Connexin30 deficiency causes instrastrial fluid-blood barrier disruption within the cochlear stria vascularis.

The endocochlear potential (EP) is essential to hearing, because it provides approximately half of the driving force for the mechanoelectrical transduction current in auditory hair cells. The EP is produced by the stria vascularis (SV), a vascularized bilayer epithelium of the cochlea lateral wall. The absence of the gap junction protein connexin30 (Cx30) in Cx30(-/-) mice results in the SV failure to produce an EP, which mainly accounts for the severe congenital hearing impairment of these mice. Here, we show that the SV components of the EP electrogenic machinery and the epithelial barriers limiting the intrastrial fluid space, which are both necessary for the EP production, were preserved in Cx30(-/-) mice. In contrast, the endothelial barrier of the capillaries supplying the SV was disrupted before EP onset. This disruption is expected to result in an intrastrial electric shunt that is sufficient to account for the absence of the EP production. Immunofluorescence analysis of wild-type mice detected Cx30 in the basal and intermediate cells of the SV but not in the endothelial cells of the SV capillaries. Moreover, dye-coupling experiments showed that endothelial cells were not coupled to the SV basal, intermediate, and marginal cells. SV transcriptome analysis revealed a significant down-regulation of betaine homocysteine S-methyltransferase (Bhmt) in the Cx30(-/-) mice, which was restricted to the SV and resulted in a local increase in homocysteine, a known factor of endothelial dysfunction. Disruption of the SV endothelial barrier is a previously undescribed pathogenic process underlying hearing impairment.

Effects of catechin on homocysteine metabolism in hyperhomocysteinemic mice.

We have recently focused on the interaction between hyperhomocysteinemia, defined by high plasma homocysteine levels, and paraoxonase-1 expression and found a reduced activity of paraoxonase-1 associated with a reduced gene expression in the liver of cystathionine beta synthase (CBS) deficient mice, a murine model of hyperhomocysteinemia. As it has been demonstrated that polyphenolic compounds could modulate the expression level of the paraoxonase-1 gene in vitro, we have investigated the possible effect of flavonoid supplementation on the impaired paraoxonase-1 gene expression and activity induced by hyperhomocysteinemia and have evaluated the link with homocysteine metabolism. High-methionine diet significantly increased serum homocysteine levels, decreased hepatic CBS activity, and down-regulated paraoxonase-1 mRNA and its activity. However, chronic administration of catechin but not quercetin significantly reduced plasma homocysteine levels, attenuated the reduction of the hepatic CBS activity, and restored the decreased paraoxonase-1 gene expression and activity induced by chronic hyperhomocysteinemia. These data suggest that catechin could act on the homocysteine levels by increasing the rate of catabolism of homocysteine.

Hyperhomocysteinemia due to cystathionine beta synthase deficiency induces dysregulation of genes involved in hepatic lipid homeostasis in mice.

BACKGROUND/AIMS: Cystathionine beta synthase (CBS) deficiency leads to severe hyperhomocysteinemia, which confers diverse clinical manifestations, notably fatty liver. Recently, abnormal lipid metabolism has been demonstrated in CBS-deficient mice, a murine model of severe hyperhomocysteinemia. To gain further insights into effects of CBS deficiency on hepatic cholesterol metabolism, the expression of hepatic genes involved in biosynthesis, uptake and efflux was determined in CBS-deficient mice. METHODS: Gene expression analysis was performed on liver of CBS-deficient mice using quantitative real-time PCR. RESULTS: We found that CBS-deficiency in liver mice significantly increases expression of genes induced by endoplasmic reticulum stress and genes that regulate the expression of enzymes required for cholesterol and fatty acid biosynthesis and uptake, notably the scavenger receptor class B type I (SR-BI), concomitant with overexpression of SR-BI at the protein level. Moreover, we also found increased mRNA levels of ABCG5, ABCG8, ABCG1 and ABCA1, which play important roles in reverse cholesterol transport, associated with an upregulation of liver X receptors and a downregulation of the peroxisome proliferators-activated receptor alpha. CONCLUSIONS: We found that several ATP-binding cassette transporters and nuclear hormone receptors involved in liver lipid homeostasis are differentially expressed in liver of CBS-deficient mice.

Thiol compounds metabolism in mice, rats and humans: comparative study and potential explanation of rodents protection against vascular diseases.

BACKGROUND: Rodents are often used as animal models to dissect mechanisms underlying hyperhomocysteinemia atherogenicity in humans. However, neither wild-type rodents nor cystathionine beta-synthase deficient mice develop spontaneous atherosclerosis. We investigated whether species-specific differences in thiols metabolism may explain the respective sensitivity of rodents and humans to hyperhomocysteinemia. METHODS: Thiols and vitamins B levels were determined in normohomocysteinemic humans and rodents, and in hyperhomocysteinemic mice. RESULTS: In basal status, although plasma homocysteine, cysteine and cysteinylglycine levels were lower, glutathione levels were higher in mice than in humans (4.0+/-1.6 vs. 7.9+/-2.2, P<0.0005; 147.4+/-40.3 vs. 278.5+/-50.0, P<0.0001; 2.3+/-0.7 vs. 36.6+/-7.3, P<0.0001; and 70.9+/-20.1 vs. 4.6+/-1.6, P<0.0001). Serum vitamin B12 and folate levels were 2.5- and 7.7-fold higher in rats than in humans. In wild-type mice, the increase in plasma Hcy levels induced by methionine-enriched diet was accompanied by a proportional increase in GSH levels. CONCLUSION: GSH levels are enough to modulate Hcy effects in normo- and hyperhomocysteinemic mice but not in humans. This rodents characteristic, likely supported by species differences in the relative contribution of remethylation and transsulfuration pathways, may partly explain their protection against atherosclerosis.

Evaluation of Advia Centaur automated chemiluminescence immunoassay for determining total homocysteine in plasma.

BACKGROUND: A lot of methods are now available for total plasma homocysteine (tHcy) determination. HPLC with fluorescence detection were the most widely used methods until recently but immunoassays, easier to use, currently supplant in-house laboratory methods. METHODS: The latest commercial automated chemiluminescence immunoassay (ICL) for tHcy, developed on the ADVIA Centaur analyzer (Bayer Diagnostics), was fully evaluated and compared with a current HPLC method. RESULTS: The ICL-ADVIA Centaur method had a detection limit <0.9 micromol/l and was linear for tHcy between 2.4 and 58.8 micromol/l. The within- and between assay imprecision was <6% and <7%, respectively. The analytical recovery ranged from 93.5% to 109.7%. The comparison of 168 clinical plasma specimens indicated a good correlation between the two methods (r=0.96, p<0.0001) but a systematic positive bias for ICL-ADVIA Centaur method (mean difference=3.0 micromol/l). CONCLUSIONS: The ADVIA Centaur method is a useful and practicable alternative to HPLC methods for tHcy determination. The ICL method is even more convenient than chromatographic methods for routine use because of its significantly higher throughput (200 samples/h vs. 40 per 24 h). However, the agreement among methods is insufficient to allow them to be used interchangeably.

Methods for homocysteine analysis and biological relevance of the results.

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.

Homocysteine decreases endothelin-1 expression by interfering with the AP-1 signaling pathway.

The increased cardiovascular risk associated with hyperhomocysteinemia has been linked to homocysteine-induced endothelial cell (EC) dysfunction. Endothelin-1 is a vasoactive peptide, synthesized mainly by vascular ECs. We have previously shown that homocysteine decreases endothelin-1 biosynthesis. Here we addressed the molecular mechanism of endothelin-1 regulation by homocysteine. Experiments with the transcription inhibitor actinomycin D indicated that the decrease in preproendothelin-1 mRNA content in homocysteine-treated cells did not result from transcript destabilization. Transient transfection assays demonstrated that homocysteine downregulated endothelin-1 at the transcriptional level by decreasing preproendothelin-1 promoter activity. Mutation of the activator protein-1 (AP-1) site of the promoter eliminated the repression induced by homocysteine. Western blot analysis showed that the homocysteine-induced decrease in promoter activity was not associated with reduced expression of the AP-1 components c-Fos and c-Jun. The inhibitory action of homocysteine on preproendothelin-1 mRNA expression was not prevented by cycloheximide. Electrophoretic mobility shift assays demonstrated that homocysteine reduced the binding activity of ECs nuclear extracts to an AP-1 consensus site. These results indicate that homocysteine downregulates endothelin-1 synthesis by inhibiting AP-1 activity, and that the AP-1 signaling pathway may be of major importance in homocysteine-induced endothelial dysfunction.

Homocysteine is the only plasma thiol associated with carotid artery remodeling.

Several authors have reported that moderate hyperhomocysteinemia is related to asymptomatic carotid arterial wall remodeling, but few data are available on other thiol compounds with potential vascular toxicity. We, therefore, investigated the relationships between major plasma thiol compounds (homocysteine, cysteine and glutathione) and the structural phenotype of the common carotid artery in a cohort of 123 subjects with no evidence of cardiovascular disease. Fasting levels of thiol compounds were measured by high-performance liquid chromatography, and arterial geometry was evaluated using high-resolution echotracking devices. In univariate regression analysis, plasma homocysteine and plasma cysteine concentrations were positively associated with carotid artery internal diameter (P=0.0001 and 0.002, respectively) and intima media thickness (P=0.003 and 0.004), but the plasma glutathione concentration was not. In multivariate analysis, plasma homocysteine was independently and positively associated with carotid artery internal diameter (P<0.005) and intima media thickness (P<0.05), but plasma cysteine was not. These data suggest that homocysteine is the only plasma thiol compound that may be considered as a risk factor for preclinical cardiovascular disease.