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Thiamine deficiency leads to reduced nitric oxide production and vascular dysfunction in rats
Nutrition, Metabolism & Cardiovascular Diseases (2014) 24, 183e188
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/nmcd
Thiamine deficiency leads to reduced nitric oxide production and vascular dysfunction in rats C.R. Gioda a,1,2, L.S.A. Capettini b,2, J.S. Cruz c,3, V.S. Lemos a,*,3 a
Departamento de Fisiologia e Biofı´sica, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil b Departamento de Farmacologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil c Departamento de Bioquı´mica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Received 13 March 2013; received in revised form 7 June 2013; accepted 10 June 2013 Available online 6 October 2013
KEYWORDS Thiamine deficiency; Endothelial dysfunction; eNOS; Nitric oxide
Abstract Background and aims: Thiamine deficiency is a condition that is known to cause damage to the nervous and cardiovascular systems because it interferes with cellular metabolism. It is well known that the control of vascular function is highly dependent on the production of nitric oxide (NO) by NO synthases. Studies exploring the physiological relevance of NO signaling under conditions of thiamine deficiency are scarce. The present study sought to investigate whether chronic metabolic changes would cause alterations in vascular responsiveness. Methods and results: By removing thiamine from the diet, we observed a reduced acetylcholine-mediated relaxation and an increased phenylephrine-mediated vasoconstriction in the aortas containing functional endothelium. Removal of the endothelium or the pretreatment of vessels with L-NAME restored the contractile responses to the level of controls. Conversely, indomethacin did not modify phenylephrine-mediated contractions. We also used carbon microsensors to continually measure NO production in situ while simultaneously measuring the vascular tone. The results revealed a significant decrease in NO production. Western blot analysis showed a decreased expression of the total eNOS in the thiaminedeficient aorta compared to the control. Concentrationeresponse curves for phenylephrine indicated no difference between the control and deficient groups in the presence and absence
* Corresponding author. Instituto de Cie ˆncias Biolo ´gicas, Universidade Federal de Minas Gerais, Av. Anto ˆnio Carlos 6627, 31970-901 Belo Horizonte, MG, Brazil. Tel.: þ55 31 3409 2950; fax: þ55 31 3409 2934. E-mail addresses: [email protected], [email protected] (V.S. Lemos). 1 Present address: Biological Science Institute, Federal University of Rio Grande, Rio Grande, RS, Brazil. 2 Co-first authors. 3 Co-senior authors. 0939-4753/$ - see front matter ª 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.numecd.2013.06.010
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C.R. Gioda et al. of SOD or Tyron. The NO donor DEA-NONOate produced a concentration-dependent relaxation response in the endothelium-denuded vessels that did not differ between the control and thiamine-deficient rats. Conclusion: Thiamine deficiency modulates eNOS-dependent NO production, leading to a decreased vasorelaxation and an increased contractile response in the rat aorta. ª 2013 Elsevier B.V. All rights reserved.
Introduction Thiamine (vitamin B1) deficiency, which is responsible for beriberi and WernickeeKorsakoff syndrome, is commonly associated with malnutrition and chronic alcoholism [1,2]. These syndromes are characterized by nervous and cardiovascular dysfunction [1,3e5]. Thiamine deficiency has been associated with inappropriate effects or the uncoupling of metabolic enzymes, such as pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and transketolase [6e8]. These effects likely occur because thiamine pyrophosphate, the active form of thiamine, is an important cofactor for the catalytic activity of these enzymes. Despite these metabolic and neuronal effects of thiamine deficiency, little is known of the effects of thiamine deficiency on the cardiovascular system. Some evidence indicates that cardiac contractility is profoundly affected by thiamine deficiency [4,9,10]. In terms of heart function, thiamine is considered to be a clinically important factor because its deficiency has been reported to cause heart failure and alterations in the myocardial antioxidant systems [9,11,12]. To our knowledge, there are no available data demonstrating that alterations in vascular function can be primarily caused by a thiamine deficiency. Cardiovascular homeostasis relies on the integrity of the vascular endothelium, which is able to modify the vascular tonus throughout the release of a multitude of active molecules, inducing relaxation or constriction in the smooth muscle and consequently modulating the blood flow and arterial pressure [13,14]. Disorders in the systemic vasculature are thought to originate in endothelial dysfunction induced by oxidative stress, which results in a reduction in NO availability and metabolic and hormonal alterations that can lead to several pathologies, including hypertension, heart failure, atherosclerosis and diabetes [15e17]. The significance of thiamine deficiency in vascular biology is therefore an important question that remains unanswered. Here, we hypothesized that thiamine is required to maintain proper vascular function. We provide pharmacological and biochemical evidence showing that thiamine deficiency provokes a substantial impairment of endothelial function that ultimately leads to vascular dysfunction.
Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats from the Animal Care facilities at UFMG (200e250 g) were kept in a 25 C room (12-h lightedark cycle) with free access to food and water. The rats were acclimated for 7 days and were then fed a thiamine-deficient diet for 35 days.
Chow The control and thiamine-deficient chow were made as previously described [3,11]. The two diets were isocaloric and the difference between them was the presence or absence of thiamine in the vitamin mixture used to prepare the chow. Both diets contained 20% protein, 1% cellulose, 5% mixture of salts, 1% mixture of vitamins, 5% corn oil, 0.4% choline and 67.6% cornstarch.
Isolated rat aortic rings Rings from the thoracic aorta (4e5 mm long) were mounted in an organ bath, containing KrebseHenseleit solution (in mM: NaCl 110.8, KCl 5.8, NaHCO3 25, MgSO4 1.07, CaCl2 2.49, NaH2PO4 2.33 and glucose 11.53), gassed with a 95% O2 and 5% CO2 mixture and equilibrated under a resting tension of 1 g for 1 h, at 37 C. The mechanical activity was recorded isometrically using a force transducer [18]. When necessary, the endothelial layer was removed mechanically. The vessels were contracted using phenylephrine (0.3 mM). The presence or absence of endothelium was verified by adding acetylcholine (10 mM).
Simultaneous measurements of NO and vascular function Acetylcholine-induced NO production was measured simultaneously with vascular function using NO-sensitive carbon microsensors (ISO-NOPF100, World Precision Instruments) placed next to the lumen of the vessels [19,20]. The generated currents (nA) were measured using the microsensors. NO concentrations were determined by comparison to calibration curves of known concentrations of the NO donor S-nitroso-N-acetylpenicillamine (SNAP).
Methods
Western blot analyses
Animals
The thoracic aorta were homogenized in ice-cold buffer (50 mM TriseHCl containing 150 mM NaCl, 5 mM EDTA-Na2, 1 mM MgCl2, 1% Nonidet P40, 0.3% triton X-100, 0.5% sodium deoxycholate, 100 mM dithiothreitol, 100 mM phenylmethylsulphonyl fluoride) and a cocktail of protease
Our investigations were approved by the institutional ethical committee for animal experimentation in accordance with the Guide for the Care and Use of Laboratory
Thiamine deficiency e reduced NO and vascular dysfunction inhibitors (SigmaFast). The protein samples were mixed with denaturing SDS-PAGE buffer, transferred to nitrocellulose membrane and subjected to a routine Western immunoblotting protocol [21] using the following antibodies: rabbit anti-eNOS (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a peroxidase-conjugated secondary antibody (1:10,000; Millipore, Billerica, MA, USA). The signal was detected using an ECL Western blotting detection system (GE Healthcare, PA, USA). The images generated were quantitatively analyzed for the protein levels using the ImageJ software. The molecular weights of the protein were determined by comparison to the molecular markers (GE Healthcare).
Drugs Acetylcholine chloride, phenylephrine chloride, superoxide dismutase, L-NAME (NG-nitro-L-arginine-methyl-ester), DEANONOate, Tyron, and indomethacin were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Statistical analysis The data were analyzed using GraphPad Prismª software, version 4.02 (GraphPad Software Inc., USA). Two-way ANOVA was used to compare the concentrationeresponse curves. Student’s t-test was used in the Western blot experiments.
Results Thiamine level was measured by the quantitative spectrophotometric transketolase activity to validate the model [4]. Average blood thiamine levels in micromoles of ribose5-phosphate consumed per hour per milliliter of blood were 27.4 3.8 (control) and 14.0 8.1 (thiamine deficient, 50% decrease) and sedoheptulose production per hour per milliliter was 1.9 0.3 (control) and 0.6 0.1 (thiamine deficient, w70% decrease). The expected decrease in transketolase activity in humans subjected to thiamine deprivation is about 50e60% [22,23], which is similar to the values found in our model. Vessels from thiamine-deficient (TD) rats showed a reduction in the acetylcholine-induced relaxation compared
185 to that of the controls (Fig. 1A). In addition, the TD rings showed an increased phenylephrine-induced contraction in the preparations that contained a functional endothelium (Fig. 1B). This response was paralleled by either endothelium removal (Fig. 1B) or pre-incubation with L-NAME (300 mM) (Fig. 1C), suggesting that the NO released by the endothelial layer is involved in the increased aorta contractile response. To investigate the participation of cyclooxygenases (COX) in the endothelial dysfunction associated with thiamine deficiency, we used indomethacin (10 mM), a non-selective inhibitor of COX. Indomethacin did not modify the vasorelaxant response to acetylcholine (Fig. 2A) or the contractile response to phenylephrine (Fig. 2B), indicating that prostanoids were not involved in the aorta endothelial dysfunction caused by thiamine deficiency. Therefore, it was important to evaluate the participation of eNOS in the endothelial dysfunction provoked by thiamine deficiency. Western blot analysis revealed that there was a 50% decrease in the expression levels of eNOS in TD rat aortas compared to controls (Fig. 3). These results suggest that protein expression is decreased in the TD rat aorta. Corroborating the previous data, the simultaneous measurement of NO and vascular function revealed a decreased level of NO production (Fig. 4) in response to acetylcholine in aortic rings from TD rats compared to controls. Superoxide production is commonly associated with endothelial dysfunction and we have previously shown that thiamine deficiency perturbs the oxidative balance in heart cells [11]. Consequently, we verified whether superoxide anion production was involved in the observed effects. Treatment with superoxide dismutase (SOD) at 300 U/mL did not alter the phenylephrine-induced contractions (Fig. 5A). In addition, this response was not modified by 10 mM Tyron (Fig. 5B), an intracellular superoxide radical scavenger [24], suggesting that the superoxide anion may not be directly involved in the endothelial dysfunction elicited by thiamine deprivation in rats. We next evaluated the sensitivity of vascular smooth muscle cells (VSMC) to NO using DEA-NONOate, an NO donor. DEA-NONOate produced a concentration-dependent vasorelaxant response that did not vary between the TD and control rats (Fig. 5C). This result indicates that the sensitivity of VSMC to NO is not modified by thiamine deprivation.
Figure 1 (A) Acetylcholine-induced vasodilation in endothelium-containing aortic rings from control and thiamine-deficient (TD) rats. (B) Contractile effect elicited by phenylephrine in the presence (Eþ) or absence (E) of endothelium. (C) Concentrationeresponse curves to phenylephrine in endothelium-intact aortic rings pre-treated with L-NAME (300 mM). The values are expressed as the mean SEM from five experiments. ***P < 0.001.
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Figure 2 Concentrationeresponse curves to acetylcholine (A) and phenylephrine (B) in endothelium-intact aortas pre-treated with indomethacin from control and thiamine-deficient (TD) rats. The values are expressed as the mean SEM from five experiments. ***P < 0.001.
Discussion Thiamine deficiency causes cardiac damage, which can be associated with impaired cardiomyocyte contractility [9], cardiac myocyte apoptosis [11] and heart failure [9,11,12]. The changes in vascular function caused by thiamine deficiency are largely unknown. In the present work, we offer the first report that thiamine deficiency causes a decrease in eNOS protein expression, leading to vascular endothelium dysfunction characterized by a decreased relaxant response and increased contractility. As the major regulator of vascular homeostasis, the endothelium not only modulates the tone of the underlying VSMC but also inhibits VSMC proliferation and migration, platelet aggregation, the oxidation of low density lipoproteins, monocyte and platelet adhesion, and the synthesis of inflammatory cytokines, thereby exhibiting important pathophysiological roles [25]. Most of these effects are primarily mediated by NO.
Figure 3 Western blot analysis of eNOS expression in aortas from control and thiamine-deficient (TD) rats. Each gel is representative of 9 samples from 3 independent experiments. The results were normalized to the b-actin content in the samples. **P < 0.01.
Direct measurements of NO revealed a decrease in NO production in thiamine-deficient aortas. The decrease in NO production was accompanied by a reduction in the acetylcholine-induced vascular relaxation. Our results also showed an increased contractile response in aortas from thiamine-deficient rats, which returned to the control response levels following endothelium removal or the inhibition of NOS with L-NAME. Taken together, these data indicate an NO-dependent vascular dysfunction in the aortas of thiamine-deficient rats. To investigate whether the reduction in NO production originates from a reduction in eNOS protein expression levels, we performed Western blot experiments. Interestingly, our results clearly demonstrate a reduction in the eNOS expression levels. A modification in the sensitivity of VSMC to NO is unlikely because vascular relaxation to the NO donor DEA-NONOate was similar between the control and thiamine-deficient vessels. Previous work had shown that thiamine deficiency induced by pyrithiamine in rat decreases NOS activity in the thalamus and cerebellum but not in the hippocampus and striatum [26]. In our study, we demonstrated for the first time that thiamine deficiency induces an important endothelial dysfunction through a reduction in the eNOS
Figure 4 Simultaneous measurements of vasorelaxation and NO production in aortas from control and thiamine-deficient (TD) rats stimulated with acetylcholine. The values are expressed as the mean SEM from five experiments. Continuous line: vasodilation, left axis; dotted line: NO production, right axis. ***P < 0.001.
Thiamine deficiency e reduced NO and vascular dysfunction
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Figure 5 Concentrationeresponse curves to phenylephrine in the presence and absence of SOD (300 U/ml) (A) and Tyron (10 mM) (B) in aortas from control and thiamine-deficient (TD) rats. (C) Concentrationeresponse curves to DEA-NONOate in endotheliumdenuded aortas pre-contracted with phenylephrine. The values are expressed as the mean SEM from five experiments. ***P < 0.001.
expression levels in a large-conductance blood vessel. We and others have demonstrated that thiamine deficiency altered cellular cardiac function via a reduction in cardiomyocyte contractility [9], induced cardiac hypotrophy [27], elicited bradycardia [10] and led to heart failure [11,12]. Taken together, the decreased vasorelaxation and increased aorta contractility would be expected to contribute to an aggravation of the consequences of cardiac malfunctioning in thiamine-deficient rats due to an augmentation of the after-load. Vascular dysfunction is commonly associated with alterations in the production of prostanoids and reactive oxygen species (ROS) in several pathologies [15]. However, the inhibition of COX with indomethacin did not modify the vascular response, ruling out the participation of prostanoids in vascular dysfunction in a model of thiamine deficiency. In addition, the changes observed in the aorta responsiveness of thiamine-deficient rats were most likely not dependent on ROS because SOD and Tyron were ineffective in restoring the vascular response to the control level. In conclusion, taken together, our results indicate that thiamine deficiency leads to an NO-mediated vascular dysfunction.
Acknowledgments This work was supported by FAPEMIG/PRONEX, CNPq/Brazil and PRPq/UFMG.
References [1] Stepuro II. Thiamine and vasculopathies. Prostaglandins Leukot Essent 2005;72:115e27. [2] Butterworth RF. Thiamin deficiency and brain disorders. Nutr Res Rev 2003;16:277e83. [3] Oliveira FA, Galan DT, Ribeiro AM, Cruz JD. Thiamine deficiency during pregnancy leads to cerebellar neuronal death in rat offspring: role of voltage-dependent Kþ channels. Brain Res 2007;1134:79e86. [4] Oliveira FA, Guatimosim S, Castro CH, Galan DT, LautonSantos S, Ribeiro AM, et al. Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats. Am J Physiol Heart Circ Physiol 2007;293:394e401.
[5] Pires RGW, Pereira SRC, Oliveira-Silva IF, Franco GC, Ribeiro AM. Cholinergic parameters and the retrieval of learned and re-learned spatial information: a study using a model of Wernicke-Korsakoff Syndrome. Behav Brain Res 2005;162:11e21. [6] Bubber P, Zun-Ji K, Gibson GE. Tricarboxylic acid cycle enzymes following thiamine deficiency. Neurochem Int 2004;45: 1021e8. [7] Gibson GE, Zhang H. Interactions of oxidative stress with thiamine homeostasis promote neurodegeneration. Neurochem Int 2002;40:493e504. [8] Zangen A, Shainberg A. Thiamine deficiency in cardiac cells in culture. Biochem Pharmacol 1997;54:575e82. [9] Gioda CR, Roman-Campos D, Carneiro-Ju ´nior MA, Silva KA, Souza MO, Mendes LJ, et al. Impaired cellular contractile function in thiamine-deficient rat cardiomyocytes. Eur J Heart Fail 2009;11:1126e8. [10] Cappelli V, Bottinelli R, Polla B, Reggiani C. Altered contractile properties of rat cardiac muscle during experimental thiamine deficiency and food deprivation. J Mol Cell Cardiol 1990;22:1095e106. [11] Gioda CR, Barreto TO, Primola-Gomes TN, Lima DC, Campos APP, Capettini LSA, et al. Cardiac oxidative stress is involved in heart failure by thiamine deprivation in rats. Am J Physiol Heart Circ Physiol 2010;298:H2039e45. [12] Da Cunha S, Bastos JC, Salles JB, Silva MC, Bastos VL. Cardiac alterations in furosemide-treated thiamine-deprived rats. J Card Fail 2007;13:774e83. [13] Durante W, Johnson FK, Johnson RA. Arginase: a critical regulator of nitric oxide synthesis and vascular function. Clin Exp Pharmacol Physiol 2007;34:906e11. [14] Brutsaert DL. Cardiac endothelial-myocardial signaling: role in cardiac growth, contractile performance, and rhythmicity. Physiol Rev 2003;83:59e115. [15] Heitzer T, Schlinzig T, Krohn K, Meinertz T, Mu ¨nzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 2001;104:2673e8. [16] Ballinger SW, Patterson C, Yan C, Doan R, Burow DL, Young CG, et al. Hydrogen peroxide- and peroxynitriteinduced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res 2000; 86:960e6. [17] Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 1997;100:2153e7. [18] Cruz AJ, Lemos VS, dos Santos MH, Nagem TJ, Cortes SF. Vascular effects of 7-epiclusianone, a prenylated benzophenone from Rheedia gardneriana, on the rat aorta. Phytomedicine 2006;72:300e3.
188 [19] Capettini LS, Cortes SF, Silva JF, Alvarez-Leite JI, Lemos VS. Decreased production of nNOS-derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis. Br J Pharmacol 2011;164:1738e48. [20] Capettini LS, Cortes SF, Lemos VS. Relative contribution of eNOS and nNOS to endothelium-dependent vasodilation in the mouse aorta. Eur J Pharmacol 2010;643:260e6. [21] Capettini LS, Cortes SF, Gomes MA, Silva GA, Pesquero JL, Lopes MJ, et al. Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor. Am J Physiol Heart Circ Physiol 2008;295: H2503e11. [22] Macias-Matos C, Rodriguez-Ojea A, Chi N, Jimenez S, Zulueta D, Bates CJ. Biochemical evidence of thiamine depletion during the Cuban neuropathy epidemic, 1992-1993. Am J Clin Nutr 1996;64:347e53.
C.R. Gioda et al. [23] Singleton CK, Pekovich SR, McCool BA, Martin PR. The thiamine-dependent hysteretic behavior of human transketolase: implications for thiamine deficiency. J Nutr 1995; 125:189e94. [24] Hamilton CA, Berg G, Mcintyre M, Mcphaden AR, Reid JL, Dominiczak AF. Effects of nitric oxide and superoxide on relaxation in human artery and vein. Atherosclerosis 1997;133:77e86. [25] Vanhoutte PM. The endothelium and arterial reactivity. J Mal Vasc 1986;11:213e22. [26] Rao VLR, Mousseaou DD, Butterworth RF. Nitric oxide synthase activities are selectively decreased in vulnerable brain regions in thiamine deficiency. Neurosci Lett 1996;208:17e20. [27] Roman-Campos D, Campos AC, Gioda CR, Campos PP, Medeiros MA, Cruz JS. Cardiac structural changes and electrical remodeling in a thiamine-deficient model in rats. Life Sci 2009;84:817e24.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/nmcd
Thiamine deficiency leads to reduced nitric oxide production and vascular dysfunction in rats C.R. Gioda a,1,2, L.S.A. Capettini b,2, J.S. Cruz c,3, V.S. Lemos a,*,3 a
Departamento de Fisiologia e Biofı´sica, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil b Departamento de Farmacologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil c Departamento de Bioquı´mica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil Received 13 March 2013; received in revised form 7 June 2013; accepted 10 June 2013 Available online 6 October 2013
KEYWORDS Thiamine deficiency; Endothelial dysfunction; eNOS; Nitric oxide
Abstract Background and aims: Thiamine deficiency is a condition that is known to cause damage to the nervous and cardiovascular systems because it interferes with cellular metabolism. It is well known that the control of vascular function is highly dependent on the production of nitric oxide (NO) by NO synthases. Studies exploring the physiological relevance of NO signaling under conditions of thiamine deficiency are scarce. The present study sought to investigate whether chronic metabolic changes would cause alterations in vascular responsiveness. Methods and results: By removing thiamine from the diet, we observed a reduced acetylcholine-mediated relaxation and an increased phenylephrine-mediated vasoconstriction in the aortas containing functional endothelium. Removal of the endothelium or the pretreatment of vessels with L-NAME restored the contractile responses to the level of controls. Conversely, indomethacin did not modify phenylephrine-mediated contractions. We also used carbon microsensors to continually measure NO production in situ while simultaneously measuring the vascular tone. The results revealed a significant decrease in NO production. Western blot analysis showed a decreased expression of the total eNOS in the thiaminedeficient aorta compared to the control. Concentrationeresponse curves for phenylephrine indicated no difference between the control and deficient groups in the presence and absence
* Corresponding author. Instituto de Cie ˆncias Biolo ´gicas, Universidade Federal de Minas Gerais, Av. Anto ˆnio Carlos 6627, 31970-901 Belo Horizonte, MG, Brazil. Tel.: þ55 31 3409 2950; fax: þ55 31 3409 2934. E-mail addresses: [email protected], [email protected] (V.S. Lemos). 1 Present address: Biological Science Institute, Federal University of Rio Grande, Rio Grande, RS, Brazil. 2 Co-first authors. 3 Co-senior authors. 0939-4753/$ - see front matter ª 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.numecd.2013.06.010
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C.R. Gioda et al. of SOD or Tyron. The NO donor DEA-NONOate produced a concentration-dependent relaxation response in the endothelium-denuded vessels that did not differ between the control and thiamine-deficient rats. Conclusion: Thiamine deficiency modulates eNOS-dependent NO production, leading to a decreased vasorelaxation and an increased contractile response in the rat aorta. ª 2013 Elsevier B.V. All rights reserved.
Introduction Thiamine (vitamin B1) deficiency, which is responsible for beriberi and WernickeeKorsakoff syndrome, is commonly associated with malnutrition and chronic alcoholism [1,2]. These syndromes are characterized by nervous and cardiovascular dysfunction [1,3e5]. Thiamine deficiency has been associated with inappropriate effects or the uncoupling of metabolic enzymes, such as pyruvate dehydrogenase, a-ketoglutarate dehydrogenase and transketolase [6e8]. These effects likely occur because thiamine pyrophosphate, the active form of thiamine, is an important cofactor for the catalytic activity of these enzymes. Despite these metabolic and neuronal effects of thiamine deficiency, little is known of the effects of thiamine deficiency on the cardiovascular system. Some evidence indicates that cardiac contractility is profoundly affected by thiamine deficiency [4,9,10]. In terms of heart function, thiamine is considered to be a clinically important factor because its deficiency has been reported to cause heart failure and alterations in the myocardial antioxidant systems [9,11,12]. To our knowledge, there are no available data demonstrating that alterations in vascular function can be primarily caused by a thiamine deficiency. Cardiovascular homeostasis relies on the integrity of the vascular endothelium, which is able to modify the vascular tonus throughout the release of a multitude of active molecules, inducing relaxation or constriction in the smooth muscle and consequently modulating the blood flow and arterial pressure [13,14]. Disorders in the systemic vasculature are thought to originate in endothelial dysfunction induced by oxidative stress, which results in a reduction in NO availability and metabolic and hormonal alterations that can lead to several pathologies, including hypertension, heart failure, atherosclerosis and diabetes [15e17]. The significance of thiamine deficiency in vascular biology is therefore an important question that remains unanswered. Here, we hypothesized that thiamine is required to maintain proper vascular function. We provide pharmacological and biochemical evidence showing that thiamine deficiency provokes a substantial impairment of endothelial function that ultimately leads to vascular dysfunction.
Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats from the Animal Care facilities at UFMG (200e250 g) were kept in a 25 C room (12-h lightedark cycle) with free access to food and water. The rats were acclimated for 7 days and were then fed a thiamine-deficient diet for 35 days.
Chow The control and thiamine-deficient chow were made as previously described [3,11]. The two diets were isocaloric and the difference between them was the presence or absence of thiamine in the vitamin mixture used to prepare the chow. Both diets contained 20% protein, 1% cellulose, 5% mixture of salts, 1% mixture of vitamins, 5% corn oil, 0.4% choline and 67.6% cornstarch.
Isolated rat aortic rings Rings from the thoracic aorta (4e5 mm long) were mounted in an organ bath, containing KrebseHenseleit solution (in mM: NaCl 110.8, KCl 5.8, NaHCO3 25, MgSO4 1.07, CaCl2 2.49, NaH2PO4 2.33 and glucose 11.53), gassed with a 95% O2 and 5% CO2 mixture and equilibrated under a resting tension of 1 g for 1 h, at 37 C. The mechanical activity was recorded isometrically using a force transducer [18]. When necessary, the endothelial layer was removed mechanically. The vessels were contracted using phenylephrine (0.3 mM). The presence or absence of endothelium was verified by adding acetylcholine (10 mM).
Simultaneous measurements of NO and vascular function Acetylcholine-induced NO production was measured simultaneously with vascular function using NO-sensitive carbon microsensors (ISO-NOPF100, World Precision Instruments) placed next to the lumen of the vessels [19,20]. The generated currents (nA) were measured using the microsensors. NO concentrations were determined by comparison to calibration curves of known concentrations of the NO donor S-nitroso-N-acetylpenicillamine (SNAP).
Methods
Western blot analyses
Animals
The thoracic aorta were homogenized in ice-cold buffer (50 mM TriseHCl containing 150 mM NaCl, 5 mM EDTA-Na2, 1 mM MgCl2, 1% Nonidet P40, 0.3% triton X-100, 0.5% sodium deoxycholate, 100 mM dithiothreitol, 100 mM phenylmethylsulphonyl fluoride) and a cocktail of protease
Our investigations were approved by the institutional ethical committee for animal experimentation in accordance with the Guide for the Care and Use of Laboratory
Thiamine deficiency e reduced NO and vascular dysfunction inhibitors (SigmaFast). The protein samples were mixed with denaturing SDS-PAGE buffer, transferred to nitrocellulose membrane and subjected to a routine Western immunoblotting protocol [21] using the following antibodies: rabbit anti-eNOS (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a peroxidase-conjugated secondary antibody (1:10,000; Millipore, Billerica, MA, USA). The signal was detected using an ECL Western blotting detection system (GE Healthcare, PA, USA). The images generated were quantitatively analyzed for the protein levels using the ImageJ software. The molecular weights of the protein were determined by comparison to the molecular markers (GE Healthcare).
Drugs Acetylcholine chloride, phenylephrine chloride, superoxide dismutase, L-NAME (NG-nitro-L-arginine-methyl-ester), DEANONOate, Tyron, and indomethacin were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Statistical analysis The data were analyzed using GraphPad Prismª software, version 4.02 (GraphPad Software Inc., USA). Two-way ANOVA was used to compare the concentrationeresponse curves. Student’s t-test was used in the Western blot experiments.
Results Thiamine level was measured by the quantitative spectrophotometric transketolase activity to validate the model [4]. Average blood thiamine levels in micromoles of ribose5-phosphate consumed per hour per milliliter of blood were 27.4 3.8 (control) and 14.0 8.1 (thiamine deficient, 50% decrease) and sedoheptulose production per hour per milliliter was 1.9 0.3 (control) and 0.6 0.1 (thiamine deficient, w70% decrease). The expected decrease in transketolase activity in humans subjected to thiamine deprivation is about 50e60% [22,23], which is similar to the values found in our model. Vessels from thiamine-deficient (TD) rats showed a reduction in the acetylcholine-induced relaxation compared
185 to that of the controls (Fig. 1A). In addition, the TD rings showed an increased phenylephrine-induced contraction in the preparations that contained a functional endothelium (Fig. 1B). This response was paralleled by either endothelium removal (Fig. 1B) or pre-incubation with L-NAME (300 mM) (Fig. 1C), suggesting that the NO released by the endothelial layer is involved in the increased aorta contractile response. To investigate the participation of cyclooxygenases (COX) in the endothelial dysfunction associated with thiamine deficiency, we used indomethacin (10 mM), a non-selective inhibitor of COX. Indomethacin did not modify the vasorelaxant response to acetylcholine (Fig. 2A) or the contractile response to phenylephrine (Fig. 2B), indicating that prostanoids were not involved in the aorta endothelial dysfunction caused by thiamine deficiency. Therefore, it was important to evaluate the participation of eNOS in the endothelial dysfunction provoked by thiamine deficiency. Western blot analysis revealed that there was a 50% decrease in the expression levels of eNOS in TD rat aortas compared to controls (Fig. 3). These results suggest that protein expression is decreased in the TD rat aorta. Corroborating the previous data, the simultaneous measurement of NO and vascular function revealed a decreased level of NO production (Fig. 4) in response to acetylcholine in aortic rings from TD rats compared to controls. Superoxide production is commonly associated with endothelial dysfunction and we have previously shown that thiamine deficiency perturbs the oxidative balance in heart cells [11]. Consequently, we verified whether superoxide anion production was involved in the observed effects. Treatment with superoxide dismutase (SOD) at 300 U/mL did not alter the phenylephrine-induced contractions (Fig. 5A). In addition, this response was not modified by 10 mM Tyron (Fig. 5B), an intracellular superoxide radical scavenger [24], suggesting that the superoxide anion may not be directly involved in the endothelial dysfunction elicited by thiamine deprivation in rats. We next evaluated the sensitivity of vascular smooth muscle cells (VSMC) to NO using DEA-NONOate, an NO donor. DEA-NONOate produced a concentration-dependent vasorelaxant response that did not vary between the TD and control rats (Fig. 5C). This result indicates that the sensitivity of VSMC to NO is not modified by thiamine deprivation.
Figure 1 (A) Acetylcholine-induced vasodilation in endothelium-containing aortic rings from control and thiamine-deficient (TD) rats. (B) Contractile effect elicited by phenylephrine in the presence (Eþ) or absence (E) of endothelium. (C) Concentrationeresponse curves to phenylephrine in endothelium-intact aortic rings pre-treated with L-NAME (300 mM). The values are expressed as the mean SEM from five experiments. ***P < 0.001.
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Figure 2 Concentrationeresponse curves to acetylcholine (A) and phenylephrine (B) in endothelium-intact aortas pre-treated with indomethacin from control and thiamine-deficient (TD) rats. The values are expressed as the mean SEM from five experiments. ***P < 0.001.
Discussion Thiamine deficiency causes cardiac damage, which can be associated with impaired cardiomyocyte contractility [9], cardiac myocyte apoptosis [11] and heart failure [9,11,12]. The changes in vascular function caused by thiamine deficiency are largely unknown. In the present work, we offer the first report that thiamine deficiency causes a decrease in eNOS protein expression, leading to vascular endothelium dysfunction characterized by a decreased relaxant response and increased contractility. As the major regulator of vascular homeostasis, the endothelium not only modulates the tone of the underlying VSMC but also inhibits VSMC proliferation and migration, platelet aggregation, the oxidation of low density lipoproteins, monocyte and platelet adhesion, and the synthesis of inflammatory cytokines, thereby exhibiting important pathophysiological roles [25]. Most of these effects are primarily mediated by NO.
Figure 3 Western blot analysis of eNOS expression in aortas from control and thiamine-deficient (TD) rats. Each gel is representative of 9 samples from 3 independent experiments. The results were normalized to the b-actin content in the samples. **P < 0.01.
Direct measurements of NO revealed a decrease in NO production in thiamine-deficient aortas. The decrease in NO production was accompanied by a reduction in the acetylcholine-induced vascular relaxation. Our results also showed an increased contractile response in aortas from thiamine-deficient rats, which returned to the control response levels following endothelium removal or the inhibition of NOS with L-NAME. Taken together, these data indicate an NO-dependent vascular dysfunction in the aortas of thiamine-deficient rats. To investigate whether the reduction in NO production originates from a reduction in eNOS protein expression levels, we performed Western blot experiments. Interestingly, our results clearly demonstrate a reduction in the eNOS expression levels. A modification in the sensitivity of VSMC to NO is unlikely because vascular relaxation to the NO donor DEA-NONOate was similar between the control and thiamine-deficient vessels. Previous work had shown that thiamine deficiency induced by pyrithiamine in rat decreases NOS activity in the thalamus and cerebellum but not in the hippocampus and striatum [26]. In our study, we demonstrated for the first time that thiamine deficiency induces an important endothelial dysfunction through a reduction in the eNOS
Figure 4 Simultaneous measurements of vasorelaxation and NO production in aortas from control and thiamine-deficient (TD) rats stimulated with acetylcholine. The values are expressed as the mean SEM from five experiments. Continuous line: vasodilation, left axis; dotted line: NO production, right axis. ***P < 0.001.
Thiamine deficiency e reduced NO and vascular dysfunction
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Figure 5 Concentrationeresponse curves to phenylephrine in the presence and absence of SOD (300 U/ml) (A) and Tyron (10 mM) (B) in aortas from control and thiamine-deficient (TD) rats. (C) Concentrationeresponse curves to DEA-NONOate in endotheliumdenuded aortas pre-contracted with phenylephrine. The values are expressed as the mean SEM from five experiments. ***P < 0.001.
expression levels in a large-conductance blood vessel. We and others have demonstrated that thiamine deficiency altered cellular cardiac function via a reduction in cardiomyocyte contractility [9], induced cardiac hypotrophy [27], elicited bradycardia [10] and led to heart failure [11,12]. Taken together, the decreased vasorelaxation and increased aorta contractility would be expected to contribute to an aggravation of the consequences of cardiac malfunctioning in thiamine-deficient rats due to an augmentation of the after-load. Vascular dysfunction is commonly associated with alterations in the production of prostanoids and reactive oxygen species (ROS) in several pathologies [15]. However, the inhibition of COX with indomethacin did not modify the vascular response, ruling out the participation of prostanoids in vascular dysfunction in a model of thiamine deficiency. In addition, the changes observed in the aorta responsiveness of thiamine-deficient rats were most likely not dependent on ROS because SOD and Tyron were ineffective in restoring the vascular response to the control level. In conclusion, taken together, our results indicate that thiamine deficiency leads to an NO-mediated vascular dysfunction.
Acknowledgments This work was supported by FAPEMIG/PRONEX, CNPq/Brazil and PRPq/UFMG.
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