Abstract
Purpose :
Peptide Lv is an endogenous pro-angiogenic peptide that is upregulated in the retina of diabetic animals and patients with early diabetic retinopathy. Although peptide Lv promotes pathological angiogenesis, the mechanisms of its angiogenic action remains unclear. Activation of endothelial potassium channels, including intermediate conductance calcium-dependent potassium (KCa3.1) channels, promotes angiogenesis. We hypothesize that peptide Lv augments endothelial KCa3.1 to promote endothelial proliferation and pathological angiogenesis.
Methods :
Membrane potentials and KCa3.1 currents were recorded from human umbilical vein endothelial cells (HUVEC) treated with peptide Lv (500 ng/ml). TRAM 34 was perfused at the end of each recording to isolate the KCa3.1 current from other outward currents. Western blots for KCa3.1, small conductance calcium-dependent potassium (KCa2.3), and ATP-sensitive potassium (Kir6.1) were performed after HUVECs and human retinal endothelial cells (HRECs) were treated with peptide Lv (500 ng/ml) or PBS (vehicle control) for 4 hours. For testing the endothelial proliferation, HUVECs were seeded onto 24-well plates and allowed to adhere overnight. Peptide Lv (500 ng/ml), VEGF (5 ng/ml), DMH4 (5 µM), TRAM 34 (10 µM) and PBS/0.1% DMSO (vehicle controls) were added to cultures for another 48 hrs. Then the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) colorimetric assays were performed on these cultures. The absorbance at 560 nm was measured using the Chromate microplate reader.
Results :
Endothelial cells treated with peptide Lv had significant increase in the mRNA and protein expression of KCa3.1 but not other types of K channels. Peptide Lv hyperpolarized the endothelial cells and increased the KCa3.1 current densities. Blocking KCa3.1 with TRAM 34 attenuated peptide Lv-elicited endothelial cell proliferation.
Conclusions :
The angiogenic property of peptide Lv is in part through the augmentation of endothelial KCa3.1.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.