Katina M. Wilson

Hydrogen Peroxide Promotes Aging-Related Platelet Hyperactivation and Thrombosis

Background— The incidence of thrombotic events increases during aging, but the mechanisms are not well understood. To investigate the prothrombotic role of oxidative stress during aging, we tested the hypothesis that aged mice overexpressing the antioxidant enzyme glutathione peroxidase-1 (Gpx1) are protected from experimental thrombosis. Methods and Results— Susceptibility to carotid artery thrombosis was first examined in wild-type C57BL/6J mice. After photochemical injury of the carotid artery, the time to stable occlusion was significantly shorter in 12- and 18-month-old mice compared with 4-month-old mice (P<0.01). Unlike wild-type mice, transgenic mice overexpressing Gpx1 (Gpx1 Tg) did not exhibit shortened times to occlusion of the carotid artery at 12 or 18 months of age. Wild-type mice also exhibited increased susceptibility to venous thrombosis after inferior vena cava ligation at 12 or 18 months of age (P<0.05 versus 4 months of age). Gpx1 Tg mice were protected from this aging-related enhanced susceptibility to venous thrombosis. Age-dependent platelet hyperactivation, evidenced by increased hydrogen peroxide, fibrinogen binding, and activation of fibrinogen receptor &agr;IIb&bgr;3, was observed in thrombin-activated platelets from wild-type but not Gpx1 Tg mice (P<0.05). Enhanced platelet activation responses in aged mice were also prevented by polyethylene glycol–catalase or apocynin, an inhibitor of NADPH oxidase. Aged mice displayed increased intraplatelet expression of p47phox and superoxide dismutase-1, suggesting a mechanistic pathway for increased hydrogen peroxide generation. Conclusions— Our findings demonstrate that hydrogen peroxide is a key mediator of platelet hyperactivity and enhanced thrombotic susceptibility in aged mice.

Prothrombotic Effects of Hyperhomocysteinemia and Hypercholesterolemia in ApoE-Deficient Mice

Objective—We tested the hypothesis that hyperhomocysteinemia and hypercholesterolemia promote arterial thrombosis in mice. Methods and Results—Male apolipoprotein E (Apoe)-deficient mice were fed one of four diets: control, hyperhomocysteinemic (HH), high fat (HF), or high fat/hyperhomocysteinemic (HF/HH). Total cholesterol was elevated 2-fold with the HF or HF/HH diets compared with the control or HH diets (P<0.001). Plasma total homocysteine (tHcy) was elevated (12 to 15 &mgr;mol/L) with the HH or HF/HH diets compared with the control or HF diets (4 to 6 &mgr;mol/L; P<0.001). Aortic sinus lesion area correlated strongly with total cholesterol (P<0.001) but was independent of tHcy. At 12 weeks of age, the time to thrombotic occlusion of the carotid artery after photochemical injury was >50% shorter in mice fed the HF diets, with or without hyperhomocysteinemia, compared with the control diet (P<0.05). At 24 weeks of age, carotid artery thrombosis was also accelerated in mice fed the HH diet (P<0.05). Endothelium-dependent nitric oxide–mediated relaxation of carotid artery rings was impaired in mice fed the HF, HH, or HF/HH diets compared with the control diet (P<0.05). Conclusions—Hyperhomocysteinemia and hypercholesterolemia, alone or in combination, produce endothelial dysfunction and increased susceptibility to thrombosis in Apoe-deficient mice.

Effect of mechanical ventilation on carotid artery thrombosis induced by photochemical injury in mice.

Summary.  Background: Increasing use of transgenic and gene targeting techniques for the investigation of hemostasis and vascular biology has generated interest in experimental models of carotid artery thrombosis in mice. Objectives: We tested the hypothesis that hypoventilation in anesthetized mice may cause hypercapnia, increased carotid artery blood flow, and altered thrombotic responses to photochemical injury of the carotid artery. Methods: Arterial blood gases and carotid artery blood flow were measured in pentobarbital‐anesthetized BALB/c or C57BL/6 J mice with and without mechanical ventilation. Photochemical injury of the carotid artery was induced using the rose bengal method. Results: Compared with ventilated mice, unventilated mice had a 45% increase in carotid artery blood flow (0.74 ± 0.04 vs. 0.41 ± 0.03 mL min−1; P < 0.001) that was associated with an elevation of arterial PCO2 (58 ± 4 vs. 33 ± 4 mmHg; P < 0.05) and a decrease in arterial pH (7.18 ± 0.05 vs. 7.32 ± 0.03; P < 0.05). Time to first occlusion of the carotid artery after photochemical injury was shorter in ventilated than in unventilated mice (29 ± 6 vs. 73 ± 9 min; P < 0.001). Time to stable occlusion was also shorter in ventilated mice (49 ± 8 vs. 81 ± 6 min; P < 0.05). Elevated carotid artery blood flow, hypercarbic acidosis, and prolonged occlusion times also were observed in mice ventilated with supplemental carbon dioxide. Conclusions: General anesthesia without mechanical ventilation has the potential to confound studies of experimental thrombosis in vivo by producing hypoventilation, hypercapnia, acidosis, and altered carotid artery blood flow. Mechanical ventilation with maintenance of normal blood gases may enhance the physiological insight gained from experimental models of carotid artery thrombosis in mice.

Overexpression of Dimethylarginine Dimethylaminohydrolase Protects Against Cerebral Vascular Effects of Hyperhomocysteinemia

Rationale: Hyperhomocysteinemia is a cardiovascular risk factor that is associated with elevation of the nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA). Objective: Using mice transgenic for overexpression of the ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1), we tested the hypothesis that overexpression of DDAH1 protects from adverse structural and functional changes in cerebral arterioles in hyperhomocysteinemia. Methods and Results: Hyperhomocysteinemia was induced in DDAH1 transgenic (DDAH1 Tg) mice and wild-type littermates using a high methionine/low folate (HM/LF) diet. Plasma total homocysteine was elevated approximately 3-fold in both wild-type and DDAH1 Tg mice fed the HM/LF diet compared with the control diet (P<0.001). Plasma ADMA was approximately 40% lower in DDAH1 Tg mice compared with wild-type mice (P<0.001) irrespective of diet. Compared with the control diet, the HM/LF diet diminished endothelium-dependent dilation to 10 &mgr;mol/L acetylcholine in cerebral arterioles of both wild-type (12±2 versus 29±3%; P<0.001) and DDAH1 Tg (14±3 versus 28±2%; P<0.001) mice. Responses to 10 &mgr;mol/L papaverine, a direct smooth muscle dilator, were impaired with the HM/LF diet in wild-type mice (30±3 versus 45±5%; P<0.05) but not DDAH1 Tg mice (45±7 versus 48±6%). DDAH1 Tg mice also were protected from hypertrophy of cerebral arterioles (P<0.05) but not from accelerated carotid artery thrombosis induced by the HM/LF diet. Conclusions: Overexpression of DDAH1 protects from hyperhomocysteinemia-induced alterations in cerebral arteriolar structure and vascular muscle function.

ADAMTS13 modulates atherosclerotic plaque progression in mice via a VWF-dependent mechanism.

ADAMTS13 reduces the adhesiveness of hyperactive ultra‐large von Willebrand factor (ULVWF) multimers by cleaving them into smaller, less active multimers. Recently, we and others have demonstrated that ADAMTS13 reduces atherosclerosis in hypercholesteremic apolipoprotein E (ApoE−/−) deficient mice. It is not known whether ADAMTS13 modulates atherosclerosis directly or indirectly by cleaving ULVWF multimers.

Endothelial PPAR-γ Protects Against Vascular Thrombosis by Downregulating P-Selectin Expression

Objective— We tested the hypothesis that endothelial peroxisome proliferator–activated receptor-&ggr; protects against vascular thrombosis using a transgenic mouse model expressing a peroxisome proliferator–activated receptor-&ggr; mutant (E-V290M) selectively in endothelium. Approach and Results— The time to occlusive thrombosis of the carotid artery was significantly shortened in E-V290M mice compared with nontransgenic littermates after either chemical injury with ferric chloride (5.1±0.2 versus 10.1±3.3 minutes; P=0.01) or photochemical injury with rose bengal (48±9 versus 74±9 minutes; P=0.04). Gene set enrichment analysis demonstrated the upregulation of NF-&kgr;B target genes, including P-selectin, in aortic endothelial cells from E-V290M mice (P<0.001). Plasma P-selectin and carotid artery P-selectin mRNA were elevated in E-V290M mice (P<0.05). P-selectin–dependent leukocyte rolling on mesenteric venules was increased in E-V290M mice compared with nontransgenic mice (53±8 versus 25±7 per minute; P=0.02). The shortened time to arterial occlusion in E-V290M mice was reversed by administration of P-selectin–blocking antibodies or neutrophil-depleting antibodies (P=0.04 and P=0.02, respectively) before photochemical injury. Conclusions— Endothelial peroxisome proliferator–activated receptor-&ggr; protects against thrombosis through a mechanism that involves downregulation of P-selectin expression and diminished P-selectin–mediated leukocyte–endothelial interactions.

Myeloperoxidase Is Increased in Human Cerebral Aneurysms and Increases Formation and Rupture of Cerebral Aneurysms in Mice

Background and Purpose— Cerebral aneurysm (CA) affects 3% of the population and is associated with hemodynamic stress and inflammation. Myeloperoxidase, a major oxidative enzyme associated with inflammation, is increased in patients with CA, but whether myeloperoxidase contributes to CA is not known. We tested the hypotheses that myeloperoxidase is increased within human CA and is critical for formation and rupture of CA in mice. Methods— Blood was drawn from the lumen of CAs and femoral arteries of 25 patients who underwent endovascular coiling of CA, and plasma myeloperoxidase concentrations were measured with ELISA. Effects of endogenous myeloperoxidase on CA formation and rupture were studied in myeloperoxidase knockout mice and wild-type (WT) mice using an angiotensin II–elastase induction model of CA. In addition, effects of myeloperoxidase on inflammatory gene expression in endothelial cells were analyzed. Results— Plasma concentrations of myeloperoxidase were 2.7-fold higher within CA than in femoral arterial blood in patients with CA. myeloperoxidase-positive cells were increased in aneurysm tissue compared with superficial temporal artery of patients with CA. Incidence of aneurysms and subarachnoid hemorrhage was significantly lower in myeloperoxidase knockout than in WT mice. In cerebral arteries, proinflammatory molecules, including tumor necrosis factor-&agr;, cyclooxygenase-2 (COX2), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C motif) ligand (XCL1), matrix metalloproteinase (MMP) 8, cluster of differentiation 68 (CD68), and matrix metalloproteinase 13, and leukocytes were increased, and &agr;-smooth muscle actin was decreased, in WT but not in myeloperoxidase knockout mice after induction of CA. Myeloperoxidase per se increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells. Conclusions— These findings suggest that myeloperoxidase may contribute importantly to formation and rupture of CA.

Smooth Muscle Peroxisome Proliferator–Activated Receptor γ Plays a Critical Role in Formation and Rupture of Cerebral Aneurysms in Mice In Vivo

Vascular inflammation plays a critical role in the pathogenesis of cerebral aneurysms. Peroxisome proliferator–activated receptor &ggr; (PPAR&ggr;) protects against vascular inflammation and atherosclerosis, whereas dominant-negative mutations in PPAR&ggr; promote atherosclerosis and vascular dysfunction. We tested the role of PPAR&ggr; in aneurysm formation and rupture. Aneurysms were induced with a combination of systemic infusion of angiotensin-II and local injection of elastase in (1) mice that received the PPAR&ggr; antagonist GW9662 or the PPAR&ggr; agonist pioglitazone, (2) mice carrying dominant-negative PPAR&ggr; mutations in endothelial or smooth muscle cells, and (3) mice that received the Cullin inhibitor MLN4924. Incidence of aneurysm formation, rupture, and mortality was quantified. Cerebral arteries were analyzed for expression of Cullin3, Kelch-like ECH-associated protein 1, nuclear factor (erythroid-derived 2)-like 2, NAD(P)H dehydrogenase (quinone)1 (NQO1), and inflammatory marker mRNAs. Neither pioglitazone nor GW9662 altered the incidence of aneurysm formation. GW9662 significantly increased the incidence of aneurysm rupture, whereas pioglitazone tended to decrease the incidence of rupture. Dominant-negative endothelial-specific PPAR&ggr; did not alter the incidence of aneurysm formation or rupture. In contrast, dominant-negative smooth muscle–specific PPAR&ggr; resulted in an increase in aneurysm formation (P<0.05) and rupture (P=0.05). Dominant-negative smooth muscle–specific PPAR&ggr;, but not dominant-negative endothelial-specific PPAR&ggr;, resulted in significant decreases in expression of genes encoding Cullin3, Kelch-like ECH-associated protein 1, and nuclear factor (erythroid-derived 2)-like 2, along with significant increases in tumor necrosis factor-&agr;, monocyte chemoattractant protein-1, chemokine (C-X-C motif) ligand 1, CD68, matrix metalloproteinase-3, -9, and -13. MLN4924 did not alter incidence of aneurysm formation, but increased the incidence of rupture (P<0.05). In summary, endogenous PPAR&ggr;, specifically smooth muscle PPAR&ggr;, plays an important role in protecting from formation and rupture of experimental cerebral aneurysms in mice.

Human Thrombomodulin Knock-In Mice Reveal Differential Effects of Human Thrombomodulin on Thrombosis and Atherosclerosis

Objective—We sought to develop a murine model to examine the antithrombotic and antiinflammatory functions of human thrombomodulin in vivo. Methods and Results—Knock-in mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knock-in mice exhibited decreased protein C activation in the aorta (P<0.01) and lung (P<0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knock-in mice compared with wild-type mice (P<0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knock-in mice (12±3 minutes) than in wild-type mice (31±6 minutes; P<0.05). No differences in serum cytokine levels were detected between knock-in and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E–deficient mice and fed a Western diet, knock-in mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis. Conclusion—Expression of human thrombomodulin in place of murine thrombomodulin produces viable mice with a prothrombotic phenotype but unaltered responses to systemic inflammatory or atherogenic stimuli. This humanized animal model will be useful for investigating the function of human thrombomodulin under pathophysiological conditions in vivo.

Deficiency of Superoxide Dismutase Impairs Protein C Activation and Enhances Susceptibility to Experimental Thrombosis

Objective—Clinical evidence suggests an association between oxidative stress and vascular disease, and in vitro studies have demonstrated that reactive oxygen species can have prothrombotic effects on vascular and blood cells. It remains unclear, however, whether elevated levels of reactive oxygen species accelerate susceptibility to experimental thrombosis in vivo. Approach and Results—Using a murine model with genetic deficiency in superoxide dismutase-1 (SOD1), we measured susceptibility to carotid artery thrombosis in response to photochemical injury. We found that SOD1-deficient (Sod1−/−) mice formed stable arterial occlusions significantly faster than wild-type (Sod1+/+) mice (P<0.05). Sod1−/− mice also developed significantly larger venous thrombi than Sod1+/+ mice after inferior vena cava ligation (P<0.05). Activation of protein C by thrombin in lung was diminished in Sod1−/− mice (P<0.05 versus Sod1+/+ mice), and generation of activated protein C in response to infusion of thrombin in vivo was decreased in Sod1−/− mice (P<0.05 versus Sod1+/+ mice). SOD1 deficiency had no effect on the expression of thrombomodulin, endothelial protein C receptor, or tissue factor in lung or levels of protein C in plasma. Exposure of human thrombomodulin to superoxide in vitro caused oxidation of multiple methionine residues, including critical methionine 388, and a 40% decrease in thrombomodulin-dependent activation of protein C (P<0.05). SOD and catalase protected against superoxide-induced methionine oxidation and restored protein C activation in vitro (P<0.05). Conclusions—SOD prevents thrombomodulin methionine oxidation, promotes protein C activation, and protects against arterial and venous thrombosis in mice.