Abstract
Purpose :
Diabetic retinopathy (DR) results in part from chronically increased expression of vascular endothelial growth factor (VEGF). VEGF can increase the proliferation of endothelial cells (ECs), a critical step in abnormal angiogenesis that contributes to the pathophysiology of DR. We have previously found that dopamine 2 receptor (D2R) activation inhibits VEGF-induced effects on human retinal microvascular ECs (HRMECs) and that HRMECs express DRD1, 2, 3, and 5, but not DRD4. D2R activation normally inhibits the activity of PKA. Here, we explored the mechanisms by which D2R activation suppresses VEGF-induced proliferation of HRMECs.
Methods :
HRMECs (CSC, Inc.), were plated in 10% serum media. Starved (0.5% serum) cells were pretreated as 3 sets, each with a control group (C), VEGF group (V), VEGF + drug group, and drug group (without VEGF). The drugs were quinpirole (Q; 50 nM), 14-22 Amide (PKI; 1 µM), and forskolin (F; 10 µM in DMSO), respectively. The VEGF + drug groups had drug added 15 min prior to VEGF. Proliferation as fold-change was determined using WST-1 assay after 48-hour incubation. Additional HRMECs were grown and were treated as C, V, V + Q, and Q. Protein extracts were collected using RIPA buffer. The ratios of phosphorylated to total protein for VEGFR2, AKT, FAK, and eNOS were determined from the protein extracts.
Results :
VEGF induced proliferation of HRMECs (1.21 ± 0.13 vs 1, normalized to C), which was blocked by Q and PKI treatment (0.96 ± 0.12 and 0.94 ± 0.12 vs 1.21 ± 0.13 respectively, p<0.05). Paradoxically, it was also blocked by F treatment. Treatment with Q, PKI, or F without VEGF had no effect on proliferation compared to control (1.10 ± 0.13, 0.97 ± 0.18, and 1.05 ± 0.01, respectively). Ratios of phosphorylation of specifically probed protein sites compared to total protein for VEGFR2, AKT, FAK, and eNOS, respectively, did not differ in the presence of drug.
Conclusions :
The D2R agonist quinpirole, the PKA inhibitor 14-22 Amide, or the AC activator forskolin each suppressed VEGF-induced HRMEC proliferation to control levels. Q did not suppress VEGF-induced phosphorylation of tested protein angiogenesis mediators. These results suggest not only that specific dopamine receptor activation but also targeting of the cAMP and PKA signaling systems in ECs may be a useful adjunct treatment for blocking VEGF-induced EC activation in DR.
This is a 2020 ARVO Annual Meeting abstract.