Richard Starke

Endothelial von Willebrand factor regulates angiogenesis

The 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.

Remission in acute refractory and relapsing thrombotic thrombocytopenic purpura following rituximab is associated with a reduction in IgG antibodies to ADAMTS-13

Thrombotic thrombocytopenic purpura (TTP) is a life‐threatening disorder and plasma exchange (PEX) remains the primary treatment modality. Twenty‐five patients with acute refractory/relapsing idiopathic TTP received rituximab in conjunction with PEX because of progressive clinical disease or deterioration in laboratory parameters, despite intensive standard therapy. In relapsing TTP, rituximab was started if antibody to ADAMTS‐13 (a disintegrin and metalloproteinase with thrombospondin motif‐13) was demonstrated during previous episodes. All 25 patients attained complete clinical and laboratory remission in a median of 11 d after initiating rituximab. In 21 cases, ADAMTS‐13 activity was within the normal range following rituximab. Inhibitors were detected in 24/25 patients by mixing studies and/or immunoglobulin G (IgG) antibodies to ADAMTS‐13 pre‐rituximab. There was no evidence of inhibitors and/or IgG activity <10% in 23/25 patients following rituximab. In acute refractory cases, the median number of PEX pre‐rituximab and following the first rituximab infusion was 13 and 9, respectively. There have been no infectious complications, despite low CD 19 levels and no relapses. In patients with acute refractory/relapsing idiopathic TTP, rituximab appears to be a safe, effective, targeted therapy with a significant reduction in the requirement for PEX.

Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis.

Dramatic activation of the coagulation cascade has been extensively documented for pulmonary fibrosis associated with acute and chronic lung injury. In addition to its role in hemostasis, thrombin exerts profibrotic effects via activation of the major thrombin receptor, protease-activated receptor-1. In this study, we examined the effect of the direct thrombin inhibitor, UK-156406 on fibroblast responses in vitro and on bleomycin-induced pulmonary fibrosis in rats. UK-156406 significantly inhibited thrombin-induced fibroblast proliferation, procollagen production, and connective tissue growth factor (CTGF) mRNA levels when used at equimolar concentration to the protease. Thrombin levels in bronchoalveolar lavage fluid and expression of thrombin and protease-activated receptor-1 in lung tissue were increased after intratracheal instillation of bleomycin. The characteristic doubling in lung collagen in bleomycin-treated animals (38.4 ± 2.0 mg versus 17.1 ± 1.4 mg, P 1 (I) procollagen and CTGF mRNA levels (3.0 ± 0.4-fold and 6.3 ± 0.4-fold respectively, ( P P 1 (I) procollagen and CTGF mRNA levels by 50% and 35%, respectively ( P

Successful management of pregnancy in women with a history of thrombotic thrombocytopaenic purpura.

Pregnancy is an initiating event for acute thrombotic thrombocytopaenic purpura (TTP). There is a high risk of relapse during pregnancy and of foetal morbidity. We describe five cases with successful maternal and foetal outcomes in patients with a history of TTP. Cases 1 and 2 presented with TTP in their first pregnancy and had second-trimester foetal losses. Case 3 had four TTP relapses and soon after achievement of clinical remission became pregnant. Case 4 presented with TTP and left-sided stroke in pregnancy. ADAMTS-13 activity was less than 5% at presentation in four patients and prior to therapy during pregnancy in cases 1–4. Case 5, who had a single acute episode of TTP and became pregnant 6 years later, had normal ADAMTS-13 activity throughout pregnancy. Only case 3 had evidence of an inhibitor on mixing studies. All five patients underwent close haematological and obstetric monitoring and continued low-dose aspirin throughout pregnancy. Patients 1–4 had regular plasma exchange and received low molecular weight heparin during pregnancy. Patient 4 also received rituximab during the third trimester with no observed maternal or neonatal toxicity. Live healthy infants were delivered in all five cases in the third trimester. These findings suggest that successful pregnancy outcome is achievable in patients with a history of TTP and that patients with severely reduced ADAMTS-13 activity at the onset of pregnancy, necessitates regular plasma exchange during pregnancy.

Prevalence of the ADAMTS‐13 missense mutation R1060W in late onset adult thrombotic thrombocytopenic purpura

Summary.  Background: Thrombotic thrombocytopenic purpura (TTP) is most commonly associated with deficiency or inhibition of von Willebrand factor‐cleaving protease (ADAMTS‐13) activity. ADAMTS‐13 mutations and polymorphisms have been reported in childhood congenital TTP, but their significance in adult onset TTP remains unclear. Objectives: We sought to identify common ADAMTS‐13 mutations in adults with late onset TTP and to investigate whether they may predispose acute clinical episodes of the disorder in adulthood. Patients/Methods/Results: We detected a missense mutation (C3178T) in exon 24 of ADAMTS‐13 in 6/53 (11.3%) adult onset TTP patients, but no normal controls (n = 100). Three of the patients had pregnancy‐associated TTP; three had chronic relapsing acute idiopathic TTP. C3178T encodes an arginine to tryptophan (R1060W) substitution in the TSP1‐7 domain of ADAMTS‐13. In vitro expression of mutant and wild‐type ADAMTS‐13 demonstrated that R1060W caused severe intracellular retention of ADAMTS‐13 (<5% secretion) without affecting its metalloprotease activity. One homozygous and five heterozygous patients were identified. No other causative mutations were discovered, yet all six patients had ADAMTS‐13 activity levels <5% at presentation (normal: 66–126%). Antibodies/inhibitors to ADAMTS‐13 were detected in three/five heterozygous patients, and all six patients had subnormal antigen levels. Six asymptomatic first‐degree relatives, including those of two probands with antibodies, were also heterozygous for C3178T; all but one had subnormal ADAMTS‐13 activity. Conclusion: The high prevalence of R1060W ADAMTS‐13 in adult onset TTP, together with its absence in childhood congenital TTP cases reported elsewhere, suggests it may be a factor in the development of late onset TTP.

Cellular and molecular basis of von Willebrand disease: studies on blood outgrowth endothelial cells

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

Thrombotic thrombocytopaenic purpura in HIV-infected patients.

Thrombotic thrombocytopaenic purpura (TTP) results from deficiency of von Willebrand factor-cleaving protease (vWF-cp) activity. Eight HIV-infected patients presented with TTP, representing 12.5% of all TTP treated at this centre. In four patients presentation with TTP revealed underlying HIV infection, the other four patients were previously known to be HIV infected, with plasma exchange and highly active antiretroviral therapy (HAART) all recovered. Normalization of vWF-cp activity was associated with recovery. Relapse occurred in two patients who discontinued HAART against medical advice, suggesting that HIV has a causal role in this condition. Given the clear benefit from HAART in addition to plasma exchange, these data suggest that all patients presenting with TTP should undergo HIV testing.

The VWF/ADAMTS13 axis in the antiphospholipid syndrome: ADAMTS13 antibodies and ADAMTS13 dysfunction

Autoantibodies to ADAMTS13 (a disintegrin‐like and metalloprotease with thrombospondin type I motif, member 13) play an important role in the development of microthrombosis in thrombotic thrombocytopenic purpura (TTP). In severe cases of antiphospholipid syndrome (APS), microthrombosis can occur similar to that seen in TTP, suggesting possible mutual pathogenic factors. However, the role of ADAMTS13 in APS is unknown. We hypothesised that aberrations in ADAMTS13 may occur in APS and evaluated ADAMTS13 and von Willebrand factor (VWF) in 68 patients with antiphospholipid antibodies (aPA) including 52 with APS. Thirty‐three (49%) had IgG anti‐ADAMTS13 with 12 of these patients having reduced ADAMTS13 activity, suggesting neutralising antibodies. Low ADAMTS13 activity (median 34%) was demonstrated in 22/68 (33%), all with normal ADAMTS13 antigen levels consistent with dysfunctional ADAMTS13. Reduced ADAMTS13 activity was not secondary to elevated von Willebrand factor (VWF), or increased VWF secretion (normal VWF propeptide), although a reduced VWF clearance was noted in APS. Analysis found no associations between the ADAMTS13 abnormalities and any aPA profile or thrombotic/obstetric complications, although this study was not adequately powered to address clinical associations. Nevertheless, these findings highlight that ADAMTS13 autoantibodies and ADAMTS13 dysfunction can occur in APS, and although the clinical significance remains undetermined, ADAMTS13 dysfunction may be contributory to thrombogenesis in autoimmune conditions other than TTP.

Von Willebrand factor, angiodysplasia and angiogenesis

The large multimeric glycoprotein Von Willebrand factor (VWF) is best known for its role in haemostasis; however in recent years other functions of VWF have been identified, indicating that this protein is involved in multiple vascular processes. We recently described a new role for VWF in controlling angiogenesis, which may have significant clinical implications for patients with Von Willebrand disease (VWD), a genetic or acquired condition caused by the deficiency or dysfunction of VWF. VWD can be associated with angiodysplasia, a condition of degenerative blood vessels often present in the gastrointestinal tract, linked to dysregulated angiogenesis. Angiodysplasia can cause severe intractable bleeding, often refractory to conventional VWD treatments. In this review we summarise the evidence showing that VWF controls angiogenesis, and review the angiogenic pathways which have been implicated in this process. We discuss the possible mechanisms though which VWF regulates angiopoietin-2 (Ang-2) and integrin αvβ3, leading to signalling through vascular endothelial growth factor receptor-2 (VEGFR2), one of the most potent activators of angiogenesis. We also review the evidence that links VWF with angiodysplasia, and how the newly identified function of VWF in controlling angiogenesis may pave the way for the development of novel therapies for the treatment of angiodysplasia in congenital VWD and in acquired conditions such as Heyde syndrome.

The expression of prion protein by endothelial cells: a source of the plasma form of prion protein?

Summary. The neuronal prion protein (PrPC) is also expressed within peripheral tissues including human blood. The majority of blood PrPC is found within the plasma fraction. We hypothesized that the vascular endothelium could be a source of this PrPC. Reverse transcription polymerase chain reaction demonstrated that both human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC‐1) expressed PrPC mRNA. Flow cytometry confirmed PrPC expression on HMEC‐1s and HUVECs (120 900 ± 15 058 and 58 327 ± 4577 molecules PrPC/cell respectively), with no upregulation following cellular activation. Confocal immunofluorescence microscopy confirmed that HMEC‐1s and HUVECs were positive for PrPC on the plasma membrane. Time‐resolved dissociation‐enhanced fluoroimmunoassay (DELFIA®) analysis of cell culture medium demonstrated a slow constitutive release of soluble PrPC not associated with activation. In contrast to von Willebrand factor antigen, PrPC plasma levels in vivo decrease following desmopressin therapy in patients with von Willebrand disease. Measurement of PrPC plasma levels in patients with varying blood counts demonstrated no association between cell count and PrPC concentration. However, there was a higher level of PrPC in plasma from patients with end‐stage renal failure. In conclusion, endothelial cells of both macrovascular and microvascular origin expressed high levels of PrPC which can be constitutively released into the cell culture medium.