MiR-18a-5p aggravates homocysteine-induced myocardial injury via autophagy. / MiR-18a-5pé€šè¿‡ä»‹å¯¼è‡ªå™¬åŠ é‡åŒåž‹åŠèƒ±æ°¨é ¸è¯±å¯¼çš„å¿ƒè‚ŒæŸä¼¤.

OBJECTIVES: Hyperhomocysteinaemia (Hcy) is an independent risk factor for cardiovascular and cerebrovascular diseases. MicroRNA (miR)-18a-5p is closely related to cardiovascular diseases. This study aims to investigate the effects of miR-18a-5p on homocysteine (Hcy)-induced myocardial cells injury. METHODS: H9c2 cells were transfected with miR-18a-5p mimic/miR-18a-5p mimic negative control (NC) or combined with Hcy for intervention, and untreated cells were set as a control group. The transfection efficiency was verified by real-time RT-PCR, and cell counting kit-8 (CCK-8) assay was used to determine cell viability. Flow cytometry was used to detect apoptosis and reactive oxygen species (ROS) levels. Western blotting was performed to measure the protein levels of microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin1, p62, Bax, Bcl-2, and Notch2. Dual luciferase reporter assay was used to detect the interaction of miR-18a-5p with Notch2. RESULTS: Compared with the control, treatment with Hcy or transfection with miR-18a-5p mimic alone, or combined treatment with Hcy and miR-18a-5p mimic/miR-18a-5p mimic NC significantly reduced the H9c2 cell viability, promoted apoptosis and ROS production, up-regulated the expressions of Bax and Beclin, down-regulated the expressions of Bcl-2, p62, and Notch2, and increased the ratio of LC3-II/LC3-I (all P<0.05). Compared with the combined intervention of miR-18a-5p mimic NC and Hcy group, the above indexes were more significantly changed in the combined intervention of miR-18a-5p mimic and Hcy group, and the difference between the 2 groups was statistically significant (all P<0.05). There is a targeted binding between Notch2 and miR-18a-5p. CONCLUSIONS: MiR-18a-5p could induce autophagy and apoptosis via increasing ROS production in cardiomyocytes, and aggravate Hcy-induced myocardial injury. Notch2 is a target of miR-18a-5p.

Exogenous administration of hydrogen sulfide alleviates homocysteine induced inflammation in ARPE-19 cells.

Plasma homocysteine (Hcy) is an independent risk factor for Age related macular degeneration (AMD) and an inducer of inflammation. Homocysteine catabolism releases hydrogen sulfide (H2S). H2S has controversial effects on inflammation. In this study we have analysed the endogenous and exogenous H2S in modulating inflammation using adult retinal pigment epithelial (ARPE-19) cells as an in vitro model for AMD. ARPE-19 cells were treated with various concentrations of Hcy (15, 30 and 50 µM) for 3 h. Expression of Hcy transulfuration genes (CBS, CSE) by qPCR and western blot. H2S levels were measured using Free Radical Analyzer System (WPI, USA). The inflammatory markers (IL-6 and IL-8) were evaluated using real-time PCR and ELISA. Hcy exposure increased CBS protein expression, hydrogen sulfide levels and pro-inflammatory cytokines, modulating CBS by silencing did not alter H2S levels, but inhibition of CSE with PAG inhibited H2S production and decreased cytokine (IL-6 and IL-8) levels. On the contrary exogenous supply of hydrogen sulfide with NaHS and by compound 1c showed anti-inflammatory effects even in the presence of Hcy. This study shows that exogenous delivery of H2S decreases inflammation in retinal pigment epithelial cells on exposure to Hcy in ARPE-19 cells.

Homocysteine induced a calcium-mediated disruption of mitochondrial function and dynamics in endothelial cells.

Homocysteine (Hcy) is a sulfur-containing amino acid that originated in methionine metabolism and the elevated level of Hcy in plasma is considered to be an independent risk factor for cardiovascular diseases (CVD). Endothelial dysfunction plays a major role in the development of CVD, while the potential mechanism of Hcy-induced endothelial dysfunction is still unclear. Here, in Hcy-treated endothelial cells, we observed the destruction of mitochondrial morphology and the decline of mitochondrial membrane potential. Meanwhile, the level of ATP was reduced and the reactive oxygen species was increased. The expressions of dynamin-related protein 1 (Drp1) and phosphate-Drp1 (Ser616) were upregulated, whereas the expression of mitofusin 2 was inhibited by Hcy treatment. These findings suggested that Hcy not only triggered mitochondrial dysfunction but also incurred an imbalance of mitochondrial dynamics in endothelial cells. The expression of mitochondrial calcium uniporter (MCU) was activated by Hcy, contributing to calcium transferring into mitochondria. Interestingly, the formation of mitochondria-associated membranes (MAMs) was increased in endothelial cells after Hcy administration. The inositol 1,4,5-triphosphate receptor (IP3R)-glucose-regulated protein 75 (Grp75)-voltage-dependent anion channel (VDAC) complex, which was enriched in MAMs, was also increased. The accumulation of mitochondrial calcium could be blocked by inhibiting with the IP3R inhibitor Xestospongin C (XeC) in Hcy-treated cells. Then, we confirmed that the mitochondrial dysfunction and the increased mitochondrial fission induced by Hcy could be attenuated after Hcy and XeC co-treatment. In conclusion, Hcy-induced mitochondrial dysfunction and dynamics disorder in endothelial cells were mainly related to the increase of calcium as a result of the upregulated expressions of the MCU and the IP3R-Grp75-VDAC complex in MAMs.

A GLP-1 analog lowers ER stress and enhances protein folding to ameliorate homocysteine-induced endothelial dysfunction.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases and increases mortality in type 2 diabetic patients. HHcy induces endoplasmic reticulum (ER) stress and oxidative stress to impair endothelial function. The glucagon-like peptide 1 (GLP-1) analog exendin-4 attenuates endothelial ER stress, but the detailed vasoprotective mechanism remains elusive. The present study investigated the beneficial effects of exendin-4 against HHcy-induced endothelial dysfunction. Exendin-4 pretreatment reversed homocysteine-induced impairment of endothelium-dependent relaxations in C57BL/6 mouse aortae ex vivo. Four weeks subcutaneous injection of exendin-4 restored the impaired endothelial function in both aortae and mesenteric arteries isolated from mice with diet-induced HHcy. Exendin-4 treatment lowered superoxide anion accumulation in the mouse aortae both ex vivo and in vivo. Exendin-4 decreased the expression of ER stress markers (e.g., ATF4, spliced XBP1, and phosphorylated eIF2α) in human umbilical vein endothelial cells (HUVECs), and this change was reversed by cotreatment with compound C (CC) (AMPK inhibitor). Exendin-4 induced phosphorylation of AMPK and endothelial nitric oxide synthase in HUVECs and arteries. Exendin-4 increased the expression of endoplasmic reticulum oxidoreductase (ERO1α), an important ER chaperone in endothelial cells, and this effect was mediated by AMPK activation. Experiments using siRNA-mediated knockdown or adenoviral overexpression revealed that ERO1α mediated the inhibitory effects of exendin-4 on ER stress and superoxide anion production, thus ameliorating HHcy-induced endothelial dysfunction. The present results demonstrate that exendin-4 reduces HHcy-induced ER stress and improves endothelial function through AMPK-dependent ERO1α upregulation in endothelial cells and arteries. AMPK activation promotes the protein folding machinery in endothelial cells to suppress ER stress.

Implication of N-Methyl-d-Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-ß-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient's serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.

Homocysteine accelerates atherosclerosis by inhibiting scavenger receptor class B member1 via DNMT3b/SP1 pathway.

Homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is characterized by lipid accumulation in the atherosclerotic plaque. Increasing evidence supports that as the main receptor of high-density lipoprotein, scavenger receptor class B member 1 (SCARB1) is protective against atherosclerosis. However, the underlying mechanism regarding it in Hcy-mediated atherosclerosis remains unclear. Here, we found the remarkable inhibition of SCARB1 expression in atherosclerotic plaque and Hcy-treated foam cells, whereas overexpression of SCARB1 can suppress lipid accumulation in foam cells following Hcy treatment. Analysis of SCARB1 promoter showed that no significant change of methylation level was observed both in vivo and in vitro under Hcy treatment. Moreover, it was found that the negative regulation of DNMT3b on SCARB1 was due to the decreased recruitment of SP1 to SCARB1 promoter. Thus, we concluded that inhibition of SCARB1 expression induced by DNMT3b at least partly accelerated Hcy-mediated atherosclerosis through promoting lipid accumulation in foam cells, which was attributed to the decreased binding of SP1 to SCARB1 promoter. In our point, these findings will provide novel insight into an epigenetic mechanism for atherosclerosis.

Betaine-rich sugar beet molasses protects from homocysteine-induced reduction of survival in Caenorhabditis elegans.

PURPOSE: Homocysteine (Hcy) in humans represents a blood-borne biomarker which predicts the risk of age-related diseases and mortality. Using the nematode Caenorhabditis elegans, we tested whether feeding betaine-rich sugar beet molasses affects the survival under heat stress in the presence of Hcy, in spite of a gene loss in betaine-homocysteine methyltransferase. METHODS: Knockdown of the genes relevant for remethylation or transsulfuration of Hcy was achieved by RNA interference (RNAi). Survival assay was conducted under heat stress at 37 °C and Hcy levels were determined by enzyme-linked immunosorbent assay. RESULTS: Addition of 500 mg/l betaine-rich sugar beet molasses (SBM) prevented the survival reduction that was caused by exposure to Hcy at 37 °C. Although SBM was no longer capable of reducing Hcy levels under RNAi versus homologues for 5, 10-methylenetetrahydrofolate reductase or cystathionine-ß-synthase, it still enabled the survival extension by SBM under exposure to Hcy. In contrast, RNAi for the small heat shock protein hsp-16.2 or the foxo transcription factor daf-16 both prevented the extension of survival by betaine-rich molasses in the presence of Hcy. CONCLUSIONS: Our studies demonstrate that betaine-rich SBM is able to prevent survival reduction caused by Hcy in C. elegans in dependence on hsp-16.2 and daf-16 but independent of the remethylation pathway.

The anti-inflammatory effect of microRNA-383-3p interacting with IL1R2 against homocysteine-induced endothelial injury in rat coronary arteries.

MicroRNAs (miRs) are widely reported to be novel biomarkers involved in the process of coronary atherosclerosis (CAS). Hence, this study aims to explore the function of miR-383-3p targeting IL1R2 on inflammatory injury of coronary artery endothelial cells (CAECs) in CAS. The underlying regulatory mechanisms of miR-383-3p were analyzed in concert with the treatment of miR-383-3p mimics, miR-383-3p inhibitors, and the combination of miR-383-3p inhibitors and siRNA against IL1R2 in homocysteine (HCY)-induced CAECs. MTT, Hoechst 33258 staining, and tube formation assay were employed in order to measure cell viability, apoptosis, and tube formation, respectively. The levels of IL-1ß, IL-6, IL-10, and IL-18 were determined by ELISA. IL1R2 was verified as the target gene of miR-383-3p by dual-luciferase reporter gene assay. MiR-383-3p was down-regulated in myocardial tissues of AS rats while IL1R2 was the reciprocal. The up-regulation of miR-383-3p decreased the levels of IL1R2, caspase-1, IL-1ß, IL-6, and IL-18 expressions, as well as cell apoptosis rate in the HCY-induced CAECs, while IL-10 expression, cell viability, and tube formation ability were increased. These results were contraindicated in the HCY-induced CAECs treated by miR-383-3p inhibitors. In conclusion, miR-383-3p mediating IL1R2 prevents HCY-induced apoptosis and inflammation injury in CAECs through the inhibition of the activation of inflammasome signaling pathway. These findings highly indicate that miR-383-3p may be beneficial in the prevention of CAS and other cardiovascular diseases.

Epigallocatechin gallate protects against homocysteine-induced vascular smooth muscle cell proliferation.

Epigallocatechin gallate (EGCG), a bioactive ingredient of green tea, plays a protective role in the cardiovascular system. Homocysteine (Hcy) is a major risk factor for chronic kidney disease and cardiovascular disease. The present study aimed to investigate the role of EGCG in Hcy-induced proliferation of vascular smooth muscle cells (VSMCs) and its underlying mechanism. We also explored the roles of rennin-angiotensin system (RAS), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) in this process. Human aortic smooth muscle cells (HASMCs) were treated with different drugs for different periods. The proliferation rate of HASMCs was detected using the CCK-8 and BrdU labeling assays. The Western blot assay was used to determine the expression levels of angiotensin II type 1 receptor (AT-1R), ERK1/2, and p38 MAPK. Compared with the control group, the HASMCs treated with Hcy at different doses (100, 200, 500, and 1000 µM) showed significantly increased proliferation. Hcy increased the expression of AT-1R, whereas EGCG decreased the protein expression of AT-1R. Furthermore, we found that Hcy-induced expression of p-ERK1/2 and p-p38MAPK was dependent on AT-1R. Compared with Hcy (500 µM)-treated cells, EGCG (20 µM)-treated cells showed decreased proliferation as well as expression of AT-1R, p-ERK1/2, and p-p38MAPK. In addition, HASMC proliferation was suppressed by the addition of an AT-1R blocker (olmesartan), an ERK1/2 inhibitor (PD98059), and a p38MAPK inhibitor (SB202190). EGCG can inhibit AT-1R and affect ERK1/2 and p38MAPK signaling pathways, resulting in the decrease of VSMC proliferation induced by Hcy.

Homocysteine, Liver Function Derangement and Brain Atrophy in Alcoholics.

AIMS: Hyperhomocysteinemia may be involved in the development of brain atrophy in alcoholics. Its pathogenesis is multifactorial. In the present study, we analyse the relationship between homocysteine levels and brain atrophy, and the relative weight of co-existing factors such as liver function impairment, the amount of ethanol consumed, serum vitamin B12, B6, and folic acid levels on homocysteine levels and brain alterations in alcoholic patients. METHODS: We included 59 patients admitted to this hospital for major withdrawal symptoms and 24 controls. The mini-mental state examination test and a brain computed tomography (CT) scan were performed and several indices were calculated. Serum levels of homocysteine, folic acid, vitamin B6 and vitamin B12 were determined. Liver function was assessed by Child-Pugh score. The daily consumption of ethanol in grams per day and years of addiction were recorded. RESULTS: A total of 83.6% and 80% of the patients showed cerebellar or frontal atrophy, respectively. Patients showed altered values of brain indices, higher levels of homocysteine and vitamin B12, but lower levels of folic acid, compared with controls. Homocysteine, B12 and liver function variables showed significant correlations with brain CT indices. Multivariate analyses disclosed that Pugh's score, albumin and bilirubin were independently related to cerebellar atrophy, frontal atrophy, cella index or ventricular index. Serum vitamin B12 was the only factor independently related to Evans index. It was also related to cella index, but after bilirubin. Homocysteine levels were independently related to ventricular index, but after bilirubin. CONCLUSION: Vitamin B12 and homocysteine levels are higher among alcoholics. Liver function derangement, vitamin B12 and homocysteine are all independently related to brain atrophy, although not to cognitive alterations. SHORT SUMMARY: Hyperhomocysteinemia has been described in alcoholics and may be related to brain atrophy, a reversible condition with an obscure pathogenesis. We studied 59 patients and found that liver function derangement, vitamin B12 and homocysteine levels are all independently related to brain atrophy assessed by computed tomography, although we found no association between these parameters and cognitive alterations.

The Protective Effects of Icariin against the Homocysteine-Induced Neurotoxicity in the Primary Embryonic Cultures of Rat Cortical Neurons.

Icariin, an ingredient in the medicinal herb Epimedium brevicornum Maxim (EbM), has been considered as a potential therapeutic agent for neurodegenerative diseases such as Alzheimer's disease (AD). Hyperhomocysteinaemia is a risk factor for AD and other associated neurological diseases. In this study we aim to investigate whether icariin can reverse homocysteine (Hcy)-induced neurotoxicity in primary embryonic cultures of rat cortical neurons. Our findings demonstrated that icariin might be able restore the cytoskeleton network damaged by Hcy through the modulation of acetyl-α-tubulin, tyrosinated-α-tubulin, and phosphorylation of the tubulin-binding protein Tau. In addition, icariin downregulated p-extracellular signal-regulated kinase (ERK) which is a kinase targeting tau protein. Furthermore, icariin effectively restored the neuroprotective protein p-Akt that was downregulated by Hcy. We also applied RT² Profiler PCR Arrays focused on genes related to AD and neurotoxicity to examine genes differentially altered by Hcy or icariin. Among the altered genes from the arrays, ADAM9 was downregulated 15 folds in cells treated with Hcy, but markedly restored by icariin. ADAM family, encoded α-secreatase, plays a protective role in AD. Overall, our findings demonstrated that icariin exhibits a strong neuroprotective function and have potential for future development for drug treating neurological disorders, such as AD.

[The effect of miR-124 on homocysteine-induced atherosclerosis via promoter region DNA methylation in ApoE(-/-) mice].

The aim of the present study is to explore the role of miR-124 and its promoter region DNA methylation in homocysteine (Hcy)-induced atherosclerosis. ApoE(-/-) mice were fed with hypermethionine diet for 16 weeks to duplicate hyperhomocysteinemia model. Meanwhile, a normal control group (C57BL/6J mice fed with normal diet, N-control) and a model control group (ApoE(-/-) mice fed with normal diet, A-control) were set. The degree of atherosclerosis was observed by HE and oil red O staining. Automatic biochemical analyzer was used to detect the serum levels of Hcy. Foam cell model was duplicated and oil red O staining was used to confirm whether the model was successfully established. And foam cells were stimulated with 0, 50, 100, 200, 500 µmol/L Hcy and 50 µmol/L Hcy + 10 µmol/L AZC respectively. Real-time quantitative PCR (RT-qPCR) was used to detect the expressions of miR-124 in mice aorta and foam cells; Nested landing methylation specific PCR (nMS-PCR) was used to detect the levels of miR-124 promoter DNA methylation in mice aorta and foam cells. Meanwhile, the effects of DNA methylation inhibitor AZC on miR-124 expression were observed at the cellular level. The effect of miR-124 promoter DNA methylation status on lipid accumulation in foam cells was observed by oil red O staining. The results showed that compared with model control group, the serum levels of Hcy in high methionine group were significantly increased (P < 0.01) and developed aortic atherosclerotic plaque, the expression of miR-124 was markedly decreased (P < 0.01), while the levels of miR-124 promoter DNA methylation were significantly increased (P < 0.01). Given different levels of Hcy, the expression of miR-124 in foam cells was decreased, while the levels of miR-124 promoter DNA methylation were increased in a dose-dependent manner (P < 0.05, P < 0.01). AZC reversed the results of mentioned indices as above markedly (P < 0.05). Downregulation of miR-124 may play a role in Hcy-induced atherosclerosis and its promoter DNA methylation status may be an important mechanism in this process.

[Ad-hVEGF165 reverses homocysteine-induced endothelial dysfunction by regulating nitric oxide system].

OBJECTIVE: To investigate the therapeutic effect of Ad-hVEGF165 on the endothelial cells dysfunction induced by homocysteine (Hcy) and related molecular mechanisms. METHODS: Human umbilical vein endothelial cells CRL-1730 were treated with Hcy at different concentrations (0, 0.05, 1.00 mmol/L) for 24 h. The same concentration of Hcy, Ad-Track and Ad-hVEGF165 were added to the cells in the following groups: blank group, Hcy0.05 group, Hcy1.00 group, Ad-Track group, Hcy0.05+Ad-Track group, Hcy1.00+Ad-Track group, Ad-hVEGF165 group, Hcy0.05+Ad-hVEGF165 group, Hcy1.00+ Ad-hVEGF165 group for 48 h. The mRNA and protein expressions of eNOS and DDAH2 were detected by real-time PCR and Western blot. The correlations of mRNA and protein expressions between endothelial nitric oxide synthase (eNOS) and dimethylarginine dimthylaminohydrolase (DDAH)2 were evaluated by Pearson correlation analysis. RESULTS: Compared with blank group and Ad-hVEGF165 group, the mRNA and protein expressions of eNOS were decreased in Hcy0.05 group and Hcy0.05+Ad-hVEGF165 group (both P < 0.05), and the mRNA and protein expressions of DDAH2 in cells treated with 0.05 mmol/L and 1.00 mmol/L Hcy were reduced as well (all P < 0.05). DDAH2 mRNA and protein expression are increased (all P < 0.05) in Ad-hVEGF165 group compared with the blank group and Ad-Track, Hcy0.05 + Ad-hVEGF165 and Hcy0.05 group compared with Hcy0.05+Ad-Track group, Hcy1.00+Ad-hVEGF165 and Hcy1.00 group compared with Hcy1.00+Ad-Track group. The mRNA and protein expressions of eNOS and DDAH2 were uncorrelated under the effect of Hcy (r = 0.057 and 0.449, both P > 0.05) and VEGF (r = 0.284 and 0.432, both P > 0.05). CONCLUSION: Recombinant adenovirus Ad-hVEGF165 could reverse Hcy-induced endothelial cells dysfunction via upregulating the expressions of eNOS and DDAH2.

Endoplasmic reticulum oxidoreductin 1α mediates hepatic endoplasmic reticulum stress in homocysteine-induced atherosclerosis.

Endoplasmic reticulum (ER) stress is emerging as an important modulator of different pathological process and as a mechanism contributing to homocysteine (Hcy)-induced hepar injury. However, the molecular event that Hcy-induced ER stress in the hepar under the atherosclerosis background is currently unknown. Endoplasmic reticulum oxidoreductin 1α (ERO1α) plays a crucial role in maintaining ER stress function. In this study, we determined the expression of ERO1α in the hepar in hyperhomocysteinemia and the effect of ERO1α in hepacytes ER stress in the presence of Hcy. HHcy model was established by feeding the methionine diet in apolipoprotein-E-deficient (ApoE-/-) mice, and the hepatocytes were incubated with folate and different concentrations of Hcy. Our results showed that Hcy triggered ER stress characterized by an increased contents of glucose-regulated protein 78 (GRP78), protein kinase RNA-like ER kinase (PERK), activating transcription factor (ATF) 6 and X-box binding protein-1 (XBP-1). The ERO1α expressions in HHcy mice and Hcy-treated hepatocytes were decreased compared with those in ApoE-/- group and control hepacytes (P < 0.05), respectively. Knocking-down the expression of ERO1α with small-interfering RNA significantly augmented Hcy-induced ER stress. Meanwhile, the expressions of ER stress-related factor including GRP78, PERK, ATF6 and XBP-1, were significantly decreased when the ERO1α gene was over-expressed in hepacytes. Our results suggested that ERO1α may be involved in Hcy-induced hepar ER stress, and the inhibition of ERO1α expression can accelerate this process.

Exercise attenuates the perioperative neurocognitive disorder induced by hyperhomocysteinemia in mice.

The perioperative neurocognitive disorder (PND) is a severe complication that affects millions of surgical patients each year. Homocysteine (Hcy) is known to increase the risk of developing PND in both young and elderly mice. However, whether Hcy alone can induce cognitive deficits in middle-aged mice (12-month-old), whether exercise can attenuate Hcy-induced hippocampus-related cognitive deficits after surgery through suppressing neuroinflammation, synaptic elimination, and the level of Hcy remains unknown. The present study aimed to answer these questions through testing the possibility of establishing a PND model using 12-month-old mice which received homocysteine injections before exploratory laparotomy and the therapeutic mechanism of exercise. In the present study, it was found that levels of serum homocysteine were age-dependently increased in mice with a significant difference between that of 18-month-old mice and 6-week, 6-month, and 12-month-old mice. PND occurred in 18-month but not in 12-month-old mice after exploratory laparotomy under isoflurane anesthesia. Intraperitoneal injection of Hcy for 3 consecutive days before surgery rendered 12-month-old mice to develop PND after abdominal laparotomy under isoflurane anesthesia at a minimal dosage of 20 mg/kg. Neuroinflammation and synaptic elimination was present in 12-month-old preoperative Hcy-injected mice. Preoperative voluntary wheel exercise could prevent PND in 12-month-old mice that have received Hcy injection before surgery, which might be related to the decreased level of serum Hcy. Activation of glial cells, proinflammatory phenotype markers and synaptic elimination were attenuated in the hippocampus of 12-month-old preoperative Hcy-injected mice by this exercise. These results provide direct evidence that hyperhomocysteinemia can induce postoperative cognitive deficits in middle-aged mice. Pre-surgery exercise can effectively prevent Hcy-precipitated postoperative cognitive dysfunction.

Effects of alcohol, lithium, and homocysteine on nonmuscle myosin-II in the mouse placenta and human trophoblasts.

OBJECTIVE: Mouse embryonic exposure to alcohol, lithium, and homocysteine results in intrauterine growth restriction (IUGR) and cardiac defects. Our present study focused on the placental effects. We analyzed the hypothesis that expression of nonmuscle myosin (NMM)-II isoforms involved in cell motility, mechanosensing, and extracellular matrix assembly are altered by the 3 factors in human trophoblast (HTR8/SVneo) cells in vitro and in the mouse placenta in vivo. STUDY DESIGN: After exposure during gastrulation to alcohol, homocysteine, or lithium, ultrasonography defined embryos exhibiting abnormal placental blood flow. RESULTS: NMM-IIA/NMM-IIB are differentially expressed in trophoblasts and in mouse placental vascular endothelial cells under pathological conditions. Misexpression of NMM-IIA/NMM-IIB in the affected placentas continued stably to midgestation but can be prevented by folate and myoinositol supplementation. CONCLUSION: It is concluded that folate and myoinositol initiated early in mouse pregnancy can restore NMM-II expression, permit normal placentation/embryogenesis, and prevent IUGR induced by alcohol, lithium, and homocysteine.

Homocysteine in occlusive vascular disease: a risk marker or risk factor.

Homocysteine has emerged as a significant marker for occlusive vascular disease, but there has been some debate as to whether it is just an association (risk marker) or actually a causative factor (risk factor). To elucidate this, a retrospective statistical analysis was done of data generated in the course of our study on homocysteine and vascular disease. Homocysteine, lipid profile components and lipoprotein(a) were estimated in fasting blood samples drawn from 252 controls and 536 patients of occlusive vascular disease. The data were analyzed by SPSS version 17. Mean homocysteine levels were significantly higher (p < 0.001) in all patients categories, as compared to controls. In fact, homocysteine level was the most significant biochemical risk factor for vascular disease. The odds ratios due to hyperhomocysteinemia varied from 3.170-4.153. When the cut-off was increased by 5 micromol/L, the odds ratio became almost three-fold. The prevalence of hyperhomocysteinemia increased by approximately equal to 20%, when the cut-off was reduced by 5 micromol/L. Statistical analysis of our data revealed that homocysteine conformed to Hill's criteria of causation. Moreover, hyperhomocysteinemia was treatable by the administration of B-vitamins, even if the cause was genetic. Hence morbidity due to vascular disease could be reduced by identification and treatment of hyperhomocysteinemia.

[Protective effect of ligustrazine hydrochloride on homocysteine-injured ECV304 cells].

OBJECTIVE: To detect the protective effect of ligustrazine hydrochloride on homocysteine-injured ECV304 cells. METHOD: In the in vitro study, human umbilical vein endothelial cells were selected as objects, with homocysteine as the molding agent, to judge the injury degree by monitoring NOS and NO contents. Based on that, the best homocysteine concentration in ECV304 cells, the best reaction time could be determined, and an endothelial cell injury model was established. After adding ligustrazine hydrochloride, NOS and NO contents in injured endothelial cells were determined to observe the protective effect of ligustrazine hydrochloride. RESULT: It was proved that the optimal concentration of homocysteine on injured ECV304 cell was 1 mmol x L(-1), the best reaction time was 48 h. An injured endothelial cell model was established. At the same time, positive drug nitroglycerin and ligustrazine hydrochloride displayed a protection effect on injured ECV304 cells, NOS and NO formation were significantly increased compared with the model group. CONCLUSION: Ligustrazine hydrochloride has a protective effect on homocysteine-injured ECV304 cells. The model established in this study can be used to screen anti-myocardial ischemia drugs targeting at an endothelial cell protective agent.

Homocysteine-Induced Disturbances in DNA Methylation Contribute to Development of Stress-Associated Cognitive Decline in Rats.

Chronic stress is generally accepted as the main risk factor in the development of cognitive decline; however, the underlying mechanisms remain unclear. Previous data have demonstrated that the levels of homocysteine (Hcy) are significantly elevated in the plasma of stressed animals, which suggests that Hcy is associated with stress and cognitive decline. To test this hypothesis, we analyzed the cognitive function, plasma concentrations of Hcy, and brain-derived neurotropic factor (BDNF) levels in rats undergoing chronic unpredicted mild stress (CUMS). The results showed that decreased cognitive behavioral performance and decreased BDNF transcription and protein expression were correlated with hyperhomocysteinemia (HHcy) levels in stressed rats. Diet-induced HHcy mimicked the cognitive decline and BDNF downregulation in the same manner as CUMS, while Hcy reduction (by means of vitamin B complex supplements) alleviated the cognitive deficits and BDNF reduction in CUMS rats. Furthermore, we also found that both stress and HHcy disturbed the DNA methylation process in the brain and induced DNA hypermethylation in the BDNF promoter. In contrast, control of Hcy blocked BDNF promoter methylation and upregulated BDNF levels in the brain. These results imply the possibility of a causal role of Hcy in stress-induced cognitive decline. We also used ten-eleven translocation (TET1), an enzyme that induces DNA demethylation, to verify the involvement of Hcy and DNA methylation in the regulation of BDNF expression and the development of stress-related cognitive decline. The data showed that TET1-expressing viral injection into the hippocampus inhibited BDNF promoter methylation and significantly mitigated the cognitive decline in HHcy rats. Taken together, novel evidence from the present study suggests that Hcy is likely involved in chronic stress-induced BDNF reduction and related cognitive deficits. In addition, the negative side-effects of HHcy may be associated with Hcy-induced DNA hypermethylation in the BDNF promoter. The results also suggest the possibility of Hcy as a target for therapy and the potential value of vitamin B intake in preventing stress-induced cognitive decline.

Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors.

PURPOSE: Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)--the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself--as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model. METHODS: A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 µM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 µM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 µM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells. RESULTS: Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsin⁺ photoreceptors and the calbindin⁺ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 µM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10⁺ or the Islet-1⁺ bipolar cells and the Pax-6⁺ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP⁺ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection. CONCLUSIONS: Excessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.