Tß4 Increases Neovascularization and Cardiac Function in Chronic Myocardial Ischemia of Normo- and Hypercholesterolemic Pigs.

Translations of new therapeutic options for cardiovascular disease from animal studies into a clinical setting have been hampered, in part by an improper reflection of a relevant patient population in animal models. In this study, we investigated the impact of thymosin ß4 (Tß4), which promotes collateralization and capillarization, during hypercholesterolemia, a known risk factor of coronary artery disease. Initial in vitro results highlighted an improved endothelial cell function upon Tß4 treatment under control conditions and during hypercholesterolemic stress (scratch area [pixels]: oxidized low-density lipoprotein [oxLDL], 191,924 ± 7,717; and oxLDL + Tß4, 105,621 ± 11,245). To mimic the common risk factor of hypercholesterolemia in vivo, pigs on regular (NC) or high-fat (HC) diet underwent chronic myocardial ischemia followed by recombinant adeno-associated virus (rAAV)-mediated transduction of Tß4 or LacZ as a control. We show that Tß4 overexpression improves capillarization and collateralization (collaterals: NC + rAAV.LacZ, 2.1 ± 0.5; NC + rAAV.Tß4, 6.7 ± 0.5; HC + rAAV.LacZ, 3.0 ± 0.3; and HC + rAAV.Tß4, 6.0 ± 0.4), ultimately leading to an improved myocardial function in both diet groups (ejection fraction [EF] at day 56 [%]: NC + rAAV.LacZ, 26 ± 1.1; NC + rAAV.Tß4, 45 ± 1.5; HC + rAAV.LacZ, 26 ± 2.5; and HC + rAAV.Tß4, 41 ± 2.6). These results demonstrate the potency of Tß4 in a patient-relevant large animal model of chronic myocardial ischemia.

Steerable Induction of the Thymosin ß4/MRTF-A Pathway via AAV-Based Overexpression Induces Therapeutic Neovascularization.

Viral vectors have been frequently used in a variety of preclinical animal models to deliver genetic constructs into tissues. Among the vectors used, adeno-associated viral vectors (AAVs) may be targeted to specific tissues, depending on the serotype used. Moreover, they show robust expression for prolonged periods of time and have a low immunogenic potential. Furthermore, AAVs, unlike other vector systems, only display a low rate of genomic integration. However, to ensure efficient transgene production, expression is typically driven by constitutively active promoters, such as the cytomegalovirus (CMV) promoter. Tetracyclin responsive promoters represent a promising alternative to unregulated promoters. The present study compares AAVs encoding either constitutively active CMV or tet-off promoter regions in the preclinical models of hindlimb and chronic myocardial ischemia. Therapeutically, mediators regulating vessel maturation, specifically thymosin beta 4 (Tß4) and the downstream signaling molecule myocardin-related transcription factor A (MRTF-A) as well as the endothelial activator angiopoietin-2 (Ang2) were overexpressed via AAVs using both promotors. In the model of rabbit hindlimb ischemia, temporary (tet-off) expression of Tß4 improved capillary density, collateralization, and perfusion in the ischemic hindlimb, with no detectable difference to constitutive Tß4 overexpression. Similarly, constitutive overexpression of MRTF-A alone was able to improve capillarization, collateralization and perfusion. Temporary expression of Ang2 for 7 days further increased capillary density and pericyte coverage compared with MRTF-A alone, without further improving collateralization or perfusion. In the pig model of chronic myocardial ischemia constitutive expression of Tß4 for 4 weeks induced capillary and collateral growth similarly to a pulsed expression (2 day expression per week for 3 weeks). Taken together these findings demonstrate for two models of preclinical interventions that temporary gene expression may lead to similar results as constitutive expression, highlighting the potential of controlled temporary gene expression for induction of vascular growth as a therapeutic approach.

MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2.

Gradual occlusion of coronary arteries may result in reversible loss of cardiomyocyte function (hibernating myocardium), which is amenable to therapeutic neovascularization. The role of myocardin-related transcription factors (MRTFs) co-activating serum response factor (SRF) in this process is largely unknown. Here we show that forced MRTF-A expression induces CCN1 and CCN2 to promote capillary proliferation and pericyte recruitment, respectively. We demonstrate that, upon G-actin binding, thymosin ß4 (Tß4), induces MRTF translocation to the nucleus, SRF-activation and CCN1/2 transcription. In a murine ischaemic hindlimb model, MRTF-A or Tß4 promotes neovascularization, whereas loss of MRTF-A/B or CCN1-function abrogates the Tß4 effect. We further show that, in ischaemic rabbit hindlimbs, MRTF-A as well as Tß4 induce functional neovascularization, and that this process is inhibited by angiopoietin-2, which antagonizes pericyte recruitment. Moreover, MRTF-A improves contractile function of chronic hibernating myocardium of pigs to a level comparable to that of transgenic pigs overexpressing Tß4 (Tß4tg). We conclude that MRTF-A promotes microvessel growth (via CCN1) and maturation (via CCN2), thereby enabling functional improvement of ischaemic muscle tissue.

Inhibition of microRNA-92a protects against ischemia/reperfusion injury in a large-animal model.

BACKGROUND: MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. METHODS AND RESULTS: Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid-modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly (P<0.01) reduced miR-92a expression in the infarct zone regardless of the application venue. However, catheter-based delivery, but not intravenous infusion, of LNA-92a significantly (P<0.05) reduced the infarct size compared with control LNA-treated pigs, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure (P<0.05). Histochemistry revealed that LNA-92a increased capillary density but decreased leukocyte influx and cardiac cell death. Complete loss of miR-92a in mice attenuated the infarct-related myocardial dysfunction to a larger extent than cardiomyocyte-specific miR-92a deletion. In vitro, LNA-92a protected against hypoxia/reoxygenation-induced cardiomyocyte cell death. CONCLUSIONS: Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia.