Abstract
Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI). Mesenchymal stem cells (MSCs) are multipotent secretory cells that are suitable for clinical DLLI treatment, but their use has been hampered by poor survival after injection. Hypoxia can significantly enhance the capacity of MSCs to secrete angiogenic factors. We investigated transient hypoxia pretreatment of MSCs to facilitate revascularization in DLLI. Rat bone marrow MSCs (BM-MSCs) were cultured at different oxygen concentrations for varying time periods. The results indicated that transient pretreatment (5% O2, 48 h) not only increased the expression of VEGF-1α, ANG, HIF-1α and MMP-9 in BM-MSCs as assessed by real-time RT-PCR, but also increased the expression of Bcl-2 as determined by western blotting. The transplantation of pretreated BM-MSCs into rats with DLLI demonstrated accelerated vascular reconstruction when assayed by angiography and immunohistochemistry. CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days. The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle. Double immunofluorescence studies confirmed that the pretreated BM-MSCs promoted the proliferation and inhibited the apoptosis of endothelial cells. In vitro, pretreated BM-MSCs increased the migratory and tube forming capacity of endothelial cells (ECs). Hypoxia pretreatment of BM-MSCs significantly improved angiogenesis in response to tissue ischemia by ameliorating endothelial cell dysfunction and is a promising therapeutic treatment for DLLI.
Highlights
Lower limb ischemia, a common complication in Type 2 diabetes (T2D), afflicts approximately 15% of the diabetic patient population worldwide [1, 2]
The results demonstrated that the expression of VEGF-1a, HIF-1a, HGF, bFGF, MMP-9, and PDGF was enhanced in BM-Mesenchymal stem cells (MSCs) cultured under hypoxia (2%, 5%, or 7%), as determined by real time RT-PCR
We further examined the phenotypic changes of bone marrow MSCs (BM-MSCs) under 5% oxygen pretreatment and found that the phenotype of BM-MSCs showed no obvious change after hypoxic preconditioning (S1 Fig)
Summary
A common complication in Type 2 diabetes (T2D), afflicts approximately 15% of the diabetic patient population worldwide [1, 2]. A key strategy for restoring blood flow is the repair of dysfunctional ECs. Previous reports have demonstrated the positive effects of ECs in vascular network reconstruction in terms of angiogenesis for lower limb ischemic lesions [6]. Some strategies attempt to recover the mobilization, adhesion, tube formation and proliferation of ECs by modifying relevant molecular pathways, including the eNOS, CXCR-4 and hemeoxygenase-1 (HO-1) pathways [7,8,9]. Other proangiogenesis factors such as VEGF, HGF, and proangiogenesis protein MMP-9 repair endothelial dysfunction by improving the ability of ECs to migrate and proliferate [10,11,12]. Due to defective VEGF signaling that results in the low bioavailability of VEGF, the effect of providing VEGF to promote angiogenesis should further explored
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