Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats.

Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.     

P38 MAPK激活在高血糖致大鼠微血管通透性增高过程中的作用

目的研究P38 MAPK信号传导通路在糖尿病大鼠微血管内皮细胞通透性增高中的作用机制。方法SD雄性大鼠,腹腔注射链脲佐菌素(STZ 55 mg/kg)制备糖尿病模型。分正常组、糖尿病组、溶剂对照组、MKK6b(A)组和MKK6b(E)组。溶剂组:给予相同容量的柠檬酸缓冲液;MKK6b(A)组:在糖尿病模型基础上,实验前24 h尾静脉注射MKK6b(A)2.5 mL/kg;MKK6b(E)组:在实验前24 h给正常大鼠尾静脉注射MKK6b(E)2.5 mL/kg。用荧光强度检测微血管通透性;用荧光染色法观察微血管内皮细胞骨架蛋白的形态。结果糖尿病大鼠的微血管通透性明显高于正常组;注射MKK6b(E)可以增高大鼠微血管的通透性,而注射MKK6b(A)后微静脉的高通透性则被抑制。结论P38 MAPK信号转导系统参与介导了糖尿病微血管通透性增高的反应过程。     

Permeability of blood-brain and blood-nerve barriers in experimental diabetes mellitus in the anaesthetized rat.

Diabetes was induced with streptozotocin in rats weighing about 160 g. These were maintained with age-matched controls for up to 14 months, blood glucose being periodically monitored. Half the diabetic and control rats received the aldose reductase inhibitor, Ponalrestat, in their diet. At 3 weeks, 6-7 months and 13-14 months, the vascular permeability in regions of brain, and in optic and sciatic nerves, were measured by maintaining radiotracers in the bloodstream--125I-albumin (100 min), [14C]sucrose (60 min) and 131I-albumin (5 min)--followed by tissue sampling and counting at termination. 131I-albumin estimated residual intravascular plasma. Diabetes of up to 13-14 weeks caused no measurable increase in the sucrose permeability of microvessels in eight different brain regions, in optic or in sciatic nerve. At 3 weeks of diabetes, sucrose permeability in all brain regions and in optic nerve was reduced relative to that in controls. Extravascular albumin entry into different regions of brain and optic nerve was insignificant and insensitive to diabetes, except in the hypothalamus and optic nerves where it was raised with increasing duration of diabetes. In sciatic nerve, extravascular albumin distribution was markedly increased by diabetes, but sucrose permeability was not demonstrably affected. At the level used in the diet, Ponalrestat reduced the sorbitol content of diabetic sciatic nerve but did not protect again the increased permeability to albumin.     

Increased in vivo production of tumor necrosis factor after development of diabetes in nontreated, long-term diabetic BB rats.

We have recently reported that chronic and systemic administration of tumor necrosis factor alpha (TNF) inhibits development of autoimmune diabetes in NOD mice and BB rats, animal models of insulin-dependent diabetes mellitus (IDDM). During these experiments, we unexpectedly found that in vivo production of TNF stimulated by a single injection of lipopolysaccharide was enhanced approximately 10 times in the long-term diabetic BB rats (P less than 0.0001), whose mean duration of diabetes with more than 16.8 mM (300 mg/dl) of nonfasting blood glucose level was 26.2 +/- 2.1 days, as compared to that in the rats of nondiabetes and in the rats at the onset of diabetes, whose mean duration of diabetes was 1.4 +/- 0.6 days. The long-term diabetic, but not short-term-diabetic, rats were also associated with increased levels of serum fructosamine/albumin (P less than 0.01) and triglyceride (P less than 0.01) and with a decreased level of serum albumin (P less than 0.01). The in vivo TNF productivity in the diabetic rats, including the short-term- and long-term-diabetic rats, was correlated positively with the level of fructosamine/albumin (P less than 0.05) and negatively with the level of serum albumin (P less than 0.05), but not with levels of blood glucose. None of these correlations were observed in nondiabetic rats. The increased LPS-induced serum TNF activity in the long-term diabetic state was observed not only in BB rats but also in NOD mice and GK rats, a model of non-IDDM, irrespective of sexes and ages, indicating that the enhancement of in vivo TNF production was a result of long-term diabetes. These findings indicate that some factor(s) associated with the long-term-diabetic state may prime macrophages in vivo to produce TNF. Further study is needed to reveal a mechanism of the enhanced TNF production and its possible relevance to various abnormalities associated with the chronic hyperglycemic state.     

Effect of ethinyl estradiol on endothelial permeability to 125I-labeled albumin in female rats

Endothelial permeability to albumin was studied in the thoracic aorta and cerebral capillaries of oophorectomized rats injected intramuscularly with ethinyl estradiol daily for 10 days. One hour prior to sacrifice, treated animals and their pair-fed controls were each injected intravenously with 1.3 μg of ¹²⁵I-bovine serum albumin. Its passage into the aortic wall and the cerebral cortex was determined by autoradiography. Net grain counts were obtained separately in the inner and outer zones of the tunica media and in the ventral and dorsal regions of individual segments of the thoracic aorta, excluding branching sites. Separate counts were also obtained in sections of the frontal, right lateral and left lateral areas of the cerebral cortex, excluding the lumen of cerebral blood vessels.In the aorta, the average grain count was found to be significantly increased in estrogen treated rats in all except the ascending segment. This increase was comparable in the ventral and dorsal regions of the aortic wall and its magnitude did not differ significantly in the various segments, indicating a rather generalised effect of the hormone. The average grain count was significantly higher in the brain of treated rats than in the control. The magnitude of this increase was comparable in the three different areas of the cerebral cortex. Such findings point to a change in the blood-brain barrier leading to extravascular leakage of albumin into the adjoining cortical tissue.The present data indicate that synthetic estrogen administration led to enhanced endothelial permeability to albumin in the aorta and in cerebral capillaries. This confirms the results of an earlier study on the permeability of the aortic endothelium to perfused Evans blue-bound albumin in female rats.     

The Cellular Pathology of Experimental Hypertension: VI. Alterations in Retinal Vasculature

The ultrastructure of retinal arterial vessels from rats with severe renal hypertension has been studied. The permeability of retinal vessels has also been examined by means of vascular labeling technics utilizing horseradish peroxidase and microperoxidase as tracer substances. Small retinal arteries and arterioles exhibit foci of smooth muscle necrosis characterized initially by fragmentation of medial smooth muscle cells, and subsequently by loss of myofilaments and release of free vesicles, vacuoles and other cytoplasmic organelles extracellularly. Evidence for increased permeability is observed occasionally in retinal capillaries and less frequently in arteries and arterioles. The enzymatic tracers penetrate the tight junctions of the endothelial cells and are found in the basement membranes adjacent to endothelial and smooth muscle cells, as well as in expanded extracellular spaces around the capillaries. The alterations in the ultrastructure and permeability of retinal vessels in experimental hypertension have been compared with that of visceral and cerebral cortical vessels.     

An ultrastructural study of the mechanisms of proteinuria in rat nephrotoxic nephritis.

Nephrotoxic nephritis was induced in Sprague-Dawley and Munich-Wistar rats by the injection of rabbit antirat kidney serum. A biphasic pattern of proteinuria was induced: the heterologous phase with a peak of proteinuria occurring at 10 to 16 hours, and the autologous phase with a peak at 10 to 15 days. For morphologic studies, glomeruli were fixed by perfusion, or by drip-fixation during good blood flow. In the heterologous phase, glomerular endothelial detachment or loss and leukocytic infiltration were prominent. In the autologous phase, focal detachment of glomerular endothelium and epithelium was commonly found. At sites of endothelial loss, in both phases, endogenous albumin (demonstrated by an ultrastructural immunoperoxidase technique), but not intravenously injected ferritin, showed abnormally deep penetration into the glomerular basement membrane. At sites of epithelial loss, found in the autologous phase, both albumin and ferritin were detected throughout the glomerular basement membrane. It is proposed that, in glomerular disease, leakage of plasma proteins may occur across the glomerular basement membrane at sites of endothelial or epithelial detachment.     

Albumin permeability and electrical resistance as means of assessing endothelial monolayer integrity in vitro

Summary Methods were developed to measure albumin permeability and electrical resistance of bovine aortic endothelial cell (BAEC) monolayers cultured on porous polycarbonate filters. Permeability to 1% bovine serum albumin (Pe) was quantified by measuring the flux of fluorescent-labeled albumin with an apparatus in which there were no transmural oncotic or hydrostatic pressure gradients. The effect of passage of BAEC monolayers in culture on permeability was studied using 60 BAEC monolayers of Passage 6 to 10. There was no significant difference in Pe between passages, and the mean Pe of all monolayers was 4.5 ± 0.5 (SEM) × 10^–6 cm/s. Using these same BAEC monolayers, a fluorescent technique was developed to examine en face permeability patterns. Most BAEC monolayers demonstrated diffuse permeability across the monolayer, whereas others had focal regions of enhanced permeability despite similar Pe values. In those monolayers with punctate permeability, there were 5.4 ± 0.6 (SEM) focal regions of enhanced permeability per 1000 cells. To study the effect of culture time on monolayer integrity, electrical conductivities of nine BAEC monolayers were measured daily using a Millipore electrical resistance system. Electrical resistance increased from 4.5 ohm·cm² at Day 2 to a peak level of 11.4 ohm·cm² at Day 7 and then decreased daily to 4.0 ohm·cm² by Day 12. The in vitro BAEC monolayer has many of the transport characteristics of intact vessels, making these techniques useful in physiologic studies of the endothelial transport barrier. These methods provide relatively simple means of assessing the integrity of endothelial cell monolayers grown on porous substrates.     

In vitro assessment of human endothelial cell permeability: effects of inflammatory cytokines and dengue virus infection

Electrical resistance across human umbilical vein endothelial cells (HUVECs) was measured using an electrical cell sensor system. The transendothelial electrical resistance (TEER) value was used to estimate the permeability through endothelial cells in vitro. Decrease in the TEER value was associated with increase in the passage of albumin through endothelial cells in the albumin permeability assay. The effects of cytokines and dengue virus infection on the permeability of HUVECs were examined by measuring the TEER value. Tumor necrosis factor alpha (TNF-alpha) at 1 and 0.1 microg/ml decreased the TEER value, but TNF-alpha at lower dose did not. Interferon-gamma (IFN-gamma) at 1 microg/ml also decreased the TEER value. In contrast, interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) or interferon-beta (IFN-beta) did not decrease the TEER value. The decrease in the TEER value was associated with the morphological changes of HUVECs. Dengue virus infection at a multiplicities of infection (m.o.i.) of 5 pfu/cell decreased the TEER value. Infection at an m.o.i. of 0.5 pfu/cell did not decrease the TEER value; however, addition of 0.01 microg/ml of TNF-alpha to these infected endothelial cells decreased the TEER value. The results suggest that TNF-alpha and dengue virus infection decrease synergistically the TEER value of endothelial cells. The TEER method is easy, reliable and can be applicable to further analysis of the increase in the permeability of endothelial cells in vitro induced by inflammatory cytokines and dengue virus infection.     

Significant glomerular basement membrane thickening in hyperglycemic and normoglycemic diabetic-prone BB Wistar rats

The diabetic-prone BioBreeding Wistar rat (BB/DP) is an autoimmune model of insulin-dependent diabetes mellitus. Approximately 80-90% of the animals are hyperglycemic (BB/DP(h)) by 90-120 days of age while those that do not become diabetic in adolescence (BB/DP(n)) remain normoglycemic for life. Likewise, rats in the diabetes-resistant (BB/DR) strain are normoglycemic. Although renal morphological studies have been carried out in this model, ultrastructural observations of age- and diabetes-related extracellular matrix (ECM) changes, including glomerular basement membrane (GBM) morphometry, are not available. Moreover, possible renal changes in the relatively uncommon BB/DP(n) control animals have not been reported. The current electron microscopic study was carried out to investigate temporal changes in detergent-treated acellular ECM in BB/DP(h) rats at 2 weeks, 3 months, 6 months, and 1 year postonset of moderate hyperglycemia. Age-matched BB/DR and BB/DP(n) control animals were also examined. Our data demonstrate age- and diabetes-related alterations in mesangial matrix distributions and GBM widths and show for the first time significant increases in GBM thickening in both hyperglycemic (BB/DP(h)) and normoglycemic (BB/DP(n)) rats when compared to age-matched BB/DR controls. Surprisingly, the rate of increase is greatest in BB/DP(n) animals. Although the pathogenesis of diabetic basement membrane disease is not completely understood, GBM thickening is widely regarded as a morphological consequence of hyperglycemia. However, data in the current investigation show that ECM alterations, including significantly increased GBM thickness, may occur in genetically diabetic animals in the absence of hyperglycemia.     

Decreased anionic groups and increased permeability precedes deposition of immune complexes in the glomerular capillary wall.

To verify whether an increase in glomerular permeability precedes the deposition of immune complexes in the capillary wall, the following were studied in mice with lupus nephritis: 1) urinary proteins; 2) glomerular transfer of IgG, albumin, and anionic ferritin (AF) (isoelectric point, 4.2-4.6); and 3) anionic groups of the capillary wall as detected by binding of cationized ferritin (CF) (isoelectric point, 7.5-8.6). The glomeruli were investigated by immunofluorescence, immunoelectron, and transmission electron-microscopic studies. Urinary proteins were quantitated and characterized by electrophoresis. When mice were 5 months of age, (the time at which pathologic proteinuria was first detected), immune deposits were few and were confined to the mesangium. Although AF molecules were largely retained at the level of the lamina rara interna, focal leakage of albumin and IgG was detected across capillary walls that were not found to contain immune deposits. Focal and irregular loss of anionic groups, reflected by decreased binding of CF molecules, occurred in the laminae rarae of the basement membrane. Foot processes of glomeruli incubated with CF showed no loss of polyanion. We conclude that the increase in glomerular permeability that precedes deposition of immune complexes is due, in part, to focal loss of anionic groups of the basement membrane.     

1-磷酸鞘氨醇对血小板活化因子所致大鼠肠系膜微血管通透性增高的影响

目的:探讨1-磷酸鞘氨醇(S1P)对血小板活化因子(PAF)引起的大鼠肠系膜微血管通透性增高的影响。方法:本研究拟采用大鼠在体肠系膜微血管灌注的方法,通过测定微静脉的静水传导性(Lp),观察S1P对外源性PAF引起的微血管通透性增高的影响;并利用激光共聚焦显微镜技术,观察S1P对PAF引起的微血管荧光强度变化以及血管内皮细胞钙粘蛋白(VE-cadherin)变化的影响。结果:给予10nmol/LPAF作用后,大鼠肠系膜微血管Lp值明显增高,而经1μmol/L S1P预处理后,再给予PAF并未引起Lp的明显变化;PAF作用微血管后可见微血管内皮细胞间隙打开,微血管荧光强度明显增加,大量红色荧光微球(FMs)分布于内皮细胞间隙之中,S1P预处理后并未见内皮细胞间隙打开及FMs的明显积聚,微血管荧光强度与正常对照值比较无显著差异。结论:PAF可增加微血管的通透性,改变内皮细胞VE-cadherin正常结构,导致粘附连接断裂,细胞间隙形成,血管通透性增加可能与此结构变化有关。S1P能改善PAF引起的血管通透性增高,其作用与加强内皮细胞间粘附连接,抑制细胞间隙打开有关。     

Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery

Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.     

内毒素诱发肝性脑病大鼠脑超微结构观察

目的：观察内毒素诱发肝硬化大鼠发生肝性脑病时血脑屏障超微结构变化。方法：用小剂量内毒素（３ｍｇ／ｋｇＢＷ）一次性腹腔内注射诱发肝硬化大鼠发生肝性脑病。取部分大脑皮层组织制备超薄切片,在电子显微镜下观察其超微结构变化。结果：肝性脑病时血脑病大鼠脑微血管明显扩张。内皮皱折增多,内皮下基底膜电子密度降低。甚至局部溶解消失。微血管周围星状胶质细胞突起及皮层内胶质细胞明显肿胀。细胞器成分减少,基质解聚。结论     

Characterization of methylated bovine serum albumin-induced allergic inflammation in rats.

An air pouch type allergic inflammation in rats was induced using an insoluble cationic protein, methylated bovine serum albumin (MeBSA), as an antigen. Changes in vascular permeability, local tissue edema, histamine contents in the pouch fluid, and number of infiltrated leukocytes and chemotactic activity in the pouch fluid were analyzed during an 8-hour period after injecting the antigen solution into the air pouch of the immunized and nonimmunized rats. Vascular permeability during the first 30-min interval in the immunized rats was higher than that in the nonimmunized rats, reflecting a higher histamine level in the pouch fluid. However, both the increase in vascular permeability and histamine level in the immunized rats in this period were much lower than those induced by a soluble, noncationic antigen, azobenzenearsonate-conjugated acetyl bovine serum albumin. In the MeBSA-induced allergic inflammation model, a second peak of vascular permeability was induced at 2 h, and local tissue edema formation became apparent at 2 h, reaching a plateau at 4 h. A prominent increase in leukocyte infiltration, especially neutrophils, into the pouch fluid was induced at 4 h in accordance with an increase in chemotactic activity in the pouch fluid. These observations indicate that the acute phase of MeBSA-induced allergic inflammation is characterized by a weak anaphylactic response and a prominent neutrophil infiltration.     

Morphological aspects and observations about the permeability of the rat mesentery to the osmium-amine

In a previous work we felt that many authors were interested in observing the permeability and structure of the mesentery employing many techniques and analysing the material from different points of view. Our collaboration, at that time, was limited to the study of the permeability of the mesentery to the lanthanum nitrate and we came to a final conclusion that the penetration of the heavy salt is impeded by the presence of a basement membrane, localized between the mesothelial and connective tissue layers and that it was continuous to the basal lamina, located between neighbouring mesothelial cells; we could then observe that the lanthanum traveled through the cytoplasm and not in between mesothelial cells. In this paper we analyze the behavior of the mesentery in relation to the osmium-amine and we compare it to the observations made in our previous work. Generally speaking, we were unable to observe any reaction in any of the structures of the mesentery, except for some special cases. In those not very common cases, employing special techniques, we came to the conclusion that the osmium-amine reacts in the periphery (outer surface) of the mesentery and also penetrates, sometimes with no visible reaction, all the way to the elastic layer, where a reaction can be detected.     

Choroidal vasculature in diabetic rats

This study aims to evaluate the diabetic influence on the choroidal vessels morphology. Twenty Wistar rats were divided into a control (CG) and a diabetic group (DG). The animals had the diabetes induced by an intra-venous injection of Alloxan (42 mg/kg). Transmission electron microscopy analysis focusing the choroidal vessels was done one (T2) and twelve (T3) months after the diabetes induction. The CG rats in T3 showed vesicles and dense bodies in the endothelial and pericytic cells; the same structures were observed in the DG at T2. The DG rats in T3 had even more and intense changes than the T2DG rats. The morphological evaluation indicates that the choroidal vessels are affected in diabetes and the disease accelerates degenerative processes in the rat choroidal vasculature.     

Endothelial alterations and colloidal carbon permeability in the peripheral vasculature of the spontaneously hypertensive rat

Ultrastructural aortic endothelial cell changes were examined in 14- and 25-week spontaneously hypertensive (SHR) and normotensive (WKY) rats. Scanning and transmission electron microscopy revealed the presence of blister-like protrusions and cavity-like defects along intercellular junctional areas, resulting from dilatation of the intercellular space, subsequent elevation of the overlying marginal fold, and cellular breakage of this attenuated cellular extension. Junctional complexes, despite the presence of dilatated intercellular spaces, appeared intact. This form of endothelial damage, therefore, does not appear to result in abnormal, indiscriminate vascular permeability, at least to large macromolecules. This was confirmed by examination of representative segments of various SHR and WKY vessels after intravenous injection of colloidal carbon tracers. Only SHR mesenteric postcapillary venules (diameter 9–32 μm) showed permeability alterations, mediated through the formation of interendothelial gaps. In addition, these vessels were extensively fibrinized and occasionally showed evidence of hyalinization. The absence of permeability to colloidal carbon in arteries and arterioles in SHR may reflect the protective function of adaptive wall changes found in chronically hypertensive animals. In addition to changes in the media, these adaptive wall changes may also include endothelial cell alterations such as elongation of marginal folds, an increase in cellular overlap, and an increase in the complexity of intercellular connections, as observed in this study.     

Glomerular basement membrane thickness following islet transplantation in the diabetic rat.

Glomerular basement membrane (GBM) thickness was measured in diabetic rats prior to and following islet transplantation. Over the course of the experiment (from 7 to 13 months of diabetes) GBM thickness in diabetic animals exceeded that of littermate controls. After 7 months of diabetes a group of animals received successful intraportal transplants of neonatal pancreatic tissue. GBM thickness at 2 and 6 months following islet transplantation matched the thickness in nontransplanted diabetic rats and exceeded that in control animals. Failure to reverse GBM thickening in diabetic rats following islet transplantation may be due to very slow rates of GBM tunover in the rat. Previous work has demonstrated normalization of urinary albumin excretion after islet transplantation, suggesting that GBM thickening, per se, is not a significant factor causing albuminuria in rats with longstanding diabetes.     

Injury induces increase of von Willebrand factor in rat endothelial cells.

This study examined the effects of injury on the content of von Willebrand factor (vWF) in rat aortic endothelium. Endothelial cells from normal, endotoxin-treated, and balloon-injured rats were stained with vWF antibodies and visualized using a biotinylated secondary antibody and avidin-tagged peroxidase. Endotoxin treatment and balloon injury caused a threefold increase in intracellular vWF, and immunoelectron microscopy showed the endoplasmic reticulum to stain heavily by the vWF antibody. Weibel-Palade bodies were not observed in all the cell profiles examined. The basement membrane of the endothelialized vessels showed no vWF staining; however, after endothelial denudation this matrix was clearly stained by the antibody. These results suggest that endothelial injury leads to an increased intracellular content of vWF that is localized primarily in the endoplasmic reticulum.