Roles Of Hypoxia Induced Proliferation In Human Pulmonary Arterial Smooth Muscle Cells Through Notch3 Pathway

Background: Pulmonary hypertension(PH) is a disease characterized by elevation of pulmonary vascular resistance(PVR) with consequent right ventricular failure and death. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are four major causes for the elevated PVR in patients with PH. Pulmonary arterial smooth muscle cells(PASMCs) and endothelial proliferation lead to vessel thickening and lumial occlusion due to structural remodeling of small pulmonary arteries and arterioles, which result in PH. Partial FPAH and IPAH can be associated with mutations in gene encoding bone morphogenic protein receptor II(BMPR2), as well as most PH with various other risk factors. Although several stimuli and conditions, such as hypoxia is associated with this disease, the exact molecular mechanism of how the lung remodels its vascular smooth muscle cell architecture in the arteriolar bed in most of PH is not known. Genes of Notch family encode single-pass trans-membrane receptors that transduce signals through cell-cell interactions. Notch signaling plays a critical role in controlling proliferation and differentiation of PASMCs. NOTCH3 is overexpressed at m RNA and protein levels in the lungs of humans with PH.NOTCH3-HES5 signaling pathway is crucial for the development of human PH and hypoxic PH in rodents. Hypoxia requires functional Notch signaling to maintain cells in an undifferentiated state due to crosstalk between Notch signal and HIF1α. Hypoxia induced proliferation of PASMCs is the most important pathology change in hypoxia pulmonary hypertension(HPH). It was supposed that hypoxia induced over-expression of Notch3 pathway, which enhanced CCE and cell proliferation in HPASMCs, led to pulmonary vascular remodeling and genesis of PH.Objective: The hypoxia treated HPASMCs were used as the hypoxia cell models. Expression level of Notch3, cell proliferation of HPASMCs, CCE and protein level of m TOR pathway were observed in order to understand the potential roles of Notch3 signaling in the proliferation of hypoxia induced HPASMCs in genesis of PH.Methods: Human pulmonary artery smooth muscle cells(HPASMCs) were cultured under hypoxia(3% O2) in the presence or absence of Notch3 signaling inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT) or Notch3-si RNA. The proliferation of HPASMCs was quantified by MTT or Brd U incorporation. Cytosolic free Ca2+ concentration( [Ca2+]cyt) and CCE were detected by using fura-2 AM fluorescence and digital fluorescence imaging system. Real-time PCR was used to investigate the m RNA level of Notch3 signaling. Western blot was measured to detect the expression of Notch3 and m TOR signaling pathways in HPASMCs.Results: Hypoxia significantly induced proliferation of HPASMCs. Protein and m RNA level of Notch3 was also increased significantly by hypoxia, as well as protein level of HES5. Hypoxia-induced proliferation is attenuated by inhibition of the Notch3 pathway using DAPT. Notch3-si RNA could decrease proliferation of HPASMCs induced by hypoxia. Hypoxia cause a rise of [Ca2+]cyt and CCE in HPASMCs. Inhibition of Notch3 by DAPT could reduce the increasing of intracellular [Ca2+] and CCE. Knockdown Notch3 by DAPT could decrease hypoxia-induced m TOR pathway phosphorylation including p-mTOR,p-p70S6 K and p-4EBP1. Notch3-si RNA could attenuate protein expression of p-p70S6 K.Conclusion: Hypoxia induced over-expression of Notch3 pathway, which enhanced CCE and cell proliferation in HPASMCs. Knockdown of Notch3 could reduce CCE and inhibit cell proliferation of HPASMCs induced by hypoxia. m TOR pathway may be involved in those functional regulations.