Abstract
Purpose :
Vascular adhesion molecules on endothelial cells (ECs) are key players in immune cell recruitment during inflammation. We have recently shown that limbal vessels are in close proximity with sensory nerves and that ciliary nerve axotomy results in upregulation of adhesion molecules on the vascular endothelium. The purpose of this study is to dissect the molecular mechanism involved in the neuronal modulation of vascular adhesion molecules by trigeminal ganglia (TG) sensory neuron.
Methods :
CD31+CD45—Lyve-1— blood ECs and CD31+CD45—Lyve-1+ lymphatic ECs were sorted by flow cytometry (FC) from normal corneal limbus and assessed for the expression of neuropeptide receptors (NP-Rs): NK1R, VIP-R1, VIP-R2, CRLR by qRT-PCR. TG sensory neurons were obtained from C57BL/6 mice pups and HUVECs were purchased from ATCC. Co-cultures were performed by seeding TG neurons in a transwell plate with HUVECs in presence of recombinant TNF-α. The antagonist for NP-Rs for VIP, SP and CGRP were used to block neuropeptides. HUVECs were then assessed for adhesion molecule expression by qRT-PCR or FC
Results :
Sorted limbal blood ECs showed high expression levels of NK1R, VIP-R1, VIP-R2 and CRLR compared to lymphatic ECs (800-, 7400-, 6200- and 69-fold change respectively (P<0.05). mRNA Expression of adhesion molecules (MadCAM-1, and E-selectin) on HUVECs was reduced with TG neuron in presence of recombinant TNF-α by 94% and 90% respectively (P<0.05). qRT-PCR showed that MadCAM-1 and E-selectin inhibition by TG neurons was reversed in the presence of SP and VIP antagonists (P<0.05). Similarly, FC histograms confirmed the reversal of the inhibitory effect of TG neurons on MadCAM-1 by using VIP and SP antagonists. Additionally, E-selectin inhibition by TG neurons was reversed by VIP and CGRP antagonists (P<0.05) by qRT-PCR.
Conclusions :
Corneal limbal blood ECs show high levels of NP-Rs expression, suggesting the potential role of NPs on regulating their functions. We observed that the expression of adhesion molecules was inhibited in co-cultures with TG neurons. Moreover, this inhibitory effect could be reversed blocking NPs, demonstrating a selective NP effect on regulating the expression of adhesion molecules. Collectively, these findings suggest that TG sensory neurons regulates the expression of adhesion molecules on ECs through NPs during inflammation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.