Luis Cuniberti

Plasmid-mediated VEGF gene transfer induces cardiomyogenesis and reduces myocardial infarct size in sheep

We have recently reported that in pigs with chronic myocardial ischemia heart transfection with a plasmid encoding the 165 isoform of human vascular endothelial growth factor (pVEGF165) induces an increase in the mitotic index of adult cardiomyocytes and cardiomyocyte hyperplasia. On these bases we hypothesized that VEGF gene transfer could also modify the evolution of experimental myocardial infarct. In adult sheep pVEGF165 (3.8 mg, n=7) or empty plasmid (n=7) was injected intramyocardially 1 h after coronary artery ligation. After 15 days infarct area was 11.3±1.3% of the left ventricle in the VEGF group and 18.2±2.1% in the empty plasmid group (P<0.02). The mechanisms involved in infarct size reduction (assessed in additional sheep at 7 and 10 days after infarction) included an increase in early angiogenesis and arteriogenesis, a decrease in peri-infarct fibrosis, a decrease in myofibroblast proliferation, enhanced cardiomyoblast proliferation and mitosis of adult cardiomyocytes with occasional cytokinesis. Resting myocardial perfusion (99mTc-sestamibi SPECT) was higher in VEGF-treated group than in empty plasmid group 15 days after myocardial infarction. We conclude that plasmid-mediated VEGF gene transfer reduces myocardial infarct size by a combination of effects including neovascular proliferation, modification of fibrosis and cardiomyocyte regeneration.

Entrance in mitosis of adult cardiomyocytes in ischemic pig hearts after plasmid-mediated rhVEGF165 gene transfer.

Replacement of the cell loss occurring after acute myocardial infarction has been proposed as a potential treatment to prevent heart remodeling and failure. On account that cardiomyocytes express VEGF receptors and that VEGF triggers mitogen-activated protein kinases, we investigated if VEGF gene transfer may induce cardiomyocyte replication. In a pig model of chronic myocardial ischemia achieved by Ameroid occlusion of the left circumflex coronary artery, we observed that direct intramyocardial injection of a plasmid encoding human VEGF165 induced a several-fold increase in cardiomyocyte mitotic index and in the number of cardiomyocyte nuclei per unit volume as compared with pigs receiving plasmid devoid of gene. Despite images of conventional cytokinesis were not observed, the fact that caryokinesis is an obligatory step for cell division suggests that our finding may contribute to the issue of heart regeneration and may potentially widen the therapeutic spectrum of VEGF gene transfer.

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.

Hyperhomocysteinemia Induces Renal Hemodynamic Dysfunction: Is Nitric Oxide Involved?

Hyperhomocysteinemia is associated with endothelial dysfunction, although the underlying mechanism is unknown. Previous studies have shown that nitric oxide (NO) plays an important role in the regulation of systemic and renal hemodynamics. This study investigated whether hyperhomocysteinemia induces renal oxidative stress and promotes renal dysfunction involving disturbances of the NO-pathway in Wistar rats. During 8 wk, control (C) and hyperhomocysteinemic (HYC) groups had free access to tap water and homocysteine-thiolactone (HTL, 50 mg/kg per d), respectively. At 8 wk, plasma homocysteine concentration, renal superoxide anion (O(2)), nitrotyrosine, and nitrite+nitrate levels, and renal function were measured. To assess NO involvement, the responses to L-Arginine (L-Arg, 300 mg/kg) and N(G)-nitro-L-arginine-methyl-ester (L-NAME, 20 microg/kg per min for 60 min) were analyzed. The HYC group showed higher homocysteine concentration (7.6 +/- 1.7 versus 4.9 +/- 1.0 micromol/L; P < 0.001), (O(2) production (157.92 +/- 74.46 versus 91.17 +/- 29.03 cpm. 10(3)/mg protein), and nitrite+nitrate levels (33.4 +/- 5.1 versus 11.7 +/- 4.3 micro mol/mg protein; P < 0.001) than the control group. Western blot analyses showed a nitrotyrosine mass 46% higher in the HYC group than in the controls. Furthermore, the HYC group showed lower GFR, renal plasma flow (RPF), and higher renal vascular resistance (RVR) than the controls. After L-Arg administration, the responses of GFR, RPF, and RVR were attenuated by 36%, 40%, and 50%, respectively; after L-NAME, the responses of RPF and RVR were exaggerated by 79% and 112%, respectively. This suggests a reduced NO bioavailability to produce vasodilation and an enhanced sensitivity to NO inhibition. In conclusion, hyperhomocysteinemia induces oxidative stress, NO inactivation, and renal dysfunction involving disturbances on the NO-pathway.

Determination of bezafibrate, ciprofibrate and fenofibric acid in human plasma by high-performance liquid chromatography

A selective high-performance liquid chromatographic method to assess either bezafibrate, ciprofibrate or fenofibric acid plasma levels is described. Drugs are extracted with diethyl ether, after acidification with HCL. An isocratic acetonitrile 0.02 M H3PO4 (55:45) mobile phase, a C18 microns) column and UV detection are used. The LOQ found was 0.25 microgram/ml for the three fibrates. Intra- and inter-assay accuracy ranges were 90-107% and 82-111%: 96-115% and 94-107%: 94-114% and 94-126% for bezafibrate, ciprofibrate and fenofibric acid, respectively. Intra- and inter-assay precision (C.V.% ranges) were 1.72-3.06% and 2.66-7.67%: 1.88-4.64% and 0.62-2.99%: 1.26-4.69% and 3.56-7.17% for the three fibrates studied. Its sensitivity, accuracy and precision make it a useful tool for monitoring plasma levels of these drugs in a clinical setting and for research purposes.

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

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.

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 μg/mg wet weight, placebo: 7.1±0.9 μg/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.

Brief Report: The Potential Role of Epigenetics on Multipotent Cell Differentiation Capacity of Mesenchymal Stromal Cells†‡§

Human umbilical cord perivascular cells (HUCPVCs) are a readily available source of mesenchymal stromal cells (MSCs) for cell therapy. We were interested in understanding how differences from human bone marrow (BM)‐derived MSCs might yield insights into MSC biology. We found that HUCPVCs exhibited increased telomerase activity and longer telomeres compared with BM‐MSCs. We also observed enhanced expression of the pluripotency factors OCT4, SOX2, and NANOG in HUCPVCs. The methylation of OCT4 and NANOG promoters was similar in both cell types, indicating that differences in the expression of pluripotency factors between the MSCs were not associated with epigenetic changes. MSC methylation at these loci is greater than reported for embryonic stem cells but less than in dermal fibroblasts, suggesting that multipotentiality of MSCs is epigenetically restricted. These results are consistent with the notion that the MSC population (whether BM‐ or HUCPV‐derived) exhibits higher proliferative capacity and contains more progenitor cells than do dermal fibroblasts. STEM Cells2013;31:215–220

Cardiomyocytes of Chronically Ischemic Pig Hearts Express the MDR-1 Gene-encoded P-glycoprotein

The multidrug-resistant (MDR)-1 gene-encoded P-glycoprotein (Pgp-170) is not normally present in the cardiomyocyte. Given that in other tissues Pgp-170 is not found under normoxic conditions but is expressed during hypoxia, we searched for Pgp-170 in chronically ischemic porcine cardiomyocytes. Pgp-170 was detected and localized via immunohistochemistry in ischemic and nonischemic cardiomyocytes of eight adult pigs 8 weeks after placement of an Ameroid constrictor at the origin of the left circumflex artery (Cx). Regional myocardial ischemia in the Cx bed was documented with nuclear perfusion scans. Pgp-170 mass was quantified using Western blot analysis. In all pigs, Pgp-170 was consistently present in the sarcolemma and T invaginations of the cardiomyocytes of the ischemic zone. Pgp-170 expression decreased toward the border of the ischemic zone and was negative in nonischemic regions as well as in the myocardium of sham-operated animals. Western blot analysis yielded significantly higher Pgp-170 mass in ischemic than in nonischemic areas. We conclude that Pgp-170 is consistently expressed in the cardiomyocytes of chronically ischemic porcine myocardium. Its role in the ischemic heart as well as in conditions such as myocardial hibernation, stunning, and preconditioning may have potentially relevant clinical implications and merits further investigation.

Repeated, but not single, VEGF gene transfer affords protection against ischemic muscle lesions in rabbits with hindlimb ischemia.

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.