investigating-von-willebrand-factor-pathophysiology-using-flow

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage ...

Queen's University

Fps/Fes and Fer comprise a distinct subfamily of cytoplasmic protein-tyrosine kinases, and have both been implicated in the regulation of innate immunity. Previous studies showed that Fps/Fes-knockout mice were hypersensitive to systemic lipopolysaccharide (LPS) challenge, and Fer-deficient mice displayed enhanced recruitment of leucocytes in response to localized LPS challenge. We show here for the first time, a role for Fps in the regulation of leucocyte recruitment to areas of inflammation. Using the cremaster muscle intravital microscopy model, we observed increased leucocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-knockout mice relative to wild-type animals subsequent to localized LPS challenge. There was also a decreased vessel wall shear rate in the post-capillary venules of LPS-challenged Fps/Fes-knockout mice, and an increase in neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry to quantify the expression of surface molecules, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex vivo with LPS. These observations provide important insights into the observed in vivo behaviour of leucocytes in LPS-challenged Fps/Fes-knockout mice and provide evidence that the Fps/Fes kinase plays an important role in the innate immune response.

The Fps/Fes kinase regulates leucocyte recruitment and extravasation during inflammation.

Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), leukotriene C(4) synthase, and additional downstream enzymes, starting with arachidonic acid substrate. CysLTs produced by macrophages, eosinophils, mast cells, and other inflammatory cells activate 3 different high-affinity CysLT receptors: CysLT(1)R, CysLT(2)R, and GPR 17. We sought to investigate vascular sites of CysLT(2)R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT(2)R was investigated by reporter gene expression in a novel CysLT(2)R deficient-LacZ mouse model. CysLT(2)R was expressed in small, but not large, vessels in mouse brain, bladder, skin, and cremaster muscle. Intravital, in addition to confocal and electron, microscopy investigations using FITC-labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT-mediated permeability, which was blocked by application of BAY-u9773, a dual CysLT(1)R/CysLT(2)R antagonist or by CysLT(2)R deficiency. Endothelial human CysLT(2)R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT(2)R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca(2+) signaling. Our results reveal that CysLT(2)R can mediate inflammatory reactions in a vascular bed-specific manner by altering transendothelial vesicle transport-based vascular permeability.

Cysteinyl leukotriene 2 receptor-mediated vascular permeability via transendothelial vesicle transport.

Selective cyclooxygenase-2 (COX-2) inhibitors (coxibs) increase the incidence of cardiovascular and cerebrovascular events. Complete disruption of the murine gene encoding COX-2 (Ptgs2) leads to renal developmental problems, as well as female reproductive anomalies and patent ductus arteriosus of variable penetrance in newborns, thus rendering this genetic approach difficult to compare with coxib administration. Here, we created hypomorphic Ptgs2 (COX-2(Neo/Neo)) mice in which COX-2 expression is suppressed to an extent similar to that achieved with coxibs, but not eliminated, in an attempt to circumvent these difficulties. In LPS-challenged macrophages and cytokine-stimulated endothelial cells obtained from COX-2(Neo/Neo) mice, COX-2 expression was reduced 70-90%, and these mice developed a mild renal phenotype compared with COX-2 mice possessing an active site mutation (COX-2(Y385F/Y385F)), with minimal signs of renal dysfunction as measured by FITC-inulin clearance and blood urea nitrogen. These COX-2 knockdown mice displayed an increased propensity for thrombogenesis compared with their wild-type (COX-2(+/+)) littermates observed by intravital microscopy in cremaster muscle arterioles upon ferric chloride challenge. Measurement of urinary prostanoid metabolites indicated that COX-2(Neo/Neo) mice produced 50% less prostacyclin but similar levels of PGE(2) and thromboxane compared with COX-2(+/+) mice in the absence of any blood pressure and ex vivo platelet aggregation abnormalities. COX-2(Neo/Neo) mice, therefore, provide a genetic surrogate of coxib therapy with disrupted prostacyclin biosynthesis that predisposes to induced arterial thrombosis.

Renal and cardiovascular characterization of COX-2 knockdown mice.

We previously reported postacrosomal sheath WW domain binding protein (PAWP) as a candidate sperm borne, oocyte-activating factor. PAWP enters the oocyte during fertilization and induces oocyte activation events including meiotic resumption, pronuclear formation, and egg cleavage. However, in order to provide proof that PAWP is a primary initiator of zygotic development it is imperative to show that PAWP initiates intracellular calcium signaling, which is considered essential for oocyte activation. Utilizing Xenopus laevis as our model, we injected recombinant PAWP or Xenopus sperm into metaphase II-arrested oocytes and observed a significant rise in intracellular calcium levels over controls. Concurring intensities and durations of PAWP and sperm-induced calcium waves, detected by infrared two-photon laser-scanning fluorescence microscopy, were prevented by coinjection of a competitive PPGY-containing peptide derived from PAWP but not by the point-mutated form of this peptide. This study also correlates PAWP and sperm-induced calcium release with meiotic resumption in Xenopus. The similar mode of oocyte activation, and the ability of the competitive peptide in blocking both sperm- and PAWP-induced calcium release, provide evidence for the first time that sperm-anchored PAWP is a primary initiator of zygotic development.

Sperm-borne protein, PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release.

Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbalpha binding. These "gain of function" mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF(-/-) mice, using hydrodynamic injection. The resultant phenotype was examined, and a ferric chloride-induced injury model was used to examine the thrombogenic effect of the 2B VWD variants in mice. Reconstitution of only the plasma component of VWF resulted in the generation of the 2B VWD phenotype in mice. Variable thrombocytopenia was observed in mice expressing 2B VWF, mimicking the severity seen in 2B VWD patients: mice expressing the V1316M mutation showed the most severe thrombocytopenia. Ferric chloride-induced injury to cremaster arterioles showed a marked reduction in thrombus development and platelet adhesion in the presence of circulating 2B VWF. These defects were only partially rescued by normal platelet transfusions, thus emphasizing the key role of the abnormal plasma VWF environment in 2B VWD.

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions.

Transgenic overexpression of the human cysteinyl leukotriene receptor 2 (CysLT2R) in murine endothelium exacerbates vascular permeability and ischemia/reperfusion injury. Here, we explore the underlying mechanisms of CysLT2R activation-mediated inflammation and delineate the relative contributions of endogenous murine CysLT2R and the transgene-derived receptor.

Multiple-site activation of the cysteinyl leukotriene receptor 2 is required for exacerbation of ischemia/reperfusion injury.

Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen-binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches. Five loss-of-function (p.S1731T, p.W1745C, p.S1783A, p.H1786D, A3 deletion) and 1 gain-of-function (p.L1757A) variants were generated in the mouse VWF complementary DNA. The results of these various assays were consistent, although the magnitude of the effects were different: the gain-of-function (p.L1757A) variant showed consistent enhanced collagen binding whereas the loss-of-function mutants showed variable degrees of functional deficit. We further analyzed the impact of direct platelet-collagen binding by blocking glycoprotein VI (GPVI) and integrin α2β1 in our ferric chloride murine thrombosis model. The inhibition of GPVI demonstrated a comparable functional defect in thrombosis formation to the VWF(-/-) mice whereas α2β1 inhibition demonstrated a milder bleeding phenotype. Furthermore, a delayed and markedly reduced thrombogenic response was still evident in VWF(-/-), GPVI, and α2β1 blocked animals, suggesting that alternative primary hemostatic mechanisms can partially rescue the bleeding phenotype associated with these defects.

Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation.

Omega-3 polyunsaturated fatty acids (ω3 PUFAs) suppress inflammation through activation of free fatty acid receptor 4 (FFAR4), but this pathway has not been explored in the context of cardiovascular disease. We aimed to elucidate the involvement of FFAR4 activation by ω3 PUFAs in the process of vascular inflammation and neointimal hyperplasia in mice.

Endogenously generated omega-3 fatty acids attenuate vascular inflammation and neointimal hyperplasia by interaction with free fatty acid receptor 4 in mice.

The maternal system is challenged with many physiological changes throughout pregnancy to prepare the body to meet the metabolic needs of the fetus and for delivery. Many pregnancies, however, are faced with pathological stressors or complications that significantly impact maternal health. A shift in this paradigm is now beginning to investigate the implication of pregnancy complications on the fetus and their continued influence on offspring disease risk into adulthood. In this investigation, we sought to determine whether maternal hypertension during pregnancy alters the cerebral response of adult offspring to acute ischemic stroke. Atrial natriuretic peptide gene-disrupted (ANP(-/-)) mothers exhibit chronic hypertension that escalates during pregnancy. Through comparison of heterozygote offspring born from either normotensive (ANP(+/-WT)) or hypertensive (ANP(+/-KO)) mothers, we have demonstrated that offspring exposed to maternal hypertension exhibit larger cerebral infarct volumes following middle cerebral artery occlusion. Observation of equal baseline cardiovascular measures, cerebrovascular structure, and cerebral blood volumes between heterozygote offspring suggests no added influences on offspring that would contribute to adverse cerebral response post-stroke. Cerebral mRNA expression of endothelin and nitric oxide synthase vasoactive systems demonstrated up-regulation of Et-1 and Nos3 in ANP(+/-KO) mice and thus an enhanced acute vascular response compared to ANP(+/-WT) counterparts. Gene expression of Na(+)/K(+) ATPase channel isoforms, Atp1a1, Atp1a3, and Atp1b1, displayed no significant differences. These investigations are the first to demonstrate a fetal programming effect between maternal hypertension and adult offspring stroke outcome. Further mechanistic studies are required to complement epidemiological evidence of this phenomenon in the literature.

Maternal hypertension programs increased cerebral tissue damage following stroke in adult offspring.

Hypoxic pulmonary vasoconstriction (HPV) is a homeostatic mechanism that is intrinsic to the pulmonary vasculature. Intrapulmonary arteries constrict in response to alveolar hypoxia, diverting blood to better-oxygenated lung segments, thereby optimizing ventilation/perfusion matching and systemic oxygen delivery. In response to alveolar hypoxia, a mitochondrial sensor dynamically changes reactive oxygen species and redox couples in pulmonary artery smooth muscle cells (PASMC). This inhibits potassium channels, depolarizes PASMC, activates voltage-gated calcium channels, and increases cytosolic calcium, causing vasoconstriction. Sustained hypoxia activates rho kinase, reinforcing vasoconstriction, and hypoxia-inducible factor (HIF)-1α, leading to adverse pulmonary vascular remodeling and pulmonary hypertension (PH). In the nonventilated fetal lung, HPV diverts blood to the systemic vasculature. After birth, HPV commonly occurs as a localized homeostatic response to focal pneumonia or atelectasis, which optimizes systemic Po2 without altering pulmonary artery pressure (PAP). In single-lung anesthesia, HPV reduces blood flow to the nonventilated lung, thereby facilitating thoracic surgery. At altitude, global hypoxia causes diffuse HPV, increases PAP, and initiates PH. Exaggerated or heterogeneous HPV contributes to high-altitude pulmonary edema. Conversely, impaired HPV, whether due to disease (eg, COPD, sepsis) or vasodilator drugs, promotes systemic hypoxemia. Genetic and epigenetic abnormalities of this oxygen-sensing pathway can trigger normoxic activation of HIF-1α and can promote abnormal metabolism and cell proliferation. The resulting pseudohypoxic state underlies the Warburg metabolic shift and contributes to the neoplasia-like phenotype of PH. HPV and oxygen sensing are important in human health and disease.

Hypoxic Pulmonary Vasoconstriction: From Molecular Mechanisms to Medicine.

Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer-like phenotype is promoted by increased cytosolic calcium ([Ca(2+)]cyto), aerobic glycolysis, and mitochondrial fission.

MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype.

Right ventricular (RV) function determines prognosis in pulmonary arterial hypertension (PAH). We hypothesize that ischemia causes RV dysfunction in PAH by triggering dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. RV function was compared in control rats (n = 50) versus rats with monocrotaline-induced PAH (MCT-PAH; n = 60) both in vivo (echocardiography) and ex vivo (RV Langendorff). Mitochondrial membrane potential and morphology and RV function were assessed before or after 2 cycles of ischemia-reperfusion injury challenge (RV-IR). The effects of Mdivi-1 (25 μM), a Drp1 GTPase inhibitor, and P110 (1 μM), a peptide inhibitor of Drp1-Fis1 interaction, were studied. We found that MCT caused RV hypertrophy, RV vascular rarefaction, and RV dysfunction. Prior to IR, the mitochondria in MCT-PAH RV were depolarized and swollen with increased Drp1 content and reduced aconitase activity. RV-IR increased RV end diastolic pressure (RVEDP) and mitochondrial Drp1 expression in both control and MCT-PAH RVs. IR depolarized mitochondria in control RV but did not exacerbate the basally depolarized MCT-PAH RV mitochondria. During RV IR mdivi-1 and P110 reduced Drp1 translocation to mitochondria, improved mitochondrial structure and function, and reduced RVEDP. In conclusion, RV ischemia occurs in PAH and causes Drp1-Fis1-mediated fission leading to diastolic dysfunction. Inhibition of mitochondrial fission preserves RV function in RV-IR.

Ischemia-induced Drp1 and Fis1-mediated mitochondrial fission and right ventricular dysfunction in pulmonary hypertension.

Bleeding associated with angiodysplasia is a common, often intractable complication in patients with von Willebrand disease (VWD). von Willebrand factor (VWF), the protein deficient or defective in VWD, is a negative regulator of angiogenesis, which may explain the pathologic blood vessel growth in VWD. This study explores the normal range of angiogenesis in blood outgrowth endothelial cells (BOECs) derived from healthy donors and compares this to angiogenesis in BOECs from VWD patients of all types and subtypes. BOECs were assessed for VWF and angiopoietin-2 (Ang-2) gene expression, secretion, and storage. To explore angiogenic potential, we characterized cellular proliferation, matrix protein adhesion, migration, and tubule formation. We found great angiogenic variability in VWD BOECs with respect to each of the angiogenesis parameters. However, type 1 and 3 VWD BOECs had higher Ang-2 secretion associated with impaired endothelial cell migration velocity and enhanced directionality. Type 2A and 2B BOECs were the most proliferative and multiple VWD BOECs had impaired tubule formation in Matrigel. This study highlights the angiogenic variability in BOECs derived from VWD patients. Abnormal cell proliferation, migration, and increased Ang-2 secretion are common features of VWD BOECs. Despite the many abnormalities of VWD BOECs, significant heterogeneity among individual VWD phenotypes precludes a simple description of relationship between VWD type and in vitro surrogates for angiodysplasia.

Article	Year
The Fps/Fes kinase regulates leucocyte recruitment and extravasation during inflammation. Immunology\| PubMed ID: 17627769	2007
Cysteinyl leukotriene 2 receptor-mediated vascular permeability via transendothelial vesicle transport. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 18779380	2008
Renal and cardiovascular characterization of COX-2 knockdown mice. American journal of physiology. Regulatory, integrative and comparative physiology\| PubMed ID: 19357295	2009
Sperm-borne protein, PAWP, initiates zygotic development in Xenopus laevis by eliciting intracellular calcium release. Molecular reproduction and development\| PubMed ID: 20017143	2010
Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions. Blood\| PubMed ID: 20371742	2010
Multiple-site activation of the cysteinyl leukotriene receptor 2 is required for exacerbation of ischemia/reperfusion injury. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 24285579	2014
Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation. Blood\| PubMed ID: 25051961	2014
Endogenously generated omega-3 fatty acids attenuate vascular inflammation and neointimal hyperplasia by interaction with free fatty acid receptor 4 in mice. Journal of the American Heart Association\| PubMed ID: 25845931	2015
Maternal hypertension programs increased cerebral tissue damage following stroke in adult offspring. Molecular and cellular biochemistry\| PubMed ID: 26169981	2015
Hypoxic Pulmonary Vasoconstriction: From Molecular Mechanisms to Medicine. Chest\| PubMed ID: 27645688	2017
MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype. American journal of respiratory and critical care medicine\| PubMed ID: 27648837	2017
Ischemia-induced Drp1 and Fis1-mediated mitochondrial fission and right ventricular dysfunction in pulmonary hypertension. Journal of molecular medicine (Berlin, Germany)\| PubMed ID: 28265681	2017
Abnormal angiogenesis in blood outgrowth endothelial cells derived from von Willebrand disease patients. Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis\| PubMed ID: 28362648	2017

Jeffrey Mewburn

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