Involvement Of S100A4 And Related Proteins In The Protective Effect Of Fluoxetine Against MCT-induced Pulmonary Hypertension In Rats

Objective: Pulmonary arterial hypertension(PAH)is a fatal disease characterized by a progressive elevation of pulmonary arterial resistance,which induce right ventricular(RV)failure and ultimately lead to death.Pulmonary vascular remodeling is an important pathological feature of pulmonary arterial hypertension.Serotonin remodels the pulmonary vasculature associated with PAH by vasoconstriction,promotion of platelet aggregation,and pulmonary arterial smooth muscle cell proliferation.S100A4/Mts1,a member of the S100 proteins family,as a potential modifier gene,transgenic mice >1 year of age,that overexpressing S100A4/Mts1,develops plexogenic lesions in pulmonary vascular similar to those shown in patients with PVD and in chronic hypoxia-induced PAH mice,S100A4 m RNA/protein was significantly upregulated,S100A4 localized to smooth muscle cells,furthermore,S100A4 showed in newly muscularized small vessels after prolonged hypoxic exposure.By interacting with the receptor for advanced glycation end products(RAGE),extracellular S100A4/Mts1 also induces human pulmonary artery smooth muscle cell(h PASMC)migration and proliferation.Additionally,several studies have shown that S100A4/Mts1 expression may induce MMP expression,promoting cells invasion.Both of those involves in the pulmonary arterial hypertension(PAH)remodeling.Moreover,S100A4/Mts1 is observed in the pulmonary arteries smooth muscle cells of plexiform lesions and detected up-regulated in the neointima and adventitia of occlusive in PAH patients.Recently,serotonin(5-HT)has been shown to upregulate expression of S100A4 m RNA/protein and to stimulate release of S100A4/Mts1 with co-operative interaction of its receptor and transporter.All above suggest S100A4/Mts1 play an important role in the pathogenesis of PAH.Lots of evidence show serotonin(5-hydroxytryptamine,5-HT)have been associated with the development of pulmonary arterial hypertension.By uptake of serotonin via the serotonin transporter,serotonin promotes pulmonary arterial smooth muscle cell proliferation which results in vascular remodeling.Selective serotonin reuptake inhibitors(SSRIs)inhibite serotonin-induced pulmonary artery smooth muscle cells proliferation through blocking the function of the serotonin transporter.Intracellular S100A4/Mts1 regulates the p53 tumor suppressor protein.S100A4 binds the C-terminal end of p53 and modulates transcription of p53-responsive genes,such as MDM2.P53 is the most typical tumor suppressor that activates many genes via its transcriptional regulatory property.P53 protein is activated via phosphorylation and binds to p53-responsive genes include genes for apoptosis induction,DNA repair,and cell-cycle repression.P53 increased transcription,then propagation of damaged cells is repressed at G1 phase.This arresting period is required for damaged cells to be repaired or to enter an apoptotic pathway.In addition,p53 can trigger apoptosis through binding to either pro-or antiapoptotic members of the Bcl-2 family.S100A4 interacts with the C terminus of p53 and inhibits protein kinase C(PKC)phosphorylation of the tumor suppressor.S100A4 inhibits p53 from binding to its consensus DNA-binding sequence,thus it was expected that S100A4 would be a general inhibitor of p53 functions.Extracellular S100A4 binds to RAGE and activates the RAGE signaling cascade.Recent studies have shown that extracellular S100A4 can induce the up-regulation of several MMPs and result in remodeling of the extracellular matrix.Both vascular remodelling and right ventricular hypertrophy are PAH major pathology changes,which include imbalance of cells proliferation/apoptosis and disorder of extracellular matrix(ECM)arrangement.Accumulating experiments were about S100A4/Mts1 influence to PAH,however most of them were focused on transgenic rodents or cells in vitro.Whether S100A4/Mts1 was involved in the progression of primary PAH and the mechanisms of S100A4/Mts1 influence that were not clear.In general,pulmonary vascular remodeling of MCTinduced PAH rats mimics that occurs in humans with primary PAH.Fluoxetine,as a SSRI(selective serotonin re-uptake inhibitors)has commonly used in clinical antidepressant treatment.Moreover,we have reported fluoxetine protective against MCT-induced PAH in previous.Thus,in the present study,we utilized MCT-induced PAH rat model to study the expression of S100A4/Mts1 and associated proteins in primary PAH and analyze the effects of those proteins to fluoxetine against PAH.Methods: Establishment of PH model,experiments were performed on male Wistar rats(180±10g)from the Animal Resource Center,China Medical University(certificate number: Liaoning 034).Sixty male Wistar rats were randomly assigned into four groups(n = 15 each): control group(CONT),MCT,MCT+fluoxetine at 2mg/kg(MCT+F2),and MCT+fluoxetine at 10mg/kg(MCT+F10).The MCT group and two fluoxetine-treated groups were injected intraperitoneally(ip)with a single dose(60 mg/kg)of MCT(Sigma–Aldrich,St Louis,MO,USA)to induce PH as previously described,meanwhile,CONT group received an same volume solution of ethanol and physiological saline(0.9%)(1:4).Fluoxetine(Cadila pharmaceuticals limited,Ankleshwar)was dissolved in distilled water.Beginning on the MCT injection day,two fluoxetine-treatment group rats were administered by gavage once daily with 2mg/kg and 10mg/kg of fluoxetine respectively,and the control and MCT group rats were treated by gavage with an equal volume of vehicle(distilled water)everyday for 21 days.Then these rats were maintained on a standard laboratory diet and tap water,and exposed to a 12 h / 12 h light-dark cycle at 20 ± 2°C and humidity(50–70%)throughout experiments(21 days).All animals were cared for in accordance with the guidelines for the Institutional Animal Care and Use Committee of China Medical University(Heping District,Shenyang,China).Hemodynamic studies,assessment of right ventricular hypertrophy,the ratio of RV weight to left ventricle plus septum weight [RV/(LV+S)] was calculated as an index of RVH,morphological analysis of pulmonary arteries,hematoxyline-eosine(H&E)stains performed,determination of m RNA expression of MDM2 by quantitative Real Time PCR.Western blotting procedure to determine the proteins: S100A4,RAGE,P53,p-P53,MMP13,MMP9 and MMP2 in lung,RV and pulmonary artery.Results: 1.Hemodynamic Parameters,the mean pulmonary arterial pressure(m PAP)in the MCT group was 30±3.9mm Hg showing a significant increase in comparison to16.2±2.2mm Hg mm Hg in the control group(P<0.001,MCT vs control).These differences between MCT group and MCT+F2 group were not statistically significant.The value decreased to 22.7±2.2mm Hg mm Hg in the MCT+F10 group(P<0.01, MCT+F10 vs MCT).No significant differences were observed of the mean systemic arterial pressure(m PAP)measured in all groups.2.Estimation of Right Ventricular Hypertrophy,RVI(%)of the MCT group was 53.3±4.3% showing significant RV hypertrophy,which was significantly higher than that 29.8±2.8% in the control group(P<0.001,MCT vs control).RVI was reduced to 49.8±3.6% in MCT+F2 group and 39.6±4.1% in MCT+F10 group(P<0.001,MCT+ F10 vs MCT).Both mean pulmonary arterial pressure(m PAP)and RVI(%)in the MCT group were elevated significantly,that indicated the models of MCT-induced PAH were successful.The decreasing values in the fluoxetine-treat groups were dose-dependently,which showed fluoxetine had the effects to prevent development of MCT-induced PAH.3.Morphometric analysis of the pulmonary arteries,the thickness of pulmonary arterial vessels medial wall increased markedly in MCT group.Compared with other groups,the percentage of medial wall thickness in MCT group increased from34.4±3.4% to 46.5±2.6%,(P<0.001,MCT vs control)and decreased to 41.6±2.8% in MCT+F2 group(P<0.001,MCT vs control)and to 38.4±3.6% in MCT+F10 group(P<0.001,MCT vs MCT+F10),respectively.All above data showed that MCT induced remarkable pulmonary artery remodeling,yet,fluoxetine prevented the change induced by MCT.4.m RNA expression of MDM2 in rat lung,Real-Time PCR was utilized to detect m RNA of MDM2 in rat lung tissue.Compared with the control group,MDM2 m RNA decreased in MCT groups,whereas it was increased in MCT+F2 groups and in MCT+F10 groups.5.Expression of S100A4 and RAGE,expressions of S100A4 and RAGE in rat pulmonary arteries,lung and RV tissues were measured by western blotting,respectively.S100A4 expression of rat pulmonary artery,lung and RVwere upregulated significantly in MCT group compared with the control group,whereas it was decreased in MCT+F2 and MCT+F10 groups.The levels of RAGE from these tissues were also obviously increased in MCT group compared with control group,and these levels were decreased in MCT+F2 and MCT+F10 groups.The slides in immunohistochemistry stained reveal that S100A4 located in the medial wall of the pulmonary arteries,alveolar lining cells.Compared with the control group,S100A4 expression was significantly increased in rat lungs in the MCT group.Compared with the MCT group,the level in rat lungs was not markedly changed in the MCT+F2 group,but was significantly decreased in MCT+F10 group.These results proved S100A4 and RAGE expression elevated markedly after MCT treatment compared with the control group.Fluoxetine administration reduced that increased expressions by dose-dependently.6.Expression of P53,P-P53,and MMP13 in rat lung,MMP2,MMP9 and MMP13 in RV were measured by western blotting,respectively.P-P53 protein in MCT groups decreased compared with control groups,it increased in MCT+F2 groups and in MCT+F10 groups.The other proteins in MCT groups were increased compared with control groups,whereas decreased in MCT+F2 groups and in MCT+F10 groups.The section in immunohistochemistry stained show MDM2 located in the medial wall of the pulmonary arteries expression was significantly decreased in the MCT group.There were not remarkable different between the the MCT group and MCT+F2 group,but was significantl increased in MCT+F10 group.Conclusion: S100A4 and RAGE expression was elevated in MCT induced PAH rat.Inhibition of S100A4 expression is associated with the effect of fluoxetine to protect againest pulmonary arterial hypertension in MCT-treated rat.