Homocysteine causes cerebrovascular leakage in mice

Abstract

Elevated plasma homocysteine (Hcy) is associated with cerebrovascular disease and activates matrix metalloproteinases (MMPs), which lead to vascular remodeling that could disrupt the blood-brain barrier. To determine whether Hcy administration can increase brain microvascular leakage secondary to activation of MMPs, we examined pial venules by intravital video microscopy through a craniotomy in anesthetized mice. Bovine serum albumin labeled with fluorescein isothiocyanate (BSA-FITC) was injected into a carotid artery to measure extravenular leakage. Hcy (30 μM/total blood volume) was injected 10 min after FITC-BSA injection. Four groups of mice were examined: 1) wild type (WT) given vehicle; 2) WT given Hcy (WT + Hcy); 3) MMP-9 gene knockout given Hcy (MMP-9−/− + Hcy); and 4) MMP-9−/− with topical application of histamine (10 −4 M) (MMP-9 −/− + histamine). In the WT + Hcy mice, leakage of FITC-BSA from pial venules was significantly (P < 0.05) greater than in the other groups. There was no significant leakage of pial microvessels in MMP-9−/− + Hcy mice. Increased cerebrovascular leakage in the MMP-9−/− + histamine group showed that microvascular permeability could still increase by a mechanism independent of MMP-9. Treatment of cultured mouse microvascular endothelial cells with 30 μM Hcy resulted in significantly greater F-actin formation than in control cells without Hcy. Treatment with a broad-range MMP inhibitor (GM-6001; 1 μM) ameliorated Hcy-induced F-actin formation. These data suggest that Hcy increases microvascular permeability, in part, through MMP-9 activation.

Ablation of MMP9 Gene Ameliorates Paracellular Permeability and Fibrinogen–Amyloid Beta Complex Formation during Hyperhomocysteinemia

“…Earlier we have shown that an elevated blood level of Hcy causes cerebrovascular protein leakage in mice and an increase in the formation of filamentous actin in cultured ECs. 17 Later we demonstrated that HHcy downregulates adherence junction protein, VE-cadherin and tight junction-associated protein, zonula occludin-1 (ZO-1) in cultured cells. 37 In the recent study, we found that injection of Hcy decreased messenger RNA of tight junction protein, occludin, and ZO-1 in mouse brain samples.…”

“…Brain pial microcirculation was prepared for observations as previously described. 17,[29][30][31] Briefly, a mouse was placed on a stereotaxic apparatus (World Precision Instruments, Sarasota, FL, USA). The scalp and connective tissues were removed over the parietal cranial bone above the left hemisphere.…”

“…16 We have already shown that high level of Hcy mediates macromolecular leakage in mouse brain pial microvessels and involves activation of matrix metalloproteinases. 17 Matrix metalloproteinases, zinc-dependent endoproteinases, are expressed in various cell types including ECs. Matrix metalloproteinases are involved in both physiologic and pathologic processes, especially in the subendothelial matrix (SEM) degradation and vascular remodeling that disrupt the BBB.…”

Role of MicroRNA29b in Blood–Brain Barrier Dysfunction during Hyperhomocysteinemia: An Epigenetic Mechanism

“…Homocysteine-induced BBB disruption model in bEnd.3 cells was confirmed by analyzing paracellular and transcellular transport systems using Lucifer yellow and BSA-647 fluorescence probes leakage at different time points (10,20,40,60, 120 minutes) in four treatment groups. In Hcy-treated cells, significant endothelial cell permeability was observed, as evident through increased leakage of the Lucifer yellow (at 40 minutes and 60 minutes; Figure 1A) and BSA-647 (at 40 minutes to 120 minutes; Figure 1B) fluorescent dyes, which suggested that both paracellular and transcellular transport systems are affected during endothelial disruption.…”

“…It has been reported that subclinical elevation in Hcy levels (from baseline of B3 mmol/L to B12 mmol/L) results in considerable leakage of plasma proteins (albumin and endogenous immunoglobulins) in rodent brains. [9][10][11] We earlier reported that CBS þ / À mice have high levels of Hcy (21 ± 4 mmol/L) as compared with WT (5 ± 2 mmol/L). 12 Homocysteine instigates BBB disruption by inducing redox stress and upregulating matrix metalloproteinases (MMPs), which are zinc-dependent endopeptidases that have a critical role in matrix remodeling and lead to blood-endothelial barrier leakage.…”