The influence of peripheral circulating monocytes from nAMD and DR patients on endothelial cell migration

Abstract

Purpose : Vascular endothelial growth factor (VEGF) is known to play an important role in both the onset and progression of neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). 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 aimed to investigate whether peripheral blood monocytes from AMD and DR patients could influence the angiogenic behaviours of endothelial cells (EC) in vitro.

Methods : Monocytes were isolated from blood collected from 25 nAMD patients (with or without diabetes), 6 with non-proliferative [NP] diabetic retinopathy (NPDR) and 5 healthy controls using CD14⁺ magnetic bead purification. We undertook an human umbilical vein EC migration assay across a polycarbonate porous membrane (8µm pores) with1nM recombinant human (rh) VEGF-A₁₆₅a with or without 50,000 monocytes and either human IgG or a neutralising antibody to VEGF-A₁₆₅b isoform. EC migration was assessed by counting cells on the abluminal side of the membrane after staining with DAPI.

Results : VEGF-A₁₆₅a induced EC migration in this model. EC migration was significantly enhanced by rhVEGF-A₁₆₅a (156±9% of cells not treated with VEGF-A₁₆₅a). Monocytes from nAMD patients did not significantly alter VEGF-A₁₆₅a induced EC migration (143±10%). Also, no significant difference was found after adding 1or 5 μg/ml anti-VEGF- A₁₆₅b antibody (155±13%, 169±15%) when all patients were considered. But monocytes from diabetic nAMD patients significantly enhanced EC migration (163±22%) compared with those from non-diabetic AMD patients (123±7%) (P<0.05). For NPDR group, monocytes significantly inhibited EC migration (122±23%) (P<0.01) which was reversed by 5 μg/ml anti-VEGF- A₁₆₅b antibody (156±27%) (P<0.01). For healthy people, EC migration was not significantly inhibited (126±24%), and no significant difference was shown after adding 1μg/ml (124±21%) but there was when adding 5μg/ml anti-VEGF- A₁₆₅b antibody (157±27%, p<0.05).

Conclusions : Peripheral circulating monocytes from patients with NPDR inhibit angiogenic behavior in ECs by production of VEGF-A₁₆₅b. In contrast peripheral circulating monocytes from diabetic nAMD patients can enhance angiogenic behavior in endothelial cells.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Monocytes of NPDR patients inhibited HUVECs migration.

 

Moncytes of nAMD with diabetic patients enhanced HUVECs migration.