Sapna Gupta

Hyperhomocysteinemia Promotes Inflammatory Monocyte Generation and Accelerates Atherosclerosis in Transgenic Cystathionine β-Synthase–Deficient Mice

Background— Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. Methods and Results— We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine &bgr;-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE−/− Cbs−/− mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-&agr; and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE−/− Cbs−/− mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE−/− Cbs−/− mice and another severe HHcy mouse model (Tg-S466L Cbs−/−) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6Chi and Ly-6Cmid monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs−/− and Tg-hCBS apoE−/− Cbs−/− mice. These changes were exacerbated in Tg-S466L Cbs−/− mice with aging. Addition of l-homocysteine (100 to 500 &mgr;mol/L), but not l-cysteine, maintained the Ly-6Chi subset and induced the Ly-6Cmid subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6Cmid subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. Conclusion— HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase–mediated oxidant stress.

Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice.

Genetic disorders of homocysteine (Hcy) or folate metabolism or high‐methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy‐thiolac‐tone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine ς‐synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy‐thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (A‐Hcy‐protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how A–Hcy‐protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma A‐Hcy‐protein was elevated 10‐fold in mice fed a high‐methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton‐coupled folate transporter (Pcft) gene resulted in a 10‐to 30‐fold increase in plasma or serum AHcy‐protein levels. Liver AHcy‐protein was elevated 3.4‐fold in severely and 11‐fold in extremely hyperhomocysteinemic Cbs‐deficient mice, 3.6‐fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr‐deficient animals, suggesting that mice have a capacity to prevent accumulation of A‐Hcy‐protein in their organs. These findings provide evidence that A‐Hcy‐protein is an important metabolite associated with Hcy pathophysiology in the mouse.—Jakubowski, H.,Peria‐Kajan, J., Finnell, R.H., Cabrera, R.M., Wang, H., Gupta, S., Kruger, W.D., Kraus, J.P., Shih, D.M. Genetic or nutritional disorders in homocysteine or folate metabolism increase protein A‐homocysteinylation in mice. FASEB J. 23, 1721–1727 (2009)

Mouse models of cystathionine β-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia

Untreated cystathionine β‐synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 µΜ), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 µΜ). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg‐hCBS Cbs–/– mice, with a mean serum tHcy of 169 µΜ, and Tg‐I278T Cbs–/– mice, with a mean tHcy of 296 µΜ. Only Tg‐I278T Cbs–/– animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg‐I278T Cbs–/– mice have significantly elevated levels of free oxidized homocysteine but not protein‐bound homocysteine in serum and elevation of all forms of homocysteine and S‐adenosyl‐homocysteine in the liver compared to Tg‐hCBS Cbs–/– mice. RNA profiling of livers indicate that Tg‐I278T Cbs–/– and Tg‐hCBS Cbs–/– mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.— Gupta, S., Kuhnisch, J., Mustafa, A., Lhotak, S., Schlachterman, A., Slifker, M. J., Klein‐Szanto, A., High, K. A., Austin, R. C., Kruger, W. D. Mouse models of cystathionine β‐synthase deficiency reveal significant threshold effects of hyperhomocysteinemia. FASEB J. 23, 883–893 (2009)

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor–mediated vasorelaxation in transgenic cystathionine beta synthase–deficient mice

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 μM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.

Methionine-deficient diet induces post-transcriptional downregulation of cystathionine β-synthase.

OBJECTIVE Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transsulfuration pathway by the action of cystathionine β-synthase (CBS). We have examined the metabolic response in mice of a shift from a methionine-replete to a methionine-free diet. METHODS AND RESULTS We found that shifting 3-mo-old C57BL6 mice to a methionine-free diet caused a transient increase in tHcy and an increase in the tHcy/methionine ratio. Because CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 d on the methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicated that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability. CONCLUSION Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway by an S-adenosylmethionine-independent mechanism that signals a rapid downregulation of CBS protein.

Cystathionine Beta-Synthase Deficiency Causes Fat Loss in Mice

Cystathionine beta synthase (CBS) is the rate-limiting enzyme responsible for the de novo synthesis of cysteine. Patients with CBS deficiency have greatly elevated plasma total homocysteine (tHcy), decreased levels of plasma total cysteine (tCys), and often a marfanoid appearance characterized by thinness and low body-mass index (BMI). Here, we characterize the growth and body mass characteristics of CBS deficient TgI278T Cbs−/− mice and show that these animals have significantly decreased fat mass and tCys compared to heterozygous sibling mice. The decrease in fat mass is accompanied by a 34% decrease in liver glutathione (GSH) along with a significant decrease in liver mRNA and protein for the critical fat biosynthesizing enzyme Stearoyl CoA desaturase-1 (Scd-1). Because plasma tCys has been positively associated with fat mass in humans, we tested the hypothesis that decreased tCys in TgI278T Cbs−/− mice was the cause of the lean phenotype by placing the animals on water supplemented with N-acetyl cysteine (NAC) from birth to 240 days of age. Although NAC treatment in TgI278T Cbs−/− mice caused significant increase in serum tCys and liver GSH, there was no increase in body fat content or in liver Scd-1 levels. Our results show that lack of CBS activity causes loss of fat mass, and that this effect appears to be independent of low serum tCys.

Serum Amino Acid Levels as a Biomarker for Renal Cell Carcinoma

PURPOSE Prognosis in renal cell carcinoma is dependent on tumor stage at presentation, with significant differences in survival between early and late stage disease. Currently to our knowledge no screening tests or biomarkers have been identified for the early detection of kidney cancer. Therefore, we investigated whether serum amino acid profiles are a potentially useful biomarker in patients with renal cell carcinoma. MATERIALS AND METHODS The concentrations of 26 amino acids were determined in serum taken preoperatively from 189 patients with renal cell carcinoma, and from 104 age and sex matched controls. RESULTS Statistically significant changes were observed in patient levels of 15 amino acids, with 13 being decreased and 2 being increased. A logistic regression model using 8 amino acids including cysteine, ornithine, histidine, leucine, tyrosine, proline, valine and lysine was created to distinguish cases from controls. A receiver operator curve based on this model had an area under the curve of 0.81. This same model also had predictive value in terms of overall survival and tumor recurrence in patients with renal cell carcinoma. CONCLUSIONS Our findings suggest that serum amino acid levels may be useful as a screening tool for the identification of individuals with renal cell carcinoma and the prediction of outcomes.

Valproic acid increases expression of methylenetetrahydrofolate reductase (MTHFR) and induces lower teratogenicity in MTHFR deficiency

Valproate (VPA) treatment in pregnancy leads to congenital anomalies, possibly by disrupting folate or homocysteine metabolism. Since methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of folate interconversion and homocysteine metabolism, we addressed the possibility that VPA might have different teratogenicity in Mthfr+/+ and Mthfr+/− mice and that VPA might interfere with folate metabolism through MTHFR modulation. Mthfr+/+ and Mthfr+/− pregnant mice were injected with VPA on gestational day 8.5; resorption rates and occurrence of neural tube defects (NTDs) were examined on gestational day 14.5. We also examined the effects of VPA on MTHFR expression in HepG2 cells and on MTHFR activity and homocysteine levels in mice. Mthfr+/+ mice had increased resorption rates (36%) after VPA treatment, compared to saline treatment (10%), whereas resorption rates were similar in Mthfr+/− mice with the two treatments (25–27%). NTDs were only observed in one group (VPA‐treated Mthfr+/+). In HepG2 cells, VPA increased MTHFR promoter activity and MTHFR mRNA and protein (2.5‐ and 3.7‐fold, respectively). Consistent with cellular MTHFR upregulation by VPA, brain MTHFR enzyme activity was increased and plasma homocysteine was decreased in VPA‐treated pregnant mice compared to saline‐treated animals. These results underscore the importance of folate interconversion in VPA‐induced teratogenicity, since VPA increases MTHFR expression and has lower teratogenic potential in MTHFR deficiency. J. Cell. Biochem. 105: 467–476, 2008.

Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70.

Missense mutant proteins, such as those produced in individuals with genetic diseases, are often misfolded and subject to processing by intracellular quality control systems. Previously, we have shown using a yeast system that enzymatic function could be restored to I278T cystathionine β-synthase (CBS), a cause of homocystinuria, by treatments that affect the intracellular chaperone environment. Here, we extend these studies and show that it is possible to restore significant levels of enzyme activity to 17 of 18 (94%) disease causing missense mutations in human cystathionine β-synthase (CBS) expressed in Saccharomyces cerevisiae by exposure to ethanol, proteasome inhibitors, or deletion of the Hsp26 small heat shock protein. All three of these treatments induce Hsp70, which is necessary but not sufficient for rescue. In addition to CBS, these same treatments can rescue disease-causing mutations in human p53 and the methylene tetrahydrofolate reductase gene. These findings do not appear restricted to S. cerevisiae, as proteasome inhibitors can restore significant CBS enzymatic activity to CBS alleles expressed in fibroblasts derived from homocystinuric patients and in a mouse model for homocystinuria that expresses human I278T CBS. These findings suggest that proteasome inhibitors and other Hsp70 inducing agents may be useful in the treatment of a variety of genetic diseases caused by missense mutations.

Cystathionine β-Synthase p.S466L Mutation Causes Hyperhomocysteinemia in Mice

Missense mutations in the cystathionine β‐synthase (CBS) gene are the most common cause of clinical homocystinuria in humans. The p.S466L mutation was identified in a homocystinuric patient, but enzymatic studies with recombinant protein show this mutant to be highly active. To understand how this mutation causes disease in vivo, we have created mice lacking endogenous mouse CBS and expressing either wild‐type (Tg‐hCBS) or p.S466L (Tg‐S466L) human CBS under control of zinc inducible metallothionein promoter. In the presence of zinc, we found that the mean serum total homocysteine (tHcy) of Tg‐S466L mice was 142±55 µM compared to 16±13 µM for hCBS mice. Tg‐S466L mice also had significantly higher levels of total free homocysteine and S‐adenosylhomocysteine in liver and kidney. Only 48% of Tg‐S466L mice had detectable CBS protein in the liver, whereas all the Tg‐hCBS animals had detectable protein. Surprisingly, CBS mRNA was significantly elevated in Tg‐S466L animals compared to Tg‐hCBS, implying that the reduction in p.S466L protein was occurring due to posttranscriptional mechanisms. In Tg‐S466L animals with detectable liver CBS, the enzyme formed tetramers and was active, but lacked inducibility by S‐adenosylmethionine (AdoMet). However, even in Tg‐S466L animals that had in vitro liver CBS activity equivalent to Tg‐hCBS animals there was significant elevation of serum tHcy. Our results show that p.S466L causes homocystinuria by affecting both the steady state level of CBS protein and by reducing the efficiency of the enzyme in vivo. Hum Mutat 0,1–7, 2008.