The Role And Mechanism Of ZIP12 In Vascular Remodeling In Rats With Monocrotaline-induced Pulmonary Arterial Hypertension

BackgroundPulmonary hypertension(PH)is a severe and fatal disease,characterized by vascular remodeling of small pulmonary arteries,increased pulmonary vascular resistance and secondary right ventricular failure.A large volume of literature proves that hyperproliferation and migration of pulmonary arterial smooth muscle cells(PASMCs)were critical pathological processes in the development of PH.ZIP12 is a membrane-spanning protein that transports zinc ions into the cytoplasm from the extracellular space.The latest research found that upregulation of ZIP12 was involved in the elevation of cytosolic free zinc ions and PASMCs excessive proliferation induced by hypoxia.ZIP 12 knockdown was found to inhibit hypoxia-induced the influx of zinc ions into cells and their subsequent proliferation,ameliorate pulmonary arterial remodeling and suppress elevated pulmonary arterial pressure.However,the expression of ZIP 12 and its role in pulmonary arterial hypertension(PAH)rats induced by monocrotaline(MCT)has not previously been described.ObjectivesThe aim of the present study was to examine the expression of ZIP 12 in MCT-induced PAH,and to investigate its role and underlying mechanisms of proliferation and migration in PASMCs.MethodsPart Ⅰ.The expression of ZIP12 in rats with monocrotaline-induced pulmonary arterial hypertensionRats were administered twice intraperitoneal injections of 20mg/kg MCT at one-week interval according to the earlier established protocol.Hemodynamics,right ventricular hypertrophy index(RVHI),and morphological changes were measured 4 weeks after MCT administration.Immunofluorescence and immunohistochemical staining were performed to localize the expression of ZIP12 in lung tissue.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression of ZIP12.The protein levels of ZIP12,p-AKT,AKT,p-ERK1/2,ERK1/2,PCNA and cyclin D1 in lung tissue were determined by Western blot.Part Ⅱ.The role of ZIP12 in the proliferation and migration of pulmonary arterial smooth muscle cells in monocrotaline-induced pulmonary arterial hypertensionPASMCs were isolated from pulmonary arteries of MCT-induced PAH or control rats.The cells were identified by morphology and α-SMA immunostaining.Localization of ZIP12 was determined by double-label immunofluorescence using DiO,the cell membrane green fluorescent probe.ZIP 12 knockdown lentivirus and overexpressing lentivirus were constructed and transfected into PAH-PASMCs and Ctrl-PASMCs,respectively.5-ethynyl-2’-deoxyuridine(EdU)incorporation assay was performed to analyze the cell proliferation.Cell migration was evaluated via a scratch assay.The protein levels of ZIP12,PCNA,and cyclin D1 were assessed by Western blot.Part Ⅲ.The mechanism of ZIP12 mediated in the proliferation and migration of pulmonary arterial smooth muscle cells in monocrotaline-induced pulmonary arterial hypertensionTo further investigate the molecular mechanism involved in ZIP12-regulated cell proliferation and migration,the phosphorylation of AKT and ERK1/2 stimulated with 10%fetal bovine serum(FBS)in Ctrl-PASMCs and PAH-PASMCs were first detected by Western blot.ZIP12 knockdown lentivirus and overexpressing lentivirus were transfected into PAH-PASMCs and Ctrl-PASMCs,respectively.The effects of ZIP12 silencing and overexpression on the phosphorylation of AKT and ERK1/2 were examined.Then,PI3K/AKT inhibitor(LY294002,10μM)and ERK inhibitor(U0126,10μM)were used respectively.PASMCs were divided into blank control,negative control,overexpression ZIP12,overexpression ZIP12+LY294002 pre-treated groups,and overexpression ZIP12+U0126 pre-treated groups.Cell proliferation was assessed using an EdU incorporation assay.The migration property was assessed via the scratch method.Western blot was performed to determine the levels of p-AKT,AKT,p-ERK1/2,ERK1/2,PCNA,and cyclin D1.ResultsPart Ⅰ.The expression of ZIP12 in rats with monocrotaline-induced pulmonary arterial hypertensionAfter 4 weeks of administration of MCT,mean pulmonary arterial pressure(mPAP),right ventricular hypertrophy index(RVHI),percent wall area(WA%),and percent wall thickness(WT%)were significantly increased when compared with the control group(mPAP:Ctrl 19.07±2.53mmHg,PAH 31.18±2.67mmHg,P<0.05;RVHI:Ctrl25.60±4.78%,PAH 50.55±6.77%,P<0.05;WA%:Ctrl 61.93±5.03,PAH 94.08±2.49,P<0.05;WT%:Ctrl 23.15±5.80,PAH 72.05±6.96,P<0.05).The degree of distal pulmonary arteries muscularization was increased after MCT administration(non-muscularized vessels:Ctrl 66.57±10.39%,PAH 18.38±9.04%,P<0.05;partially-muscularized vessels:Ctrl 20.18±7.45%,PAH 30.15±7.34%,P<0.05;fully-muscularized vessels:Ctrl 13.25±7.17%,PAH 50.57±12.53,P<0.05).The exposure of MCT showed a strong increase of PCNA-positive cells in pulmonary arterioles.Masson staining showed that MCT treatment promoted collagen deposition around pulmonary arterioles.The cardiomyocyte cross-sectional areas were significantly greater in the MCT rat(cardiomyocyte cross-sectional areas:Ctrl 174 ±31.44μm2,PAH 443.88±69.89μm2,P<0.05).The expression of ZIP12 at mRNA level and protein level in the MCT rat lung tissue were significantly increased(P<0.05).Results of immunofluorescence and immunohistochemical staining demonstrated that the expression of ZIP 12 was noticeably enhanced in pulmonary smooth muscle layer in the MCT rat.Moreover,there was a significantly higher protein expression of p-AKT,p-ERK1/2,PCNA,and cyclin D1 in the lung tissue of MCT rat(P<0.05).However,the expression of AKT and ERK1/2 did not demonstrate significant difference(P>0.05).Part Ⅱ.The role of ZIP12 in the proliferation and migration of pulmonary arterial smooth muscle cells in monocrotaline-induced pulmonary arterial hypertensionAfter isolation and culture,the PASMCs showed long spindle-shaped with clear boundaries,immunofluorescent staining for α-SMA confirmed that isolated primary cells were smooth muscle cells.ZIP12 was distributed on the cell membrane,as revealed by immunofluorescent analysis.The proliferation and migration rates of PAH-PASMCs were significantly increased compared with Ctrl-PASMCs(cell proliferation rate:Ctrl-PASMCs 11.06±1.32%,PAH-PASMCs 25.23±3.62%,P<0.05;cell migration rate:24h:Ctr1-PASMCs 21.24±2.90%,PAH-PASMCs 46.74±6.93%,P<0.05;48h:Ctrl-PASMCs 39.01±6.74%,PAH-PASMCs 77.93±11.76%,P<0.05).These results were corroborated by western blot analysis of PCNA and cyclin D1(P<0.05).All the above effects were significantly reversed by silencing ZIP 12(cell proliferation rate:Ctrl-PASMCs 12.11±1.62%,PAH-PASMCs 21.53±2.47%,PAH-PASMCs+LV-shNC 19.93±4.56%,PAH-PASMCs+LV-shZIP12 14.14±1.85%,P<0.05;cell migration rate:24h:Ctrl-PASMCs 23.02±3.62%,PAH-PASMCs 43.36±4.87%,PAH-PASMCs+LV-shNC 42.20±6.71%,PAH-PASMCs+LV-shZIP1229.95±2.41%,P<0.05;48h:Ctrl-PASMCs 40.89±3.60%,PAH-PASMCs 82.34±7.06%,PAH-PASMCs+LV-shNC 76.46±5.60%,PAH-PASMCs+LV-shZIP1250.23±5.55%,P<0.05).Conversely,ZIP12 overexpression significantly promoted cell proliferation,migration(cell proliferation rate:Ctrl-PASMCs 12.46±1.23%,Ctrl-PASMCs+LV-NC 10.90±1.61%,Ctrl-PASMCs+LV-ZIP12 18.03±0.77%,P<0.05;cell migration rate:24h:Ctrl-PASMCs 26.11±3.39%,Ctrl-PASMCs+LV-NC 20.92±8.79%,Ctrl-PASMCs+LV-ZIP12 60.28±9.81%,P<0.05;48h:Ctrl-PASMCs 44.58±3.80%,Ctrl-PASMCs+LV-NC 37.67±10.65%,Ctrl-PASMCs+LV-ZIP1277.03±10.06%,P<0.05),and upregulated the expression of PCNA and cyclin D1(P<0.05).Part Ⅲ.The mechanism of ZIP12 mediated in the proliferation and migration of pulmonary arterial smooth muscle cells in monocrotaline-induced pulmonary arterial hypertensionWestern blot analysis showed that stimulation of serum-starved PAH-PASMCs with 10%fetal bovine serum(FBS)induces a stronger and longer phosphorylation of AKT and ERK1/2 compared with Ctrl-PASMCs(P<0.05).Silencing of ZIP12 in PAH-PASMCs reduced AKT and ERK1/2 phosphorylation(P<0.05),whereas overexpression of ZIP12 in Ctrl-PASMCs enhanced it(P<0.05).Selective inhibition of AKT phosphorylation by LY294002 abolished the role of ZIP12 overexpression in enhancing cell proliferation and migration,and partially suppressed the increase in ERK1/2 phosphorylation due to ZIP12 overexpression;however,inhibition of ERK activity by U0126 resulted in the partial but not complete reversal of this effect,and did not affect the increase in AKT phosphorylation due to ZIP12 overexpression(cell proliferation rate:Ctrl-PASMCs 11.49±1.05%,Ctrl-PASMCs+LV-NC 11.32±1.36%,Ctrl-PASMCs+LV-ZIP12 19.85±0.60%,Ctrl-PASMCs+LV-ZIP12+LY2940024.81±1.30%,Ctrl-PASMCs+LV-ZIP12+U0126 15.31±0.39%,P<0.05;cell migration rate:24h:Ctrl-PASMCs 30.29±3.22%,Ctrl-PASMCs+LV-NC 20.94±6.01%,Ctrl-PASMCs+LV-ZIP12 57.48±8.76%,Ctrl-PASMCs+LV-ZIP12+LY294002 24.53±3.31%,Ctrl-PASMCs+LV-ZIP12+U0126 44.53±7.03%,P<0.05;48h:Ctrl-PASMCs 41.92±8.98%,Ctrl-PASMCs+LV-NC 36.06±9.18%,Ctrl-PASMCs+LV-ZIP12 78.99±5.73%,Ctrl-PASMCs+LV-ZIP12+LY29400236.74±2.15%,Ctrl-PASMCs+LV-ZIP12+U0126 60.91±8.04%,P<0.05).Conclusions1.The expression of ZIP 12 was upregulated in the MCT-induced PAH rat model.2.ZIP12 was implicated in the excessive proliferation and migration of PASMCs in rats with MCT-induced PAH.3.ZIP12 can promote the proliferation and migration of PASMCs through the AKT/ERK signal pathway.