The splicing of VEGF-A in monocytes from people with neovascular age related macular degeneration (nAMD) and diabetic retinopathy is altered by inhibition of SRPK1 and CLK

Abstract

Purpose : VEGF-A pre-mRNA is alternatively spliced at exon 8 to generate two families of isoforms: VEGF-A₁₆₅b and VEGF-A₁₆₅a, which are highly expressed by monocytes. We have previously shown that isoform expression is different between patients with nAMD (more VEGF-A₁₆₅a) and proliferative diabetic retinopathy (more VEGF-A₁₆₅b) Splicing has previously been shown to be dependent on the activity of the splicing factor kinases SRPK1 and CLK. We aimed to investigate whether SRPK1 and Clk inhibition have the potential to change the splicing of VEGF-A in peripheral blood monocytes from AMD and DR patients.

Methods : Monocytes were isolated from blood collected from nAMD patients (with or without diabetes), DR patients, PAD (peripheral artery disease) and healthy controls using CD14⁺ magnetic bead purification. Isolated monocytes (1X10⁶) were treated with 1%DMSO, 3μM Sphinx31 (SRPK1 inhibitor) or 3μM of a CLK inhibitor (Hawtrey, TJ, The development of novel chemical scaffolds as potent and selective inhibitors of splicing kinases, PhD Thesis, UNSW, 2018). Protein and RNA was extracted after 16 hours treatment. Elisa and RTPCR was applied to test the concentration of VEGF₁₆₅a and VEGF₁₆₅b.

Results : Overall VEGF₁₆₅b (22.04±4.5) protein secreted by monocytes was higher than VEGF₁₆₅a (9.73±1.73) (P=0.0027), but there was a wide range of VEGF₁₆₅a and VEGF₁₆₅b expression in monocytes (ratio ranged from 0.11-2.2). Overall VEGF₁₆₅b RNA expression (4.65±0.42x10³ relative to GAPDH) was also higher than VEGF₁₆₅a (2.1±0.29) (P<0.01). In monocytes in which the VEGF₁₆₅a RNA was less than 50% that of VEGF-A₁₆₅b, SRPK1 inhibition reduced the ratio of VEGF₁₆₅b to VEGF₁₆₅a (enhancing VEGF₁₆₅a, p<0.01), but CLK inhibition had no effect. In monocytes in which the VEGF₁₆₅a RNA was greater than 50% that of VEGF-A₁₆₅b, CLK inhibition (p<0.05), and SRPK1 inhibition (p<0.01) increased the ratio of VEGF₁₆₅b to VEGF₁₆₅a. In monocytes from patients with nAMD the, ratio of VEGF₁₆₅b and VEGF₁₆₅a RNA was reduced by SRPK1 inhibition (P=0.047).

Conclusions :
In monocytes with low VEGF₁₆₅a expression, SRPK1 inhibition enhances VEGF₁₆₅a. In monocytes with high VEGF₁₆₅a expression, SRPK1 inhibition and CLK inhibition both enhance VEGF₁₆₅b. These results suggest that the effect of splicing factor kinase inhibition in monocytes is dependent on the splicing state of the cell.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.