Nox4-ROS-TRPM2 Pathway In The Regulation Of Cell Proliferation、Migration And Apoptosis In PASMCs Of The Chronic Hypoxic Pulmonary Hypertension

Continuous increasing of pulmonary artery pressure and vascular remodeling are the important characters of pulmonary hypertension（PH）.It was mainly caused by the disequilibrium of calcium homeostasis and the imbalance of proliferation and apoptosis.Studies have shown that pulmonary inflammation in patients with severe PH is associated with oxidative stress.As the main source of reactive oxygen species（ROS）,the expression of NADPH oxidase（No_x）in the cardiovascular system was upregulated,so as TRPM2,which is the unique oxidation-sensitive channel in the TRP family.However,the interaction between No_x family and TRPM2 in the development of PH still remains obscure.The present study firstly detected the expression of No_x family and TRPM2 from PAs of the rats exposed to normoxia and chronic hypoxia respectively.Then we explored the role of No_x family protein and TRPM2 in the proliferation,migration and apoptosis of the PASMCs induced by either 5-HT or CH and the relationship in the development of chronic hypoxia-induced pulmonary hypertension（CHPH）,we put a new insight into the pathogenesis and potential therapeutic targets for the disease.1.The effects of different No_x subtypes on cell proliferation、migration and apoptosis in CHPH rats.Objective: To detect the expression of No_x family genes and their effects on the proliferation,migration and apoptosis in PASMCs.Methods: The CHPH rat model was constructed by 3-weeks of CH exposure,then subsequence test was conducted as follows :（1）Hemodynamic detection was used to determine right ventricular systolic pressure（RVSP）,meanwhile the right ventricular weight index（RVMI）was recorded;（2）The expression of No_x was tested by PCR and western blot in rat Pas without endothelium.（3）MTS and Ed U incorporation were used to observe the effect of CHPH on PASMCs proliferation and effects of No_x inhibitors（No_x1 inhibitors: ML171;No_x2 inhibitors: gp91ds-tat.;No_x4 inhibitors:GKT137831）on 5-HT and CH-mediated PASMCs proliferation.（4）Scratch assay and Transwell chambers were used to observe the role of CHPH in migration and the effects of three inhibitors on 5-HT and CH induced migration;（5）Annexin V FITC/PI Kit was used to detect the effect of CHPH on the apoptosis in PASMCs and the influence of three inhibitors on 5-HT and CH group,respectively.（6）The role of No_x4 on the proliferation,migration and apoptosis of PASMCs were verified by No_x4 specific knockdown.Results: Compared with the control group（CON）,（1）the RVSP in the CHPH group rose from 24.8±0.5 mm Hg to 50.8±0.9 mm Hg,and the RVMI from 26.3±0.5% to36.5±1.4%,suggesting that the PH rat model was successfully constructed.（2）The m RNA level of No_x1、2 and 4 in the PAs increased significantly,while the upregulation of proteins were mainly No_x1 and No_x4,suggesting that No_x1 and No_x4 were involved in the development of PH.（3）The numbers of the PASMCs in CHPH rats were significantly higher than that in the CON group,with an increased OD value from 0.79±0.01 to 1.34±0.09.The incorporation rate of Ed U increased significantly in the PASMCs of CHPH rats from 0.31±0.02 to 0.45±0.02,suggesting the enhanced proliferation in the CHPH group.The migration area augmented significantly in CH group,and the number of cells in the Transwell chamber which migrated to the lower chamber increased significantly in the CHPH group from71.8±4.34 to 146.91±5.05,suggesting a significant increase in the cell migration levels in the CHPH group,and there was a remarkable decrease in the binding rate of Annexin V/ FITC markers in CHPH rats,and the apoptosis rate was suspended by the CH group.（4）Similar to the CH induced cell proliferation,migration and apoptosis,5-HT can also promote the proliferation and migration of PASMCs and reduce the cell apoptosis.（5）In the 5-HT and CHPH induced PASMCs,ML171 inhibited the increase of OD value the increased rate of Ed U incorporation caused by 5-HT,but had no significant inhibitory effect on CH group.Meanwhile ML171 can obviously inhibit the migration of PASMCs caused by 5-HT and CH.GKT137831 had significant inhibitory effects on the proliferation and migration of PASMCs caused by 5-HT and CH,but gp91 ds-tat had no significant effect neither.As for the cell apoptosis,ML171,Gp91ds-tat and GKT137831 can further promote apoptosis resistance caused by 5-HT.While in the CH group,three inhibitors can reduce apoptosis resistance to normal levels caused by CH.It was suggested that among the three kinds of No_x subtypes,No_x4 may play a major role in the CHPH process.（6）The specific knockdown of No_x4 gene can inhibit the proliferation,migration and apoptosis of normal PASMCs,which is consistent with the effect of the aforementioned inhibitors,suggesting that No_x4 plays an indispensable role in the proliferation,migration and apoptosis of PASMCs.Conclusion: The 3 weeks of CH exposure can successfully induce PH and right heart hypertrophy in rats,and the mechanism may involve a significant increase in the expression of No_x1 and No_x4 caused by 5-HT in the No_x-ROS-TRPM2 signaling pathway of CHPH rats,espercially the No_x4 gene,resulting in an increase in the source of ROS.It also further caused the changes in proliferation,migration and apoptosis,promoting PAs vascular remodeling,even the development of PH.2.The effects of TRPM2 on cell proliferation、migration and apoptosis in CHPH rats.Objective: To observe the expression alternation of TRPM2 in the No_x-ROS-TRPM2 signaling pathway in CHPH group,and the influence of TRPM2 on PASMCs proliferation,migration and apoptosis induced by 5-HT and CH exprosure,then further discuss the joint role of No_x4 and TRPM2 on cell proliferation,migration and apoptosis,which can contribute to implying the role of the No_x-ROS-TRPM2 pathway during the pathogenesis of CHPH.Methods:（1）Real-time fluorescence quantitative PCR and Western blot detection method to determine the expression of TRPM2 in rat non-endothelial PAs;（2）Preincubation with three kinds of TRPM2 inhibitors（2APB:100μM,Aca:30μM,PJ34:100μM）for 24 h,their effects on the proliferation,migration and apoptosis level of induced by 5-HT and CH were observed by MTS and Ed U incorporation method,Scratching and Transwell chamber method,and Annexin V FITC/PI Kit,respectively.（3）Specific knockdown of the TRPM2 gene through si RNA interference was used to observed its effort on the proliferation,migration and apoptosis of PASMCs.（4）Appling GKT137831 combined with ACA,2APB and PJ34 respectively to inhibit the effort of No_x4 and TRPM2,then observe the proliferation and migration induced by5-HT treatment and CH exposure.Results:（1）Compared with the CON group,the expression of TRPM2 in CH group increased significantly,suggesting that TRPM2 was involved in the development of CHPH.（2）2APB and PJ34 can markedly inhibit the increased cell proliferation and migration caused by in 5-HT and CH.ACA had a lesser effect on 5-HT and CH induced proliferation and migration.But three inhibitors can promote the apoptosis of PASMCs caused by 5-HT and CH.It was suggested TRPM2 was involved in the regulation of PASMCs proliferation、migration apoptosis in the process of CHPH.（3）Specific knockout of TRPM2 gene can inhibit the proliferation、migration and apoptosis of PASMCs,which is consistent with the effect of the inhibitors,suggesting that TRPM2 plays an indispensable role in the proliferation、migration and apoptosis of PASMCs.（4）The combination of GKT137831 and ACA,2APB and PJ34 respectively can inhibit the proliferation and migration of PASMCs caused by 5-HT and CH in different degrees,suggesting that No_x4 and TRPM2 play a synergistic role in the proliferation and migration caused by CH in process of CHPH.Conclusion: TRPM2 expression was significantly enhanced in the process of CHPH.Inhibiting TRPM2 channel can significantly inhibit the proliferation and migration of PASMCs,and promote apoptosis,indicating that TRPM2 involved in regulation of proliferation、migration and apoptosis in the development of CHPH.In the same time with No_x4 inhibition preincubation,5-HT and CH induced proliferation and migration can also be inhibited,suggesting that No_x4 and TRPM2 have synergies.TRPM2 may be downstream signals of No_x4 to regulate cell proliferation、migration and apoptosis.3.Effect of No_x4-ROS-TRPM2 signaling pathway in pulmonary hypertensionObjective: To observe the effect of No_x4 gene knockout(No_x4^-/-)on CHPH and the effect of No_x4^-/-on 5-HT-mediated PASMCs proliferation、migration and apoptosis.To observe the changes of TRPM2 expression after No_x4 gene knockout.To further clarify the interaction of No_x4 and TRPM2 channels and their regulation on PASMCs function in the process of CHPH.Method:（1）Agarose gel electrophoresis、Western Blot and PCR were used to verify the success knockout of No_x4^-/-mice.（2）To observe the changes of hemodynamics and RVMI in the CH model of No_x4^-/-mice,compared with the model of wild-type（WT）mice.（3）MTS method、scratching method and Annexin V kit were used to observe the effect of proliferation and migration on the PASMCs of No_x4^-/-mice induced by 5-HT.（4）Western blot and PCR technology were used to detect the expression of TRPM2 in No_x4^-/-mice.Results:（1）Agarose electrophoresis showed that the DNA stripe of No_x4^-/-mice was around 300 bp,higher than that of WT mice,suggesting that the genotype of No_x4^-/-mice was the pure zygote.Western blot and PCR showed that there were few levels of m RNA and protein expression of No_x4 in No_x4^-/-mice compared with the lung tissue of WT mice,suggesting that No_x4 gene was successful knockout and the accuracy of the experiment was guaranteed.（2）Compared with the CH model of WT mice,the RVSP and RVMI was decreased significantly in the CH model of No_x4^-/-mice,suggesting that the absence of No_x4 can reduce the increase of RVSP and RVMI caused by CH.It indicated that No_x4^-/-is beneficial to the prevention of CHPH.（3）Compared with the PASMCs in WT mice,No_x4^-/-can effectively inhibit the proliferation and migration,and further promote the apoptosis caused by 5-HT.It was suggested that No_x4 did have an indispensable role in proliferation,migration and apoptosis in the process of CHPH.（4）Compared with the lung tissue of WT mice,the expression level of TRPM2 in No_x4^-/-mice increased,suggesting that there was mutual regulation between No_x4 and TRPM2.To sum up,the increase of oxidative stress levels in PASMCs leads to the enhancement of No_x expression and functional activity,as well as TRPM2,which plays an important role in the process of CHPH pathogenesis.CH raised the expression of the main role subtype No_x4 of the No_x family through 5-HT,and then regulates PASMCs [Ca²⁺]i changes by producing an overdose of ROS to activate the TRPM2 channel,causing changes in cell proliferation、migration and apoptosis levels,leading to vascular remodeling and promoting the development of PH.In addition,the vascular remodeling caused by CH can be effectively alleviated,and the cell proliferation and migration induced by 5-HT was also significantly reduced in No_x4^-/-mice,which further indicates that No_x4 played an important role in the oxidative stress response in the development of CHPH.The increase of TRPM2 expression in No_x4^-/-mice showed that TRPM2 played a role in the downstream in No_x4 dependence.The above results provide a new scientific basis for the elucidation of PH pathogenesis and its targeted treatment.