The Role Of DNA Methylation In Hypoxia-induced Pulmonary Hypertension

Background : One of the important characteristic of pulmonary vascular remodeling and right heart remodeling in hypoxic pulmonary arterial hypertension were their reversibility after reoxygenation, but the mechanism is still unkown. DNA mythylation is one of the important epigenetic regulation mechanism. DNA methylation participates in various pathophysiological process. Under the external stimuli, DNA methylation will be dynamic change, so does gene expression controled by DNA methylation. DNA methylation is mainly affected by the regulation of DNA methyltransferases(DNMTs), and it is known that DNMTs play important roles in vascular disease and cancer. However, few studies focused on the role of DNMTS in pulmonary hypertension.Objective:This study aims to build hypoxia-induced pulmonary hypertension rat model and revered pulmonary hypertension after reoxygenation and to investigate the expression level and potential role of DNMTs in hypoxia-induced pulmonary vascular remodeling and right heart remodeling and their reversibility.Method:In order to build hypoxia-induced pulmonary hypertension rats model, we put the rats into hypobaric cabin for three weeks. After the hypoxia-induced pulmonary hypertension rats model is ready, we took the rats out of the hypobaric cabin, and put the rats into normal external environment for six weeks to build the revered pulmonary hypertension rat model. In the hypoxia and reoxygenation continuous time points, we used the echocardiography, right cardiac catheterization and histopathological examination to identify the phenotype of the rat model. Western blot and immunohistochemistry were used to explore the DNMTs protein expression in the lung and right ventricle tissues of normal control, hypoxia 3 weeks and recovery 3 weeks groups.Result:1. Identification of sustained hypoxia-induced pulmonary hypertension rats model.The PAAT was gradually decreased with the hypoxia time extended. The PAAT in hypoxia 3w group(n=8, 14.4±1.8ms) was less than normal control group(n=6,33.4±3.7ms) by 57%(P<0.001). While, the right ventricular free wall thickness(RVWT), m PAP, RVSP and RV/LV+S were gradually increased with the hypoxia time extended. RVWT in hypoxia 3w group(n=8, 1.04±0.26mm) was 1.5 times of normal control group(n=6, 0.70 ± 0.14mm)(P<0.01). The m PAP in hypoxia 3w group(n=8, 31.68±5.67 mm Hg) was 2.2 times of the normal control group(n=6, 14.63 ±2.39 mm Hg)(P<0.01). RVSP in hypoxia 3w group(n=8, 47.86±5.54 mm Hg) was 2.1 times of normal control group(n=6, 23.33±1.90 mm Hg)(P<0.001). RV/LV+S in hypoxia 3w group(n=8,0.412±0.08) was 1.8 times of normal control group(n=6,0.225±0.021)(P<0.05). The pathological staining suggests that there exists an obvious pulmonary arterial remodeling and right heart remodeling in hypoxia 3w group. The thickness of the pulmonary arterioles percentage in hypoxia 3w group(n=3, 0.42±0.02%) was 1.8 times of normal control group(n=3, 0.23±0.04%)(P<0.05), and the pulmonary arteriole area percentage in hypoxia 3w group(n=3, 0.72±0.08%) was 1.8 times of normal control group(n=3, 0.49±0.11%)(P<0.05).2. Identification of reversed pulmonary hypertension rats model after reoxygenation.The PAAT was gradually increased as the reoxygenation time extended.. PAAT in recovery 6w group(n=8, 23.2±3.7ms) was 1.6 times of hypoxia 3w group(n=8, 14.4±1.8ms)(P<0.001), and the PAAT in recovery 6w group was significantly higher than normal control group(n=6,33.4±3.7ms)(P<0.001). While, RVWT, m PAP, RVSP and RV/LV+S were gradually increased as the reoxygenation time extended. The RVWT in recovery 3w group decreaseed to 0.65±0.05 mm, which has no significant difference with normal control group(n=6, 0.70±0.14ms). The m PAP in recovery 6w group(n=6, 19.25±3.54 mm Hg) has no significant difference with normal control group(n=6, 14.63 ± 2.39 mm Hg). While, RVSP in hypoxia 6w group(n=8, 30.75±7.28 mm Hg) was higher than normal control group(n=6,23.33±1.90 mm Hg)(P<0.001). RV/LV+S in recovery 3w group reduced to 0.248±0.070, which has no significant difference with normal control and recovery 3w group. The pathological staining suggests that the right heart remodeling has been completely reversed in recovery 3w group. However, pulmonary arterial remodeling was partly reversed in recovery 3w group. Comparied with hypoxia 3w group, the WT% in recovery 3w group decreased by 24%(P<0.05), WT% in hypoxia 3w group was also signicantly higher than normal control group(P<0.05). The WA% in recovery 3w group(n=3, 0.54±0.08%) reduced to 75% of hypoxia 3w group(P<0.05), and the WA% in recovery 3w group has no difference with normal control group(n=3, 0.49±0.11%)(P>0.05).3. The expression of DNMTs in lung tissue of normal control group, hypoxia 3w group, and recovery 3w group.The expression of Dnmt1 and Dnmt3 a protein have no significant difference among the 3 groups. The expression of Dnmt3 b protein in hypoxia 3w group and recovery 3w group were both twice higher than normal control group(P<0.05). While, there was no significant difference in Dnmt3 b protein expression between recovery 3w group and normal control group(P<0.05). The expression of Dnmt3 L protein in hypoxia 3w group was 59% of normal control group(P<0.05). The expression of Dnmt3 L protein in recovery 3w group was twice than hypoxia 3w group(P<0.05). When comparied to normal control group, there has no significant difference(P<0.05).Immunohistochemistry showed that Dnmt3 b protein mainly located in pulmonary vascular and bronchial smooth muscles. The expression of Dnmt3 b protein in hypoxia 3w group is significantly higher than normal control group and recovery 3w group.4. The expression of DNMTs in the right ventricle of normal control group, hypoxia 3w group, and recovery 3w group.The expression of Dnmt1 and Dnmt3 L protein in the right ventricle have no significant difference among the 3 groups. There was also no significant difference of Dnmt3 a expression in the right ventricle between hypoxia 3w group and normal control group(P>0.05). While, the expression of Dnmt3 a protein in the right ventricle of recovery 3w group was less than hypoxia 3w group by 40%(P<0.05). The expression of Dnmt3 b protein in the right ventricle of hypoxia 3w group was 1.88 times of normal control group(P<0.001). What’s more, the expression level of Dnmt3 b protein in recovery 3w group was less than hypoxia 3w group by 41%(P<0.01), and there was no significant difference of Dnmt3 b expression level between recovery 3w group and normal control group(P>0.05).Conclusion:1. The RVSP and the RV/LV+S ratio in hypoia 3 weeks group increased 2 times,which indicated that the hypoxia induced-pulmonary hypertension rats model wassuccessfully constructed.2. The pulmonary vascular remodeling and right heart remodeling was partly orcompletely reversed in recovery 3 weeks group.3. Dnmt3 b, a member of DNMTs family, was significantly increased in pulmonary vascular remodeling and right heart remodeling, which indicated that Dnmt3 b may play an important role in pulmonary vascular remodeling and right heart remodeling process.