mTOR is required for pulmonary arterial vascular smooth muscle cell proliferation under chronic hypoxia

Abstract

Pulmonary arterial vascular smooth muscle (PAVSM) cell proliferation is a key pathophysiological component of vascular remodeling in pulmonary arterial hypertension (PAH) for which cellular and molecular mechanisms are poorly understood. The goal of our study was to determine the role of mammalian target of rapamycin (mTOR) in PAVSM cell proliferation, a major pathological manifestation of vascular remodeling in PAH. Our data demonstrate that chronic hypoxia promoted mTOR(Ser-2481) phosphorylation, an indicator of mTOR intrinsic catalytic activity, mTORC1-specific S6 and mTORC2-specific Akt (Ser-473) phosphorylation, and proliferation of human and rat PAVSM cells that was inhibited by siRNA mTOR. PAVSM cells derived from rats exposed to chronic hypoxia (VSM-H cells) retained increased mTOR(Ser-2481), S6, Akt (Ser-473) phosphorylation, and DNA synthesis compared to cells from normoxia-exposed rats. Suppression of mTORC2 signaling with siRNA rictor, or inhibition of mTORC1 signaling with rapamycin and metformin, while having little effect on other complex activities, inhibited VSM-H and chronic hypoxia-induced human and rat PAVSM cell proliferation. Collectively, our data demonstrate that up-regulation of mTOR activity and activation of both mTORC1 and mTORC2 are required for PAVSM cell proliferation induced by in vitro and in vivo chronic hypoxia and suggest that mTOR may serve as a potential therapeutic target to inhibit vascular remodeling in PAH.

HIPPO–Integrin-linked Kinase Cross-Talk Controls Self-Sustaining Proliferation and Survival in Pulmonary Hypertension

“…A proliferative/antiapoptotic role of mTORAkt and Notch3 intracellular domain in PAH PAVSMCs had been reported by us and others (5,28,29), and we confirmed activation of HIF1 and Wnt/b-catenin using respective activation assays (see Figure E7). Supporting the bioinformatic predictions, inactivation of LATS1 in nondiseased human PAVSMCs significantly up-regulated mTOR-Akt, as evidenced by increase in P-S473 Akt, mTORC2-specific P-T2481 mTOR, and mTORC1-specific P-S6 (5,14,(30)(31)(32), and led to accumulation of b-catenin, Notch3 intracellular domain, and HIF1a, whereas "restoration" of P-T1079 LATS1 or suppression of Yap in PAH PAVSMCs by LATS1 T1079D or VP had the opposite effect ( Figure 3; see Figure E8). Collectively, these data show that LATS1 inactivation up-regulates proliferative/prosurvival signaling and promotes human PAH PAVSMC proliferation and survival via Yap.…”

“…Immunohistochemical, Immunocytochemical, and Immunoblot Analyses and Transfection, Infection, DNA Synthesis, Apoptosis, Migration, and Cell Count Assays These were performed as described (5,14,15). siRNAs and shRNAs were purchased from Dharmacon (Lafayette, CO) and Origen (Rockville, MD).…”

“…Treatment with Cpd22 (20 mg/kg, intraperitoneally 5 d/wk) (EMD Millipore, Billerica, MA) or vehicle was performed at Days 22-35. Negative control subjects included normoxia-maintained male agematched animals (5,14). Hemodynamic and histochemical analyses were performed as described (5,19).…”

A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse Lung

“…mPMVEC and A549 human lung epithelial cells were grown at 37°C in Dulbecco's modified Eagle's medium (DMEM)-low glucose medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT), 25 mM HEPES, and 5 mM L-glutamine without antibiotics; for endothelial cells, growth supplement from bovine neural tissue was added at 1.5 mg/ 100 ml (E2759; Sigma-Aldrich). Immortalized hPASMCs, a gift from Dr. Elena Goncharova (Krymskaya et al, 2011), were grown in DMEM-low glucose medium supplemented with 5% FBS, 2 mM L-glutamine with antibiotics, and 1% insulin, transferrin, and selenium.…”

Hypoxia and Pulmonary Hypertension

“…By contrast, the vasculature responds to long-term hypoxia by promoting new blood vessel growth-angiogenesis, which in turn, restores O 2 to deprived tissues. Hypoxic stress is a key driving force in the vascular remodelling observed in pulmonary hypertension, and HIFs activate pulmonary artery endothelial and smooth muscle cell proliferation, which is mediated by both mTORC1 and mTORC2 [83][84][85]. Currently, the mechanisms by which hypoxia/HIFs signal to activate mTOR in ECs and VSMCs are poorly understood [86][87][88][89][90].…”