PKA Inhibition Mimics Dopamine 2 receptor‐driven Inhibition of VEGF‐induced Proliferation of Human Retinal Microvascular Endothelial Cells

Abstract

Diabetic retinopathy (DR), a leading cause of blindness, results in part from chronically increased expression of vascular endothelial growth factor (VEGF) in the eye in the diabetic state. VEGF increases proliferation of endothelial cells, a critical step in abnormal angiogenesis that contributes to the pathophysiology of DR. We have previously found that activation of dopamine receptors, specifically using a D2‐type receptor (D2R) agonist, inhibits VEGF signaling on human retinal microvascular endothelial cells (HRMECs) and that HRMECs express the dopamine receptor genes DRD1, 2, 3, and 5, but not DRD4. D2R activation normally inhibits the activity of PKA via activation of Gi‐alpha subunits. Here, we determined whether inhibition of PKA, the canonical downstream target of D2R‐agonist activation, suppresses VEGF‐induced proliferation of HRMECs.HRMECs, obtained from CSC, Inc. (Kirkland, WA), were plated in CSC 10% serum complete media in 96‐well plates. After overnight starvation in 0.5% serum media, wells were pretreated with either the D2R agonist quinpirole (Q: 50 nM), the PKA inhibitor 14–22 Amide (PKI: 1 μM), or only 0.5% serum media (C). This and all consecutive steps were performed in 0.5% serum media. After 15 min, VEGF (V: 25 ng/mL) was added to some wells pre‐treated with Q or PKI, resulting in the following groups: C, V, V+PKI, V+Q, Q, and PKI. Proliferation as a percent of control was determined using a WST‐1 assay after 48 hours. As anticipated, VEGF induced proliferation of HRMECs compared to control (1.21 ± 0.13 vs 1, normalized values). This proliferation was blocked by either Q or PKI treatment (0.96 ± 0.12 and 0.94 ± 0.12 vs 1.21 ± 0.13 respectively, p<0.05). Treatment with Q or PKI without VEGF had no effect on proliferation compared to control (1.10 ± 0.13 and 0.97 ± 0.18 respectively).In conclusion, application of the D2R agonist quinpirole or of the PKA inhibitor 14–22 Amide, suppressed VEGF‐induced HRMEC proliferation to control levels. These results suggest that targeting not only specific dopamine receptor activation but also cAMP and PKA signaling systems in HRMECs may be a useful adjunct treatment for blocking VEGF‐induced HRMEC activation in DR.Support or Funding InformationNIH R01 EY023290 and UIWRSO Faculty Development Grant