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Methylenetetrahydrofolate reductase (MTHFR) synthesizes 5-methyltetrahydrofolate, a methyl donor for conversion of homocysteine to methionine. A common thermolabile variant causes mild MTHFR deficiency, induces mild hyperhomocysteinemia when plasma folate levels are low and increases risk for neural tube defects (NTD) and pregnancy loss. To increase our understanding of Mthfr regulation, the 5' and 3' regions of the mouse cDNA and gene were characterized. These studies revealed two major promoters, an internal coding exon in the 5'UTR, alternative transcriptional and translational start sites and alternative splicing and polyadenylation. These data suggest that Mthfr regulation is likely to be complex. To investigate the role of Mthfr in NTD, several approaches were taken. First, folate and MTHFR co-factor, flavin adenine dinucleotide, were shown to stabilize normal and thermolabile MTHFR during heat inactivation, suggesting that folate might prevent hyperhomocysteinemia in individuals with thermolabile enzyme through protein stabilization. Next, in situ hybridization of neurulating mouse embryos showed that Mthfr is expressed in the forebrain, hindbrain, branchial arches, blood vessels, gut, and importantly, in the ventral part of the neural tube. Mthfr+/- mice were then used as a model of mild deficiency to address the effects of maternal and embryonic Mthfr deficiency on development. When combined with inadequate dietary folate, Mthfr +/- pregnant females showed a two-fold higher rate of pregnancy loss than Mthfr+/+ pregnant females. As well, a percentage of day 10.5 embryos from only the Mthfr+/- pregnant females were underdeveloped by 2 days. These effects were not apparent when dietary folate was sufficient, consistent with a genetic-nutritional interactive effect. Finally, folate metabolism was investigated in an NTD model, the curly-tail (ct) mouse, since the ct defect and Mthfr were mapped in close proximity. However, Mthfr sequence in ct mice was simila

Background and hypothesis. Edentulous patients may be at risk for malnutrition and cardiovascular disease even with well made dentures. Improvements in methods used to assess nutritional status suggest that functional biomarkers such as plasma homocysteine (tHcy), in addition to traditional methods, will provide a better quality assessment. The hypothesis is that there is an association between functional biomarkers of nutritional status and traditional cardiovascular disease risk factors in a community dwelling edentulous elderly population. / Methods. This was a cross-sectional study of the baseline characteristics of a convenient sample of 254 edentulous community dwelling elderly over 65 years. Measurements included anthropometric, body composition, homocysteine, vitamins and relevant blood components. Correlations, multiple regression models and Adjusted Odds Ratios (AOR) for tertiles were used to assess the relationship between vitamins and other parameters with hyperhomocystenemia, defined as THcy value of ≥ 14mmol/L. The independent effect of edentulism on hyperhomocysteinemia was sought using the NHANES III data. / Results. The prevalence of hyperhomocysteinemia was 49.2%. Effect of folate on hyperhomocysteinemia was significant (p = 0.037). Low folate tertile group had a 2.45 times higher odds (CI: 1.23, 4.87) of hyperhomocysteinemia than patients in the higher tertile (p = 0.019). Groups with normal folate did not have higher odds of hyperhomocysteinemia regardless of levels of vitamin B6 or B12. AOR for vitamin B12 tertiles in relation to hyperhomocysteinemia were 2.36 (CI: 1.18, 4.75) and 2.12 (1.07, 4.22) for lower vs. high. A weak inverse relationship existed between tHcy and vitamin C (r= -0.11, p= 0.045). A borderline negative correlation existed between HDL and THcy adjusted for age (r = - 0.12; P = 0.05). Significant correlation existed between THcy and BMI (r = 0.15, P = 0.02), small waist circumference (r = 0.22; p = 0.0004) and waist/hip ratio (r = 0.23, p = 0.0003). In a multivariate analyses, edentulism was associated with hyperhomocysteinemia (p = 0.012). / Conclusions. In this sample, homocysteine levels appeared much higher than one would expect with folate fortification, and were related to several cardiovascular risk factors. Using data from NHANES III, edentulism was found to independently predict hyperhomocysteinemia. The inverse relationship between homocysteine and vitamin C and the effect of folate on hyperhomocysteinemia suggests that increasing dietary intake of fruit and vegetables in edentulous individuals might be beneficial. Furthermore, the latter are at risk of developing hyperhomocysteinemia, a condition associated with cognitive impairment, dementia and coronary artery disease risk.

Hyperhomocysteinemia is a risk factor for cardiovascular disease and stroke. Nutritional and/or genetic disruptions in homocysteine metabolism can cause hyperhomocysteinemia. Mild methylenetetrahydrofolate reductase (MTHFR) deficiency due to the 677C → T mutation in the MTHFR gene is the most common genetic cause of hyperhomocysteinemia. The 677C → T variant is associated with an increased risk for neural tube defects, pregnancy complications, schizophrenia and Down syndrome, and with a decreased risk for colon cancer and leukemia. This variant is also a potential risk factor for vascular disease. Severe MTHFR deficiency results in homocystinuria, an inborn error of metabolism with neurological and vascular complications. We have generated mice with a knockout of the Mthfr gene. The Mthfr-deficient mice exhibit hyperhomocysteinemia and decreased methylation capacity. The Mthfr+/- mice appear normal, whereas the Mthfr-/- mice are smaller and have reduced survival. Abnormal external granule neuron development associated with increased cell death in the cerebellum was observed in the Mthfr-/- mice. / Evidence for cardiovascular pathology was obtained in several ways. Impaired aortic relaxation response to acetylcholine was seen in the Mthfr +/- mice fed a high methionine diet. Both Mthfr+/- and Mthfr-/- mice fed a low folate high methionine diet developed myocardial fibrosis in the left ventricle. Abnormal lipid deposition in the proximal portion of the aorta was observed in older Mthfr+/- and Mthfr-/- mice. After crossing Mthfr -deficient mice with apoE-null mice, we demonstrated that MTHFR deficiency promoted atherogenesis and its progression in the apoE-null mice. / Gene expression in brain of Mthfr-deficient mice was investigated via microarray analysis. Five genes with altered expression in the brain of Mthfr-/- mouse were validated by RT-PCR. In biochemical studies of human MTHFR, both FAD and folate were shown to stabilize the purified recombinant wild type and mutant MTHFRs from the baculovirus expression system against heat inactivation. The effect of folate appeared to be secondary to that of FAD, and S-adenosylmethionine (SAM) inhibited purified wild type and mutant MTHFRs with similar efficiency. / This dissertation will significantly contribute to our understanding of the role of MTHFR in human disease.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-10-26T14:13:48Z No. of bitstreams: 2 Dissertação - Carla Cristina de Morais - 2015.pdf: 3178195 bytes, checksum: 8ef80dde024e6522fcf444b8134bf176 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-26T14:43:30Z (GMT) No. of bitstreams: 2 Dissertação - Carla Cristina de Morais - 2015.pdf: 3178195 bytes, checksum: 8ef80dde024e6522fcf444b8134bf176 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-26T14:43:30Z (GMT). No. of bitstreams: 2 Dissertação - Carla Cristina de Morais - 2015.pdf: 3178195 bytes, checksum: 8ef80dde024e6522fcf444b8134bf176 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-27 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Objective: to investigate the prevalence of alterations in homocysteinemia, lipid profile and vitamins B6, B12 and folic acid concentrations, as well as the possible relationships among C677T and A1298C polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene and blood concentrations of these biomarkers in adolescents with cardiovascular risk factors. Material and Methods: cross-sectional study with adolescents (10-19 years old) from a public school in Goiânia, Goiás. Screening was performed with 454 students in this age group. Those who were overweight, or presented risk factors associated with family history, or had been previously diagnosed with dyslipidemia were recruited, totaling a final sample of 115 individuals. Blood concentrations of homocysteine, vitamin B6 and vitamin B12, folic acid, oxidized low density lipoprotein (ox-LDL) and lipids were analyzed. The sample was stratified according to tertiles of plasma homocysteine concentrations. Genotyping of the above mentioned polymorphisms was performed. Analysis of differences between means was performed using Student’s t test, Mann Whitney, ANOVA or Kruskal-Wallis’s test. Correlations among variables were evaluated with the Pearson’s correlation or Spearman’s test. Results: hyperhomocysteinemia was observed in 19.1% of the sample and alterations in prevalence of alterations in the lipid profile were 30.4%, with 48.0% to high-density lipoprotein, and vitamin B6 deficiency was 23, 5%. Individuals in the highest tertile for homocysteine showed blood concentrations of folic acid and HDL-c higher compared to those of the middle and lower tertile, and waist circumference greater than in those of the lower tertile. There were no significant differences in anthropometric and biochemical variables when analyzed according to the three possible genotypes realtives to the C677T and A1298C polymorphisms. However, when heterozygous and homozygous for the variant alleles for C677T polymorphism were grouped, carriers of the variant ones showed higher plasma concentrations of oxidized low density lipoprotein (p < 0.01) and vitamin B6 plasma levels (p < 0.02). Conclusion: Despite the high prevalence of hyperhomocysteinemia, it was not observed it was not observed relationship of C677T and A1298C polymorphisms with plasma concentrations of homocysteine. However, the results of the study revealed the influence of MTHFR C677T genotype on vitamin B6 and oxidized low density lipoprotein levels. / Objetivo: investigar a prevalência de alterações na homocisteinemia, no perfil lipídico e nas concentrações de vitaminas B6, B12 e ácido fólico, bem como as possíveis relações entre os polimorfismos C677T e A1298C no gene que codifica a metilenotetra-hidrofolato redutase (MTHFR) e as concentrações sanguíneas destes biomarcadores em adolescentes com fatores de risco cardiovascular. Material e métodos: estudo observacional, transversal, com adolescentes (10-19 anos de idade) de uma escola pública de Goiânia, Goiás. Realizou-se triagem inicial com 454 alunos e aqueles com excesso de peso e/ou dislipidemia previamente diagnosticada foram recrutados, com amostra final de 115 indivíduos. As concentrações plasmáticas de homocisteína e vitamina B6 e séricas de vitamina B12, ácido fólico, lipoproteína de baixa densidade oxidada (LDL-ox) e perfil lipídico foram analisadas. Realizou-se a genotipagem em relação aos polimorfismos MTHFR C677T e A1298C. Análises de diferenças de médias foram feitas com o teste t de Student, Mann Whitney, ANOVA ou Kruskal-Wallis. Correlações entre as variáveis foram avaliadas por meio do teste de correlação de Pearson ou Spearman. Resultados: Foi observada hiper-homocisteinemia em 19,1% da amostra, prevalência de alterações no perfil lipídico de 30,4%, com destaque para 48,0% em relação à lipoproteína de alta densidade, e deficiência de vitamina B6 e 23,5%. Os indivíduos do tercil superior em relação a homocisteína apresentaram concentrações sanguíneas de ácido fólico e HDL-c maiores em relação àqueles do tercil médio e inferior, e circunferência da cintura maior em relação àqueles do tercil inferior. A circunferência da cintura Não houve diferenças significativas para antropometria e exames bioquímicos quando analisados em função dos três possíveis genótipos referentes aos polimorfismos C677T e A1298C. Entretanto, ao agrupar os genótipos heterozigotos e homozigotos para a variante do polimorfismo C677T, carreadores do alelo variante apresentaram maiores concentrações séricas de lipoproteína de baixa densidade oxidada (p<0,01) e de concentrações plasmáticas de vitamina B6 (p<0,02). Conclusão: Apesar da alta prevalência de hiper-homocisteinemia, não houve relação dos polimorfismos C677T e A1298C com as concentrações plasmáticas de homocisteína. Entretanto, os resultados do estudo revelaram a influência do genótipo em relação ao polimorfismo C677T no gene da MTHFR sobre as concentrações de vitamina B6 e de lipoproteína de baixa densidade oxidada.

Homocysteine (Hcy) has been identified as a primary risk factor for atherosclerosis as it induces endothelial cell (EC) activation/dysfunction and thus potentially initiating atherosclerotic plaque formation. There is accumulating evidence indicating a key role for oxidative stress in mediating Hcy atherogenic effects. The aim of this study was to evaluate the effects of chronic treatment with Hcy on EC activation and to explore the role of oxidative stress in these effects. Human umbilical vein endothelial cells (HUVEC) were cultured and treated chronically with DL-Hcy for 5-9 days. An in vitro flow system was also used to characterize the different types of interactions between DL-Hcy-treated HUVEC and neutrophils under physiological flow conditions. EC activation was studied by characterizing the activation of the JNK pathway and the up-regulation of different cell adhesion molecules (CAM) and cytokines, using different techniques including western blot, immunohistochemical staining, enzyme-linked immunosorbent assay and polymerase chain reaction. The role of oxidative stress was investigated by measuring the production of ROS and evaluating the efficiency of antioxidants. Furthermore, the role of nitric oxide and nitric oxide synthase in modulating Hcy effects was investigated. Chronic treatment with DL-Hcy did not kill the EC however, it inhibited cell proliferation. Furthermore, this treatment induced EC activation/dysfunction which was characterized by sustained activation of the JNK pathway, which in turn mediated up-regulation of E-selectin, ICAM-1 and to lesser extent P-selectin. Furthermore, DL-Hcy induced production of IL-8 protein. These CAM and chemokines collectively mediated different interactions between DL-Hcy-treated HUVEC and neutrophils under flow conditions including tethering, rolling, adherence and transmigration. DL-Hcy was also shown to induce significant ROS generation which mediated activation of the JNK pathway. Antioxidants restored DL-Hcy-induced interactions under flow to the basal level. DL-Hcy was shown to induce eNOS uncoupling which mediated, at least in part, the DL-Hcy-induced ROS production. Furthermore, short term treatment with NO inhibited DL-Hcy-induced HUVEC:neutrophil interactions in a cGMP-independent manner. In summary, this research showed that DL-Hcy has several proatherogenic effects, mediated at least in part by the JNK pathway, and induces EC activation/dysfunction priming for atherosclerosis initiation. The data supports that oxidative stress mediates the majority of Hcy atherosclerotic effects. Antioxidants tested, JNK inhibitors and NO showed promising results in reversing all DL-Hcy effects and restoring EC normal status. ¿

No description available.

Homocysteine (Hcy) has been identified as a primary risk factor for atherosclerosis as it induces endothelial cell (EC) activation/dysfunction and thus potentially initiating atherosclerotic plaque formation. There is accumulating evidence indicating a key role for oxidative stress in mediating Hcy atherogenic effects. The aim of this study was to evaluate the effects of chronic treatment with Hcy on EC activation and to explore the role of oxidative stress in these effects. Human umbilical vein endothelial cells (HUVEC) were cultured and treated chronically with DL-Hcy for 5-9 days. An in vitro flow system was also used to characterize the different types of interactions between DL-Hcy-treated HUVEC and neutrophils under physiological flow conditions. EC activation was studied by characterizing the activation of the JNK pathway and the up-regulation of different cell adhesion molecules (CAM) and cytokines, using different techniques including western blot, immunohistochemical staining, enzyme-linked immunosorbent assay and polymerase chain reaction. The role of oxidative stress was investigated by measuring the production of ROS and evaluating the efficiency of antioxidants. Furthermore, the role of nitric oxide and nitric oxide synthase in modulating Hcy effects was investigated. Chronic treatment with DL-Hcy did not kill the EC however, it inhibited cell proliferation. Furthermore, this treatment induced EC activation/dysfunction which was characterized by sustained activation of the JNK pathway, which in turn mediated up-regulation of E-selectin, ICAM-1 and to lesser extent P-selectin. Furthermore, DL-Hcy induced production of IL-8 protein. These CAM and chemokines collectively mediated different interactions between DL-Hcy-treated HUVEC and neutrophils under flow conditions including tethering, rolling, adherence and transmigration. DL-Hcy was also shown to induce significant ROS generation which mediated activation of the JNK pathway. Antioxidants restored DL-Hcy-induced interactions under flow to the basal level. DL-Hcy was shown to induce eNOS uncoupling which mediated, at least in part, the DL-Hcy-induced ROS production. Furthermore, short term treatment with NO inhibited DL-Hcy-induced HUVEC:neutrophil interactions in a cGMP-independent manner. In summary, this research showed that DL-Hcy has several proatherogenic effects, mediated at least in part by the JNK pathway, and induces EC activation/dysfunction priming for atherosclerosis initiation. The data supports that oxidative stress mediates the majority of Hcy atherosclerotic effects. Antioxidants tested, JNK inhibitors and NO showed promising results in reversing all DL-Hcy effects and restoring EC normal status.

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The aim of the present study sought to determine the signaling cascade that triggers glomerular injury and sclerosis during hHcys mediated by Vav2, a guanine nucleotide exchange factor (GNEF). Using both genetic and pharmacological interventions of Vav2, we first tested whether this GNEF is involved in hHcys-induced NLRP3 inflammasome activation in podocytes by its role in activation of the Rac-1-NOX complex. Further, we explored whether pharmacological targeting of Vav2 activation may regulate NLRP3 inflammasome signaling pathway during hHcys-induced glomerular injury. We found that mice with hHcys (on the FF diet) or oncoVav2 (a constitutively active form of Vav2) transfection in the kidney exhibited increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated IL-1β levels in glomeruli, indicating the formation and activation of the NLRP3 inflammasome. This glomerular NLRP3 inflammasome activation was accompanied by podocyte dysfunction and glomerular injury, even sclerosis. Local transfection of Vav2 shRNA plasmids significantly attenuated hHcys-induced NLRP3 inflammasome activation, podocyte injury, and glomerular sclerosis. In cultured podocytes, Hcys treatment and oncoVav2 transfection increased NLRP3 inflammasome formation and activation. This NLRP3 activation was inhibited by Vav2 shRNA, associated with reduction of Rac-1 activity and ROS production. Administration of NSC23766, a Rac-1 inhibitor substantially attenuated inflammasome formation, desmin expression and decreased podocin expression in glomeruli of hHcys mice. These results suggest that elevated Hcys levels activate Vav2 and thereby increase NOX activity, leading to ROS production. ROS trigger NLRP3 inflammasome activation, podocyte dysfunction and glomerular injury. Therefore, the present study defines a novel mechanism underlying hHcys-induced NLRP3 inflammasome activation and its progression to ESRD.