Mohammad Abdul Hai Siddique

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary Hypertension: A Possible Novel Therapeutic Target.

Rationale: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. Objective: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Methods and Results: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator–activated receptor-&agr; agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. Conclusions: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.

Selenoprotein P Promotes the Development of Pulmonary Arterial Hypertension: A Possible Novel Therapeutic Target

Background: Excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) are key mechanisms of pulmonary arterial hypertension (PAH). Despite the multiple combination therapy, a considerable number of patients develop severe pulmonary hypertension (PH) because of the lack of diagnostic biomarker and antiproliferative therapies for PASMCs. Methods: Microarray analyses were used to identify a novel therapeutic target for PAH. In vitro experiments, including lung and serum samples from patients with PAH, cultured PAH-PASMCs, and high-throughput screening of 3336 low-molecular-weight compounds, were used for mechanistic study and exploring a novel therapeutic agent. Five genetically modified mouse strains, including PASMC-specific selenoprotein P (SeP) knockout mice and PH model rats, were used to study the role of SeP and therapeutic capacity of the compounds for the development of PH in vivo. Results: Microarray analysis revealed a 32-fold increase in SeP in PAH-PASMCs compared with control PASMCs. SeP is a widely expressed extracellular protein maintaining cellular metabolism. Immunoreactivity of SeP was enhanced in the thickened media of pulmonary arteries in PAH. Serum SeP levels were also elevated in patients with PH compared with controls, and high serum SeP predicted poor outcome. SeP-knockout mice (SeP–/–) exposed to chronic hypoxia showed significantly reduced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling compared with controls. In contrast, systemic SeP-overexpressing mice showed exacerbation of hypoxia-induced PH. Furthermore, PASMC-specific SeP–/– mice showed reduced hypoxia-induced PH compared with controls, whereas neither liver-specific SeP knockout nor liver-specific SeP-overexpressing mice showed significant differences with controls. Altogether, protein levels of SeP in the lungs were associated with the development of PH. Mechanistic experiments demonstrated that SeP promotes PASMC proliferation and resistance to apoptosis through increased oxidative stress and mitochondrial dysfunction, which were associated with activated hypoxia-inducible factor-1&agr; and dysregulated glutathione metabolism. It is important to note that the high-throughput screening of 3336 compounds identified that sanguinarine, a plant alkaloid with antiproliferative effects, reduced SeP expression and proliferation in PASMCs and ameliorated PH in mice and rats. Conclusions: These results indicate that SeP promotes the development of PH, suggesting that it is a novel biomarker and therapeutic target of the disorder.

SmgGDS Prevents Thoracic Aortic Aneurysm Formation and Rupture by Phenotypic Preservation of Aortic Smooth Muscle Cells

Background: Thoracic aortic aneurysm (TAA) and dissection are fatal diseases that cause aortic rupture and sudden death. The small GTP-binding protein GDP dissociation stimulator (SmgGDS) is a crucial mediator of the pleiotropic effects of statins. Previous studies revealed that reduced force generation in aortic smooth muscle cells (AoSMCs) causes TAA and thoracic aortic dissection. Methods: To examine the role of SmgGDS in TAA formation, we used an angiotensin II (1000 ng·min−1·kg−1, 4 weeks)–induced TAA model. Results: We found that 33% of Apoe−/−SmgGDS+/− mice died suddenly as a result of TAA rupture, whereas there was no TAA rupture in Apoe−/− control mice. In contrast, there was no significant difference in the ratio of abdominal aortic aneurysm rupture between the 2 genotypes. We performed ultrasound imaging every week to follow up the serial changes in aortic diameters. The diameter of the ascending aorta progressively increased in Apoe−/−SmgGDS+/− mice compared with Apoe−/− mice, whereas that of the abdominal aorta remained comparable between the 2 genotypes. Histological analysis of Apoe−/−SmgGDS+/− mice showed dissections of major thoracic aorta in the early phase of angiotensin II infusion (day 3 to 5) and more severe elastin degradation compared with Apoe−/− mice. Mechanistically, Apoe−/−SmgGDS+/− mice showed significantly higher levels of oxidative stress, matrix metalloproteinases, and inflammatory cell migration in the ascending aorta compared with Apoe−/− mice. For mechanistic analyses, we primary cultured AoSMCs from the 2 genotypes. After angiotensin II (100 nmol/L) treatment for 24 hours, Apoe−/−SmgGDS+/− AoSMCs showed significantly increased matrix metalloproteinase activity and oxidative stress levels compared with Apoe−/− AoSMCs. In addition, SmgGDS deficiency increased cytokines/chemokines and growth factors in AoSMCs. Moreover, expressions of fibrillin-1 (FBN1), &agr;-smooth muscle actin (ACTA2), myosin-11 (MYH11), MYLLK, and PRKG1, which are force generation genes, were significantly reduced in Apoe−/−SmgGDS+/− AoSMCs compared with Apoe−/− AoSMCs. A similar tendency was noted in AoSMCs from patients with TAA compared with those from control subjects. Finally, local delivery of the SmgGDS gene construct reversed the dilation of the ascending aorta in Apoe−/−SmgGDS+/− mice. Conclusions: These results suggest that SmgGDS is a novel therapeutic target for the prevention and treatment of TAA.

Different roles of myocardial ROCK1 and ROCK2 in cardiac dysfunction and postcapillary pulmonary hypertension in mice

Significance Our data suggest opposite roles for ROCK1 and ROCK2 in cardiomyocytes. Additional studies identified downstream targets of ROCK1 and ROCK2 related to calcium handling, mitochondrial function, and oxidative stress. In particular, our findings indicate that cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary pulmonary hypertension (PH) in cROCK1−/− mice, while their expressions were lower in cROCK2−/− mice. Finally, screening of the public chemical library in the Drug Discovery Initiative enabled us to identify compounds that reduce the expressions of CyPA and Bsg. Among them, celastrol suppressed the expression of both CyPA and Bsg in heart and lungs, thereby ameliorating both heart failure and postcapillary PH in mice. Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1−/−) and ROCK2-deficient (cROCK2−/−) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1−/− mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2−/− mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1−/− hearts and down-regulated in cROCK2−/− hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1−/− mice, whereas their expressions were significantly lower in cROCK2−/− mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary HypertensionNovelty and Significance

Rationale: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. Objective: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Methods and Results: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator–activated receptor-&agr; agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. Conclusions: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.

Activated TAFI Promotes the Development of Chronic Thromboembolic Pulmonary HypertensionNovelty and Significance: A Possible Novel Therapeutic Target

Rationale: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients. Objective: To determine the role of activated TAFI (TAFIa) in the development of CTEPH. Methods and Results: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator–activated receptor-&agr; agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats. Conclusions: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH.