Developing a novel stem cell and gene therapy system based on resveratrol-induced HSP70 promoter-regulated VEGFA expression

Autor: Young-Bin Chen, 陳永斌
Rok vydání: 2015
Druh dokumentu: 學位論文 ; thesis
Popis: 103
Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or mesenchymal stem cell (MSC) transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. Furthermore, several studies have demonstrated that MSC-based cell therapy results in improvement in the treatment of emphysema in rat models. MSCs secrete anti-apoptotic and anti-inflammatory paracrine factors and reduce lung injuries. Despite reported successes, it was demonstrated that the amount of engrafted MSCs decreased dramatically after 24 hours of transplantation due to exposure to toxic and oxidative microenvironments. In additions, several possible side effects that may arise from clinical applications of VEGFA should be closely monitored. To avoid the VEGFA complications, we propose to further examine the protective effect of a conditional overexpression of VEGFA (under Resveratrol induced HSP70 promoter regulation) in two animal models. In this work, we aimed to test that VEGFA expression increased by RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects in a mouse angiogenesis model and protects against elastase-induced pulmonary emphysema in a mouse model by using stem cell and gene therapy. In the first study, HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, to develop MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. We investigated the protective effects of our approach by using ex vivo aortic ring co-culture system and 3D scaffolds in vivo model. Results of first investigation demonstrated that HSP70 promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by c-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. In the second study, this same stem cell line was further evaluated for its protective capacity to alleviate elastase-induced pulmonary emphysema in mice. Results of second study showed that c-RSV-treatment of HSP-VEGFA-MSC exhibited synergy between HSP70 transcription activity and induced expression of antioxidant-related genes when challenged by cigarette smoke extracts. Eight weeks after jugular-vein injection of HSP-VEGFA-MSC into mice with elastase-induced pulmonary emphysema, followed by c-RSV treatment to induce transgene expression, significant improvement was observed in respiratory functions. Expression of VEGFA, endogenous Nrf2 and MnSOD was significantly increased in the lung tissues of the c-RSV-treated mice. Histopathologic examination of treated mice revealed gradual but significant abatement of emphysema and restoration of airspace volume. In conclusion, the present investigation demonstrates that resveratrol-regulated VEGFA expression in HSP-VEGFA-MSC significantly improved the therapeutic effects on the treatment of ischemic disease and COPD in the mouse, possibly avoiding side effects associated with constitutive VEGFA expression.
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