Browsing by Subject "Insulin-Like Growth Factor I"
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Item The Impact of Diurnal Changes and Inter-Visit Variability on the Concentration of Insulin-Like Growth Factor-1 in Human Tears(2015-01-26) Patel, Roshni; Zhu, Meifang; Robertson, Danielle M.INTRODUCTION: There is a growing body of research focused on the use of tear film-derived proteins as biomarkers of disease. Previous studies have reported quantitative changes in tear-derived growth factors and related proteins associated with various systemic and ocular diseases. Major challenges when working with human tears however, includes sample volume limitation and the high potential for reflex tearing. One method of tear collection that is increasingly being reported involves the use of microcapillary tubes to draw tears from the inferior tear reservoir. The purpose of this study was to investigate the impact of diurnal changes and inter-visit variability on the concentration of a known growth factor present in human tears, the insulin-like growth factor-1 (IGF-1). METHODS: Nine healthy volunteers without any reported symptoms of dry eye were recruited for this study. At visit 1 (baseline), all participants underwent a standard dry eye examination to assess tear volume, tear film break up time (TFBUT), and tear production. Subjects were asked to return to clinic for an additional 5 visits (morning and afternoon on a total of 3 days). Tears were collected at the start of each visit from the inferior temporal tear meniscus of both eyes using 1 - 10 μl glass microcapillary tubes and frozen at -80C until use. Total protein was measured for each patient using a bicinchoninic assay. IGF-1 levels wear assessed using ELISAs. RESULTS: 8.8 ± 2.1 μg/μl of total protein was obtained from each subject. Total protein was unchanged at each visit. There was no difference in IGF-1 between morning and afternoon. Tear levels of IGF-1 did vary with visit, with the final visit showing a 2 fold-increase over baseline (p<0.05). Tear levels of IGF-1 were correlated with TFBUT (R=0.856, p=0.007). DISCUSSION: While diurnal variation did not affect basal levels of IGF-1 in tears, there was a visit-dependent increase. This increase was likely due to a reduction in reflex tearing during tear collection as patients became more comfortable with the technique. Similarly, the decrease in IGF-1 that corresponded with increased tear evaporation was likely due to changes in reflex tearing. Together, these findings suggest that low abundant proteins, such as IGF-1, are highly susceptible to changes in reflex tearing. These findings also suggest that a participant training phase may be required.Item Proteomic Discovery of Functionally Important Pathways in Myocardial Ischemia-Reperfusion Injury(2016-04-01) Ahmed, Kamran; Hill, Joseph A.; Rosenzweig, Anthony; Munshi, Nikhil; Sadek, Hesham A.BACKGROUND: Coronary heart disease, a source of myocardial ischemia-reperfusion injury (IRI), is the world's leading cause of death and disability. Insulin-like growth factor 1 (IGF1) transgenic (Tg) mouse hearts are protected from IRI, whereas Akt-Tg mouse hearts recover poorly from IRI. Surprisingly, Akt is a downstream component of IGF1 signaling. The Akt-Tg phenotype can be rescued by cardiac gene transfer of activated PI3-kinase (PI3K), another component of the IGF1 pathway, suggesting that PI3K-dependent but Akt-independent pathways are key determinants of IRI. To discern such pathways, we analyzed the proteomic and phosphoproteomic changes in wild-type (WT) mouse hearts subjected to IRI ex vivo, and IGF1-TG and Akt-Tg mouse hearts in order to identify 20 differentially regulated candidates as potential modifiers of IRI, and began testing their functional roles in an in vitro model. OBJECTIVE: We hypothesize that the cardioprotection observed in IGF1 overexpression is a result of PI3K-dependent but Akt-independent signaling pathways. METHODS: WT hearts were collected at 4 time points of ex-vivo Langendorff IRI and analyzed with liquid chromatography-tandem mass spectrometry to determine protein abundance and phosphorylation changes. IGF1-Tg and Akt-Tg hearts were analyzed at baseline. Protein network analysis was performed using Cytoscape software. The functional effects of candidates with abundance or phosphorylation differences ≥2-fold were assessed in rat neonatal ventricular myocytes using in vitro redox-based viability assays and cell proliferation studies. RESULTS: In the WT IRI studies, 6403 proteins and 22833 phosphopeptides were quantified. During IRI, no proteins changed in abundance, 10 phosphopeptides were upregulated, and 330 phosphopeptides were downregulated. In the IGF1-Tg and Akt-Tg hearts, 6700 proteins and 23000 phosphopeptides were quantified. Out of the significantly regulated proteins, in vitro knockdown of rho-associated protein kinase 2 (ROCK2) increased the viability signal by 17% in normoxia and 33% in simulated IRI (p<0.05) and increased EdU incorporation from 28.9% to 40.15% (p<0.00001). Network analysis of Akt-Tg hearts revealed significant downregulation of 24 out of 45 subunits of Complex I of the electron transport chain (p<0.05). CONCLUSION: Dephosphorylation of the cardiac phosphoproteome is the dominant pattern in IRI, which may reflect phosphatase activation or reduced ATP levels inhibiting kinase activity. ROCK2 knockdown increased the viability signal by stimulating proliferation in vitro. Whether ROCK2 is involved in cardiomyogenesis in the adult heart will be addressed in future studies. Akt-Tg hearts may be susceptible to IRI due to a reduced ATP reserve caused by Complex I downregulation.Item Regulation of the Insulin-like Growth Factor 1-Secretory Clusterin Expression Axis in Genomic Instability and Cell Stress(2009-09-04) Goetz, Eva Marie; Boothman, David A.Secretory clusterin (sCLU) is a pro-survival factor that is up-regulated in human tumors and after exposure to cell stress. Understanding the regulation of sCLU expression in cancer, and after exposure to therapeutic agents, could reveal new therapeutic targets for cancer treatment. A DNA damage induced signaling cascade leading from ATM to sCLU expression mediated by IGF-1/IGF-1R/MAPK activation was uncovered. IGF-1 ligand promoter activity, mRNA, and protein expression induced after exposure to ionizing radiation (IR), hydrogen peroxide, or topoisomerase I and II-alpha poisons matched sCLU expression. Elevated basal IGF-1-sCLU signaling was noted in genomically unstable cells, whether they were deficient in DNA repair factors or telomerase function. ATM function was necessary for induction of sCLU after IR, and for maintaining elevated expression of sCLU in genomically unstable cells. p53 suppressed IGF-1 promoter activity, leading to decreased mRNA and protein expression, and abrogated induction of IGF-1 and sCLU by IR. Loss of p53 by knockdown or knockout enhanced IGF-1 and sCLU induction. Mutations in the p53 DNA binding domain found in cancer did not repress IGF-1 and sCLU. An NF-Y binding site in the IGF-1 promoter was essential for p53 suppression, and both p53 and NF-YA bound to the IGF-1 promoter. Nutlin-3, an Mdm2-p53 inhibitor, stabilized p53 expression, leading to dramatically decreased sCLU expression. Nutlin-3 treatment sensitized wild-type p53 cells to IR exposure. Finally, exogenous IGF-1 exposure led to serine 1981 auto-phosphorylation of ATM, and enhanced DNA damage repair and abrogated cell death after IR exposure. These studies uncovered key molecules important for the regulation of IGF-1-sCLU expression axis after IR exposure, and supported the use of IGF-1 or sCLU expression inhibitors for cancer chemotherapy.