Abstract
Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.
Highlights
Despite significant advances in the medical management of heart failure, ischemia/reperfusion injury is still a leading cause of death in developed countries [1]
Expression under hypoxia/serum deprivation (SD) using qRT-PCR, we confirmed the baseline expression of Angiopoietin-like 4 (ANGPTL4) messenger RNA (mRNA) under hypoxia/SD conditions increased approximately 3 folds compared with the control (Fig. 1A)
It was shown that the presence of ANGPTL4 prevented cells undergoing apoptosis, and the most evident protection came from the condition in which cells were pre-incubated with ANGPTL4 for 1 hr prior to the induction of hypoxia/SD, and continuously incubated with ANGPTL4 for 24 hr (2.6360.40 vs. 7.4860.58, P,0.05 ) (Fig. 2)
Summary
Despite significant advances in the medical management of heart failure, ischemia/reperfusion injury is still a leading cause of death in developed countries [1]. MSCs treatment before transplantation, including treatment with growth factors and cytokines, preconditioning, and genetic modification, has been attempted to enhance the MSCs survival [5,6,7]. This pretreatment strategy has proven to be successful in several studies, it showed little benefit to ischemic environment-induced detachment of matrix, and subsequently the apoptotic death of graft cells [8]. Enhancement of the anti-apoptotic ability of graft MSCs during the initial settlement to resist apoptosis in harsh conditions and pass survival signals, even in the absence of cellextracellular matrix (ECM) interaction, would be an attractive approach in development of cell transplantation techniques
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