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N-Methyl-D-aspartate receptor activation, novel mechanism of homocysteine-induced blood-retinal barrier dysfunction.

ABSTRACT: Elevated levels of amino acid homocysteine (Hcy) recognized as hyperhomocysteinemia (HHcy) was reported in several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of blood-retinal barrier (BRB) is concomitant with vision loss in DR and AMD. We previously reported that HHcy alters BRB. Here, we tested the hypothesis that HHcy alters BRB via activation of N-methyl-D-aspartate receptor (NMDAR). Human retinal endothelial cells subjected to high level of Hcy and mouse model of HHcy were used. We injected Hcy intravitreal and used a mouse model of HHcy that lacks cystathionine-β-synthase (CBS). RT-PCR, western blot, and immunofluorescence showed that retinal endothelial cells (RECs) express NMDAR at the gene and protein levels both in vitro and in vivo and this was increased by HHcy. We assessed BRB function and retinal morphology using fluorescein angiogram and optical coherence tomography (OCT) under HHcy with and without pharmacological inhibition of NMDAR by (MK801) or in mice lacking endothelial NMDAR (NMDAR^E-/- mouse). Additionally, retinal albumin leakage and tight junction proteins ZO-1 and occludin were assessed by western blotting analysis. Inhibition or elimination of NMDAR was able to improve the altered retinal hyperpermeability and morphology under HHcy as indicated by significant decrease in retinal albumin leakage and restoration of tight junction proteins ZO-1 and occludin. Our findings underscore a potential role for endothelial NMDAR in mediating Hcy-induced breakdown of BRB and subsequently as a potential therapeutic target in retinal diseases associated with HHcy such as DR and AMD. KEY MESSAGES: • Elevated levels of homocysteine (Hcy) are defined as hyperhomocysteinemia (HHcy). • HHcy is implicated in diabetic retinopathy and age-related macular degeneration. • HHcy alters BRB via activation of N-methyl-D-aspartate receptor.

SUBMITTER: Tawfik A

PROVIDER: S-EPMC7785674 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1β, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.

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N-Methyl-D-aspartate receptor activation, novel mechanism of homocysteine-induced blood-retinal barrier dysfunction.

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