Avaliação do potencial pró-angiogênico/arteriogênico de células-tronco/estromais mesenquimais humanas geneticamente modificadas para superexpressar o fator inibidor da leucemia em modelo de isquemia crítica de membros

Abstract

INTRODUCTION: Peripheral arterial disease (PAD) is a chronic vascular condition whose most extreme outcome, critical lower limb ischemia (CLI), is one of the main causes of morbidity and mortality worldwide. Despite its high prevalence, PAD still has no effective treatment to recover the lost vasculature. Mesenchymal stem/stromal cells (MSCs) are cells capable of secreting molecules that mediate biological effects such as angiogenesis and arteriogenesis, representing a therapeutic option against CLI. The therapeutic potential of MSCs can be increased through the overexpression of factors of interest, via genetic modification. Leukemia inhibitory factor (LIF) has been described as a cytokine capable of increasing the pro-angiogenic potential when overexpressed in MSCs. AIMS: To generate a human MSC line overexpressing LIF (MSC_LIF) and evaluate its pro-angiogenic/arteriogenic potential through in vitro and in vivo models. METHODS: MSCs were isolated from umbilical cord and transduced using second-generation lentiviral vectors expressing the LIF gene. Transduction efficiency and LIF protein production were confirmed by RT-qPCR and ELISA, respectively. The generated cells were characterized by differentiation into the three mesodermal lineages and immunophenotyping. Cell doubling was assessed by population doubling and CFU-F assay. Gene expression analysis of the main markers of angiogenesis, perivascular cells and arteriogenesis was performed by RT-qPCR. The potential for microvessel formation in vitro was investigated by the aortic ring sprouting assay. The Matrigel® plug assay investigated the potential for recruitment and migration of vasculature in vivo. The critical hindlimb ischemia model evaluated the potential of MSC_LIF in the formation of collateral arterial vessels and tissue protection against ischemia in vivo. RESULTS: MSC_LIF showed increased expression and secretion of LIF compared to non-transduced cells, indicating successful genetic modification. Imunophenotyping and mesodermal differentiation assay showed that MSC_LIF maintained the characteristic MSC phenotype and multipotency. CFU-F and PD assays demonstrated increased cell duplication in MSC_LIF. Gene expression analysis demonstrated upregulation of genes essential for angiogenesis, arteriogenesis and perivascular markers. The aortic ring sprouting assay showed that MSC_LIF promoted increased microvessel sprouting from arterial tissue. The Matrigel® plug assay revealed that MSC_LIF was more effective in inducing migration of CD31+ blood vessels and aSMA+ arterioles into the plugs. Treatment with MSC_LIF restored blood flow through the formation of aSMA+ arterioles, improved ischemic score and functional performance, and promoted greater tissue protection against ischemia in the critical limb ischemia model. CONCLUSION: LIF overexpression is a promising strategy to enhance the angiogenic/arteriogenic and tissue repair potential of MSC for the treatment of CLI.