CTGF; stromal vascular fraction; Pericytes; perfusion bioreactor; cardioprotection; cardiac ischemia; angiogenesis ; paracrine effects
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Background. In chronic cardiac ischemia, the effective induction of microvascular networks is crucial to rescue the local hibernating cardiomyocytes and ultimately restore the cardiac function. The current surgical revascularization strategies restore mostly the macro-circulation and rarely the damaged micro-vasculature. Mesenchymal stem cell- (MSC) based therapies showed some benefits in stabilizing cardiac function in preclinical studies, but the outcomes of clinical trials are still controversial. The therapeutic potential of this approach is mainly related to paracrine effects and is strongly limited by the low in vivo cell survival associated to the cell delivery method (intra-myocardial injections). The implantation of cells previously organized in vitro in three-dimensional (3D) engineered tissues with a rapid vascularization potential could overcome this issue. The applicant main aim is to use 3D engineered patches generated by multiple cell types to improve their in vivo engraftment and sustain the release of key factors. Rationale. Among the investigated MSC, the stromal vascular fraction (SVF) freshly isolated from adipose tissue has recently gained interest since it is a unique source of relevant multiple cell types, including both mesenchymal stromal and endothelial/mural cells, with a wide secretion profile of relevant factors. Chronic ischemic heart treated with freshly generated SVF-sheets showed some promising results in improving angiogenesis and stabilizing the cardiac function, but donor variability is still a major drawback, holding back its real therapeutic potential. Our in vitro findings showed that, compared to static, perfusion culture of 8/8 SVF donors in 3D scaffolds reproducibly amplified a particular SVF subpopulation, namely the pericytes, and increased the release of pro-angiogenic factors and of the connective tissue growth factor (CTGF, 16 times higher). Pericytes might play a pivotal role in cardiac repair, since they are a key component of the cardiac unit and take a fundamental part in angiogenesis and possibly also in cardiac homeostasis and regeneration via -cytokine / -microRNA signaling. Moreover, CTGF might be a crucial factor (i) in the reproducible enrichment of in vitro patches during perfusion culture acting as mitogen for the pericytes; and (ii) in cardiac repair due to its possible involvement in cardiomyocyte hypertrophy/survival, vascular remodeling and fibrosis. In this project, we aim to investigate the molecular mechanisms underlying the pericyte growth during dynamic culture and to investigate the role of the pericytes and the paracrine SVF effects in angiogenesis, cardiomyocyte survival and fibrosis reduction. Specific aims. This proposal targets to investigate 1. the mechanisms at the molecular (CTGF role) and cellular (pericyte role) level possibly underlying the reproducible generation of SVF-patches with high angiogenic potential following perfusion culture (Aim 1); 2. the in vivo angiogenic dynamics (Aim 2); 3. the in vitro release of cardio-protective factors (Aim 3a) and their effects on 3D functional human cardiac in vitro models (Aim 3b) and 4. the overall efficacy on angiogenesis, fibrosis reduction and recovery of cardiac function in ischemic heart rat model of the pericyte-enriched SVF-based patches (Aim 4).Experimental design. The CTGF in vitro role will be assessed by using a CTGF neutralizing antibody during the in vitro culture. To investigate pericyte role, freshly isolated human SVF cells depleted or not of pericytes will be cultured on collagen scaffolds either in a perfusion-based bioreactor or in static conditions. The specific role of the perfusion will be investigated in terms of 1. angiogenesis induction in an ectopic site; 2. cardiomyocyte survival/phenotype and function in a relevant 3D human micro-engineered cardiac tissue model; and 3. fibrosis reduction, micro-circulation recovery, cardiomyocyte survival/phenotype and heart function in a cardiac ischemic rat model.Expected value of the proposed project. We expect to expand our scientific knowledge on the effects of SVF-based therapies and, in particular, on the role of pericytes in cardiac repair. The acquired insights will represent a significant step towards the sound clinical translation of this approach by understanding the mode of action of SVF-based patches, which is a pre-requisite to improve the standardization in their manufacture and reproducibility in their effectiveness.