The Role Of KLF4 In Cigarette Smoke-induced Pulmonary Hypertension

Part 1:Expression of KLF4 in lung vessels of rats exposed to cigarette smoke and its role in pulmonary vessel remodelingObjective:To explore the change of the KLF4 expression in the pulmonary vessels of rats exposed to cigarette smoke and its role in pulmonary vessel remodeling.Methods:Twenty rats were randomly assigned to one of two groups:control group(n=9)and group exposed to CS(smoking group;n=11).The smoking group underwent whole body exposure to CS in a ventilated chamber,and the control animals were exposed to air.All animals were sacrificed on the 120th day.Pathomorphology of pulmonary vessels was analyzed using hematoxylin and eosin staining.Pulmonary vascular remodeling was assessed by medial wall thickness and the extent of muscularization occurred in small arteries was measured with a-smooth muscle actin(?-SMA)immunochemistry experiment.KLF4 expression was measured with immunofluorescence method,and smooth muscle cells of small lung arteries exhibiting proliferation were assessed by immunostaining with proliferating cell nuclear antigen(PCNA).Results:Compared with the control group,the general condition of the group exposed to cigarette smoke was worse.Their pulmonary arteriole wall was thickened significantly with the medial wall thickness increased significantly,and the total muscularization rate of pulmonary artery increased significantly.Compared with the control group,the percentage of non-muscularized pulmonary vessels in smoking rats decreased,while the percentage of partially muscularized and completely muscularized pulmonary vessels increased significantly.The expression of KLF4 and the number of PCNA positive cells in the lung vessels of the smoke model rats were significantly higher than those in the control group.Conclusions:KLF4 is upregulated in pulmonary vessels in CS-induced pulmonary vessel remodeling,and it is associated with an increase in proliferation of smooth muscle cells of small lung arteries.Part 2:Changes in the expression of KLF4 in the enhanced human pulmonary artery smooth muscle cell(HPASMC)proliferation and migration induced by cigarette smoke extract(CSE)and the role of KLF4 in this processObjective:To explore whether KLF4 regulates the proliferation and migration of HPASMCs induced by CSE.Methods:HPAMSCs were stimulated by CSE of different concentrations for 48h.The optimal CSE concentration in promoting HPAMSCs proliferation was measured by CCK-8 assay.KLF4 protein expression of HPASMCs stimulated with CSE for different times was measured by western blot.KLF4-siRNA was applied to silence KLF4 expression in CSE-induced proliferation and migration of HPAMSCs.The proliferation of HPAMSCs was detected by CCK-8,cell counting and PCNA protein expression,and Transwell migration assay was performed to evaluate HPAMSC migration.P-AKT and AKT were detected to determine whether KLF4 might affect the activation of the AKT signaling pathway,mediating its effect on cell proliferation and migration.Results:2%CSE has the strongest effect on the proliferation of HPASMCs for 48 hours.KLF4 expression were up-regulated in HPAMSCs under 2%CSE stimulation.KLF4-siRNA did not only down-regulated PCNA expression,but also suppressed the proliferation and migration significantly in CSE-administrated HPAMSCs.At the same time,it inhibited the CSE-induced increase of AKT phosphorylation.Conclusion:KLF4 may promote the CSE-administrated proliferation and migration of HPASMCs by acting on the AKT signaling pathway.Part 3:Prevention of cigarette smoke-induced pulmonary hypertension by intratracheal delivery of AAV1-KLF4-shRNAObjective:To assess the feasibility of sustained pulmonary vascular KLF4 gene knockdown using intratracheal delivery of adeno-associated virus type 1(AAV1)in an animal model of cigarette smoke-induced PH and to determine whether gene transfer of KLF4-shRNA via AAV1-KLF4-shRNA could prevent the development of PH.Methods:Animals were randomized to control group(sham,n=8)and intratracheal administration of AAV1 carrying the KLF4-shRNA(AAV1-KLF4-shRNA group,n=12)or AAV1 carrying control vector(AAV1-control vector group,n=12)or saline(n=12).30 days after the intratracheal administration,the latter three groups underwent whole body exposure to CS for another 4 months.We tested hemodynamic measurements using right ventricular catheterization,calculated the right ventricular hypertrophy index(Fulton's index),and observed the degree of pulmonary vascular remodeling.The protein levels of KLF4 and PCNA in pulmonary arteries were determined by immunochemistry staining.Results:KLF4 expression and some hemodynamic measurements(such as RVESP,dP/dt max)were increased significantly in remodeled pulmonary arteries from the rat CS-induced PH model(both AAV 1-control vector group and saline group)in comparison with controls.While in the prevention group(AAV1-KLF4-shRNA group),all the indexes above which are associated with systolic function of right ventricle decreased and the degree of pulmonary vascular remodeling relieved compared to PH models.Conclusions:Targeted vascular gene knockdown of KLF4 via AAV1-KLF4-shRNA could prevent the development of PH.Part 4:Therapeutic efficacy of AAV1-KLF4-shRNA in CS-induced pulmonary hypertensionObjective:To determine whether gene knockdown of KLF4 via AAV1-KLF4-shRNA had therapeutic efficacy in established PH.Methods:Rats were randomly assigned to one of two groups:control group and group exposed to CS.The smoking group underwent whole body exposure to CS in a ventilated chamber,and the control animals were exposed to air.3 months after the CS exposure,rats from the CS group were randomly assigned to 1 of 3 treatment groups:saline,AAVl-control vector,or AAV1-KLF4-shRNA.Therapeutic efficacy was evaluated 1 months after the treatment above.We tested hemodynamic measurements using right ventricular catheterization,calculated the right ventricular hypertrophy index(Fulton's index),and observed the degree of pulmonary vascular remodeling.The levels of KLF4 and PCNA in pulmonary arteries were determined by immunochemistry staining.Results:KLF4 expression and some hemodynamic measurements(such as RVESP,dP/dt max)were increased significantly in remodeled pulmonary arteries from the rat CS-induced PH model(both AAV 1-control vector group and saline group)in comparison with controls.Intratracheal delivery of AAV1-KLF4-shRNA improved pulmonary hemodynamics and pulmonary vascular remodeling in the rat model of PH.Conclusions:Upregulation of KLF4 plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PH.Selective pulmonary vascular KLF4 gene knockdown may offer benefit as a therapeutic intervention in PH.