Aprotinin decreases release of 6-keto-prostaglandin F1 alpha and increases release of thromboxane B2 in cultured human umbilical vein endothelial cells.

Use of the proteinase inhibitor aprotinin significantly improves hemostasis and reduces bleeding after operations in which extracorporeal circulation is used. The mechanism of action, however, has been only partially clarified. In this work we investigated whether aprotinin influenced the production and release of the eicosanoids prostacyclin, measured as the stable metabolite 6-keto-prostaglandin F1 alpha, and thromboxane A2, measured as the stable metabolite thromboxane B2, from endothelial cells. Human umbilical vein endothelial cells were incubated with different concentrations of aprotinin (5.5, 20, 55, and 100 mumol/L). The levels of 6-keto-prostaglandin F1 alpha and thromboxane B2 were measured at baseline and after thrombin stimulation. A concentration-dependent effect of aprotinin on 6-keto-prostaglandin F1 alpha synthesis was demonstrated. After incubation with 100 mumol/L of aprotinin, a 90% reduction in 6-keto-prostaglandin F1 alpha production was seen (31.69 versus 307.44 picograms per million cells; p less than 0.001). Conversely, thromboxane B2 production showed a 345% increase after incubation with aprotinin (287.80 versus 83.82 picograms per million cells; p less than 0.0001). Since 6-keto-prostaglandin F1 alpha inhibits and thromboxane B2 strongly enhances platelet aggregation, it appears that one mechanism of the clinically observed effectiveness of aprotinin lies in the altered ratio of 6-keto-prostaglandin F1 alpha: thromboxane B2 in endothelial cells, which leads to enhanced platelet aggregation and improved vessel sealing.     

Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig-to-human pulmonary xenotransplantation

Abstract: Background: In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Methods: Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E‐selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA‐treated blood (n = 5), and ATA and lepirudin‐treated blood (n = 5). Results: Aurintricarboxylic acid significantly inhibited TNF‐α‐ or lipopolysaccharide‐induced endothelial E‐selectin expression in a dose‐dependent manner. ATA also prevented human serum induced‐E‐selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin‐induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex‐vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Conclusions: Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.     

The cleaved peptide of PAR1 is a more potent stimulant of platelet-endothelial cell adhesion than is thrombin

PURPOSE: Platelet-endothelial cell adhesion is an important pathologic response to vessel injury or inflammation. On binding to its endothelial or platelet G protein-linked seven-transmembrane domain receptor, protease-activated receptor-1 (PAR1), thrombin releases a 41-amino acid peptide (TR(1-41)). We examined the effect of TR(1-41) on platelet activation and on platelet-endothelial cell adhesion. METHODS: A monolayer of confluent human saphenous vein endothelial cells was incubated with washed human platelets. Platelets were stimulated with either TR(1-41), TR(21-41), scrambled TR(1-41), adenosine diphosphate (ADP)-epinephrine (EPI), thrombin, or thrombin receptor activating peptide (TRAP). Platelet activation was identified with flow cytometry. The magnitude of platelet-endothelial cell adhesion was determined with a laser scanning cytometer that scanned the monolayer of endothelial cells and identified fluorescently bound platelets. RESULTS: Maximal thrombin stimulation (0.1 U/mL) induced a threefold increase in platelets bound to endothelial cells compared with buffer alone. Stimulation with TR(1-41) (20 mmol/L) tripled the number of platelets bound to endothelial cells compared with thrombin. Scrambled sequence of TR(1-41) (20 mmol/L) and TR(21-41) (20 mmol/L), neither of which induces platelet activation, had minimal effect on platelet adhesion. Both TRAP (20 mmol/L) and ADP-EPI (20 mmol/L) induced less platelet-endothelial cell adhesion than did thrombin. TR(1-41)-induced platelet-endothelial cell adhesion was partially blocked by glycoprotein (GP)IIb-IIIa-specific monoclonal antibody, 10E5 (10 mg/mL). CONCLUSIONS: TR(1-41), the cleaved peptide of PAR1, is a more potent stimulant of platelet-endothelial cell adhesion than is thrombin, TRAP, or ADP-EPI, and this adhesion is at least in part mediated by the platelet GPIIb-IIIa receptor.     

Precoating expanded polytetrafluoroethylene grafts alters production of endothelial cell-derived thrombomodulators.

Although the use of extracellular matrix proteins to precoat small-caliber vascular grafts before endothelial cell seeding has been shown to improve cell attachment, proliferation, and adherence, the effect of precoating on the thrombomodulatory properties of the seeded cells is unknown. The use of vascular prostheses lined with confluent endothelial cell monolayers expressing optimal thromboresistant properties may enhance patency rates. In this study human saphenous vein endothelial cells were seeded onto expanded polytetrafluoroethylene (ePTFE) graft material, both unmodified and precoated with fibronectin, type I collagen, or fibronectin and type I collagen (fibronectin/type I collagen). After 3 days of in vitro cultivation, endothelial cell production of prostacyclin, tissue plasminogen activator, and plasminogen activator inhibitor was evaluated under basal conditions and after stimulation with arachidonate or thrombin. Production of tissue plasminogen activator by endothelial cells cultured on fibronectin-ePTFE was significantly greater compared with production by endothelial cells grown on noncoated or fibronectin/type I collagen-ePTFE under basal conditions (p values less than 0.01 and less than 0.05, respectively) and in response to thrombin (p values less than 0.002 and less than 0.003, respectively). Plasminogen activator inhibitor-1 production was not detected in any of the four experimental groups. Endothelial cells cultured on fibronectin-ePTFE also synthesized significantly more prostacyclin than endothelial cells grown on type I collagen- or fibronectin/type I collagen-ePTFE, under basal conditions (p values less than 0.02 and less than 0.01, respectively) and in response to arachidonate (p values less than 0.03 and less than 0.002, respectively) and thrombin (p values less than 0.003 and less than 0.002, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of Angiogenic Potential of Human Microvascular Endothelial Cells and Human Umbilical Vein Endothelial Cells

Summary BACKGROUND: Endothelial cells cultured in vitro are commonly used as a model for testing the effects of therapeutic or detrimental agents on endothelium. Cells originating from different vascular beds display, however, a heterogeneity of function and phenotype. Here we compared the production of angiogenic growth factors and the sensitivity to exogenous growth factor stimulation of two popular endothelial cell types. METHODS: Experiments were performed on human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) incubated in optimal conditions for 24–48 h. RESULTS: The profile of the spontaneously produced growth factors differed significantly between the two different cell lines tested. HUVEC did not produce detectable amounts of VEGF, whereas HMEC-1 released 24.9 pg/ml and this amount was significantly increased in response to IL-1. Instead, HUVEC produced high concentrations of soluble form of VEGF receptor-1 (VEGF-R1), whereas the release of VEGF-R1 from HMEC-1 was 10-fold lower. Small amounts of bFGF were found in media from both cell types, but higher levels were detected in HMEC-1 cultures. In contrast, the secretion of interleukin-8 (IL-8) and matrix metalloproteinase-1 (MMP-1) were 30- to 40-fold higher in HUVEC than in HMEC-1. The cell types differed also in their sensitivity to exogenous growth factors. The basal proliferation of HUVEC was very low but could be effectively stimulated by supplementation with VEGF or bFGF. HMEC-1 proliferated spontaneously and their proliferation rate was not further augmented by growth factors. Similarly, the spontaneous outgrowth of capillaries was negligible in HUVEC but well pronounced in HMEC-1. CONCLUSIONS: Production of angiogenic agents and sensitivity to exogenous growth factors is cell-type dependent. HUVEC, which do not release VEGF, can be easily stimulated with exogenous factors, whereas HMEC-1, which are able to produce VEGF, do not respond well to the additional stimulation. Our study demonstrates that conclusions resulting from in vitro experiments performed on only one type of endothelial cells can be misleading.     

Endothelial cells incubated with platelet‐rich plasma express PDGF‐B and ICAM‐1 and induce bone marrow stromal cell migration

Platelet‐rich plasma (PRP) is used to accelerate bone repair through the growth factors released by platelets. The purpose of this study was to evaluate if PRP induce human umbilical vein endothelial cells (HUVEC) to express mRNA for osteogenic growth factors and stimulate the migration of bone marrow stromal cell (BMSC). The effects of PRP were compared to those induced by vascular endothelial growth factor‐A (VEGF‐A) or, as a negative control, by platelet poor plasma (PPP). After incubation with PRP, but not with PPP, HUVEC showed an increased expression of mRNA for platelet derived growth factor‐B (PDGF‐B), and this effect was not inhibited by an anti‐VEGF‐A antibody. The migration of BMSC was more stimulated by HUVEC incubated with PRP than by HUVEC incubated with low serum medium or PPP. Besides, PRP increased the expression of intercellular adhesion molecule‐1 (ICAM‐1) and osteoprotegerin, but did not affect the expression either of the receptor activator for nuclear factor κB ligand (RANKL) or of RANK. These findings support the hypothesis that PRP contribute to bone repair by favoring the pro‐osteogenic function of endothelial cells, including the recruitment of osteoblast precursors and the expression of adhesion molecules for monocyte/macrophages, while inhibiting their pro‐osteolytic properties. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1493–1498, 2009     

Aprotinin inhibits leukocyte-endothelial cell interactions after hemorrhage and reperfusion

BACKGROUND: The serine protease inhibitor aprotinin has been successfully used to reduce blood loss in patients undergoing cardiac operations. We studied aprotinin for its ability to modulate leukocyte-endothelial cell interactions after ischemia and reperfusion. METHODS: The effects of aprotinin on leukocyte-endothelial cell interactions were observed by intravital microscopy in the rat mesenteric microcirculation and immunohistochemical analysis. The inflammatory cascade (leukocyte rolling, firm adherence, and transmigration) was studied after thrombin stimulation and after hemorrhage and reperfusion. RESULTS: Intravenous bolus administration of aprotinin treatment (20,000 U/kg) significantly reduced leukocyte rolling from 55 +/- 8 to 17 +/- 3 cells/min (p < 0.01) and adherent cells from 12 +/- 2 to 7 +/- 1.4 cells (p < 0.05) along the venous endothelium of the rat mesentery after thrombin activation. In addition, aprotinin pretreatment significantly inhibited transmigration of leukocytes from 11.3 +/- 1.2 to 6.0 +/- 1.1 cells (p < 0.05) through the microvascular endothelial wall. Similarly, aprotinin decreased leukocyte-endothelium interaction after hemorrhagic shock. Moreover, immunohistochemistry demonstrated that aprotinin significantly attenuated P-selectin expression by the intestinal vascular endothelium. CONCLUSIONS. Our data demonstrate that aprotinin potently inhibits recruitment of leukocytes in the microvasculature by interfering with endothelial cell-polymorphonuclear neutrophil interaction, and is a potent endothelial protective agent in clinically relevant doses. Thus, aprotinin pretreatment may be useful for primary prevention of inflammatory tissue injury mediated by ischemia-reperfusion injury such as shock, trauma, open heart operation, or other extensive vascular surgical procedures.     

Tacrolimus prevents von Willebrand factor secretion by allostimulated human glomerular endothelium

Little is known about the endothelial injury caused directly by circulating donor‐specific antibodies (DSAs) during antibody‐mediated rejection. von Willebrand factor (vWF) is a highly thrombotic glycoprotein stored in Weibel‐Palade bodies in endothelial cells. It has been shown that its secretion is triggered by allostimulation. Calcineurin‐like phosphatases regulate pathways involved in vWF secretion. Therefore, we hypothesized that tacrolimus would prevent alloantibody‐induced glomerular lesions, in part via inhibition of vWF secretion from endothelial cells. Here, we used a human in vitro model of glomerular endothelium expressing HLA class I and II antigens and demonstrated that anti–HLA class II antibodies elicit a higher endothelial release of vWF than do anti–HLA class I antibodies in cell supernatants. We observed that tacrolimus treatment decreased vWF secretion after stimulation with both classes of anti‐HLA antibodies and decreased platelet adhesion on allostimulated endothelial cells in a microfluidic chamber. In kidney recipients, tacrolimus trough levels were negatively associated with vWF blood levels. These results indicate that direct disruption of hemostasis via vWF secretion is a potential mechanism of antibody‐mediated injury in patients with DSAs. Our results further suggest that the targeting of microcirculation hemostasis may be beneficial to prevent the development of microangiopathic lesions in antibody‐mediated rejection.     

Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro. Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothelial cells (HUVEC) were prepared in suspension (5 × 10⁶/ml) using EDTA-collagenase. Human platelet rich plasma (PRP) and washed platelets with platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors. All aggregation tests used a two-sample, four-channel, model 560 Ca Lumi-Aggregometer (Chronolog Corporation, Havertown, PA). Selected assays for complement (C3a; C5b-C9; CH50; and AP50) were then performed on supernatant fluids. To test the effects of complement inhibition and thrombin antagonists, the following agents were pre-incubated with PRP (or PPP) in various titrations for 10 min at 37°C prior to combination with pEC in the aggregometer: soluble complement receptor typel (sCR1); Cobra Venom Factor (CVF), heparin, hirudin, and anti-CD31 (anti-PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10–20% human PPP; supernatant fluids were harvested at various time points and used for platelet activation assays and for functional tests of thrombin or levels of thrombin-antithrombin complexes (TAT). Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platelet activation could be inhibited by pre-incubation of samples with CVF (2–5 IU/ml to deplete complement components) and to a variable extent with sCRl (1–2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient samples also supported platelet activation. Aggregation of platelets was inhibited by preincubation of PRP with concentrations of hirudin (1 unit/ml) and heparin (5–10 units/ml) sufficient to neutralize thrombin generated. Supernatant fluids containing PPP removed from pEC were found to have increased levels of thrombin and thrombin-antithrombin complexes and could also activate platelets in a hirudin sensitive manner. Discussion: Platelet and pEC combinations underwent aggregation only in the presence of complement components. This process appeared independent, at least in part, of the assembly of terminal complement components. Consequent pEC generation of thrombin appeared adequate to trigger platelet aggregation which could in turn be inhibited by hirudin or heparin in vitro.     

The effects of thrombin on bovine aortic endothelial and smooth muscle cells

Recent evidence suggests that thrombin interacts with various cell types, stimulating cellular proliferation and protein and prostanoid production. To further delineate its role in vascular healing, we have studied the effects of thrombin on proliferation and matrix production by the cells of the vessel wall. The addition of thrombin (1 unit/ml) to cultures of bovine aortic smooth muscle cells resulted in an increase in cell proliferation (p less than 0.01) and number (p less than 0.03), whereas in cultures of bovine aortic endothelial cells thrombin produced a decrease in cell proliferation (p less than 0.01) and number (p less than 0.02). Thrombin also altered matrix composition in cultures of these cells. In both bovine aortic endothelial cells and bovine aortic smooth muscle cell cultures grown in the presence of thrombin, total protein content was significantly increased when compared to controls (p less than 0.03). In bovine aortic endothelial cell cultures the addition of thrombin resulted in a decrease in collagen content (p less than 0.01) and an increase in sulfated glycosaminoglycan content (p less than 0.02). In contrast, in bovine aortic smooth muscle cell cultures thrombin resulted in an increase in collagen content (p less than 0.03), whereas glycosaminoglycan content was unaffected. These findings suggest that thrombin may significantly influence vascular healing and function by altering cell number and matrix composition.     

Effect of advanced glycation end-products on gene expression and synthesis of TNF-alpha and endothelial nitric oxide synthase by endothelial cells

BACKGROUND: Advanced glycation end-products (AGEs), which are formed in aging, diabetes mellitus, and kidney failure are implicated in the occurrence of vascular complications. We, thus, evaluated in cultured endothelial cells, the AGEs' effect on gene expression and synthesis of tumor necrosis factor-alpha (TNF-alpha) and endothelial nitric oxide synthase (eNOS) mRNA and protein expression, which may be involved in vascular remodeling. METHODS: Human umbilical vein cords endothelial cells (HUVEC) were stimulated with AGE-specific compounds [AGE-human serum albumin (AGE-HSA), N(epsilon)-carboxymethylysine (CML), AGE-beta2 microglobulin (AGE-beta2m)], and thereafter, incubated with interleukin1-alpha, lipopolysaccharide, and interferon-gamma. RESULTS: mRNA expression and secretion of TNF-alpha were significantly enhanced after incubation with AGE-HSA, CML, and AGE-beta2m compared to that found in HUVEC incubated with HSA or beta2m. AGE-HSA, CML, and AGE-beta2m induced a significant decrease in eNOS protein and mRNA expression. CONCLUSION: AGEs promote mRNA expression and secretion of TNF-alpha and reduce eNOS mRNA and protein expression in HUVEC. Such changes may play a role in the vascular dysfunction and the development of vasculopathy seen in diabetes, uremia, and old age.     

Insulin inhibition of endothelial prostacyclin production

Graft thrombosis is a common cause of early graft failure in pancreatic transplantation, even in the absence of rejection. Altered endothelial cell (EC) production of thromboactive substances may play a primary role in this and other settings of thrombosis where intraluminal insulin concentrations are high. We therefore investigated the effect of insulin on EC production of prostacyclin (PGI2), a potent endogenous antagonist of platelet aggregation and vasodilator. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated for 15 min in Hanks'/Hepes buffer containing 0, 50, 250, or 500 microU/ml of human insulin or 0, 500, 1000, or 2000 microU/ml of porcine insulin. PGI2 production was assessed by exposing the monolayers to either 20 microM arachidonic acid (stimulated) or arachidonic acid vehicle (basal) for an additional 15 min. Test buffers were then assayed by RIA for 6-keto-PGF1 alpha, the stable metabolite of PGI2. The results of these experiments indicate that human insulin inhibits both basal and arachidonic acid-stimulated production of PGI2 from HUVEC in a dose-dependent manner. Inhibition of stimulated production was significant at concentrations of 250-500 microU/ml. Porcine insulin also inhibited arachidonic acid-stimulated production of PGI2 from HUVEC in a dose-dependent manner. However, HUVEC were less sensitive to porcine insulin than to human insulin and a concentration of 2000 microU/ml was required for significant inhibition. We therefore conclude that insulin, in locally high concentrations, inhibits endothelial PGI2 production in vitro. The ability of insulin to alter the production of thromboresistant substances from endothelium may facilitate thrombosis in circumstances where counterregulatory mechanisms are disturbed by injury or transplantation.     

A preliminary study on the effects of exercising to maximum walking distance on platelet and endothelial function in patients with intermittent claudication.

BACKGROUND: Platelet and endothelial activation has been shown to be increased in patients with intermittent claudication (IC). Recent studies have suggested that exercise may induce further platelet activation. The aims of this study were to investigate the effect of exercising to maximum walking distance on platelet and endothelial function in patients with intermittent claudication who were receiving statin and aspirin therapy compared with age matched healthy controls. METHODS: Platelet aggregation through COX-mediated and thrombin receptor activator peptide (TRAP)-stimulated GPIIb/IIIa pathways was measured by the Ultegra point of care system in 20 patients with IC on aspirin and 20 healthy volunteers before, immediately and 1h after exercising to treadmill maximal walking distance (MWD). Soluble P-selectin, vWF and sICAM were measured using an enzyme linked immuno-sorbent assay technique. RESULTS: Baseline platelet aggregation was significantly reduced in patients with IC compared to volunteers (p<0.05). In patients, exercising to MWD significantly reduced platelet aggregation (COX, median -5% [range -24 to 13%]; p = 0.02; GPIIIa/IIb, median -13% [range -72 to 33%]; p = 0.02) immediately post-exercise which returned to baseline values at 1 h. There was no change in the healthy volunteers following the same median duration of exercise. Baseline sP-selectin levels were higher in the patients with IC compared to the healthy volunteers [Median values (interquartile range), 42.72 (33.28-54.24) versus 29.16 (24.40-34.10), p = 0.0003] but there were no differences in vWF levels. Both sP-selectin and vWF levels increased significantly in the control and patient group following exercise (p<0.005). sICAM were higher at baseline in the patients with IC but were unchanged following exercise [Median values (interquartile range),560.9 (405.5-739.4) versus 467.0 (325.7-643.4), p<0.05]. CONCLUSION: This study is the first to show that platelet aggregation is reduced immediately following treadmill exercise to maximum walking distance in patients with IC despite a rise in sP-selectin and vWF, suggesting endothelial activation. The inhibition of platelet aggregation after exercise in subjects on antiplatelet and statin therapy suggests that exercise is unlikely to exacerbate platelet thrombus formation in patients with IC.     

Secretory activity of the coronary artery endothelial cells in conditions of the peritoneal dialysis

IntroductionEndothelial dysfunction is frequent in patients treated with peritoneal dialysis and may lead to cardiac complications. We evaluated the effect of effluent dialysates and serum on the function of coronary artery endothelial cells (CAEC).MethodsHuman CAEC in in vitro culture were exposed to serum and dialysates from 24 patients treated with continuous ambulatory peritoneal dialysis (CAPD) and secretion of interleukin-6 (IL6), von Willebrand factor (vWF), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured. Modulation of the secretory activity of CAEC by Sulodexide, mixture of glycosaminoglycans: heparin sulfate and dermatan sulfate, was studied.ResultsSerum from CAPD patients stimulated synthesis of IL6 (+93%), vWF (+18%), and PAI-1 (+20%) and did not change t-PA secretion in CAEC. Dialysates stimulated secretion of IL6 (+89%), vWF (+29%), and PAI-1 (+31%) and did not change t-PA synthesis. Dialysates collected in 12 patients after 6 months more strongly stimulated synthesis of IL6 (+37%) and PAI-1 (+7%). Sulodexide suppressed the secretory activity of CAEC stimulated by the studied sera: IL6 (–38%), vWF (–19%), t-PA (–13%), and PAI-1 (–12%).ConclusionsSerum and the dialysate from CAPD patients induce inflammatory and prothrombotic reaction in coronary arterial endothelial cells. The general pattern of the observed effects for serum and dialysates was similar but the intensity of the effects was not identical. Sulodexide reduced these effects.     

p38 MAPK在LPS诱导内皮细胞表达ICAM-1中的作用

目的 研究p38丝裂原激活蛋白激酶(MAPK)信号转导通路在脂多糖(LPS)诱导人脐静脉内皮细胞(HUVEC)表达细胞间粘附分子-1(ICAM—1)中的作用。方法 脐静脉内皮细胞培养后分为两组：(1)刺激组,设不同时相点分别用LPS刺激内皮细胞;(2)预处理组,在LPS刺激前2h,用SB203580预处理内皮细胞。观察ICAM—1蛋白和mRNA表达的变化,检测内皮细胞p38MAPK活性变化。结果 LPS刺激后,内皮细胞表面ICAM—1分子在8～36h显增加,胞浆中mRNA在2h即有显增加;LPS刺激HUVEC后15min,p38MAPK活性即有升高,30～60min达高峰。p38抑制剂SB203580可显抑制LPS的诱导作用。结论 LPS可能通过激活p38MAPK信号转导通路,调节HUVEC的ICAM—1基因和蛋白表达。     

Platelet-activating factor synthesis and response on pancreatic islet endothelial cells: relevance for islet transplantation

BACKGROUND: Recent data suggest that donor intraislet endothelial cells may survive islet transplantation and participate to the events that influence islet engraftment. However, the mechanisms that regulate islet endothelial behavior in this setting are poorly known. METHODS: We obtained immortalized human (hIECs) and mouse (mIECs) islet endothelial cells by transfection with SV40-T-large antigen and studied the synthesis and response to Platelet-activating factor (PAF), a multipotent phospholipid that acts as endothelial mediator of both inflammation and angiogenesis. RESULTS: HIECs showed typical endothelial markers such as expression of vWF, CD31, and CD105, uptake of acetylated-LDL and binding to ULE-A lectin. Moreover, they expressed nestin, the PAF-receptor and possess surface fenestrations and in vitro angiogenic ability of forming tubular structures on Matrigel. Likewise, mIECs showed expression of vWF, CD31, nestin, PAF-receptor and CD105, and uptake of acetylated-LDL. HIECs and mIECs rapidly produced PAF under stimulation with thrombin in a dose-dependent way. Exogenous PAF or thrombin-induced PAF synthesis increased leukocyte adhesion to hIECS and mIECs and cell motility of both endothelial cell lines. Moreover, PAF or thrombin-induced PAF synthesis accelerated in vitro formation of vessel-like tubular structures when hIECs are seeded on Matrigel. Notably, gene-microarray analysis detected up-regulation of beta3 integrin gene on hIECs stimulated with PAF, that was confirmed at the protein level. CONCLUSIONS: Based on the novel development of immortalized islet endothelium, these results suggest that PAF may have a dual role that links inflammation to angiogenesis in the early events of islet transplantation.     

烧伤血清对内皮细胞核因子-κB核移位的影响

目的　了解烧伤血清对内皮细胞核因子 κB (NF κB)异二聚体 p5 0 /p6 5核移位 ,以及核抑制因子κB(IκB)α降解的影响 ,进一步探讨烧伤血清对内皮细胞的活化作用。方法　采用体外培养的人脐静脉内皮细胞株ECV 30 4 ,分别用正常人血清、烧伤患者血清、烧伤患者血清 吡咯烷二硫代氨基甲酸盐 (PDTC)刺激内皮细胞 ,并以正常培养的内皮细胞为对照。应用激光共聚焦显微镜观察刺激 30、6 0、12 0、4 80min后内皮细胞 p5 0 /p6 5核移位情况 ,采用Western印迹法检测刺激 30、6 0、90、12 0min后内皮细胞IκBα蛋白降解的情况。　结果　与对照组比较 ,烧伤血清刺激内皮细胞 30min后 ,p5 0、p6 5即发生核移位 ,30～ 6 0min达高峰 ,2h后恢复至刺激前状态 ;而烧伤血清刺激 30min后 ,IκBα发生降解 (P <0 .0 1) ,刺激 4 5～ 6 0min后最为明显 ,2h后表达逐渐恢复。PDTC能有效抑制烧伤血清作用 30、6 0min后 ,p5 0、p6 5的核移位和IκBα降解。 　结论　烧伤血清可导致内皮细胞NF κBp5 0、p6 5发生核移位 ,并使IκBα降解 ,进而活化NF κB ,诱导内皮细胞分泌细胞因子。PDTC对这一系列变化可能有抑制作用     

Thiamine reverses hyperglycemia-induced dysfunction in cultured endothelial cells.

BACKGROUND: High levels of glucose have previously been shown to inhibit endothelial cell migration and increase secretion of the von Willebrand factor (vWF), a marker of endothelial cell damage. This study investigates whether thiamine, an important coenzyme in intracellular glucose metabolism, improves endothelial cell migration and decreases von Willebrand factor secretion under hyperglycemic conditions. METHODS: Bovine aortic endothelial cells (BAECs) were grown under physiological glucose (5.5 mmol/L) and hyperglycemic (13.8 mmol/L and 27.7 mmol/L) conditions with or without thiamine (200 micromol/L) supplementation. Endothelial cell migration was investigated in monolayers of BAECs that were wounded by scraping. The distance of migration, the number of migrating cells, and the surface area covered by the migrating cells were measured. Secretion of vWF by BAECs under physiological glucose and high glucose conditions with or without thiamine (200 micromol/L) supplementation was studied with enzyme-linked immunosorbent assay. RESULTS: Under hyperglycemic conditions, there was a significant decrease in the number of endothelial cells and an increase in the secretion of vWF (P <.001). Thiamine treatment limited this inhibitory effect of elevated glucose levels on BAECs. Glucose (27.7 mmol/L) significantly decreased the migration distance of BAECs into the wounded area to 4.0 +/- 1.4 cm, as compared with 6.2 +/- 0.3 cm in the control. Thiamine supplementation restored the migration distance by BAECs (6.94 +/- 0.7 cm) and the wound surface area covered (47.7 +/- 5.6 cm(2)) (P <.001). CONCLUSIONS: Hyperglycemia activates BAECs and promotes secretion of vWF, a marker of endothelial cell damage. Thiamine inhibits this endothelial cell activation and the effects of hyperglycemia on endothelial cell migration. This beneficial effect of thiamine limiting endothelial cell dysfunction is possibly through the diversion of glucose flux from anaerobic to aerobic pathways. The data from this study lead to the speculation that thiamine intake may mitigate or delay vascular complications of diabetes.     

Oxidized LDL induces proliferation and hypertrophy in human umbilical vein endothelial cells via regulation of p27Kip1 expression: role of RhoA

Oxidized LDL (OxLDL) induces proliferation in human umbilical vein endothelial cells (HUVEC). The influence of OxLDL on the cyclin-dependent kinase inhibitor p27(Kip1), on the activity of the small GTPase RhoA as a known regulator of p27(Kip1), and on resulting cell proliferation and hypertrophy was studied. HUVEC were stimulated with OxLDL (1 to 50 mug/ml). Proliferation was quantified by (3)H-thymidine incorporation, colorimetric 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazolium bromide assay, and cell count and was compared with proliferation of HUVEC that were transfected with dominant negative RhoA or treated with the Rho-kinase inhibitor Y27632. Hypertrophy was quantified by (3)H-leucine incorporation and by planimetry. p27(Kip1) expression was determined by Western blot analysis. p27(Kip1) was downregulated by transient transfection with antisense oligonucleotides. Low concentrations of OxLDL induced proliferation of HUVEC, paralleled by a persistent decrease of p27(Kip1) expression. With the use of antisense oligonucleotides, further downregulation of p27(Kip1) expression enhanced the OxLDL-induced proliferative response. High concentrations of OxLDL resulted in cellular hypertrophy and caused a delayed increase in p27(Kip1) expression after initial downregulation. Concomitant, OxLDL caused a significant activation of the small GTPase RhoA. In cells that were transfected with dominant negative RhoA, the effect of OxLDL on p27(Kip1) expression and on cellular proliferation was abolished. HUVEC that were preincubated with the Rho-kinase inhibitor Y27632 also showed a significantly decreased proliferative response to OxLDL stimulation. In summary, OxLDL has a dual effect on cell-cycle progression via regulation of p27(Kip1) expression, resulting in cellular proliferation and hypertrophy, involving activation of RhoA. OxLDL may importantly contribute to vascular hyperplasia in atherosclerosis and other diseases associated with increased levels of OxLDL.     

Differential responsiveness of early- and late-passage endothelial cells to shear stress

BACKGROUND: The incidence of vascular disease increases with age. Because atherosclerosis and neointimal hyperplasia colocalize in areas of disturbed shear stress, the effects of orbital shear stress (SS) on endothelial cell proliferation, protein kinase B (Akt) activation, and functional activity were analyzed using a senescence model. METHODS: Early- (p3 to 7) and late- (p28 to 32) passage bovine aortic endothelial cells were exposed to orbital SS (210 rpm) or static conditions (0 to 5 days). Cell proliferation was directly counted and confirmed with proliferating cell nuclear antigen reactivity. Phosphorylated and total Akt were assessed with Western blotting. Endothelial cell-induced smooth muscle cell migration was assessed with a Boyden chamber. RESULTS: Late-passage endothelial cells demonstrated no increase in orbital SS stimulated proliferation compared with early-passage cells (P = .42). Late-passage endothelial cells demonstrated decreased Akt phosphorylation in response to SS compared with early passage cells (n = 6, P = .01). Late-passage cells induced 26% less smooth muscle cell migration than early-passage cells (n = 3, P = .03). CONCLUSIONS: Late-passage endothelial cells demonstrate decreased proliferation, Akt phosphorylation, and secretion of smooth muscle cell chemoattractants in response to orbital SS compared with early passage cells. These results suggest that late-passage endothelial cells respond to SS differently than early-passage cells and confirm the utility of the in vitro senescence model.