Effects Of Imidaprilat On Matrix Metalloproteinase-2 And TIMP-2 In Cardiac Fibroblasts Induced By IL-1β And The Study Of Possible Mechanism

BackgroundInflammatory factor plays an important role in myocardial extracellular matrix (ECM)remodeling.The synthase and degradation of ECM are regulated by matrix metalloproteinases(MMPs).Among MMPs,MMP-2 has the widest distribution in the myocardium and type 2 tissue inhibitor of matrix metalloproteinase(TIMP-2)is an endogenous inhibitor of MMP-2.Inhibition or knockout of MMP-2 and MMP-9 in mouse suppressed ventricular remodeling,myocardial dysfunction and progression of heart failureInterleukin-1β(IL-1β)is a key pro-inflammatory cytokine during heart failure. It was testified that IL-1βstimulated the expression and transcription of type 1A angiotensin receptor in rat cardiac fibroblasts and induced protein synthase and activity of MMP-9 and MMP-2 of cardiac fibroblasts,leading to ECM remodeling. Nitric oxide(NO)is an important cellular messenger,produced by nitric oxide synthases.Inducible NOS(iNOS),one of NOS,isn't usually expressed in normal tissue,however,LPS or pro-inflammatory cytokines,such as IL-1β,can induce expression of iNOS that generates large quantities of NO,resulting in cardiac pathophysiological change including stimulating the expression and activity of MMP-2.It has been observed that IL-1βcould affect the iNOS-NOS system in cardiac fibroblasts.But up to now,there are no evidences whether IL-1βcan affect MMPs or TIMPs by intracellular iNOS-NOS pathway of cardiac fibroblasts.Angiotensin converting enzyme inhibitor(ACEI)is widely used in the treatment of heart failure because of the attenuation of ventricular remodeling. However,the respond mechanisms remain to be elucidated.Recently,Kobayasi et al found that ACEI depressed the ventricular remodeling and heart failure by inhibiting expression of iNOS protein in myocardium of salt-sensitive Dahl rat,meanwhile, ACEI suppressed the gene expression and activity of MMPs at the predilatation stage and prevented the transition to congestive heart failure in spontaneously hypertensive rat.These effects of ACEI against remodeling are difference from traditional mechanisms:blocking the release of tissue and circulatory angiotensinâ…¡and decreasing the breakdown of bradykinin.But it is little report about how ACEI regulates ventricular remodeling except traditional mechanisms.Corresponding questions mentioned,we are ready to explore whether IL-1βhas effects on MMPs /TIMPs balance by iNOS-NOS of cardiac fibroblasts;whether ACEI can regulate the unbalance of MMPs/TIMPs by iNOS-NOS system.Objective(1)Observe effects of IL-1βon the transcription and activity of MMP-2,MMP-9 and the transcription of TIMP-2 in cultured human cardiac fibroblasts;(2)Investigate effects of imidaprilat,an ACEI,on MMP-2,MMP-9 and TIMP-2 in cultured human cardiac fibroblasts;(3)Observe effects of imidaprilat,an ACEI,on MMP-2,MMP-9 and TIMP-2 in cultured human cardiac fibroblasts induced by IL-1βand the possible mechanism involved.MethodsCulture of primary human cardiac fibroblasts Primary human cardiac fibroblasts were purchased at passage 2 from Cell Systems.It was plated in 6-well tissue culture plates(5×10⁴ cells/well).All cells were harvested at passage 3 to 6 and used at 80% to 90%confluence for experiments.The culture medium was replaced with CS-C medium(CSM)without serum and growth factor for 24h.Cells were washed two times with PBS and then cells were cultured in CSM in the absence or the presence of various treatments.RT-PCR Subconfluent human cardiac fibroblasts were cultured in CSM in the absence or the presence of various treatments 12h later.Total RNA was isolated from cultured cells and one microgram of total RNA was reversely transcripted with Super Scriptâ…¡^TM-reverse transcriptase.PCR amplification of cDNA was done using the primers of MMP-9,MMP-2,TIMP-2 and GAPDH respectively that were designed according to the sequence in GENEBANK.The resulting densities of the MMP-2, TIMP-2 and MMP-9 bands were expressed relative to the corresponding density of the GAPDH band from the same RNA sample.Zymography Subconfluent cells were serum deprived and treated with IL-1β(1ng/ml～10ng/ml)and/or imidaprilat(10^-9M～10^-6M)for 24h.Medium samples were harvested and the activity of MMPs was analyzed by zymography.Clear zones against the blue background indicated the presence of gelatinase.To quantify the amount of gelatinase production,the stained zymograms were scanned on a densitograph.NO assessment Assessment of NO production in the culture medium was performed using the Griess reagent that measures the level of nitrite(NO₂),100μl Griess reagent(a mixture of one part of Griess reagent A and one part of Griess reagent B)was added to 100μl of sample or standard(sodium nitrite served as the standard)in each well of a 96-well plate.After incubation at room temperature for 10 min,the optical density at 540 nm(OD540)was measured and the amount of nitrite was calculated from a standard curve.Western blot Cardiac fibroblasts were treated for 24h,the protein of cells were collected.The expression of iNOS protein level was detected by Western blot analysis.Statistical analysis Each experiment was repeated four times.Data are presented as means±standard deviation(SD).Statistical analysis was performed with the Statistical Package for the Social Sciences(SPSS version 11.5).Differences among all data were analyzed for significance by one-way analysis of variance(ANOVA) followed by unpaired Student t test.A probability level of P＜0.05 were considered significant.Results(1)MMP-2 can be detected in human cardiac fibroblasts by zymography and RT-PCR.TIMP-2 can be also detected by RT-PCR;however,it was difficulty to detect MMP-9 in human cardiac fibroblasts even if we tried to maximal sample volumn during zymography and adjusted many protocols of PCR.(2)After cardiac fibroblasts were incubated with IL-βfor 24h,MMP activities in supernatant were measured using zymogrpahy.We observed that IL-1βincreased MMP-2 activity.4ng/ml IL-1βincreased more evidently MMP activity(foldchange versus control,170.24±13.12%,P＜0.01).There was no significant difference between MMP-2 activity in 4ng/ml IL-1βgroup and 10ng/ml IL-1βgroup(P＞0.05).(3)RT-PCR analysis demonstrated that expression of MMP-2 mRNA was increased after 12h incubation of IL-1β.The significant change in MMP-2 mRNA compared with control group was detectable at 4ng/ml concentration of IL-1β.There was no significant difference between MMP-2 mRNA expressions in 4ng/ml IL-1βgroup and 10ng/ml IL-1βgroup(P＞0.05)(4)After cardiac fibroblasts were incubated with 4ng/ml IL-βfor 0.5h～24h,MMP-2 activity in conditioned media was measured by zymogrpahy.We observed that IL-1βstimulated MMP-2 activity through a time-dependent method and the effect of IL-1βon MMP-2 activity reached maximum after 24h incubation (P＜0.01).(5)After cardiac fibroblasts were incubated with 4ng/ml IL-βfor 24h,the level of NO in conditioned media was measured by Griess method.The result showed that IL-1βincreased NO accumulation in supematant(P＜0.01);however, inclusion of N^G-methyl L-Arginine(L-NMMA,10^-3M),a competitive inhibitor of NO synthase(NOS),significantly prevented the effect of IL-1βon cardiac fibroblasts including the increasing of NO(P＜0.01)and the increasing of MMP-2 activity(P＜0.01)and transcription(P＜0.01).Western blot analysis showed that iNOS did not detected in human cardiac fibroblasts;however,IL-1βstimulated significantly the expression of iNOS protein in cardiac fibroblasts.(6)Using RT-PCR and zymography,we found that imidaprilat alone with experimental concentration had no effect on the activity(P＞0.05)and transcription((P＞0.05)of MMP-2 in cardiac fibroblasts.(7)After human cardiac fibroblasts were incubated with 4ng/ml IL-βand imidaprilat with different concentrations for 24h,It was demonstrated that imidaprilat(10^-9～10^-6M)significantly inhibited the increasing of MMP-2 activity induced by IL-1β(4ng/ml)in a dose-dependent manner.(8)After human cardiac fibroblasts were incubated with 4ng/ml IL-βand imidaprilat (10^-7M)for 24h,Griess method showed that imidaprilat inhibited the accumulation of supematant NO induced by IL-1β(P＜0.01)and Western blot testified imidaprilat inhibited expression of iNOS protein in cardiac fibroblasts induced by IL-1β(P＜0.01).Sodium nitroprusside(SNP,10^-5M),an exogenous NO donor,blunted the inhibition effect of imidaprilat on IL-1β-induced MMP-2 transcription(P＜0.01)and activity(P＜0.01).(9)RT-PCR analysis of TIMP-2 showed that after 12h incubation of IL-1β(4ng/ml) or imidaprilat(10^-7M),neither of them could significantly change expression of TIMP-2 mRNA in human cardiac fibroblasts(P＞0.05).(10)We demonstrated that imidaprilat(10^-7M)didn't affect MMP-2 activity in vitro system due to forming chelating complexes(P＞0.05),however EDTA chelated metal ion and resulting in inactivity of MMP-2(P＜0.01).Conclusions1.IL-1βstimulated MMP-2 activity and transcription of human cardiac fibroblasts and iNOS-NO system was possibly involved with effects of IL-1β.2.Imidaprilat alone had no significant effects on MMP-2 transcription and activity of human cardiac fibroblasts without medications,but it significantly inhibited the increasing of MMP-2 mRNA expression and activity induced by IL-1β.Inhibition to iNOS-NOS system induced by IL-1βwas possibly related with effects of imidaprilat on MMP-2 induced by IL-1β.3.There were no significant effects of IL-1βor imidaprilat on TIMP-2 mRNA expression in human cardiac fibroblasts.