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The Effects of Reactive Oxygen Species on Hypoxic Inducible Factor 1α and Cell Proliferation in Rat Pulmonary Arterial Smooth Muscle Cells under the Hypoxic Condition

Author: ZhouZhiGang
Tutor: ZhaoJianPing
School: Huazhong University of Science and Technology
Course: Department of Respiratory Medicine
Keywords: Hypoxia Pulmonary arterial smooth muscle cells Reactive oxygen species Hypoxia inducible factor Proliferation
CLC: R363
Type: Master's thesis
Year: 2007
Downloads: 111
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Abstract


Objective1: To observe the changes of reactive oxygen species (ROS) in rat pulmonary arterial smooth muscle cells (PASMCs) under hypoxic condition;2: To test if hypoxia-caused smooth muscle cell proliferation in pulmonary artery was mediated by ROS, which could regulate the HIF-1 expression.Methods and MaterialsPASMCs were isolated and cultured, which were divided into three groups randomly: normal group(cells are cultured under hypoxia for 24 h, A group), hypoxia group(cells are cultured under normoxia for 24 h, B group) and hypoxia+Mn-TBAP group (cells are cultured under hypoxia with Mn-TBAP, a ROS scavenger, for 24 h, C group). The level of ROS was determined by a laser scanning confocal microscope. The expression of HIF-1αmRNA was tested by semi-quantitative reverse transcription PCR (RT-PCR). The HIF-1αprotein was detected using immunohistochemical staining (Streptomycete avidin-Peroxidase jointing, SP), and the proliferation of PASMCs was examined by MTT colorimetric assay.Results1. The level of ROS in hypoxia groups(69.59±3.50)was significantly increased as compared with that in the normal group(27.39±2.25, n=6, P<0.05); however, the level of ROS in hypoxia+Mn-TBAP groups(41.09±2.47)was significantly decreased as compared with that in hypoxia groups (n=6, P<0.05), but was still higher than that in normal groups(n=6, P<0.05). 2. The expression of HIF-1αmRNA and protein in hypoxia groups(HIF-1αmRNA: 0.43±0.02, protein: 146.93±14.48 ) and hypoxia+Mn-TBAP groups(HIF-1αmRNA: 0.25±0.03, protein: 160.07±7.21 ) were increased as compared with those in the normal groups (HIF-1αmRNA: 0.16±0.03, protein: 186.46±5.13 , both P<0.05). These changes were more significant in hypoxia groups than those in hypoxia+Mn-TBAP groups (n=6, P<0.05).3. Compared with normal group(0.12±0.01), the cell proliferation (A value) was significantly increased in hypoxia group(0.20±0.01)and hypoxia+Mn-TBAP group (0.16±0.01,both P<0.05), and the change was more significant in hypoxia groups compared with that in hypoxia+Mn-TBAP groups (n=6, P<0.05).ConclusionThe levels of ROS increase under hypoxia in rat pulmonary arterial smooth muscle cells; Hypoxia could significantly enhance the expressions of HIF-1αmRNA , and obviously promoted the proliferation of rat PASMCs. But when the levels of ROS were decreases by Mn-TBAP, the above enhancements of the expression of HIF-1αand the proliferation of PASMCs could be suppressed. These suggest: 1. At least in rat PASMCs, ROS, probably as a signaling molecule, could participate hypoxic signal transduction ways; 2. At least in rat PASMCs, hypoxia regulate HIF-1αat the levels of transcription, translation and post-translation; 3. The changes of ROS, HIF-1αand cell proliferation are coincident in rat PASMCs. So, The level of ROS may be associated with the expression of HIF-1αand the proliferation of PASMCs. ROS could control HIF-1αas a signaling molecule, through participating in some signal transduction ways or having the potential to interfere with hydroxylase activity. Beside, ROS maybe control the proliferation of PASMCs through regulating the HIF-1 expression. Therefore, ROS may play an important role in the pathogenesis of pulmonary hypertension and hypoxic signal transductions.

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