Ger M.J. Janssen

Chronic Hypoxia Stimulates Periarterial Sympathetic Nerve Development in Chicken Embryo

BackgroundEpidemiological findings suggest an association between low-for-age birth weight and the risk to develop coronary heart diseases in adulthood. During pregnancy, an imbalance between fetal demands and supply may result in permanent alterations of neuroendocrine development in the fetus. We evaluated whether chronic prenatal hypoxia increases arterial sympathetic innervation. Methods and ResultsChicken embryos were maintained from 0.3 to 0.9 of the 21-day incubation period under normoxic (21% O2) or hypoxic conditions (15% O2). At 0.9 incubation, the degree of sympathetic innervation of the embryonic femoral artery was determined by biochemical, histological, and functional (in vitro contractile reactivity) techniques. Chronic hypoxia increased embryonic mortality (32% versus 13%), reduced body weight (21.9±0.4 versus 25.4±0.6 g), increased femoral artery norepinephrine (NE) content (78.4±9.4 versus 57.5±5.0 pg/mm vessel length), and increased the density of periarterial sympathetic nerve fibers (14.4±0.7 versus 12.5±0.6 counts/104 &mgr;m2). Arteries from hypoxic embryos were less sensitive to NE (pD2, 5.99±0.04 versus 6.21±0.10). In the presence of cocaine, however, differences in sensitivity were no longer present. In the embryonic heart, NE content (156.9±11.0 versus 108.1±14.7 pg/mg wet wt) was also increased after chronic hypoxia. ConclusionsIn the chicken embryo, chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system. In humans, an analogous mechanism may increase the risk for cardiovascular disease in adult life.

High Sodium Intake Increases Blood Pressure and Alters Renal Function in Intrauterine Growth–Retarded Rats

A suboptimal fetal environment increases the risk to develop cardiovascular disease in the adult. We reported previously that intrauterine stress in response to reduced uteroplacental blood flow in the pregnant rat limits fetal growth and compromises renal development, leading to an altered renal function in the adult offspring. Here we tested the hypothesis that high dietary sodium intake in rats with impaired renal development attributable to intrauterine stress, results in increased blood pressure, altered renal function, and organ damage. In rats, intrauterine stress was induced by bilateral ligation of the uterine arteries at day 17 of pregnancy. At the age of 12 weeks, the offspring was given high-sodium drinking water (2% sodium chloride). At the age of 16 weeks, rats were instrumented for monitoring of blood pressure and renal function. After intrauterine stress, litter size and birth weight were reduced, whereas hematocrit at birth was increased. Renal blood flow, glomerular filtration rate, and the glomerular filtration fraction were increased significantly after intrauterine stress. High sodium intake did not change renal function and blood pressure in control animals. However, during high sodium intake in intrauterine stress offspring, renal blood flow, glomerular filtration rate, and the filtration fraction were decreased, and blood pressure was increased. In addition, these animals developed severe albuminuria, an important sign of renal dysfunction. Thus, a suboptimal fetal microenvironment, which impairs renal development, results in sodium-dependent hypertension and albuminuria.

Reduced Uteroplacental Blood Flow Alters Renal Arterial Reactivity and Glomerular Properties in the Rat Offspring

Fetal malnutrition and hypoxia may modify organ system maturation and result in cardiovascular diseases in the adult. We tested whether intrauterine stress (IUS) leads to persistent alterations of renal biology. In rats, intrauterine stress was induced by ligation of the uterine arteries at day 17 of pregnancy. Renal arteries of the 21-day-old male offspring were isolated to study pharmacological reactivity. Kidneys were dissected to analyze renal structure and β-adrenoceptor expression. At 21 days of age, half of the animals underwent unilateral left nephrectomy. At the age of 12 weeks, rats were instrumented for blood pressure monitoring, blood sampling, and renal function measurements. After IUS, litter size and birth weight were reduced, whereas the hematocrit was increased. Renal arterial responses to β-adrenergic stimulation and sensitivity to adenylyl cyclase activation were increased, along with the renal expression of β2-adrenoceptors. At 21 days and at 6 months of age, the number and density of the glomeruli were reduced, whereas their size was increased. The filtration fraction and urinary albumin concentration were increased 12 weeks after intrauterine stress. In control rats, removal of the left kidney at 21 days of age did not affect kidney function and blood pressure. However, after IUS, the remaining right kidney failed to compensate for the loss of the left kidney, and blood pressure was increased. In conclusion, prenatal stress transiently modifies renal arterial reactivity and results in long-lasting adverse effects on renal structure and function and on renal compensatory mechanisms.

Impaired Autonomic Regulation of Resistance Arteries in Mice With Low Vascular Endothelial Growth Factor or Upon Vascular Endothelial Growth Factor Trap Delivery

Background— Control of peripheral resistance arteries by autonomic nerves is essential for the regulation of blood flow. The signals responsible for the maintenance of vascular neuroeffector mechanisms in the adult, however, remain largely unknown. Methods and Results— Here, we report that VEGF∂/∂ mice with low vascular endothelial growth factor (VEGF) levels suffer defects in the regulation of resistance arteries. These defects are due to dysfunction and structural remodeling of the neuroeffector junction, the equivalent of a synapse between autonomic nerve endings and vascular smooth muscle cells, and to an impaired contractile smooth muscle cell phenotype. Notably, short-term delivery of a VEGF inhibitor to healthy mice also resulted in functional and structural defects of neuroeffector junctions. Conclusions— These findings uncover a novel role for VEGF in the maintenance of arterial neuroeffector function and may help us better understand how VEGF inhibitors cause vascular regulation defects in cancer patients.

α1-Adrenoceptor subtypes in rat aorta and mesenteric small arteries are preserved during left ventricular dysfunction post-myocardial infarction

Objective: In heart failure, homologous downregulation of β-adrenoceptors contributes to impaired adrenergic responsiveness of the myocardium. We evaluated α1,-adrenoceptors (α1-AR) in a sparsely innervated and a densely innervated peripheral artery in an experimental model of left ventricular dysfunction post-myocardial infarction. Methods: [3H]Prazosin binding was determined in arterial segments of Wistar-Kyoto rats (WKY), and of Wistar rats 5 weeks after myocardial infarction (MI) or sham operation (SHAM). Results: In the thoracic aorta (TAO) of WKY, specific prazosin binding was: (i) prevented by the irreversible α 1 B -AR and relatively selective α1 D -AR antagonist, chloroethylclonidine (CEC); (ii) displaced with low affinity (p K i 6.25) by the α1 A -AR selective ligand, (+)-niguldipine; and (iii) displaced with both high (p K i 10.4) and low (p K i 7.37) affinity by the α1 D -AR antagonist, BMY 7378. In mesenteric small arteries (MSA) of WKY, prazosin binding was: (i) reduced 50% by CEC; (ii) displaced in a biphasic fashion by (+)-niguldipine (p K i 8.60 and p K i 6.22); and (iii) displaced by BMY 7378 with low affinity only (p K i 6.86). Also in TAO of SHAM, prazosin binding was prevented by CEC, but neither 30 nM (+)-niguldipine nor 1 nM BMY 7378 affected it. In MSA of SHAM, prazosin binding was virtually abolished in the presence of 30 nM (+)-niguldipine and was not reduced by 1 nM BMY 7378. In TAO and MSA of MI, compared to SHAM, the density of binding sites tended to be increased rather than decreased and neither the affinity for the ligand nor the effects of α1-AR subtype selective tools were significantly modified. Conclusions: These findings indicate that: (i) radioligand binding can be applied in intact arterial segments to quantify and characterize α1-AR; (ii) although differences seem to exist between rat strains, α1 B -AR and α1D-AR predominate in rat thoracic aorta and α1 A -AR and α1B-AR in mesenteric small arteries; and (iii) α1-AR density is not reduced in the poorly innervated aorta and the densely innervated mesenteric small arteries of rats with heart failure due to myocardial infarction.

Twenty-Four-Hour Exposure to Altered Blood Flow Modifies Endothelial Ca2+-Activated K+ Channels in Rat Mesenteric Arteries

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (KCa), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [−90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of KCa channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.

Reduced responsiveness of rat mesenteric resistance artery smooth muscle to phenylephrine and calcium following myocardial infarction

We evaluated responses of peripheral resistance arterial smooth muscle to α1‐adrenoceptor stimulation in a rat model of heart failure in relation to neurohumoral changes, wall structure, receptor density and cellular calcium handling. Plasma samples and third order mesenteric artery side‐branches were obtained from Wistar rats after induction of left ventricular infarction (MI) or sham surgery. Vessels were denuded of endothelium, sympathectomized, depleted of neuropeptides, and mounted in a myograph for recording of isometric force development in response to calcium, agonist and high potassium. Also, the morphology of these preparations was determined. Separate vessel segments were used in radioligand binding assays with [3H]‐prazosin. At 1 week after MI, circulating plasma levels of adrenaline, angiotensin II, atrial natriuretic factor (ANF) and vasopressin were significantly elevated. At 5 weeks only a significant elevation of ANF persisted. At 5 weeks after MI, the structure of the vessels and responsiveness to high potassium or Bay K 8466 (10−6 mol l−1) were not modified. Yet, at this stage, sensitivity to phenylephrine was increased (pD2: 6.24±0.04 vs 5.98±0.04 for controls) while maximal contractile responses to phenylephrine in the presence of 2.5 mmol l−1 calcium (2.26±0.28 vs 3.53±0.34 N m−1) and the sensitivity to calcium in the presence of phenylephrine (pD2: 2.81±0.22 vs 3.74±0.16) were reduced. Responses to the agonist in calcium‐free solution and the calcium sensitivity in the presence of 125 mmol l−1 potassium or of phorbol myristate acetate (PMA, 10−6 mol l−1) were not altered. At 5 weeks after MI, the density of prazosin binding sites was not reduced (4.04±1.40 vs 2.29±0.21 fmol μg−1 DNA in controls). In conclusion, myocardial infarction leads in the rat to a reduction of contractile responses of mesenteric resistance arterial smooth muscle to α1‐adrenoceptor stimulation. This seems to involve impaired agonist‐stimulated calcium influx.

Phosphodiesterase 1 regulation is a key mechanism in vascular aging.

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; β=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; β=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy.

Mechanisms Leading to Increased Vasodilator Responses to Calcitonin-Gene-Related Peptide in Mesenteric Resistance Arteries of Early Pregnant Rats

The objective of this study was to explore the mechanism responsible for the higher relaxing responses of mesenteric arteries to calcitonin-gene-related peptide (CGRP) in pregnancy. We performed myograph and ligand binding studies to determine the role of matrix metalloproteinase-2 (MMP-2) and CGRP receptor density. MMP activity was manipulated in isolated arteries by exposing them to the blocking effects of doxycycline. Vascular activity of MMP-2 was studied by gelatin zymography, and CGRP receptor density was determined by ligand binding analysis. Compared to nonpregnant rats, CGRP elicited stronger arterial relaxation in pregnant rats. The latter effect was neither accompanied by a change in relaxing responses to direct activation of adenylyl cyclase by forskolin nor by a change in the response to stimulation of G-protein-coupled adrenergic receptors by isoproterenol. Doxycycline did not affect the stronger arterial relaxation in pregnancy in spite of the observed more than threefold higher arterial MMP-2 activity. Density of binding sites for [125I]CGRP in arteries from pregnant rats (64 ± 14 fmol/mg protein) and from virgin rats (54 ± 5 fmol/mg protein) were comparable. The results of this study provide evidence for increased coupling of CGRP receptors to adenylyl cyclase in early pregnancy.

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress

BACKGROUND Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.