Platelet accumulation and turnover on de-endothelialized aortae in rats.

Previous studies indicate that the subendothelium of rabbit aortae de-endothelialized with a balloon catheter rapidly becomes covered with a monolayer of platelets; after 60 min few additional platelets accumulate and although most platelets are lost from the injured surface by 4 days, there is a substantial delay before re-endothelialization. We examined the dynamics of platelet accumulation on rat aortae de-endothelialized with a balloon catheter to determine if the response to this type of injury is similar to rabbit aortae. When 51Cr-platelets were injected prior to aortic de-endothelialization, 25,500 +/- 2,750 platelets/mm2 accumulated on rat subendothelium in the first 15 min. After 60 and 92 h, fewer platelets remained on the surface (13,740 +/- 2,400 and 5,020 +/- 1,330 platelets/mm2, respectively). When 51Cr-platelets were injected into rats 30 min after injury, platelet accumulation in a 30-min period was 8,610 +/- 1,230 platelets/mm2. By 4 days rat aortae did not accumulate newly injected platelets significantly in a 30-min period, but in a 24-h period 20,600 +/- 3,490 platelets/mm2 accumulated. Morphologically, the non-endothelialized areas of rat aortae were almost completely covered with platelets 4 days after injury. Fourteen days after injury, rat aortae did not accumulate newly injected platelets and, morphologically, no platelets were present on the surface which was almost re-endothelialized. Thus, in rats, as with rabbits, platelets rapidly accumulate on de-endothelialized aortae and the ability to attract newly introduced platelets is considerably reduced shortly after injury. In contrast to rabbits, however, de-endothelialized aortae in rats remain attractive to new platelets up to 4 days following injury, but less so than at the time of injury. Also, in contrast to rabbits, 14 days after injury to rat aortae the surface is almost completely re-endothelialized. Thus, there are species differences in platelet interactions with de-endothelialized vessels.     

Platelet interaction with damaged rabbit aorta.

We have quantified the accumulation of 51Cr-labeled washed rabbit platelets on the subendothelium of rabbit aortae following injury with a balloon catheter. The amount of radioactivity that became associated with the damaged wall within 10 minutes of the injury did not change appreciably during the following 24 hours, indicating that there was little turnover of platelets on the injured vessel wall. In addition, by injecting 51Cr-labeled platelets into rabbits at different times after injury, it was possible to estimate the reactivity of the exposed surface to newly injected platelets. Scanning electron microscopy showed that a monolayer of platelets initially formed on the injured surface; the number of platelets associated with the surface decreased over the 7-day observation period. The amount of 51Cr associated with the injured vessel wall also diminished during this period. The ability of the damaged surface to attract fresh platelets gradually decreased during the 7 days following injury. Platelet survival in rabbits was not significantly reduced following the removal of the aortic endothelium (balloon catheter injury 66.3 +/- 12.2 hours, sham operated 72.1 +/- 7.2 hours, untreated controls 76.2 +/- 3.8 hours). Thus, in rabbits, it cannot be assumed that platelet survival provides an estimate of endothelial injury in all circumstances.     

Endothelium modifies the altered metabolism of the injured aortic wall.

Results of previous experiments in this laboratory indicate that lipids, especially cholesterol and cholesteryl ester, preferentially accumulate in re-endothelialized, as compared with de-endothelialized, areas of aorta (Am J Pathol 1980, 99:81-104). In the experiments reported here, the hypothesis that this lipid accumulation results from alterations in arterial wall metabolism induced by injury and modified by endothelium was tested. Activities of the two cholesterol-ester-metabolizing enzymes acyl CoA: cholesterol acyltransferase and acid cholesteryl esterase were assayed in uninjured aortas and in de-endothelialized and re-endothelialized areas of balloon-catheter-injured aortas from normocholesterolemic and hypercholesterolemic rabbits. Activities of marker enzymes for major cell organelles were also assayed. Our results indicate that acyl CoA: cholesterol acyltransferase activity was similarly increased in re-endothelialized and de-endothelialized areas of injured aortas. Activity of acid cholesteryl esterase was also increased; however, it was significantly less in re-endothelialized as compared with de-endothelialized areas. Activities of several marker enzymes were changed in injured aortas, particularly in de-endothelialized as compared with re-endothelialized areas. These findings suggest that 1) injury predisposes to general metabolic changes in the aorta that are modified by endothelium and 2) increased cholesteryl ester accumulation in re-endothelialized aortas occurs at least in part from increased synthesis and decreased hydrolysis.     

Increased platelet, but unaltered fibrinogen, accumulation in experimental thrombi in alloxan-induced diabetic rabbits.

Platelets from diabetic humans and animals have been found previously to be hypersensitive to agonists, including thrombin, in vitro but it is unclear if this hypersensitivity also occurs in vivo and leads to a greater thrombotic tendency. In the present study, the effect of diabetes was examined on thrombus formation and vessel wall responses which result from continuous intimal injury induced by indwelling aortic catheters in rabbits. Platelet and fibrin(ogen) associated with the thrombus and damaged aortae were examined. Control or alloxan-induced diabetic rabbits (9-12 months after initial treatment) were injected with 51Cr-labeled autologous platelets and 125I-labeled fibrinogen (prepared from control rabbits) before insertion of indwelling aortic catheters. The anesthetized rabbits were perfused-fixed after 20 hr or 4 days. The dry weight of thrombus that formed was determined and platelet and fibrin(ogen) accumulation in thrombi and on injured aortae were calculated from the associated 51Cr and 125I, respectively. In diabetic rabbits, more platelets accumulated in the thrombi which formed after either 20 hr or 4 days, although the weight of thrombus and net fibrin(ogen) incorporation into the thrombus were not different from corresponding control rabbits. Net platelet and fibrin(ogen) association with the injured aortae were not different between control and diabetic rabbits. It is likely that the increased platelet accumulation in arterial thrombi in diabetic rabbits which results from continuous injury to aortae is a consequence of hypersensitivity of these platelets to thrombin generated in the thrombus and at the sites of vessel injury.     

Distribution of cellular responses in rabbit aortae following one and two injuries with a balloon catheter

To investigate the mechanisms involved in the cellular reactions to arterial injuries, we studied the distribution of the deposits on the injured intima and the pattern of neointimal thickening following single and double injuries of rabbit aortae with a balloon catheter. Thirty minutes after the first injury most, but not all, of the inner surface of the aortae was covered by adherent, spread platelets. Seven days following the first injury areas of neointima, mainly proliferating smooth muscle cells, had formed around and opposite the orifices of branch vessels. The rest of the inner aortic surface consisted of acellular subendothelial matrix. Thirty minutes after the second injury, 7 days after the first, single platelets adhered once more to parts of the reinjured subendothelium, mostly between the orifices. Numerous fibrin-rich, platelet thrombi were present mainly on the surface of the injured neointima. Thirty minutes after both the first and second injury polymorphonuclear leucocytes adhered to the inner surface downstream from the orifices of branch vessels and in longitudinally oriented zones opposite the orifices. Four days following the second injury, the neointima was restored with the same distribution as before the second injury, and few thrombi, adherent platelets, or leucocytes remained.     

Distribution of cellular responses in rabbit aortae following one and two injuries with a balloon catheter.

To investigate the mechanisms involved in the cellular reactions to arterial injuries, we studied the distribution of the deposits on the injured intima and the pattern of neointimal thickening following single and double injuries of rabbit aortae with a balloon catheter. Thirty minutes after the first injury most, but not all, of the inner surface of the aortae was covered by adherent, spread platelets. Seven days following the first injury areas of neointima, mainly proliferating smooth muscle cells, had formed around and opposite the orifices of branch vessels. The rest of the inner aortic surface consisted of acellular subendothelial matrix. Thirty minutes after the second injury, 7 days after the first, single platelets adhered once more to parts of the reinjured subendothelium, mostly between the orifices. Numerous fibrin-rich, platelet thrombi were present mainly on the surface of the injured neointima. Thirty minutes after both the first and second injury polymorphonuclear leucocytes adhered to the inner surface downstream from the orifices of branch vessels and in longitudinally oriented zones opposite the orifices. Four days following the second injury, the neointima was restored with the same distribution as before the second injury, and few thrombi, adherent platelets, or leucocytes remained.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Ultrastructural changes in re-endothelialized and non-endothelialized rabbit aortic neo-intima following re-injury with a balloon catheter.

The response by normal rabbit aortas to the removal of the endothelium with a balloon catheter, was compared to the response to the removal of regenerated endothelium from rabbit aortas that had been previously de-endothelialized. De-endothelialization results in the formation of a neo-intima. Thrombus formation following a second balloon catheter injury was compared among injured neo-intima that had been re-endothelialized, non-re-endothelialized neo-intima, and the subendothelium of normal vessels following a single injury. Rabbit aortas were examined by scanning electron microscopy of full circumference segments of the aorta and by transmission electron microscopy. Thirty minutes after a single de-endothelialization injury with a balloon catheter the luminal surface is covered by a monolayer of platelets adhering to the subendothelial connective tissues. Two weeks later there is neo-intimal formation and endothelial regeneration around branch vessel orifices. The remainder of the luminal surface is composed of smooth muscle cells (SMC). A balloon catheter injury to a vessel injured 2 weeks previously results in fibrin formation and platelet-fibrin microthrombi on the aortic intimal surface. The response of the aortic wall to re-injury does not seem to be related to the prior existence of endothelium. Both single and repeated injuries result in a distribution of formed elements which may depend, in part, on haemodynamic factors.     

Enhancement of cholesterol and cholesteryl ester accumulation in re-endothelialized aorta.

The purpose of the experiments reported here was to determine chemically the character and quantity of lipid in re-endothelialized and de-endothelialized areas of rabbit aortas. The aortas of 22 rabbits, Groups I and II, were de-endothelialized with a balloon catheter, and the rabbits were maintained on a lipid-poor diet for 4 weeks. Thirteen rabbits, Group II, were then fed an egg-supplemented diet for 6 weeks. Nine rabbits, Group I, were continued on the lipid-poor diet for an additional 6 weeks. Control rabbits with uninjured aortas were fed a lipid-poor diet for 10 weeks (Group III) or an egg-supplemented diet for 6 weeks (Group IV). Nonesterified cholesterol and fatty acids, cholesteryl esters, triacylglycerols, and squalene were quantitated in re-endothelialized and de-endothelialized aorta by thin-layer chromatography and fluorometric analysis. The results indicate 1) that there was approximately three times as much nonesterified cholesterol and cholesteryl ester in re-endothelialized aorta of Groups I and II as compared with adjacent de-endothelialized aorta and 2) that in re-endothelialized aorta of Group II the amount of total cholesterol correlated with serum cholesterol concentration in contrast to adjacent de-endothelialized aorta, with no correlation over a range of nearly 900 mg/100 ml. These studies indicate that the presence of endothelium favors accumulation of aortic cholesteryl esters. The results suggest that arterial lipid accumulation is not simply a result of passive filtration but may result from metabolic differences in the re-endothelialized neointima.     

Survival of rabbit platelets exposed to immune complexes

Rabbits injected with human serum albumin (HSA) formed detectable immune complexes after 5 days; complex formation was maximal between 11 and 14 days after which the complexes were cleared from the circulation. Platelets from control rabbits or HSA-injected rabbits had a reduced survival upon injection into rabbits in which complexes were forming. Platelets from HSA-animals tended to survive for a longer period upon injection into control rabbits than when they were injected into HSA-rabbits, raising the possibility that some of the immune complexes may have eluted from their surface. Platelets prepared from either control animals or from HSA-treated animals at the time when complexes were being cleared from the circulation (14-21 days) did not have a shortened life span in HSA- or control rabbits. When platelet survival was reduced, it could not be attributed to platelet accumulation at sites of vessel wall injury or to accumulation in kidneys damaged by immune complexes, since the tissues (aorta and kidney) appeared to be morphologically normal and free of thrombi. The reduction in platelet survival likely results from the interactions of immune complexes with the surface of platelets leading to the platelets being recognized as "foreign" and cleared from the circulation by the reticuloendothelial system.     

Role of endothelium and hypercholesterolemia in intimal thickening and lipid accumulation.

In experiments reported here we tested the hypothesis that persistent absence of endothelium favors intimal thickening, lipid accumulation, and atherosclerosis. Rabbit aortas were de-endothelialized with a balloon catheter at Day 0. Initially, all rabbits were fed a diet low in lipid. Some rabbits (Group I) were continued on a diet low in lipid for 8 to 20 weeks after de-endothelialization. Beginning 4 to 9 weeks after de-endothelialization, other rabbits were fed semisynthetic lipid-rich diets (Group II) or cholesterol-supplemented diets (Groups III) for 4 to 20 weeks. Lipid accumulation in all groups was significantly greater in the re-endothelialized intima than in adjacent intima lacking an endothelial lining. In aortas of Groups I, II, and III the degree of intimal thickening was significantly greater in re-endothelialized areas than in adjacent areas lacking endothelium. Intimal thickness was enhanced in re-endothelialized areas of hypercholesterolemic rabbits of Group III compared with normocholesterolemic rabbits of Group I but not in areas lacking endothelium. Thus, results of these experiments do not support the hypothesis that the absence of endothelium particularly favors intimal thickening and intimal lipid accumulation. Results indicate that intima covered by regenerated endothelium is significantly thicker and more likely to accumulate lipid.     

Acute administration of ethanol to rabbits inhibits thrombus formation induced by indwelling aortic catheters

Platelet aggregation and secretion of granule contents in response to specific agonists are inhibited by acute ethanol (1 to 4 mg/ml in vitro or 4 to 5 mg/ml ex vivo). However, acute administration of ethanol, giving blood levels of 4 to 5 mg/ml, does not affect platelet adherence to de-endothelialized rabbit aortae in vivo, which is the initial step in thrombus formation. Whether ethanol inhibits subsequent reactions in thrombus formation induced by indwelling catheters in rabbit aortae was investigated. Before insertion of the catheters, the rabbits received injections of 51Cr-labeled platelets (and, in some experiments, [125I]fibrinogen), and were given ethanol by stomach tube, to achieve blood levels of approximately 4 mg/ml. Thrombus formation after 3 hours was assessed by determining the number of platelets and the amount of radiolabeled fibrin(ogen) associated with the aortae, and by determining the thrombus weights; all three measurements indicated a 64 to 84% reduction in thrombosis. Thus, experimentally induced thrombus formation in the aorta is significantly reduced by ethanol.     

Time course of arterial repair following endothelial denudation in the rat carotid artery

Summary Repair processes in the intima and media of the rat carotid artery were studied morphometrically for time intervals of up to 28 days after injury induced by air-drying. Air-drying injury included endothelial denudation as well as medial necrosis.Repair was most rapid between days 9 and 11 after injury as regards the increase in myointimal lesion size, the extent of repopulation of the media and re-endothelialization. After day 11, myointimal lesion size continued to increase until day 28. Medial repair, however, almost completely ceased at day 11, 25% of the inner media and 5% of the outer media remaining necrotic. At day 21, the vessels were almost completely re-endothelialized; however, even at day 28, about 10% of the middle of the vessel was still permeable to Evans Blue.The response of Sprague Dawley rats to injury differed from that of Wistar rats. Compared with Wistar rats, Sprague Dawley rats showed larger myointimal lesions, less medial necrosis and slower endothelial repair. It is suggested that the extent of medial necrosis and the speed of endothelial regeneration affect the arteriosclerotic response in rats     

Platelet Kinetics in Canine Ehrlichiosis: Evidence for Increased Platelet Destruction as the Cause of Thrombocytopenia

A significant (P < 0.025) increase in the mean platelet diameter occurred in five Ehrlichia canis-infected dogs when platelet numbers decreased to 100,000/mul or less. Maximal incorporation of [(75)Se]selenomethionine into platelets of six uninfected dogs was 0.080 +/- 0.019% (mean +/- standard error) and occurred 5 to 6 days after dosage, whereas maximal incorporation was 0.036 +/- 0.004% within 2 to 3 days after dosage in seven chronically infected dogs that had thrombocytopenia. Analysis of the [(75)Se]selenomethionine curves yielded a platelet lifespan of 9 days in uninfected dogs versus 4 days in chronically infected dogs. Thus, megakaryocyte maturation and/or platelet release occurred at an accelerated rate in infected dogs, whereas increased destruction of newly produced labeled platelets diminished their number of peripheral blood. [(51)Cr]sodium chromate-labeled platelet survival was exponential, with a half-life of approximately 1 day in two dogs at 2 to 4 days postinfection and three chronically infected dogs. Platelet survival time was 8 days and rectilinear in four uninfected dogs. Platelet recovery was 39.43 +/- 2.86% in infected dogs as compared with 68.2 +/- 10.72% in uninfected dogs. Whole-body scans of one dog prior to and 7 days after infection showed that labeled platelets were destroyed primarily in the spleen. It is concluded that the thrombocytopenia in E. canis-infected dogs is the result of increased platelet destruction which begins within a few days after infection.     

Deendothelialization in vivo initiates a thrombogenic reaction at the rabbit aorta surface. Correlation of uptake of fibrinogen and antithrombin III with thrombin generation by the exposed subendothelium.

Purified radiolabeled fibrinogen and antithrombin III (ATIII) were injected intravenously into rabbits before a deendothelializing injury to the aorta, and allowed to circulate for 0.1 to 6 hours before exsanguination, excision of the aorta, and quantification of each protein/unit area of subendothelium (intima-media). Uptake of fibrinogen was rapid (saturation 10 minutes after injury was approximately 13.0 pmol/cm2) compared with that of ATIII (45 to 60 minutes; 3.5 to 4.3 pmol/cm2). Both proteins associated primarily (greater than 90%) with the subendothelium rather than the platelet monolayer. The avidity of the deendothelialized vessel of these proteins was measured after a 20-minute circulation time at various intervals after injury. Whereas turnover of fibrinogen was fairly constant (approximately 100% per hour), that of ATIII was maximal (approximately 200% per hour) at 1 hour, decreasing to approximately 105% per hour at 5 hours after injury. The profile of ATIII turnover mirrored that of thrombin released in vitro from the deendothelialized aorta up to 10 days after injury, whereas the uninjured aorta and the aorta deendothelialized ex vivo adsorbed fibrinogen poorly and released negligible thrombin. Pretreatment of the aorta, deendothelialized ex vivo with thrombin in vitro increased fibrinogen uptake significantly. It is possible that, after deendothelialization in vivo, fibrinogen adsorption is determined largely by thrombin generation at the vessel wall. ATIII binding is limited by the availability of binding sites in the subendothelium, although the rate of thrombin generation influences ATIII turnover.     

The relation among vessel injury, thrombus formation, and platelet survival in rabbits

Continuous or repeated injury of rabbit aortae by indwelling vascular catheters caused the deposition of platelets on the injured vessels and the formation of thrombi rich in platelets and fibrin at sites where flow was most disturbed and injury was most extensive. Incorporation of 51Cr platelets into the thrombi reached a maximum between 3 and 24 hr. The platelet-fibrin-rich thrombi remained reactive to circulating platelets for at least 14 days. Continuing reactivity of thrombi and the turnover of platelets in the thrombi were accompanied by an increase in the proportion of platelets that separated in the least dense fraction on Stractan density gradients. Platelet survival was also shortened (43.5 +/- 5.9 hr in animals with catheters, compared with 62.6 +/- 4.5 hr in animals with a sham operation), indicating that some platelets that had taken part in thrombus formation or had interacted with the injured vessel wall were rapidly cleared from the circulation. Platelets from rabbits that had had indwelling aortic catheters in place for 3 or 6 days survived significantly longer than those from animals with a sham operation upon injection of the platelets into normal animals; thus, continuous turnover of platelets on injured vessels and thrombi, and the clearance of altered platelets, leads to a population of younger platelets that survive longer. The continuing reactivity of thrombi may in part account for repeated occlusive episodes in vascular disease. The contribution of thrombin generation and fibrin formation to the platelet-rich thrombi is substantial and warrants the ongoing evaluation of treatment with a combination of anticoagulant and antiplatelet agents in arterial thrombosis and in thrombus formation on vascular catheters.     

Kinetics of cellular proliferation after arterial injury. V. Role of acute distension in the induction of smooth muscle proliferation

Arteries denuded by the passage of a balloon catheter demonstrate marked smooth muscle cell (SMC) proliferation, whereas arteries denuded by a fine wire do not. We examined the possibility that vessel distension in addition to endothelial loss accounted for the SMC proliferation in the balloon model. Segments of rat left carotid isolated by temporary ligatures were distended hydrostatically with tissue culture medium at 300 mm Hg pressure via an external carotid cannula. Animals received either continuous [3H]thymidine infusion (intraperitoneally, Alzet pump, Alz Corp., Palo Alza, California) for 14 days or pulsed with [3H]thymidine at 1, 2, 3, or 7 days, and SMC proliferation was assessed by autoradiography. Distended vessels demonstrated patchy denudation acutely and complete endothelial regeneration by 3 days. No SMC were present in the intima at later times. Nevertheless, cumulative SMC proliferation over 14 days was 21 +/- 3% (perfused but not distended control: 0.8 +/- 0.6%; partially de-endothelialized but not distended control: 0.3 +/- 0.1%). Pulse-labeling demonstrated a peak of SMC proliferation between 2 and 3 days after injury. These results suggest that acute distension can contribute to the induction of SMC proliferation after endothelial denudation.     

Vascular wound healing and neointima formation induced by perivascular electric injury in mice.

Vascular interventions for atherothrombotic disease frequently induce neointima formation, which can contribute to restenosis of blood vessels. As the molecular mechanisms of this process remain largely unknown, quantitative models of arterial injury in transgenic animals may be useful to study this process at the genetic level. Here, an injury model is proposed in which surgically exposed femoral arteries in mice were injured perivascularly via a single delivery of an electric current. Transmission electron microscopy, light microscopy, and immunohistochemistry revealed that electric injury destroyed all medial smooth muscle cells, denuded the injured segment of intact endothelium, and transiently induced platelet-rich mural thrombosis. A vascular wound-healing response resulted that was characterized by degradation of the mural thrombus, transient infiltration of the vessel wall by inflammatory cells, and progressive removal of the necrotic debris. Topographic analysis revealed repopulation of the media and accumulation in the neointima of smooth muscle cells originating from the uninjured borders and progressing into the necrotic center. Within 3 weeks after injury, a neointima of 0.026 +/- 0.003 mm2 (n = 7 arteries) was formed that contained a maximum of 12 +/- 1 layers of smooth muscle alpha-actin-immunoreactive cells. Evans blue staining in five electrically injured arteries revealed a denuded distance of 2.8 +/- 0.2 mm immediately after injury, which became progressively re-endothelialized from the uninjured borders to 2.2 +/- 0.08 mm (P = 0.013 vs freshly injured by analysis of variance), 0.8 +/- 0.22 mm (P < 0.001), and 0.005 +/- 0.003 mm (P < 0.001) within 2, 7, and 14 days after injury, respectively. Analysis of 5'-bromo-2'-deoxyuridine incorporation revealed that a maximum of 35 +/- 10% endothelial cells proliferated within 2 days after injury and that in the media and neointima, a maximum of, respectively, 12 +/- 2% and 18 +/- 3% smooth muscle cells proliferated within 2 weeks after injury. Thus, electric injury of arteries provides a model of vascular wound healing with arterial neointima formation and re-endothelialization that may be useful for the genetic analysis of its molecular mechanisms in transgenic mice.     

Platelets mediate glomerular cell proliferation in immune complex nephritis induced by anti-mesangial cell antibodies in the rat.

We investigated whether platelets, which are rich in growth factors, could mediate glomerular cell proliferation in immune complex glomerulonephritis (GN) in the rat induced with an antibody directed against the Thy-1 antigen present on mesangial cells. Rats were depleted of platelets (mean platelet count less than 20,000/mm3) with goat anti-rat platelet IgG before induction of GN and platelet depletion was maintained for 48 hours. At 72 hours sections were immunostained for cyclin, an S-phase-related nuclear antigen, to identify proliferating cells, and for the common leukocyte antigen (CD45) to identify infiltrating leukocytes. Platelet depleted rats had fewer proliferating resident glomerular cells (CD45-, cyclin+) compared to controls (0.8 +/- 0.5 vs. 2.8 +/- 1.4 cells/glom cross section, P less than 0.01) and better renal function (creatinine 1.07 +/- 0.12 vs. 1.27 +/- 0.15 mg/dl, P less than 0.05). These effects were not due to changes in circulating or glomerular leukocyte counts, complement, or glomerular antibody binding. These studies provide the first direct evidence that platelets mediate glomerular (probably mesangial cell) proliferation in antibody-mediated GN.     

Sequence of cellular responses in rabbit aortas following one and two injuries with a balloon catheter.

In order to further elucidate the pathogenesis of intimal proliferation and increased thrombogenesis following repeated arterial injuries we studied the sequence of the cellular changes following two injuries of rabbit aortas with a balloon catheter. Following the first injury, the de-endothelialized surface was covered by a platelet monolayer. Polymorphonuclear leucocytes adhered to the inner surface of this monolayer and did not appear to penetrate the vessel wall. By 4 to 7 days, areas of neointima had formed. Within seconds after the reinjury at 7 days after the de-endothelialization small platelet aggregates formed on injured neointimal smooth muscle cells. Within I min platelet thrombi and fibrin strands formed. At 30 min most of the platelet thrombi had become fibrin-rich. Polymorphonuclear leucocytes had accumulated and many had begun to penetrate into the neointimal tissue. The number and extent of penetration of leucocytes into the inner parts of the arterial wall increased with time. Four days after the injury the neointimal cushions were restored and thickened. Both following the first and second injury the formation of neointimal cushions was accompanied by a change in the polarity of the inner layers of medial smooth muscle cells, some of which appeared to have migrated into the neointima.