Jeffrey Mewburn

Renal and cardiovascular characterization of COX-2 knockdown mice.

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.

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

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. Mol. Reprod. Dev. 77: 249–256, 2010.

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

Cysteinyl leukotrienes (CysLTs) are potent mediators of inflammation synthesized by the concerted actions of 5‐lipoxygenase (5‐LO), 5‐LO‐activating protein (FLAP), leukotriene C4 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: CysLT1R, CysLT2R, and GPR 17. We sought to investigate vascular sites of CysLT2R expression and the role and mechanism of this receptor in mediating vascular permeability events. Vascular expression of CysLT2R was investigated by reporter gene expression in a novel CysLT2R deficient‐LacZ mouse model. CysLT2R 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 FIT C‐labeled albumin in cremaster postcapillary venule preparations indicated rapid CysLT‐mediated permeability, which was blocked by application of BAY‐u9773, a dual CysLT1R/CysLT2R antagonist or by CysLT2R deficiency. Endothelial human CysLT2R overexpression in mice exacerbated vascular leakage even in the absence of exogenous ligand. The enhanced vascular permeability mediated by CysLT2R takes place via a transendothelial vesicle transport mechanism as opposed to a paracellular route and is controlled via Ca2+ signaling. Our results reveal that CysLT2R can mediate inflammatory reactions in a vascular bed‐specific manner by altering transendothelial vesicle transport‐based vascular permeability.— Moos, M. P. W., Mewburn, J. D., Kan, F. W. K., Ishii, S., Abe, M., Sakimura, K., Noguchi, K., Shimizu, T., Funk, C. D. Cysteinyl leukotriene 2 receptor‐mediated vascular permeability via transendothelial vesicle transport. FASEB J. 22, 4352–4362 (2008)

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

Rationale: 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 ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAHs cancer‐like phenotype. Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore‐forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid‐mediated up‐regulation, as well as through modulation of the upstream microRNAs (miRs) miR‐138 and miR‐25. In vivo, nebulized anti‐miRs were administered to rats with monocrotaline‐induced PAH. Measurements and Main Results: Impaired MCUC function, resulting from down‐regulation of MCU and up‐regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAHs pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR‐138) down‐regulate MCU directly and also by decreasing MCUs transcriptional regulator cAMP response element‐binding protein 1. Nebulized anti‐miRs against miR‐25 and miR‐138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model. Conclusions: These results highlight miR‐mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted.

Hypoxic Pulmonary Vasoconstriction: From Molecular Mechanisms to Medicine

&NA; 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)‐1&agr;, 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‐1&agr; 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.

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

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.

Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis.

Essentials Dysregulated DNA and histone release can promote pathological immunothrombosis. Weibel‐Palade bodies (WPBs) are sentinel‐like organelles that respond to proinflammatory stimuli. Histones induce WPB exocytosis in a caspase, calcium and charge‐dependent mechanism. A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis.

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

Background 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. Methods and Results We used mice with disruption of FFAR4 (Ffar4−/−), along with a strain that synthesizes high levels of ω3 PUFAs (fat‐1) and a group of crossed mice (Ffar4−/−/fat‐1), to elucidate the role of FFAR4 in vascular dysfunction using acute and chronic thrombosis/vascular remodeling models. The presence of FFAR4 in vascular‐associated cells including perivascular adipocytes and macrophages, but not platelets, was demonstrated. ω3 PUFAs endogenously generated in fat‐1 mice (n=9), but not in compound Ffar4−/−/fat‐1 mice (n=9), attenuated femoral arterial thrombosis induced by FeCl3. Neointimal hyperplasia and vascular inflammation in the common carotid artery were significantly curtailed 4 weeks after FeCl3 injury in fat‐1 mice (n=6). This included greater luminal diameter and enhanced blood flow, reduced intima:media ratio, and diminished macrophage infiltration in the vasculature and perivascular adipose tissue compared with control mice. These effects were attenuated in the Ffar4−/−/fat‐1 mice. Conclusions These results indicate that ω3 PUFAs mitigate vascular inflammation, arterial thrombus formation, and neointimal hyperplasia by interaction with FFAR4 in mice. Moreover, the ω3 PUFA–FFAR4 pathway decreases inflammatory responses with dampened macrophage transmigration and infiltration.

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

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.

In vitro and in vivo evaluation of the effect of elevated factor VIII on the thrombogenic process

Factor VIII (FVIII), a procoagulant cofactor, plays a crucial role in the intrinsic coagulation cascade. A causal association between elevated FVIII levels and venous thrombosis incidence has been established; no such association has been confirmed with arterial thrombosis. The independent role of elevated FVIII levels in arteriolar thrombosis was evaluated in a mouse model to determine the thrombogenic potential of elevated levels of FVIII. The in vitro thrombogenic effect of elevated FVIII levels was examined using thrombin-antithrombin (TAT) complex generation and thromboelastography (TEG) assays. The thrombogenic potential of acute and extended elevation of circulating FVIII levels was assessed using ferric chloride induced injury of the cremaster arterioles. The rate of TAT complex formation, and the final concentration of TAT complexes, significantly increased as FVIII levels were elevated from 100% to 400% FVIII activity. TEG analysis of fibrin and clot formation showed that as FVIII levels were elevated, the time to initial fibrin formation decreased and the rate of fibrin formation increased. The acute elevation of circulating FVIII to 400% FVIII activity resulted in significantly decreased times to vessel occlusion. Prolonged elevation of FVIII activity did not significantly affect time to vessel occlusion. In conclusion, acute elevations in FVIII levels result in a non-linear thrombogenic effect, with non-significant increases in thrombogenic risk within the physiological range (FVIII levels up to 200%). Prolonged elevation of plasma FVIII did not further increase the thrombogenic potential of elevated FVIII levels.