Mechanism Of Integrin-linked Kinase In Hypoxia-induced Pulmonary Vascular Remodeling

Part I Expression and distribution characteristics of integrin-linked kinase in hypoxic pulmonary hypertension ratsObjective: Hypoxic pulmonary hypertension(HPH)is one of the serious complications of chronic hypoxic pulmonary disease and a global common cause of disability and death.Hypoxia-induced phenotypic transition and proliferation of pulmonary artery smooth muscle cells(PASMCs)are important pathological processes in the development of HPH.Integrin-linked kinase(ILK)is an early hypoxia response factor and is a key gene that maintains the contractile phenotype of vascular smooth muscle cells(VSMCs).This section aims to explore the expression and distribution characteristics of ILK in HPH rats.Methods: Sprague-Dawley(SD)rats were placed in a hypoxic chamber to establish an HPH model;hypoxic intervention was performed at 0,2,3,and 4 weeks.The mean pulmonary artery pressure(m PAP)of the rats in each group was measured by right heart catheterization.The left ventricle(LV),septum(S),and right ventricle(RV)of each group of rats were extracted to measure the right ventricular index [RV /(LV + S)].The HE staining method was used to observe pulmonary remodeling and calculate the ratio of the wall thickness to the outer diameter of the blood vessel(WT %)and the ratio of the wall area to the total area of the blood vessel(WA %).Serum ILK levels in each group were detected by ELISA.The pulmonary arteries of each group were extracted and the expression levels of ILK and myocardin were detected by Western blot and RT-PCR.The distribution of ILK in the rats was observed by immunohistochemistry.Results:(1)Rats in the hypoxic group had significantly increased m PAP levels when compared with those in the control group;there was a time-dependent increase in hypoxia(P < 0.05).The RV /(LV + S)level of rats in the hypoxic 2-week group tended to increase when compared with that in the control group;however,this was not statistically different(P > 0.05).After 3 weeks of hypoxia,RV /(LV + S)levels increased significantly in the rats,there was also a time-dependent increase in hypoxia(P < 0.05).(2)In the hypoxic rats,the levels of WT % and WA % in the pulmonary arteries increased significantly when compared with those in the control group;there was a time-dependent increase of hypoxia(P < 0.05).(3)Serum ILK levels significantly decreased in the hypoxic group when compared with that in the control group.After 4 weeks of hypoxia,the ILK levels further reduced(P < 0.05).(4)The expression levels of ILK and myocardin in the pulmonary arteries of the hypoxic rats were significantly reduced as compared with the normal control group;the expression levels of these 2 parameters decreased further with the extension of hypoxic time(P < 0.05).(5)ILK is widely expressed in rat lungs and is mainly localized in the cytoplasm.Conclusion: Chronic hypoxia can promote pulmonary vascular remodeling in rats.Expression of ILK in serum and pulmonary arteries of rats showed a time-dependent decrease under hypoxia.ILK is widely expressed in rat lungs and is mainly localized in the cytoplasm of cells.Part II Mechanisms of integrin-linked kinases involved in hypoxiainduced phenotypic transition of PASMCsObjective: Under hypoxic conditions,the transition of PASMCs from contractile/differentiated to synthetic/dedifferentiated is an important part of the HPH process occurrence and development.As a key cofactor for serum response factor(SRF),myocardin plays a key role in regulating the transcription of PASMCs differentiation under hypoxic conditions.The mechanism of ILK as an early hypoxia response factor and key gene that maintains the contraction phenotype of VSMCs in hypoxia-induced phenotypic transition PASMCs is unknown.We hypothesize that ILK participates in the regulation of hypoxia-induced phenotypic transition of PASMCs by acting on myocardin.Methods: Rat primary PASMCs were extracted for culture and identification.Hypoxia intervention was performed on PASMCs to detect ILK,myocardin,ETS-like protein 1(ETS-like1,Elk-1),p-Elk-1,SRF,smooth muscle ?-actin(SM ?-actin),calponin,and osteopontin expression changes.A reaction system using Myelin Basic Protein(MBP)as a substrate was established to detect changes in ILK kinase activity.Co-immunoprecipitation(Co-IP)and Chromatin immunoprecipitation(Ch IP)methods were used to detect the binding differences between myocardin,Elk-1,and SRF,SM ?-actin gene promoters.The immunofluorescence method was used to observe the distribution of ILK in PASMCs.Small interfering RNA(Si RNA)technology was used to knock down the expression of ILK and Myocardin.The effects on myocardin and contraction\synthetic phenotype protein in PASMCs were observed.The effect of reduced expression of ILK on the morphology of PASMCs was examined by phalloidin staining.An adenovirus(Ad)vector was established to overexpress ILK;its effects on the expression of myocardin,contraction\synthetic phenotype protein,and binding of myocardin to the SM ?-actin gene promoter in PASMCs were observed.Results:(1)Compared with the control group,1 and 6 h of hypoxia had no effect on the expression of ILK and myocardin in PASMCs(P > 0.05).The expressions of ILK and myocardin were significantly reduced after 24 h of hypoxia;both further reduced after 48 and 72 h of hypoxia,with a time-dependent decrease in hypoxia.(2)Compared with those in the control group,hypoxia for 1 h could significantly promote Elk-1 phosphorylation and reduce ILK kinase activity(P < 0.05).(3)Compared with those in the control group,hypoxia or QLT0267 intervention in PASMCs for 1 h caused a decrease in binding of myocardin to the SRF and SM ?-actin gene promoters,with an increase in the binding of Elk-1 to SRF and SM ?-actin gene promoters(P < 0.05).(4)Compared with the control group,hypoxia for 24 hours had no significant effect on the protein expression of p-Elk-1,Elk-1,and SRF(P > 0.05),but could increase the expression of Elk-1 and SRF m RNA(P < 0.05).We also found that 24 h hypoxia could decrease protein and m RNA expression of SM ?-actin and calponin and increase protein and m RNA expression of osteopontin in PASMCs(P < 0.05).(5)ILK is widely expressed in PASMCs and is mainly localized in the cytoplasm of PASMCs.(6)Decreased expression of myocardin can reduce the expression of SM ?-actin and calponin in PASMCs,and increase the expression of osteopontin(P < 0.05).(7)Decreased expression of ILK can reduce the expression of SM ?-actin and calponin in PASMCs,and increase the expression of osteopontin.Additionally,decreased expression of ILK could reduce myocardin levels(P < 0.05).Decreased expression of ILK can transform PASMCs from a typical spindle structure to a flat shape;the length of PASMCs is significantly shorter,but the surface area is significantly increased(P < 0.05).(8)ILK overexpression can significantly reverse the decrease of myocardin expression caused by hypoxia,significantly reverse the decrease of SM ?-actin and calponin expression,and increase osteopontin expression caused by hypoxia(P < 0.05).Additionally,ILK overexpression can significantly reverse the hypoxia-induced decrease in binding of myocardin to the SM ?-actin gene promoters.Conclusion: Early hypoxia causes a decrease in ILK kinase activity,which in turn reduces the myocardin binding to SRF and SM ?-actin gene promoters.The decreased ILK expression can be targeted to reduce myocardin levels and promote phenotypic transition of PASMCs.Overexpression of ILK can significantly reverse the reduction of myocardin expression and binding to the SM ?-actin gene promoter caused by hypoxia.Part III Mechanisms of integrin-linked kinases involved in hypoxiainduced proliferation of PASMCsObjective: The increase of basal intracellular Ca²⁺ concentration([Ca²⁺]i)in PASMCs caused by hypoxia is an important molecular signal to promote the proliferation of PASMCs.Under hypoxic conditions,the hypoxia-inducible factor-1?(HIF-1?)can promote the expression of transient receptor potential canonical(TRPC)proteins through the Rho A / Rho-associated kinase(ROCK)signaling pathway,causing a rise in basal [Ca²⁺]i.HIF-1? can participate in the occurrence of endothelialto-mesenchymal transition(End MT)by regulating the expression of ILK.In addition,ILK can participate in the regulation of cellular signals by regulating the Rho A / ROCK signaling pathway.We speculate that ILK may be a downstream factor of HIF-1?,which in turn regulates the expression of TRPC protein through the Rho A / ROCK signaling pathway,and then affects basal [Ca²⁺]i participation in the hypoxiainduced proliferation of PASMCs.Methods: Cell counting,MTT,and flow cytometry were used to detect the effects of ILK overexpression on the proliferation of PASMCs.Co Cl2 was used to interfere with the PASMCs,and the effects on the expression of ILK,myocardin and ILK kinase activity were observed.Si RNA technology was used to knock down HIF-1? expression and observe the effect on ILK and myocardin expression.Overexpression of ILK was used to observe the effects on TRPC1,TRPC6,basal [Ca²⁺]i,and Rho A / ROCK signaling pathways.Results:(1)Hypoxic intervention in PASMCs for 48 h can significantly increase the number,absorbance of PASMCs,and percentage of PASMCs in the S+G2/M phase,while decreasing the percentage of PASMCs in the G0/G1 phase.Overexpression of ILK can significantly reverse this phenomenon(P < 0.05).(2)Co Cl2(100 ?M)could significantly reduce the expression of ILK and myocardin after 24 h of PASMCs intervention.With extension of the intervention time,the expression of ILK and myocardin decreased further(P < 0.05).Additionally,Co Cl2 intervention in PASMCs for 1 h could significantly reduce ILK kinase activity(P < 0.05).(3)The levels of ILK and myocardin in PASMCs were significantly reduced after 48 h of hypoxia,and a reduction in the expression of ILK and myocardin caused by hypoxia was reversed after knocking down HIF-1? expression(P < 0.05).(4)After 48 h of hypoxia intervention in PASMCs,TRPC1,TRPC6,basal [Ca²⁺]i increased significantly.However,ILK overexpression can obviously reverse the increase of TRPC1,TRPC6,and basal [Ca²⁺]i caused by hypoxia(P < 0.05).(5)Hypoxia intervention in PASMCs for 24 h and 48 h can significantly increase the expression of Rho A,ROCK1,and ROCK2.ILK overexpression can significantly reverse the increase of Rho A,ROCK1,and ROCK2 expression caused by hypoxia(P <0.05).Conclusion: Chronic hypoxia can reduce the expression of ILK and myocardin by increasing the level of HIF-1?.ILK may regulate the expression of TRPC1 and TRPC6 through the Rho A / ROCK signaling pathway and then regulate the basal [Ca2⁺]i to participate in the proliferation of PASMCs induced by hypoxia.