Teodoro Bottiglieri Ph.D.

McCullough, P. A., A. Vasudevan, M. Sathyamoorthy, J. M. Schussler, C. E. Velasco, L. R. Lopez, C. Swift, M. Peterson, J. Bennett-Firmin, R. Schiffmann and T. Bottiglieri (2017). “Urinary 11-dehydro-thromboxane b2 and mortality in patients with stable coronary artery disease.” Am J Cardiol: 2017 Jan [Epub ahead of print].

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Antiplatelet therapy with aspirin has been shown to reduce adverse outcomes in patients with coronary artery disease (CAD). Aspirin irreversibly inhibits platelet cyclooxygenase-1 and attenuates thromboxane A2 (TXA2)-mediated platelet aggregation, but there is variable suppression of cyclooxygenase-1. From a cohort of patients with stable CAD, we performed blinded, detailed chart abstraction, and measured urinary 11-dehydro-thromboxane B2 (11dhTxB2), an inactive metabolite of TxA2 from frozen samples. There were 327 men (73%) and 122 women (27%) with a mean age (+/-SD) of 67 +/- 10 and 65 +/- 10 years, respectively. A positive linear trend for age was observed among tertiles of 11dhTxB2 (p trend = 0.01). Higher proportions of women (p = 0.001), chronic obstructive pulmonary disease (p trend = 0.0003), and heart failure (p trend = 0.003) were observed in the upper tertile of 11dhTxB2. Sixty-seven patients (14.9%) died over a median follow-up of 1,149 days and 87.5% of the deaths were due to cardiovascular causes. Twenty-six nonsurvivors (38.8%) were treated with P2Y12 receptor antagonists versus 161 survivors (42.2%; p = 0.61). By stepwise Cox proportional hazards analysis, we identified that patients in the middle (hazard ratio 7.14; 95% CI 2.46 to 20.68) and upper tertiles (hazard ratio 9.91; 95% CI 3.45 to 28.50) had higher risks for mortality after adjusting for age and co-morbidities. In conclusion, urinary concentration of 11dhTxB2 was a strong independent risk factor for all-cause mortality among patients with stable CAD on aspirin therapy and may be a marker for patients with CAD who require more intensive secondary prevention measures.

Shen, J. S., E. Arning, M. L. West, T. S. Day, S. Chen, X. L. Meng, S. Forni, N. McNeill, O. Goker-Alpan, X. Wang, P. Ashcraft, D. F. Moore, S. H. Cheng, R. Schiffmann and T. Bottiglieri (2017). “Tetrahydrobiopterin deficiency in the pathogenesis of fabry disease.” Hum Mol Genet: 2017 Feb [Epub ahead of print].

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Fabry disease is caused by deficient activity of α-galactosidase A and subsequent accumulation of glycosphingolipids (mainly globotriaosylceramide, Gb3), leading to multisystem organ dysfunction. Oxidative stress and nitric oxide synthase (NOS) uncoupling are thought to contribute to Fabry cardiovascular diseases. We hypothesized that decreased tetrahydrobiopterin (BH4) plays a role in the pathogenesis of Fabry disease. We found that BH4 was decreased in the heart and kidney but not in the liver and aorta of Fabry mice. BH4 was also decreased in the plasma of female Fabry patients, which was not corrected by enzyme replacement therapy (ERT). Gb3 levels were inversely correlated with BH4 levels in animal tissues and cultured patient cells. To investigate the role of BH4 deficiency in disease phenotypes, 12-month-old Fabry mice were treated with gene transfer-mediated ERT or substrate reduction therapy (SRT) for 6 months. In the Fabry mice receiving SRT but not ERT, BH4 deficiency was restored, concomitant with ameliorated cardiac and renal hypertrophy. Additionally, glutathione levels were decreased in Fabry mouse tissues in a sex-dependent manner. Renal BH4 levels were closely correlated with glutathione levels and inversely correlated with cardiac and kidney weight. In conclusion, this study showed that BH4 deficiency occurs in Fabry disease and may contribute to the pathogenesis of the disease through oxidative stress associated with a reduced antioxidant capacity of cells and NOS uncoupling. This study also suggested dissimilar efficacy of ERT and SRT in correcting pre-existing pathologies in Fabry disease.

McCullough, P. A., A. Vasudevan, L. R. Lopez, C. Swift, M. Peterson, J. Bennett-Firmin, R. Schiffmann and T. Bottiglieri (2016). “Oxidative stress reflected by increased F2-isoprostanes is associated with increasing urinary 11-dehydro thromboxane B2 levels in patients with coronary artery disease.” Thromb Res 148: 85-88.

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Our study demonstrated that: 1) CAD patients with higher levels of 8-isoPGF2α had higher levels of 11dhTxB2, which could be related to poor inhibition of COX-1 pathway in spite of adequate ASA treatment, 2) 8-isoPGF2α is an independent determinant of 11dhTxB2 and 3) 8-isoPGF2α levels were significantly higher in females and patients with diabetes and COPD. Patients with diabetes and central obesity have been observed to have an incomplete ASA response manifested as incomplete inhibition of thromboxane production, in a pro-inflammatory background with enhanced oxidative stress. Oxidative stress enhances the production of platelet isoprostanes and is believed to mitigate the aspirin mediated TxA2 inhibition among diabetic patients on low-dose aspirin [9]. Among F2-isoprostanes metabolites, 8-isoPGF2α is a marker of in vivo oxidative stress, which has been shown to stimulate the activation of platelets by direct binding to the thromboxane platelet receptor. Elevated 8-isoPGF2α levels in our ASA-treated CAD patients indicate the presence of an active oxidative stress environment that is not affected by ASA treatment. Because ASA inhibits over 95% of platelet COX-1 activity in practically all subjects, the residual platelet activation in poor ASA responders can be explained by alternative sources of TxA2 produced by non-platelet inflammatory COX-2 pathways. Our findings are congruent with the notion that oxidative stress mechanisms play an important role in platelet activation in addition to their role on the initiation, progression, and consequences of atherogenesis. Excessive production of ROS may damage lipoproteins creating an inflammatory and atherogenic background and also enhance the arachidonic acid production of F2-isoprostanes that are capable of activating platelets and making them resistant to the therapeutic effect of ASA. These observations support the concept that oxidative stress maintains platelet hyperactivity linking proatherogenic mechanisms to platelet dysfunction in patients with stable CAD. Considering that thromboxanes are not the only factor contributing to platelet activation and atherothrombosis, it is not surprising that a single anti-platelet agent such as ASA does not prevent all adverse events. Cyclo-oxygenase-1 derived TxA2 activates the same and nearby platelets in an autocrine signaling fashion. Due to the very short half-life of TxA2 (20–30 s) and low concentrations (1–60 pg/mL), a constant production of TxA2 is necessary to maintain a homeostatic (physiologic) control of platelet activity. However, the half-life of 8-isoPGF2α is much longer (10 min), with concentrations that are thirty-fold higher. If 8-isoPGF2α can bind and stimulate thromboxane platelet receptors with similar affinity, its longer half-life and concentration makes it a relevant agonist for platelet activation in patients with an underlying oxidative process. Because ASA blocks most of COX-1 activity reducing the production of TxA2, it is possible that the thromboxane platelet receptor can still be activated by TxA2 produced via the 8-isoPGF2α pathway. In this situation, blocking 8-isoPGF2α TPR stimulation could be a potential therapeutic target instead of increasing the dose of ASA. Sex related differences in platelet function and ASA pharmacokinetics exist with female gender associated with elevated 11dhTxB2. We found that females had significantly higher 8-isoPGF2α levels, suggesting an enhanced oxidative stress and lesser attenuation of TxA2. Elevated 11dhTxB2 was found to increase the risk of adverse events in patients with stable CAD and myocardial infarction. (Excerpt from text, p. 86-87.)

Vasudevan, A., T. Bottiglieri, K. M. Tecson, M. Sathyamoorthy, J. M. Schussler, C. E. Velasco, L. R. Lopez, C. Swift, M. Peterson, J. Bennett-Firmin, R. Schiffmann and P. A. McCullough (2016). “Residual thromboxane activity and oxidative stress: Influence on mortality in patients with stable coronary artery disease.” Coron Artery Dis: 2016 Dec [Epub ahead of print].

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BACKGROUND: Aspirin use is effective in the prevention of cardiovascular disease; however, not all patients are equally responsive to aspirin. Oxidative stress reflected by F2-isoprostane [8-iso-prostaglandin-F2alpha (8-IsoPGF2alpha)] is a potential mechanism of failure of aspirin to adequately inhibit cyclooxygenase-1. The objective was to examine the relation between all-cause mortality and the concentrations of urinary 11-dehydro thromboxane B2 (11dhTxB2) and 8-IsoPGF2alpha in patients with stable coronary artery disease (CAD). METHODS: The data for this analysis are from a prospective study in which patients were categorized into four groups based on the median values of 11dhTxB2 and 8-IsoPGF2alpha. RESULTS: There were 447 patients included in this analysis with a median (range) age of 66 (37-91) years. The median (range) values of 11dhTxB2 and 8-IsoPGF2alpha were 1404.1 (344.2-68296.1) and 1477.9 (356.7-19256.3), respectively. A total of 67 (14.9%) patients died over a median follow-up of 1149 days. The reference group for the Cox proportional hazards survival analysis was patients with values of 11dhTxB2 and 8-IsoPGF2alpha below their corresponding medians. Adjusting for the age and sex, patients with values of 11dhTxB2 greater than the median had a significantly higher risk of mortality when compared with the reference group (high 11dhTxB2 and low 8-IsoPGF2alphaadj: hazard ratio: 3.2, 95% confidence interval: 1.6-6.6, P=0.002; high 11dhTxB2 and 8-IsoPGF2alphaadj: hazard ratio: 3.6, 95% confidence interval: 1.8-7.3, P<0.001). The findings were similar when we adjusted for the comorbidities of cancer, kidney function, and ejection fraction. CONCLUSION: We found that 11dhTxB2 appears to be a better prognostic marker for mortality as compared with 8-IsoPGF2alpha, suggesting aspirin resistance itself is a stronger independent determinant of death in CAD patients treated with aspirin.

Christensen, K. E., W. Hou, R. H. Bahous, L. Deng, O. V. Malysheva, E. Arning, T. Bottiglieri, M. A. Caudill, L. A. Jerome-Majewska and R. Rozen (2016). “Moderate folic acid supplementation and mthfd1-synthetase deficiency in mice, a model for the r653q variant, result in embryonic defects and abnormal placental development.” Am J Clin Nutr 104(5): 1459-1469.

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BACKGROUND: Moderately high folic acid intake in pregnant women has led to concerns about deleterious effects on the mother and fetus. Common polymorphisms in folate genes, such as methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase (MTHFD1) R653Q, may modulate the effects of elevated folic acid intake. OBJECTIVES: We investigated the effects of moderate folic acid supplementation on reproductive outcomes and assessed the potential interaction of the supplemented diet with MTHFD1-synthetase (Mthfd1S) deficiency in mice, which is a model for the R653Q variant. DESIGN: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed a folic acid-supplemented diet (FASD) (5-fold higher than recommended) or control diets before mating and during pregnancy. Embryos and placentas were assessed for developmental defects at embryonic day 10.5 (E10.5). Maternal folate and choline metabolites and gene expression in folate-related pathways were examined. RESULTS: The combination of FASD and maternal MTHFD1-synthetase deficiency led to a greater incidence of defects in E10.5 embryos (diet x maternal genotype, P = 0.0016; diet x embryonic genotype, P = 0.054). The methylenetetrahydrofolate reductase (MTHFR) protein and methylation potential [ratio of S-adenosylmethionine (major methyl donor):S-adenosylhomocysteine) were reduced in maternal liver. Although 5-methyltetrahydrofolate (methylTHF) was higher in maternal circulation, the methylation potential was lower in embryos. The presence of developmental delays and defects in Mthfd1S+/- embryos was associated with placental defects (P = 0.003). The labyrinth layer failed to form properly in the majority of abnormal placentas, which compromised the integration of the maternal and fetal circulation and presumably the transfer of methylTHF and other nutrients. CONCLUSIONS: Moderately higher folate intake and MTHFD1-synthetase deficiency in pregnant mice result in a lower methylation potential in maternal liver and embryos and a greater incidence of defects in embryos. Although maternal circulating methylTHF was higher, it may not have reached the embryos because of abnormal placental development; abnormal placentas were observed predominantly in abnormally developed embryos. These findings have implications for women with high folate intakes, particularly if they are polymorphic for MTHFD1 R653Q.