Specific induction of tumor neovasculature death by modified murine PPE-1 promoter armed with HSV-TK.

BACKGROUND: One strategy to increase tissue specificity of gene therapy is to use promoters or enhancers. OBJECTIVES: (1) To enhance the selectivity of a murine preproendothelin-1 (PPE-1) promoter in tumor angiogenesis by using a positive endothelial transcription-binding element. (2) To test the specificity and efficiency of the modified PPE-1 promoter [PPE-1(3X)] in vitro and in vivo by using reporter genes, and the therapeutic gene herpes simplex virus-thymidine kinase (HSV-TK) in a mouse model of Lewis lung carcinoma (LLC). RESULTS: The modified PPE-1 promoter specifically induced expression in the tumor angiogenic vascular bed with a 35-fold higher expression compared to the normal vasculare bed of the lung. Thus, when the HSV-TK gene controlled by the modified PPE-1 promoter was used systemically, it induced tumor-specific necrosis, apoptosis and mononuclear infiltrates, leading to massive destruction of the neovasculature of the pulmonary metastasis, which suppressed metastasis development. CONCLUSIONS: These results show that an adenoviral vector armed with HSV-TK controlled by the endothelial-selective murine PPE-1(3X) promoter is efficient and safe to target tumor neovasculature.

Tissue-specific gene therapy directed to tumor angiogenesis.

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of 'vascular-directed' gene therapy will be applicable to both vascular diseases and cancer.

Cytotoxic T lymphocytes are activated following myocardial infarction and can recognize and kill healthy myocytes in vitro.

The damage of myocardial infarction (MI) is often progressive. A possible mechanism for subsequent myocardial damage and heart failure after MI is immune response against cardiac self-antigens. The purpose of our study was to test the hypothesis that cytotoxic T lymphocytes are activated following acute MI and may have a role in producing further myocardial damage. Rats were allocated into three experimental groups: acute MI, Sham MI and non-operated control. One, two and three weeks after surgery, lymphocytes were obtained from rat spleens and incubated with neonatal cardiac myocytes. Lymphocyte proliferation was assessed by a thymidine incorporation assay and calculated as proliferation index (PI). Myocyte destruction was measured by a crystal-violet staining assay and expressed as percentage of cell destruction. Proliferation index was significantly higher among lymphocytes obtained from MI animals (44. 3+/-5.8 and 44.9+/-5.1, at 2 and 3 weeks after MI, respectively) than sham MI (29.3+/-5.3, 27.1+/-4.7) (P<0.05) or control animals (17.1+/-2.5, 16.2+/-2.8) (P=0.03). Cytotoxic activity of the MI lymphocytes against the cultured cardiomyocytes was significantly higher 2 and 3 weeks after MI, (36.4+/-7.3%, 69.3+/-4.9%) compared to sham MI (17.9+/-3.14%, 36.6+/-5.3%) (P<0.001) and control animals respectively (13.3+/-5.4%, 17.4+/-6.1%) (P<0.001). The cytotoxic activity against healthy cardiomyocytes was myocyte-specific, induced by CD8 lymphocytes and major-histocompatibility complex (MHC) restricted. Cytotoxic T lymphocytes (CD8) are activated following MI and can recognize and kill normal cardiomyocytes in vitro. The newly described pathophysiological insights may provide novel oportunities to prevent death of non-ischemic cardiomyocytes and heart failure following myocardial infarction.

The effect of low molecular weight heparin (fragmin) on myocardial neutrophil accumulation and infarct size in a rat model of myocardial infarction.

BACKGROUND: Heparin molecules possess immunomodulating properties, which are thought to complement their established antithrombotic activity. The purpose of this study was to evaluate whether the antiinflammatory properties of low molecular weight heparin (LMWH) can attenuate polymorphonuclear neutrophil accumulation and infarct size in a rat model of myocardial infarction. METHODS: Myocardial infarction was induced by ligating the left main coronary artery. LMWH (fragmin 500 anti-FXa u/kg) or vehicle (saline) were administered subcutaneously thirty minutes prior to coronary artery occlusion. Significant anticoagulant activity was attained with LMWH for more than eight hours. Twenty-four hours later, neutrophil accumulation and infarct size were determined by measuring left ventricular free wall myeloperoxidase and residual creatine kinase activity, respectively. RESULTS: As compared with rats administered vehicle, myeloperoxidase activity was insignificantly decreased in rats treated with LMWH (1.24 +/- 0.28 u/g vs 1.66 +/- 0.15 u/g, P = 0.16. Infarct size was also not significantly different between the groups (62.48 +/- 3.5% and 50.67 +/- 7.2% of left ventricular free wall with vehicle and LMWH, respectively, P = 0.1). CONCLUSION: The authors conclude that LMWH does not significantly reduce myocardial neutrophil accumulation and infarct size twenty-four hours after myocardial infarction in the rat.

Exogenous insulin-like growth factor II enhances post-infarction regional myocardial function in swine.

OBJECTIVES: Insulin-like growth factor II (IGF-II) promotes cardiac myocyte growth and contractility in vitro. This study was designed to investigate the effect of exogenous IGF-II on regional myocardial function at the area of infarct in the pig. METHODS: Myocardial infarction was induced in 12 female anaesthetized pigs by affigel blue beads, embolizing microvessels of the left anterior descending coronary artery distribution. In the experimental group (n = 6), IGF-II (0.12 microgram.kg-1 in two animals and 0.6 microgram.kg-1 in four) was incorporated into the beads and delivered by them to the infarct area. Myocardial function was followed echocardiographically, and the excised heart was analysed immunohistochemically and histopathologically. RESULTS: Myocardial function in injured zones, inversely related to an echocardiographic segmental wall motion score (mean +/- SEM), was similar between the two groups at baseline, but at 4 weeks post-infarction was significantly (P = 0.008) reduced in the control group (0.58 +/- 0.38 vs 3.42 +/- 0.84), in contrast to nearly baseline values in the experimental group (0.58 +/- 0.33 vs 1.17 +/- 0.42, P = 0.41). Cardiac performance in injured segments was significantly better after myocardial injury in the experimental group (P = 0.04). Tissue samples from both groups (4 weeks post-infarction), stained with haematoxylin and eosin demonstrated peri-infarct myocyte hypertrophy, corresponding to regions selectively stained by an antibody for CD56, which highlights growing cardiac myocytes. By image analysis semi-quantification, staining for CD56 was significantly (P = 0.04) higher in the peri-infarct region of the experimental group, as compared with controls (106.5 +/- 2.8 vs 92 +/- 4.4 gray level units). Microvessels stained for von-Willebrand factor were similar in number in both groups (P = 0.8), as were mesenchymal cells stained for vimentin (P = 0.7). CONCLUSIONS: Exogenous IGF-II, delivered to the infarct area ameliorates regional cardiac function in the pig, perhaps by inducing peri-infarct myocyte growth.

Noninvasive evaluation of microcirculatory hemodynamic changes during hemorrhage followed by saline or blood transfusion.

The purpose of the study was to examine the ability of a system combining laser Doppler flowmetry (LDF), photoplethysmograph (PPG), and transcutaneous oxygen tension (tc-PO2) to follow changes in the microcirculation during hemorrhage and following blood or saline return, and to test the hypothesis that such changes precede and might predict changes in the systemic blood pressure. Measurements were performed on the skin of anesthetized rabbits (n = 10) during mild (0-8%), moderate (9-24%), and severe (25-30% of blood volume) hemorrhage, and following complete volume restitution by blood or saline. We found the following: 1) hemorrhage caused typical changes in the LDF, PPG, and tc-PO2 signals that could be formulated by mathematical models, 2) these signals identified blood as being more efficient than saline for volume restitution following hemorrhage, and 3) microcirculatory changes precede and might predict systemic hemodynamic events.

A closed-chest pig model of sustained ventricular tachycardia.

The goal of this study was to develop and explore a closed-chest animal model of sustained VT. Seven of 11 domestic pigs had successful induction of myocardial infarction by injection of agarose gel microbeads into the left anterior descending coronary artery through an inflated balloon angioplasty catheter. Four of the first five pigs died and seem to represent a "learning experience." During a 3- to 50-day follow-up period, each pig underwent 1-3 electrophysiological studies. Sustained, monomorphic VT was induced 1-4 times in 5 of the 7 pigs (a total of 19 episodes), was reproducible during the same study in all pigs, and could be repetitively induced during successive studies in some. Ventricular fibrillation was induced less frequently (nine episodes) and was successfully terminated by DC shock in eight episodes. We conclude that a closed-chest pig model of VT is feasible and is associated with a relatively high induction rate of sustained, monomorphic, and reproducible VT and a relatively low mortality rate.

Failure of captopril to attenuate myocardial damage, neutrophil accumulation, and mortality following coronary artery occlusion and reperfusion in rat.

Captopril, a sulfhydryl-containing angiotensin-converting enzyme inhibitor, has been suggested as possessing antiischemic and antiinflammatory properties. To test the hypothesis that captopril may prevent neutrophil-induced myocardial injury during acute myocardial infarction (AMI), the authors subjected rats to coronary occlusion for thirty minutes and reperfusion for twenty-four hours (MI) or to sham operation (sham MI). Oral captopril (100 mg/kg) or vehicle was administered thirty minutes before coronary occlusion. The effect of captopril on mean arterial blood pressure was assessed in separate group of animals (n = 8). Infarct size and neutrophil accumulation in myocardium were determined by measuring creatine phosphokinase depletion and myeloperoxidase (MPO) activity, respectively, in the left ventricular free wall (LVFW). Animals treated with 100 mg/kg of captopril exhibited significant reduction in mean arterial blood pressure compared with vehicle-treated animals (P < 0.01). Compared with vehicle-treated animals, administration of 100 mg/kg of captopril to MI animals attenuated neither twenty-four-hour mortality (56% vs 52%, respectively), nor infarct size (36 +/- 7% vs 34% +/- 7% respectively), nor MPO activity (1.0 +/- 0.17 vs 1.26 +/- 0.19). Thus, in the present experiment captopril did not reduce neutrophil-induced myocardial damage following coronary occlusion and reperfusion. These findings may be partly explained by the negative effect of captopril on arterial blood pressure during AMI.

Electrical injury in the femoral artery of rabbits as a model for arterial thrombosis: a pilot study.

Electrical stimulation was delivered to the femoral artery of 20 rabbits to examine whether endothelial injury results in a consistent formation of arterial thrombosis. The arterial patency was monitored throughout the experiment by flowmeter and was visualized by femoral angiography in 5/20 cases. The arterial segments remained totally occluded in 7/20 (35%), partially occluded in 9/20 (45%), and patent in 4/20 (20%) rabbits following stimulation with 200 microA anodal current for 180 minutes. The average time of electrical stimulation needed to achieve total occlusion (n = 7) was 110 +/- 49 minutes. Alternating occlusion and recanalization of the artery (cyclic flow variation) was observed in 12 rabbits, with total occlusion ensuing in 6/12 cases. Intravenous administration of recombinant tissue-type plasminogen activator (20 micrograms/kg/min for sixty minutes) resulted in femoral reflow and subsequent reocclusion in 2/5 cases. Histopathologic examination disclosed arterial thrombi composed of platelets, fibrin, and red blood cells. Thus, according to these data this technique was found to induce arterial thrombosis following electrical stimulation of the rabbit femoral artery but was inconsistent regarding the arterial patency.

Intracoronary injection of basic fibroblast growth factor enhances angiogenesis in infarcted swine myocardium.

OBJECTIVES: This study was performed to examine the effect of intracoronary exogenous basic fibroblast growth factor (bFGF) on angiogenesis in infarcted myocardial regions. BACKGROUND: Exogenous bFGF is a potent promoter of angiogenesis. Little information is available on its effect on myocardial angiogenesis. METHODS: Myocardial infarction was induced in 10 pigs by intracoronary injection of microscopic beads. Four pigs served as a control group; in six pigs slow-release bFGF was delivered by the beads. Cardiac performance was evaluated by repeated echocardiographic measurement and angiogenesis was evaluated by immunohistochemical studies 14 days later. RESULTS: As compared with control pigs, pigs treated with bFGF had higher microvessel counts (mean +/- SEM) in both viable tissue (141 +/- 27 per field vs. 39 +/- 4, p = 0.01) and nonviable tissue (329 +/- 26 per field vs. 95 +/- 7, p < 0.001) within the infarct area. No significant differences in total regional left ventricular wall motion were noted between the two groups throughout the 14-day study period. CONCLUSIONS: In the swine, direct intracoronary application of bFGF to infarcted myocardium enhances myocardial neovascularization within 2 weeks.