Does complement factor 5a induce endothelial production of soluble vascular endothelial growth factor receptor-1 and could this cause proteinuria?

Broeren, S.G.M. (Stefan) (2014) Does complement factor 5a induce endothelial production of soluble vascular endothelial growth factor receptor-1 and could this cause proteinuria? thesis, Medicine.

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Abstract

Introduction: Vascular endothelial growth factor (VEGF) is the dominant growth factor for blood vessels and is presumably crucial for maintaining glomerular health. VEGF receptor-2 (VEGFR-2) mediates the angiogenic effects of VEGF, while VEGFR-1 prevents VEGF from interacting with VEGFR-2 and is therefore anti-angiogenic. Soluble VEGFR-1 (sVEGFR-1) is a non-membrane-bound form of VEGFR-1. It can either be produced directly by synthesis or indirectly by shedding of VEGFR-1. There are two known condition in which reduced availability of VEGF leads to proteinuric kidney disease: (1) in preeclampsia, where VEGF is sequestered by sVEGFR-1; and (2) in cancer treatment with anti-VEGF antibodies. In animal studies VEGF deprivation also causes proteinuria. In various animal models, inhibition of the complement system decreases serum sVEGFR-1 concentration and increases angiogenesis, while administration of exogenous complement factor 5a (C5a) has opposite effects. Here we investigate whether C5a induces endothelial production of sVEGFR-1. This could mean that glomerular endothelium contributes to inflammatory proteinuric kidney disease by secreting sVEGFR-1. Material and methods: Three different isolates of human umbilical vein endothelial cells (HUVEC) were acquired. Cells were incubated for 6 hours with different concentrations of C5a (0 – 25 nM) or for different amounts of time (0 – 9 hours) with 6.25 nM C5a. Afterwards the medium was stored and RNA was isolated from the cells. The HUVEC were not serum starved before the experiments. The sVEGFR-1 concentration in the experimental medium was determined by ELISA. VEGFR-1 and sVEGFR-1 expression was determined with quantitative PCR (qPCR). Statistical analysis was done with one-way repeated measurements ANOVA with post-hoc analysis for linear trend. Results: A significant, but small effect of C5a concentration on sVEGFR-1 secretion was found (p < 0.05, R2 ≈ 0.11). Increasing duration of stimulation increased sVEGFR-1 concentration significantly (p < 0.001, R2 ≈ 0.84). In qPCR analysis no effect of C5a concentration on either VEGFR-1 or sVEGFR-1 expression was found. Conclusion: C5a had a significant effect on the sVEGFR-1 secretion. The effect was small compared to secretion without C5a stimulation and duration of stimulation had a much larger effect on sVEGFR-1 concentration. This suggests continuous secretion of sVEGFR-1 by HUVEC and a small augmenting effect of C5a on this secretion. No effect of C5a concentration on gene expression was found. The latter could mean that shedding of VEGFR-1 is the more important mechanism of sVEGFR-1 secretion.

Item Type:	Thesis (Thesis)
Supervisor name:	Seelen, Dr M. and Experimental Nephrology and University Medical Centre Groningen
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 11:03
Last Modified:	25 Jun 2020 11:03
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/2327

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