Our researach focuses on the molecular mechanisms of oxidative stress and pharmacological agents of cytoprotection. Oxidants are byproducts of aerobic metabolism. The level of oxidants increases as a result of radiation, intoxication of certain xenobiotics and disease states involving ischemic reperfusion or inflammatory response. Oxidative stress has been shown to contribute to aging, cancer and heart disease. Our research projects are: 1) Molecular Mechanisms of Oxidative Stress Response using human fibroblasts as an experimental model system. We characterize genes and proteins that are upregulated or altered by oxidants using genomic and proteomic approaches to identify critical molecules that control a series of cellular changes resulting from oxidant exposure such as premature senescence and apoptosis; 2) The Role of Oxidative Stress in Heart Failure. Pathological analyses often reveal apoptotic cardiomyocytes, hypertrophy of remaining cardiomyocyte, and fibrosis or hyperplasia of fibroblasts in failing hearts. We isolate cardiomyocytes and fibroblasts from the heart of experimental animals to determine the cellular and molecular changes that are produced by oxidants in these cell types.Our studies indicate that oxidants can induce hypertrophy of cardiomyocytes in culture. Thorough analyses indicate a role of phosphatidylinositol 3 kinase, MAP kinases, AP-1 transcription factors, and cyclooxygenase-2 in oxidant-induced cardiomyocyte hypertrophy. In contrast to cardiomyocyte hypertrophy, fibrosis is a disease involving proliferation of fibroblasts and changes in the expression of extracellular matrix proteins and secreted proteases by fibroblasts. Genomic profiling, proteomic mining and transcription factor measurements are ongoing to determine the influence of cell type on molecular and cellular changes induced by oxidants and to search for critical targets that play a role in fibrosis or cardiomyocyte hypertrophy associated with oxidative stress.An important emphasis of our laboratory is searching for pharmacological agents that serve as cytoprotectants. Recent studies have pointed to a role of apoptosis in heart failure associated with cardiomyopathy and myocardial infarction. Our laboratory has found that corticosteroids can prevent cardiomyocytes to undergo apoptosis in vitro. Genomic and proteomic analyses show elevation of certain antioxidant proteins and anti-apoptotic proteins induced by corticosteroid. Currently we are exploring the transcription factors regulating cell survival responses and validating our in vitro findings with in vivo experimental models of heart failure.
Inhibition Of Apoptosis By Progesterone In Cardiomyocytes. Source: Aging Cell
While gender-based differences in heart disease have raised the possibility that estrogen (ES) or progesterone (PG) may have cardioprotective effects, recent controversy regarding hormone replacement therapy has questioned the cardiac effects of these steroids. Using cardiomyocytes, we tested whether ES or PG has protective effects at the cellular level. We found that PG but not ES protects cardiomyocytes from apoptotic cell death induced by doxorubicin (Dox). PG inhibited apoptosis in a dose-dependent manner, by 12 ± 4.0% at 1 μm and 60 ± 1.0% at 10 μm. The anti-apoptotic effect of PG was also time dependent, causing 18 ± 5% or 62 + 2% decrease in caspase-3 activity within 1 h or 72 h of pretreatment. While PG causes nuclear translocation of its receptor within 20 min, the cytoprotective effect of PG was canceled by mifepristone (MF), a PG receptor antagonist. Analyses using Affymetrix high-density oligonucleotide array and RT-PCR found that PG induced Bcl-xL, metallothionine, NADPH quinone oxidoreductase 1, glutathione peroxidase-3, and four isoforms of glutathione S-transferase. Western blot analyses revealed that PG indeed induced an elevation of Bcl-xL protein in a dose- and time-dependent manner. Nuclear run-on assay indicated that PG induced Bcl-xL gene transcription. Inhibiting the expression of Bcl-xL using siRNA reduced the cytoprotective effect of PG. Our data suggests that PG induces a cytoprotective effect in cardiomyocytes in association with induction of Bcl-xL gene.<br><br>
Cystatin C Increases In Cardiac Injury: A Role In Extracellular Matrix Protein Modulation. Source: Cardiovascular Research
AIMS:<br>Numerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology.<br><br>METHODS AND RESULTS:<br>Cardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H(2)O(2) for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H(2)O(2) caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III.<br><br>CONCLUSION:<br>Oxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.<br><br>