Mild hyperhomocysteinemia induces blood–brain barrier dysfunction but not neuroinflammation in the cerebral cortex and hippocampus of wild-type mice

This study explored the potential effects of mild hyperhomocysteinemia (HHcy) on the blood–brain barrier (BBB) and neuroinflammation. Seven-week-old male wild-type C57BL/6 mice were fed normal mouse chow (the control group) or a methionine-enriched diet (the HHcy group) for 14 weeks. Mice in the HHcy group exhibited a slight increase in serum Hcy levels (13.56 ± 0.61 μmol/L). Activation of the ERK signaling pathway, up-regulation of matrix metalloproteinase-9 (MMP-9), and degradation of tight junction proteins (occludin and claudin-5) were observed in both the cerebral cortex and hippocampus of mice with mild HHcy. However, microglia were not activated and the levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were not changed in either the cerebral cortex or hippocampus of mice with mild HHcy. Moreover, the signaling activity of STAT3 also did not differ significantly between the two groups. These findings demonstrate that the BBB is highly vulnerable to homocysteine insult. Even a slight increase in serum homocysteine levels up-regulates MMP-9 expression and disrupts the BBB integrity. Meanwhile, microglia activation or the STAT3 pathway might not contribute to the effects of mild HHcy on the brain.

Diet-Induced Mild Hyperhomocysteinemia in Mice Fed a Ketogenic Diet Does Not Alter the Development of Atherosclerosis nor Specific Epigenetic Content

Objectives Elevated plasma homocysteine (Hcy), or hyperhomocysteinemia (HHcy), is a risk factor for atherosclerosis by mechanisms still elusive. A possibility includes the alteration of specific epigenetic tags at lysine 27 of histone H3 (H3K27) due to hypomethylating stress. Similarly, ketogenic diets (KD), or very-low carbohydrate diets, which stimulate ketosis, and may also affect the epigenetic content on the H3K27 residue. Studies connecting the effects of dietary ketosis, mild HHcy, and specific epigenetic dysregulation are lacking. We hypothesize that diet-induced HHcy and ketosis will induce H3K27 hypomethylation combined with increased acetylation to produce a pro-atherogenic phenotype. Methods Seven-week-old male apoe−/− (apolipoprotein E-deficient) mice, a model for human atherosclerosis, were fed ad libitum a KD (in %kcal: fat, 81; carbohydrate, 1; protein, 18; n = 4–6) or HHcy-KD (same macronutrients, with added methionine and reduced methyl donors; n = 4). After 4, 8 and 12 wk of diet treatment, plasma was collected to quantify ketosis via beta-hydroxybutyrate levels (OH-But) by a colorimetric assay, and measure Hcy by HPLC. At the endpoint, mice were euthanized and aortas were collected for quantification of the vascular methylation index, S-adenosylmethionine to S-adenosylHcy ratio by LC-MS-MS; 3-D analysis of the atherosclerotic plaque burden by magnetic resonance imaging; and quantification of the epigenetic tags H3K27me3 and H3K27ac using immunohistochemistry. Results A sustained ketosis was detected through elevated OH-But levels in both KD and HHcy-KD mice. HHcy was mildly but significantly (P < 0.05) elevated in HHcy-KD-mice compared to KD-mice after 4 wk (19.5 ± 2.3 vs 4.5 ± 0.6 µM) and 12 wk (17.2 ± 2.1 vs 4.4 ± 0.9 µM). Nevertheless, no significant differences were observed in aortic methylation index, plaque accumulation, or content of the H3K27me3 or H3K27ac epigenetic tags between the two groups of mice. Conclusions While mild HHcy was achieved in HHcy-KD mice, this phenotype failed to induce vascular hypomethylation, atherosclerosis progression or specific epigenetic dysregulation, suggesting that a more severe Hcy accumulation may be necessary to cause vascular toxicity and specific epigenetic dysregulation. Funding Sources Huck Institutes of the Life Sciences

Mild Hyperhomocysteinemia Induced by a Hypomethylating Diet Does Not Favor Aortic Plaque Formation in apoE Knockout Mice (P24-037-19)

Objectives Hyperhomocysteinemia is an independent risk factor for atherosclerosis and cardiovascular disease through mechanisms still incompletely defined. We investigated the impact of mild diet-induced accumulation of homocysteine on atherosclerotic plaque formation in apoE knockout (KO) mice, a model for atherosclerosis in humans. We hypothesize that diet induced hyperhomocysteinemia will promote plaque development. Methods 7 wk-old male apoE-KO mice (n = 5) were fed a methyl-deficient diet for 16 wk. The diet consisted of a purified high (40%)-fat high-methionine diet with restricted levels of B vitamins and choline (HypoMet). A second group of animals (control, n = 5) was fed a normal-methyl matched diet. After 4 and 12 wk, plasma homocysteine was quantified by reverse phase HPLC with fluorometric detection, and a panel of inflammatory cytokines (MCP-1; TNF-; IL-17A/F; IL-2; IL-6, IL-10; KC/GRO; MIP-1; IFN-) were assayed (U-plex, Meso Scale Discovery). After 16 wk, mice were euthanized and perfusion-fixed aortas were stained for lipids (Oil Red-O) and subjected to 2-D-quantification of stained plaque (Image J software), and to 3-D analysis by magnetic resonance imaging (MRI, Agilent 14-tesla microimaging system). Standard 3-D gradient echo imaging yielded a resolution of 20 microns isotropic. Data were reconstructed using Matlab and segmented to obtain plaque volumes using Avizo 9.0. Results HypoMet-mice had significantly higher plasma homocysteine (µM), when compared to controls, at 4 wk (10.6 ± 4.5 vs 2.3 ± 0.8, P < 0.05) and 12 wk (7.6 ± 1.5 vs 2.5 ± 1.1, P < 0.05). No significant differences were observed in inflammatory cytokines. Surprisingly, Oil Red-O staining revealed that HypoMet-mice did not display more plaque coverage (37 ± 6.9%) than controls (54.4 ± 10.4%). 14-T MRI results (Fig.1) confirmed that HypoMet-mice did not present higher plaque volumes than controls (0.61 ± 0.18 mm3 vs 1.2 ± 0.35 mm3, for HypoMet mice vs controls). Conclusions A mild accumulation of homocysteine, induced by a methyl-deficient diet, did not favor atherosclerosis formation in the aortas of apoE-KO mice. Future analysis of intracellular hypomethylation may explain the observed effects of mild hyperhomocysteinemia on plaque formation. Funding Sources Graduate Program and University Funding. Supporting Tables, Images and/or Graphs