Endothelial cell growth supplement (ecgs)

All reagents were purchased from Sigma (Oakville, Ontario, Canada) unless specified. Human retinal microvascular endothelial cells (HRECs) were obtained from Olaf Pharmaceuticals. HRECs were grown in endothelial cell basal medium 2 (EBM‐2, Lonza) supplemented with 5% fetal bovine serum, endothelial cell growth supplement (Bio‐Whittaker), and 100 μg/ml penicillin/streptomycin. Cells were plated at a density of 1 × 10⁵ cells/ml. All cells were maintained in a humidified atmosphere containing 5% CO₂ at 37°C incubation. The cells were cultured in six‐well plates (Corning) and were treated with various concentrations of D‐glucose (5 mmol/L, representing normal glucose [NG], or 25 mmol/L, representing high glucose [HG]) or 25 mmol/L L‐glucose (osmotic control) for 48 hr. As for the miRNA‐based experiments, cells were transfected with either a miRIDIAN miRNA antagomir for miR‐1, miR‐19b, miR‐320a or a negative control miRNA (100 nM for each miRNA) (Thermo Fisher Scientific) using the transfection reagent Lipofectamine 2000 (Invitrogen). After transfection for 24 hr, the cells were changed into serum‐free media and treated with HG for another 48 hr. For the resveratrol experiments, cells were treated with 10 µM resveratrol (Sigma) dissolved in ethanol and cultured in HG media for 48 hr (Chen et al., 2010; Feng et al., 2016; Mortuza et al., 2013).

Human lung microvascular endothelial cells (HL-MVEC), cell medium and endothelial cell growth supplement were purchased from Lonza (East Rutherford, NJ). Cells were cultured in humidified 5% CO₂ at 37°C using the appropriate culture medium.

KYOUDXR0109B Human Induced Pluripotent Stem Cells (hiPSCs) [201B7] (ATCC^® ACS1023™) cell line 201B7 were purchased from the American Type Culture Collection (ATCC) and cultivated in a feeder-free system in the presence of laminin (LN)-511 E8 fragments (iMATRIX-511, Tokyo, Japan) and animal component-free culture medium (TeSR-E8; Stem cell technologies, Cologne, Germany).
Organ cultured human corneoscleral tissues (n = 25) were obtained from the LIONS Cornea Bank Baden-Württemberg, Eye Center, University Medical Center Freiburg. Informed consent for research use of tissue remnant after transplant excision had been given by the donors or their next of kin. To isolate primary limbal cells, organ cultured corneoscleral rims remaining after penetrating keratoplasty were used (n = 25, Supplementary Table S1). The study was approved by the institutional review board of the University of Freiburg (25/20) and followed the tenets of the Declaration of Helsinki. Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Basel, Switzerland) and were cultured in endothelial growth medium (EGM) containing endothelial basal medium and endothelial cell growth supplement (Lonza, Basel, Switzerland).
All the experiments were done with multiple batches of LEPC independently derived from 201B7 cell lines.

HUVECs (CC-2519; Lonza, Basel, Switzerland) were cultured in DMEM/F12 medium (11320033; Gibco™), supplemented with antibiotics, endothelial cell growth supplement (50 μg mL⁻¹; Lonza) and 10% FBS (16000044; Gibco™) until confluency. HUVECs at passages four to seven were used for experiments. HUVECs were transfected with miR-181b mimics (20 nmol L⁻¹), miR-181b inhibitor (50 nmol L⁻¹) or negative control (50 nmol L⁻¹) by using Lipofectamine RNAiMax (Invitrogen) following the manufacturer’s instructions. The cells were transfected and cultured for 24 h before pharmacological treatment. For pharmacological incubation, HUVECs were incubated with AGEs (200 μg mL⁻¹, 12 h), the AMPK activator AICAR (2 mmol L⁻¹, 4–12 h), and the AMPK inhibitor 9-beta-d-arabinofuranoside (ara-A; 1 mmol L⁻¹, 12 h; Sigma-Aldrich).