Klaus Witte

Resveratrol, a Polyphenolic Phytoalexin Present in Red Wine, Enhances Expression and Activity of Endothelial Nitric Oxide Synthase

Background—Estrogens can upregulate endothelial nitric oxide synthase (eNOS) in human endothelial cells by increasing eNOS promoter activity and enhancing the binding activity of the transcription factor Sp1. Resveratrol, a polyphenolic phytoalexin found in grapes and wine, has been reported to act as an agonist at the estrogen receptor. Therefore, we tested the effect of this putative phytoestrogen on eNOS expression in human endothelial cells. Methods and Results—Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol for 24 to 72 hours upregulated eNOS mRNA expression in a time- and concentration-dependent manner (up to 2.8-fold). eNOS protein expression and eNOS-derived NO production were also increased after long-term incubation with resveratrol. Resveratrol increased the activity of the eNOS promoter (3.5-kb fragment) in a concentration-dependent fashion, with the essential trans-stimulated sequence being located in the proximal 263 bp of the promoter sequence. In addition, eNOS mRNA was stabilized by resveratrol. The effect of resveratrol on eNOS expression was not modified by the estrogen receptor antagonists ICI 182780 and RU 58668. In electrophoretic mobility shift assays, nuclear extracts from resveratrol-incubated EA.hy 926 cells showed no enhanced binding activity of the eNOS promoter-relevant transcription factors Sp1, GATA, PEA3, YY1, or Elf-1. In addition to its long-term effects on eNOS expression, resveratrol also enhanced the production of bioactive NO in the short-term (after a 2-minute incubation). Conclusions—In concert with other effects, the stimulation of eNOS expression and activity may contribute to the cardiovascular protective effects attributed to resveratrol.

Supplementation With Oral Vitamin D3 and Calcium During Winter Prevents Seasonal Bone Loss: A Randomized Controlled Open-Label Prospective Trial†

Bone metabolism follows a seasonal pattern with high bone turnover and bone loss during the winter. In a randomized, open‐label 2‐year sequential follow‐up study of 55 healthy adults, we found that supplementation with oral vitamin D3 and calcium during winter abolished seasonal changes in calciotropic hormones and markers of bone turnover and led to an increase in BMD. Supplementation with oral vitamin D3 and calcium during the winter months seems to counteract the effects of seasonal changes in vitamin D and thus may be beneficial as a primary prevention strategy for age‐related bone loss.

Experimental heart failure in rats: effects on cardiovascular circadian rhythms and on myocardial β-adrenergic signaling

OBJECTIVES Patients with chronic heart failure frequently show blunted circadian blood pressure profiles. The mechanisms involved in the loss of physiological day-night variation are still unclear, but a continuously active sympathetic nervous system could play a role. The present study evaluated long-term consequences of rat heart failure on cardiovascular circadian patterns in vivo, and on density and function of cardiac beta-adrenoceptor subtypes in vitro, as a marker of cardiac adrenergic drive. METHODS Heart failure in rats was induced by coronary artery ligation leading to infarct sizes of >30% of left ventricular circumference. Blood pressure and heart rate were monitored for 10 weeks after infarction using radiotelemetry. Density and function of cardiac beta(1) and beta(2)-adrenoceptors were measured by radioligand binding and adenylyl cyclase stimulation. RESULTS During the activity period at night blood pressure and heart rate were lower in rats with heart failure than in sham controls, leading to reduced night-day variation in the heart failure group. Depression of circadian rhythmicity in blood pressure was found over the whole study period, while that in heart rate occurred with a lag-time of several weeks. In failing left ventricles beta-adrenoceptors showed reduced high affinity agonist binding, a shift in the beta(1):beta(2) ratio towards the beta(2)-subtype, and decreased beta(1)-adrenergic stimulation of adenylyl cyclase. In right ventricles no differences were found between failing and control rats. The blunted nocturnal increase in blood pressure and heart rate as well as beta(1)-adrenergic desensitization were correlated with the severity of left ventricular dysfunction. CONCLUSIONS Heart failure in rats leads to disturbed circadian patterns in blood pressure and heart rate, and to desensitization of cardiac beta(1)-adrenoceptors, indicating chronic sympathetic overactivity.

Circannual Rhythms and Interactions of Vitamin D Metabolites, Parathyroid Hormone, and Biochemical Markers of Skeletal Homeostasis: A Prospective Study

Recent studies suggest a circannual pattern of bone turnover. To further investigate the underlying mechanisms, 41 healthy subjects (25‐80 years old) living in a southwestern German city were studied prospectively over a period of 18 months. Participants were examined every 4 weeks, and blood and urine samples were obtained on each visit. The following parameters were measured: serum 25‐hydroxyvitamin D3 [25(OH)D3], 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], and parathyroid hormone (PTH), as regulators, and serum total alkaline phosphatase (TAP), bone‐specific alkaline phosphatase (BAP), urinary total pyridinoline (PYD), deoxypyridinoline (DPD), and the aminoterminal telopeptide of collagen type I (NTX), as biochemical markers of bone turnover. The presence of significant circannual rhythms for the various markers was tested using the Pharmfit method. In the total group, 25(OH)D3, 1,25(OH)2D3, and PTH as well as BAP, PYD, DPD, and NTX showed a significant seasonal variation. 25(OH)D3 revealed the highest amplitude (38.0%) with an acrophase in August. Levels of the biochemical markers and of PTH were highest in winter with amplitudes of up to 17.7% (DPD). Results were most pronounced in premenopausal women, in subjects <50 years of age, and in subjects who did show a significant individual rhythm in 25(OH)D3 levels. No differences were found regarding other anthropometric or life style factors. Correlation analyses revealed strongest associations between the amplitudes of a vitamin D metabolite and a biochemical marker in premenopausal women. We conclude that specific markers of bone turnover show significant circannual rhythms. These changes are related directly to variations in the hormonal regulation of skeletal homeostasis. In postmenopausal women and in men, other effects may superimpose the circannual variation of biomarkers of bone turnover.

Twenty-four-hour ambulatory blood pressure profiles in pediatric patients after renal transplantation

Abstract. Ambulatory blood pressure monitoring was applied in 27 pediatric patients aged 6.3 – 24.3 (median 15.0) years who had been transplanted 1.5 – 8.4 years previously. Daytime values were compared with the mean of 10 concomitant casual blood pressure recordings. At the time of the study, antihypertensive drugs were given to 17 patients. Inulin clearance ranged from 18 to 116 (median 66) ml/min per 1.73 m2. Ambulatory blood pressure monitoring confirmed hypertension or normotension determined by casual blood pressure measurements in 63% of patients. The physiological nocturnal dip in blood pressure was attenuated or reversed in 8 of 27 patients. It was reduced in all 3 patients with renal artery stenosis of the graft, in 3 of 4 patients with chronic rejection, in the only patient with recurrent focal segmental glomerulosclerosis, and in 1 of 6 patients with past acute rejection. The dipping was not related to inulin clearance. In conclusion, casual blood pressure measurements do not accurately reflect blood pressure in pediatric patients transplanted more than 1.5 years previously. A reduced nocturnal dip in blood pressure may indicate an underlying renovascular or renoparenchymal pathology. Ambulatory blood pressure monitoring should regularly be applied in patients with renal transplants.

Effects of SCN-lesions on circadian blood pressure rhythm in normotensive and transgenic hypertensive rats

Transgenic hypertensive TGR(mREN2)27 (TGR) rats, carrying an additional mouse renin gene, have been found to show inverse circadian blood pressure profiles compared to normotensive Sprague-Dawley rats. In order to evaluate the contributions of the suprachiasmatic nucleus (SCN) and the neurohormone melatonin to cardiovascular circadian regulation in TGR(mREN2)27 rats and Sprague-Dawley (SPRD) controls, we investigated the effects of melatonin agonist and antagonist treatment in SCN-lesioned and nonlesioned rats, which were kept under conditions of alternating light and darkness (LD). After destruction of the SCN, circadian rhythmicity in blood pressure, heart rate (HR), and motor activity (MA) was almost abolished in rats of both strains. One week of treatment with a synthetic melatonin agonist S-21634 was not able to restore circadian variation in the parameters monitored. In nonlesioned TGR(mREN2)27 rats and Sprague-Dawley control rats, the melatonin antagonist S-22365 had no suppressive effect on LD-synchronized circadian rhythmicity, indicating that LD itself may have a stronger influence on the SCN than endogenous melatonin.

CIRCADIAN RHYTHMS IN THE RENIN-ANGIOTENSIN SYSTEM AND ADRENAL STEROIDS MAY CONTRIBUTE TO THE INVERSE BLOOD PRESSURE RHYTHM IN HYPERTENSIVE TGR(mREN-2)27 RATS

The transgenic TGR(mREN-2)27 rat is not only characterized by fulminant hypertension, but also by a disturbance in circadian blood pressure regulation, resulting in inverse circadian blood pressure profiles. The reasons for these alterations are not very well understood at present. We therefore investigated the circadian rhythms in several hormones participating in blood pressure regulation. From TGR and Sprague-Dawley (SPRD) control rats synchronized to 12h light and 12h dark (LD 12:12) blood was collected at different circadian times (07, 11, 15, 19, 23, 03, and 07 again, 5 rats per strain and time). The activities of plasma renin and converting enzyme, as well as plasma concentrations of corticosterone and aldosterone, were determined by radioimmunoassay (RIA). SPRD rats showed significant circadian rhythms in all variables except plasma renin activity, with maxima occurring during the day. TGR rats showed significant circadian rhythmicity in plasma renin activity and corticosterone and daily variation in aldosterone; angiotensin-converting enzyme (ACE) activity did not reach statistical significance. In TGR rats, 24h means in plasma renin activity and aldosterone were approximately sevenfold and fourfold higher, respectively, than in SPRD rats. Peak concentrations in corticosterone around 15h were more than two times higher in TGR rats than in SPRD rats, whereas no differences were observed during the night. It is concluded that, in TGR rats, the overall increase in plasma renin activity and aldosterone may contribute to the elevated blood pressure. The comparatively high levels in corticosterone and plasma renin activity during daytime may be involved in the inverse circadian blood pressure profiles in the transgenic animals. (Chronobiology International, 17(5), 645–658, 2000)

Effects of the angiotensin II receptor antagonist losartan on 24-hour blood pressure profiles of primary and secondary hypertensive rats

Primary and secondary hypertension differ with regard to circadian blood pressure (BP) profiles. To evaluate the contribution of the renin-angiotensin system (RAS) to circadian BP regulation, we studied cardiovascular effects of the angiotensin II (AII) receptor antagonist losartan and the angiotensin-converting enzyme (ACE) inhibitor enalapril in animal models of primary and secondary hypertension after morning and evening dosing. Systolic/diastolic BP (SBP/DBP) and heart rate (HR) were measured telemetrically in spontaneously hypertensive rats (SHR) and transgenic hypertensive rats (TGR[mRen-2]27). Losartan (0.3 to 30 mg/kg) or enalapril maleate (10 mg/kg) were injected intraperitoneally (i.p.) either at 0700 or 1900 h. Baseline SBP/DBP and HR showed significant circadian rhythmicity in both strains. The 24-h means in SBP/DBP were 190/127 mm Hg in SHR and 200/139 mm Hg in TGR. TGR showed a reversed circadian profile in BP, with peaks occurring during the daily resting period, whereas HR peaked at night. Losartan reduced BP dose dependently; reductions in TGR were significantly greater and obtained at 30-fold lower doses than in SHR. Maximum decreases induced by losartan were similar to those induced with enalapril 10 mg/kg. Both drugs reduced BP in TGR more effectively when applied at 0700 than at 1900 h, resulting in a normalized circadian BP profile. Our results demonstrate that the RAS is involved in both the pathomechanism of hypertension and in the inverse circadian BP pressure pattern in TGR.

Development of Inverse Circadian Blood Pressure Pattern in Transgenic Hypertensive Tgr(mREN2)27 Rats

TGR(mREN2)27 (TGR) rats are transgenic animals with an additional mouse renin gene, which leads to overactivity of the renin-angiotensin system. Adult TGR rats are characterized by fulminant hypertension, hypertensive end-organ damage, and an inverse circadian blood pressure pattern. To study the ontogenetic development of cardiovascular circadian rhythms, telemetric blood pressure transmitters were implanted in male Sprague-Dawley (SPRD, n = 5) and heterozygous, transgenic TGR rats before 5 weeks of age. The TGR received either drinking water or enalapril 10 mg/L in drinking water (n = 5 per group). Drug intake was measured throughout the study by computerized monitoring of drinking volume. Circadian patterns in blood pressure and heart rate were analyzed from 5 to 11 weeks of age. In the first week after transmitter implantation, blood pressure did not differ among SPRD, untreated, and enalapril-treated TGR rats. In parallel with the rise in blood pressure of untreated TGR rats, a continuous delay of the circadian acrophase (time of fitted blood pressure maximum) was observed, leading to a complete reversal of the rhythm in blood pressure at an age of 8 weeks. Enalapril reduced blood pressure at night, but was less effective during the day, presumably due to the drinking pattern of the animals, which ingested about 90% of their daily water intake during the nocturnal activity period. After discontinuation of treatment, blood pressure returned almost immediately to values found in untreated TGR rats. In conclusion, the inverse circadian blood pressure profile in TGR rats develops in parallel with the increase in blood pressure. Direct effects of the brain renin-angiotensin system may be involved in the disturbed circadian rhythmicity in TGR(mREN2)27 rats.

Transgenic TGR(mREN2)27 Rats as a Model for Disturbed Circadian Organization at the Level of the Brain, the Heart, and the Kidneys

In transgenic hypertensive TGR(mREN2)27 rats (TGR) harboring the murine Ren‐2 gene an inverse 24h blood pressure (BP) profile was described in relation to a normal pattern in heart rate (HR) and motility (MA), normotensive Sprague‐Dawley rats (SDR) were used as controls. Transgenic rats as an animal model of human secondary hypertension (non‐dipper) was studied in detail at different levels: (1) Radiotelemetry was applied to document gross circadian rhythms/rhythm disturbances in cardiovascular functions, MA and body temperature under normal LD conditions, under DD and after a light pulse. (2) Signal transduction of the overexpressed renin‐angiotensin in TGR was studied by determation of AT1‐receptors in kidney glomeruli together with kidney functions. (3) Expression of key processes involved in increased sympathetic regulation in TGR, mRNAs, the tyrosine‐hydroxylase (TH) and norepinephrine (NE) reuptake1‐carrier were determined. (4) In the SCN mRNA of c‐fos and c‐jun were determined under LD and after light pulse. (5) In primary cultures of pinealocytes the effects of adrenergic agonists and antagonists were evaluated on second messenger (cAMP, cGMP) accumulation and melatonin release. The results of these studies clearly demonstrate that the additional mouse renin genin in TGR greatly affected not only the renin‐angiotensin‐system and led—as expected—to an increased BP in this rat but also disturbed circadian rhythms from the BP pattern down to the level of hormones, processes of signal transduction, and expression of transcription factors and clock genes. In conclusion, the expression of a single additional gene is able to disturb the circadian system of an animal in a highly complex way. These findings are of importance for chronobiologic as well as pharmacologic research.