Telomere length and cardiovascular risk in hypertensive patients with left ventricular hypertrophy: the LIFE study.

Short telomeres are associated with aging and age-related diseases. Our aim was to determine whether short leukocyte telomere length is associated with risk factors and cardiovascular diseases in a high-risk hypertensive population. We measured leukocyte telomere lengths at recruitment in 1271 subjects with hypertension and left ventricular hypertrophy (LVH) participating in the Lifestyle Interventions and Independence for Elders (LIFE) study. At baseline, short mean telomere length was associated with coronary artery disease in males (odds ratio (OR) 0.61, 95% confidence interval (CI) 0.39-0.95), and transient ischemic attack in females (OR 0.62 95% CI 0.39-0.99). Proportion of short telomeres (shorter than 5 kb) was associated with Framingham risk score (r=0.07, P<0.05), cerebrovascular disease (OR 1.18, 95% CI 1.01-1.15) and type 2 diabetes in men (OR 1.07, 95% CI 1.02-1.11). During follow-up, proportion of short telomeres was associated with combined cardiovascular mortality, stroke or angina pectoris (hazard ratio 1.04, 95% CI 1.01-1.07). Telomere length was not associated with smoking, body mass index, pulse pressure or self-reported use of alcohol. Our data suggest that reduced leukocyte telomere length is associated with cardiovascular risk factors and diseases as well as type 2 diabetes, and is a predictor of cardiovascular disease in elderly patients with hypertension and LVH.

Telomere length and progression of diabetic nephropathy in patients with type 1 diabetes.

OBJECTIVES: To determine whether short telomere length of blood leucocytes from patients with type 1 diabetes is associated with or predictive of progression of diabetic nephropathy. DESIGN AND METHODS: Two consecutive DNA samples were obtained from 132 patients from the nationwide Finnish Diabetic Nephropathy Study with type 1 diabetes. Control DNA samples were taken from 44 healthy blood donors. Telomere length was measured by Southern blot. Patients were divided into three groups according to their urinary albumin excretion rate (AER): 48 patients with normoalbuminuria (AER < 20 microg min(-1)); seven patients with microalbuminuria (AER > or = 20 microg min(-1) <200 microg min(-1)) and 77 patients with macroalbuminuria (AER > or = 200 microg min(-1)). Progression was defined as a change in albuminuria to a higher level. RESULTS: Progression occurred in 21 patients. Progressors had shorter mean telomere length (8.1 +/- 0.7 kb, mean +/- SD; P = 0.017) and higher percentage of short telomeres (32.0 +/- 8%, P = 0.002) than nonprogressors (8.5 +/- 0.7 kb and 27 +/- 7.2%, respectively). Thus, both shorter telomeres (HR = 0.190, 95%CI 0.065-0.558, P = 0.0025) and higher proportion of short telomeres (HR = 1.115, 1.039-1.195, P =0.0023) were independent predictors of diabetic nephropathy. Telomere length was not associated with the degree of albuminuria and was not different in patients with type 1 diabetes compared with healthy controls. CONCLUSIONS: Short telomeres are independent predictors of progression of diabetic nephropathy in patients with type 1 diabetes.

Renin-angiotensin system revisited.

New components and functions of the renin-angiotensin system (RAS) are still being unravelled. The classical RAS as it looked in the middle 1970s consisted of circulating renin, acting on angiotensinogen to produce angiotensin I, which in turn was converted into angiotensin II (Ang II) by angiotensin-converting enzyme (ACE). Ang II, still considered the main effector of RAS was believed to act only as a circulating hormone via angiotensin receptors, AT1 and AT2. Since then, an expanded view of RAS has gradually emerged. Local tissue RAS systems have been identified in most organs. Recently, evidence for an intracellular RAS has been reported. The new expanded view of RAS therefore covers both endocrine, paracrine and intracrine functions. Other peptides of RAS have been shown to have biological actions; angiotensin 2-8 heptapeptide (Ang III) has actions similar to those of Ang II. Further, the angiotensin 3-8 hexapeptide (Ang IV) exerts its actions via insulin-regulated amino peptidase receptors. Finally, angiotensin 1-7 (Ang 1-7) acts via mas receptors. The discovery of another ACE2 was an important complement to this picture. The recent discovery of renin receptors has made our view of RAS unexpectedly complex and multilayered. The importance of RAS in cardiovascular disease has been demonstrated by the clinical benefits of ACE inhibitors and AT1 receptor blockers. Great expectations are now generated by the introduction of renin inhibitors. Indeed, RAS regulates much more and diverse physiological functions than previously believed.

Downregulation of angiotensin-converting enzyme by tumor necrosis factor-alpha and interleukin-1beta in cultured human endothelial cells.

Angiotensin-converting enzyme (ACE) and cytokines are considered to play an important role in the pathophysiology of cardiovascular diseases such as atherosclerosis. In the present study, the effects of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on ACE in cultured human umbilical vein endothelial cells (HUVECs) was studied. TNF-alpha (0.1-10 ng/ml) and IL-1beta (0.1-10 ng/ml) caused a dose- and time-dependent decrease in the amount of ACE in intact endothelial cell membranes and decreased levels of ACE mRNA. TNF-alpha and IL-1beta activated p44/42 and p38 mitogen-activated protein kinases (MAPKs) in HUVECs; this was inhibited by the specific inhibitors of these kinases, PD98059 and SB202190, respectively. Pretreatment of endothelial cells with the specific p38 MAPK inhibitor SB202190 (5 microM) or hydrocortisone (5 microM) partly reversed the suppression of ACE by TNF-alpha or IL-1beta, whereas the specific p44/42 MAPK inhibitor PD98059 (40 microM) was without effect. Vascular endothelial growth factor (1 ng/ml) caused an increase in membrane-bound ACE and ACE mRNA levels which was inhibited by pretreatment of the cells with TNF-alpha (1 ng/ml) or IL-1beta (1 ng/ml). In summary, the cytokines TNF-alpha and IL-1beta downregulated ACE in cultured human endothelial cells, which effect was probably mediated by the p38 MAPK pathway. Downregulation of ACE by TNF-alpha and IL-1beta locally in the vascular wall may be a counterbalancing mechanism in inflammatory processes such as atherosclerosis, leading to decreased production of angiotensin II and accumulation of bradykinin.

Upregulation of angiotensin-converting enzyme by vascular endothelial growth factor.

The role of vascular endothelial growth factor (VEGF), a potent endothelium-specific angiogenic factor, in the regulation of angiotensin-converting enzyme (ACE) in cultured human umbilical vein endothelial cells (HUVECs) was studied. VEGF (0.07-1.2 x 10(-6) mmol/l) caused a dose-dependent increase in ACE measured in intact endothelial cells and increased the expression of ACE mRNA. The stimulatory effect of VEGF was inhibited by pretreatment of endothelial cells with the tyrosine kinase inhibitor herbimycin (4.35 x 10(-5) mmol/l). The stimulatory effect of VEGF was potentiated by the selective cGMP phosphodiesterase inhibitor zaprinast (0.1 mmol/l). The nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 5.4 mmol/l) suppressed the stimulatory effect of VEGF. The nonselective cyclooxygenase (COX) inhibitor indomethacin (5 microM) and the selective COX-2 inhibitor NS-398 (5 microM) potentiated the stimulatory effect of VEGF, whereas the selective COX-1 inhibitor resveratrol (5 microM) was without effect. ACE induction by VEGF was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (2.5 x 10(-3) mmol/l) and by downregulating PKC with phorbol 12-myristate 13-acetate. In summary, VEGF induced ACE in cultured HUVECs. Intracellular events such as tyrosine kinase activation, PKC activation, and increase of cGMP were probably involved in ACE induction by VEGF. Nitric oxide may partially contribute to ACE induction by VEGF. The powerful capacity of VEGF to increase ACE in endothelial cells shown here suggests a synergistic relation between VEGF and the renin-angiotensin system in vascular biology and pathophysiology.

Induction of angiotensin-converting enzyme by oncostatin m in human endothelial cells.

OBJECTIVE: To examine the role of oncostatin M (OSM) in the regulation of angiotensin converting enzyme (ACE) in endothelial cells. METHODS: Cultured endothelial cells were incubated with OSM (25-200 pM) for 24 h. Incubations were performed without or with the tyrosine kinase inhibitor, herbimycin (87 nM), or the selective MAP kinase kinase inhibitor, PD98059 (50 microM). ACE amount in intact endothelial cells was measured by an inhibitor binding assay and ACE mRNA levels by RNase protection assay. RESULTS: OSM caused a dose dependent increase in ACE amount and increased the expression of ACE mRNA. The stimulatory effect of OSM was inhibited by pretreatments with herbimycin or PD98059. CONCLUSIONS: OSM induced ACE in cultured HUVECs. Tyrosine kinase and MAPK activation were probably involved in ACE induction. Local induction of ACE by OSM in the vascular wall may be a consequence of inflammatory processes leading to locally increased production of angiotensin II and breakdown of bradykinin.

Endothelin in migraine patients.

Endothelins (ETs) are the most potent vasoconstrictors known, and may be the mediators of the vasoconstrictive phase in migraine attacks. We studied 31 previously selected migraine patients with (9) and without (22) aura ictally and interictally to determine their plasma ET-1 values. The mean interictal and ictal values were 5.3 pg/ml (SD 1.8) and 6.4 pg/ml (SD 3.9), respectively. The ictal values were markedly elevated at the beginning of the migraine attack (<2 h) and declined to interictal or even lower level later (4 to 6 h) in the course of an attack. The local vasoconstriction at the beginning of a migraine attack might be ET-mediated secondarily to serotonin activation.

Oncostatin M regulates endothelin-1 production in human endothelial cells.

The effect of the macrophage- and T-lymphocyte-derived cytokine oncostatin M (OSM) on endothelin-1 (ET-1) production in cultured human umbilical cord vein endothelial cells (HUVEC) was studied. OSM (2.5-10 ng/ml) stimulated ET-1 production and the expression of preproendothelin-1 mRNA. The stimulatory effect of OSM was reversed by anti-interleukin (IL)-6 IgG (33 microg/ml). IL-6 (10 ng/ml) was shown to stimulate ET-1 production. The tyrosine kinase inhibitors herbimycin (250-500 ng/ml) and genistein (1-4 microg/ml) suppressed basal ET-1 production and reversed the stimulatory effect of OSM, whereas daidzein (1-8 microg/ml), a less active analog of genistein, had no effect on basal ET-1 production and only partly reversed the stimulatory effect of OSM. The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibited ET-1 production. Downregulation of protein kinase C (PKC) with PMA (1 microM) preincubation potentiated OSM-induced ET-1 production. In summary, OSM stimulated ET-1 production in cultured HUVEC. The stimulation was probably mediated by IL-6. Furthermore, the present data suggest that tyrosine kinase activation was involved in ET-1 stimulation and that PKC activation leads to suppression of basal and OSM-stimulated ET-1 production.

Upregulation of angiotensin converting enzyme by atrial natriuretic peptide and cyclic GMP in human endothelial cells.

OBJECTIVE: To examine the role of atrial natriuretic peptide (ANP) and cyclic GMP in the regulation of angiotensin converting enzyme (ACE) in cultured human endothelial cells. METHODS: Cultured endothelial cells from human umbilical veins (HUVEC) were treated with ANP (0.3-30 nM), 8-Br-cGMP (1-100 microM), Rp-8-Br-PET-cGMPS (1 microM), or the phosphodiesterase inhibitors, zaprinast (10-100 microM), dipyridamole (1-10 microM), or isobutyl methyl xanthine (IBMX, 0.1-0.5 mM). ACE amounts were measured by inhibitor binding assay and cellular cGMP levels by radioimmunoassay. RESULTS: ANP caused a dose dependent increase in ACE measured in intact endothelial cell culture. The stimulatory effect of ANP was blocked by Rp-8-Br-PET-cGMPS, a protein kinase G inhibitor. The cyclic GMP analog, 8-Br-cGMP and the cyclic GMP specific phosphodiesterase inhibitor, zaprinast, both increased ACE. Increase of ACE was also caused by nonspecific phosphodiesterase inhibitors, dipyridamole and IBMX. Intracellular cGMP levels were shown to increase by ANP, and phosphodiesterase inhibitors. CONCLUSIONS: These data suggest that cGMP is an intracellular mediator regulating ACE and that ANP induced increase of ACE is mediated via a cGMP dependent mechanism.

Carvedilol and its metabolites suppress endothelin-1 production in human endothelial cell culture.

Carvedilol (0.25-25 microM), an antihypertensive drug is shown here to reduce endothelin-1 (ET-1) production in cultured human umbilical cord endothelial cells. Two of its metabolites, M14 and M21 (2.5-25 microM) also suppressed ET-1 production, less potently, however, than carvedilol. Carvedilol is a multiple-acting compound with non-selective beta-adrenoceptor and selective alpha 1-adrenoceptor blocking activity, calcium channel blocking and anti-oxidant activity. To study whether these activities were related to suppressed ET-1 production, endothelial cells were treated with a beta 1-blocker, metoprolol (1-10 microM), a non-selective beta-blocker, propanolol (1-10 microM), an alpha 1-blocker, prazosin (1-10 microM), a calcium channel antagonist, nicardipine (1-10 microM), or with the antioxidative compounds probucol (1-100 microM) and ascorbic acid (1-100 microM). None of these compounds modified ET-1 production. The inhibitory effects of carvedilol, M14 or M21 on ET-1 production were not reversed by N Nitro-L-arginine methyl ester (L-NAME) (1.9 mM), or by indomethacin (1.5 microM), suggesting that mechanisms other than the stimulation of nitric oxide or prostacyclin production were involved.

Purinergic agonists stimulate the secretion of endothelin-1 in rat thyroid FRTL-5 cells.

The aim of the present study was to investigate the mechanisms regulating endothelin-1 (ET-1) secretion in rat thyroid FRTL-5 cells. ET-1 was found to be secreted after stimulation with adenosine and ATP. The release of ET-1 was sensitive to pertussis toxin, indicating a role of G-proteins in the stimulus-secretion coupling. The stimulation evoked by ATP or adenosine was inhibited by the P1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and in the presence of adenosine deaminase the adenosine- and ATP-mediated ET-1 secretion was abolished. These evidences suggest a role of a P1-adenosine receptor in the secretion of ET-1. Increasing cyclic AMP with forskolin decreased the adenosine-mediated secretion. In addition, the intracellular calcium chelator BAPTA or inhibition of calcium entry with Ni2+ prevented the response. Protein kinase C (PKC) is also partly involved in ET-1 secretion in FRTL-5 cells. Activation of PKC with the phorbol ester phorbol 12-myristate 13-acetate (PMA) stimulated the secretion of ET-1 in a time- and dose-dependent manner. Furthermore, downregulation of PKC decreased the secretion of ET-1 stimulated by adenosine. In conclusion, ET-1 secretion in FRTL-5 cells is stimulated via a pertussis toxin-sensitive P1-receptor pathway which is modulated by several signal transduction mechanisms including cAMP, Ca2+, and PKC.

Tissue-specific regulation of angiotensin-converting enzyme by angiotensin II and losartan in the rat.

Angiotensin II (Ang II) may regulate the release of components of the renin-angiotensin system in a tissue-specific manner. In order to study: (1) the effect of Ang II on gene expression and tissue levels of angiotensin-converting enzyme (ACE), and (2) the mechanism of the possible Ang II effect, we treated normal rats with Ang II and Losartan, an angiotensin AT1-receptor antagonist. Forty normal rats received Ang II (n = 20) at a rate of 200 ng kg-1 min-1 or 0.9% NaCl (n = 20) subcutaneously for 3 days using osmotic Alzet minipumps. Ten rats in both groups received Losartan (15 mg kg-1 day-1) in their drinking water, while the rest received tap water. ACE activity and mRNA levels were measured from pulmonary, cardiac, and renal tissue. Ang II treatment resulted in significant increases in blood pressure and heart weight as well as an increase in plasma Ang II concentration and a decrease in plasma renin activity. Simultaneous treatment with Losartan reduced the Ang II-induced effects on blood pressure and heart weight, and attenuated the Ang II-induced decrease in plasma renin activity. Pulmonary ACE activity and mRNA levels decreased during Ang II treatment, and these effects were not modified by simultaneous treatment with Losartan. Cardiac and kidney ACE activities and mRNA levels did not change significantly during Ang II treatment, but Losartan increased cardiac ACE activity (and decreased pulmonary ACE activity). The data indicate that Ang II regulates gene expression and activity of ACE in a tissue-specific manner in the rat, an effect probably involving angiotensin receptor subtype(s) different from the AT1-receptor.

Perioperative plasma endothelin-1 concentrations and vasoconstriction during prolonged plastic surgical procedures.

The role of endothelin-1, a potent vasoconstrictor released by vascular endothelium, in the vasoconstriction that develops after prolonged operations is not clear. This study was performed in order to determine if there was any relationship between endothelin-1 and the degree of vasoconstriction during prolonged plastic surgery. Plasma concentrations of endothelin-1, skin-forearm temperature gradient (Tgrad), rectal temperature, mean arterial pressure (MAP) and heart rate (HR) were measured at nine predetermined times before, during and after operation in nine women undergoing breast reconstruction with a pedicled transverse rectus abdominis musculocutaneous flap. Development of cutaneous or fat necrosis of the flap was assessed clinically and with ultrasound. Concentrations of endothelin-1 before induction were increased (median 8.9 (25-75% quartiles 5.5-12.5) pg ml-1). During operation they were approximately 3 pg ml-1 and after operation approximately 5 pg ml-1. Tgrad was approximately 4 degrees C before induction and after operation, indicating marked vasoconstriction; during operation it was about zero, indicating vasodilatation. There was a statistically significant correlation between endothelin-1 concentrations and Tgrad (Spearman non-linear correlation) (r = 0.32, P = 0.004) and between endothelin-1 and MAP (r = 0.25, P = 0.02), but not between endothelin-1 and HR or development of minor cutaneous or fat necrosis of the flap (five patients). We conclude that increased plasma concentration of endothelin-1 is associated with the extent of peripheral vasoconstriction.

Degradation of endothelin-1 by extracts of rat lung, kidney, and liver.

Degradation of 125I-labeled endothelin-1 (125I-ET-1) when incubated 120 min at 37 degrees C with rat lung, kidney and liver plasma membrane extracts was examined using HPLC. Lung and kidney extracts showed degrading enzyme activity, but none was found in liver extract. EDTA almost abolished degradation of 125I-ET-1 in lung and kidney extracts. Phosphoramidon and SCH 39370, both inhibitors of neutral endopeptidase 24.11 (NEP), markedly inhibited degradation of 125I-ET-I in lung extract and clearly less in kidney extract. Soybean trypsin inhibitor (STI) and elastase inhibitor partly inhibited degradation in lungs and in kidney extract. Leupeptin had no inhibitory effect neither in lung nor in kidney extract. Our results suggest: (1) at least two types of enzymes degrade ET-1 in lung and kidney extracts, namely metallo-proteinases and serine proteinases. (2) The ET-1 degrading effect appears to be different in lungs and kidneys, metallo-proteinases being more important in pulmonary than in renal degradation of ET-1.

Serum concentrations of erythropoietin in healthy and anaemic pregnant women.

We have found serum erythropoietin (EPO) concentration significantly (p < 0.01) increased during normal pregnancy. Erythropoietin concentration was significantly (p < 0.05) higher before the 24th gestational week than after it. In pregnant women with iron deficiency anaemia serum EPO concentration was significantly (p < 0.001) higher than in healthy pregnant women. In anaemic women significant (p < 0.001) linear correlation between haemoglobin (hgb) and log serum EPO concentrations was observed. In pregnant women including both healthy and anaemic women log serum EPO concentrations correlated inversely with hgb concentrations (p < 0.01). We conclude that erythropoietin secretion is raised in normal pregnancy and is at highest in the first and second trimesters, when hgb mass begins to grow. In pregnancy with concomitant anaemia a more extensive demand for erythropoietin secretion is obvious.

Insulin increases the release of endothelin in endothelial cell cultures in vitro but not in vivo.

Endothelin-1 (ET-1), a potent vasoconstrictor and mitogenic peptide for vascular smooth muscle cells, may be a marker for development of vascular disorders in diabetic patients. The aim of this study was to elucidate the possible role of insulin in the regulation of ET-1 production. The effect of hyperinsulinemia (with and without concomitant hyperglycemia) on the release of ET-1 was studied in 23 healthy men in vivo, as well as in human umbilical cord vein endothelial cell (HUVEC) cultures in vitro. Plasma glucose and insulin were maintained at four desired levels (from 5 to 22 mmol/L and 0.065 to 12.9 nmol/L, respectively) during the in vivo studies. The mean (SEM) plasma ET-1 during normoglycemia and a fasting insulin concentration in healthy men was 3.8 (0.4) pg/mL, and ET-1 levels did not change in response to changes in the concentration of glucose (from 5.0 to 22 mmol/L) or insulin (from 0.065 to 12.9 nmol/L). The ET-1 concentration in HUVEC culture medium increased linearly during 24 hours, and insulin further enhanced the release of ET-1 dose-dependently. ET-1 release was stimulated by angiotensin II, thrombin, and transforming growth factor-beta (TGF-beta), whereas treatment with glucose and insulin-like growth factor-1 (IGF-1) was not associated with changed ET-1 levels in culture medium. Our results show that although high insulin concentrations stimulate ET-1 release in vitro, hyperinsulinemia is not associated with increased plasma ET-1 levels in healthy men in vivo. The role of insulin in the regulation of ET-1 production in vivo, if any, remains unsettled.

Endothelin antiserum decreases volume-stimulated and basal plasma concentration of atrial natriuretic peptide.

BACKGROUND: Endothelin-1 (ET-1) is the most powerful factor known to release atrial natriuretic peptide (ANP) in vivo and in cultured cardiac myocytes or preparations of atrium. We tested the role of endogenous ET-1 in the regulation of ANP release by passive immunization in anesthetized rats. METHODS AND RESULTS: Intravenous injection of antiserum against ET-1 was shown to decrease basal and volume-stimulated plasma concentrations of ANP, whereas control serum was without effect. Antiserum generated in rabbits cross-reacted 100% with endothelin-2 and -3. In pentobarbital-anesthetized Wistar rats treated with ET-1 antiserum, plasma ANP concentration measured by radioimmunoassay was reduced by 37% from starting level after 10 minutes and by 30% after 60 minutes. Control rat serum had no effect on plasma ANP. Rapid intravenous infusion of 8 mL of 0.9% NaCl caused a sixfold increase of plasma ANP concentration in control rats but only twofold in rats pretreated with ET-1 antiserum (P < .01). This effect of ET-1 antiserum was dose dependent. ET-1 antiserum changed neither blood pressure nor heart rate significantly in anesthetized rats. Pretreatment with ET-1 antiserum did not affect the initial hypotensive response to intravenous ET-1 0.5 nmol/kg but significantly attenuated the subsequent hypertensive response to endothelin. CONCLUSIONS: Endothelin may be a physiological modulator of both basal and stimulated ANP release.

Raised plasma endothelin-1 concentration in patients with nephropathia epidemica.

Nephropathia epidemica (NE) with renal syndrome, caused by the Puumala-virus, is manifested clinically by the triad of fever, hemorrhage and renal failure. We observed raised plasma concentrations of endothelin-1 (ET-1) and atrial natriuretic peptide (ANP) in 23 patients during the acute phase of NE. They all developed transient renal failure and all displayed characteristics of NE, also verified by a rapid IgG antibody test. Blood pressure was normal or low in all subjects during the acute phase of the disease. Plasma ET-1 and ANP levels returned to normal following recovery one month later. The cause of increased ET-1 synthesis in NE remains unknown. It may be related to vascular damage or extravasation of blood. ET-1 may participate in the pathogenesis of acute renal failure of NE. Raised plasma ANP levels were most likely caused by fluid retention during the acute phase of NE. However, high levels of circulating ET-1 might have contributed to increased release of ANP.

Raised plasma endothelin during acute migraine attack.

Endothelins are the most potent vasoconstrictor peptides known. Plasma endothelin (ET-1) concentrations were measured in eight migraine patients (mean age 44.5 years), two during an acute migraine attack with aura and six during an attack without aura. The mean ET-1 values were elevated in all migraine patients above the range of normal subjects, and were 10.6 (range 6.0-16.0) pg/ml in migraine patients and 3.8 (range 0.7-5.8) pg/ml in controls. We hypothesize that ET-1 may constrict cerebral vessels during the initial stage of the migraine attack.