Therapy with ribozyme to the proliferating cell nuclear antigen-ribozyme and 5-fluorouracil of experimental choroidal neovascularization in rats.

PURPOSE: The aim of this study was to evaluate the efficacy of hammerhead ribozyme to the proliferating cell nuclear antigen (PCNA-Rz) and 5-fluorouracil (5-FU) in experimental choroidal neovascularization (CNV) model in rats. METHODS: Laser was used to induce CNV in each eye of 44 rats. For angiography studies, injections of either a mixture of PCNA-Rz 10 microg/microL and 5-FU 1.5 microg/microL, versus the same dose of either drug alone versus a control injection of Hanks' Balanced Salt Solution (HBSS) were performed. We also studied this regimen to evaluate scar size and volume. RESULTS: There was significantly less angiographic leakage for the treated eyes compared to the controls by 3.53 grading points (P = 0.0005); CNV leakage was reduced in the combination group compared to 5-FU alone by 1.75 grading units (P = 0.04) and compared to PCNARz by 2.22 grading units (P = 0.07). The scar size and volume were smaller (diameter 354.6 +/- 174.2 microm vs 477.3 +/- 157.0 microm), (thickness 52.7 +/- 43.0 microm versus 79.6 +/- 46.2 microm) with a reduction in scar volume of 44.8%. CONCLUSIONS: Subretinal injection of PCNA-Rz and 5-FU mixture is more effective as treatment of laser-induced CNV, than either drug alone. The majority of the antiangiogenic effect is a result of 5-FU activity with a contribution by the PCNA ribozyme.

Thyrotropin-releasing hormone is induced in the left ventricle of rats with heart failure and can provide inotropic support to the failing heart.

BACKGROUND: We reported previously that left ventricular gene expression for thyrotropin-releasing hormone (TRH) precursor was increased in rats with heart failure 8 weeks after myocardial infarction (MI) and that early ACE inhibition tended to cause further myocardial induction of this gene. METHODS AND RESULTS: Here, we show that after MI, the expression of pro-TRH is induced in the heart coordinately with the protease PC1, an important enzyme in TRH biosynthesis. Pro-TRH gene expression was induced in cardiac interstitial cells after MI, and this effect was restricted to the heart, because no increase in TRH mRNA abundance was observed in the hypothalamus, kidney, or lung. Transcript abundance of pro-TRH can be increased in cultured cardiac fibroblasts by several adrenergic agonists, indicating that the adrenergic axis may play a regulatory role in cardiac TRH production. Acute intravenous administration of TRH to rats with ischemic cardiomyopathy caused a significant increase in heart rate, mean arterial pressure, cardiac output, stroke volume, and cardiac contractility. CONCLUSIONS: Taken together, these results indicate that TRH is specifically induced in the heart after MI and that it can increase cardiac performance in rats with ischemic cardiomyopathy. Thus, in addition to catecholamine and angiotensin II, pro-TRH/TRH may be another important axis that affects hemodynamics and cardiac function in heart failure.

Transcriptional profiling of the transition from normal intestinal epithelia to adenomas and carcinomas in the APCMin/+ mouse.

Mutations in the adenomatous polyposis coli (APC) gene that result in excessive beta-catenin-induced cell signaling are implicated in the risk of colon cancer. Although the mechanism of APC-mediated tumorigenesis is known, the pathways that translate beta-catenin signaling into tumor growth in vivo are undefined. To address this, gene expression profiles of normal intestinal epithelial cells were compared with those from adenomas and carcinomas from APC(Min/+) mice, a model of APC-related colorectal cancer. The gene expression profiles of adenomas and carcinomas were very similar, which is consistent with the theory that carcinomas progress from adenomas in this model system. Tumors had altered transcript abundance for members of several pathways that influence cell growth and proliferation including growth factors/receptors, molecules involved in apoptosis, and protein processing and catabolism enzymes. Comparison of gene expression between adenomas and carcinomas revealed nine differentially expressed transcripts. These included members of three growth-regulating pathways, and the results are consistent with the increased growth potential of carcinomas. SRY-box containing gene 17 (Sox 17), a negative regulator of beta-catenin signaling, and calbindin-D9K, a factor that enhances calcium transport, were more highly expressed in adenomas than carcinomas (approximately 4-fold and 15- to 22-fold, respectively). Transcript abundance for insulin-like growth factor binding protein 5, which mediates insulin-like growth factor function, was 2.6-fold greater in carcinomas. Because the changes in gene expression observed in this study are directly associated with a deficiency in APC, the data provide new insights into how loss of this important tumor suppressor translates into benign and malignant tumor growth.

Time course of skeletal muscle repair and gene expression following acute hind limb ischemia in mice.

DNA microarrays were used to measure the time course of gene expression during skeletal muscle damage and regeneration in mice following femoral artery ligation (FAL). We found 1,289 known sequences were differentially expressed between the FAL and control groups. Gene expression peaked on day 3, and the functional cluster "inflammation" contained the greatest number of genes. Muscle function was depressed for 3 days postligation, but returned to normal by day 7. Decreased muscle function was accompanied by reduced expression of genes involved in mitochondrial energy production, muscle contraction, and calcium handling. The induction of MyoD on day 1 denoted the beginning of muscle regeneration and was followed by the reemergence of the embryonic forms of muscle contractile proteins, which peaked at day 7. Transcriptional analysis indicated that the ischemic skeletal muscle may transition through a functional adaptation stage with recovery of contractile force prior to full regeneration. Several members of the insulin-like growth factor axis were coordinately induced in a time frame consistent with their playing a role in the regenerative process.

The anti-ischemia agent ranolazine promotes the development of intestinal tumors in APC(Min/+) mice.

Ranolazine was shown to improve exercise parameters in patients with chronic angina. It works by switching myocardial energy metabolism from fatty acids to glucose, thus increasing the efficiency of ATP production under hypoxic conditions. Tumors are hypoxic and may also respond to ranolazine. We found that ranolazine caused a dose-dependent increase in tumor number in APC(Min/+) mice, a model of spontaneous intestinal tumorigenesis. Tumors from drug-treated mice were also more dysplastic and invasive than those from untreated mice. These findings have implications for the use of ranolazine in patients with a history of malignant neoplasms or adenomatous polyps.

Effects of early treatment with growth hormone on infarct size, survival, and cardiac gene expression after acute myocardial infarction.

OBJECTIVE: This study examined the effects of growth hormone (GH) on infarct size, survival, and cardiac gene expression in rats with acute myocardial infarction. DESIGN: Animals randomly received sc injection of either saline vehicle (n = 98) or GH (2mg/kg/day, n = 105) for 14 days commencing the day of left coronary artery ligation. Infarct size was determined by morphometric analysis at the time of death or at 52 weeks post-surgery. Gene expression was analyzed by real-time RT-PCR after 2-week treatment. RESULTS: GH decreased infarct size by 18% (P < 0.01) and increased survival by 36% at 52 weeks. GH also significantly reduced cardiac expression of atrial natriuretic factor, beta-myosin heavy chain, alpha-smooth muscle actin, collagen I, collagen III, fibronectin, and pro-inflammatory cytokines. CONCLUSIONS: Treatment with GH for 2 weeks beginning on the day of myocardial infarction produced beneficial effects that were associated with reductions in cardiac gene expression symptomatic of pathological remodeling.

Inhibitory effects of interferon-gamma on myocardial hypertrophy.

Prostaglandin F(2alpha) (PGF(2alpha)) plays an important role in pathologic cardiac growth. After testing several immune cytokines, we found that interferon-gamma (IFN-gamma) inhibited responsiveness of adult myocytes to PGF(2alpha). The present study was designed to test the hypothesis that IFN-gamma inhibits cardiac hypertrophy induced by PGF(2alpha). Incubation of cultured adult rat cardiac myocytes with PGF(2alpha) caused cell spreading, which was inhibited by IFN-gamma. The inhibitory effect was not affected by nitric oxide (NO) synthase inhibitors. In addition, administration of fluprostenol, a more selective agonist at the PGF(2alpha) receptor, induced cardiac hypertrophy in rats. Chronic treatment with IFN-gamma inhibited this myocardial growth, and the inhibitory effect of IFN-gamma was not accompanied by an increase in myocardial NO synthase gene expression. Further, abdominal aortic constriction resulted in a substantial increase in heart, ventricular and left ventricular weights to BW ratio that was significantly attenuated by treatment with IFN-gamma. The results demonstrate that IFN-gamma inhibits the in vitro and in vivo effects of PGF(2alpha) on cardiac hypertrophy, and that the mechanism of action is likely independent of NO production. IFN-gamma also attenuated cardiac hypertrophy induced by pressure overload, suggesting that PGF(2alpha) plays a role in the pathogeneses of this severe type of cardiac hypertrophy.

Early treatment with hepatocyte growth factor improves cardiac function in experimental heart failure induced by myocardial infarction.

Plasma levels of hepatocyte growth factor (HGF) are increased within hours of cardiac ischemia/reperfusion in rats, and HGF has been shown to be cardioprotective toward acute ischemic injury. Myocardial levels of HGF mRNA and protein are increased for several days after myocardial infarction (MI), however, indicating a possible additional protective effect of HGF toward the progression of MI to heart failure. The purpose of this study was to determine whether HGF administration during the time course of endogenous cardiac HGF induction would lead to long-term improvement in cardiac function in rats with MI. MI was induced by 2-h occlusion of the left coronary artery, followed by reperfusion. HGF was given by intravenous infusion at 0.45 mg/kg/day for 6 days beginning on the day after surgery. Cardiac function and hemodynamic parameters were measured by using indwelling catheters and perivascular flow probes in conscious animals 8 weeks post-MI. Myocardial infarcts were approximately 30% of the left ventricle, and there was no difference in infarct size between the vehicle-treated and HGF-treated groups. Compared with untreated sham-operated rats, vehicle-treated MI animals had significantly lower cardiac index and stroke volume index and higher systemic vascular resistance, indicating heart failure developed. Treatment with HGF caused a significant increase in cardiac index and stroke volume index and a reduction in systemic vascular resistance in rats with MI, restoring these parameters close to those observed in sham-operated control animals. These results provide direct evidence that HGF may be of benefit to cardiovascular function in ischemic cardiomyopathy.

Stanniocalcin 1 is an autocrine modulator of endothelial angiogenic responses to hepatocyte growth factor.

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a "stop signal" or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis.