Podocyte-specific silencing of acid sphingomyelinase gene to abrogate hyperhomocysteinemia-induced NLRP3 inflammasome activation and glomerular inflammation.

Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1-/- mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1-/- mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect.NEW & NOTEWORTHY In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects.

Association of MTHFR C677T, MTHFRA1298C, and MTRRA66G Gene Polymorphisms with Hyperhomocysteinemia and Its Modulation by the Combined Effect of Vitamin B12 and Folate in Chinese Population with Hypertension.

BACKGROUND: In China, the MTHFR 677T allele, unlike in most Western populations, is a rare genetic variant linked to various disorders. The contributing nutritional and genetic factors to this genetic risk remain unclear. OBJECTIVE: This study aimed to elucidate the interactions between genetic variations in total homocysteine (tHcy) pathway genes, serum tHcy concentrations, and nutritional factors in a Chinese population with hypertension. METHODS: This study analyzed 1304 Chinese adults with hypertension aged ≥18 y enrolled in the China Precision Nutrition and Health KAP Real World Study (CPNAS). Serum concentrations of vitamin B12 and folate were measured using the magnetic microparticle chemiluminescence method, and tHcy concentrations were measured using Hcy Assay kits. Identification of the MTHFR C677T, MTHFR A1298C, and MTRR A66G polymorphisms was performed via time-of-flight nucleic spectrometry. RESULTS: Our findings revealed significant sex differences in tHcy concentrations, with males exhibiting higher tHcy concentrations than females (13.95 µmol/L vs. 11.15 µmol/L, P < 0.001). Individuals deficient in both vitamin B12 and folate had an increased risk of hyperhomocysteinemia (H-Hcy) (57.4%). In contrast, the prevalence of H-Hcy was lower among those deficient in either vitamin B12 (31.1%) or folate (23.2%) alone. Significant associations were identified between the MTHFR C677T and A1298C polymorphisms and elevated serum tHcy concentrations, particularly in individuals homozygous for the T allele. Conversely, the MTRR A66G genotype did not show a significant correlation with tHcy concentrations. Optimal vitamin B12 concentrations significantly modulated the genotypic effect on tHcy concentrations, with individuals having adequate vitamin B12 and folate exhibiting low tHcy concentrations, even among high-risk genotypes (TT). CONCLUSIONS: Adequate concentrations of folate and vitamin B12 significantly reduce serum tHcy concentrations and mitigate the genotypic impact on tHcy concentrations, highlighting the potential for targeted nutritional interventions to manage cardiovascular risks associated with H-Hcy. This trial was registered at clinicaltrials.gov as ChiCTR2100051983.

Hydrogen Sulfide Ameliorated Endothelial Dysfunction in hyperhomocysteinemia Rats: Mechanism of IRE1α/JNK pathway-mediated Autophagy.

Previous studies showed that hyperhomocysteinemia (HHcy) induced endothelial dysfunction by endoplasmic reticulum (ER) stress induction and autophagy stimulation. This study aimed to determine the effect of hydrogen sulfide (H2S) in homocysteine (Hcy)-induced endothelial dysfunction and observe the possible mechanism involved.Male Wistar rats (160-180g) were used and randomly divided into four groups: Control group, HHcy group, HHcy+Sodium hydrosulfide (NaHS) group and NaHS group. Rats were fed with 2% high methionine diet for 8 weeks to set up HHcy model. Plasma concentration of Hcy was measured by ELISA.Endothelium-dependent and non-endothelium-dependent vasodilation of rat renal arteries were determined by myograph.The protein expression of cystathionine-γ-lyase (CSE), ER stress-and autophagy-related proteins in renal arteries or human umbilical vein endothelial cells (HUVECs) were analyzed by western blotting. The endothelial function was impaired in HHcy rats and HUVECs. NaHS supplementation could improve the ACh-induced vasodilation, however it was eliminated by ER stress inducer Tunicamycin (TM) or autophagy inducer Rapamycin. Western blotting in renal arteries showed that Glucose-regulated protein 78 (GRP78) and three branches of ER stress (p-IRE1α, p-PERK, ATF6, GRP78) and p-JNK1+p-JNK2 was downregulated by NaHS administration, simultaneously the autophagy marker Beclin1, LC3BII/LC3BI ratio was decreased and p62 was increased in HHcy rats. In HUVECs, IRE1α-JNK induced autophagy was involved in HHcy-induced endothelial dysfunction, while NaHS stimulation decreased the protein expression in IRE1α/JNK-autophagy pathway with Hcy incubation. This study might suggest that endothelial dysfunction induced by HHcy might be correlate with IRE1α-JNK-autophagy axis pathway, which was suppressed by exogenous supplementation of H2S donor NaHS.

H2S prevents the disruption of the blood-brain barrier in rats with prenatal hyperhomocysteinemia.

Elevation of the homocysteine concentration in the plasma called hyperhomocysteinemia (hHCY) during pregnancy causes a number of pre- and postnatal developmental disorders. The aim of our study was to analyze the effects of H2S donors -NaHS and N-acetylcysteine (NAC) on blood-brain barrier (BBB) permeability in rats with prenatal hHCY. In rats with mild hHCY BBB permeability assessed by Evans Blue extravasation in brain increased markedly throughout life. Administration of NaHS or NAC during pregnancy attenuated hHCY-associated damage and increased endogenous concentrations of sulfides in brain tissues. Acute application of dl-homocysteine thiolactone induced BBB leakage, which was prevented by the NMDA receptor antagonist MK-801 or H2S donors. Rats with hHCY demonstrated high levels of NO metabolite - nitrites and proinflammatory cytokines (IL-1ß, TNF-α, IL-6) in brain. Lactate dehydrogenase (LDH) activity in the serum was higher in rats with hHCY. Mitochondrial complex-I activity was lower in brain of hHCY rats. NaHS treatment during pregnancy restored levels of proinflammatory cytokines, nitrites and activity of the respiratory chain complex in brain as well as the LDH activity in serum. Our data suggest that H2S has neuroprotective effects against prenatal hHCY-associated BBB disturbance providing a potential strategy for the prevention of developmental impairments in newborns.

Catalpol inhibits HHcy-induced EndMT in endothelial cells by modulating ROS/NF-κB signaling.

BACKGROUND: Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis (AS). Endothelial mesenchymal transition (EndMT) refers to the process in which endothelial cells lose endothelial cell morphology and characteristic gene expression, and acquire phenotypic characteristics and gene expression related to mesenchymal cells. Numerous studies have confirmed that EndMT is involved in the formation of atherosclerosis. Catalpol is one of the active components of Rehmannia, which has antioxidant, anti-inflammatory, anti-tumor, neuroprotective and other biological activities. Studies have shown that catalpol can reduce atherosclerotic plaque induced by high sugar or fat. However, the effect of catalpol on HHCY-induced EndMT is unclear. METHODS AND RESULTS: In vitro HHcy-treated primary human umbilical vein endothelial cells (HUVECs) were used to construct a cell model, and the antioxidants N-acetylcysteine (NAC) and catalase alcohol were administered. In vivo C57BL/6N mice were given a diet fed with 4.4% high methionine chow to construct a HHcy mice model and were treated with catalpol. The results showed that hhcy could induce morphological transformation of endothelial cells into mesenchymal cells, increase intracellular ROS content, up-regulate α-SMA, N-cadherin, p-p65 protein expression, down-regulate VE-cadherin, CD31 protein expression, induce pathological changes of aortic root endothelium, and increase aortic endothelial ROS content. Catalpol reversed these hhcy induced outcomes. CONCLUSIONS: Catalpol inhibits HHcy-induced EndMT, and the underlying mechanism may be related to the ROS/NF-κB signaling pathway. Catalpol may be a potential drug for the treatment of HHcy-related AS.

Analysis of MTHFR C677T genotype and related factors in H-type hypertension in Tibet, China.

BACKGROUND AND AIMS: H-type hypertension is essential hypertension combined with high homocysteine, and both synergistically increase the risk of cardiovascular and cerebrovascular events. The aim of this study was to investigate the risk factors of H-type hypertension in Tibetan plateau population and correlation with MTHFR C677T gene. METHODS AND RESULTS: A multi-stage cluster random sampling method was used to select the research subjects in Tibet Autonomous Region from June 2020 to November 2021. Among Tibetans, the incidence of H-type hypertension accounted for 84.31% of hypertensive patients. The logistic regression analysis demonstrated that age, uric acid (UA), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were risk factors for the prevalence of H-type hypertension, the OR (95% CI) was 1.083(1.073-1.094), 1.002(1.001-1.004), 1.240(1.050-1.464) and 2.274(1.432-3.611), respectively. MTHFR C677T TT genotype patients with H-type hypertension OR (95% CI) was 1.629(1.004-2.643). Based on this, a nomogram model was established, and the reliability of the model was proved by area under ROC curve, Brier score and average absolute error. The model's results indicate that for every five years of age, the score increases by 6 points; for a 2mmol/L increase in TG, the score increases by 5.5 points; for a 1mmol/L increase in LDL-C, the score increases by 10 points; and individuals with the TT genotype receive 8 points. The higher the score, the greater the risk of disease. CONCLUSION: The MTHFR C677T TT genotype is a risk locus for Tibetan patients with H-type hypertension, with age, TG, and LDL-C were identified as risk factors for the disease.

Adult-onset combined methylmalonic acidemia and hyperhomocysteinemia, cblC type with aortic dissection and acute kidney injury: a case report.

BACKGROUND: Combined methylmalonic acidemia (MMA) and hyperhomocysteinemia, cobalamin C (cblC) type, also named cblC deficiency is a rare autosomal recessive genetic metabolic disease. It progressively causes neurological, hematologic, renal and other system dysfunction. The clinical manifestations are relatively different due to the onset time of disease. CASE PRESENTATION: This report describes a rare case of a 26 year old man with cblC deficiency who developed life-threatening aortic dissection and acute kidney injury (AKI) and showed neuropsychiatric symptoms with elevated serum homocysteine and methylmalonic aciduria. After emergent operation and intramuscular cobalamin supplementation therapy, the male recovered from aortic dissection, neurological disorder and AKI. Finally, two previously published compound heterozygous variants, c.482G > A (p.R161Q) and c.658_660del (p.K220del) in the MMACHC gene were detected in this patient and he was confirmed to have cblC deficiency. CONCLUSIONS: Poor cognizance of presenting symptoms and biochemical features of adult onset cblC disease may cause delayed diagnosis and management. This case is the first to depict a case of adult-onset cblC deficiency with aortic dissection. This clinical finding may contribute to the diagnosis of cblC deficiency.

Tryptophan Metabolism in Obesity: The Indoleamine 2,3-Dioxygenase-1 Activity and Therapeutic Options.

Obesity activates both innate and adaptive immune responses in adipose tissue. Adipose tissue macrophages are functional antigen-presenting cells that promote the proliferation of interferon-gamma (IFN-γ)-producing cluster of differentiation (CD)4+ T cells in adipose tissue of obese subjects. The increased formation of neopterin and degradation of tryptophan may result in decreased T-cell responsiveness and lead to immunodeficiency. The activity of inducible indoleamine 2,3-dioxygenase-1 (IDO1) plays a major role in pro-inflammatory, IFN-γ-dominated settings. The expression of several kynurenine pathway enzyme genes is significantly increased in obesity. IDO1 in obesity shifts tryptophan metabolism from serotonin and melatonin synthesis to the formation of kynurenines and increases the ratio of kynurenine to tryptophan as well as with neopterin production. Reduction in serotonin (5-hydroxytryptamine; 5-HT) production provokes satiety dysregulation that leads to increased caloric uptake and obesity. According to the monoamine-deficiency hypothesis, a deficiency of cerebral serotonin is involved in neuropsychiatric symptomatology of depression, mania, and psychosis. Indeed, bipolar disorder (BD) and related cognitive deficits are accompanied by a higher prevalence of overweight and obesity. Furthermore, the accumulation of amyloid-ß in Alzheimer's disease brains has several toxic effects as well as IDO induction. Hence, abdominal obesity is associated with vascular endothelial dysfunction. kynurenines and their ratios are prognostic parameters in coronary artery disease. Increased kynurenine/tryptophan ratio correlates with increased intima-media thickness and represents advanced atherosclerosis. However, after bariatric surgery, weight reduction does not lead to the normalization of IDO1 activity and atherosclerosis. IDO1 is involved in the mechanisms of immune tolerance and in the concept of tumor immuno-editing process in cancer development. Serum IDO1 activity is still used as a parameter in cancer development and growth. IDO-producing tumors show a high total IDO immunostaining score, and thus, using IDO inhibitors, such as Epacadostat, Navoximod, and L isomer of 1-methyl-tryptophan, seems an important modality for cancer treatment. There is an inverse correlation between serum folate concentration and body mass index, thus folate deficiency leads to hyperhomocysteinemia-induced oxidative stress. Immune checkpoint blockade targeting cytotoxic T-lymphocyte-associated protein-4 synergizes with imatinib, which is an inhibitor of mitochondrial folate-mediated one-carbon (1C) metabolism. Antitumor effects of imatinib are enhanced by increasing T-cell effector function in the presence of IDO inhibition. Combining IDO targeting with chemotherapy, radiotherapy and/or immunotherapy, may be an effective tool against a wide range of malignancies. However, there are some controversial results regarding the efficacy of IDO1 inhibitors in cancer treatment.

Homocysteine fibrillar assemblies display cross-talk with Alzheimer's disease ß-amyloid polypeptide.

High levels of homocysteine are reported as a risk factor for Alzheimer's disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated ß-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between ß-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.

Relationship between homocysteine and chronic total coronary occlusion: a cross-sectional study from southwest China.

BACKGROUND: Chronic total coronary occlusion is among the most complex coronary artery diseases. Elevated homocysteine is a risk factor for coronary artery diseases. However, few studies have assessed the relationship between homocysteine and chronic total coronary occlusion. METHODS: 1295 individuals from Southwest China were enrolled in the study. Chronic total coronary occlusion was defined as complete occlusion of coronary artery for more than three months. Homocysteine was divided into quartiles according to its level. Univariate and multivariate logistic regression models, receiver operating characteristic curves, and subgroup analysis were applied to assess the relationship between homocysteine and chronic total coronary occlusion. RESULTS: Subjects in the higher homocysteine quartile had a higher rate of chronic total coronary occlusion (P < 0.001). After adjustment, the odds ratio for chronic total coronary occlusion in the highest quartile of homocysteine compared with the lowest was 1.918 (95% confidence interval 1.237-2.972). Homocysteine ≥ 15.2 µmol/L was considered an independent indicator of chronic total coronary occlusion (odds ratio 1.53, 95% confidence interval 1.05-2.23; P = 0.0265). The area under the receiver operating characteristic curve was 0.659 (95% confidence interval, 0.618-0.701; P < 0.001). Stronger associations were observed in elderly and in those with hypertension and diabetes. CONCLUSIONS: Elevated homocysteine is significantly associated with chronic total coronary occlusion, particularly in elderly and those with hypertension and diabetes.

A micronutrient supplement modulates homocysteine levels regardless of vitamin B biostatus in elderly subjects.

Elevated homocysteine (Hcy) levels (≥15 µmol/L) in the elderly are frequently associated with a higher risk of cardiovascular disease and cognitive decline. Several studies have already shown an Hcy-lowering effect of B vitamin supplementation in cohorts deficient in these nutrients. The aim of this randomized, double-blinded 12-week intervention study was to investigate whether Hcy levels in healthy elderly subjects (75.4±4.5 years, n=133) could be lowered with a micronutrient supplement (i.e., 400 µg folic acid, 100 µg cobalamin). Difference in mean initial Hcy levels between intervention (17.6±7.1 µmol/L, n=65) and placebo group (18.9±6.1 µmol/L, n=68) was not significant. The prevalence of cobalamin and folate deficiency in the total study population was low: 27% had serum-cobalamin levels ≤150 pmol/L, 12% holo-transcobalamin (Holo-TC) levels ≤50 pmol/L, 13% low cobalamin status using the aggregated cobalamin marker 4cB12 and 10% red blood cell (RBC) folate ≤570 nmol/L. Nevertheless, the treated subjects still showed improved cobalamin and folate biostatus (serum cobalamin Δt12-t0: 63±48 pmol/L; Holo-TC Δt12-t0: 17±19 pmol/L; RBC folate Δt12-t0: 326±253 nmol/L) and Hcy levels (Δt12-t0: -3.6±5.7 µmol/L). The effects were statistically significant compared to the placebo group with p=0.005 (serum cobalamin), p=0.021 (Holo-TC), p=0.014 (RBC-folate) and p<0.001 (Hcy). The Hcy-lowering effect was dependent on the initial Hcy levels (p<0.001). Our findings suggest that elevated Hcy levels in elderly subjects can be lowered regardless of the initial cobalamin and folate biostatus.

Effects of Aerobic Treadmill Training on Oxidative Stress Parameters, Metabolic Enzymes, and Histomorphometric Changes in Colon of Rats with Experimentally Induced Hyperhomocysteinemia.

The aim of this study was to investigate the effects of aerobic treadmill training regimen of four weeks duration on oxidative stress parameters, metabolic enzymes, and histomorphometric changes in the colon of hyperhomocysteinemic rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C, 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.) 2x/day; H, homocysteine 0.45 µmol/g b.w./day s.c. 2x/day; CPA, saline (0.9% NaCl 0.2 mL/day s.c. 2x/day) and an aerobic treadmill training program; and HPA, homocysteine (0.45 µmol/g b.w./day s.c. 2x/day) and an aerobic treadmill training program. The HPA group had an increased level of malondialdehyde (5.568 ± 0.872 µmol/mg protein, p = 0.0128 vs. CPA (3.080 ± 0.887 µmol/mg protein)), catalase activity (3.195 ± 0.533 U/mg protein, p < 0.0001 vs. C (1.467 ± 0.501 U/mg protein), p = 0.0012 vs. H (1.955 ± 0.293 U/mg protein), and p = 0.0003 vs. CPA (1.789 ± 0.256 U/mg protein)), and total superoxide dismutase activity (9.857 ± 1.566 U/mg protein, p < 0.0001 vs. C (6.738 ± 0.339 U/mg protein), p < 0.0001 vs. H (6.015 ± 0.424 U/mg protein), and p < 0.0001 vs. CPA (5.172 ± 0.284 U/mg protein)) were detected in the rat colon. In the HPA group, higher activities of lactate dehydrogenase (2.675 ± 1.364 mU/mg protein) were detected in comparison to the CPA group (1.198 ± 0.217 mU/mg protein, p = 0.0234) and higher activities of malate dehydrogenase (9.962 (5.752-10.220) mU/mg protein) were detected in comparison to the CPA group (4.727 (4.562-5.299) mU/mg protein, p = 0.0385). Subchronic treadmill training in the rats with hyperhomocysteinemia triggers the colon tissue antioxidant response (by increasing the activities of superoxide dismutase and catalase) and elicits an increase in metabolic enzyme activities (lactate dehydrogenase and malate dehydrogenase). This study offers a comprehensive assessment of the effects of aerobic exercise on colonic tissues in a rat model of hyperhomocysteinemia, evaluating a range of biological indicators including antioxidant enzyme activity, metabolic enzyme activity, and morphometric parameters, which suggested that exercise may confer protective effects at both the physiological and morphological levels.

The Etiology of the Thrombotic Phenomena Involved in the Process of Coronary Artery Disease-What Is the Role of Thrombophilic Genes in the Development of This Pathology?

Cardiovascular diseases, among which includes coronary artery disease, represent one of the most important causes of mortality and morbidity worldwide. Research aimed at determining the risk factors involved recognizes a group of "traditional" risk factors, but also more recent studies identified over 100 "novel" ones which may have a role in the disease. Among the latter is the thrombophilia profile of a patient, a pathology well-established for its involvement in venous thromboembolism, but with less studied implications in arterial thrombosis. This paper reviews the literature, explaining the pathophysiology of the thrombophilia causes associated most with coronary thrombosis events. Results of several studies on the subject, including a meta-analysis with over 60,000 subjects, determined the significant involvement of factor V Leiden, prothrombin G20210A mutation, plasminogen activator inhibitor-1 and antiphospholipid syndrome in the development of coronary artery disease. The mechanisms involved are currently at different stages of research, with some already established and used as therapeutic targets.

Homocysteine promotes atherosclerosis through macrophage pyroptosis via endoplasmic reticulum stress and calcium disorder.

BACKGROUND: Elevated plasma homocysteine levels, known as hyperhomocysteinemia, have been identified as an independent risk factor for atherosclerosis and related cardiovascular diseases. Macrophage pyroptosis-mediated inflammation is crucial in the development of atherosclerosis, but the underlying mechanisms remain unclear. METHODS: A hyperhomocysteinemia atherosclerotic model with ApoE-/- mice fed with a high-methionine diet was constructed to investigate the role of plasma homocysteine in atherosclerosis. THP-1-derived macrophages were used to investigate the mechanisms by which Hcy regulates pyroptosis. RESULTS: We found that hyperhomocysteinemia resulted in larger atherosclerotic plaques and more secretion of inflammatory cytokines, while these effects were attenuated in Caspase-1 knockdown mice. Likewise, in vitro experiments demonstrated that treatment of macrophages with homocysteine resulted in NLRP3 inflammasome activation and pyroptosis, as evidenced by cleavage of Caspase-1, production of downstream IL-1ß, elevation of lactate dehydrogenase activity, and extensive propidium iodide-positive staining of cells. These were all inhibited by Caspase-1 inhibitor. In addition, excessive generation of reactive oxygen species was associated with mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential and ATP synthesis. Moreover, further experiments revealed that homocysteine induced endoplasmic reticulum stress, enhanced communication between the endoplasmic reticulum and mitochondria, and consequently contributed to calcium disorder. Furthermore, the endoplasmic reticulum stress inhibitor, 4PBA, the calcium chelator, BAPTA, and calcium channel inhibitor, 2-APB significantly improved macrophage pyroptosis. CONCLUSION: Homocysteine accelerates atherosclerosis progression by enhancing macrophages pyroptosis via promoting endoplasmic reticulum stress, endoplasmic reticulum-mitochondria coupling, and disturbing of calcium disorder.

Atorvastatin rescues hyperhomocysteinemia-induced cognitive deficits and neuroinflammatory gene changes.

BACKGROUND: Epidemiological data suggests statins could reduce the risk of dementia, and more specifically, Alzheimer's disease (AD). Pre-clinical data suggests statins reduce the risk of dementia through their pleiotropic effects rather than their cholesterol lowering effects. While AD is a leading cause of dementia, it is frequently found co-morbidly with cerebral small vessel disease and other vascular contributions to cognitive impairment and dementia (VCID), which are another leading cause of dementia. In this study, we determined if atorvastatin ameliorated hyperhomocysteinemia (HHcy)-induced VCID. METHODS: Wild-type (C57Bl6/J) mice were placed on a diet to induce HHcy or a control diet each with or without atorvastatin for 14 weeks. Mice underwent novel object recognition testing before tissue collection. Plasma total cholesterol and total homocysteine as well as related metabolites were measured. Using qPCR and NanoString technology, we profiled glial cell-associated gene expression changes. Finally, microglial morphology, astrocyte end feet, and microhemorrhages were analyzed using histological methods. RESULTS: Atorvastatin treatment of HHcy in mice led to no changes in total cholesterol but decreases in total homocysteine in plasma. While HHcy decreased expression of many glial genes, atorvastatin rescued these gene changes, which mostly occurred in oligodendrocytes and microglia. Microglia in HHcy mice with atorvastatin were trending towards fewer processes compared to control with atorvastatin, but there were no atorvastatin effects on astrocyte end feet. While atorvastatin treatment was trending towards increasing the area of microhemorrhages in HHcy mice in the frontal cortex, it only slightly (non-significantly) reduced the number of microhemorrhages. Finally, atorvastatin treatment in HHcy mice led to improved cognition on the novel object recognition task. CONCLUSIONS: These data suggest that atorvastatin rescued cognitive changes induced by HHcy most likely through lowering plasma total homocysteine and rescuing gene expression changes rather than impacts on vascular integrity or microglial changes.

Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C > T and c.1286A > C variants affect fibrin clot properties in Polish venous thromboembolic patients.

BACKGROUND AND AIMS: Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients. METHODS: In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness. RESULTS: There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 µM (n = 71, 18.3%), compared to patients with tHcy ≤15 µM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 µM compared to tHcy ≤15 µM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 µM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 µM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05). CONCLUSIONS: Our study indicates that patients with MTHFR variants and tHcy >15 µM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Comprehensive transcriptomics and metabolomics analyses reveal that hyperhomocysteinemia is a high risk factor for coronary artery disease in a chinese obese population aged 40-65: a prospective cross-sectional study.

BACKGROUND: Clinical observations suggest a complex relationship between obesity and coronary artery disease (CAD). This study aimed to characterize the intermediate metabolism phenotypes among obese patients with CAD and without CAD. METHODS: Sixty-two participants who consecutively underwent coronary angiography were enrolled in the discovery cohort. Transcriptional and untargeted metabolomics analyses were carried out to screen for key molecular changes between obese patients with CAD (CAD obese), without CAD (Non-CAD obese), and Non-CAD leans. A targeted GC-MS metabolomics approach was used to further identify differentially expressed metabolites in the validation cohorts. Regression and receiver operator curve analysis were performed to validate the risk model. RESULTS: We found common aberrantly expressed pathways both at the transcriptional and metabolomics levels. These pathways included cysteine and methionine metabolism and arginine and proline metabolism. Untargeted metabolomics revealed that S-adenosylhomocysteine (SAH), 3-hydroxybenzoic acid, 2-hydroxyhippuric acid, nicotinuric acid, and 2-arachidonoyl glycerol were significantly elevated in the CAD obese group compared to the other two groups. In the validation study, targeted cysteine and methionine metabolomics analyses showed that homocysteine (Hcy), SAH, and choline were significantly increased in the CAD obese group compared with the Non-CAD obese group, while betaine, 5-methylpropanedioic acid, S-adenosylmethionine, 4-PA, and vitamin B2 (VB2) showed no significant differences. Multivariate analyses showed that Hcy was an independent predictor of obesity with CAD (hazard ratio 1.7; 95%CI 1.2-2.6). The area under the curve based on the Hcy metabolomic (HCY-Mtb) index was 0.819, and up to 0.877 for the HCY-Mtb.index plus clinical variables. CONCLUSION: This is the first study to propose that obesity with hyperhomocysteinemia is a useful intermediate metabolism phenotype that could be used to identify obese patients at high risk for developing CAD.

Non-Traditional Risk Factors as Contributors to Cardiovascular Disease.

Cardiovascular disease (CVD) remains one of the primary causes of morbidity and mortality worldwide. Classic cardiovascular risk factors, such as hypertension, diabetes mellitus (DM), hyperlipidemia, and smoking, have been well identified and given increased attention in clinical practice. However, the incidence and prevalence of CVD remains high, especially in developing countries. Therefore, there has been more attention to non-traditional CVD risk factors such as gut microbiota, sleep disorders, dietary structure, and psychosocial factors in their important roles in the development of CVD. In this review we summarize the association of non-traditional risk factors with CVD with the aim of further reducing the risk of CVD.

Parkinson's Disease Risk and Hyperhomocysteinemia: The Possible Link.

Parkinson's disease (PD) is one of the most common degenerative brain disorders caused by the loss of dopaminergic neurons in the substantia nigra (SN). Lewy bodies and -synuclein accumulation in the SN are hallmarks of the neuropathology of PD. Due to lifestyle changes and prolonged L-dopa administration, patients with PD frequently have vitamin deficiencies, especially folate, vitamin B6, and vitamin B12. These disorders augment circulating levels of Homocysteine with the development of hyperhomocysteinemia, which may contribute to the pathogenesis of PD. Therefore, this review aimed to ascertain if hyperhomocysteinemia may play a part in oxidative and inflammatory signaling pathways that contribute to PD development. Hyperhomocysteinemia is implicated in the pathogenesis of neurodegenerative disorders, including PD. Hyperhomocysteinemia triggers the development and progression of PD by different mechanisms, including oxidative stress, mitochondrial dysfunction, apoptosis, and endothelial dysfunction. Particularly, the progression of PD is linked with high inflammatory changes and systemic inflammatory disorders. Hyperhomocysteinemia induces immune activation and oxidative stress. In turn, activated immune response promotes the development and progression of hyperhomocysteinemia. Therefore, hyperhomocysteinemia-induced immunoinflammatory disorders and abnormal immune response may aggravate abnormal immunoinflammatory in PD, leading to more progression of PD severity. Also, inflammatory signaling pathways like nuclear factor kappa B (NF-κB) and nod-like receptor pyrin 3 (NLRP3) inflammasome and other signaling pathways are intricate in the pathogenesis of PD. In conclusion, hyperhomocysteinemia is involved in the development and progression of PD neuropathology either directly via induction degeneration of dopaminergic neurons or indirectly via activation of inflammatory signaling pathways.

Homozygous whole body Cbs knockout in adult mice features minimal pathology during ageing despite severe homocysteinemia.

Deficiencies in Cystathionine-ß-synthase (CBS) lead to hyperhomocysteinemia (HHCy), which is considered a risk factor for cardiovascular, bone and neurological disease. Moreover, CBS is important for the production of cysteine, hydrogen sulfide (H2 S) and glutathione. Studying the biological role of CBS in adult mice has been severely hampered by embryological disturbances and perinatal mortality. To overcome these issues and assess the effects of whole-body CBS deficiency in adult mice, we engineered and characterized a Cre-inducible Cbs knockout model during ageing. No perinatal mortality occurred before Cbs-/- induction at 10 weeks of age. Mice were followed until 90 weeks of age and ablation of Cbs was confirmed in liver and kidney but not in brain. Severe HHCy was observed in Cbs-/- (289 ± 58 µM) but not in Cbs+/- or control mice (<10 µM). Cbs-/- showed impaired growth, facial alopecia, endothelial dysfunction in absence of increased mortality, and signs of liver or kidney damage. CBS expression in skin localized to sebaceous glands and epidermis, suggesting local effects of Cbs-/- on alopecia. Cbs-/- showed increased markers of oxidative stress and senescence but expression of other H2 S producing enzymes (CSE and 3-MST) was not affected. CBS deficiency severely impaired H2 S production capacity in liver, but not in brain or kidney. In summary, Cbs-/- mice presented a mild phenotype without mortality despite severe HHCy. The findings demonstrate that HHCy is not directly linked to development of end organ damage.