Martin Tepel

Activation of TRPV1 by Dietary Capsaicin Improves Endothelium-Dependent Vasorelaxation and Prevents Hypertension

Some plant-based diets lower the cardiometabolic risks and prevalence of hypertension. New evidence implies a role for the transient receptor potential vanilloid 1 (TRPV1) cation channel in the pathogenesis of cardiometabolic diseases. Little is known about impact of chronic TRPV1 activation on the regulation of vascular function and blood pressure. Here we report that chronic TRPV1 activation by dietary capsaicin increases the phosphorylation of protein kinase A (PKA) and eNOS and thus production of nitric oxide (NO) in endothelial cells, which is calcium dependent. TRPV1 activation by capsaicin enhances endothelium-dependent relaxation in wild-type mice, an effect absent in TRPV1-deficient mice. Long-term stimulation of TRPV1 can activate PKA, which contributes to increased eNOS phosphorylation, improves vasorelaxation, and lowers blood pressure in genetically hypertensive rats. We conclude that TRPV1 activation by dietary capsaicin improves endothelial function. TRPV1-mediated increase in NO production may represent a promising target for therapeutic intervention of hypertension.

Acetylcysteine Reduces Plasma Homocysteine Concentration and Improves Pulse Pressure and Endothelial Function in Patients With End-Stage Renal Failure

Background—Increased oxidative stress, elevated plasma homocysteine concentration, increased pulse pressure, and impaired endothelial function constitute risk factors for increased mortality in patients with end-stage renal failure. Methods and Results—We investigated the metabolic and hemodynamic effects of intravenous administration of acetylcysteine, a thiol-containing antioxidant, during a hemodialysis session in a prospective, randomized, placebo-controlled crossover study in 20 patients with end-stage renal failure. Under control conditions, a hemodialysis session reduced plasma homocysteine concentration to 58±22% predialysis (mean±SD), whereas in the presence of acetylcysteine, the plasma homocysteine concentration was significantly more reduced to 12±7% predialysis (P <0.01). The reduction of plasma homocysteine concentration was significantly correlated with a reduction of pulse pressure. A 10% decrease in plasma homocysteine concentration was associated with a decrease of pulse pressure by 2.5 mm Hg. Analysis of the second derivative of photoplethysmogram waveform showed changes of arterial wave reflectance during hemodialysis in the presence of acetylcysteine, indicating improved endothelial function. Conclusions—Acetylcysteine-dependent increase of homocysteine removal during a hemodialysis session improves plasma homocysteine concentration, pulse pressure, and endothelial function in patients with end-stage renal failure.

Increased plasma phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression

NO prevents atherogenesis and inflammation in vessel walls by inhibition of cell proliferation and cytokine-induced endothelial expression of adhesion molecules and proinflammatory cytokines. Reduced NO production due to inhibition of either eNOS or iNOS may therefore reinforce atherosclerosis. Patients with end-stage renal failure show markedly increased mortality due to atherosclerosis. In the present study we tested the hypothesis that uremic toxins are responsible for reduced iNOS expression. LPS-induced iNOS expression in mononuclear leukocytes was studied using real-time PCR. The iNOS expression was blocked by addition of plasma from patients with end-stage renal failure, whereas plasma from healthy controls had no effect. Hemofiltrate obtained from patients with end-stage renal failure was fractionated by chromatographic methods. The chromatographic procedures revealed a homogenous fraction that inhibits iNOS expression. Using gas chromatography/mass spectrometry, this inhibitor was identified as phenylacetic acid. Authentic phenylacetic acid inhibited iNOS expression in a dose-dependent manner. In healthy control subjects, plasma concentrations were below the detection level, whereas patients with end-stage renal failure had a phenylacetic acid concentration of 3.49 +/- 0.33 mmol/l (n = 41). It is concluded that accumulation of phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression. That mechanism may contribute to increased atherosclerosis and cardiovascular morbidity in patients with end-stage renal failure.

Effect of continuous positive airway pressure therapy on 24-hour blood pressure in patients with obstructive sleep apnea syndrome.

BACKGROUND Patients with obstructive sleep apnea syndrome (OSAS) are subject to an increased cardiovascular morbidity including systemic hypertension. Little is known about the effects of treatment with nasal continuous positive airway pressure (CPAP) on systemic hypertension. METHODS Automated ambulatory 24-h blood pressure (BP) monitoring was performed in 88 consecutive patients who were referred for evaluation of snoring or suspected OSAS. In addition, the long-term effects of CPAP therapy on 24-h BP were assessed. RESULTS A total of 62 patients had OSAS and 26 habitual snoring. Patients with OSAS had significantly higher mean arterial BP values than snorers (102.7 +/- 10.7 v 94.0 +/- 10.2 mm Hg; P < .01). Multiple stepwise linear regression analysis disclosed that the degree of systemic hypertension was independently associated with the severity of OSAS as determined by the apnea/hypopnea index (R = 0.43; P < .001), but not with age, body mass index, or smoking habits. Of the 62 patients with OSAS, 52 were treated with CPAP and reevaluated after 9 months. The CPAP resulted in a significant decrease in mean arterial BP (from 103.7 +/- 10.4 to 99.1 +/- 10.8 mm Hg; P < .05). For those patients with systemic hypertension whose BP improved with CPAP therapy, 24-h mean pulse pressure at baseline (r = -0.36; P < .05) as well as average heart rate during the day (r = -0.35; P < .05) turned out as predictors. CONCLUSIONS Obstructive sleep apnea syndrome contributes, at least in part, to the development of systemic hypertension, and CPAP may improve BP values in treated OSAS patients. Predictors of a beneficial CPAP effect on BP are a high heart rate and a high pulse pressure before treatment.

Activation of TRPV1 reduces vascular lipid accumulation and attenuates atherosclerosis

AIMS Activation of transient receptor potential vanilloid type-1 (TRPV1) channels may affect lipid storage and the cellular inflammatory response. Now, we tested the hypothesis that activation of TRPV1 channels attenuates atherosclerosis in apolipoprotein E knockout mice (ApoE(-/-)) but not ApoE(-/-)TRPV1(-/-) double knockout mice on a high-fat diet. METHODS AND RESULTS Both TRPV1 mRNA and protein expression were identified in vascular smooth muscle cells (VSMC) and in aorta from C57BL/6J mice using RT-PCR, immunoblotting, and immunohistochemistry. In vitro, activation of TRPV1 by the specific agonists capsaicin and resiniferatoxin dose-dependently increased cytosolic calcium and significantly reduced the accumulation of lipids in VSMC from C57BL/6J mice but not from TRPV1(-/-) mice. TRPV1 activation increased ATP-binding cassette transporter A1 (ABCA1) expression and reduced low-density lipoprotein-related protein 1 (LRP1) expression in VSMC by calcium-dependent and calcineurin- and protein kinase A-dependent mechanisms. These results showed increased cellular cholesterol efflux and reduced cholesterol uptake. In vivo, long-term activation of TRPV1 by capsaicin for 24 weeks increased ABCA1 and reduced LRP1 expression in aorta from ApoE(-/-) mice on a high-fat diet. Long-term activation of TRPV1 significantly reduced lipid storage and atherosclerotic lesions in the aortic sinus and in the thoracoabdominal aorta from ApoE(-/-) mice but not from ApoE(-/-)TRPV1(-/-) mice on a high-fat diet. These findings indicated that TRPV1 activation ameliorates high-fat diet-induced atherosclerosis. CONCLUSION Activation of TRPV1 may be a novel therapeutic tool to attenuate atherosclerosis caused by a high-fat diet.

Reactive oxygen species in essential hypertension and non–insulin-dependent diabetes mellitus

To evaluate whether increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of essential hypertension (EH) and non-insulin-dependent diabetes mellitus (NIDDM), both resting and stimulated levels of intracellular ROS were measured in lymphocytes from patients with EH (n = 10), NIDDM (n = 16) and age-matched healthy individuals (control subjects, n = 19). ROS was monitored with the dye, dihydrorhodamine-123 (DHR; 1 micromol/L) in the presence or absence of superoxide dismutase (superoxide scavenger), sodium azide (singlet oxygen/hydrogen peroxide scavenger), genistein (tyrosine kinase inhibitor), or bisindolylmaleimide (protein kinase C inhibitor). Simultaneous monitoring of cytosolic [Ca2+]i was done with fura-2. Resting ROS levels were significantly higher in NIDDM (4.71+/-0.25 nmol/10(6) cells; mean +/- SEM, P<.05) compared with EH (4.03+/-0.22 nmol/10(6) cells) or controls (4.05+/-0.15 nmol/10(6) cells). The formyl-Met-Leu-Phenylalanine-(fMLP)-induced ROS generation was significantly higher in NIDDM (21.92+/-2.23 nmol/10(6) cells; P<.05) compared with EH (14.58+/-1.90 nmol/10(6) cells) or control (16.06+/-1.22 nmol/10(6) cells). The fMLP-induced ROS increase was significantly reduced in the presence of sodium azide in all groups (P<.01) but was largely unaffected in the presence of SOD. Genistein and bisindolylmaleimide significantly inhibited the fMLP-induced ROS in all groups. The fMLP-induced [Ca2+]i increase was significantly higher in NIDDM (71+/-12 nmol/L, P <.01) compared with EH (42+/-4 nmol/L) and control subjects (35+/-3 nmol/L). Phytohemagglutinin was more effective in increasing [Ca2+]i than ROS. It is concluded that ROS may play a role in the metabolic syndrome of NIDDM but not in EH.

Identification and characterization of diadenosine 5′,5‴-P1,P2 -diphosphate and diadenosine 5′,5‴-P1,P3-triphosphate in human myocardial tissue

We examined whether human cardiac tissue contains diadenosine polyphosphates and investigated their physiological role. Extracts from human cardiac tissue from transplant recipients were fractionated by size exclusion‐, affinity‐, anion exchange‐ and reversed‐phase chromatography. MALDI‐MS analysis of two absorbing fractions revealed molecular masses of 676.2 Da and 756.0 Da. The UV spectra of both fractions were identical to that of adenosine. Postsource decay MALDI mass spectrometry indicated that the molecules with a mass of 676.2 Da and 757.0 Da contained AMP and ATP, respectively. As shown by enzymatic cleavage, both molecules consist of two adenosines interconnected by either two or three phosphates in 5′‐positions of the riboses. Two substances can be identified as 5′,5‴‐P1,P2‐diphosphate (Ap2A) and 5′,5‴‐P1,P3‐triphosphate (Ap3A). Ap2A and Ap3A, together with ATP and ADP, are stored in myocardial‐specific granules in biologically active concentrations. In the isolated perfused rat heart, Ap2A and Ap3A caused dose‐dependent coronary vasodilations. In myocardial preparations, Ap2A and Ap3A attenuated the effect of isoproterenol, exerting a negative inotropic effect. The calcium current of guinea pig ventricular myocytes, stimulated by isoproterenol, was also attenuated by Ap2A and Ap3A. The presence of Ap2A and Ap3A in cardiac‐specific granules and the actions of these substances on the myocardium and coronary vessels indicate a role for these substances as endogenous modulators of myocardial functions and coronary perfusion.—Luo, J., Jankowski, J., Knobloch, M., van der Giet, M., Gardanis, K., Russ, T., Vahlensieck, U., Neumann, J., Schmitz, W., Tepel, M., Deng, M. C., Zidek, W., Schlüter, H. Identification and characterization of diadenosine 5′,5‴‐P1,P2‐diphosphate and diadenosine 5′,5‴‐P1,P3‐triphosphate in human myocardial tissue. FASEB J. 13, 695–705 (1999)

Evidence of Altered Homocysteine Metabolism in Chronic Renal Failure

The fasting serum concentrations of total homocysteine and metabolites of transsulfuration (cystathionine, cysteine, methylmalonic acid, 2-methylcitric acid) and remethylation (methionine) were determined by gas chromatography-mass spectrometry in 40 nondialyzed patients with chronic renal disease and in 50 patients with end-stage renal disease requiring chronic maintenance hemodialysis. The nondialyzed patients and 28 of the dialysis patients did not receive additional vitamin supplementations. Twenty-two of the dialysis patients received daily oral vitamin preparations containing 10 mg pyridoxine (vitamin B6), 6 µg cyanocobalamin (vitamin B12), and 1 mg folic acid. In the nondialyzed patients, linear regression analysis showed positive correlations between serum concentrations of creatinine and total homocysteine (r = 0.68, p < 0.0001), cystathionine (r = 0.73, p < 0.0001), methylmalonic acid (r = 0.77, p < 0.0001), and 2-methylcitric acid (r = 0.81, p < 0.0001). Serum homocysteine was positively correlated with serum concentrations of cystathionine (r = 0.59, p < 0.0001), cysteine (r = 0.69, p = 0.004), methylmalonic acid (r = 0.64, p = 0.0001), and 2-methylcitric acid (r = 0.64, p < 0.0001). There was no significant correlation between serum concentrations of homocysteine and methionine (r = –0.14, p = 0.63). In the hemodialysis patients receiving oral vitamin supplementation, serum homocysteine and cystathionine concentrations were significantly lower than in hemodialysis patients not receiving vitamins (homocysteine 21.8 ± 1.1 vs. 33.2 ± 3.7 µmol/l, p = 0.0004; cystathionine 2,075.9 ± 387.1 vs. 3,171.3 ± 680.2 nmol/l, p = 0.02; mean ± SEM). In summary, our results show increased intermediate products of the transsulfuration pathway, but no increase in remethylation of homocysteine in chronic renal disease, including end-stage renal disease requiring chronic maintenance dialysis.

Isoforms of Retinol binding protein 4 (RBP4) are increased in chronic diseases of the kidney but not of the liver

BackgroundThe levels of retinol-binding protein 4 (RBP4) – the carrier protein for Vitamin A in plasma – are tightly regulated under healthy circumstances. The kidney, the main site of RBP4 catabolism, contributes to an elevation of RBP4 levels during chronic kidney disease (CKD) whereas during chronic liver disease (CLD) RBP4 levels decrease. Little is known about RBP4 isoforms including apo-RBP4, holo-RBP4 as well as RBP4 truncated at the C-terminus (RBP4-L and RBP4-LL) except that RBP4 isoforms have been reported to be increased in hemodialysis patients. Since it is not known whether CLD influence RBP4 isoforms, we investigated RBP4 levels, apo- and holo-RBP4 as well as RBP4-L and RBP4-LL in plasma of 36 patients suffering from CKD, in 55 CLD patients and in 50 control subjects. RBP4 was determined by ELISA and apo- and holo-RBP4 by native polyacrylamide gel electrophoresis (PAGE). RBP4-L and RBP4-LL were analyzed after immunoprecipitation by mass spectrometry (MALDI-TOF-MS).ResultsRBP4 isoforms and levels were highly increased in CKD patients compared to controls (P < 0.05) whereas in CLD patients RBP4 isoforms were not different from controls. In addition, in hepatic dysfunction RBP4 levels were decreased whereas the amount of isoforms was not affected.ConclusionThe occurrence of RBP4 isoforms is not influenced by liver function but seems to be strongly related to kidney function and may therefore be important in investigating kidney function and related disorders.

Identification and Characterization ofP 1,P 7-Di(adenosine-5′)-heptaphosphate from Human Platelets

Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H]+) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap7A) demonstrated that the isolated substance was identical to Ap7A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5′-positions of the riboses. With thrombin-induced platelet aggregation, Ap7A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap7A on the vasculature of the isolated perfused rat kidney Ap7A was slightly less than that of Ap6A. The threshold of the vasoconstrictive action of Ap7A was 10−5mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid, suggesting an activation of P2x receptors. Furthermore, Ap7A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap7A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.