Resveratrol Inhibits Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Pathological Neovascularization

PURPOSE: To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice. MATERIALS AND METHODS: ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1alpha, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1alpha were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4. RESULTS: In ARPE-19 cells, resveratrol significantly inhibited HIF-1alpha and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1alpha degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner. CONCLUSION: Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization.

Protective Effect of Survivin in Doxorubicin-Induced Cell Death in H9c2 Cardiac Myocytes

BACKGROUND AND OBJECTIVES: Apoptosis has been known to be an important mechanism of doxorubicin-induced cardiotoxicity. Survivin, which belongs to the inhibitor of apoptosis protein family, is associated with apoptosis and alteration of the cardiac myocyte molecular pathways. Therefore, we investigated the anti-apoptotic effect and cellular mechanisms of survivin using a protein delivery system in a doxorubicin-induced cardiac myocyte injury model. MATERIALS AND METHODS: We constructed a recombinant survivin which was fused to the protein transduction domain derived from HIV-TAT protein. In cultured H9c2 cardiac myocytes, TAT-survivin (1 microM) was added for 1 hour prior to doxorubicin (1 microM) treatment for 24 hours. Cell viability and apoptosis were evaluated by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, caspase-3 activity, and terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay. We measured the expression levels of several apoptosis-related signal proteins. RESULTS: The survivin level was significantly reduced in a dose dependent manner up to 1 microM of doxorubicin in concentration. Purified recombinant TAT-survivin protein was efficiently delivered to H9c2 cardiac myocytes, and its transduction showed an anti-apoptotic effect, demonstrated by reduced caspase-3 activity and the apoptotic index, concomitantly with increased cell viability against doxorubicin injury. The phosphorylation of p38 mitogen-activated protein (MAP) kinase and the release of Smac from mitochondria were suppressed and the expression levels of Bcl-2 and cAMP response element-binding protein (CREB), the transcription factor of Bcl-2, were recovered following TAT-survivin transduction, indicating that survivin had an anti-apoptotic effect against doxorubicin injury. CONCLUSION: Our results suggest that survivin has a potentially cytoprotective effect against doxorubicin-induced cardiac myocyte apoptosis through mechanisms that involve a decrease in the phosphorylation of p38 MAP kinase, mitochondrial Smac release, and increased expression of Bcl-2 and CREB.

Cathepsin L derived from skeletal muscle cells transfected with bFGF promotes endothelial cell migration

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.

Heat shock protein 90 regulates IkappaB kinase complex and NF-kappaB activation in angiotensin II-induced cardiac cell hypertrophy

Heat shock protein 90 (HSP90), one of the most abundant proteins in the cardiac cells is essential for cell survival. Previous studies have shown that angiotensin II induces cardiac cell hypertrophy. However, the role of HSP90 in the angiotensin II-induced cardiac hypertrophy is unclear. In this study, we showed that HSP90 regulated angiotensin II-induced hypertrophy via maintenance of the IkappaB kinase (IKK) complex stability in cardiac cells. An HSP90 inhibitor, geldanamycin (GA), significantly suppressed angiotensin II-induced [3H]leucine incorporation and atrial natriuretic factor expression in cardiac cells. GA also inhibited the NF-kappaB activation induced by angiotensin II. Importantly, treatment with GA caused a degradation of IKKalpha/beta; on the other hand, a proteasome-specific inhibitor restored the level of IKKalpha/beta. We also found that GA prevented HSP90-IKKs complex induced by angiotensin II in cardiac cells. The small interfering RNA (siRNA)-mediated knockdown of HSP90 expression significantly inhibited angiotensin II-induced cell hypertrophy and NF-kappaB activation. These results suggest that angiotensin II-induced cardiac hypertrophy requires HSP90 that regulates the stability and complex of IKK.

Cardiac expression profiles of the naked DNA vectors encoding vascular endothelial growth factor and basic fibroblast growth factor

We investigated expression profiles and biological effects of the naked DNA vectors in the heart. To this end, naked DNA vector was injected into the apex of the beating rat heart after thorocotomy. When the expression of LacZ reporter was examined by reverse transcription-PCR and histochemical staining for b-galactosidase, LacZ expression was detected only in the heart, suggesting limited dissemination of the injected vector in vivo. Even within the heart, LacZ expression was limited to the injection area (apex). Similar observations were made with other transgenes such as VEGF and basic fibroblast growth factor (bFGF), where 77% and 69% of the total transgene exprssion were detected in the heart segments containing the apex. Although VEGF and bFGF expressions were detected until 2 weeks after DNA injection, the highest levels of VEGF and bFGF were observed on day 5 and day 1, respectively. The optimal doses of the vectors were 10 mg and 25 mg for the VEGF and bFGF vectors, respectively. Interestingly, injection of bFGF vector led to 50% increase in the level of endogenous murine VEGF expression. Consistent with this finding, the number of vessels that stained positive for alpha-smooth muscle actin was increased in the bFGF vector-injected heart. These results suggest that simple injection of naked DNA vector may be sufficient to induce significant angiogenesis in the myocardium and that naked DNA gene therapy may be a feasible approach for the treatment of ischemic heart disease.