| Background: Valvular heart disease(VHD) is a common and growing problem in clinics and makes up a large proportion of the global disease burden.At present stage, artificial heart valve replacement has become the most effective treatment for VHD. However, prosthetic valves are not flawless and have limitations in its application. As a consequence, autologous tissue-engineered heart valves(TEHVs) have become the most attractive replacement valves because they can overcome the limitations of mechanical andbioprosthetic valves with their ability to remodel, regenerate, and grow. However, the current TEHVs do not adapt well to high flow shear stress(FSS) when transplanted in vivo, which limits its application. Adiponectin(APN) is an adipokine secreted by adipose tissues and some other cells. APN exhibits protective effects in various cellular processes. In particular, APN can maintain myocardial cells survival.It protects heart from ischemia reperfusion injury(IRI) pressure overload-induced dysfunction, and reverse the structural and metabolic remodeling. APN protects the bone marrow mesenchymal stem cells from FSSI and the underlying mechanism are still unknown. In this experiment, APN was introduced to the research of TEHVs.This study explored the cell proliferation and apoptosis of BMSCs under interaction of APN and attempted to explore the apoptotic signaling pathway and molecular mechanism of APN, which protects BMSCs from the FSSI.Objective: This experiment aims to investigate the effects of FSSI on BMSCs and the roles of APN in this process as well as the underlying mechanism.We investigated the apoptotic signaling pathway and the role of AMP-activated protein kinase(AMPK) signaling in this process. Therefore, the FSS for BMSCs is carried out in a parallel plate flow chamber in order for the sake of long-term research study in the new key molecules and targets to protect the seed cells in the process of cultivating TEHVs.Methods: 1. The isolation and culture of BMSCs were performed as described previousl. Cultured cells were subjected to fluorescence-activated cell sorting(FACS) analysis to assess the expression of various signature antigen markers of BMSCs. Cultured BMSCs at passage three were used for the following experiments. 2. Shear stress stimulation of BMSCs is carried out in a parallel plate flow chamber with a flow loop apparatus in previous studies. The BMSCs are isolated and cultured in parallel plate flow chambers. This experiment included 4 groups: 0 dyne/cm2 group,7.5 dyne/cm2group,15 dyne/cm2 group and 30 dyne/cm2 group. Four different magnitudes of FSS wasapplied to the MSCs for 24 hours.Cell proliferation analysis was measured using cell counting kit-8.Apoptosis rate was assessed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling(TUNEL) assay. Then, the samples were analyzed for the expression of Bax,Bcl-2,AMPK,p-AMPK,ACC and p-ACC by Western Blot. The b FGF, TGF-b, VEGF, and PDGF concentrations in the regenerated uterus were measured using an enzyme-linked immunosorbent assay(ELISA) kit. 3. The cells are exposed to the FSS environment with or without APN pretreatment at different concentrations(10, 20, and 30 ?g/m L) for 18 hours and 15 dynes/cm2 FSS are applied to the BMSCs for 24 hours. This experiment included 5 groups:(1)0 dyne/cm2 group,(2)15 dyne/cm2 group,(3)10 ?g/m L APN+15 dyne/cm2 group,(4)20 ?g/m L APN+15 dyne/cm2 group and(5)30 ?g/m L APN+15 dyne/cm2 group. FSS was applied to the MSCs for 24 hours.Cell proliferation analysis was measured using cell counting kit-8.Apoptosis rate was assessed using TUNEL assay. Then, the samples were analyzed for the expression of Bax,Bcl-2,AMPK,p-AMPK,ACC and p-ACC by Western Blot. The b FGF, TGF-b, VEGF, and PDGF concentrations in the regenerated uterus were measured using an ELISA kit. 4. To investigate the role of AMPK on the protective effects of APN, AMPK was silenced by si RNA. This experiment included 4 groups:(1)Control si RNA+15 dyn/cm2 group,(2)Control si RNA+APN+15 dyn/cm2 group,(3)AMPK si RNA+APN+15 dyn/cm2 group,(4) AMPK si RNA+15 dyn/cm2 group. FSS was applied to the MSCs for 24 hours.Cell proliferation analysis was measured using cell counting kit-8. Then, the samples were analyzed for the expression of Bax,Bcl-2,AMPK,p-AMPK,ACC and p-ACC by Western Blot. The b FGF, TGF-b, VEGF, and PDGF concentrations in the regenerated uterus were measured using an ELISA kit.Results: 1. Most cultured adherent cells showed the fibroblastic morphology that is characteristicof BMSCs, particularly in the third-generation cells. FACS analysis demonstrated that BMSCs were 99.4% pure for CD90 and 99.6% pure for CD29. The percentages of contaminated populations of hematopoietic stem cells positive for CD34, CD45, and CD106 were 2.2%, 2.6%, and 2.2%, respectively. 2. Compared with the control group(without FSS), stimulation of BMSCs for 24 hours with 7.5, 15, or 30 dynes/cm2 FSS inhibited cell viability in an intensity-dependent manner(P<0.05). Microscopy images indicated that the FSS stimulation resulted in significant cell shrinkage and reduced the rate of cellular attachment compared with the control group. Compared with the control group(without FSS), FSS stimulation inhibited cell viability, decreased the expression of Bcl-2 and increased the expression of Bax(P<0.05) in an intensity-dependent manner. The apoptotic index increased, showing an intensity-dependent increase in apoptosis induction(P<0.05).TGF-?, b FGF, VEGF, and PDGF levels can be decreased by shear stress in an intensity-dependent manner(P<0.05). 3. APN promoted the cellular viability of and reduced apoptosis in FSS-cultured rat BMSCs(P < 0.05),showing a concentration-dependent correlation. Additionally,APN increased the phosphorylation of AMPK and ACC, increased the expression of Bcl2 and decreased the expression of Bax(P<0.05),showing a concentration-dependent correlation.TGF-?, b FGF, VEGF, and PDGF levels can be decreased by shear stress in an intensity-dependent manner(P<0.05). 4. Silencing AMPK reversed the protective effects of APN against FSS-induced BMSC death(P<0.05). Inhibition of AMPK by si RNA also reduced the productions of cytokines(P<0.05), such as b FGF, TGF-?, VEGF, and PDGF, in rat BMSCs.Conclusion: 1. The Classic marrow adherent culture method can be used to produce high purity of BMSCs. 2. After stimulation with different intensities of FSS,the cell viability decreased and the apoptotic index increased. TGF-?, b FGF, VEGF, and PDGF levels were reduced.FSS stimulation decreased the expression of Bcl-2 and increased the expression of Bax.3. APN increased the expression of Bcl-2 and decreased the expression of Bax. APN restores the local production levels of b FGF, TGF-?, VEGF, and PDGF in FSS-cultured rat BMSCs.APN can contribute to the survival of and antiapoptosis function in BMSCs and neutralizes the influence of FSS on BMSCs in TEHVs. 4. The study found that the protective effects of APN are attributed to the activation of AMPK, which can further promote ACC phosphorylation, Bcl-2 up-regulation, and Bax down-regulation. APN exerts protective roles in BMSCs resistance to FSSI. |
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