G-CSF augments small vessel and cell density in canine myocardial infarciton.

We recently reported that granulocyte-colony stimulating factor (G-CSF) prevented cardiac remodeling by mobilization and differentiation of bone marrow-derived cells in murine experimental myocardial infarction (MI). Little is known, however, whether these findings can be reproduced in large animals. The aim of this study is to investigate the effect of G-CSF after MI in canine model. MI was generated in twenty-six beagle dogs by ligation of left anterior descending artery. They were divided into two groups: G-CSF group which received subcutaneous injection of G-CSF (10 microg/kg/day) for 10 days, and the control group with saline injection. After six weeks, they were subjected to echocardiography and catheterization to measure hemodynamic parameters, and histological analysis was performed. No dogs died during the period. No hemodynamic changes were observed between these two groups probably due to the smaller size of the MI than we expected. We found significant increase in wall thickness and higher cell density in G-CSF group. Immunohistochemical staining against alpha-smooth muscle actin and CD31 revealed increased vessel density mainly in the epicardium in G-CSF group. The number of survived cardiomyocytes in G-CSF group was slightly greater than that in the control group, although it was not statistically significant. These findings suggested G-CSF prevented cardiac remodeling in canine model not by increasing the cardiomyocytes but by increasing the vessel density and cell numbers in the infarcted area.

Angiogenic gene cell therapy using suicide gene system regulates the effect of angiogenesis in infarcted rat heart.

BACKGROUND: Although angiogenic gene therapy has been reported to be effective in restoring ischemic heart function, there are several obstacles to its clinical application, such as unreliable efficiency of transfection and uncontrollable expression. We developed human HGF (hHGF)-producing cells that regulated hHGF production using the thymidine kinase gene of Herpes Simplex Virus (TK) and the Ganciclovir (GCV) system. We tested whether these cells induced and regulated angiogenic effects in infarcted myocardium. METHODS: NIH3T3 cells were stably transfected with an hHGF cDNA expression plasmid (NIH/HGF). Next, the NIH/HGF cells were stably transfected with TK (NIH/HGF/TK). The left anterior descending artery was ligated in the heart of severe combined immunodeficiency rats, and four materials were transplanted: 1) NIH/HGF (n=10), 2) NIH/HGF/TK, with orally administered GCV (n=10), 3) NIH3T3 (n=10), and 4) culture medium (n=10). RESULTS: In vitro, the proliferation of NIH/HGF/TK cells was suppressed by GCV. In vivo, significant increases in cardiac performance and angiogenesis were observed in the NIH/HGF and NIH/HGF/TK groups 4 weeks after transplantation. Although tumorous lesions were detected in the NIH/HGF group, their growth was completely controlled in the NIH/HGF/TK group. CONCLUSIONS: Angiogenic gene cell therapy using the TK-GCV suicide gene system induces and regulates angiogenesis under the control of cell growth, suggesting it as a promising system for therapeutic angiogenesis.

Cardiomyocytes undergo cells division following myocardial infarction is a spatially and temporally restricted event in rats.

Dividing cardiomyocytes are observed in autopsied human hearts following recent myocardial infarction, however there is a lack of information in the literature on the division of these cells. In this study we used a rat model to investigate how and when adult mammalian cardiomyocytes proliferate by cell division after myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. The rats were sacrificed periodically up to 28 days following induced myocardial infarction, and the hearts subjected to microscopic investigation. Cardiomyocytes entering the cell cycle were assayed by observation of nuclear morphology and measuring expression of Ki-67, a proliferating cell marker. Ki-67 positive cardiomyocytes and dividing nuclei were observed initially after 1 day. After 2 days dividing cells gradually increased in number at the ischemic border zone, reaching a peak increase of 1.12% after 3 days, then gradually decreasing in number. Dividing nuclei increased at the ischemic border zone after 3 days, peaked by 0.14% at day 5, and then decreased. In contrast, Ki-67 positive cells and dividing nuclei were limited in number in the non-ischemic area throughout all experiments. In conclusion, mitogenic cardiomyocytes are present in the adult rat heart following myocardial infarction, but were spatially and temporally restricted.

Powerful and controllable angiogenesis by using gene-modified cells expressing human hepatocyte growth factor and thymidine kinase.

OBJECTIVES: This study investigated the possibility of achieving angiogenesis by using gene-modified cells as a vector. BACKGROUND: Although gene therapy for peripheral circulation disorders has been studied intensively, the plasmid or viral vectors have been associated with several disadvantages, including unreliable transfection and uncontrollable gene expression. METHODS: Human hepatocyte growth factor (hHGF) and thymidine kinase (TK) expression plasmids were serially transfected into NIH3T3 cells, and permanent transfectants were selected (NIH3T3 + hHGF + TK). Unilateral hindlimb ischemia was surgically induced in BALB/c nude mice, and cells were transplanted into the thigh muscles. All effects were assessed at four weeks. RESULTS: The messenger ribonucleic acid expression and protein production of hHGF were confirmed. Assay of growth inhibition by ganciclovir revealed that the 50% (median) inhibitory concentration of NIH3T3 + hHGF + TK was 1000 times lower than that of NIH3T3 + hHGF. The NIH3T3 + hHGF + TK group had a higher laser Doppler blood perfusion index, higher microvessel density, wider microvessel diameter, and lower rate of hindlimb necrosis, as compared with the plasmid- and adenovirus-mediated hHGF transfection groups or the NIH3T3 group. The newly developed microvessels were accompanied by smooth muscle cells, as well as endothelial cells, indicating that they were on the arteriolar or venular level. Laser Doppler monitoring showed that the rate of blood perfusion could be controlled by oral administration of ganciclovir. The transplanted cells completely disappeared in response to ganciclovir administration for four weeks. CONCLUSIONS: Gene-modified cell transplantation therapy induced strong angiogenesis and collateral vessel formation that could be controlled externally with ganciclovir.

Endothelin-1 regulates cardiac sympathetic innervation in the rodent heart by controlling nerve growth factor expression.

The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1-induced NGF augmentation was mediated by the endothelin-A receptor, Gibetagamma, PKC, the Src family, EGFR, extracellular signal-regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein delta element. Either conditioned medium or coculture with endothelin-1-stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1-deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1-deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1-deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.