MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death.

Reactive oxygen species (ROS) are critical in tissue responses to ischemia-reperfusion. The enzyme methionine sulfoxide reductase-A (MsrA) is capable of protecting cells against oxidative damage by reversing damage to proteins caused by methionine oxidation or by decreasing ROS through a scavenger mechanism. The current study employed adenovirus mediated over-expression of MsrA in primary neonatal rat cardiac myocytes to determine the effect of this enzyme in protecting against hypoxia/reoxygenation in this tissue. Cells were transduced with MsrA encoding adenovirus and subjected to hypoxia/reoxygenation. Apoptotic cell death was decreased by greater than 45% in cells over-expressing MsrA relative to cells transduced with a control virus. Likewise total cell death as determined by levels of LDH release was dramatically decreased by MsrA over-expression. These observations indicate that MsrA is protective against hypoxia/reoxygenation stress in cardiac myocytes and point to MsrA as an important therapeutic target for ischemic heart disease.

5-hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries: importance of the 5-HT1B receptor.

1. The 5-hydroxytryptamine (5-HT) receptors mediating vasoconstriction in isolated human small muscular pulmonary arteries (SMPAs) were determined using techniques of wire myography and reverse transcription-polymerase chain reaction (RT - PCR). 2. The agonists 5-HT, 5-carboxamidotryptamine (5-CT, unselective for 5-HT1 receptors) and sumatriptan (selective for 5-HT1B/D receptors) all caused contraction and were equipotent (pEC50s: 7.0+/-0.2, 7.1+/-0.3 and 6.7+/-0.1, respectively) suggesting the presence of a 5-HT1 receptor. 3. Ketanserin (5-HT2A-selective antagonist, 0.1 microM) inhibited 5-HT-induced contractions only at non-physiological/pathological concentrations of 5-HT (>0.1 microM) whilst GR55562 (5-HT1B/1D-selective antagonist, 1 microM) inhibited 5-HT-induced contractions at all concentrations of 5-HT (estimated pKB=7.7+/-0.2). SB-224289 (5-HT1B-selective antagonist, 0.2 microM) inhibited sumatriptan-induced contractions (estimated pKB=8.4+/-0.1) whilst these were unaffected by the 5-HT1D-selective antagonist BRL15572 (0.5 microM) suggesting that the 5-HT1B receptor mediates vasoconstriction in this vessel. 4. RT - PCR confirmed the presence of substantial amounts of mRNA for the 5-HT2A and 5-HT1B receptor subtypes in these arteries whilst only trace amounts of 5-HT1D receptor message were evident. 5. These findings suggest that a heterogeneous population of 5-HT2A and 5-HT1B receptors co-exist in human small muscular pulmonary arteries but that the 5-HT1B receptor mediates 5-HT-induced vasoconstriction at physiological and pathophysiological concentrations of 5-HT. These results have important implications for the treatment of pulmonary hypertension in which the 5-HT1B receptor may provide a novel and potentially important therapeutic target.

Gene transfer and models of gene therapy for the myocardium.

1. Gene transfer into the myocardium can be achieved through direct injection of plasmid DNA or through the delivery of viral vectors, either directly or through the coronary vasculature. Direct DNA injection has proven extremely valuable in studies aimed at characterizing the activities of promoter elements in cardiac tissue and for examining the influence of the pathophysiological state of the myocardium on expression of transferred foreign genes. 2. Viral vectors, in particular adenoviruses and adeno-associated virus, are capable of transfecting genetic material with high transduction efficiencies and have been applied to a range of model systems for in vivo gene transfer. Efficient gene transfer has been achieved into the coronary vessels and surrounding myocardium by intracoronary infusion of adenovirus. 3. Because the immunogenicity of viral vectors can limit transgene expression, much attention has been paid to strategies for circumventing this, including the development of new modified adenovirus and adeno-associated virus vectors that do not elicit significant inflammatory responses. While cellular transplantation may prove valuable for the repair of myocardial tissue, confirmation of its value awaits establishment of a functional improvement in the myocardium following cell grafting. 4. Because gene transfer into the myocardium can now be achieved with high efficiency in the absence of significant inflammatory responses, the ability to regulate foreign gene expression in response to an endogenous disease phenotype will enable the development of new effective viral vectors with direct clinical applicability for specified therapeutic targets.

Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model.

OBJECTIVE: We have shown previously that there is a relative nitric oxide deficiency at the level of vascular endothelium in the stroke-prone spontaneously hypertensive rat (SHRSP), a model of human essential hypertension, as compared to its normotensive reference strain Wistar Kyoto (WKY) rat. The aim of the current study was to investigate whether adenoviral-mediated gene transfer of an endothelial nitric oxide synthase (eNOS) cDNA (AdCMVeNOS) into carotid arteries of the SHRSP may improve endothelial function. METHODS: Enzyme activity of the recombinant eNOS protein encoded by AdCMVeNOS was tested using a Griess assay in endothelial cells in culture. Left carotid arteries of SHRSP were surgically isolated and exposed to either the AdCMVeNOS or control beta-galactosidase-containing virus, (2 x 10(9) pfu/ml) ex vivo and in vivo. The vessels were harvested 24 h after surgery and analysed by Western blotting, immunohistochemistry and by examining endothelial function ex vivo. RESULTS: Cultured endothelial cells showed almost 100% transduction with both viruses and a dose response of eNOS expression showed a five-fold increase in nitrite production for AdCMVeNOS with no change for beta-galactosidase-containing virus. Western blotting demonstrated a significant increase of eNOS expression in vessels infused with AdCMVeNOS when compared to controls. Immunohistochemistry showed highly positive staining with monoclonal antibodies against eNOS in the intact endothelial cells of the AdCMVeNOS infused vessels. The areas under the curve of the concentration responses to phenylephrine (10(-9) to 3 x 10(-6) M) in the absence and presence of NG-nitroarginine methyl ester (100 microM) showed increased basal nitric oxide bioavailability in the carotid arteries infused with AdCMVeNOS compared to the control (n = 6 for each; P = 0.0069; 95% CI, 0.864 to 3.277). CONCLUSIONS: Our results show that AdCMVeNOS is an effective tool for vascular gene transfer and that it can improve endothelial NO availability in the SHRSP, a genetic model of essential hypertension and endothelial dysfunction.

Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain.

cDNA clones encoding neural cell adhesion molecule (N-CAM) mRNAs of 6.7, 5.2, and 4.3 kb from human skeletal muscle cells were isolated. A 6.7 kb mRNA encodes a transmembrane N-CAM isoform, present predominantly in mononucleate myoblasts, that shows sequence homology with chick brain N-CAM-140 and is down-regulated during myotube formation. In contrast, the 5.2 and 4.3 kb mRNAs encode nontransmembrane N-CAM isoforms that greatly increase during myoblast fusion. Furthermore, a discrete muscle-specific sequence domain (MSD1) was detected in the extracellular coding regions of the 5.2 and 4.3 kb mRNAs. This encodes a unique run of 37 amino acids and is not expressed in 7.2 and 6.7 kb mRNAs from human or chick brain or in the corresponding 6.7 kb muscle transcript. The MSD1 is also absent from chick and mouse brain mRNAs of 4.0 and 2.9 kb. These results show that diversity in N-CAM primary structure can be found in the extracellular domain in a tissue-specific manner.

Nerve growth factor-induced changes in neural cell adhesion molecule (N-CAM) in PC12 cells.

The effects of nerve growth factor (NGF) on the expression of neural cell adhesion molecule (N-CAM) in PC12 cells were determined. A quantitative immunoassay was used to show that NGF induces a 4- to 5-fold increase in relative N-CAM levels over a 3-day period. This increase could not be mimicked by cholera toxin suggesting that it is not a simple consequence of morphological differentiation. The changes in N-CAM levels induced by NGF were accompanied by changes in N-CAM molecular forms. The 140-kd N-CAM species is the major N-CAM expressed by naive PC12 cells, while NGF-treated cultures express N-CAM species of 180 kd and 140 kd. Northern analysis showed that naive cells express a 6.7-kd N-CAM mRNA species only, while NGF-treated cultures express both a 6.7-kb and a 7.2-kb transcript. As the 6.7-kb and 7.2-kb mRNAs are alternative spliced transcripts of a single gene, this result shows that NGF can activate a neuron-specific splicing mechanism. This is the first description of control of N-CAM expression by a growth factor.

Identification and characterization of neuron-specific and developmentally regulated gene transcripts in the chick embryo spinal cord.

Clones corresponding to neuron-specific and developmentally regulated messenger RNA species in the chick have been isolated from a complementary DNA library prepared using polyadenylated RNA from 7-day embryonic spinal cord. The library was initially screened by differential complementary DNA hybridization procedures for clones identifying polyadenylated RNAs present in embryonic spinal cord but absent from or at low abundance in liver tissue. A high proportion of selected recombinant plasmids were found to identify different RNA species which, although present in 14-day embryonic spinal cord, could not be detected in a corresponding region of the developing chick CNS that is devoid of neuronal cell bodies, the optic nerve. The neuron-specific assignment of these mRNAs within the developing neuroectoderm was confirmed using bulk-isolated neuronal and glial-enriched cell fractions from 7-day embryonic spinal cord. In addition, several distinctive patterns of developmentally regulated expression of neuron-specific messenger RNA species have been observed in the chick spinal cord. The studies lay a foundation for detailed examination of the regional and temporal distribution and control of neuronal gene expression in the chick spinal cord during embryogenesis.