Mesenchymal stem/stromal cells overexpressing leukemia inhibitory factor (LIF) promote arteriogenesis and functional recovery in a mouse model of critical hindlimb ischemia.

[BACKGROUND] Critical limb ischemia (CLI) is the most severe manifestation of peripheral arterial disease (PAD), for which there are still no effective therapeutic options. Mesenchymal stem/stromal cells (MSCs) can secrete bioactive molecules capable of promoting tissue regeneration and neovascularization. The therapeutic potential of MSCs can be enhanced by genetic modification to overexpress molecules of interest. Here, we produced an umbilical cord-derived MSC line overexpressing leukemia inhibitory factor (LIF), MSC_LIF, and evaluated its pro-angiogenic/arteriogenic potential.

[METHODS] MSCs were transduced using a second-generation lentiviral system to express LIF. Transgene overexpression was confirmed by qRT-PCR and ELISA. The transcriptional and secretory profile of MSC_LIF was accessed by qRT-PCR and angiogenic protein array. The pro-angiogenic/arteriogenic potential of MSC_LIF was tested by counting microvessel sprouting in the in vitro 3D aortic ring model, in addition to measuring hemoglobin with Drabkin's reagent and quantifying CD31 and αSMA vessels by fluorescence microscopy in the in vivo Matrigel plug assay. In the critical hind limb ischemia model developed in C57Bl/6 mice, ischemic score and exercise test were evaluated as functional parameters. Blood perfusion was quantified using a fluorescent vascular probe. Blood vessel distribution was assessed by quantification of CD31 and αSMA markers, and tissue damage was assessed by histological staining and qRT-PCR.

[RESULTS] Gene expression analysis demonstrated that MSC_LIF upregulates strategic genes in the neovascularization process and the secretion of angiogenic proteins. MSC_LIF increased the number of microvessel sprouting in aortic rings and promoted enhanced migration of endogenous vasculature into the Matrigel plugs. Intramuscular administration of MSC_LIF was able to restore blood flow in the ischemic paw due to the formation of CD31 blood vessels and αSMA arterioles. The treatment promoted protection against tissue damage and modulation of the ischemic environment through the expression of genes related to angiogenesis/arteriogenesis (such as Vegf, Mmp2 and Mmp9), inflammation (such as Il10) and myoprotection (Myog, Myod and Pax7).

[CONCLUSION] LIF overexpression is a promising strategy to increase the potential of MSC in the treatment of CLI and for tissue repair.