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Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells

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Abstract

Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5 % O2) or normoxic (21 % O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

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Acknowledgments

This work was supported by the Science and Technology Development Fund of Shanghai Pudong New Area (PKj2011-y36) and the National Nature Science Foundation of China (No. 81272145).

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    Authors and Affiliations

    1. Department of Anesthesiology, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, No. 600, Yishan Road, Xuhui District, Shanghai, 200233, China

      Yongmei Cao, Zhen Zeng, Yujing Liu & Yingchuan Li

    2. Department of Anesthesiology, Dongfang Hospital, Tongji University, Shanghai, 200120, China

      Yongmei Cao, Zhen Jiang, Yuchun Gu, Yingying Ji & Yupeng Zhao

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    Yongmei Cao and Zhen Jiang contributed equally to this study.

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    Cao, Y., Jiang, Z., Zeng, Z. et al. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells. Apoptosis 21, 69–84 (2016). https://doi.org/10.1007/s10495-015-1184-3

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