VEGF regulates local inhibitory complement proteins in the eye and kidney
Lindsay S. Keir, … , Moin A. Saleem, Martin Friedlander
Lindsay S. Keir, … , Moin A. Saleem, Martin Friedlander
Published December 5, 2016
Citation Information: J Clin Invest. 2017;127(1):199-214. https://doi.org/10.1172/JCI86418.
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Research Article Nephrology Ophthalmology

VEGF regulates local inhibitory complement proteins in the eye and kidney

Abstract

Outer retinal and renal glomerular functions rely on specialized vasculature maintained by VEGF that is produced by neighboring epithelial cells, the retinal pigment epithelium (RPE) and podocytes, respectively. Dysregulation of RPE- and podocyte-derived VEGF is associated with neovascularization in wet age-related macular degeneration (ARMD), choriocapillaris degeneration, and glomerular thrombotic microangiopathy (TMA). Since complement activation and genetic variants in inhibitory complement factor H (CFH) are also features of both ARMD and TMA, we hypothesized that VEGF and CFH interact. Here, we demonstrated that VEGF inhibition decreases local CFH and other complement regulators in the eye and kidney through reduced VEGFR2/PKC-α/CREB signaling. Patient podocytes and RPE cells carrying disease-associated CFH genetic variants had more alternative complement pathway deposits than controls. These deposits were increased by VEGF antagonism, a common wet ARMD treatment, suggesting that VEGF inhibition could reduce cellular complement regulatory capacity. VEGF antagonism also increased markers of endothelial cell activation, which was partially reduced by genetic complement inhibition. Together, these results suggest that VEGF protects the retinal and glomerular microvasculature, not only through VEGFR2-mediated vasculotrophism, but also through modulation of local complement proteins that could protect against complement-mediated damage. Though further study is warranted, these findings could be relevant for patients receiving VEGF antagonists.

Authors

Lindsay S. Keir, Rachel Firth, Lyndsey Aponik, Daniel Feitelberg, Susumu Sakimoto, Edith Aguilar, Gavin I. Welsh, Anna Richards, Yoshihiko Usui, Simon C. Satchell, Valeryia Kuzmuk, Richard J. Coward, Jonathan Goult, Katherine R. Bull, Ruchi Sharma, Kapil Bharti, Peter D. Westenskow, Iacovos P. Michael, Moin A. Saleem, Martin Friedlander

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Figure 3

VEGF-induced changes in CFH were mediated by PKC signaling via CREB.

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VEGF-induced changes in CFH were mediated by PKC signaling via CREB. RPE...
RPE cells treated with either PKC (GF109203X) or PKA inhibitor (H-89) before treatment with VEGF failed to produce an increase in CFH RNA transcript when compared with VEGF treatment alone. This was comparable to the effect of bevacizumab and VEGF treatment (A, n = 4). Inhibiting p38 MAPK (SB202190) prior to VEGF stimulation had no effect on the increase in CFH transcript. VEGF caused greater phosphorylation of the CREB transcription factor (top panel), while anti-VEGF treatment reduced this phosphorylation (B, n = 4). Total CREB was also measured in these samples (lower panel). Human RPE cells and podocytes treated with the PKC activator (PMA) or PKA/adenylate cyclase activator (forskolin) showed increased CFH transcript compared with control (C, n = 3). The effect was more pronounced with PKC activation. Human podocytes were then treated with PKC inhibitor (GF109203X) or bevacizumab prior to VEGF stimulation, and they also showed reduced CFH RNA compared with VEGF treatment alone (D, n = 3). Podocytes with a CRISPR-induced knockout of PKC-α showed reduced CREB phosphorylation after VEGF stimulation compared with control cells (E, n = 3), showing that podocyte VEGF stimulation produced CREB phosphorylation via PKC-α. Finally, podocytes with a CRISPR-induced knockout of CREB showed significantly reduced CFH RNA compared with control VEGF–stimulated podocytes (F, n = 3). One-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.