Abstract
Purpose :
Epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is critical event in proliferative vitreoretinopathy (PVR). Presently, no pharmacotherapies have proven efficacious in PVR. Pyruvate kinase 2 (PKM2) a key regulatory enzyme of glycolysis, exhibits metabolic and non-metabolic functions driven by its oligomeric state. Importantly, PKM2 is implicated in cellular glycolytic reprogramming, which is an important hallmark of RPE EMT. Chemical modulator, ML-265 induces PKM2 tetramerization and mitigates glycolytic reprogramming. Here, using a human primary RPE culture model of EMT, we tested the necessity of PKM2 in the initiation and maintenance of RPE EMT and the therapeutic potential of ML-265 in the treatment of PVR.
Methods :
To mimic loss of cell contact observed in PVR and stimulate EMT, primary human fetal RPE (hfRPE) were seeded at 10% density. Expression of EMT markers, PKM2, and mediators of glycolytic reprogramming and proliferation were assessed. We assessed role of PKM2 RPE EMT by transient knockdown(siRNA) and permanent knockdown (shRNA). An enzyme-coupled assay measured PK activity. The oligomeric state of PKM2 was assessed via native polyacrylamide gel electrophoresis. The therapeutic potential of ML-265 was evaluated using several in vitro assays. ML-265 toxicity was assessed via transepithelial electrical resistance (TEER).
Results :
Low seeding density of hfRPE demonstrated a fibroblastic-like PVR phenotype (hfRPE EMT) and induction of EMT markers. Along with PKM2 (but not PKM1) we observed increased expression of key transcription factors MYC and HIF-1a. Knockdown of PKM2 in hfRPE EMT induced cell death. Despite increased PKM2 expression, PK activity was reduced in hfRPE EMT and accordingly, there was a shift away from the high activity tetramer to the low activity PKM2 dimer. ML-265 shifted the PKM2 oligomeric state towards the tetramer and increased PK activity in hfRPE EMT. ML-265 decreased the proliferation of EMT hfRPE cells, expression of MYC and HIF-1a, inhibited EMT hfRPE-mediated gel contraction with a reduction in α-SMA expression, and reduced EMT-induced hfRPE cell migration in a wound healing assay. Exposure to ML-265 was non-toxic in well- differentiated hfRPE.
Conclusions :
We demonstrate that PKM2 is a critical regulator of RPE EMT and pharmacologically inducing PKM2 tetramerization may be an innovative therapeutic strategy for PVR.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.