Reduced folate carrier 1 is present in retinal microvessels and crucial for the inner blood retinal barrier integrity

Abstract

Background Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known. Methods We used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically. Results Our analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia. Conclusion In conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia–immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

Case series of retinal vein occlusions showing early recovery using oral l-methylfolate

“… 74 Recent observations that aspects of early DR may be reversed with the use of a high dose-methylfolate cocktails suggest a practical way to overcome central and retinal folate insufficiency due to deficient intake, methylation impairment, or transport. 23 , 78 This has important implications for RVO ischemia.…”

“…Ocufolin ® has been shown to lower homocysteine, lower RVP, lower systolic blood pressure, increase conjunctival and retinal perfusion, increase retinal venous blood flow, and increase capillary density in the macula. 32 – 35 , 78 The ingredients of Ocufolin ® ( Table 1 ) have previously been published. 32 Additionally, coupled with anti-VEGF therapy, Ocufolin ® has sufficient B12 to maintain optimal anti-VEGF effectiveness.…”

Pericytes: jack-of-all-trades in cancer-related inflammation

“…Moreover, the analysis of single-cell RNA expression profiles derived from different human and mouse brain regions using a high-throughput and low-cost single-cell transcriptome sequencing method called EasySci revealed that highly pericyte-enriched expression was notable for SLC6A12 and SLC19A1. The immunohistochemical technique of staining for both antibodies was strongly positive in small blood vessels and was far more effective than a PDGFR-β antibody at staining pericyte-like cells in human brain sections ( Gurler et al, 2023 ; Sziraki et al, 2023 ).…”

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.…”