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Joseph F. Arboleda-Velasquez, David Leyton, Santiago Delgado-Tirado, Leo A Kim; Role of PIM3 kinase/Runx1 modulation in aberrant retinal neovascularization. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3560.
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© ARVO (1962-2015); The Authors (2016-present)
Pathologic neovascularization (NV) is a common feature of proliferative diabetic retinopathy (PDR). PIM kinases are a family of proto-oncogenes with serine/threonine activity that have been implicated in apoptosis, cell survival and protein translation. We hypothesize that PIM kinases regulate pathologic NV and are a potential therapeutic target.
Transcriptomes were obtained via RNA-Seq from endothelial cells (ECs) from patient-derived PDR epiretinal membranes (n=8) and compared to normal retinal ECs from donor eyes (n=4). Subsequently, in vitro analysis of human retinal microvascular endothelial cells (HRMECs) exposed to high glucose was performed, and PIM3 protein levels were quantified by Western Blot. Then PIM3 inhibition with AZD1208 (Selleckchem), a small molecule inhibitor of PIM3 kinase activity was characterized. Gene expression, cell migration and cell proliferation assays in HRMEC were evaluated by qRT-PCR, scratch assay and Ki67 staining, respectively. PIM3 siRNA knockdown and PIM3 inhibition (PIM3 inhibitor VII, EMD Millipore) were also performed to evaluate the effect of PIM3 inhibition on Runx1 modulation.
Transcriptome analysis detected a 7-fold increase in PIM3 expression in pathological ECs when compared to normal retinal ECs. Upregulation of PIM3 in HRMECs exposed to high glucose in a dose-dependent manner was found. PIM3 siRNA knockdown revealed an 88% reduction of PIM3 expression compared to controls. Scratch assay showed in HRMECs transfected with PIM3 siRNA, a reduction of 35% in cell migration compared to scramble siRNA (p<0.01). Cell migration was reduced by 39% in HRMECs treated with AZD1208 compared to control (p<0.01). We discovered a 43% reduction in proliferation of HRMECs treated with AZD1208 compared to control (p<0.01). Transfection of HRMECs with siRNA targeting PIM3 significantly reduced both PIM3 and Runx1 expression (p<0.001). PIM3 inhibition with PIM3 inhibitor VII showed a reduction in Runx1 protein levels in HRECs under normal and high glucose conditions.
Our results show an elevated expression of PIM3 in pathologic EC from patient-derived specimens. PIM3 expression is glucose dependent, and regulates EC migration and proliferation. Finally, PIM3 inhibition affects Runx1 expression, a transcription factor linked to retinal NV. Future work will evaluate the role of PIM3 using in vivo models of retinal NV.
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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