Characterization of Vascular Cell Receptors that Regulate Blood Perfusion of the Mouse Retina

Abstract

Purpose : To identify the vascular cell signaling pathways that regulate blood perfusion of the retina.

Methods : SMA-GFP transgenic (a.k.a. “OTO2-10”) mice, which express GFP under the acta2 promoter, were acquired from the National Eye Institute. Their retinas were dissected and dissociated with papain into single cell suspensions. VSMCs were purified by the collection of GFP positive cells with the use of a fluorescence activated cell sorter (FACS). Western blots (WB) from the sorted GFP positive cells were performed to confirm the purification of vascular cells, and liquid chromatography-mass spectrometry (LC-MS) was used to identify cell signaling components that regulate contractility. Immunohistochemistry of retinal flat mounts was used to validate key targets identified by our exploratory LC-MS profile.

Results : Confocal microscopy of SMA-GFP retinal flat mounts found GFP expression to be exclusive to the vascular tree. WB of GFP positive cells showed enrichment of alpha-smooth muscle actin (α-SMA), smooth muscle-myosin heavy chain (SM-MHC), neuron-glial antigen 2 (NG2), and depletion of neurofilament and glial fibrillary acidic protein (GFAP), consistent with the purification of VSMCs and pericytes via FACS. LC-MS yielded a total of 6,175 proteins within three replicates of SMA-GFP mice with 100 high confidence proteins (FDR < 0.01), 18 medium confidence proteins (FDR 0.01 - 0.05), and 6057 low confidence proteins (FDR > 0.05). LC-MS identified proteins that potentially regulate cell contractility include: cell surface receptors (such as glutamate and acetylcholine receptors), generators of intracellular calcium, cyclic AMP, ATP, and nitric oxide (second messengers), and effector proteins of contractility (such as myosin light chain kinase, myosin light chain phosphatase, and Rho-kinase). The localization of key regulators of contractility within the retinal vasculature is shown with flat mount immunohistochemistry.

Conclusions : Neurovascular coupling and regulation of blood flow in the retina remain poorly understood. We used mass spectrometry to identify VSMC cell surface receptors and downstream signaling pathways that regulate vascular tone to further understand neurovascular coupling in the mouse retinaVSMC receptors and signaling pathways that regulate contractility play a key role in vascular resistance in the retina. Our findings shed light on the molecular regulation of ocular perfusion.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.