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Susmit Suvas, Pushpa Rao; Hypoxia associated glycolytic reprogramming in the pathogenesis of herpes stromal keratitis (HSK). Invest. Ophthalmol. Vis. Sci. 2018;59(9):518. doi: https://doi.org/.
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We previously reported the development of hypoxia in HSK lesions. In this study, we detemined the expression of hypoxia associated glycolytic genes in HSK lesions, and tested if in vivo blocking of transcriptional activity of hypoxia inducible factor (HIF) reduces the severity of HSK lesions.
Expression of hypoxia associated glycolytic genes in HSK lesions were detected by PCR Array and real time RT-PCR assay. Confocal microscopy and flow cytometry was carried out to determine the localization and cell specific expression of hypoxia associated glycolytic protein and HIF molecules in corneas with HSK lesions. Corneal opacity and angiogenesis was measured with hand-held slit lamp, and the extent of glycolysis in HSK lesions was measured by colorimteric lactate assay.
PCR-array of hypoxia associated gene was carried out on naive and HSV-1 infected corneas (5-day and 10-day post-infection). Our results showed several fold increase in the expression level of genes involved in promoting glycolysis such as GLUT1, GLUT3, MCT4, HK2, PFKB3, and PFKP in corneas with HSK lesions on 10-day post-infection. Real time RT-PCR assay confirmed the results obstained from PCR Array assay. The functional involvement of these genes in promoting glycolysis in HSK lesions was determined by measuring the level of lactate in HSK lesions. Our results showed significant increase in the amount of lactate in the corneas with HSK lesions. Many of hypoxia associated genes involved in glycolytic reprograming are transcriptionally regulated by HIF-1α or HIF-2α. Hypoxia is known to cause the stabilization of HIF. Our confocal microscopy results showed the stabilization and nuclear localization of HIF-2α in eptihelial cells of the corneas with HSK lesions. On the other hand, flow cytometric analysis showed HIF-1α accumulation in myeloid, neutrophils and CD4 T cells in HSK lesions. Lastly, in vivo blocking of HIF transcriptional activity reduced the expression of glycolysis associated genes in infected corneas, and reduced the severity of HSK lesions.
Development of hypoxia in HSK lesions are associated with an increased glycolysis, which is mediated by hypoxia inducible factors, and blocking of HIF transcriptional activity reduced the expression of glycolytic genes in infected corneas and thus reduced the severity of HSK.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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