Metabolic plasticity in retinal microvascular endothelial cells exposed to diabetes-related conditions in vitro

Abstract

Purpose : Endothelial cells have a remarkable dependency on glycolytic metabolism although the degree of flux through this pathway in some non-retinal endothelial cell-types has been shown to vary in disease conditions. In the context of diabetic retinopathy (DR), retinal microvascular endothelial cells experience increasing hyperglycemia as disease progresses. This study has assessed the nature of glycolysis and related plasticity in human retinal microvascular endothelial cells (HRMEC) exposed to in vitro conditions relevant to DR.

Methods : HRMEC were obtained from human donors and the expression of glycolysis-related gene transcripts assessed by quantitative PCR. HRMECs were also exposed to 25 mM D-glucose (or 25 mM L-glucose as a control) for up to 24 hours. Metabolic flux based on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was assessed using the Seahorse XF analyser. To understand the in vitro angiogenic potential, proliferation, and tubulogenesis assays were conducted. The B3 isozyme phosphofructokinase-2/fructose-2,6-bisphosphatase (PFKFB3) is a key regulator of glycolysis in endothelial cells (De Bock et al. Cell 2013 Aug 1;154(3):651-63) and its role in HRMECs was assessed using the inhibitor 3PO ((2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one).

Results : A range of glycolysis-related transcripts are expressed in HRMECs including PFKFB3, ALDO A, PGK1, and PGAM4. When compared to L-glucose controls, 25mM D-glucose treated HRMECs demonstrated a decrease in glycolytic ATP production capacity from 320.2 to 134.2 pmol/min (P ≤ 0.05), as well as reduced mitochondrial ATP production capacity from 57.4 to 16.8 pmol/min (P ≤ 0.05). Furthermore, a reduction in OCR from 22 to 7.4 (pmol/min) was observed and ECAR was reduced from 44.9 to 17.21 (mpH/min). 25mM D-glucose -exposed HRMECs displayed decreased tubulogenic capacity (P ≤ 0.01), along with decreased proliferative potential (P≤ 0.001). In addition, HRMEC exposed to 3PO showed impaired glycolysis with a concomitant decrease in their ability to replicate (P≤ 0.001).

Conclusions : HRMEC show glycolytic plasticity following in vitro exposure to high glucose conditions. The glycolysis regulatory enzyme PFKFB3 is active in HRMECs and regulates metabolism and key functional readouts in diabetic-like conditions.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.