Patricia Cabeza Meckert

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.

Cardiomyocyte hypertrophy, oncosis, and autophagic vacuolization predict mortality in idiopathic dilated cardiomyopathy with advanced heart failure.

OBJECTIVES The aim of this study was to identify the remodeling parameters cardiomyocyte (CM) damage or death, hypertrophy, and fibrosis that may be linked to outcomes in patients with advanced heart failure (HF) in an effort to understand the pathogenic mechanisms of HF that may support newer therapeutic modalities. BACKGROUND There are controversial results on the influence of fibrosis, CM hypertrophy, and apoptosis on outcomes in patients with HF; other modalities of cell damage have been poorly investigated. METHODS In endomyocardial biopsy specimens from 100 patients with idiopathic dilated cardiomyopathy and advanced HF, CM diameter and the extent of fibrosis were determined by morphometry. The proportion of CMs with evidence of apoptosis, autophagic vacuolization (AuV), and oncosis was investigated by immunohistochemical methods and by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling. Those parameters were correlated with mortality in 3 years of follow-up by univariate analysis and with multivariate models incorporating the clinical variables more relevant to the prediction of outcomes. RESULTS CM AuV occurred in 28 patients (0.013 ± 0.012%) and oncosis in 41 (0.109 ± 0.139%). Nineteen patients showed both markers. Apoptotic CM nuclei were observed in 3 patients. In univariate analysis, CM diameter and AuV, either alone or associated with oncosis, were predictors of mortality. In multivariate analysis, CM diameter (hazard ratio: 1.37; 95% confidence interval: 1.12 to 1.68; p = 0.002) and simultaneous presence in the same endomyocardial biopsy specimen of AuV and oncosis (hazard ratio: 2.82; 95% confidence interval: 1.12 to 7.13; p = 0.028) were independent predictors of mortality. CONCLUSIONS CM hypertrophy and AuV, especially in association with oncosis, are predictors of outcome in patients with idiopathic dilated cardiomyopathy and severe HF.

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.

Inhibitory Receptors Are Expressed by Trypanosoma cruzi-Specific Effector T Cells and in Hearts of Subjects with Chronic Chagas Disease

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4+ T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4+CTAL-4+ T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4+LIR-1+ among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3+ T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease.

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.

Presence of vulnerable coronary plaques in middle-aged individuals who suffered a brain death

AIMS Vulnerable plaques in coronary arteries are frequently found in individuals who died suddenly or due to an acute coronary syndrome. The prevalence and characteristics of these plaques in the middle-aged apparently healthy population are unknown. METHODS AND RESULTS From a total of 652 hearts from transplant donors collected between 1996 and 2007, we selected those from apparently healthy individuals older than 40 years old who died of head trauma or stroke and had no evidence of prior vascular diseases. The coronary arteries were examined by serial sectioning at 3 mm intervals, and all areas of cross-sectional luminal narrowing were processed for histological, immunohistochemical, and morphometric studies. The atherosclerotic plaques were classified according to the American Heart Association Report. A total of 160 hearts were examined. Mean age was 50.3 +/- 5.8 years. Sixty-eight hearts had no advanced coronary atherosclerotic lesions (Type I, II, and III of the American Heart classification). In the remaining 92 hearts, we found 179 plaques considered high-risk lesions (American Heart Association Type IV, V, and VI). These plaques were more frequently found in males (P < 0.001) and in those with a higher heart weight (P < 0.001). The median (25th and 75th percentiles) vascular narrowing value using a planimetric analysis was 32% (21-53). No significant association with the cause of death was found (P = 0.09). CONCLUSION High-risk coronary artery plaques not associated with significant vascular lumen reduction exist in 57% of patients who suffered a brain death with a mean of 1.11 lesions prone to rupture per individual.

Coronary Microcirculation Remodeling in Patients with Idiopathic Dilated Cardiomyopathy

Background: In patients with idiopathic dilated cardiomyopathy (IDCM), the myocardial blood flow is markedly depressed. The presence of alterations in the number and length of the coronary microcirculation has not been explored. Methods: In explanted hearts of 6 patients with IDCM and in 6 normal control hearts, the arteriolar length density and capillary numerical density were determined in sections stained with orcein (vessels 50–200 µm in diameter), for smooth muscle actin (vessels 6–50 µm in diameter) and CD34 (capillaries). Results: No difference with normal hearts was observed in capillary numerical density and in length density of vessels above 50 µm in diameter. In IDCM, more than 50% of arterioles below 50 µm in diameter displayed an incomplete smooth muscle wall, and length density, especially for arterioles between 6 and 20 µm in diameter, was significantly lower (42.84 ± 8.51 mm/mm3) than in controls (75.34 ± 3.05 mm/mm3, p = 0.001). Conclusions: In IDCM, arterioles below 50 µm in diameter display an incomplete smooth muscle wall and a significant decrease in length density. These observations provide an anatomical basis for a decreased coronary flow reserve in IDCM.

Effect of acriflavin on the fine structure of the heart muscle cell mitochondria of normal and exercised rats

Abstract The administration of neutral acriflavin to rats provoked the appearance of numerous myocardial mitochondria containing DNA fibres. The highest incidence of DNA-containing mitochondria was found at 4 and 6 h after drug injection. The association of acriflavin administration and acute exercise induced the appearance of a higher number of DNA-containing mitochondria as compared with the non-exercised controls, and prevented the increase in the volume fraction of mitochondria that occurs after exercise. These findings suggest that the appearance of intramitochondrial DNA fibres after acriflavin administration is the result of an inhibitory effect of acriflavin on the replication of mitochondrial DNA. On the basis of this interpretation it was suggested that acriflavin could be used as an “antimitotic drug” for mitochondria, and that both “mitotic index” and “mitotic time” could be calculated for the mitochondrial population in the same way as they are determined for whole cells after mitotic arrest with spindle poisons.

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·min−1·m−2, 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.