Evaluation and Treatment of Endoplasmic Reticulum (ER) Stress in Right Ventricular Dysfunction during Monocrotaline-Induced Rat Pulmonary Arterial Hypertension.

PURPOSE: Endoplasmic reticulum (ER) stress contributes to pulmonary artery hypertension (PAH). However, the exact roles of ER stress in right ventricular (RV) dysfunction, which is strongly associated with PAH, are largely unknown. Here, we aimed to explore how ER stress affects RV function in a rat PAH model and evaluated the effects of an ER stress inhibitor on RV dysfunction. METHODS: We examined expression changes of an ER marker: chaperone glucose-regulated protein 78 (GRP78), three ER stress sensor proteins: activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and protein kinase RNA-like endoplasmic reticulum kinase (PERK), and a key ER stress-induced apoptosis indicator: CCAAT/enhancer-binding protein homologous protein (CHOP), with inflammation indicators: interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs) in RV at 3, 7, 14 and 28 days following a single dose of monocrotaline (MCT) injection, with or without a preventive treatment [4-phenylbutyric acid (PBA)]. RV function was evaluated by histological, molecular and echocardiographic analysis. RESULTS: 1) GRP78 protein expression started to increase (1.5 ± 0.06 fold change) at 3d post MCT injection, even before the formation of PAH. 2) ATF6, IRE1, and PERK showed distinctive expression patterns post MCT injection. 3) CHOP expression remained low at day 3 & 7, but significantly increased at day 14 (p < 0.05), along with the peak of RV cardiomyocytes apoptosis. 4) PBA inhibited ER stress and alleviated remodeling and dysfunction in the RV. CONCLUSIONS: The early phase of ER stress might benefit RV function, whereas the extended phase led to RV cardiomyocyte apoptosis and dysfunction. Inhibition of ER stress by PBA during PAH directly improved RV function.

Monocrotaline-induced pulmonary arterial hypertension is attenuated by TNF-α antagonists via the suppression of TNF-α expression and NF-κB pathway in rats.

Inflammation is involved in various types of human pulmonary arterial hypertension (PAH), especially in PAH-associated connective tissue diseases. Although the pathogenesis of pulmonary hypertension has still remained largely unclear, TNF-α has been reported as a key pro-inflammatory cytokine in severe pulmonary hypertension and emphysema. The aim of this study was to investigate the effect of a TNF-α antagonist, recombinant TNF-α receptor II:IgG Fc fusion protein (rhTNFRFc), on the development of monocrotaline (MCT)-induced PAH in rats. Our results revealed that treatment of rhTNFRFc in these rats had favorable effects on mPAP levels, hemodynamics and pulmonary vascular remodeling, preventing PAH development at 3weeks following MCT. Furthermore, rhTNFRFc treatment resulted in markedly reduced expression of TNF-α via the inhibition of NF-κB activity in rat lungs. These results demonstrated that rhTNFRFc attenuated the process of MCT-induced PAH through its anti-inflammatory property. Although further studies are needed to define the appropriate treatment regimen, our findings suggest that rhTNFRFc might provide therapeutic benefits for PAH patients.

Nicorandil prevents right ventricular remodeling by inhibiting apoptosis and lowering pressure overload in rats with pulmonary arterial hypertension.

BACKGROUND: Most of the deaths among patients with severe pulmonary arterial hypertension (PAH) are caused by progressive right ventricular (RV) pathological remodeling, dysfunction, and failure. Nicorandil can inhibit the development of PAH by reducing pulmonary artery pressure and RV hypertrophy. However, whether nicorandil can inhibit apoptosis in RV cardiomyocytes and prevent RV remodeling has been unclear. METHODOLOGY/PRINCIPAL FINDINGS: RV remodeling was induced in rats by intraperitoneal injection of monocrotaline (MCT). RV systolic pressure (RVSP) was measured at the end of each week after MCT injection. Blood samples were drawn for brain natriuretic peptide (BNP) ELISA analysis. The hearts were excised for histopathological, ultrastructural, immunohistochemical, and Western blotting analyses. The MCT-injected rats exhibited greater mortality and less weight gain and showed significantly increased RVSP and RV hypertrophy during the second week. These worsened during the third week. MCT injection for three weeks caused pathological RV remodeling, characterized by hypertrophy, fibrosis, dysfunction, and RV mitochondrial impairment, as indicated by increased levels of apoptosis. Nicorandil improved survival, weight gain, and RV function, ameliorated RV pressure overload, and prevented maladaptive RV remodeling in PAH rats. Nicorandil also reduced the number of apoptotic cardiomyocytes, with a concomitant increase in Bcl-2/Bax ratio. 5-hydroxydecanoate (5-HD) reversed these beneficial effects of nicorandil in MCT-injected rats. CONCLUSIONS/SIGNIFICANCE: Nicorandil inhibits PAH-induced RV remodeling in rats not only by reducing RV pressure overload but also by inhibiting apoptosis in cardiomyocytes through the activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. The use of a mitoK(ATP) channel opener such as nicorandil for PAH-associated RV remodeling and dysfunction may represent a new therapeutic strategy for the amelioration of RV remodeling during the early stages of PAH.

The effects of siRNA against RPL22 on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells.

The aim of this study was to investigate the effects of small interference RNA (siRNA) against ribosomal protein L22 (RPL22) on ET-1-induced proliferation of human pulmonary arterial smooth muscle cells (HPASMCs). HPASMCs were transfected with siRNA against RPL22, incubated in smooth muscle cell culture medium (SMCM) and ET-1. RPL22 gene expression and protein levels of HPASMCs were measured by real-time PCR and western blotting, respectively. PCNA, CCK-8 immunohistochemistry and flow cytometry analysis were used to evaluate HPASMC proliferation. Cyclin D1 (CCND1) expression was also assayed. Following transfection with siRNA against RPL22, RPL22 expression was significantly inhibited in the control and ET-1 groups. HPASMC proliferation was significantly suppressed by transfection with siRNA against RPL22. Moreover, CCND1 was also downregulated by inhibiting RPL22 expression. In conclusion, these data suggest that inhibition of RPL22 expression can suppress HPASMC proliferation and CCND1 expression. The effects of siRNA against RPL22 on HPASMC proliferation are considered to be mediated through inhibition of CCND1 expression.

Usefulness of intravenous adenosine in idiopathic pulmonary arterial hypertension as a screening agent for identifying long-term responders to calcium channel blockers.

Although intravenous adenosine is recommended for acute vasodilator testing in patients with pulmonary hypertension, long-term outcomes in acute responders treated with calcium channel blockers (CCBs) who are identified by adenosine remain unknown. In this study, the value of adenosine for identifying long-term responders to CCBs was investigated in patients with idiopathic pulmonary arterial hypertension (IPAH). All acute responders were subsequently treated with high-dose CCB monotherapy, and 6-minute walk distances, hemodynamic data, and World Health Organization functional classifications were followed. Nine of 104 patients exhibited an acute response with intravenous adenosine (8.7%, 95% confidence interval 3.2 to 14.2). After 12 months of follow-up, all acute responders were still alive; however, only 6 patients showed sustained hemodynamic improvement (5.8%, 95% confidence interval 2 to 13). Three patients had failed CCB monotherapy and bosentan was added to their treatment. Mean tolerated dose of intravenous adenosine was 142 ± 49 µg/kg/min. No life-threatening adverse events were observed and only 2 patients of the nonresponders exhibited a 20% decrease in mean systemic arterial pressure. In nonresponders, 1- and 3-year survival rates were 89% and 75%, respectively. In conclusion, acute vasodilator testing with intravenous adenosine was safe and able to screen responders to CCB therapy in patients with IPAH. Long-term CCB responders accounted for about 5.8% of patients with IPAH.