VEGF Upregulates EGFR Expression to Stimulate Chemotactic Behaviors in the rMC-1 Model of Müller Glia

Abstract

Progressive vision loss in adults has become increasingly prevalent worldwide due to retinopathies associated with aging, genetics, and epigenetic factors that damage the retinal microvasculature. Insufficient supply of oxygen and/or nutrients upregulates factors such as vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), which can induce abnormal angiogenesis and damage the structural arrangement of the retinal blood barrier (BRB). Müller glia (MG) regulate the diffusion of essential compounds across the BRB and respond to retinal insults via reactive gliosis, which includes cell hypertrophy, migration, and/or proliferation near areas of elevated VEGF concentration. Increasing concentrations of exogenous VEGF, upregulated by retinal pigmented epithelium cells, and endogenous epidermal growth factor receptor (EGF-R) stimulation in MG, implicated in MG proliferative and migratory behavior, often lead to progressive and permanent vision loss. Our project examined the chemotactic responses of the rMC-1 cell line, a mammalian MG model, toward VEGF and EGF signaling fields in transwell assays, and within respective concentration gradient fields produced in the glia line (gLL) microfluidic system previously described by our group. rMC-1 receptor expression in defined ligand fields was also evaluated using quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Results illustrate dramatic increases in rMC-1 chemotactic responses towards EGF gradient fields after pre-treatment with VEGF. In addition, qPCR illustrated significant upregulation of EGF-R upon VEGF pre-treatment, which was higher than that induced by its cognate ligand, EGF. These results suggest interplay of molecular pathways between VEGF and EGF-R that have remained understudied in MG but are significant to the development of effective anti-VEGF treatments needed for a variety of retinopathies.

Harnessing the Neuroprotective Behaviors of Müller Glia for Retinal Repair

“…Most recently, bioengineering groups have built upon these in vitro systems to model the extracellular landscape using micropatterned substrates [174][175][176] based on the different stages of gliosis. Likewise, the chemotactic ability of MG has been quantified using transwell assays, which have the ability to retain concentration gradients of cytokines overtime [177][178][179]. In addition, complex engineering systems such as microfluidic devices have served as tunable platforms to study migratory patterns of MG under specific biochemical conditions, as well as providing a medium to study cellular interconnectivity through the use of microchannels in compartmentalized chambers.…”

“…The migration of MG has been particularly characterized in response to the upregulation of growth factors such as EGF and VEGF [ 13 , 177 , 197 ]. Concentration gradients of VEGF have demonstrated to increase the chemotactic response of MG in vitro, which correlated to the increased concentration of VEGF in later stages of neovascular retinopathies in vitro (21484851, 24412518).…”

Microfluidic and Microscale Assays to Examine Regenerative Strategies in the Neuro Retina

“…( B ) Schematic of transwell assay (e.g., Boyden Chamber) where cells are inserted into a top, or sink, reservoir and migrate within the pores of a semi-permeable membrane towards the bottom, or source, reservoir. Motile cells then adhere to the underside of the membrane, are stained, and counted to estimate numbers of motile cells (reproduced with permission from [ 189 ]). ( C ) Rendering of a micro-patterned substrate, comprised of a flexible (or rigid) substrate that is functionalized with a densely-packed array of polymer substances in a pre-designed arrangement.…”

“…Experiments further used a large range of concentration gradient fields and commercial inhibitors to highlight mechanistic signaling from the MAPK pathway in the chemotaxis of Müller glia towards EGF [ 190 ]. Recent work from this same group has examined the interplay of the EGF receptor and VEGF ligand within gradient fields of EGF in microfluidic environments [ 189 ]. Surprisingly, the results demonstrated that VEGF upregulated EGF-R expression nearly 10-fold more than the cognate EGF ligand to increase Müller glia chemotaxis.…”

“…Retinal glia are mechanosensors that maintain homeostasis as well as initiate neuroprotective responses to retinal insults. Müller glia are well known to ensheath retinal vasculature, provide nutrients and neurotransmitters, and act as light scattering cells for enhanced visual acuity [ 146 , 189 ]. However, their direct communication with the different groups of retinal neurons remains incompletely understood.…”