Vascular endothelial growth factor (VEGF) gene transfer-mediated angiogenesis has been proposed for peripheral artery disease. However, protocols using single administration have shown little benefit. Given that the transient nature of VEGF gene expression provokes instability of neovasculature, we hypothesized that repeated administration would provide efficient tissue protection. We thus compared single vs repeated transfection in a rabbit model of hindlimb ischemia by injecting a plasmid encoding human VEGF₁₆₅ (pVEGF₁₆₅) at 7 (GI, n=10) or 7 and 21 (GII, n=10) days after surgery. Placebo animals (GIII, n=10) received empty plasmid. Fifty days after surgery, single and repeated administration similarly increased saphenous peak flow velocity and quantity of angiographically visible collaterals. However, microvasculature increased only with repeated transfection: capillary density was 49.4±15.4 capillaries per 100 myocytes in GI, 84.6±14.7 in GII (P<0.01 vs GI and GIII) and 49.3±13.6 in GIII, and arteriolar density was 1.9±0.6 arterioles per mm² in GI, 3.0±0.9 in GII (P<0.01 vs GI and GIII) and 1.5±0.6 in GIII. Muscle lesions were reduced only within repeated transfection. With single administration, gene expression peaked at 7 days and declined rapidly, but with repeated administration, it remained positive at 50 days. At 90 days of repeated transfection (additional animals), gene expression decreased significantly, but neovessel densities did not. Thus, repeated, but not single, VEGF gene transfection resulted in increased microvasculature, which, in turn, afforded effective protection against ischemic muscle damage.

Angiogenic gene overexpression has been the main strategy in numerous vascular regenerative gene therapy projects. However, most have failed in clinical trials. CRISPRa technology enhances gene overexpression levels based on the identification of sgRNAs with maximum efficiency and safety. CRISPick and CHOP CHOP are the most widely used web tools for the prediction of sgRNAs. The objective of our study was to analyze the performance of both platforms for the sgRNA design to angiogenic genes (VEGFA, KDR, EPO, HIF-1A, HGF, FGF, PGF, FGF1) involving different human reference genomes (GRCH 37 and GRCH 38). The top 20 ranked sgRNAs proposed by the two tools were analyzed in different aspects. No significant differences were found on the DNA curvature associated with the sgRNA binding sites but the sgRNA predicted on-target efficiency was significantly greater when CRISPick was used. Moreover, the mean ranking variation was greater for the same platform in EPO, EGF, HIF-1A, PGF and HGF, whereas it did not reach statistical significance in KDR, FGF-1 and VEGFA. The rearrangement analysis of the ranking positions was also different between platforms. CRISPick proved to be more accurate in establishing the best sgRNAs in relation to a more complete genome, whereas CHOP CHOP showed a narrower classification reordering.

Background: Recent preclinical studies have demonstrated that bone marrow (BM)-derived Muse cells have a homing mechanism to reach damaged cardiac tissue while also being able to reduce myocardial infarct size and improve cardiac function; however, the potential of BM-Muse cells to foster new blood-vessel formation has not been fully assessed. Up to date, adipose tissue (AT)-derived Muse cells remain to be studied in acute myocardial infarction (AMI). The aim of the present study was to analyze in vitro and in vivo the neovascularization capacity of AT-Muse cells while exploring their biodistribution and differentiation potential in a translational ovine model of AMI. Methods and results: AT-Muse cells were successfully isolated from ovine adipose tissue. In adult sheep, one or more diagonal branches of the left anterior descending coronary artery were permanently ligated for thirty minutes. Sheep were randomized in two groups and treated with intramyocardial injections: Vehicle (PBS, n = 4) and AT-Muse (2x10⁷ AT-Muse cells labeled with PKH26 Red Fluorescent Dye, n = 4). Molecular characterization showed higher expression of angiogenic genes (VEGF, PGF and ANG) and increased number of tube formation in AT-Muse cells group compared to Adipose-derived mesenchymal stromal cells (ASCs) group. At 7 days post-IAM, the AT-Muse group showed significantly more arterioles and capillaries than the Vehicle group. Co-localization of PKH26+ cells with desmin, sarcomeric actin and troponin T implied the differentiation of Muse cells to a cardiac fate; moreover, PKH26+ cells also co-localized with a lectin marker, suggesting a possible differentiation to a vascular lineage. Conclusion: Intramyocardially administered AT-Muse cells displayed a significant neovascularization activity and survival capacity in an ovine model of AMI.

Background: Gene therapy has been proposed as a strategy to induce cardiac regeneration following acute myocardial infarction (AMI). Given that Tbx20, a transcription factor of the T‐box subfamily, stimulates cell proliferation and angiogenesis, we designed a baculovirus overexpressing Tbx20 (Bv‐Tbx20) and evaluated its effects in cultured cardiomyocytes and in an ovine model of AMI. Methods and Results: Cell proliferation and angiogenesis were measured in cardiomyocytes transduced with Bv‐Tbx20 or Bv‐Null (control). Subsequently, in sheep with AMI, Bv‐Tbx20 or Bv‐Null was injected in the infarct border. Cardiomyocyte cell cycle activity, angioarteriogenesis, left ventricular function, and infarct size were assessed. Cardiomyocytes transduced with BvTbx20 increased cell proliferation, cell cycle regulatory and angiogenic gene expression, and tubulogenesis. At 7 days posttreatment, sheep treated with Bv‐Tbx20 showed increased Tbx20 , promitotic and angiogenic gene expression, decreased levels of P21 , increased Ki67‐ (17.09±5.73 versus 7.77±7.24 cardiomyocytes/mm ² , P <0.05) and PHH3 (phospho‐histone H3)‐labeled cardiomyocytes (10.10±3.51 versus 5.23±2.87 cardiomyocytes/mm ² , P <0.05), and increased capillary (2302.68±353.58 versus 1694.52±211.36 capillaries/mm ² , P <0.001) and arteriolar (146.95±53.14 versus 84.06±16.84 arterioles/mm ² , P <0.05) densities. At 30 days, Bv‐Tbx20 decreased infarct size (9.89±1.92% versus 12.62±1.33%, P <0.05) and slightly improved left ventricular function. Baculoviral gene transfer‐mediated Tbx20 overexpression exerted angiogenic and cardiomyogenic effects in vitro. Conclusions: In sheep with AMI, Bv‐Tbx20 induced angioarteriogenesis, cardiomyocyte cell cycle activity, infarct size limitation, and a slight recovery of left ventricular function, suggesting that Bv‐Tbx20 gene therapy may contribute to cardiac regeneration following AMI.