PKC\u03b8-JunB axis via upregulation of VEGFR3 expression mediates hypoxia-induced pathological retinal neovascularization

Raj Kumar,Arul M Mani,Nikhlesh K Singh,Gadiparthi N Rao

doi:10.1038/s41419-020-2522-0

Abstract

Pathological retinal neovascularization is the most common cause of vision loss. PKCθ has been shown to play a role in type 2 diabetes, which is linked to retinal neovascularization. Based on these clues, we have studied the role of PKCθ and its downstream target genes JunB and VEGFR3 in retinal neovascularization using global and tissue-specific knockout mouse models along with molecular biological approaches. Here, we show that vascular endothelial growth factor A (VEGFA) induces PKCθ phosphorylation in human retinal microvascular endothelial cells (HRMVECs) and downregulation of its levels attenuates VEGFA-induced HRMVECs migration, sprouting and tube formation. Furthermore, the whole body deletion of PKCθ or EC-specific deletion of its target gene JunB inhibited hypoxia-induced retinal EC proliferation, tip cell formation and neovascularization. VEGFA also induced VEGFR3 expression via JunB downstream to PKCθ in the regulation of HRMVEC migration, sprouting, and tube formation in vitro and OIR-induced retinal EC proliferation, tip cell formation and neovascularization in vivo. In addition, VEGFA-induced VEGFR3 expression requires VEGFR2 activation upstream to PKCθ-JunB axis both in vitro and in vivo. Depletion of VEGFR2 or VEGFR3 levels attenuated VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and retinal neovascularization in vivo and it appears that these events were dependent on STAT3 activation. Furthermore, the observations using soluble VEGFR3 indicate that VEGFR3 mediates its effects on retinal neovascularization in a ligand dependent and independent manner downstream to VEGFR2. Together, these observations suggest that PKCθ-dependent JunB-mediated VEGFR3 expression targeting STAT3 activation is required for VEGFA/VEGFR2-induced retinal neovascularization.

Highlights

Retinal neovascularization is the most common cause of vision loss in various ocular diseases such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and agerelated macular degeneration (AMD)[1,2,3]
Retinal neovascularization is a clinical manifestation of diabetic retinopathy, and it was reported that genetic deletion of PKCθ protects mice against diet-induced insulin resistance[16]
SiRNA-mediated downregulation of PKCθ levels inhibited Vascular endothelial growth factor-A (VEGFA)-induced human retinal microvascular endothelial cells (HRMVECs) migration, sprouting, and tube formation with little or no effect on DNA synthesis (Fig. 1b–e). These results suggest that PKCθ activation is necessary for VEGFA-induced HRMVEC migration, sprouting, and tube formation but not proliferation

Summary

Introduction

Retinal neovascularization is the most common cause of vision loss in various ocular diseases such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and agerelated macular degeneration (AMD)[1,2,3]. VEGFA binds to both VEGFR1 and VEGFR2, the latter appears to mediate most of its VEGFR2 and VEGFR3 are required for sprouting angiogenesis as well as blood vessel development[7,8]. It was demonstrated that VEGFR3 is expressed highly in tip cells and promotes angiogenesis and vessel growth even in the absence of VEGFR2 activation[10]. In the absence of VEGFA-VEGFR2 signaling and low Notch activity, ligand-independent increase in VEGFR3 kinase activity has been shown to be sufficient in the modulation of retinal angiogenesis[10]. Contrary to these observations, it Official journal of the Cell Death Differentiation Association

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