Magnesium Lithospermate B Derived from Salvia miltiorrhiza Ameliorates Right Ventricle Remodeling in Pulmonary Hypertensive Rats via Inhibition of NOX/VPO1 Pathway.

Right ventricle (RV) remodeling is a major pathological feature in pulmonary arterial hypertension (PAH). Magnesium lithospermate B (MLB) is a compound isolated from the roots of Salvia miltiorrhiza and it possesses multiple pharmacological activities such as anti-inflammation and antioxidation. This study aims to investigate whether MLB is able to prevent RV remodeling in PAH and the underlying mechanisms. In vivo, SD rats were exposed to 10% O2 for 21 d to induce RV remodeling, which showed hypertrophic features (increases in the ratio of RV weight to tibia length, cellular size, and hypertrophic marker expression), accompanied by upregulation in expression of NADPH oxidases (NOX2 and NOX4) and vascular peroxidase 1 (VPO1), increases in hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production and elevation in phosphorylation levels of ERK; these changes were attenuated by treating rats with MLB. In vitro, the cultured H9c2 cells were exposed to 3% O2 for 24 h to induce hypertrophy, which showed hypertrophic features (increases in cellular size and hypertrophic marker expression). Administration of MLB or VAS2870 (a positive control for NOX inhibitor) could prevent cardiomyocyte hypertrophy concomitant with decreases in NOX (NOX2 and NOX4) and VPO1 expression, H2O2 and HOCl production, and ERK phosphorylation. Based on these observations, we conclude that MLB is able to prevent RV remodeling in hypoxic PAH rats through a mechanism involving a suppression of NOX/VPO1 pathway as well as ERK signaling pathway. MLB may possess the potential clinical value for PAH therapy.

Cerebellin-2 promotes endothelial-mesenchymal transition in hypoxic pulmonary hypertension rats by activating NF-κB/HIF-1α/Twist1 pathway.

AIMS: Endothelial-mesenchymal transition (EndMT) is one of the critical factors leading to vascular remodeling in pulmonary hypertension (PH). Recent studies found that the expression of Cerebellin-2 (CBLN2) is significantly increased in the lung tissue of patients with PH, suggesting that CBLN2 may be closely related to the development of PH. This study aims to investigate the role and potential mechanism of CBLN2 in the hypoxia-induced EndMT of PH rats. MATERIAL AND METHODS: Hypoxia-induced PH rat model or EndMT cell model was constructed to investigate the role of CBLN2 in the process of endothelial mesenchymal transition during PH. The effects of CBLN2 siRNA, KC7F2 (HIF-1α inhibitor), and PDTC (NF-κB inhibitor) on hypoxia-induced EndMT were observed to evaluate the potential mechanism of CBLN2 in promoting EndMT. KEY FINDINGS: The right ventricular systolic pressure and pulmonary vascular remodeling index in hypoxia-treated rats were significantly increased. The transformation of endothelial cells (marked by CD31) to mesenchymal cells (marked by α-SMA) can be observed in the pulmonary vessels of PH rats, and the expression of CBLN2 in the intima was also significantly up-regulated. In the hypoxia-induced HPAECs, endothelial cell markers such as VE-cadherin and CD31 expression were significantly down-regulated, while mesenchymal-like cell markers such as α-SMA and vimentin were increased considerably, along with the increased expressions of CBLN2, p-p65, HIF-1α, and Twist1; CBLN2 siRNA, PDTC, and KC7F2 could inhibit those phenomena. SIGNIFICANCE: CBLN2 can promote EndMT by activating NF-κB/HIF-1α/Twist1 pathway. Therefore, CBLN2 may be a new therapeutic target for PH.

Magnolol alleviates hypoxia-induced pulmonary vascular remodeling through inhibition of phenotypic transformation in pulmonary arterial smooth muscle cells.

Phenotypic transformation and excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) play an important role in vascular remodeling during pulmonary hypertension (PH). Magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl) is the major bioactive constituent isolated from the bark of Magnolia Officinalis, which has anti-inflammatory, antioxidant, and cardiovascular protection effects. However, the effect of magnolol on the phenotypic transformation of PASMCs is still unknown. This study aims to evaluate the effects of magnolol on the phenotypic transformation of PASMCs induced by hypoxia. In vivo, Sprague Dawley rats were exposed to hypoxia (10% O2) for four weeks to establish a PH model. The results showed that hypoxia treatment led to an increase in right ventricle systolic pressure, Fulton index, collagen production, accompanied by upregulation in the expression of collagen Ⅰ, collagen Ⅲ, OPN, PCNA, CyclinD1, p-JAK2, and p-STAT3, as well as decreases in expression of SM-22α; these changes were attenuated by magnolol. In vitro, the primary cultured PASMCs were exposed to 3% O2 for 48 h to induce phenotypic transformation. Consistent with the findings in vivo, magnolol treatment could prevent the phenotypic transformation and hyperproliferation of PASMCs induced by hypoxia, accompanied by downregulation in the expression of p-JAK2 and p-STAT3. In summary, this study demonstrated that the protective effect of magnolol on PH vascular remodeling is related to the inhibition of phenotypic transformation and hyperproliferation of PASMCs by inhibiting the JAK2/STAT3 pathway.

Coordination between NADPH oxidase and vascular peroxidase 1 promotes dysfunctions of endothelial progenitor cells in hypoxia-induced pulmonary hypertensive rats.

Previous studies have demonstrated that NADPH oxidase (NOX)/vascular peroxidase (VPO1) pathway - mediated oxidative stress plays an important role in the pathogenesis of multiple cardiovascular diseases. This study aims to evaluate the correlation between NOX/VPO1 pathway and endothelial progenitor cells (EPCs) dysfunctions in hypoxia-induced pulmonary hypertension (PH). The rats were exposed to 10% hypoxia for 3 weeks to establish a PH model, which showed increases in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, acceleration in apoptosis and impairment in functions of the peripheral blood derived - EPCs (the reduced abilities in adhesion, migration and tube formation), accompanied by up-regulation of NOX (NOX2 and NOX4) and VPO1. Next, normal EPCs were cultured under hypoxia to induce apoptosis in vitro. Consistent with the in vivo findings, hypoxia enhanced the apoptosis and dysfunctions of EPCs concomitant with an increase in NOX and VPO1 expression, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production; these phenomena were attenuated by NOX2 or NOX4 siRNA. Knockdown of VPO1 showed similar results to that of NOX siRNA except no effect on NOX expression and H2O2 production. Based on these observations, we conclude that NOX/VPO1 pathway-derived reactive oxygen species promote the oxidative injury and dysfunctions of EPCs in PH, which may contribute to endothelial dysfunctions in PH.

Magnesium lithospermate B prevents phenotypic transformation of pulmonary arteries in rats with hypoxic pulmonary hypertension through suppression of NADPH oxidase.

Magnesium lithospermate B (MLB) shows multiple biological activities including anti-oxidation and anti-proliferation in various diseases. However, the function of MLB in pulmonary arterial hypertension (PAH) is still unknown. This study aims to investigate the effect of MLB on hypoxia-induced phenotypic transformation of pulmonary arterial smooth muscle cells (PASMCs) and the underlying mechanisms. SD rats (or PASMCs) were exposed to 10% O2 for 3 weeks (or 3% O2 for 48 h) along with MLB or NADPH oxidase （NOX） inhibitor intervention. The effects of MLB on hemodynamics, pulmonary vascular remodeling and phenotypic transformation of PASMCs were observed first. Then, its effects on the protein levels of NOX (NOX2 and NOX4), ERK and p-ERK were examined. The results showed that MLB prevented the elevation in right ventricular systolic pressure and the increase in ratio of wall thickness to vessel external diameter of pulmonary arteries in PAH rats, and attenuated phenotypic transformation of PASMCs (decrease in α-smooth muscle actin while increase in osteopontin), accompanied by downregulation of NOX (NOX2 and NOX4) protein levels, decrease of ROS and H2O2 production, and suppression of the phosphorylation of ERK. NOX inhibitor (VAS2870) achieved similar results to that of MLB did in the hypoxia-treated PASMCs. Based on the observations, we conclude that MLB is able to prevent phenotypic transformation of pulmonary arteries in hypoxic PAH rats through suppression of NOX/ROS/ERK pathway, and MLB might have the potentials in PAH therapy.