Endothelial Cell Von Willebrand Factor Research Articles

Ralb and Exocyst Regulate Endothelial Cell Von Willebrand Factor Release

Background: Identification of novel therapeutic targets for pharmacologic modulation of circulating von Willebrand factor (vWF) would be invaluable in treatment of von Willebrand disease, but also in other pathophysiologic processes where vWF plays a vital role. These include cardiovascular disease, cancer metastasis, angiogenesis, and smooth muscle proliferation. vWF is almost exclusively produced by the endothelial cells and stored in specialized secretory granules, Weibel Palade bodies (WPBs). We have previously shown that loss of the exocyst complex, an essential multi-subunit trafficking protein, which physically tethers secretory granules to the plasma membrane in yeast, increases vWF release. Small GTPases act as regulatable molecular switches. Cycling between GTP-bound ('on') and GDP-bound ('off') states, they control all essential cellular processes. Among these, Ral GTPase has previously been shown to be important in WPB secretion and vWF release. Since Ral specifically regulates exocyst in other cellular functions, we hypothesized that exocyst is also under direct control of Ral in vWF release. Methods: All experiments were performed on human umbilical vein endothelial cells (HUVECs). RalA/B and exocyst depletion were performed via targeted siRNAs. vWF antigen was estimated using ELISA. RalB activation was determined by a commercial pulldown assay specific for the activated form of RalB. Expression of FLAG-tagged RalB was performed by lentiviral transduction. RalB mutants were generated using site-directed mutagenesis of FLAG-RalB (WT). Spinning disc confocal microscope was used for immunofluorescence (IF). Immuno-electron microscopy was performed using immune-gold labeling. FLAG-pulldown and immunoprecipitation (IP) was performed on cell lysates using antibody-labeled magnetic beads. Results: RalB but not RalA depletion, or degradation with the RalB-selective inhibitor dihydroartemisinin, significantly impaired thrombin-induced vWF exocytosis (55±7% decrease). RalB was activated (GTP-bound) and phosphorylated upon thrombin stimulation of HUVECs. Correspondingly, expression of constitutively active RalB-G23V mutant, which stays in a GTP-bound state, significantly increased vWF exocytosis compared to RalB-WT (69±4.6% increase). IF and EM confirmed co-localization of RalB and exocyst on WPBs. Unexpectedly, FLAG pulldown and protein identification by mass spectrometry demonstrated that RalB-WT (GDP-bound;“off”) but not RalB-G23V (GTP-bound;“on”) interacts with exocyst, confirmed by immunoblotting. IP of resting and thrombin-stimulated native HUVEC lysates with RalB antibodies, as well as reverse-IP with exocyst antibodies confirmed this differential binding. Exocyst binding site mutation (RalB-WTD49E) and transduction of these mutants in HUVECs resulted in significantly augmented vWF exocytosis (40±3% increase), like constitutively active RalB-G23V mutant. Moreover, IF showed uniform cytoplasmic distribution of WPB cigars in RalB-WT HUVECs, whereas in RalB-WTD49E mutants WPBs were found at the plasma membrane, like RalB-G23V. This confirms that RalB-free exocyst tethers WPB to the plasma membrane ( Figure 1). Serine phosphorylation site mutation (RalB-WTS198A), previously shown to regulate GTPase activity of RalB, hence GTP-GDP cycling, also significantly decreased RalB exocyst binding, in turn increasing vWF release. This shows that post-translational phosphorylation of RalB at serine 192, which is also accelerated by thrombin activation of endothelial cells, regulates RalB-exocyst interaction, hence vWF release. Conclusions: We have identified an important regulatory role of RalB in exocyst-dependent WPB secretion and vWF release. WPB localized exocyst is associated with RalB in resting endothelium. Endothelial cell activation switches RalB ‘on’, and exocyst, now uncoupled from GTP-bound RalB, is free to tether WPBs to the plasma membrane and facilitate vWF release.

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

Primary Myeloma Cell Induced Von Willebrand Factor Release from the Endothelium Is Mediated By VEGF and Attenuated By Heparin

IntroductionMultiple Myeloma (MM) remains an incurable disease and is associated with high rates of venous thromboembolism (VTE), the biological basis for which is not fully understood. Critically, VTE is associated with increased mortality in MM. This highlights the clinical importance of understanding cancer-coagulation crosstalk in MM. Accumulating evidence demonstrates that many solid tumours trigger endothelial cell (EC) activation with resultant von Willebrand Factor (VWF) secretion proposed to contribute to both risk of VTE and cancer metastasis. However, the interplay between VWF and MM disease biology remains poorly defined.Methods/Results100 patients with plasma cell disorders were recruited to this study. Significantly elevated plasma VWF antigen (VWF:Ag) levels were observed in patients with newly diagnosed MM (NDMM) compared to those with monoclonal gammopathy of undetermined significance (MGUS) or smouldering MM (SMM) (median 292.7 IU/dL vs 133.1 IU/dL; P<0.0001). Moreover, VWF:Ag levels in those with relapsed/refractory MM (RRMM) were further elevated (median 339 IU/dL; P<0.0001]). VWF:Ag levels also appear to correlate with disease response to therapy, with lower levels seen in patients achieving a complete response/very good partial response compared with those gaining only a partial response or worse (median 154.45 IU/dL vs 338.5 IU/dL; P<0.05).We also observed increased VWF collagen binding activity in NDMM/RRMM compared with MGUS/SMM (median 455.7 IU/dL vs 189.7 IU/dL; P<0.05). This qualitative measurement is sensitive to alterations in haemostatically active high molecular weight multimers of VWF secreted upon EC activation. Supporting this, raised plasma VWF propeptide (VWFpp) levels, a marker of acute EC activation, were seen in those with NDMM/RRMM compared with MGUS/SMM (median 182 IU/dL vs 133.01 IU/dL; P<0.05). Reduced VWFpp/VWF:Ag ratios were seen in those with NDMM/RRMM, indicating decreased circulatory clearance of VWF. We hypothesise that MM may exhibit a ‘double-hit’ in regulation of plasma VWF:Ag with increased secretion of VWF but also simultaneously reduced VWF clearance leading to sustained and markedly elevated systemic plasma VWF levels.Next we measured VWF:Ag levels in bone marrow (BM) samples from 10 patients and found that VWF is released locally within the BM niche (median 190.2 IU/dL). Co-culture of primary human ECs with supernatant from freshly isolated primary MM cells or several Human Myeloma Cell Lines (HMCLs) demonstrated that MM cells stimulate release of VWF from ECs in a rapid manner, suggestive of Weibel-Palade body exocytosis (median 34.9ng/ml vs 9.2ng/ml in untreated EC; P<0.05). Elevated levels of Vascular Endothelial Growth Factor-A (VEGF-A), a potent EC activator, were observed in MM cell supernatants. Pre-treatment of primary MM cells or HMCLs with anti-VEGF antibody bevacizumab abolished EC VWF secretion in a dose dependant manner (32.3ng/ml vs anti-VEGF treated 10.1ng/ml P<0.0001). Furthermore, clinically relevant doses of low molecular weight heparin (LMWH) also attenuated VWF secretion (29.6ng/ml vs LMWH treated 15ng/ml; P<0.05). Measurement of VEGF-A in primary MM cell and HMCL supernatant treated with LMWH or bevacizumab confirmed significant reductions in VEGF-A levels. Together these data indicate that stimulation of VWF release from the endothelium by MM cells is mediated by VEGF-A and directly inhibited by LMWH treatment.Using flow cytometry, we assessed the direct interaction of MM cells and VWF in vitro. Both primary MM cells and HMCLs bound significantly to human recombinant VWF in a dose-dependent manner. Binding was reduced by 50% following treatment with LMWH (P<0.05). Given that the VWF A1 domain contains a heparin binding motif, we speculate that MM cell adhesion to VWF may be at least in part mediated by this domain and future work focuses on elucidating the receptors involved.ConclusionThese data suggest that MM is not only associated with a marked quantitative increase in plasma VWF:Ag, but also impacts VWF functional activity. Our novel data help define the biological mechanisms underpinning elevated VWF levels in MM, with a key contribution of VEGF-A secreted directly from MM cells in the BM microenvironment. Collectively, our findings provide insights into cancer-coagulation crosstalk in MM and may help identify novel therapeutic targets to reduce VTE risk and disease progression. DisclosuresGlavey: Celgene and BMS company: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Quinn: Takeda: Honoraria. O'Sullivan: Leo Pharma: Research Funding.

α-galactosidase A deficiency promotes von Willebrand factor secretion in models of Fabry disease

Fabry disease results from loss of activity of the lysosomal enzyme α-galactosidase A (GLA), leading to the accumulation of globoseries glycosphingolipids in vascular endothelial cells. Thrombosis and stroke are life-threatening complications of Fabry disease; however, the mechanism of the vasculopathy remains unclear. We explored the relationship between GLA deficiency and endothelial cell von Willebrand factor (VWF) secretion in invivo and invitro models of Fabry disease. Plasma VWF was significantly higher at two months and increased with age in Gla-null compared to wild-type mice. Disruption of GLA in a human endothelial cell line by siRNA and CRISPR/Cas9 resulted in a 3-fold and 5-fold increase in VWF secretion, respectively. The increase in VWF levels was associated with decreased endothelial nitric oxide synthase (eNOS) activity in both invitro models. Pharmacological approaches that increase nitric oxide bioavailability or decrease reactive oxygen species completely normalized the elevated VWF secretion in GLA deficient cells. In contrast, the abnormality was not readily reversed by recombinant human GLA or by inhibition of glycosphingolipid synthesis with eliglustat. These results suggest that GLA deficiency promotes VWF secretion through eNOS dysregulation, which may contribute to the vasculopathy of Fabry disease.

Cocaine and Certain of Its Long-Acting Metabolites Stimulate Endothelial Cell Von Willebrand Factor Secretion

Abstract 1148Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures:No relevant conflicts of interest to declare.

Endothelial von Willebrand factor regulates angiogenesis

AbstractThe regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)–dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)–2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

Fluvastatin inhibits regulated secretion of endothelial cell von Willebrand factor in response to diverse secretagogues

Regulated secretion of EC (endothelial cell) vWF (von Willebrand factor) is part of the haemostatic response. It occurs in response to secretagogues that raise intracellular calcium or cAMP. Statins are cholesterol-lowering drugs used for the treatment of cardiovascular disease. We studied the effect of fluvastatin on regulated secretion of vWF from HUVEC (human umbilical-vein ECs). Secretion in response to thrombin, a protease-activated receptor-1 agonist peptide, histamine, forskolin and adrenaline (epinephrine) was inhibited. This inhibition was reversed by mevalonate or geranylgeranyl pyrophosphate, and mimicked by a geranylgeranyl transferase inhibitor, demonstrating that the inhibitory mechanism includes inhibition of protein geranylgeranylation. To investigate this mechanism further, calcium handling and NO (nitric oxide) regulation were studied in fluvastatin-treated HUVEC. Intracellular calcium mobilization did not correlate with vWF secretion. Fluvastatin increased eNOS [endothelial NOS (NO synthase)] expression, but NOS inhibitors failed to reverse the effect of fluvastatin on vWF secretion. Exogenous NO did not inhibit thrombin-induced vWF secretion. Many small GTPases are geranylgeranylated and some are activated by secretagogues. We overexpressed DN (dominant negative) Rho GTPases, RhoA, Rac1 and Cdc42 (cell division cycle 42), in HUVEC. DNCdc42 conferred inhibition of thrombin- and forskolin-induced vWF secretion. We conclude that, via inhibition of protein geranylgeranylation, fluvastatin is a broadspectrum inhibitor of regulated vWF secretion. Geranylgeranylated small GTPases with functional roles in regulated secretion, such as Cdc42, are potential targets for the inhibitory activity of fluvastatin.

Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors

Novel tubular scaffolds of marine source collagen and PLGA fibers were fabricated by freeze drying and electrospinning processes for vascular grafts. The hybrid scaffolds, composed of a porous collagen matrix and a fibrous PLGA layer, had an average pore size of 150±50 μm. The electrospun fibrous PLGA layer on the surface of a porous tubular collagen scaffold improved the mechanical strength of the collagen scaffolds in both the dry and wet states. Smooth muscle cells (SMCs)- and endothelial cells (ECs)-cultured collagen/PLGA scaffolds exhibited mechanical properties similar to collagen/PLGA scaffolds unseeded with cells, even after culturing for 23 days. The effect of a mechanical stimulation on the proliferation and phenotype of SMCs and ECs, cultured on collagen/PLGA scaffolds, was evaluated. The pulsatile perfusion system enhanced the SMCs and ECs proliferation. In addition, a significant cell alignment in a direction radial to the distending direction was observed in tissues exposed to radial distention, which is similar to the phenomenon of native vessel tissues in vivo. On the other hand, cells in tissues engineered in the static condition were randomly aligned. Immunochemical analyses showed that the expressions of SM α-actin, SM myosin heavy chain, EC von Willebrand factor, and EC nitric oxide were upregulated in tissues engineered under a mechano-active condition, compared to vessel tissues engineered in the static condition. These results indicated that the co-culturing of SMCs and ECs, using collagen/PLGA hybrid scaffolds under a pulsatile perfusion system, leads to the enhancement of vascular EC development, as well as the retention of the differentiated cell phenotype.

Characterization of Recessive Severe Type 1 and 3 von Willebrand Disease (VWD), Asymptomatic Heterozygous Carriers Versus Bloodgroup O-Related von Willebrand Factor Deficiency, and Dominant Type 1 VWD

Recessive type 3 von Willebrand disease (VWD) is caused by homozygosity or double heterozygosity for two non-sense mutations (null alleles). Type 3 VWD is easy to diagnose by the combination of a strongly prolonged bleeding time (BT), absence of ristocetine-induced platelet aggregation (RIPA), absence of von Willebrand factor (VWF) protein, and prolonged activated partial thromboplastin time (aPTT) due to factor VIII:coagulant (FVIII:C) deficiency. VWD type 3 is associated with a pronounced tendency to mucocutaneous and musculoskeletal bleedings since early childhood. Carriers of one null allele are usually asymptomatic at VWF levels of 50% of normal. Recessive severe type 1 VWD is caused by homozygosity or double heterozygosity for a missense mutation. Recessive type 1 VWD differs from type 3 VWD by the presence of detectable von Willebrand factor: antigen VWF:Ag and FVIII:C levels between 0.09 and 0.40 U/mL. Patients with recessive type 1 VWD show an abnormal VWF multimeric pattern in plasma and/or platelets consistent with severe type 2 VWD. Carriers of a missense mutation may have mild bleeding and mild VWF deficiency and can be diagnosed by a double VWF peak on cross immunoelectrophoresis (CIE). There will be cases of mild and moderate recessive type 1 VWD due to double heterozygosity of two missense mutations, or with the combination of one missense mutation with a non-sense or bloodgroup O. Mild deficiency of VWF in the range of 0.20 to 0.60 U/mL, with normal ratios of von Willebrand factor: ristocetine cofactor/antigen VWF:RCo/Ag and VWF:collagen binding/antigen (VWF:CB/Ag), normal VWF multimers, and a completely normal response to desmopressin acetate (DDAVP) with VWF level rising from below to above 1.00 U/mL are very likely cases of so-called pseudo-VWF deficiency in individuals with normal VWF protein and gene. Autosomal dominant type 1 VWD variants are in fact type 2 variants caused by a heterozygous missense mutation in the VWF gene that produces a mutant VWF protein that has a dominant effect on normal VWF protein produced by the normal VWF allele with regard to the synthesis, processing, storage, secretion, and/or proteolysis of VWF in endothelial cells. A DDAVP challenge test clearly differentiates between dominant type 1 VWD phenotype and dominant type 2 M VWD.

Aortic endothelial cell von Willebrand factor content, and circulating plasminogen activator inhibitor-1 are increased, but expression of endothelial leukocyte adhesion molecules is unchanged in insulin-dependent diabetic BB rats

Endothelial cell injury has been implicated in the increased incidence of vascular disease associated with diabetes mellitus. In diabetic humans, elevated plasma von Willebrand Factor (vWF) has been interpreted as an indication of endothelial damage. In contrast, in an animal model of inherited insulin-dependent diabetes, the bio-breeding (BB) rat, plasma vWF levels did not differ from those in age-matched control rats during the first 7 months of diabetes although morphological evidence of mild aortic endothelial alteration or injury was observed. In the present study efforts have been made to define the endothelial alterations in BB diabetic rats compared to controls more precisely over this time period. Thus, adhesion molecules: intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were evaluated by in situ immunohistochemistry, vWF content was determined by biochemical analysis of aortic extracts and by quantitative immunohistochemistry, plasma vWF levels were measured by ELISA and vWF mRNA by RNAse protection assay. Neither age nor diabetic state significantly affected either the expression of adhesion molecules, or the levels of circulating vWF. Endothelial vWF content was significantly increased in the diabetic vessels, as observed by both approaches but the vWF mRNA content was not different from that in control vessels. Plasma plasminogen activator inhibitor (PAI-1) activity was significantly increased in diabetic animals. In conclusion, endothelial alterations in BB rats associated with diabetes, together with the raised plasma PAI-1 levels, promote the thrombogenic potential of the vessel wall, and are consistent with an increased risk for vascular disease.

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

In endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules. so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Pure beta-particle-emitting stents inhibit neointima formation in rabbits.

Considerable experimental evidence exists that neointimal hyperplasia after angioplasty is inhibited by gamma-irradiation of the treated arteries. A beta-particle radiation is absorbed in tissue within a shorter distance away from the source than gamma-radiation and may be more suitable for localized vessel irradiation. This study outlines a method to implant a beta-particle-emitting radioisotope (32P; half-life, 14.3 days) into metallic stents. The effects of these stents on the inhibition of neointimal hyperplasia was compared with conventional stents in a rabbit model. 32P was produced by irradiation of red amorphous phophorus (31P) with neutrons and was implanted into Palmaz-Schatz stents (7.5 mm in length) after being kept apart from 31P in a mass separator. The radioisotope was tightly fixed to the stents, and the ion implantation process did not alter the surface texture. Stent activity levels of 4 and 13 microCi were chosen for the study. Four and 12 weeks after placement of conventional stents and 32P-implanted stents in rabbit iliac arteries, vascular injury and neointima formation were studied by histomorphometry. Immunostaining for smooth muscle cell (SMC) alpha-actin was performed to determine SMC cellularity in the neointima. SMCs were quantified by computer-assisted counting of alpha-actin immunoreactive cells. Endothelialization of the stents was evaluated by immunostaining for endothelial cell von Willebrand factor. No difference in vessel wall injury was found after placement of conventional and 32P-implanted stents. Neointima formation was potently inhibited by 32P-implanted stents only at an activity level of 13 microCi after 4 and 12 weeks. Neointimal SMC cellularity was reduced in 32P-implanted stents compared with conventional stents. Radioactive stents were endothelialized after 4 weeks, but endothelialization was less dense than in conventional stents. Neointima formation in rabbits is markedly suppressed by a beta-particle-emitting stent incorporating the radioisotope 32P. In this model, a dose-response relation with this type of radioactive stent was observed, indicating that a threshold radiation dose must be delivered to inhibit neointima formation after stent placement over the long term.

Low-dose radioactive endovascular stents prevent smooth muscle cell proliferation and neointimal hyperplasia in rabbits.

Restenosis induced by smooth muscle cell (SMC) migration and proliferation and neointimal thickening limits the clinical success of balloon angioplasty and stent implantation. In this study, the long-term effect of endovascular irradiation via low-dose radioactive stents on neointima formation was compared with conventional stent implantation in a rabbit model. Palmaz-Schatz stents were made radioactive in a cyclotron. The stents had a very low activity (maximum, 35 microCi), and thus, manipulation did not require extensive radiation protection. One, 4, 12, and 52 weeks after the implantation of nonradioactive stents and radioactive stents in rabbit iliac arteries, neointimal thickening was analyzed by quantitative histomorphometry. Immunostaining for endothelial cell von Willebrand factor, macrophages, SMC alpha-actin, collagen type I, and proliferating cell nuclear antigen (PCNA) was performed to determine radiation-induced changes in the arterial wall. SMC proliferation was quantified by computer-assisted cell counting of PCNA-immunoreactive cells. Neointima formation was markedly suppressed by the implantation of radioactive stents in a dose-dependent fashion at all observed time points. At peak proliferative activity of SMCs 1 week after nonradioactive stent implantation, 30 +/- 2% of SMCs in the neointima were proliferating, compared with 0.5 +/- 0.1% of SMCs after implantation of stents with an initial activity of 35 microCi (P < .001). The neointima covering radioactive stents was characterized by decreased smooth muscle cellularity and increased extracellular matrix formation. Further, we observed a delayed endothelialization depending on the radiation dose. No difference in vascular thrombosis was found after nonradioactive and radioactive stent implantation. The results of this study clearly indicate that low-dose radioactive endovascular stents potently inhibit SMC proliferation and neointimal hyperplasia in rabbits.

The high molecular weight form of endothelial cell von Willebrand factor is released by the regulated pathway

We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma-like multimers, and a non-secreted low molecular weight (LMW) form. In this study, the mode of vWf release and the relationship between the two forms were examined. As determined by two-dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro-vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide. Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H. Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf. Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form. These results suggest that two pools of vWf exist in endothelial cells--a LMW form of pro-vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment. Released vWf derives only from the storage pool.

Von Willebrand Factor in Cultured Human Vascular Endothelial Cells from Adult and Umbilical Cord Arteries and Veins

Endothelial cells were cultured from various human arteries and veins, obtained from adult individuals and from umbilical cords. We compared the storage and secretion of von Willebrand factor by endothelial cells from umbilical veins with that of endothelial cells cultured from a number of adult vessels, including aorta, arteria iliaca, vena saphena magna and vena cava. There were no differences in the way the cultured endothelial cells handled the von Willebrand factor they synthesized. Endothelial cells from the various vessels responded to stimuli in secreting stored von Willebrand factor. The cells also responded to thrombin and ionophore A23187 in producing enhanced amounts of prostacyclin. Thus, cultured umbilical vein endothelial cells have properties that are very similar to those of cultured endothelial cells of various other origins. It is concluded that foetal venous cells provide a representative model for studies of endothelial cell von Willebrand factor biosynthesis and prostacyclin production.

Arteriosclerosis in normal and von Willebrand pigs: long-term prospective study and aortic transplantation study.

In a long-term prospective study, five normal control pigs and five pigs with homozygous von Willebrand's disease received a nonfatty diet from age 3 months to 4 years; then the aortas were analyzed. The fibrous arteriosclerotic plaques in the distal abdominal aortic region involved an average of 28% of the surface area in control pigs and only 7% of the surface area in pigs with von Willebrand's disease (P less than 0.01). In a subsequent study of 3-month-old pigs, the distal abdominal aortic segments from nine normal pigs were cross-transplanted with segments from nine other normal pigs (control study), and aortic segments from four normal pigs were transplanted into four host pigs with von Willebrand's disease (exchange study). All pigs received a 2% cholesterol diet for up to 6 months; then the transplanted aortic segments were analyzed. The donor normal aortic segments in the host normal pigs developed arteriosclerosis that involved an average of 20% of the surface; the endothelial fluorescent pattern of von Willebrand factor was identified. In contrast, the donor normal aortic segments in the host pigs with von Willebrand's disease had arteriosclerosis that involved an average of only 4% of the surface (P less than 0.01); the endothelial cell von Willebrand factor was not identified. The long-term prospective study indicates that pigs with von Willebrand's disease are resistant to the development of spontaneous arteriosclerosis. The aortic transplantation data are compatible with the hypothesis that the absence of von Willebrand factor in pigs with von Willebrand's disease may cause impairment of platelet-arterial wall interaction and resistance to arteriosclerosis.

Synthesis of von Willebrand factor by cultured human endothelial cells.

Cultured human endothelial cells synthesize and secrete a protein(s) which has Factor VIII antigen but which lacks Factor VIII clot-promoting activity (J. Clin. Invest. 52, 2757-2764, 1973). Von Willebrand factor activity has been identified in medium from cultured human endothelial cells. This activity was demonstrated by the ability to correct the defect in platelet adhesiveness of blood obtained from patients with von Willebrand's disease. This activity also supported ristocetin-induced aggregation of washed normal human platelets. The von Willebrand factor activity from cultured endothelial cells has physicochemical and immunologic properties like those of the von Willebrand factor activity and the Factor VIII antigen present in human plasma and the Factor VIII antigen synthesized by human endothelial cells in vitro. Rabbit antibody to chromatographic fractions containing endothelial cell von Willebrand factor inhibits the platelet retention of normal blood in glass bead columns.