Activated macrophages as a feeder layer for growth of resident cardiac progenitor cells.

The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72 h after intraperitoneal injection of 12 ml thioglycollate. Cells (1 × 10(6)) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10 min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21 days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.

Presence of antigen-experienced T cells with low grade of differentiation and proliferative potential in chronic Chagas disease myocarditis.

BACKGROUND: The main consequence of chronic Trypanosoma cruzi infection is the development of myocarditis in approximately 20-30% of infected individuals but not until 10-20 years after the initial infection. We have previously shown that circulating interferon-γ-secreting T cells responsive to Trypanosoma cruzi antigens in chronic Chagas disease patients display a low grade of differentiation and the frequency of these T lymphocytes decreases along with the severity of heart disease. This study thought to explore the expression of inhibitory receptors, transcription factors of type 1 or regulatory T cells, and markers of T cell differentiation, immunosenescence or active cell cycle in cardiac explants from patients with advanced Chagas disease myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: The expression of different markers for T and B cells as well as for macrophages was evaluated by immunohistochemistry and immunofluorescence techniques in cardiac explants from patients with advanced chronic Chagas disease submitted to heart transplantation. Most infiltrating cells displayed markers of antigen-experienced T cells (CD3(+), CD4(+), CD8(+), CD45RO(+)) with a low grade of differentiation (CD27(+), CD57(-), CD45RA(-), PD(-)1(-)). A skewed T helper1/T cytotoxic 1 profile was supported by the expression of T-bet; whereas FOXP3(+) cells were scarce and located only in areas of severe myocarditis. In addition, a significant proliferative capacity of CD3(+) T cells, assessed by Ki67 staining, was found. CONCLUSIONS/SIGNIFICANCE: The quality of T cell responses and immunoregulatory mechanisms might determine the pattern of the cellular response and the severity of disease in chronic Trypanosoma cruzi infection.

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.

Presencia de angiogénesis en placas vulnerables ateroscleróticas en corazones humanos aparentemente sanos / Angiogenesis in Vulnerable Atherosclerotic Plaques in Apparently Healthy Human Hearts / Angiogenesis in Vulnerable Atherosclerotic Plaques in Apparently Healthy Human Hearts

Introducción La angiogénesis o neovascularización involucra la formación de nuevos conductos en las adyacencias de vasos preexistentes. Esta proliferación vascular es frecuente en varias circunstancias clínicas, como es el caso de la aterosclerosis. Los microvasos de las placas ateroscleróticas coronarias pueden estar vinculados a la inestabilidad de la lesión. Objetivo Correlacionar la presencia de angiogénesis en placas ateroscleróticas con los criterios de vulnerabilidad de la clasificación de la American Heart Association (AHA). Material y métodos En 121 corazones de donantes no diabéticos aparentemente sanos y mayores de 40 años destinados para homoinjertos se examinaron las arterias coronarias y todas las áreas de estrechamiento luminal se sometieron a estudios histológicos, inmunohistoquímicos y morfométricos. Para el análisis de la angiogénesis se empleó un puntaje semicuantitativo (escala 0-3). Se realizó un análisis de regresión logística univariado y multivariado para identificar factores de riesgo relacionados con la angiogénesis. Resultados Se hallaron 143 lesiones de riesgo alto (AHA tipos IV, V y VI) en las arterias descendente anterior (46,3%), circunfleja (28,9%) y coronaria derecha (43%). La angiogénesis se asoció en forma estadísticamente significativa con el grado de oclusión vascular, la infiltración de células inflamatorias, la presencia de centro lipídico, la fibrosis, la periarteritis y, sólo en la descendente anterior, con el antecedente de hipertensión arterial (p < 0,006). Se detectó angiogénesis en 1 placa tipo II, en 5 tipo III, en 21 tipo IV, en 22 tipo V y en 7 placas tipo VI (AHA). Conclusiones La angiogénesis de placas vulnerables se asoció con el grado de oclusión vascular, la infiltración de células inflamatorias, la fibrosis, la presencia de núcleo lipídico y, sólo en la descendente anterior, con el antecedente de hipertensión arterial. No se encontró asociación con la hemorragia intraplaca o la calcificación, lo cual sugiere que la angiogénesis puede anticipar la rotura de las placas.

Background Angiogenesis or neovascularization involves the formation of new blood vessels adjacent to preexisting vessels. This vascular proliferation is prevalent in various clinical conditions, such as atherosclerosis. Microvessels in coronary artery atherosclerotic plaques may contribute to plaque instability. Objectives The aim of this study was to correlate the presence of angiogenesis in atherosclerotic plaques with the criteria of plaque vulnerability used by the American Heart Association (AHA). Methods One hundred and twenty one hearts from non-diabetic and apparently healthy transplant donors older than 40 years were selected. The coronary arteries were examined and all areas of cross-sectional luminal narrowing underwent histological, immunohistochemical and morphometric studies. A semi-quantitative score (scale 0-3) was used to identify of angiogenesis. Univariate and multivariate logistic regression analysis was performed to identify angiogenesisrelated risk factors. Results On hundred and forty three high-risk lesions (AHA type IV, V and VI) in the left anterior descending coronary artery (46.3%), the circumflex coronary artery (28.9%) and the right coronary artery (43%) were identified. Angiogenesis had a statistically significant association with the severity of vascular occlusion, inflammatory cell infiltration, presence of a lipid core, fibrosis and periarteritis. A history of hypertension (HT) was associated with angiogenesis only in lesions of the left anterior descending coronary artery (LAD). According to the AHA classification angiogenesis was detected in 1 Type II, 5 Type III, 21 Type IV, 22 Type V, and 7 Type VI plaques. Conclusions Angiogenesis in vulnerable plaques was associated with the severity of vascular occlusion, inflammatory cell infiltration, fibrosis and presence of a lipid core, and with a history of HT in LAD lesions. There was no association between angiogenesis and plaque hemorrhage or calcification, suggesting that angiogenesis may anticipate plaque rupture.

Presencia de angiogénesis en placas vulnerables ateroscleróticas en corazones humanos aparentemente sanos / Angiogenesis in Vulnerable Atherosclerotic Plaques in Apparently Healthy Human Hearts / Angiogenesis in Vulnerable Atherosclerotic Plaques in Apparently Healthy Human Hearts

Introducción La angiogénesis o neovascularización involucra la formación de nuevos conductos en las adyacencias de vasos preexistentes. Esta proliferación vascular es frecuente en varias circunstancias clínicas, como es el caso de la aterosclerosis. Los microvasos de las placas ateroscleróticas coronarias pueden estar vinculados a la inestabilidad de la lesión. Objetivo Correlacionar la presencia de angiogénesis en placas ateroscleróticas con los criterios de vulnerabilidad de la clasificación de la American Heart Association (AHA). Material y métodos En 121 corazones de donantes no diabéticos aparentemente sanos y mayores de 40 años destinados para homoinjertos se examinaron las arterias coronarias y todas las áreas de estrechamiento luminal se sometieron a estudios histológicos, inmunohistoquímicos y morfométricos. Para el análisis de la angiogénesis se empleó un puntaje semicuantitativo (escala 0-3). Se realizó un análisis de regresión logística univariado y multivariado para identificar factores de riesgo relacionados con la angiogénesis. Resultados Se hallaron 143 lesiones de riesgo alto (AHA tipos IV, V y VI) en las arterias descendente anterior (46,3%), circunfleja (28,9%) y coronaria derecha (43%). La angiogénesis se asoció en forma estadísticamente significativa con el grado de oclusión vascular, la infiltración de células inflamatorias, la presencia de centro lipídico, la fibrosis, la periarteritis y, sólo en la descendente anterior, con el antecedente de hipertensión arterial (p < 0,006). Se detectó angiogénesis en 1 placa tipo II, en 5 tipo III, en 21 tipo IV, en 22 tipo V y en 7 placas tipo VI (AHA). Conclusiones La angiogénesis de placas vulnerables se asoció con el grado de oclusión vascular, la infiltración de células inflamatorias, la fibrosis, la presencia de núcleo lipídico y, sólo en la descendente anterior, con el antecedente de hipertensión arterial. No se encontró asociación con la hemorragia intraplaca o la calcificación, lo cual sugiere que la angiogénesis puede anticipar la rotura de las placas.(AU)

Background Angiogenesis or neovascularization involves the formation of new blood vessels adjacent to preexisting vessels. This vascular proliferation is prevalent in various clinical conditions, such as atherosclerosis. Microvessels in coronary artery atherosclerotic plaques may contribute to plaque instability. Objectives The aim of this study was to correlate the presence of angiogenesis in atherosclerotic plaques with the criteria of plaque vulnerability used by the American Heart Association (AHA). Methods One hundred and twenty one hearts from non-diabetic and apparently healthy transplant donors older than 40 years were selected. The coronary arteries were examined and all areas of cross-sectional luminal narrowing underwent histological, immunohistochemical and morphometric studies. A semi-quantitative score (scale 0-3) was used to identify of angiogenesis. Univariate and multivariate logistic regression analysis was performed to identify angiogenesisrelated risk factors. Results On hundred and forty three high-risk lesions (AHA type IV, V and VI) in the left anterior descending coronary artery (46.3%), the circumflex coronary artery (28.9%) and the right coronary artery (43%) were identified. Angiogenesis had a statistically significant association with the severity of vascular occlusion, inflammatory cell infiltration, presence of a lipid core, fibrosis and periarteritis. A history of hypertension (HT) was associated with angiogenesis only in lesions of the left anterior descending coronary artery (LAD). According to the AHA classification angiogenesis was detected in 1 Type II, 5 Type III, 21 Type IV, 22 Type V, and 7 Type VI plaques. Conclusions Angiogenesis in vulnerable plaques was associated with the severity of vascular occlusion, inflammatory cell infiltration, fibrosis and presence of a lipid core, and with a history of HT in LAD lesions. There was no association between angiogenesis and plaque hemorrhage or calcification, suggesting that angiogenesis may anticipate plaque rupture.(AU)

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.

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/mm(3)) than in controls (75.34 ± 3.05 mm/mm(3), 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.

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.

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.

Presencia de placas coronarias vulnerables en mujeres de mediana edad que sufrieron muerte cerebral / Presence of Vulnerable Coronary Plaques in Middle-Aged Women Who Suffered Brain Death

Introducción Las placas vulnerables de las arterias coronarias se encuentran habitualmente en varones sin sintomatología previa que fallecieron súbitamente o debido a un síndrome coronario agudo. La frecuencia y las características de estas placas en la población femenina de mediana edad se desconocen. Objetivo Indagar acerca de la presencia de placas coronarias vulnerables en mujeres de mediana edad que sufrieron muerte cerebral. Material y métodos De un total de 652 corazones de donantes para trasplante obtenidos entre 1996 y 2007, se seleccionaron los provenientes de individuos aparentemente sanos y mayores de 40 años que murieron a causa de un accidente traumático o cerebrovascular. De los 160 órganos que cumplieron los criterios de selección, se estudiaron 70 corazones femeninos en los que se examinaron las arterias coronarias en cortes consecutivos a intervalos de 3 mm. Las áreas con estrechamiento luminal se procesaron para su posterior estudio histológico e inmunohistoquímico. Las placas se clasificaron según la American Heart Association (AHA). Resultados La edad promedio de las mujeres fue de 50,7 ± 5,7 años. El grado de oclusión vascular promedio fue del 28,66% ± 17,35%. Cuarenta y un corazones poseían sólo lesiones ateroscleróticas no avanzadas (tipos I, II y III de la clasificación de la AHA). En los 29 restantes se encontraron 58 placas consideradas de alto riesgo o vulnerables (AHA tipos IV, V y VI). Éstas fueron menos frecuentes que en los varones (p < 0,001) y se asociaron con el peso del órgano (OR 1,02, IC 95% 1,01-1,04; p < 0,001) y con la edad (OR 1,11, IC 95% 1,01- 1,23; p = 0,038). No se encontró una relación significativa con la causa de la muerte (p = 0,065). Conclusiones El 41,4% de las mujeres que sufrieron muerte cerebral presentaron placas vulnerables en los vasos coronarios no asociadas con una reducción significativa de la luz. De esta serie puede inferirse que existe un promedio de 0,82 lesiones propensas a generar trombosis en mujeres de mediana edad aparentemente sanas que sufrieron muerte cerebral.

Background The presence of vulnerable plaques is a common finding in the coronary arteries of previously asymptomatic men who died suddenly or due to an acute coronary syndrome. The prevalence and the characteristics of these plaques in middle-aged women are still unknown. Objective To evaluate the presence of vulnerable coronary plaques in middle-aged women who suffered brain death. Material and Methods 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. From a total of 160 elegible organs, the coronary arteries of 70 female hearts were examined by serial sectioning at 3-mm intervals. The areas with luminal narrowing were processed for subsequent histological and immuhistochemical studies. Plaques were classified according to the American Heart Association (AHA) report. Results Mean age was 50.7±5.7 years. The degree of average vascular obstruction was 28.66%±17.35%. A total of 41 hearts had no advanced coronary atherosclerotic lesions (Type I, II, and III of the AHA classification). In the remaining 29 hearts we found 58 plaques considered high-risk lesions (AHA type IV, V and VI). These plaques were less frequent in women than in men (p<0,001) and were associated with higher heart weight (OR 1.02, 95% CI 1.01-1.04; p<0.001) and with age (OR 1.11, 95% CI 1.01-1.23; p=0.038). No significant association with the cause of death was found (p = 0.065). Conclusions In 41.4% of women who suffered brain death, we found vulnerable plaques in the coronary arteries that were not associated with significant lumen obstructions. This series suggests the presence of 0.82 coronary lesions which are prone to develop thrombosis in apparently healthy middleaged women who suffered brain death.

Expression of the MDR-1 gene-encoded P-glycoprotein in cardiomyocytes of conscious sheep undergoing acute myocardial ischemia followed by reperfusion.

We have recently reported that in chronic myocardial ischemia, adult mammalian cardiomyocytes express P-glycoprotein (P-gp). We now investigate if P-gp is also expressed in acute regional ischemia followed by reperfusion. Adult conscious sheep underwent 12-min occlusion of the mid-left anterior descending artery (inflatable cuff). Successful ischemia-reperfusion was confirmed by monitoring percent systolic left ventricular anterior wall thickening (sonomicrometry) during the whole ischemic period and every 10 min over 2 hr following cuff deflation. At 3, 24, and 48 hr after reperfusion, P-gp expression was investigated by immunohistochemistry and Western blot and MDR-1 mRNA by RT-PCR. Cardiomyocytes in the occluded artery territory (but not those in remote areas) consistently expressed P-gp at their sarcolemma. Whereas at 3 and 24 hr P-gp was mainly observed in the T tubules, at 48 hr it predominated in intercalated discs and gap junctions. RT-PCR and Western blot revealed higher expression in ischemic than in control myocardium. We conclude that in adult sheep with acute myocardial ischemia, the MDR-1 gene-encoded P-gp is expressed at the sarcolemma of the cardiomyocytes from 3 hr up to at least 48 hr after reperfusion.

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.

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.

Dilated cardiomyopathy in Erb-b4-deficient ventricular muscle.

The neuregulin receptor tyrosine kinase Erb-b4, initially linked to early cardiac development, is shown here to play a critical role in adult cardiac function. In wild-type mice, Erb-b4 protein localized to Z lines and to intercalated disks, suggesting a role in subcellular and intercellular communications of cardiomyocytes. Conditional inactivation of erb-b4 in ventricular muscle cells led to a severe dilated cardiomyopathy, characterized by thinned ventricular walls with eccentric hypertrophy, reduced contractility, and delayed conduction. This cardiac dysfunction may account for premature death in adult erb-b4-knockout mice. This study establishes a critical role for Erb-b4 in the maintenance of normal postnatal cardiac structure and function.

Expresión y efecto del gen de VEGF165 humano en músculo esquelético isquémico de conejos. ¿Necesidad de administración repetitiva para el tratamiento de la enfermedad vascular periféica? / Expression and effects after VEGF165 gene transfer: is it necessary the reiterative administration for the treatment of peripheral vascular disease?

Peripheral vascular disease is a disorder fifficult to treat. Due to the lack of an effective pharmacological therapy, the ischemia of an effective pharmacological therapy, the ischemia of the lower limbs pursues a relentless course, ending many times in the amputation...The authors present in this study a 6 years experience with this management, and recently the effects of an experimental model of peripheral vascular disease in rabbits is evaluated. The results obtained in this study are important because they could attribute to he trnasitority of the genetic expression the contradictory results founded in clinical assays of peripheral vascular disease

Expresión y efecto del gen de VEGF165 humano en músculo esquelético isquémico de conejos. ¿Necesidad de administración repetitiva para el tratamiento de la enfermedad vascular periféica? / Expression and effects after VEGF165 gene transfer: is it necessary the reiterative administration for the treatment of peripheral vascular disease?

Peripheral vascular disease is a disorder fifficult to treat. Due to the lack of an effective pharmacological therapy, the ischemia of an effective pharmacological therapy, the ischemia of the lower limbs pursues a relentless course, ending many times in the amputation...The authors present in this study a 6 years experience with this management, and recently the effects of an experimental model of peripheral vascular disease in rabbits is evaluated. The results obtained in this study are important because they could attribute to he trnasitority of the genetic expression the contradictory results founded in clinical assays of peripheral vascular disease

Reducción del tamaño del infarto de miocardio mediante la transferencia génica de VEGF en ovejas / Myocardial infarct size reduction after VEGF gene transfer in sheep

La transferencia génica de plásmido codificante para VEGF (pVEGF) induce angiogénesis, incremento del índice mitótico e hiperplasia miocítica en cerdos con isquemia miocárdica crónica. El presente trabajo se realizó con el objetivo de estudiar el efecto del pVEGF sobre el tamaño del infarto de miocardio. Una hora después de la ligadura de la descendente anterior, 28 ovejas de 23 ± 0,5 kg se agruparon al azar para recibir 10 inyecciones intramiocárdicas de 3,8 mg de pVEGF (n = 14) o placebo (n= 14) distribuídas en la periferia del infarto. La función miocárdica se estudió con SPECT gatillado y los animales se sacrificaron 7, 10 y 15 días después del tratamiento. El tamaño del infarto fue el 34 por ciento menor en el grupo tratado que en el placebo (placebo: 17,1 ± 2,1 por ciento, VEGF: 11,2 ± 1,5 por ciento, p < 0,05) a los 15 pero no a los 10 días. Aunque hubo una leve tendencia a favor del grupo tratado, la mejoría de la función miocárdica no fue diferente entre grupos (placebo: 3,3 ± 1,4; VEGF: 3,8 ± 2,4; p = NS). El estudio histológico mostró que los mecanismos involucrados fueron la respuesta angiogénica a los 7 días (placebo: 676 ± 31 capilares / mm²: VEGF: 1925 ± 262; p <0,05), la menor fibrosis periinfarto a los 10 días (contenido de colágeno; placebo: 70, 1 ± 1,7 por ciento; VEGF: 43,5 ± 4,4 por ciento; p < 0,05); la proliferación de mioblastos a los 7 y 10 días. La expresión del pVEGF fue positiva a los 3 (ARNm), 7 y 10 días. La expresión del pVEGF fue positiva a los 3 (ARNm), 7 y 10 días (proteína) posadministración. Conclusión: A los 15 días la transferencia génica de VEGF humano reduce el tamaño de infarto en ovejas mediante angiogénesis, disminución de la fibrosis e inducción de proliferación de mioblastos (miocardiogénesis).

Reducción del tamaño del infarto de miocardio mediante la transferencia génica de VEGF en ovejas / Myocardial infarct size reduction after VEGF gene transfer in sheep

La transferencia génica de plásmido codificante para VEGF (pVEGF) induce angiogénesis, incremento del índice mitótico e hiperplasia miocítica en cerdos con isquemia miocárdica crónica. El presente trabajo se realizó con el objetivo de estudiar el efecto del pVEGF sobre el tamaño del infarto de miocardio. Una hora después de la ligadura de la descendente anterior, 28 ovejas de 23 ± 0,5 kg se agruparon al azar para recibir 10 inyecciones intramiocárdicas de 3,8 mg de pVEGF (n = 14) o placebo (n= 14) distribuídas en la periferia del infarto. La función miocárdica se estudió con SPECT gatillado y los animales se sacrificaron 7, 10 y 15 días después del tratamiento. El tamaño del infarto fue el 34 por ciento menor en el grupo tratado que en el placebo (placebo: 17,1 ± 2,1 por ciento, VEGF: 11,2 ± 1,5 por ciento, p < 0,05) a los 15 pero no a los 10 días. Aunque hubo una leve tendencia a favor del grupo tratado, la mejoría de la función miocárdica no fue diferente entre grupos (placebo: 3,3 ± 1,4; VEGF: 3,8 ± 2,4; p = NS). El estudio histológico mostró que los mecanismos involucrados fueron la respuesta angiogénica a los 7 días (placebo: 676 ± 31 capilares / mm²: VEGF: 1925 ± 262; p <0,05), la menor fibrosis periinfarto a los 10 días (contenido de colágeno; placebo: 70, 1 ± 1,7 por ciento; VEGF: 43,5 ± 4,4 por ciento; p < 0,05); la proliferación de mioblastos a los 7 y 10 días. La expresión del pVEGF fue positiva a los 3 (ARNm), 7 y 10 días. La expresión del pVEGF fue positiva a los 3 (ARNm), 7 y 10 días (proteína) posadministración. Conclusión: A los 15 días la transferencia génica de VEGF humano reduce el tamaño de infarto en ovejas mediante angiogénesis, disminución de la fibrosis e inducción de proliferación de mioblastos (miocardiogénesis). (AU)

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.

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