Improvement in coronary vascular dysfunction produced with euglycaemic control in patients with type 2 diabetes.

OBJECTIVE: To determine the effect of plasma glucose lowering on coronary circulatory function in type 2 diabetes mellitus. METHODS: Twenty patients with type 2 diabetes and 18 weight-matched controls were studied. At baseline, myocardial blood flow (MBF) was measured with [(13)N]ammonia and positron emission tomography at rest, during cold pressor testing (CPT), and during adenosine hyperaemia. In diabetic patients, MBF and blood chemistry were analysed again after 3 months of glucose-lowering treatment with glyburide and metformin. RESULTS: Although hyperaemic MBF did not differ significantly between the patients and controls (1.81 (0.38) v 1.97 (0.43) ml/min/g; mean (SD)), the CPT-induced MBF increase (DeltaMBF) was significantly less in diabetic patients than in controls (0.07 (0.07) v 0.25 (0.12) ml/min/g; p<0.001). Treatment with glyburide and metformin significantly decreased plasma glucose concentrations from 207 (76) to 134 (52) mg/dl (p<0.001). This decrease in plasma glucose was paralleled by a significant increase in DeltaMBF in response to CPT (0.20 (0.16) from 0.07 (0.07) ml/min/g; p<0.001), which tended to be lower than in controls at baseline (0.20 (0.16) v 0.25 (0.12) ml/min/g; p = NS). The decrease in plasma glucose concentrations correlated significantly with the improvement in DeltaMBF in response to CPT (r = 0.67, p<0.01). CONCLUSIONS: Type 2 diabetes mellitus is associated with abnormal MBF response to CPT, which can be significantly improved by euglycaemic control with glyburide and metformin. The close association between the decrease in plasma glucose concentration and the improvement in coronary vasomotor function in response to CPT suggests a direct adverse effect of raised plasma glucose concentration on diabetes-related coronary vascular disease.

Transplantation of fetal myocardial tissue into the infarcted myocardium of rat. A potential method for repair of infarcted myocardium?

BACKGROUND: Unlike skeletal myocytes, mammalian adult cardiomyocytes cannot regenerate after injury. A possible strategy to increase viability and augment ventricular function after myocardial injury is fetal myocardial tissue transplantation. The engrafted fetal cells are a potential source of growth factors and can be used for cardiomyocyte-based gene therapy. The purpose of our study was to test the feasibility and efficiency of fetal cardiomyocyte transplantation into a model of myocardial infarction. METHODS AND RESULTS: We subjected rats after myocardial infarction to three protocols of therapy. In the first protocol, tissue fragments of cultured human fetal ventricles were injected into the scar 7 to 24 days after infarction. The rats were treated with intraperitoneal injections of 12.5 mg.kg-1.d-1 cyclosporine. In the second protocol, fragments of cultured fetal rat ventricles were injected into the scar 9 to 17 days after infarction. A third group of animals with myocardial infarction was treated with injection of saline into the scar (control). After 7 to 65 days post-transplantation, hearts were harvested and processed for electron microscopy and alpha-actin immunohistochemistry. Toluidine blue staining and electron microscopy revealed the presence of engrafted human and rat cardiomyocytes in the infarcted myocardium up to 14 and 65 days after transplantation, respectively. The morphology was similar to that of cultured fetal cardiomyocytes. The engrafted fetal tissues were also stained positive for alpha-actin, which is unusual for the adult rat myocardium. Examination of control hearts detected infarcted tissue only, and alpha-actin staining was limited to vessel walls. CONCLUSIONS: Fetal cardiomyocyte tissue can be implanted and survive in the infarcted myocardium. This experimental approach may provide a therapeutic strategy for cardiomyocyte-based gene therapy for introduction of therapeutic proteins into myocardial infarction.

Adenovirus-mediated gene transfer into infarcted myocardium: feasibility, timing, and location of expression.

Gene transfer as a therapeutic modality for the treatment of myocardial ischemia and/or infarction has been proposed as a revolutionary approach to improve collateral circulation, enhance myocardial viability and amplify healing. Our study was undertaken to assess the feasibility, efficiency, anatomic distribution, timing and localization of adenovirus-mediated gene transfer into the vicinity of infarcted myocardium in the adult mammalian heart. We induced myocardial infarction by subjecting rats to 60 min of coronary artery occlusion followed by sustained reperfusion. Gene transfer into the infarction area was performed using direct injection of a replication-defective adenovirus vector encoding the bacterial reporter gene, beta-galactosidase. A total of 5.0 x 10(9) plaque-forming units of virus was delivered into the left ventricular myocardium either immediately (n = 7) or at 7 (n = 6), 22 (n = 5) or 30 days (n = 5) after reperfusion of rat hearts. Control rats received either 50 microliters of saline 13 days after myocardial infarction (n = 2) or were not subjected to infarction and received Adenovirus carrying the beta-galactosidase gene as described above (n = 4). All rats were killed at 7 days after cardiac injection. Hearts were harvested, frozen and sectioned and stained for beta-galactosidase activity and with hematoxylin and eosin. Sections were evaluated by light microscopy. Relative beta-galactosidase activity was measured by digital planimetry and expressed as the ratio of the maximal area of beta-galactosidase staining relative to the total area of the section examined (% +/- S.E.M.). beta-galactosidase gene expression was limited mainly to viable myocytes at the border of the myocardial infarction. The area of transgene expression in the non-infarcted hearts (28 +/- 7%) was significantly higher (P = 0.02) than at any time point studied in infarcted tissues (3.4 +/- 1.2%, 1.4 +/- 1.0%, 2.8 +/- 0.8% and 3.4 +/- 0.9% at reperfusion and at 7, 22 and 30 days after myocardial infarction, respectively). Hearts injected 7 days after infarction had significantly less transgene activity (P = 0.03) with three of five samples displaying no macroscopically visible beta-gal activity. Following viral injection, an inflammatory response consisting of mononuclear cell infiltration was much less intense seven days following injection in non-infarcted control rat hearts than at any of the time points examined for infarcted hearts. Gene transfer into infarcted myocardium, while feasible, was limited by low transfection efficiency when compared to non-infarcted normal myocardium. Transgene expression in the infarcted myocardium appears restricted to residual cardiomyocytes in the periphery. Nevertheless, the ability to introduce genes into these viable peripheral cells might be a useful therapeutic strategy for enhancing neovascularization, collateral flow and healing.

Avoidance of immune response prolongs expression of genes delivered to the adult rat myocardium by replication-defective adenovirus.

BACKGROUND: Gene delivery is a rapidly expanding field with potential applications to every human organ system. Recently, adenoviruses have been used as efficient vectors for in vivo gene transfer into the myocardium. These methods, however, have shown a sharp decline of gene expression after 1 week. To test the hypothesis that an immune-effector mechanism is involved in this decline, we compared the results after injection of adenovirus-5 carrying the beta-galactosidase gene (Ad beta-gal) into the left ventricular myocardium of athymic nude rats (NDRs) versus immunocompetent Sprague-Dawley rats (SDRs). METHODS AND RESULTS: Ad beta-gal (5.0 x 10(9) PFU/mL) was injected into the left ventricle of NDRs (n = 16) and SDRs (n = 22). Hearts were harvested, embedded in paraffin, and sectioned and stained for beta-gal activity, hematoxylin and eosin and picrosirius red at 4, 21, 35, 85, and 120 days. Representative samples were immunostained with antibodies directed at inflammatory markers. beta-gal activity was quantified by digital planimetry and expressed as area of staining (% +/- SEM). Peak beta-gal activity was highest at 4 days, with NDRs displaying significantly greater staining (83 +/- 3.0% versus 54 +/- 8.0%; P = .03). SDRs sustained a rapid drop in activity, such that at 35 (1 +/- 0.19%) and 85 (1 +/- 0.4%) days, only occasional cells stained positive and by 120 days (0.3 +/- 0.0%), activity had been extinguished. NDRs continued to show transgene expression at all time periods (35 and 85 days, 25 +/- 7.1% and 7.4 +/- 2.7%, respectively) and was still readily detected at 120 days. An inflammatory response was limited in NDRs compared with SDRs, in which there was intense mononuclear cell infiltration, with collagen deposition and scar formation. Immunostaining identified the majority of these inflammatory cells as not being of lymphocyte lineage, although small numbers of lymphocytes and phagocytic and activated plasma cells were identified. CONCLUSIONS: Our data suggest that immune-effector mechanisms can severely affect the expression of genes delivered by adenovirus. The present model provides efficient gene expression for at least 120 days without significant inflammatory reaction.

Upregulation of angiotensin II type 1 receptor gene expression in chronic renovascular hypertension.

Although the renin-angiotensin system has been implicated in the pathogenesis of renovascular hypertension (RVH), blood pressure does not parallel serum levels of renin or angiotensin II (AII) in chronic RVH. Upregulation of angiotensin II type 1 receptor (AT1) gene expression may explain this paradox and clarify the pathogenesis of chronic hypertension in RVH. To investigate this hypothesis, we studied changes in AT1 mRNA levels in rat kidney in a two-kidney, one-clip (2K1C) rat model of RVH. Animals were sacrificed at 1 or 10 weeks postoperatively. Blood pressure was measured with a tail cuff photosensor. Relative gene expression was quantitated by dot blotting total RNA, hybridizing with a cDNA probe for AT1, and quantitating signal intensity with scanning densitometry. A significant increase in blood pressure (BP) was observed at 1 week postoperatively (delta BP: 2K1C = +24 mm Hg, n = 3; controls = +7 mm Hg, n = 3; P < 0.05), and at this time relative AT1 mRNA levels actually decreased in the clipped kidney (P < 0.05). Hypertension intensified 10 weeks postoperatively (delta BP: 2K1C = +46 mm Hg, n = 20; controls = -17 mm Hg, n = 7; P < 0.005) and, remarkably, was paralleled by an almost sevenfold upregulation of AT1 mRNA levels in the clipped kidney (P < 0.005) and more than eightfold in the unclipped kidney (P < 0.005) of 2K1C animals. Upregulation of renal AT1 gene expression could lead to increased AT1 receptor production, hypersensitivity to AII, and chronic hypertension in RVH.