Dataset Information

Lipopolysaccharide-Induced Functional Alteration of P-glycoprotein in the Ex Vivo Rat Inner Blood-Retinal Barrier.

ABSTRACT: At the inner blood-retinal barrier (BRB), P-glycoprotein (P-gp) contributes to maintaining the homeostasis of substance concentration in the retina by transporting drugs and exogenous toxins from the retina to the circulating blood. Under inflammatory conditions, P-gp activities have been reported to be altered in various tissues. The purpose of this study was to clarify the alterations in P-gp activity at the inner BRB due to lipopolysaccharide (LPS), an inflammatory agent, and the molecular mechanisms of the alterations induced by LPS. Ex vivo P-gp activity was evaluated as luminal accumulation of 7-nitro-2,1,3-benzoxadiazole-cyclosporin A (NBD-CSA), a fluorescent P-gp substrate, in freshly prepared rat retinal capillaries. The luminal NBD-CSA accumulation was significantly decreased in the presence of LPS, indicating that P-gp activity at the inner BRB is reduced by LPS. This LPS-induced attenuation of the luminal NBD-CSA accumulation was abolished by inhibiting toll-like receptor 4 (TLR4), a receptor for LPS. Furthermore, an inhibitor/antagonist of tumor necrosis factor receptor 1, endothelin B receptor, nitric oxide synthase, or protein kinase C (PKC) significantly restored the LPS-induced decrease in the luminal NBD-CSA accumulation. Consequently, it is suggested that the TLR4/PKC pathway is involved in the reduction in P-gp function in the inner BRB by LPS.

SUBMITTER: Daikohara K

PROVIDER: S-EPMC9779076 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Lipopolysaccharide-Induced Functional Alteration of P-glycoprotein in the Ex Vivo Rat Inner Blood-Retinal Barrier.

Daikohara Kiyotaka K Akanuma Shin-Ichi SI Kubo Yoshiyuki Y Hosoya Ken-Ichi KI

International journal of molecular sciences 20221207 24

At the inner blood-retinal barrier (BRB), P-glycoprotein (P-gp) contributes to maintaining the homeostasis of substance concentration in the retina by transporting drugs and exogenous toxins from the retina to the circulating blood. Under inflammatory conditions, P-gp activities have been reported to be altered in various tissues. The purpose of this study was to clarify the alterations in P-gp activity at the inner BRB due to lipopolysaccharide (LPS), an inflammatory agent, and the molecular me ...[more]

PMID: 36555148

Similar Datasets

Project description:BackgroundImpairment of the inner blood-retinal barrier (iBRB) leads to various blinding diseases including diabetic retinopathy (DR). The cGAS-STING pathway has emerged as a driving force of cardiovascular destruction, but its impact on the neurovascular system is unclear. Here, we show that cGAMP, the endogenous STING agonist, causes iBRB breakdown and retinal degeneration thorough P2RX7-mediated transport into microglia.MethodsExtracellular cGAMP and STING pathway were determined in tissue samples from patients with proliferative DR (PDR) and db/db diabetic mice. Histological, molecular, bioinformatic and behavioral analysis accessed effects of cGAMP on iBRB. Single-cell RNA sequencing identified the primary retinal cell type responsive to cGAMP. Specific inhibitors and P2RX7-deficienct mice were used to evaluate P2RX7' role as a cGAMP transporter. The therapeutic effects of P2RX7 inhibitor were tested in db/db mice.ResultscGAMP was detected in the aqueous humor of patients with PDR and elevated in the vitreous humor with STING activation in db/db mouse retinas. cGAMP administration led to STING-dependent iBRB breakdown and neuron degeneration. Microglia were the primary cells responding to cGAMP, essential for cGAMP-induced iBRB breakdown and visual impairment. The ATP-gated P2RX7 transporter was required for cGAMP import and STING activation in retinal microglia. Contrary to previous thought that mouse P2RX7 nonselectively transports cGAMP only at extremely high ATP concentrations, human P2RX7 directly binds to cGAMP and activates STING under physiological conditions. Clinically, cGAMP-induced microglial signature was recapitulated in fibrovascular membranes from patients with PDR, with P2RX7 being predominantly expressed in microglia. Inhibiting P2RX7 reduced cGAMP-STING activation, protected iBRB and improved neuron survival in diabetic mouse retinas.ConclusionsOur study reveals a mechanism for cGAMP-mediated iBRB breakdown and suggests that targeting microglia and P2RX7 may mitigate the deleterious effects of STING activation in retinal diseases linked to iBRB impairment.

Project description:BackgroundReduced 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.MethodsWe 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.ResultsOur 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.ConclusionIn 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.

Dataset Information

Lipopolysaccharide-Induced Functional Alteration of P-glycoprotein in the Ex Vivo Rat Inner Blood-Retinal Barrier.

Publications

Lipopolysaccharide-Induced Functional Alteration of P-glycoprotein in the Ex Vivo Rat Inner Blood-Retinal Barrier.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets