Reduced Folate Carrier 1 Is Present in Retinal Microvessels and Crucial for the Inner Blood Retinal Barrier Integrity

Abstract

The inner blood-retina barrier (BRB) resembles the blood-brain barrier (BBB) and is comprised of a single layer endothelium sealed with tight junctions whose abluminal surface is ensheathed by pericytes. The inner BRB is essential for providing a constant and controlled flow of nutrients and molecules to meet metabolic needs. Accordingly, the inner BRB harbors numerous carriers providing the exchange of molecules between blood and retina. One of these carriers is Reduced Folate Carrier 1 (RFC1), which functions as a folate transporter, and has been one of the most consistently detected genes in cerebral pericytes in a recent meta-analysis. The presence of RFC1 protein and its functions in retinal pericytes and microvessels have been overlooked so far despite the similarities between retinal and cerebral microcirculation. In this report, our purposes were to assess the presence of RFC1 protein in retinal microvessels and surrounding pericytes, and modify to increase or decrease RFC1 levels to explore its roles in healthy and ischemic retinal microvessels. Our analyses on whole mount retinas, and trypsin digest preparations from adult Swiss albino mice with concomitant pericyte and endothelial cell markers have shown the presence of RFC1 protein in the inner BRB and colocalization with pericytes. In vivo modifications of RFC1 expression levels were accomplished by intravitreal delivery of genetic and pharmacological agents. Knocking down RFC1 expression via custom-designed short interfering RNA (siRNA) resulted in microvascular dysfunction which was presented by the disintegration of tight junction proteins and the main basal membrane protein collagen-4. This was accompanied by extensive endogenous IgG extravasation suggesting impairment in the barrier functions. On the other hand, delivering RFC1 expressing lentiviral vector to overexpress RFC1 in the retina induced immunosignal increase in tight junction proteins and collagen-4 confirming the role of RFC1 in the inner BRB functions. Administration of methotrexate (MTX), the pharmacological competitive inhibitor of folate, resulted in increased levels of RFC1. In retinal ischemia models, one-hour retinal ischemia increased RFC1 levels and decreased collagen-4 and occludin levels. These effects could be partially reversed with pre-treatment by RFC1 expressing lentiviral vector. All these indicate the importance of RFC1 levels in the pathophysiology of retinal ischemia, a prevalent cause of blindness worldwide. In conclusion, our study clarifies the presence of RFC1 in inner BRB and offers a novel perspective on RFC1, other than being a folate carrier, as a regulator of inner BRB in healthy and ischemic retinas.

Response letter: Complexities in pericyte markers

“…retinal pericytes in which they described that SLC19A1 was a pericyte marker [2]. We are thankful to these investigators for bringing this to our attention.…”

Letter to the editor

“…In our recent studies, where we used mice brain sections, whole-mount retinas, and retinal and cerebral microvessel isolates, we demonstrated Slc19a1 (Mus musculus symbol for SLC19A1) protein expression in mice retinal and brain microvessels. 2,3 SLC19A1 expression at the protein level had not previously been studied in the pericytes of the retina and the brain, despite its consistent detection in the transcripts of mural cells. 4 We applied commercial primary antibodies targeted to different regions-MyBioSource (MBS9134642) and Sigma-Aldrich (AV44167)-against SLC19A1 for immunohistochemistry.…”

“…In line with the findings of Sziraki et al, we showed for the first time that Slc19a1 protein is present in the pericytes and endothelial cells of the retina comprising the inner bloodretina barrier, as well as the cerebral pericytes. 2,3 It is established in pre-clinical studies that pericytes do not have a specific marker, thus presenting certain challenges in studies focusing on their morphology and functions. The available, acceptable, and extensively used markers in literature such as platelet growth factor receptor β (PDGFR-b), NG2 (Cspg4), CD13 (aminopeptidase N), and vimentin are not entirely pericyte-specific and can only reliably identify pericytes when combined with morphological and location-based criteria.…”