it has been demonstrated that areas with intact endothelium remain macroscopically unstained, while denuded surfaces present with an intense blue colour (8).
Effects o f Glucocorticoid Treatment. Our results presented here suggest that re-endothelialization after ballooning was enhanced by prednisolone treatment. Manthorpe et al. (23) have published on the effects of glucocorticoids on vascular connective tissue during repair. These authors found that prednisolone inhibited in rabbits intimal thickening and the biosynthesis of non-dialysable [14C]-hydroxyproline. These results seem to fit very well in the hypothesis discussed by Haudenschild (personal commun.) who believes that after endo thelial injury in vivo there is competition between re-endothelialization and proliferation of smooth muscle cells. Glucocorticoid treatment would enhance re-endothelialization because the underlying smooth muscle cell proliferation and their connective tissue synthesizing capacity is delayed by prednisolone. The possibility of competition between smooth muscle cell proliferation and re-endo thelialization also allows an explanation of the resistance to experimental athero sclerosis in pigs with von Willebrand’s disease (24). It has been shown that the platelets of these pigs cannot effectively adhere to the subendothelial surfaces.
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Effects o f Hyperlipemia. Hyperlipemia resulted in increased endothelial permeability of mechanically undamaged aortic segments. This finding is in agreement with many reports in the literature, in particular with the results published by Stefanovitch and Gore (20) who found increased permeability of the aortic wall to 1311-labeled albumin in rabbits during dietary atherogenesis. In addition, Ross and Harker (21) published that chronic hyperlipemia in monkeys caused endothelial cell desquamation. Hyperlipemia might thus be considered as a condition by which the arterial endothelium is chemically injured. After injury of the endothelium with a balloon catheter, re-endothelialization takes place, starting from the branches of the intercostal arteries. We found that intimal healing occurs preferentially along the axis of the aorta. This observation was first described by Schwartz et al. (22) who demonstrated that endothelial cells regenerate faster in the direction parallel to the blood flow than perpendicular to it. The macroscopic picture of white and of blue areas in the thoracic segment (unpublished) together with the figures of the amount of Evans-blue taken up, might be interpreted as retarded re-endothelialization in hyperlipemic rabbits.
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Therefore, the amount of mitogenic factors released from platelets might be reduced, causing fewer smooth muscle cells to proliferate. Delayed smooth muscle cell proliferation would allow enhanced re-endothelialization, leading to less atherosclerosis. In vitro Studies It has been reported that 3T3-B cells in culture show many characteristics of genuine endothelial cells (14, 15). One may therefore assume that a monolayer of 3T3-B cells in a culture dish is a convenient model for intact endothelium. In that case, repopulation of artificial wounds would mimic re-endotheli alization occurring in vivo after in-arterial balloon catheterization. We showed that hyperlipemic serum reduced migration of these cells and also pig aorta endothelial cells (unpublished results) after wounding a confluent monolayer. Of particular interest seems to be the fact that we were able to follow the dietary status of our pigs. Changes in serum lipids were accompanied by changes of cell migration activity. Atherosclerotic lesions are thought to develop at sites of persisting endo thelial damage. If our migration assay in vitro gives a true measure of the ability of the blood to promote regeneration of damaged endothelium in vivo, then it may become possible to turn the migration assay into a diagnostic tool for one important factor causing atherosclerosis.
References 1 Jaffe, E.A.: Endothelial cells and the biology of factor VIII. New Engl. J. Med. 296: 377-383 (1977). 2 Schwartz, C.J.; Gerrity, R.G.; Lewis, L.J.; Chisholm, G.M., and Brotherton, K.N.: Arterial endothelial permeability to macromolecules; in Schettler, Goto, Hata and
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Baumgartner, H.R.: Zur Pathogenese der Atherosklerose. Schweiz, med. Wschr. 107: 717-722 (1977). Ross, R. and Glomset, J.A.: The pathogenesis of atherosclerosis. N. Engl. J. Med. 295: 420-425 (1976). Harker, LA.; Ross, R., and Glomset, J.: Role of the platelet in atherogcnesis. Ann. N.Y. Acad. Sei. 275: 321-329 (1976). Ross, R.: Atherosclerosis: the role of endothelial injury, smooth muscle proliferation and platelet factors. Triangle 15: 45-51 (1975). Sholley, M.M.; Gimbrone, M.A., and Cotran, R.S.: Cellular migration and replication in endothelial regeneration. Lab. Invest. 36: 18-25 (1977).
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Minick, C.R.; Stemerman, M.B., and Insull, W.: Effect of regenerated endothelium on lipid accumulation in the arterial wall. Proc. natn. Acad. Sci. 74: 1724-1728 (1977). Clopath, P.: Arteriosclerotic and atherosclerotic lesions induced in swine by single and repeated endothelial cell injury. Artery 4: 275-288 (1978). Schwartz, S.M.; Stemerman, M.B., and Benditt, E.P.: The aortic intima. Repair of the aortic lining after mechanical denudation. Am. J. Pathol. 81: 15-42 (1975). Stemerman, M.B. and R,oss, R.: Experimental arteriosclerosis. I. Fibrous plaque formation in primates, an electron microscope study. J. exp. Med. 136: 769-789 (1972). Collatz Christensen, B.; Chemnitz, J.; Tkocz, I., and Blaabjerg, O.: Repair in arterial tissue. Acta path, microbiol. scand. Sect. A 85: 297-310 (1977). Caplan, B.A.; Gerrity, R.G., and Schwartz, C.J.: Endothelial cell morphology in focal areas of in vivo Evans-blue uptake in the young pig aorta. Expl molec. Path. 21: 102-117 (1974). Boone, C.W.; Takeichi, N.; Paranjpe, M., and Gilden, R.: Vasoformative sarcomas arising from BALB/3T3 cells attached to solid substrates. Cancer Res. 36: 1626-1633 (1976). Porter, K.R.;Todaro, G.J., and Fonte, V.A.: A scanning electron microscope study of surface features of viral and spontaneous transformants of mouse BALB/3T3 cells. J. Cell Biol. 59: 633 642 (1973). Baumgartner, H.R. and Studer, A.: Gezielte Oberdehnung der Aorta abdominalis am normo- und hypercholesterinamischen Kaninchen. Pathol. Microbiol. 26: 129-148 (1963). Burk, R.R.: A factor from a transformed cell line that affects cell migration. Proc. natn. Acad. Sci. USA 70: 36 9 - 3 72 (19 73). Clopath, P.: Rapid production of advanced atherosclerosis in swine. Artery 3: 429-438(1977). Gerrity, R.G.; Richardson, M.; Somer, J.B.; Bell, F.B., and Schwartz, C.J.: Endothelial cell morphology in areas of in vivo Evans-blue uptake in the aorta of young pigs. Am. J. Pathol. 89: 313-334 (1977). Stefanovitch, V. and Gore, I.: Cholesterol diet and permeability of rabbit aorta. Expl molec. Path. 14: 20-29(1971). Ross, R. and Harker, L.: Hyperlipidemia and atherosclerosis. Science, N.Y. 193: 1094-1100 (1976). Schwartz, S.M.; Haudenschild, C.C., and Eddy, E.M.: Endothelial regeneration. Lab. Invest. 38: 568-580 (1978). Manthorpe, R.; Garbarsch, C.; Kofod, B., and Lorenzen, I.: Glucocorticoid effects on vascular connective tissue during repair. Importance of dose level and pre- and post-injury treatment. Acta endocr., Copenh. 86: 437-448 (1977). Fuster, V.; Bowie, E.J.W.; Lewis, J.C.; Fass, D.N.; Owen, C.A., and Brown, A.L.: Resistance to arteriosclerosis in pigs with von Willebrand’s disease. J. clin. Invest. 61: 722-730 (1978).
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