miR-27a promotes endothelial-mesenchymal transition in hypoxia-induced pulmonary arterial hypertension by suppressing BMP signaling.

Growing evidence suggests that endothelial-mesenchymal transition (EndMT) play key roles in pulmonary arterial remodeling during pulmonary arterial hypertension (PAH), but the underlying mechanisms have yet to be fully understood. miR-27a has been shown to promote proliferation of pulmonary arterial cells during PAH, but its role in EndMT remains unexplored. This study was designed to investigate the role and underlying mechanism of miR-27a in EndMT during PAH. Rats were exposed in hypoxia (10% O2) for 3 weeks to induce PAH, and human pulmonary artery endothelial cells (HPAECs) were exposed in hypoxia (1% O2) for 48 h to induce EndMT. Immunohistochemistry, in situ hybridization, immunofluorescence, real-time PCR and Western blot were conducted to detect the expressions of RNAs and proteins, and luciferase assay was used to verify the putative binding site of miR-27a. We found that hypoxia up-regulated miR-27a in the tunica intima of rat pulmonary arteries and HPAECs, and that inhibition of miR-27a suppressed hypoxia-induced EndMT. Furthermore, elevated expression of miR-27a suppressed bone morphogenetic protein (BMP) signaling by targeting Smad5, thereby lessening Id2-mediated repression of the 2 critical mediators of EndMT (Snail and Twist). Our data unveiled a novel role of miR-27a in EndMT during hypoxia-induced PAH. Thus, targeting of miR-27a-related pathway may be therapeutically harnessed to treat PAH.

Liu T ，Zou XZ ，Huang N ，Ge XY ，Yao MZ ，Liu H ，Zhang Z ，Hu CP ... -  《-》

Down-regulation of miR-204 attenuates endothelial-mesenchymal transition by enhancing autophagy in hypoxia-induced pulmonary hypertension.

Pulmonary arterial remodeling is a crucial cause of increased pulmonary artery pressure during pulmonary hypertension (PH). Recently, growing evidence has upheld the contribution of endothelial-mesenchymal transition (EndMT) to pulmonary arterial remodeling, but the underlying mechanisms remain largely unaddressed. miR-204 has been implicated in PH, being anti-proliferative and pro-apoptotic in pulmonary artery smooth muscles cells (PASMCs), but its role in EndMT is still unknown. Here we found that miR-204 was down-regulated by hypoxia in rat pulmonary arterial intima and human pulmonary artery endothelial cells (HPAECs), and its further down-regulation by using miR-204 inhibitor suppressed hypoxia-induced EndMT. Moreover, autophagy, evoked by hypoxia in rat pulmonary arterial intima and HPAECs, suppressed hypoxia-induced EndMT via p62-dependent degradation of Snail and Twist. Additionally, autophagy was regulated by miR-204 targeting ATG7. While down-regulation of miR-204 in PASMCs reportedly promoted monocrotaline-induced pulmonary arterial hypertension via increased cell proliferation, our data suggested an important, albeit dichotomous, role of miR-204 down-regulation in endothelial cells in the process of EndMT that it attenuated EndMT by enhancing autophagy, thereby ameliorating hypoxia-induced PH to some extent.

Liu T ，Zou XZ ，Huang N ，Ge XY ，Yao MZ ，Liu H ，Zhang Z ，Hu CP ... -  《-》

miR-181b-5p inhibits endothelial-mesenchymal transition in monocrotaline-induced pulmonary arterial hypertension by targeting endocan and TGFBR1.

Endothelial-mesenchymal transition (EndMT) is a frequent event in endothelial dysfunction, which is associated with pulmonary arterial hypertension (PAH). MiR-181 family members exert diverse effects in multiple biological processes. However, the relationships between miR-181b-5p (miR-181b) and EndMT in PAH are not well understood. In this study, Sprague-Dawley (SD) rats were injected with monocrotaline (MCT) to establish PAH model, and primary rat pulmonary arterial endothelial cells (rPAECs) were treated with TNF-α, TGFβ1 and IL-1β in combination to induce EndMT (I-EndMT). Then we explored miR-181b expression and examined its functional role in PAH. Our data showed that miR-181b was down-expressed in PAH, and its overexpression attenuated the hemodynamics, pulmonary vascular hypertrophy, right ventricular remodeling and EndMT process in MCT-induced PAH rats. In I-EndMT rPAECs, we observed that inducing miR-181b reversed the decrease of endothelial markers and increase of mesenchymal markers. However, knockdown of miR-181b induced similar effects to EndMT. In addition, endocan and TGFBR1 levels were also increased in EndMT, which were negatively regulated by miR-181b. Luciferase activity results indicated that endocan and TGFBR1 were direct target genes of miR-181b. In summary, our findings firstly demonstrate that the beneficial effect of miR-181b on PAH may be associated with endocan/TGFBR1-mediated EndMT, providing a new insight into the diagnosis and treatment of PAH.

Zhao H ，Wang Y ，Zhang X ，Guo Y ，Wang X ... -  《-》

miR-143 and miR-145 promote hypoxia-induced proliferation and migration of pulmonary arterial smooth muscle cells through regulating ABCA1 expression.

Excessive proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) play an important role in the occurrence and development of pulmonary arterial hypertension (PAH). miR-143/145 was reported to be up-regulated in the animal models and PAH patients, and deletion of miR-143/145 cluster prevented the development of hypoxia-induced pulmonary hypertension, but its underlying mechanism has not been elucidated. qRT-PCR and Western blot were performed to detect the expressions of miR-143/145 and ATP-binding cassette transporter A1 (ABCA1) in PAH patients and PASMCs under hypoxic conditions. Cell proliferation and migration were assessed by Cell Counting Kit-8 and wound-healing assay, respectively. Luciferase reporter assay and RNA immunoprecipitation were conducted to confirm the interaction between miR-143/145 and ABCA1. Hypoxia-induced PAH rat model was established to confirm the functions of miR-143/145 in the pathogenesis of PAH and its underlying mechanism in vivo. miR-143 and miR-145 were up-regulated and ABCA1 was down-regulated in PAH patients and PASMCs under hypoxic conditions for different time, as well as hypoxia-induced PAH rats. miR-143/145 inhibition and ABCA1 overexpression suppressed hypoxia-induced proliferation and migration in PASMCs. ABCA1 was identified as a direct target of miR-143/145 in PASMCs. Moreover, ABCA1 partially reversed miR-143/145-mediated promotion of hypoxia-induced cell proliferation and migration in PASMCs. Furthermore, in vivo experiments confirmed that inhibition of miR-143/145 prevents hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling by targeting ABCA1. miR-143/145 promoted hypoxia-induced proliferation and migration of PASMCs by targeting ABCA1, contributing to better understanding of the mechanism underlying the pathogenesis of PAH.

Yue Y ，Zhang Z ，Zhang L ，Chen S ，Guo Y ，Hong Y ... -  《-》

N6-methyladenosine modification of KLF2 may contribute to endothelial-to-mesenchymal transition in pulmonary hypertension.

Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-β1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

Kang K ，Xiang J ，Zhang X ，Xie Y ，Zhou M ，Zeng L ，Zhuang J ，Kuang J ，Lin Y ，Hu B ，Xiong Q ，Yin Q ，Su Q ，Liao X ，Wang J ，Niu Y ，Liu C ，Tian J ，Gou D ... -  《-》