Emerging concepts in heart failure management and treatment: focus on SGLT2 inhibitors in heart failure with preserved ejection fraction.

The role of sodium-glucose cotransporter 2 inhibitors (SLTG2i), developed initially as glucose-lowering agents, has represented a novelty in patients with heart failure (HF) and reduced ejection fraction (HFrEF) since dapagliflozin (DAPA-HF study) and empagliflozin (EMPEROR-Reduced study) were able to reduce morbidity and mortality in this setting regardless of the presence or absence of diabetes. In previous large clinical trials (EMPA-REG OUTCOME study, CANVAS, DECLARE-TIMI 58), SGLT2i have been shown to attenuate HF progression expressed by reducing the risk of HF hospitalizations in patients with type 2 diabetes mellitus mostly without HF at baseline. This benefit was then corroborated with positive results in HF outcomes (cardiovascular mortality and HF hospitalizations) in patients with HF with preserved ejection fraction (HFpEF) in the EMPEROR-Preserved (empagliflozin) and DELIVER (dapagliflozin) trials. Several biological mechanisms apart from the glycosuria are attributed to these agents in this last context, including anti-inflammatory effects, reduction of fibrosis and apoptosis, improvement of myocardial metabolism, mitochondrial function optimization, and oxidative stress protection. Moreover, SGLT2i can also improve ventricular loading conditions by forcing diuresis and natriuresis, and by enhancing vascular and renal function. In addition, SGLT2i can reduce myocardial passive stiffness (diastolic function) by enforcing the phosphorylation of myofilament modulatory proteins. This article provided an overview of the main pathophysiological characteristics of HFpEF and of the diverse mechanisms of action of SGLT2i in this setting. The supporting clinical evidence of SGLT2i in HFpEF (EMPEROR-Preserved and DELIVER trials) is also reviewed. This article is part of the Emerging concepts in heart failure management and treatment Special Issue: https://www.drugsincontext.com/special_issues/emerging-concepts-in-heart-failure-management-and-treatment.

High-dose intramyocardial HMGB1 induces long-term cardioprotection in sheep with myocardial infarction.

In rodents with acute myocardial infarction (AMI), high mobility group box 1 (HMGB1) injection has produced controversial results. Given the lack of data in large mammals, we searched the dose that would promote angiogenesis and expression of specific regenerative genes in sheep with AMI (protocol 1) and, subsequently, use this dose to study long-term effects on infarct size and left ventricular (LV) function (protocol 2). Protocol 1: Sheep with AMI received 250 µg (high-dose, n = 7), 25 µg (low-dose, n = 7) HMGB1, or PBS (placebo, n = 7) in 10 intramyocardial injections (0.2 ml each) in the peri-infarct area. Seven days later, only the high-HMGB1-dose group exhibited higher microvascular densities, Ki67-positive cardiomyocytes, and overexpression of VEGF, Ckit, Tbx20, Nkx2.5, and Gata4. Protocol 2: Sheep with AMI received HMGB1 250 µg (n = 6) or PBS (n = 6). At 60 days, HMGB1-treated sheep showed smaller infarcts (8.5 ± 2.11 vs. 12.2 ± 1.97% LV area, P < 0.05, ANOVA-Bonferroni) and higher microvascular density (capillaries, 1798 ± 252 vs. 1266 ± 250/mm2; arterioles, 18.3 ± 3.9 vs. 11.7 ± 2.2/mm2; both P < 0.01). Echocardiographic LV ejection fraction, circumferential shortening, and wall thickening increased from day 3 to 60 with HMGB1 (all P < 0.05). Conclusion: in ovine AMI, high-dose HMGB1 induces angio-arteriogenesis, reduces infarct size, and improves LV function at 2 months post-treatment.

Effect of poly (l-lactic acid) scaffolds seeded with aligned diaphragmatic myoblasts overexpressing connexin-43 on infarct size and ventricular function in sheep with acute coronary occlusion.

Diaphragmatic myoblasts (DM) are stem cells of the diaphragm, a muscle displaying high resistance to stress and exhaustion. We hypothesized that DM modified to overexpress connexin-43 (cx43), seeded on aligned poly (l-lactic acid) (PLLA) sheets would decrease infarct size and improve ventricular function in sheep with acute myocardial infarction (AMI). Sheep with AMI received PLLA sheets without DM (PLLA group), sheets with DM (PLLA-DM group), sheets with DM overexpressing cx43 (PLLA-DMcx43) or no treatment (control group, n = 6 per group). Infarct size (cardiac magnetic resonance) decreased ∼25% in PLLA-DMcx43 [from 8.2 ± 0.6 ml (day 2) to 6.5 ± 0.7 ml (day 45), p < .01, ANOVA-Bonferroni] but not in the other groups. Ejection fraction (EF%) (echocardiography) at 3 days post-AMI fell significantly in all groups. At 45 days, PLLA-DM y PLLA-DMcx43 recovered their EF% to pre-AMI values (PLLA-DM: 61.1 ± 0.5% vs. 58.9 ± 3.3%, p = NS; PLLA-DMcx43: 64.6 ± 2.9% vs. 56.9 ± 2.4%, p = NS), but not in control (56.8 ± 2.0% vs. 43.8 ± 1.1%, p < .01) and PLLA (65.7 ± 2.1% vs. 56.6 ± 4.8%, p < .01). Capillary density was higher (p < .05) in PLLA-DMcx43 group than in the remaining groups. In conclusion, PLLA-DMcx43 reduces infarct size in sheep with AMI. PLLA-DMcx43 and PLLA-DM improve ventricular function similarly. Given its safety and feasibility, this novel approach may prove beneficial in the clinic.

Allogeneic Mesenchymal Stromal Cells Overexpressing Mutant Human Hypoxia-Inducible Factor 1-α (HIF1-α) in an Ovine Model of Acute Myocardial Infarction.

BACKGROUND: Bone marrow mesenchymal stromal cells (BMMSCs) are cardioprotective in acute myocardial infarction (AMI) because of release of paracrine angiogenic and prosurvival factors. Hypoxia-inducible factor 1-α (HIF1-α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSCs engineered to overexpress mutant, oxygen-resistant HIF1-α would confer greater cardioprotection than nontransfected BMMSCs in sheep with AMI. METHODS AND RESULTS: Allogeneic BMMSCs transfected with a minicircle vector encoding mutant HIF1-α (BMMSC-HIF) were injected in the peri-infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance (CMR) imaging decreased by 71.7±1.3% (P<0.001), and left ventricular (LV) percent ejection fraction (%EF) increased near 2-fold (P<0.001) in the presence of markedly decreased end-systolic volume. Sheep receiving nontransfected BMMSCs (BMMSC; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo-treated animals (n=6), neither parameters changed over time. HIF1-α-transfected BMMSCs (BMMSC-HIF) induced angio-/arteriogenesis and decreased apoptosis by HIF1-mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin-1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long-term retention of BMMSC-HIF. CONCLUSIONS: Intramyocardial delivery of BMMSC-HIF reduced infarct size and improved LV systolic performance compared to BMMSC, attributed to increased neovascularization and cardioprotective effects induced by HIF1-mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSCs for allogeneic application, this treatment may be potentially useful in the clinic.

Vascular endothelial growth factor overexpression does not enhance adipose stromal cell-induced protection on muscle damage in critical limb ischemia.

OBJECTIVES: Critical limb ischemia complicates peripheral artery disease leading to tissue damage and amputation. We hypothesized that modifying adipose stromal cells (ASCs) to overexpress human vascular endothelial growth factor 165 (VEGF) would limit ischemic muscle damage to a larger extent than nonmodified ASCs. APPROACH AND RESULTS: Rabbits with critical hindlimb ischemia were injected with allogeneic abdominal fat-derived ASCs transfected with plasmid-VEGF165 (ASCs-VEGF; n=10). Additional rabbits received nontransfected ASCs (ASCs; n=10) or vehicle (placebo; n=10). One month later, ASCs-VEGF rabbits exhibited significantly higher density of angiographically visible collaterals and capillaries versus placebo (both P<0.05) but not versus ASCs (both P=NS). Arteriolar density, however, was increased in both ASCs and ASCs-VEGF groups (both P<0.05 versus placebo). ASCs-VEGF and ASCs showed comparable post-treatment improvements in Doppler-assessed peak systolic velocity, blood pressure ratio, and resistance index. Ischemic lesions were found in 40% of the muscle samples in the placebo group, 19% in the ASCs-VEGF group, and 17% in the ASCs groups (both P<0.05 versus placebo, Fisher test). CONCLUSIONS: In a rabbit model of critical limb ischemia, intramuscular injection of ASCs genetically modified to overexpress VEGF increase angiographically visible collaterals and capillary density. However, both modified and nonmodified ASCs increase arteriolar density to a similar extent and afford equal protection against ischemia-induced muscle lesions. These results indicate that modifying ASCs to overexpress VEGF does not enhance the protective effect of ASCs, and that arteriolar proliferation plays a pivotal role in limiting the irreversible tissue damage of critical limb ischemia.

Combined VEGF gene transfer and erythropoietin in ovine reperfused myocardial infarction.

BACKGROUND: In reperfused acute myocardial infarction (RAMI), cardioprotective treatments may enhance myocardial salvage and hence reduce the area of necrosis. Based on studies showing that plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer reduces infarct size by combining angio-arteriogenic and cardiomyogenic effects and that erythropoietin (EPO) exerts anti-apoptotic actions in animal models of AMI, we aimed to assess if their association would reduce infarct size to a larger extent than any of them individually in a large mammalian model of RAMI. METHODS: Adult sheep subjected to 90-minute coronary artery occlusion received upon reperfusion intramyocardial pVEGF 3.8 mg plus intravenous EPO 1000 IU/kg (n=8), pVEGF (n=8), EPO (n=8) or placebo (n=8). RESULTS: Fifteen days after treatment, infarct size was smaller in the 3 treatment groups (pVEGF+EPO: 8 ± 1 %; pVEGF: 16 ± 5 %; EPO: 13 ± 4 %) compared to placebo (25 ± 7 %, p<0.001). However, in the EPO+VEGF group infarct size was significantly smaller than in the groups receiving EPO or VEGF individually (p<0.05). DNA fragmentation, a hallmark of late apoptosis, was significantly lower in sheep receiving EPO. The combined treatment, while not affecting global left ventricular performance, improved regional peri-infarct function and prevented over-time expansion of the post-infarct perfusion defect. CONCLUSIONS: Combined pVEGF and EPO treatment might be clinically useful to enhance the benefits of early revascularization in patients with acute myocardial infarction.

Effect of vascular endothelial growth factor gene transfer on infarct size, left ventricular function and myocardial perfusion in sheep after 2 months of coronary artery occlusion.

BACKGROUND: In large mammalian models of acute myocardial infarction (AMI), plasmid-mediated vascular endothelial growth factor (pVEGF) gene transfer has been shown to induce angio-arteriogenesis, proliferation of myocyte precursors and adult cardiomyocyte mitosis, reducing infarct size at 15 days after coronary artery occlusion. However, it is unknown whether these effects persist at longer follow-up times, nor how they affect cardiac performance. We thus assessed infarct size, left ventricular (LV) function and perfusion in 2-month-old ovine AMI. METHODS: Adult sheep with coronary artery occlusion were randomized to blindly receive ten intramyocardial injections of 3.8 mg of pVEGF or empty plasmid distributed at the infarct border. Three and 60 days later, LV perfusion (single-photon emission computed tomography) and function (stress echocardiography) were assessed. Finally, hemodynamics (LV catheterization), scar size and peri-infarct histology were studied. RESULTS: Infarct size was 30% smaller in pVEGF-treated sheep (23.6 ± 1.9% versus 32.7 ± 2.7% of the LV; p < 0.02). Percentage fractional shortening and wall thickening at the infarct border improved after pVEGF, as did myocardial perfusion and LV wall motion under pharmacological stress. Global LV function did not differ between groups, although the force-frequency response was preserved in pVEGF group and lost in placebo animals. These effects were associated with angio-arteriogenesis and proliferation of cardiomyocyte precursors. CONCLUSIONS: In sheep with AMI, pVEGF gene transfer affords long-term infarct size reduction, yielding regional LV function and perfusion improvement and reducing remodeling progression. These results suggest the potential usefulness of this approach in the clinical setting.

Reference values for echocardiographic parameters and indexes of left ventricular function in healthy, young adult sheep used in translational research: comparison with standardized values in humans.

Ovine models of ischemic heart disease and cardiac failure are increasingly used in translational research. However, reliable extrapolation of the results to the clinical setting requires knowing if ovine normal left ventricular (LV) function is comparable to that of humans. We thus assessed for echocardiographic LV dimensions and indexes in a large normal adult sheep population and compared them with standardized values in normal human adults. Bidimensional and tissue Doppler echocardiograms were performed in 69 young adult Corriedale sheep under light sedation. LV dimensions and indexes of systolic and diastolic function were measured. Absolute and body surface areanormalized values were compared to those for normal adult humans and their statistical distribution was assessed. Normalized dimensions (except for end diastolic diameter) as well as ejection fraction and fractional shortening fell within the ranges established by the American Society of Echocardiography and European Association of Echocardiography for normal adult humans. Normalized end diastolic diameter exceeded the upper normal limit but got close to it when correcting for the higher heart mass/body surface area ratio of sheep with respect to humans. Diastolic parameters also fell within normal human ranges except for a slightly lower mitral deceleration time. All values exhibited a Gaussian distribution. We conclude that echocardiographic parameters of systolic and diastolic LV performance in young adult sheep can be reliably extrapolated to the adult human, thus supporting the use of ovine models of human heart disease in translational research.

Long-term effects of growth hormone on infarct size and left ventricular function in sheep with coronary artery occlusion.

The effects of growth hormone (GH) on infarct size and left ventricular (LV) function in experimental acute myocardial infarction (AMI) have been controversial. Moreover, little, if any, information exists regarding long-term evaluation of therapeutic doses of GH in large mammalian models of AMI. We therefore aimed to assess the effect of therapeutic doses of GH over 3.5 months on infarct size and heart function in sheep with AMI. After coronary artery ligation, sheep received subcutaneous human GH 8 IU/d (n = 8) or vehicle (n = 8) over 100 days. Infarct area was similar in GH (16.9% +/- 3% of LV area) and placebo (16.5% +/- 3.7%, P = not significant) sheep. At 3 days of treatment onset, but not at later times, GH sheep had higher LV shortening fraction (30.7% +/- 3.5% vs. 24.8% +/- 6.1%, P < 0.04), systolic anterior wall thickness (10.1 +/- 0.8 vs. 8.6 +/- 1.2 mm, P < 0.02), and cardiac index (3.8 +/- 0.6 vs. 2.8 +/- 0.7 L x min x m, P < 0.01). This evolution of function parameters paralleled that of serum insulin-like growth factor 1 levels, which differed significantly only during the first week, suggesting a direct effect of GH on LV contractility. These results may suggest the usefulness of therapeutic doses of GH at the early phases of AMI but do not support maintaining the treatment for longer time.

High-dose erythropoietin has no long-term protective effects in sheep with reperfused myocardial infarction.

High-dose erythropoietin has been claimed to be cardioprotective in experimental acute myocardial infarction. In large mammals, however, results are controversial and long-term follow-up data are lacking. We thus assessed the long-term effects of high-dose erythropoietin on left ventricular infarct size and function in an ovine model of reperfused myocardial infarction. After 90 minutes of coronary occlusion followed by reperfusion, sheep received recombinant human erythropoietin (rhEPO) 3000 units/kg on 3 consecutive days (rhEPO group, n=7) or vehicle (placebo group, n=6). Ten weeks later, ventricular function was assessed by echocardiography and catheterization. Infarct size, evaluated as percent fibrotic myocardium (morphometry) and by hydroxyproline quantification, was similar in both groups (morphometry: rhEPO: 22.1 +/- 5.5%, placebo: 18.1 +/- 3.3%, P not significant; hydroxyproline: rhEPO: 6.6 +/- 1.3 microg/mg wet weight, placebo: 7.1 +/- 0.9 microg/mg, P not significant). Ventricular function was diminished in the rhEPO group, as indicated by lower septal wall thickening at the infarct border zone (rhEPO: -1.9 +/- 16.4%, placebo: 20.5 +/- 17%, P<0.04), higher end systolic volume (rhEPO: 47 +/- 14.3 mL, placebo: 32.6 +/- 7.3 mL, P<0.05), and higher end diastolic pressure (rhEPO: 17 +/- 6.5 mm Hg, placebo: 10.1 +/- 2.8 mm Hg, P<0.03). In the rhEPO group, left ventricular endocardial area was larger, suggesting dilatation. High-dose erythropoietin has no cardioprotective effects in sheep with reperfused myocardial infarction.

Cardiomyocyte hyperplasia after plasmid-mediated vascular endothelial growth factor gene transfer in pigs with chronic myocardial ischemia.

BACKGROUND: For over 40 years it has been proposed that cardiomyocyte hyperplasia may occur in hypertrophic human hearts. While this implies that heart myocytes can undergo cytokinesis, evidence of conventional cell division has been exceptionally reported. Recently, we found that gene transfer of vascular endothelial growth factor (VEGF) displays a mitogenic effect on adult cardiomyocytes. In the present study we searched for cardiomyocyte hyperplasia as evidence of VEGF-induced cardiomyocyte cytokinesis. METHODS: Three weeks after implanting an Ameroid constrictor at the origin of the left circumflex artery, 16 pigs were randomized to receive 10 direct intramyocardial injections of 3.8 mg of plasmid encoding for VEGF (pVEGF) or empty plasmid. Five weeks later, hearts were weighed, myocyte diameter was measured in tissue sections, and myocyte length and nuclei number were studied in isolated myocytes. A resting echocardiogram was performed immediately before reoperation and before sacrifice to evaluate global and regional left ventricular function. Investigators were blinded to the study groups and nature of the injectate until the end of data analysis. RESULTS: No heart weight differences existed between groups. However, in the ischemic myocardium, pVEGF-treated hearts had 22% more cardiomyocytes per unit volume and exhibited significantly more oligonucleated (1 or 2 nuclei) cardiomyocytes than hearts receiving empty plasmid. CONCLUSIONS: In pigs with chronic myocardial ischemia, VEGF gene transfer induced cardiomyocyte cytokinesis, as revealed by cardiomyocyte hyperplasia. Our finding extends the previously reported mitogenic effect of VEGF on adult cardiomyocytes and supports the hypothesis that VEGF may have a therapeutic role in diseases characterized by myocardial cell loss.

Arteriogenesis induced by intramyocardial vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs.

Exogenous vascular endothelial growth factor (VEGF) improves tissue perfusion in large animals and humans with chronic myocardial ischemia. Because tissue perfusion is mainly dependent on the arteriolar tree, we hypothesized that the neovascularizing effect of VEGF should include arteriogenesis, an effect not as yet described in large mammalian models of myocardial ischemia. In the present study we investigated the effect of intramyocardial plasmid-mediated human VEGF(165) gene transfer (pVEGF(165)) on the proliferation of vessels with smooth muscle in a pig model of myocardial ischemia. In addition, we assessed the effect of treatment on capillary growth, myocardial perfusion, myocardial function and collateralization. Three weeks after positioning of an Ameroid constrictor (Research Instruments SW, Escondido, CA) in the left circumflex artery, pigs underwent basal perfusion (single-photon emission computed tomography [SPECT] with (99m)Tc-sestamibi) and regional function (echocardiography) studies at rest and under dobutamine stress, and were then randomly assigned to receive transepicardial injection of pVEGF(165) 3.8 mg (n = 8) or placebo (empty plasmid, n = 8). All experimental steps and data analysis were done in a blinded fashion. Five weeks later, pVEGF(165)-treated pigs showed a significantly higher density of small (8-50 microm in diameter) vessels with smooth muscle, higher density of capillaries, and improved myocardial perfusion. These results indicate an arteriogenic effect of VEGF in a large mammalian model of myocardial ischemia and encourage the use of VEGF to promote arteriolar growth in patients with severe coronary artery disease.

Inducción de arteriogénesis mediante la transferencia génica de factor de crecimiento de endotelio vascular de 165 aminoácidos en cerdos crónicamente isquémicos / Arteriogenesis induced by vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs

El factor de crecimiento de endotelio vascular (VEGF), un angiógeno considerado específico de los endoteliocitos, mejora la perfusión miocárdica en animales y en seres humanos con cardiopatía isquémica. Dado que la perfusión tisular depende principalmente de la irrigación arterial y de que los receptores para VEGF se encontraron recientemente en células musculares lisas, la administración de VEGF debería promover también la arteriogénesis. Nuestro objetivo fue el de estudiar el probable efecto arteriogénico de la administración de un nuevo plásmido codificante para VEGF recombinante humano (pCMVrhVEGF165), desarrollado y producido en la Argentina, en cerdos con isquemia miocárdica crónica. Tres semanas después de la colocación de un oclusor Ameroid en la arteria circunfleja, 16 cerdos fueron sometidos a estudios de función miocárdica (ecocardiografía estrés con dobutamina) y distribuídos al azar en un grupo tratado (n = 8) que recibió 10 inyecciones intramiocárdicas de pCMVrhVEGF165 (3,8 mg) y un grupo placebo (n = 8) que recibió el plásmido desprovisto del gen. A las 5 semanas se repitió la ecocardiografía, se realizó una cinecoronariografía y se extrajeron el corazón y los tejidos remotos para su análisis histopatológico. La clave se ocultó hasta el fin del análisis estadístico. El grupo tratado presentó, con respecto al placebo, mayor densidad de longitud (2,4 ñ 0,4 versus 1,3 ñ 0,3 mm/mm³; p< 0,02) y numérica (1 ñ 0,1 versus 0,6 ñ 0,1 mm², p <0,02) de vasos pequeños (<50 Am) provistos de túnica muscular lisa evidenciable mediante inmunohistoquímica. No se halló proliferación vascular indeseada en tejidos remotos. Concluímos que en cerdos crónicamente isquémicos la inyección intramiocárdica directa de pCMVrhVEGF165 es segura e induce arteriogénesis en el miocardio isquémico (AU)

Inducción de arteriogénesis mediante la transferencia génica de factor de crecimiento de endotelio vascular de 165 aminoácidos en cerdos crónicamente isquémicos / Arteriogenesis induced by vascular endothelial growth factor 165 gene transfer in chronically ischemic pigs

El factor de crecimiento de endotelio vascular (VEGF), un angiógeno considerado específico de los endoteliocitos, mejora la perfusión miocárdica en animales y en seres humanos con cardiopatía isquémica. Dado que la perfusión tisular depende principalmente de la irrigación arterial y de que los receptores para VEGF se encontraron recientemente en células musculares lisas, la administración de VEGF debería promover también la arteriogénesis. Nuestro objetivo fue el de estudiar el probable efecto arteriogénico de la administración de un nuevo plásmido codificante para VEGF recombinante humano (pCMVrhVEGF165), desarrollado y producido en la Argentina, en cerdos con isquemia miocárdica crónica. Tres semanas después de la colocación de un oclusor Ameroid en la arteria circunfleja, 16 cerdos fueron sometidos a estudios de función miocárdica (ecocardiografía estrés con dobutamina) y distribuídos al azar en un grupo tratado (n = 8) que recibió 10 inyecciones intramiocárdicas de pCMVrhVEGF165 (3,8 mg) y un grupo placebo (n = 8) que recibió el plásmido desprovisto del gen. A las 5 semanas se repitió la ecocardiografía, se realizó una cinecoronariografía y se extrajeron el corazón y los tejidos remotos para su análisis histopatológico. La clave se ocultó hasta el fin del análisis estadístico. El grupo tratado presentó, con respecto al placebo, mayor densidad de longitud (2,4 ñ 0,4 versus 1,3 ñ 0,3 mm/mmü; p< 0,02) y numérica (1 ñ 0,1 versus 0,6 ñ 0,1 mm², p <0,02) de vasos pequeños (<50 µm) provistos de túnica muscular lisa evidenciable mediante inmunohistoquímica. No se halló proliferación vascular indeseada en tejidos remotos. Concluímos que en cerdos crónicamente isquémicos la inyección intramiocárdica directa de pCMVrhVEGF165 es segura e induce arteriogénesis en el miocardio isquémico