Abstract
Purpose :
Endomucin (EMCN) is a 261 AA transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells. EMCN plays an important role in angiogenesis by modulating VEGF signaling, and reduced ERK1/2 activation and delayed retinal angiogenesis have been reported in Emcn knockout mice. In addition to its role in angiogenesis, EMCN has emerged as a widespread endothelial marker for histology due to the commercial availability of high-quality antibodies. Here, using a combination of histological and single cell RNA sequencing (scRNA-seq) approaches, we aimed to validate the endothelial-specific expression of EMCN in the mouse and human choroid.
Methods :
EMCN expression was evaluated in paraffin sections and whole-mounted mouse choroids obtained from formaldehyde-fixed mouse eyes by immunostaining and following single-cell RNA sequencing of whole mouse choroids at developmental timepoints ranging from embryonic day 14 to 8 weeks of age. To confirm our findings in human eyes, scRNA-seq data was obtained from the IOWA integrated retina, RPE, and choroid dataset and accessed using Spectacle (https://singlecell-eye.org/app/spectacle/).
Results :
Robust EMCN expression was observed in both mouse and human choroidal endothelial cells, including cells of the choriocapillaris, Sattler and Haller layers and vortex veins. Unexpectedly, EMCN labeling was also observed in a subset of choroidal stromal cells, co-localizing with canonical stromal marker PDGFR-β. In our mouse scRNA-seq datasets, this EMCN-positive stromal cell population was observed throughout choroidal development. EMCN/PDGFRB co-expressing cells were also observed in a published scRNA-seq dataset from human choroid, suggesting that this expression pattern is evolutionarily conserved.
Conclusions :
Outside of the endothelium, EMCN is widely reported as a marker of hematopoietic stem cells, but to our knowledge, this is the first report of an EMCN expressing stromal cell population in the eye. This result may not be wholly unexpected as the closely related glycoprotein CD34--which is also commonly used as a marker of endothelial cells--is robustly expressed on specific stromal cell populations, including some cells of the cornea. Our finding emphasizes that care must be taken when using EMCN as an endothelial marker in the choroid, as robust expression in choroidal stroma may confound results.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.