The Role Of PTEN(Phosphatase And Tensin Homologue Deleted On Chromosome 10) In The Process Of Extra-cardiomyocyte Matrix Remodeling

Background:Cardial remodeling which includes cardiomycyte remodeling and extra-cardiomycyte matrix(ECM) remodeling,is crucial for the transition by which cardiac function from adaptive to maladaptive and progression. More and more evidences have indicated that ECM is onergoing a vivid metabolic status,and participate in inner-out cell signal transporting and preservation of cardiac structure and function. The network of cardiac ECM is mainly composed with Collagen typeⅠand typeⅢ,which maintains a relative homeostasis of synthesis by cardiac fibroblast cells and degradation by matrix metalloproteinases(MMPs) secreted mainly by cardiac fibroblast cells.Tissue inhibitor of matrix metalloproteinases( TIMPs) promote fibrosis by suppressing the acitivity of MMPs. Fibroblast cells will proliferate pathologically ,upregulate the expression of collagens,and downregulate the expression of MMPs when stimulated by AngⅡ,cause the metabolic balance to be broken,and the elevated deposit rate of collagens , inducing ECM remodeling,enhancing the stiffness,and eventually impairing the pump function of heart.PTEN (Phosphatase and tensin homologue deleted on chromosome 10),a type of tumor suppressor, negatively regulate the PI3K/Akt pathyway,is involved in multiple physiological and pathological process including cell development, proliferation, differentiation, migration, and apoptosis,etc,and also play an important role in the ECM remodeling of heart. Overexpression of PTEN in tumor cells resulted in cell cycle arrest and suppression of proliferation,and suppression of MMP and the degradation of ECM as well,and eventually inhibite the migration of tumor cells.But studies have proved that overexpression of PTEN inhibited primary pulmonary interstitial fibrosis,and the mechanism was different from that in tumor cells So we supposed that overexpression of PTEN may also inhibit the cardiac interstitial fibrosis,but lack of relevant study reports.This study was aimed to elucidate the role of PTEN in the proliferation and sythesis and degradation of collagen secreted by cardiac fibroblast cells induced by AngⅡ, and furthermore the mechanism by which PTEN influence the ECM remodeling of heart.Methods:1.Identifying and amplifying adenovirus containing wild-type PTEN (Ad-PTEN) or G129E which is point-mutated to loss lipid phosphatase activity but retaining protein phosphatase activity(Ad-G129E) by infecting 293 cells repeatly.2. Primary cultured neonatal rat cardiac fibroblast cells were infected with the adenovirus containing PTEN or G129E. Following up expression of GFP, we detected the infective efficiency of virus response to different M.O.I., and the positive rate of GFP expression at 24h, 48h and 72h after adenovirus infection, then combining with viability of cardiac fibroblast cells evaluated by MTT, we ascertained the suitable infection programme and M.O.I..3. RT-PCR and Western Blot were used to detect the level of PTEN mRNA and protein in the infected cell to ensure PTEN or G129E overexpression as a result of adenovirus infection.4. Using AngⅡas pro-proliferation and pro-collagen synthetic stimuli, we observed the effects of PTEN or G129E overexpression on the cardiac fibroblast cells, such as cell proliferation ,synthesis of collagen typeⅠ,Ⅲ,MMP-2,TIMP-1 and TGF-β1, taking empty adenovirus containing GFP (Ad-GFP) as control..5. Measuring cell cycle with flow cytometer,and apoptosis with AO/EB essay.6. RT-PCR was used to detect the level of collagen typeⅠ,Ⅲ,MMP-2,TIMP-1 and TGF-β1, Western Blot was used to detect the level of Akt and p27.3H-Proline Incorporating Essay was used to detect the synthetic rate of collagens,and Gelatinase Zymography to detect the gelatinase activities.Results:1. Following repeatedly infecting the AD293 package cell line, high titer adenovirus particles yielded.2. Infecting Ad-G129E to cardiac fibroblast cells will cause Anoikis of the cells.3.After Ad-PTEN,and Ad-GFP infection, the positive rate of GFP expression in the host cardiac fibroblast cells was growing up in M.O.I.-dependent and time-dependent manner; When M.O.I. increased up to 100 or above, the percent of cardiomyocytes expressing GFP exceeded 90%; For each strata of M.O.I., there was no more increase in the positive rate of GFP expression as the expression time exceeded 48h, So we took M.O.I. 100 and 48h as appropriate standard to continue the subsequent study.4. Findings with RT-PCR and Western Blot showed that infection of adenovirus resulted in PTEN overexpression in the cardiac fibroblast cells. 5. AngⅡstimulation resulted in elevate expression of TGF-β1,inhibited PTEN expression ,and activated the PI3K/Akt pathway,and prevented the expression of P27,and eventually accelerated proliferation of cardiac fibroblast cells; Overexpression of PTEN suppressed the PI3K/Akt pathway,and promoted the expression of p27,inducing G1/S cell cycle arrest,,and eventually suppressed the proliferation of cardiac fibroblast cells.6. AngⅡstimulation was followed by significantly increased mRNA expression and protein synthetic rate of collagen typeⅠ,Ⅲ;overexpression of PTEN would inhibit the increase of collagen typeⅠ,ⅢmRNA,and lowered the protein synthetic rate significantly.7. AngⅡstimulation was followed by significantly increased mRNA expression of MMP-2 and TIMP-1 mRNA, overexpression of PTEN inhibited the transcription of MMP-2 and promoted the transcription of TIMP-1 by AngⅡ.8. AngⅡstimulation was followed by significantly decreased activity of gelatinase secreted by cardiac fibroblast cells.Overexpression of PTEN significantly suppressed the activity of gelatinase,but after stimulated by AngⅡfor 48h, the activity of gelatinase increased significantly,and more significantly after 72h.Conclusions:AngⅡstimulation promoted the expression of TGF-β1,and inhibited the expression of PTEN in cardiac fibroblast cells,relieved the suppression over PI3K/Akt,and downregulated the expression of P27,the inhibitor to CDKs,and eventually promoted the division and proliferation of cardiac fibroblast cells; overexpression of PTEN suppressed the PI3K/Akt pathway ,elevated the level of P27,and induced the G1/S cell cycle arrest,and eventually suppress the proliferation of cardiac fibroblast cells.Overexpression of PTEN can reverse the increase expression of collagen typeⅠ,Ⅲby AngⅡstimulating,inhibited the transcription of MMP-2 and promoted the transcription of TIMP-1;.Overexpression of PTEN time-dependently promoted the activity of gelatinase after AngⅡstimulating. In addition, to some degree, overexpression of G129E induced the cardiac fibroblast cells to anoikis, which implied that the apoptosis of cardiac fibroblast cells by PTEN might be associated with its protein phosphatase activity as well.