273
Atherosclerosis, 30 (1978) 273-284 @ Elsevier/North-Holland Scientific
Publishers,
Ltd.
FATTY ACIDS AND THE INITIAL EVENTS OF ENDOTHELIAL DAMAGE SEEN BY SCANNING AND TRANSMISSION ELECTRON MICROSCOPY A.W. SEDAR,
M.J. SILVER,
J.J. KOCSIS and J.B. SMITH
Department of Anatomy and Pharmacology and Cardeza Foundation, University, Philadelphia, PA 19107 (U.S.A.) (Received (Revised, (Accepted
Thomas Jefferson
30 January, 1978) received 7 April, 1978) 19 April, 1978)
A method was developed for observing changes in the endothelial cells in rabbit ear veins in vivo by scanning electron microscopy. Injection of fatty acids into the ear vein caused damage to the endothelium. The first signs of damage seen were marked bulges in the nuclei and loss of the rhomboidal shape of the endothelial cells. More severe damage included loss of nuclei, .leaving holes in the cytoplasm. Some parts of the damaged endothelium showed complete separation of cells from each other and exposure of sub-endothelial tissue to which platelets with pseudopodia were adhering. Damage to the endotheliurn was produced by arachidonic, linoleic, r-linolenic, 8,11,14-eicosatrienoic, 5,8,11,14,-eicosatetraenoic or 15-hydroperoxy-5,8,11,13_eicosatetraenoic acids. The effect of arachidonic acid was not prevented by pre-treating the animals with aspirin. It appears that damage produced by the fatty acids is nonspecific. Key words:
Endothelium
- Fatty acids - Scanning and transmission electron microscopy
Introduction Recent studies of the early stage of atherogenesis in rabbits maintained on atherogenic diets [ 1,2] and pigeons with spontaneously developing atherosclerosis [3] have revealed distinct morphological changes in the endothelial cells of the affected blood vessel walls. Areas of vessel wall were reported to contain irregularly shaped cells with an increased incidence of stigmata and stomata. Other changes included ruffling of luminal plasma membranes, endothelial desquamation and the attachment of platelets to subendothelial tissue and apparently to damaged endothelial cells. Such changes are considered to be amongst the “earliest identifiable events in atherogenesis in the pigeon” [3]. We have
274
developed a simple model system in which changes in the endothelial cells of rabbit ear veins can be rapidly produced by different agents and observed by scanning electron microscopy. In our initial studies we chose to investigate the effects of fatty acids on the endothelium. Materials and Methods Fatty acids (>99% pure) were obtained from Nuchek Prep., Inc., Elysian, MI. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid was prepared by incubating arachidonic acid with soybean lipoxygenase (Sigma Chem. Co., St. Louis, MO) under an atmosphere of oxygen. 5,8,11,14-Eicosatetraenoic acid was a gift from P. Whitman, Hoffman La Roche, Nutley, NJ. Solutions of the sodium salts were made by dissolving the free fatty acids in sodium carbonate solution as described elsewhere [ 41. Aspirin was obtained from Merck and Co., Rahway, NJ. Rabbits employed were male New Zealand whites, weighing between 2.5 and 3.5 kg. All injections of test substances or of vehicle into the lateral ear vein were made at the rate of 1 ml/min via an E-Z set, infusion set with a 23gauge needle (Deseret Pharmaceutical Co., Inc., Sandy, UT). Rabbits were anesthetized by injecting 2-3 ml of a 50 mg/ml sodium pentobarbital solution (Nembutal, Abbott Laboratories, North Chicago, IL) into the lateral ear vein of the opposite ear. Initial preparation of blood vessels for scanning electron microscopy @EM) Three min after the injection of the solution of fatty acid or the vehicle, the ear vein was clamped distal to the site of the venipuncture. An infusion of Tyrode’s solution (pH 7.4) into the ear vein was then begun. The vessel was immediately cut with a sharp scalpel blade at the point where it descends into the base of the ear and a clamp was placed across the central artery. The infusion of Tyrode’s solution was continued at a rate maintained to just keep the vessel distended. In most cases, this was about 2 ml/min. This infusion of Tyrode’s solution allowed for a gentle washing out of the residual blood in the vein. When this was accomplished, a clear effluent was seen to emerge at the cut end. In some cases, it was necessary to cut small side branches entering the vein. Following the wash with Tyrode’s solution, 1% glutaraldehyde in Tyrode’s solution was infused in similar fashion. After the infusion of 10 ml of the glutaraldehyde solution, the ear vein, full of glutaraldehyde solution, was clamped just above the cut end and just below the site of injection. The ear tissue (30-40 mm long) containing the vessel between the clamps was then cut out. Further preparation of the vessel for SEM The tissue containing the vessel was fixed overnight in 1% glutaraldehyde solution. The following day the specimen was pinned to a wax plate. The skin was then carefully removed from one surface of the vessel under a dissecting microscope. The upper half of the vessel was removed with an iridectomy scissors cutting longitudinally along each side wall of connective tissue. This technique provided a half cylinder of the vessel embedded in skin with its endothelial-luminal surface exposed. The specimen was divided into strips of skin approximately 10 mm long and fixation continued overnight in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. After rinsing 3 times with the
275
same buffer, the specimens were fixed for 1 h in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. To reduce charging of the specimens, more osmium was incorporated using a modification of the ligand binding method of Kelley et al. [ 51. This consisted of a thorough rinsing (5-10 times) for 15 min in buffer, incubation for 10 min in 1% filtered aqueous thiocarbohydrazide solution, a rinse for 15 min in several changes of distilled water and postfixation in 1% aqueous osmium tetroxide for 1 h. The specimens were washed several times in distilled water over a 15-min period before dehydration in ethanol. The absolute ethanol was replaced with ethanol : amyl acetate 50 : 50 (v : v) and then with absolute amyl acetate. Specimens were dried by the critical point method using liquid CO? as the intermediate fluid. They were then vacuum coated with 10 nm of carbon and 150 nm of gold before examination in the JEOL-JSM-50A scanning electron microscope at magnifications of X 400 to 10,000. Results (1) Control studies For controls, 1 ml of saline solution or 25 mM sodium carbonate solution (the vehicle for the fatty acids studied below) was injected into the lateral ear vein of 6 rabbits. In all cases, the endothelial lining of the vessel wall appeared as follows: The endothelial cells had a rhomboidal shape, with long axis in the direction of blood flow (Fig. 1). The center of all cells exhibited nuclear bulges (Fig. 2). The margins between adjacent cells exhibited irregular lines, presumably the association between adjacent cell membranes. At higher magnification these irregular lines between cells were seen to consist of saccules and vesicles interconnected by small tubular structures (Fig. 3). The cell margins that make up the rhomboidal pattern of endothelial cells were continuous. No interruptions were seen. Some of the cells exhibited microvilli (Fig. 3) similar to that reported for other venous endothelial cells [ 61. Some cells had more than others. Although occasional red cells were seen on the surface of the endothelium, platelets and other formed elements were not seen. A transmission electron micrograph of the endothelial cells of control rabbit ear veins is shown in Fig. 4. (2) Damage to endothelial cells caused by arachidonic acid Damage to endothelial cells induced by injection of sodium arachidonate into the ear veins of 6 rabbits is shown in Figs. 5-11. The first signs of damage to endothelial cells were seen in response to injection of a low dose (0.5 mg/kg) of arachidonic acid. In some areas of the luminal surface of the vessel, the nuclear bulges of the endothelial cells appeared to be more dense than in the controls and the nuclear outlines were more discrete (Fig. 5). Isolated areas were seen in which individual cells had lost their rhomboidal shape and the cell membranes were no longer distinct. This was clearly shown by transmission electron microscopy (Fig. 11). Higher doses (1.4 mg/kg) of arachidonic acid are lethal for rabbits (see Ref. [4]). When these amounts were injected into ear veins the damage to endothelial cells was more pronounced. The bulging of nuclei of greater density
216
Fig. 1. Appearance of endothelium after injection of sodium carbonate solution. of endothelial cells. Occasional red blood cells are seen. En face view. X 1300.
Fig. 2. Another view of a vessel wall after injection on the side wall of the vessel. X 1300.
of sodium
carbonate.
Note rhoml: Boidal Ishape
Nuclear bulges are clear15 1 seen
Fig. 3. Higher magnification of endothelium, after injection of sodium carbonate solution, showing parts of two cells. The intercellular cleft (arrow) contains a series of saccules, tubules and vesicles. The microvilli on the surface of the cells are seen to better advantage in this specimen. X 13,000.
occurred in .more cells than those exposed to a lower dose. Occasional cells were enucleated (Fig. 6) while none were observed in controls. Holes were seen on the surface of the cytoplasmic portion of other cells (Fig. 7). The rhomboidal shape of the endothelial cells was no longer present and distinct cell boundaries were not obvious (Figs. 6 and 7). In badly damaged endothelium, cells were separated from one another, exposing sub-endothelial tissue to which platelets appeared to be adhering (Figs. 8 and 9). These platelets had changed shape and exhibited pseudopodia (Fig. 10). (3) Failure of aspirin to inhibit endothelial damage caused by arachidonic acid Since aspirin blocks prostaglandin formation by platelets [7] as well as the lethality of an injection of arachidonic acid [4], we considered it of interest to determine whether pre-treatment of the animals with aspirin would also inhibit the formation of endothelial lesions produced by arachidonic acid. Rabbits were given an intraperitoneal injection of aspirin (13 mg/kg) as described elsewhere [4]. Two hours later they were challenged with an injection of arachidonic acid (1.4 mg/kg) as above. The animals were protected from death but they were not protected from the formation of endothelial lesions. Electron microscopy showed that endothelial damage similar to that caused by arachidonic acid alone also occurred in animals pre-treated with aspirin. (4) Effects of some other fatty acids 8,11,14-Eicosatrienoic acid, a prostaglandin
precursor
known
to inhibit
218
platelet aggregation [ 71 as well as fatty acids which are not prostaglandin pre(5,8,11,14_eicosatetraenoic, cursors 15-hydroperoxy-5,8,11,13-eicosatetraenoic, linolenic and y-linolenic acids) were injected into the ear veins of rabbits at a concentration of 1.4 mg/kg exactly as described for arachidonic acid. In all
Fig. 4. Transmission electron micrograph of portion of lateral ear vein after injection of sodium carbonate solution. Two endothelial cells are seen on the luminal surface. The tunica media of the vessel contains 3 layers of smooth muscle cells. X 12.000.
279
Fig. 6. After a low dose of sodium arachidonate (0.6 mg/kg). Nuclei of endothelial lined and appear to be of greater density than in sodium carbonate controls (compare
Fig. 6. A view of a portion of endothelium after injection of sodium arachidonate ting a nuclear crater and a number of presumably damaged nuclei. X 1300.
cells are clearly outto Fig. 2 ). x 13 00.
(1.4 mgkg)
demo]
280
Fig. 7. A view of a region of endothelium, after injection holes in the cytoplasm of endothelial cells. X 1300.
of sodium
arachidonate
(1.4
mg, ‘kg), showing
Fig. 8. A view of a region of endothelium after injection of sodium arachidonate (1.4 mg/k :g) shlowing behween separation between endothelial cells. In many instances, platelets appear to be sticking in regio mns endothelial cells. X 1300.
281
Fig. 9. Separated endothelial cells are seen after an injection of sodium vessel. Platelets are seen within the enlarged intercellular clefts. X 3900.
Fig. 10. View of endothelium. donate (1.4 mg/kg). In many x 6500.
arachidonate
(1.4 mg /kg) into the
at higher magnification, after vessel had been injected with SCdum araichiinstances platelets have undergone shape change and exhibit 1aseudopo bdia.
Fig. 11. Transmission electron micrograph of lateral ear vein after injection of a high dose of sodium arachidonate (1.4 mg/kg). Platelets’are seen adhering to vessel surface denuded of endothelium. X 12.000.
cases endothelial lesions similar to those produced by arachidonic acid were observed when the lumina of these vessels were examined by scanning electron microscopy.
283
Discussion Acosta and Wenzel [8] observed that free fatty acids are injurious to endothelial cells in culture and Maca and Hoak [9] reported damage to endothelial cells in the aortas of rabbits that had received an injection of adrenocorticotrophic hormone (ACTH). The changes in morphology in the latter study were associated with a 4-5 fold rise in the free fatty acid levels of the plasma, suggesting that fatty acids can damage endothelial cells. In this paper we describe a simple and rapid method for studying damage to endothelial cells in vivo and report here on the effects produced by fatty acids. Initially we observed that arachidonic acid damaged the endothelium in an apparently concentration-dependent fashion. We considered two possible ways in which such damage might occur: (1) A biochemical event might convert arachidonic acid into one of its metabolites which could damage the endothelium; (2) A physical or chemical property of arachidonic acid itself might be detrimental to endothelial cells. Arachidonic acid is present in the phospholipids of most mammalian cells. It induces platelet aggregation in vitro [ 71 and in vivo [ 41 because it is converted via the platelet cyclooxygenase pathway into PGG2, PGH? and thromboxane AZ which are extremely potent platelet-aggregating agents (see Ref. [lo]). We considered it possible that arachidonic acid might cause endothelial cell damage because it is converted into PGG2, PGH? or thromboxane Az. However, aspirin, an inhibitor of prostaglandin production by platelets [ 111, did not protect the endothelial cells from damage by arachidonic acid. These experiments did not rule out the possibility that oxygenation products of arachidonic acid formed via the platelet lipoxygenase pathway were involved. However, injection of 5,8,11,14-eicosatetraenoic acid, an inhibitor of both cyclooxygenase and lipoxygenase, produced similar changes in endothelial cells to those produced by arachidonic acid. Finally, it was possible that the transformation of arachidonic acid into high concentrations of prostacyclin by the endothelial cells might be responsible for the damage. This possibility was ruled out by showing that (1) 15hydroperoxy-5,8,11,13-eicosatrienoic acid, an inhibitor of prostacyclin synthetase [ 121, produced lesions similar to arachidonic acid and (2) the demonstration by scanning electron microscopy (Figs. 8, 9 and 10) of scattered platelet aggregates that should not occur in the presence of this powerful albeit short-lived prostaglandin. The above experiments indicated that a biochemical transformation of arachidonic acid was not necessary for endothelial cell damage. Further, the damage produced by the fatty acid inhibitors of lipoxygenase and prostacyclin synthetase suggested that fatty acids of differing chemical structures could damage the endothelium. We found that damage was also produced by linoleic, y-linolenic and 8,11,14-eicosatrienoic acid. Therefore, we suggest that endothelial lesions produced by fatty acids is non-specific, possibly resulting from the physical changes in surface tension produced by these molecules. It is possible that individual differences in cytotoxicity among the fatty acids would be seen for each fatty acid studied at lower concentrations. The local concentrations of the fatty acids in the ear vein are presumably much higher than in the general circulation.
284
The rabbit ear vein appears to be a simple model for the investigation of early lesions of endothelial cells and the effects of substances which may inhibit the formation of these lesions. References 1 Goode, T.B., Davies, P.F., Reidy, M.A. and Bowyer, D.E., Aortic endothelial cell morphology observed in situ by scanning electron microscopy during atherogenesis in the rabbit. Atherosclerosis, 27 (1977) 235. 2 Moore, S.. Thromboatherosclerosis in normalipemic rabbits - A result of continuing endothellal damage, Lab. Invest., 29 (1973) 478. 3 Lewis, J.C. and Kottke, B.A., Endothelial damage and thrombocyte adhesion in pigeon atherosclerosis, Science, 196 (1977) 1007. 4 Silver, M.J., Hoch. W., Kocsis, 5 6 7 8 9 10 11 12
J.J.,
Ingerman,
C.M. and Smith,
J.B.,
Arachidonic
acid causes sudden
death in rabbits, Science, 183 (1974) 1085. Kelley, R.C.. Dekker. B.A.F. and Bluemink, J.E., Ligand-mediated osmium binding - Its application in coating biological specimens for SEM, J. Ultrastruct. Res., 45 (1973) 254. Buss, H. and Hollweg, M.G.. Proc. Workshop on Biomedical Applications, IIT Research Institute/SEM. Vol. 2, 1977, p. 467. Silver. M.J., Smith, J.B., Ingerman. C. and Kocsis, J.J., Arachidonic acid-induced human platelet aggregation and prostaglandin formation, Prostaglandins, 4 (1973) 863. Acosta, D. and Wentel, D.G., Injury produced by free fatty acids to lysosomes and mitochondrla in cultured heart muscle and endothelial cells, Atherosclerosis, 20 (1974) 417. Maca. R.D. and Hoak, J.C., Endothellal injury and platelet aggregation associated with acute lipid mobilization, Lab. Invest., 30 (1974) 689. Smith, J.B. and Silver, M.J.. Prostaglandin synthesis by platelets and its biological significance. In: J.L. Gordon (Ed.), Platelets in Biology and Pathology. Elsevier, Amsterdam, 1976, pp. 331-352. Smith. J.B. and Willis, A.L.. Aspirin selectively inhibits prostaglandin production in human platelets, Nature (Land.), 231 (1971) 235. Moncada, S.. Gryglewskl. R.J., Bunting, S. and Vane, J.R., A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation, Prostaglandlns. 12 (1976) 715.
Atherosclerosis, 30 (1978) 273-284 @ Elsevier/North-Holland Scientific
Publishers,
Ltd.
FATTY ACIDS AND THE INITIAL EVENTS OF ENDOTHELIAL DAMAGE SEEN BY SCANNING AND TRANSMISSION ELECTRON MICROSCOPY A.W. SEDAR,
M.J. SILVER,
J.J. KOCSIS and J.B. SMITH
Department of Anatomy and Pharmacology and Cardeza Foundation, University, Philadelphia, PA 19107 (U.S.A.) (Received (Revised, (Accepted
Thomas Jefferson
30 January, 1978) received 7 April, 1978) 19 April, 1978)
A method was developed for observing changes in the endothelial cells in rabbit ear veins in vivo by scanning electron microscopy. Injection of fatty acids into the ear vein caused damage to the endothelium. The first signs of damage seen were marked bulges in the nuclei and loss of the rhomboidal shape of the endothelial cells. More severe damage included loss of nuclei, .leaving holes in the cytoplasm. Some parts of the damaged endothelium showed complete separation of cells from each other and exposure of sub-endothelial tissue to which platelets with pseudopodia were adhering. Damage to the endotheliurn was produced by arachidonic, linoleic, r-linolenic, 8,11,14-eicosatrienoic, 5,8,11,14,-eicosatetraenoic or 15-hydroperoxy-5,8,11,13_eicosatetraenoic acids. The effect of arachidonic acid was not prevented by pre-treating the animals with aspirin. It appears that damage produced by the fatty acids is nonspecific. Key words:
Endothelium
- Fatty acids - Scanning and transmission electron microscopy
Introduction Recent studies of the early stage of atherogenesis in rabbits maintained on atherogenic diets [ 1,2] and pigeons with spontaneously developing atherosclerosis [3] have revealed distinct morphological changes in the endothelial cells of the affected blood vessel walls. Areas of vessel wall were reported to contain irregularly shaped cells with an increased incidence of stigmata and stomata. Other changes included ruffling of luminal plasma membranes, endothelial desquamation and the attachment of platelets to subendothelial tissue and apparently to damaged endothelial cells. Such changes are considered to be amongst the “earliest identifiable events in atherogenesis in the pigeon” [3]. We have
274
developed a simple model system in which changes in the endothelial cells of rabbit ear veins can be rapidly produced by different agents and observed by scanning electron microscopy. In our initial studies we chose to investigate the effects of fatty acids on the endothelium. Materials and Methods Fatty acids (>99% pure) were obtained from Nuchek Prep., Inc., Elysian, MI. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid was prepared by incubating arachidonic acid with soybean lipoxygenase (Sigma Chem. Co., St. Louis, MO) under an atmosphere of oxygen. 5,8,11,14-Eicosatetraenoic acid was a gift from P. Whitman, Hoffman La Roche, Nutley, NJ. Solutions of the sodium salts were made by dissolving the free fatty acids in sodium carbonate solution as described elsewhere [ 41. Aspirin was obtained from Merck and Co., Rahway, NJ. Rabbits employed were male New Zealand whites, weighing between 2.5 and 3.5 kg. All injections of test substances or of vehicle into the lateral ear vein were made at the rate of 1 ml/min via an E-Z set, infusion set with a 23gauge needle (Deseret Pharmaceutical Co., Inc., Sandy, UT). Rabbits were anesthetized by injecting 2-3 ml of a 50 mg/ml sodium pentobarbital solution (Nembutal, Abbott Laboratories, North Chicago, IL) into the lateral ear vein of the opposite ear. Initial preparation of blood vessels for scanning electron microscopy @EM) Three min after the injection of the solution of fatty acid or the vehicle, the ear vein was clamped distal to the site of the venipuncture. An infusion of Tyrode’s solution (pH 7.4) into the ear vein was then begun. The vessel was immediately cut with a sharp scalpel blade at the point where it descends into the base of the ear and a clamp was placed across the central artery. The infusion of Tyrode’s solution was continued at a rate maintained to just keep the vessel distended. In most cases, this was about 2 ml/min. This infusion of Tyrode’s solution allowed for a gentle washing out of the residual blood in the vein. When this was accomplished, a clear effluent was seen to emerge at the cut end. In some cases, it was necessary to cut small side branches entering the vein. Following the wash with Tyrode’s solution, 1% glutaraldehyde in Tyrode’s solution was infused in similar fashion. After the infusion of 10 ml of the glutaraldehyde solution, the ear vein, full of glutaraldehyde solution, was clamped just above the cut end and just below the site of injection. The ear tissue (30-40 mm long) containing the vessel between the clamps was then cut out. Further preparation of the vessel for SEM The tissue containing the vessel was fixed overnight in 1% glutaraldehyde solution. The following day the specimen was pinned to a wax plate. The skin was then carefully removed from one surface of the vessel under a dissecting microscope. The upper half of the vessel was removed with an iridectomy scissors cutting longitudinally along each side wall of connective tissue. This technique provided a half cylinder of the vessel embedded in skin with its endothelial-luminal surface exposed. The specimen was divided into strips of skin approximately 10 mm long and fixation continued overnight in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. After rinsing 3 times with the
275
same buffer, the specimens were fixed for 1 h in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. To reduce charging of the specimens, more osmium was incorporated using a modification of the ligand binding method of Kelley et al. [ 51. This consisted of a thorough rinsing (5-10 times) for 15 min in buffer, incubation for 10 min in 1% filtered aqueous thiocarbohydrazide solution, a rinse for 15 min in several changes of distilled water and postfixation in 1% aqueous osmium tetroxide for 1 h. The specimens were washed several times in distilled water over a 15-min period before dehydration in ethanol. The absolute ethanol was replaced with ethanol : amyl acetate 50 : 50 (v : v) and then with absolute amyl acetate. Specimens were dried by the critical point method using liquid CO? as the intermediate fluid. They were then vacuum coated with 10 nm of carbon and 150 nm of gold before examination in the JEOL-JSM-50A scanning electron microscope at magnifications of X 400 to 10,000. Results (1) Control studies For controls, 1 ml of saline solution or 25 mM sodium carbonate solution (the vehicle for the fatty acids studied below) was injected into the lateral ear vein of 6 rabbits. In all cases, the endothelial lining of the vessel wall appeared as follows: The endothelial cells had a rhomboidal shape, with long axis in the direction of blood flow (Fig. 1). The center of all cells exhibited nuclear bulges (Fig. 2). The margins between adjacent cells exhibited irregular lines, presumably the association between adjacent cell membranes. At higher magnification these irregular lines between cells were seen to consist of saccules and vesicles interconnected by small tubular structures (Fig. 3). The cell margins that make up the rhomboidal pattern of endothelial cells were continuous. No interruptions were seen. Some of the cells exhibited microvilli (Fig. 3) similar to that reported for other venous endothelial cells [ 61. Some cells had more than others. Although occasional red cells were seen on the surface of the endothelium, platelets and other formed elements were not seen. A transmission electron micrograph of the endothelial cells of control rabbit ear veins is shown in Fig. 4. (2) Damage to endothelial cells caused by arachidonic acid Damage to endothelial cells induced by injection of sodium arachidonate into the ear veins of 6 rabbits is shown in Figs. 5-11. The first signs of damage to endothelial cells were seen in response to injection of a low dose (0.5 mg/kg) of arachidonic acid. In some areas of the luminal surface of the vessel, the nuclear bulges of the endothelial cells appeared to be more dense than in the controls and the nuclear outlines were more discrete (Fig. 5). Isolated areas were seen in which individual cells had lost their rhomboidal shape and the cell membranes were no longer distinct. This was clearly shown by transmission electron microscopy (Fig. 11). Higher doses (1.4 mg/kg) of arachidonic acid are lethal for rabbits (see Ref. [4]). When these amounts were injected into ear veins the damage to endothelial cells was more pronounced. The bulging of nuclei of greater density
216
Fig. 1. Appearance of endothelium after injection of sodium carbonate solution. of endothelial cells. Occasional red blood cells are seen. En face view. X 1300.
Fig. 2. Another view of a vessel wall after injection on the side wall of the vessel. X 1300.
of sodium
carbonate.
Note rhoml: Boidal Ishape
Nuclear bulges are clear15 1 seen
Fig. 3. Higher magnification of endothelium, after injection of sodium carbonate solution, showing parts of two cells. The intercellular cleft (arrow) contains a series of saccules, tubules and vesicles. The microvilli on the surface of the cells are seen to better advantage in this specimen. X 13,000.
occurred in .more cells than those exposed to a lower dose. Occasional cells were enucleated (Fig. 6) while none were observed in controls. Holes were seen on the surface of the cytoplasmic portion of other cells (Fig. 7). The rhomboidal shape of the endothelial cells was no longer present and distinct cell boundaries were not obvious (Figs. 6 and 7). In badly damaged endothelium, cells were separated from one another, exposing sub-endothelial tissue to which platelets appeared to be adhering (Figs. 8 and 9). These platelets had changed shape and exhibited pseudopodia (Fig. 10). (3) Failure of aspirin to inhibit endothelial damage caused by arachidonic acid Since aspirin blocks prostaglandin formation by platelets [7] as well as the lethality of an injection of arachidonic acid [4], we considered it of interest to determine whether pre-treatment of the animals with aspirin would also inhibit the formation of endothelial lesions produced by arachidonic acid. Rabbits were given an intraperitoneal injection of aspirin (13 mg/kg) as described elsewhere [4]. Two hours later they were challenged with an injection of arachidonic acid (1.4 mg/kg) as above. The animals were protected from death but they were not protected from the formation of endothelial lesions. Electron microscopy showed that endothelial damage similar to that caused by arachidonic acid alone also occurred in animals pre-treated with aspirin. (4) Effects of some other fatty acids 8,11,14-Eicosatrienoic acid, a prostaglandin
precursor
known
to inhibit
218
platelet aggregation [ 71 as well as fatty acids which are not prostaglandin pre(5,8,11,14_eicosatetraenoic, cursors 15-hydroperoxy-5,8,11,13-eicosatetraenoic, linolenic and y-linolenic acids) were injected into the ear veins of rabbits at a concentration of 1.4 mg/kg exactly as described for arachidonic acid. In all
Fig. 4. Transmission electron micrograph of portion of lateral ear vein after injection of sodium carbonate solution. Two endothelial cells are seen on the luminal surface. The tunica media of the vessel contains 3 layers of smooth muscle cells. X 12.000.
279
Fig. 6. After a low dose of sodium arachidonate (0.6 mg/kg). Nuclei of endothelial lined and appear to be of greater density than in sodium carbonate controls (compare
Fig. 6. A view of a portion of endothelium after injection of sodium arachidonate ting a nuclear crater and a number of presumably damaged nuclei. X 1300.
cells are clearly outto Fig. 2 ). x 13 00.
(1.4 mgkg)
demo]
280
Fig. 7. A view of a region of endothelium, after injection holes in the cytoplasm of endothelial cells. X 1300.
of sodium
arachidonate
(1.4
mg, ‘kg), showing
Fig. 8. A view of a region of endothelium after injection of sodium arachidonate (1.4 mg/k :g) shlowing behween separation between endothelial cells. In many instances, platelets appear to be sticking in regio mns endothelial cells. X 1300.
281
Fig. 9. Separated endothelial cells are seen after an injection of sodium vessel. Platelets are seen within the enlarged intercellular clefts. X 3900.
Fig. 10. View of endothelium. donate (1.4 mg/kg). In many x 6500.
arachidonate
(1.4 mg /kg) into the
at higher magnification, after vessel had been injected with SCdum araichiinstances platelets have undergone shape change and exhibit 1aseudopo bdia.
Fig. 11. Transmission electron micrograph of lateral ear vein after injection of a high dose of sodium arachidonate (1.4 mg/kg). Platelets’are seen adhering to vessel surface denuded of endothelium. X 12.000.
cases endothelial lesions similar to those produced by arachidonic acid were observed when the lumina of these vessels were examined by scanning electron microscopy.
283
Discussion Acosta and Wenzel [8] observed that free fatty acids are injurious to endothelial cells in culture and Maca and Hoak [9] reported damage to endothelial cells in the aortas of rabbits that had received an injection of adrenocorticotrophic hormone (ACTH). The changes in morphology in the latter study were associated with a 4-5 fold rise in the free fatty acid levels of the plasma, suggesting that fatty acids can damage endothelial cells. In this paper we describe a simple and rapid method for studying damage to endothelial cells in vivo and report here on the effects produced by fatty acids. Initially we observed that arachidonic acid damaged the endothelium in an apparently concentration-dependent fashion. We considered two possible ways in which such damage might occur: (1) A biochemical event might convert arachidonic acid into one of its metabolites which could damage the endothelium; (2) A physical or chemical property of arachidonic acid itself might be detrimental to endothelial cells. Arachidonic acid is present in the phospholipids of most mammalian cells. It induces platelet aggregation in vitro [ 71 and in vivo [ 41 because it is converted via the platelet cyclooxygenase pathway into PGG2, PGH? and thromboxane AZ which are extremely potent platelet-aggregating agents (see Ref. [lo]). We considered it possible that arachidonic acid might cause endothelial cell damage because it is converted into PGG2, PGH? or thromboxane Az. However, aspirin, an inhibitor of prostaglandin production by platelets [ 111, did not protect the endothelial cells from damage by arachidonic acid. These experiments did not rule out the possibility that oxygenation products of arachidonic acid formed via the platelet lipoxygenase pathway were involved. However, injection of 5,8,11,14-eicosatetraenoic acid, an inhibitor of both cyclooxygenase and lipoxygenase, produced similar changes in endothelial cells to those produced by arachidonic acid. Finally, it was possible that the transformation of arachidonic acid into high concentrations of prostacyclin by the endothelial cells might be responsible for the damage. This possibility was ruled out by showing that (1) 15hydroperoxy-5,8,11,13-eicosatrienoic acid, an inhibitor of prostacyclin synthetase [ 121, produced lesions similar to arachidonic acid and (2) the demonstration by scanning electron microscopy (Figs. 8, 9 and 10) of scattered platelet aggregates that should not occur in the presence of this powerful albeit short-lived prostaglandin. The above experiments indicated that a biochemical transformation of arachidonic acid was not necessary for endothelial cell damage. Further, the damage produced by the fatty acid inhibitors of lipoxygenase and prostacyclin synthetase suggested that fatty acids of differing chemical structures could damage the endothelium. We found that damage was also produced by linoleic, y-linolenic and 8,11,14-eicosatrienoic acid. Therefore, we suggest that endothelial lesions produced by fatty acids is non-specific, possibly resulting from the physical changes in surface tension produced by these molecules. It is possible that individual differences in cytotoxicity among the fatty acids would be seen for each fatty acid studied at lower concentrations. The local concentrations of the fatty acids in the ear vein are presumably much higher than in the general circulation.
284
The rabbit ear vein appears to be a simple model for the investigation of early lesions of endothelial cells and the effects of substances which may inhibit the formation of these lesions. References 1 Goode, T.B., Davies, P.F., Reidy, M.A. and Bowyer, D.E., Aortic endothelial cell morphology observed in situ by scanning electron microscopy during atherogenesis in the rabbit. Atherosclerosis, 27 (1977) 235. 2 Moore, S.. Thromboatherosclerosis in normalipemic rabbits - A result of continuing endothellal damage, Lab. Invest., 29 (1973) 478. 3 Lewis, J.C. and Kottke, B.A., Endothelial damage and thrombocyte adhesion in pigeon atherosclerosis, Science, 196 (1977) 1007. 4 Silver, M.J., Hoch. W., Kocsis, 5 6 7 8 9 10 11 12
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