Inhibition of Siah2 ubiquitin ligase ameliorates monocrotaline-induced pulmonary arterial remodeling through inactivation of YAP.

It has been shown that up-regulation of E3 ubiquitin ligase seven-in-absentia-homolog 2 (Siah2) and activation of Hippo signaling pathway effector yes-associated protein (YAP) are involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether Siah2 activates YAP in monocrotaline (MCT)-induced PAH rat models. Intraperitoneal injection of MCT was used to induce PAH rat models. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-SMA, Ki-67 and TUNEL staining were performed to evaluate the development of PAH. Protein levels of Siah2, Lats1/2, YAP phosphorylation and total YAP, and the subcellular localization of YAP were examined using immunoblotting. Proteasome activity was measured by an assay kit. The protein level of Siah2 was significantly increased in MCT-induced PAH rats, this was accompanied with the proteasome-dependent degradation of Lats1/2 and subsequent up-regulation and dephosphorylation of YAP and its nuclear localization. Administration of PAH rats with Siah2 inhibitor Vitamin K3 or proteasome inhibitor MG-132 dramatically suppressed MCT-induced down-regulation of Lats1/2 and activation of YAP, finally reduced RVSP, RVHI, %MT, pulmonary arterial muscularization, pulmonary arterial smooth muscle cells (PASMCs) proliferation and enhanced PASMCs apoptosis in PAH rats. Siah2 contributes to the development of MCT-induced PAH by destabilizing Lats1/2 and subsequently stimulating YAP activation. Inhibition of Siah2 or proteasome alleviates pulmonary arterial remodeling through inactivation of YAP, indicating Siah2 ubiquitin ligase as a novel target might have potential value in the management of PAH.

Wang Q ，Shi W ，Zhang Q ，Feng W ，Wang J ，Zhai C ，Yan X ，Li M ... -  《-》

Inhibition of ubiquitin proteasome function prevents monocrotaline-induced pulmonary arterial remodeling.

Previous study has indicated that inhibition of proteasome function ameliorates the development of pulmonary arterial hypertension (PAH), while its underlying mechanisms are still unclear. This study was performed to address these issues. Male Sprague-Dawley (SD) rats were divided into five groups: control group, PAH group, vehicle treated PAH group, MG-132 treated PAH group and bortezomib treated PAH group. PAH model was established by a single intraperitoneal injection of monocrotaline (MCT). MG-132 and bortezomib were administered to inhibit proteasome function. The right ventricular systolic pressure (RVSP), the right ventricle hypertrophy index (RVHI) and the percentage of medial wall thickness (%MT) were used to evaluate the development of PAH. Hematoxylin and eosin staining was performed to measure vascular remodeling. Immunoblotting was used to determine Akt phosphorylation, expression of PTEN and NEDD4, and the level of ubiquitinated-PTEN protein. MCT increased RVSP, RVHI and %MT in rats, while these changes were suppressed by treatment of PAH rats with MG-132 or bortezomib. In PAH model, expression of PTEN was decreased and phosphorylation of Akt was increased, these were accompanied by an elevation of NEDD4 protein level. Treatment of PAH model with MG-132 or bortezomib increased PTEN expression and accumulation of ubiquitinated-PTEN protein and decreased Akt phosphorylation, while didn't change NEDD4 expression. Inhibition of proteasome function ameliorates pulmonary arterial remodeling by suppressing UPS-mediated PTEN degradation and subsequent inhibition of PI3K/Akt pathway, indicating that UPS might be a novel target for prevention of PAH.

Zhu Y ，Wu Y ，Shi W ，Wang J ，Yan X ，Wang Q ，Liu Y ，Yang L ，Gao L ，Li M ... -  《-》

Inhibition of HDAC1 alleviates monocrotaline-induced pulmonary arterial remodeling through up-regulation of miR-34a.

It has been found that up-regulation of histone deacetylases 1 (HDAC1) is involved in the development of pulmonary arterial hypertension (PAH). However, it is still unclear whether inhibition of HDAC1 suppresses the development of PAH via restoring miR-34a level in monocrotaline (MCT)-induced PAH rats. PAH rat models were induced by intraperitoneal injection of MCT. HDAC1 was suppressed by intraperitoneal injection of the class I HDAC inhibitor MS-275, and miR-34a was over-expressed via tail vein injection of miR-34a agomiR. HDAC1 protein was significantly increased in MCT-induced PAH rats; this was accompanied with down-regulation of miR-34a and subsequent up-regulation of matrix metalloproteinase 9 (MMP-9)/tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-2/TIMP-2. Administration of PAH rats with MS-275 or miR-34a agomiR dramatically abolished MCT-induced reduction of miR-34a and subsequent up-regulation of MMP-9/TIMP-1 and MMP-2/TIMP-2, finally reduced extracellular matrix (ECM) accumulation, pulmonary arterial remodeling, right ventricular systolic pressure (RVSP) and right ventricle hypertrophy index (RVHI) in PAH rats. HDAC1 contributes to the development of MCT-induced rat PAH by suppressing miR-34a level and subsequently up-regulating the ratio of MMP-9/TIMP-1 and MMP-2/TIMP-2. Inhibition of HDAC1 alleviates pulmonary arterial remodeling and PAH through up-regulation of miR-34a level and subsequent reduction of MMP-9/TIMP-1 and MMP-2/TIMP-2, suggesting that inhibition of HDAC1 might have potential value in the management of PAH.

Li F ，Wang D ，Wang H ，Chen L ，Sun X ，Wan Y ... -  《RESPIRATORY RESEARCH》

Resveratrol inhibits monocrotaline-induced pulmonary arterial remodeling by suppression of SphK1-mediated NF-κB activation.

This study aims to explore the molecular mechanisms underlying sphingosine kinase 1 (SphK1) inducing pulmonary vascular remodeling and resveratrol suppressing pulmonary arterial hypertension (PAH). monocrotaline (MCT) was used to induce PAH in rats. The right ventricular systolic pressure (RVSP), right ventricle hypertrophy index (RVHI) and histological analyses including hematoxylin and eosin staining, the percentage of medial wall thickness (%MT), α-SMA staining and Ki67 staining were performed to evaluate the development of PAH. Protein levels of SphK1, nuclear factor-kappaB (NF-κB)-p65 and cyclin D1 were determined using immunoblotting. Sphingosine-1-phosphate (S1P) concentration was measured using enzyme-linked immunosorbent assay. SphK1 protein level, S1P production, NF-κB activation and cyclin D1 expression were significantly increased in MCT-induced PAH rats. Inhibition of SphK1 by PF543 suppressed S1P synthesis and NF-κB activation and down-regulated cyclin D1 expression in PAH rats. Suppression of NF-κB by pyrrolidine dithiocarbamate (PDTC) also reduced cyclin D1 expression in PAH model. Treatment of PAH rats with either PF543 or PDTC dramatically decreased RVSP, RVHI and %MT and reduced pulmonary arterial smooth muscle cells proliferation and pulmonary vessel muscularization. In addition, resveratrol effectively inhibited the development of PAH by suppression of SphK1/S1P-mediated NF-κB activation and subsequent cyclin D1 expression. SphK1/S1P signaling induces the development of PAH by activation of NF-κB and subsequent up-regulation of cyclin D1 expression. Resveratrol inhibits the MCT-induced PAH by targeting on SphK1 and reverses the downstream changes of SphK1, indicating that resveratrol might be a therapeutic agent for the prevention of PAH.

Shi W ，Zhai C ，Feng W ，Wang J ，Zhu Y ，Li S ，Wang Q ，Zhang Q ，Yan X ，Chai L ，Liu P ，Chen Y ，Li M ... -  《-》

Activation of AMPK prevents monocrotaline-induced pulmonary arterial hypertension by suppression of NF-κB-mediated autophagy activation.

It has been shown that activation of autophagy is involved in the development of pulmonary arterial hypertension (PAH). Meanwhile, activation of nuclear factor-kappaB (NF-κB) has been found to induce autophagy in several types of human diseases including cancer and cardiac diseases. However, it is still unknown whether NF-κB mediates autophagy activation in PAH, and whether activation of adenosine monophosphate-activated protein kinase (AMPK) benefits PAH by modulation of NF-κB and autophagy. Rat models of PAH were established by intraperitoneally injection of monocrotaline (MCT). The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and percentage of medial wall thickness (%MT) were performed to evaluate the development of PAH. The translocation of NF-κB p65 from cytosol to nucleus, the protein levels of LC3A, LC3B, and RND3 were determined by immunoblotting. Metformin was used to activate AMPK. NF-κB and autophagy were significantly activated in MCT-induced PAH rats, this was accompanied with the reduction of RND3. Pharmacological inhibition of NF-κB suppressed MCT-induced activation of autophagy and down-regulation of RND3 expression and reduced RVSP, RVHI, and %MT in MCT-induced PAH rats. In addition, activation of AMPK by metformin suppressed NF-κB-mediated autophagy activation and down-regulation of RND3 and therefore reduced RVSP, RVHI, and %MT in MCT-induced PAH. NF-κB-induced autophagy activation and consequent down-regulation of RND3 contributes to the development of PAH in MCT-treated rats. Activation of AMPK prevents the development of PAH by targeting on NF-κB to suppress autophagy and vascular remodeling.

Zhai C ，Shi W ，Feng W ，Zhu Y ，Wang J ，Li S ，Yan X ，Wang Q ，Zhang Q ，Chai L ，Li C ，Liu P ，Li M ... -  《-》