Proteomic and Microarray Identification of Novel Cardiac Specific Indicators of Oxidative Injury and Mechanism of Action
Committee ChairChen, Qin
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PublisherThe University of Arizona.
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AbstractCardiovascular disease (CVD) is the leading cause of death in the United States. Oxidative stress plays an important role in the pathogenesis of CVD. Heart failure is the end point of many forms of CVD. The purpose of this study is to identify novel cardiac specific indicators of oxidative injury useful for early and convenient diagnosis of heart failure.To determine the most suitable method for identification of non-invasive oxidative injury indicators in general, human diploid fibroblasts (HDFs) were treated with H2O2 for collection of mRNA, cell lysates and conditioned media to perform cDNA microarray and LC-MS/MS based Multidimensional Protein Identification Technology (MudPIT) analyses. Electron Spray Ionization (ESI)-LC-MS/MS analysis of the conditioned media led to the finding of IGFBP-6 as a non-invasive biomarker of cell oxidative injuy in vitro and in vivo. The data obtained from this study indicate that proteomic analysis of conditioned media is useful to identify non-invasive biomarkers valuable for diagnosis or management of diseases.Cardiomyocyts (CMCs) and Cardiac fibroblasts (CFs) in culture were used to identify cardiac specific indicators of oxidative stress. Increased level of Cystatin C was detected in the conditioned medium of CMCs due to H2O2 treatment. In vivo models of oxidative stress were used to validate the increase of Cystatin C. Cystatin C levels increased in the plasma of mice with doxorubicin induced cardiomyopathy and coronary artery occlusion induced myocardial infarction (MI). These data indicate that Cystatin C can be a potential indicator of CMC oxidative injury in vitro and in vivo.Cystatin C is a cysteine protease inhibitor. The finding that oxidative stress induces Cystatin C led us to investigate a novel pathway regulating cardiac extracellular matrix (ECM) with CFs in culture, increased levels of ECM protein and decreased levels of Cathepsin B (CTB) protein and activity were detected upon Cystatin C treatment. With coronary artery occlusion induced MI mouse model, increased levels of Cystatin C and ECM protein and decreased levels of CTB protein and activity were detected in the infarcted area of the myocardium. These data indicate that Cystatin C serves as a potential fibrotic factor during myocardial remodeling.
Degree ProgramMedical Pharmacology