von Willebrand factor, endothelial dysfunction, and cardiovascular disease

von Willebrand factor (VWF), a glycoprotein involved in arterial thrombus formation, is released into the circulation by secretion from endothelial cells. Plasma VWF levels are determined by genetic factors including ABO blood groups and VWF mutations, and by non-genetic factors including aging, impaired nitric oxide production, inflammation, free radical production and diabetes. Plasma VWF levels have been proposed as a risk factor for cardiovascular events. Although they are only weakly associated with the risk of coronary heart disease (CHD) in the general population, they are a more promising CHD risk factor in high-risk populations with previous cardiovascular events, diabetes or old age. However, is it still unclear whether VWF levels directly determine the rate and severity of arterial thrombus formation or whether they merely reflect alteration in other endothelial functions. The future status of VWF levels as a cardiovascular risk factor depends on additional studies on the genetic determinants of both VWF levels and cardiovascular outcomes. Further studies on VWF levels as a predictor of the risk of stroke (rather than CHD) in elderly or other high-risk population are also promising. Such studies could lead to the clinical use of plasma VWF levels to refine the estimation of the cardiovascular risk and of the expected benefit of antithrombotic agents.     

The effects of exercise stress testing on soluble E-selectin, von Willebrand factor, and circulating endothelial cells as indices of endothelial damage/dysfunction

BACKGROUND: The quantification of circulating endothelial cells (CECs) in whole blood is an increasingly recognized index of endothelial damage/dysfunction. Abnormal CECs have been linked to the severity of coronary artery disease (CAD). OBJECTIVE: We assessed the relationship of CECs to other markers of endothelial dysfunction (von Willebrand factor (vWF) and soluble E-selectin (sEsel)) during exercise stress testing (EST) in a cohort of patients with suspected CAD. METHODS: We studied a cohort of patients referred to our chest pain clinic with a history of exertional chest pain. Treadmill EST was performed, using a full Bruce exercise protocol. Blood for CECs (immunobead method), vWF and sEsel (both ELISA) were collected immediately before (pre-exercise), immediately following exercise, and at 30 minutes post-EST. RESULTS: We studied 31 patients (84% male; mean (SD) age 58.4 (9.8) years). Of the entire cohort, 14 patients (45.2%) had a positive EST. Exercise led to significant increases in levels of CECs, sEsel, vWF, white blood cells (WBC), heart rate, mean and systolic blood pressure compared with base-line (all P < 0.05). There was a significant correlation between the change (delta (immediate post-pre-exercise)) in CECs and delta vWF (r = 0.45; 95% CI 0.11-0.69: P = 0.01) and delta sEsel (r = 0.41; 0.05-0.7: P = 0.02), as well as between delta vWF and delta sEsel (r = 0.55; 0.25-0.76: P = 0.001). Neither absolute nor delta CEC counts were predictive of exercise work-load/functional capacity, nor the presence of positive EST results. CONCLUSION: EST led to a significant increase in endothelial markers (CECs, vWF, and sEsel) compared with base-line levels. The rise in CECs correlated with the increases in other endothelial markers, but was not related to the either exercise workload/capacity or to the presence of a positive EST.     

Endothelial cell synthesis of von Willebrand antigen II, von Willebrand factor, and von Willebrand factor/von Willebrand antigen II complex.

von Willebrand antigen II (vW AgII) and von Willebrand factor (vWf) are immunochemically distinct proteins that are deficient in the plasma and platelets of patients with severe von Willebrand's disease. Normal human umbilical vein endothelial cells were cultured in the presence of [35S]methionine. Crossed immunoelectrophoresis of endothelial cell supernates and detergent-solubilized endothelial cells demonstrated specific incorporation of the [35S]methionine into vW AgII. Furthermore, when endothelial cells were lysed in the presence of proteolytic inhibitors, a second, less anodal peak was identified on crossed immunoelectrophoresis. This peak represented a complex of vW AgII and vWf and demonstrated a reaction of complete identity with the vW AgII immunoprecipitate. When plasma, serum, or platelets were evaluated by crossed immunoelectrophoresis, this "complex" peak was not present. When antibodies to vWf, fibronectin, or fibrinogen were present in the first dimension of crossed immunoelectrophoresis, only the antibodies to vWf removed the complex. Radioiodinated polyclonal and monoclonal antibodies to vWf also localized vWf to this complex. Under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled immunoprecipitates indicated that the molecular weight of vW AgII is 98,000 and that vWf was present as two species of 220,000 and 260,000 mol wt, respectively. Immunofluorescent microscopy of endothelial cells demonstrated colocalization of vW AgII and vWf in endothelial cells with intense immunostaining of the same subcellular granules.     

The effects of exercise stress testing on soluble E‐selectin,von Willebrand factor,and circulating endothelial cells as indices of endothelial damage/dysfunction

Background. The quantification of circulating endothelial cells (CECs) in whole blood is an increasingly recognized index of endothelial damage/dysfunction. Abnormal CECs have been linked to the severity of coronary artery disease (CAD).

Objective. We assessed the relationship of CECs to other markers of endothelial dysfunction (von Willebrand factor (vWF) and soluble E‐selectin (sEsel)) during exercise stress testing (EST) in a cohort of patients with suspected CAD.

Methods. We studied a cohort of patients referred to our chest pain clinic with a history of exertional chest pain. Treadmill EST was performed, using a full Bruce exercise protocol. Blood for CECs (immunobead method), vWF and sEsel (both ELISA) were collected immediately before (pre‐exercise), immediately following exercise, and at 30 minutes post‐EST.

Results. We studied 31 patients (84% male; mean (SD) age 58.4 (9.8) years). Of the entire cohort, 14 patients (45.2%) had a positive EST. Exercise led to significant increases in levels of CECs, sEsel, vWF, white blood cells (WBC), heart rate, mean and systolic blood pressure compared with base‐line (all P<0.05). There was a significant correlation between the change (Δ (immediate post–pre‐exercise)) in CECs and ΔvWF (r = 0.45; 95% CI 0.11–0.69: P = 0.01) and ΔsEsel (r = 0.41; 0.05–0.7: P = 0.02), as well as between ΔvWF and ΔsEsel (r = 0.55; 0.25–0.76: P = 0.001). Neither absolute nor ΔCEC counts were predictive of exercise work‐load/functional capacity, nor the presence of positive EST results.

Conclusion. EST led to a significant increase in endothelial markers (CECs, vWF, and sEsel) compared with base‐line levels. The rise in CECs correlated with the increases in other endothelial markers, but was not related to the either exercise work‐load/capacity or to the presence of a positive EST.     

脓毒症患者外周血祖细胞和内皮祖细胞数量的变化

目的 检测脓毒症患者外周血单个核细胞(peripheral blood mononuelear cell,PBMC)中祖细胞和血管内皮祖细胞(endothelial progenitor cells,EPC)相对数量的变化,探讨感染性休克和非休克患者外周血EPC变化的特点.方法 收集2007年8月至2008年2月复大学附属中山医院急诊科收治的脓毒症患者27例进行前瞻性研究,其中感染性休克患者12例、非休克患者15例,另选10例健康成年人作为正常对照,ICU非脓毒症患者10例作为ICU对照.Ficoll梯度离心法分离外周血PBMC,通过流式细胞仪检测外周血PBMC标记的CDl33,CIY34和血管内皮牛长因子受体-2(vascular endothelialgrowth factor receptor-2.VEGFR-2)的表达情况,计算祖细胞以及内皮祖细胞的相对数量.组间比较采用单因素方差分析.结果 健康成年人外周血祖细胞、EPC数量较少,分别占PBMC的0.25%.4-0.14%和0.09%.4-0.02%;ICU非脓毒症患者祖细胞和EPC数量分别占PBMC的0.38%.4-0.29%和0.12%.4-O.02%,与正常对照组相比无明显的变化(P>0.05);脓毒症非休克组患者外周血祖细胞、EPC的数量明显增加,分别占PBMC的0.57%±0.12%和0.22%±0.10%,与正常对照组相比差异具有统计学意义(P<0.05);感染性休克患者外周血祖细胞和EPE的数量明显减少,分别占PBMC的0.20%.4-0.12%和0.04%±O.01%,与非休克组、ICU对照组和正常对照组相比差异均具有统计学意义(P相似文献   

von Willebrand factor and endothelial abnormalities in diabetic microangiopathy

We briefly summarize current knowledge on 1) the abnormalities of von Willebrand factor (vWF) as an indicator of endothelial cell (EC) dysfunction in diabetes and 2) the modifications induced in the growth of cultured ECs by high glucose in the incubation media. A MEDLINE search (1986 through Sept. 1989) was performed to update previous relevant references on vWF and ECs in healthy and diabetic subjects. Main data in the literature and personal contributions were scrutinized. Study quality, information, and relevance to the subject were assessed. vWF is synthesized and stored mainly in ECs. Its plasma levels are increased in diabetic microangiopathy but are not influenced by circulating glucose, insulin, or growth hormone, nor do they acutely affect platelet function in diabetes. Supraphysiological concentrations of glucose inhibit the replication of cultured ECs from large vessels via different possible mechanisms but appear to stimulate pathways involved in the activation of capillary ECs. vWF is a possible marker of EC damage in diabetes, and prospective studies will ascertain its role as a predictor for the development of microangiopathy. The possible dichotomy in the response of cultured ECs from large and small vessels to high glucose in the culture media may help explain some of the lesions observed in the walls of arteries and capillaries in diabetes.     

Fibrin induces release of von Willebrand factor from endothelial cells.

Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating. We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.     

Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry

The finding of angiogenic and vasculogenic cells in the peripheral circulation may have profound effects on the course of a variety of diseases ranging from cancer to cardiovascular disease. These cells are ascribed to be endothelial in nature and are generally referred to as circulating endothelial cells if mature or as endothelial progenitor cells if immature. Different approaches have been used to detect these cells, including in vitro culture, magnetic bead isolation, and flow cytometry. We review flow cytometric methods for the detection and enumeration of these cells and provide technical suggestions to promote the accurate enumeration of circulating endothelial cells and endothelial progenitor cells.     

A sticky proposition: The endothelial glycocalyx and von Willebrand factor

Von Willebrand factor (VWF) is a critical component of the hemostatic system. Basal secretion of VWF from endothelial cells is the principal determinant of an individual's baseline plasma VWF levels, while endothelial VWF release can also be induced by several biochemical agonists and biomechanical forces such as increased shear stress. However, the mechanotransduction machinery responsible for this latter response is unclear. Here we propose that the endothelial glycocalyx (EGC), a dynamic layer of proteins and carbohydrates that covers the surface of the vascular endothelium, may play a key role in mediating this response. The EGC has previously been implicated in mediating the mechanotransduction of shear stress in other shear‐responsive endothelial processes, such as nitric oxide production and stem cell differentiation. Here, we hypothesize that a similar mechanism may be responsible for the basal secretion of endothelial VWF, whereby the EGC mediates the mechanotransduction of physiological shear stress generated by flowing blood, that in turn contributes to the maintenance of physiological plasma VWF levels.     

Correlation between circulating levels of von Willebrand's antigen II and von Willebrand factor: discrimination between type I and type II von Willebrand's disease

Classification of the subtypes of von Willebrand's disease (vWd) has been based on a quantitative deficiency or an abnormal multimeric composition of von Willebrand factor (vWf). Although the co-deficiency of a second protein, von Willebrand's antigen II (vW AgII), had been previously recognized, its concentration in a relatively large number of normal individuals or patients with well-defined vWd variants had not been studied. The plasma from patients with type I, IIA, IIB, IIC, and III (severe) vWd was evaluated, and the concentrations of vW AgII and vWf were determined. Although patients with type I and III vWd had reduced levels of both proteins, the plasma vW AgII concentration was normal in patients with type II vWd. Analysis of the results indicates that type I and type II variants can be discriminated with greater than 80% accuracy by comparison of results of these two antigenic assays. The normal levels of vW AgII in type II variants suggest a possible difference in the pathophysiology of type I and type II vWd.     

Relationship between spontaneous echo contrast in the thoracic aorta and plasma von Willebrand factor

Purpose To clarify the relationships between spontaneous echo contrast (SEC) detected by transesophageal echocardiography (TEE) and coagulopathy, ultrasonographic findings that may correlate to biochemical coagulation markers were examined. Methods TEE was performed on 49 consecutive patients (mean age 64 ± 14 years; 28 men, 21 women). Blood samples were taken at the same time as TEE was carried out. Aortic SEC (Ao-SEC) and left atrial SEC (LA-SEC) were classified into three grades: absent, mild and marked. Levels of von Willebrand factor (vWF), thrombin antithrombin III complex (TAT), prothrombin fragments 1+2 (F1+2) and fibrinopeptide A (FPA) were measured. Results Mean plasma vWF levels by Ao-SEC grade were 144 ± 39% for absent, 177 ± 55% for mild and 210 ± 73% for marked, with significantly higher levels in the Ao-SEC marked group than in the Ao-SEC absent group (P < 0.05). Mean plasma vWF levels by LA-SEC were 185 ± 73% for absent, 180 ± 49% for mild and 201 ± 62% for marked, with no significant differences apparent between groups. Moreover, no relationships were identified between Ao-SEC grade and plasma levels of coagulation indicators TAT, F1+2 and FPA. Conclusion Plasma vWF levels correlated to grade of aortic SEC. Characteristics of the coagulation system differ between Ao-SEC and LA-SEC. Ao-SEC offers a clinical indicator of platelet thrombus formation.     

肺动脉高压模型大鼠循环内皮祖细胞与血浆一氧化氮水平相关性的实验研究

目的 探讨肺动脉高压(PH)模型大鼠循环内皮祖细胞(EPC)水平的变化及其与血浆一氧化氮(NO)浓度的关系.方法 利用野百合碱诱导大鼠发生肺动脉高压,使用流式细胞仪对其外周血CD45 阴性、CD34/Flk-1 双阳性的单个核细胞计数,以表示循环EPC,采用Greiss 法对其血浆NO 浓度进行检测.利用简单线性回归分析二者之间的关系.同时,对体外培养条件下循环EPC 的产量,CD34、Flk-1 的表达情况进行检测.结果 PH 模型组循环EPC 水平明显低于对照组[(0.016 ±0.007)% vs.(0.031 ±0.011)%,t ＝3.144,P ＜0.01].血浆NO 浓度亦显著下降[(19.66 ±2.78)μmol/L vs.(54.31 ±3.81)μmol/L,t ＝20.784,P ＜0.01].二者之间呈正相关(r ＝0.792,P ＜0.05).在体外培养条件下,PH 模型组EPC 的数量以及CD34、Flk-1 的表达亦较对照组明显下调.结论 PH 的发生可能与血浆NO 浓度降低导致循环EPC 水平下调有关.     

Vasopressin-induced von Willebrand factor secretion from endothelial cells involves V2 receptors and cAMP

Vasopressin and its analogue 1-deamino-8-D-arginine vasopressin (DDAVP) are known to raise plasma von Willebrand factor (vWF) levels. DDAVP is used as a hemostatic agent for the treatment of von Willebrand's disease. However, its cellular mechanisms of action have not been elucidated. DDAVP, a specific agonist for the vasopressin V2 receptor (V2R), exerts its antidiuretic effect via a rise in cAMP in kidney collecting ducts. We tested the hypothesis that DDAVP induces vWF secretion by binding to V2R and activating cAMP-mediated signaling in endothelial cells. vWF secretion from human umbilical vein endothelial cells (HUVECs) can be mediated by cAMP, but DDAVP is ineffective, presumably due to the absence of V2R. We report that DDAVP stimulates vWF secretion in a cAMP-dependent manner in HUVECs after transfection of the V2R. In addition, vasopressin and DDAVP induce vWF secretion in human lung microvascular endothelial cells (HMVEC-L). These cells (but not HUVECs) express endogenous V2R, as shown by RT-PCR. Vasopressin-induced vWF secretion is mimicked by DDAVP and inhibited by the selective V2R antagonist SR121463B. It is mediated by cAMP, since it is inhibited by the protein kinase A inhibitor Rp-8CPT-cAMPS. These results indicate that vasopressin induces cAMP-mediated vWF secretion by a direct effect on endothelial cells. They also demonstrate functional expression of V2R in endothelial cells, and provide a cellular mechanism for the hemostatic effects of DDAVP.     

Effects of immunoadsorption on endothelial function,circulating endothelial progenitor cells and circulating microparticles in patients with inflammatory dilated cardiomyopathy

Background  

Immunoadsorption (IA) is used in patients with chronic inflammatory dilative cardiomyopathy (iDCM) to remove cardiotoxic autoantibodies, and to improve myocardial function. We examined the effects of IA on endothelial function, circulating endothelial progenitor cells, and circulating microparticles, including endothelial-derived microparticles, in patients with chronic iDCM.