Modulation of the cochaperone AHA1 regulates heat-shock protein 90 and endothelial NO synthase activation by vascular endothelial growth factor.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling to endothelial NO synthase (eNOS) plays a central role in angiogenesis. In endothelial cells (ECs), heat-shock protein 90 (Hsp90) is also a regulator of eNOS activity. Our study is designed to determine whether modulation of the activator of Hsp90 ATPase 1 (AHA1) regulates the function of Hsp90 in ECs. METHODS AND RESULTS: We show that eNOS phosphorylation on Ser-1179 after VEGF stimulation is significantly reduced in ECs transfected with a small interfering RNA against AHA1. Accordingly, VEGF-stimulated NO production, endothelial permeability, cell migration, and EC invasion in Matrigel implants in mice are reduced in small interfering RNA against AHA1-treated conditions. Furthermore, the induction of eNOS association with Hsp90 after VEGF stimulation is decreased in AHA1-downregulated cells. We also demonstrate that modulation of Hsp90 activity by AHA1 regulates phosphorylation of Hsp90 on Tyr-300. Interestingly, the association of AHA1 with Hsp90 is increased after c-Src-mediated phosphorylation of Hsp90 on Tyr-300. Finally, we show that overexpression of AHA1 in ECs promotes association of eNOS and Hsp90, phosphorylation of Ser-1179 of eNOS, increases NO production, and cell migration. CONCLUSIONS: These results reveal that modulation of Hsp90 activity by AHA1 regulates VEGF signaling to eNOS and angiogenesis.

Does early mismatched nutrition predispose to hypertension and atherosclerosis, in male mice?

BACKGROUND: A link between early mismatched nutritional environment and development of components of the metabolic syndrome later in life has been shown in epidemiological and animal data. The aim of this study was to investigate whether an early mismatched nutrition produced by catch-up growth after fetal protein restriction could induce the appearance of hypertension and/or atherosclerosis in adult male mice. METHODOLOGY/PRINCIPAL FINDINGS: Wild-type C57BL6/J or LDLr-/- dams were fed a low protein (LP) or a control (C) diet during gestation. Catch-up growth was induced in LP offspring by feeding dams with a control diet and by culling the litter to 4 pups against 8 in controls. At weaning, male mice were fed either standard chow or an obesogenic diet (OB), leading to 4 experimental groups. Blood pressure (BP) and heart rate (HR) were assessed in conscious unrestrained wild-type mice by telemetry. Atherosclerosis plaque area was measured in aortic root sections of LDLr-/- mice. We found that: (1) postnatal OB diet increased significantly BP (P<0.0001) and HR (P<0.008) in 3-month old OB-C and OB-LP offspring, respectively; (2) that maternal LP diet induced a significant higher BP (P<0.009) and HR (P<0.004) and (3) an altered circadian rhythm in addition to higher plasma corticosterone concentration in 9 months-old LP offspring; (4) that, although LP offspring showed higher plasma total cholesterol than control offspring, atherosclerosis assessed in aortic roots of 6-mo old mice featured increased plaque area due to OB feeding but not due to early mismatched nutrition. CONCLUSIONS/SIGNIFICANCE: These results indicate a long-term effect of early mismatched nutrition on the appearance of hypertension independently of obesity, while no effect on atherosclerosis was noticed at this age.

Vascular endothelial growth factor receptor-2 activates ADP-ribosylation factor 1 to promote endothelial nitric-oxide synthase activation and nitric oxide release from endothelial cells.

Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates endothelial function via production and release of nitric oxide (NO), an important signaling molecule. The molecular basis leading to NO production involves phosphatidylinositiol-3 kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) activation. In this study, we have examined whether small GTP-binding proteins of the ADP-ribosylation factor (ARF) family act as molecular switches to regulate signaling cascades activated by VEGF in endothelial cells. Our results show that this growth factor can promote the rapid and transient activation of ARF1. In endothelial cells, this GTPase is present on dynamic plasma membrane ruffles. Inhibition of ARF1 expression, using RNA interference, markedly impaired VEGF-dependent eNOS phosphorylation and NO production by preventing the activation of the PI3K/Akt signaling axis. Furthermore, our data indicate that phosphorylation of Tyr(801), on VEGF receptor 2, is essential for activating Src- and ARF1-dependent signaling events leading to NO release from endothelial cells. Lastly, this mediator is known to regulate a broad variety of endothelial cell functions. Depletion of ARF1 markedly inhibits VEGF-dependent increase of vascular permeability as well as capillary tubule formation, a process important for angiogenesis. Taken together, our data indicate that ARF1 is a novel modulator of VEGF-stimulated NO release and signaling in endothelial cells.

PKD1 haploinsufficiency is associated with altered vascular reactivity and abnormal calcium signaling in the mouse aorta.

Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease (ADPKD), which leads to major cardiovascular complications. We used mice with a heterozygous deletion of Pkd1 (Pkd1+/-) and wild-type (Pkd1+/+) littermates to test whether Pkd1 haploinsufficiency is associated with a vascular phenotype in different age groups. Systolic blood pressure measured by the tail-cuff method was similar up to 20 weeks of age, but significantly higher in 30-week-old Pkd1+/- compared to Pkd1+/+. By contrast, similar telemetric recordings were obtained in unrestrained Pkd1+/- and Pkd1+/+ mice. The contractile responses evoked by KCl or phenylephrine were similar in young animals but increased in abdominal aortas of 30-week-old Pkd1+/- mice, and acetylcholine-evoked relaxation was depressed. Basal cytosolic calcium, KCl, and phenylephrine-evoked calcium signals were significantly lower in the Pkd1+/- aortas, whereas calcium release evoked by caffeine or thapsigargin was significantly larger. These changes were paralleled with a significant change in the mRNA expression of Pkd2, Trpc1, Orai1, and Serca2a in the aortas from Pkd1+/- vs. Pkd1+/+. These results are the first to indicate that haploinsufficiency in Pkd1 is associated with altered intracellular calcium homeostasis and increased vascular reactivity in the aorta with compensatory changes in transport proteins involved in the calcium signaling network.

Control of blood pressure variability in caveolin-1-deficient mice: role of nitric oxide identified in vivo through spectral analysis.

AIMS: In endothelial cells, caveolin-1 (cav-1) is known to negatively modulate the activation of endothelial nitric oxide synthase, a key regulator of blood pressure (BP). However, the impact of genetic alteration of cav-1 on vascular nitric oxide (NO) production and BP homeostasis in vivo is unknown. METHODS AND RESULTS: We used spectral analysis of systolic blood pressure (SBP) variability in mice chronically equipped with telemetry implants to identify frequency ranges (0.05-0.4 Hz; very low frequency, VLF) specifically responding to NO, independently of changes in absolute BP or systemic neurohormone levels. VLF variability was inversely correlated to aortic vasodilator-stimulated Ser(239) phosphoprotein (VASP) phosphorylation, reflecting NO bioactivity. We show that mice deficient in cav-1 have decreased VLF variability paralleled with enhanced systemic and vascular production of NO at unchanged mean SBP levels. Conversely, VLF variability was increased upon acute injection of mice, with a peptide containing the caveolin-scaffolding domain (CSD; residues 82-101) fused to an internalization sequence of antennapedia that decreased vascular and circulating NO in vivo. CONCLUSION: These data highlight the functional importance of cav-1 for the production of bioactive NO in conduit arteries and its control of central BP variability. Given the impact of the latter on target organ damage, this raises the interest for genetic, pharmacological, or molecular interventions that modulate cav-1 expression in diseases with NO-dependent endothelial dysfunction.

Rosuvastatin increases vascular endothelial PPARgamma expression and corrects blood pressure variability in obese dyslipidaemic mice.

AIMS: Statins improve atherosclerotic diseases through cholesterol-reducing effects. Whether the latter exclusively mediate similar benefits, e.g. on hypertension, in the metabolic syndrome is unclear. We examined the effects of rosuvastatin on the components of this syndrome, as reproduced in mice doubly deficient in LDL receptors and leptin (DKO). METHODS AND RESULTS: DKO received rosuvastatin (10 mg/kg/day or 20 mg/kg/day) or saline for 12 weeks. Saline-treated DKO mice had elevated blood pressure (BP) and nitric oxide-sensitive BP variability recorded by telemetry. Compared with saline, rosuvastatin (20 mg/kg/day) had no effect on weight gain and a minor effect on plasma cholesterol. Despite incomplete correction of insulin sensitivity, rosuvastatin fully corrected BP and its variability (P = 0.01), in conjunction with upregulation of PPARgamma (but not PPARalpha) in the aortic arch. Rosuvastatin similarly increased PPARgamma (P = 0.002) and SOD1 (P = 0.01) expression in isolated endothelial cells. Both GW9662, a PPARgamma-specific antagonist, and siRNA raised against PPARgamma abrogated rosuvastatin's effect, which was reproduced in PPARgamma- (but not PPARalpha-) dependent transactivation assays. CONCLUSION: Beyond partial improvement in insulin sensitivity, rosuvastatin normalized BP homeostasis in obese dyslipidaemic mice independently of changes in body weight or plasma cholesterol. Upregulation of PPARgamma and SOD1 in the endothelium may be involved as a unique vasculoprotective effect of statin treatment.

Dyslipidaemia in type II diabetic mice does not aggravate contractile impairment but increases ventricular stiffness.

AIMS: Type II diabetes, often associated with abdominal obesity, frequently leads to heart failure. Clinical and epidemiological evidence suggests that supplemental dyslipidaemia and hypertension, as clustered in the metabolic syndrome, aggravate the cardiovascular outcome. The differential impact of type II diabetes and the metabolic syndrome on left ventricular function, however, remains incompletely defined. METHODS AND RESULTS: We studied left ventricular function in vivo using pressure-volume analysis in obese diabetic mice with leptin deficiency (ob/ob) and obese diabetic dyslipidemic mice with combined leptin and low-density lipoprotein-receptor deficiency (DKO). ob/ob and DKO mice developed a diabetic cardiomyopathy, characterized by impaired contractility and relaxation, from the age of 24 weeks onwards. This was-at least partially-explained by increased apoptosis and disturbed Ca(2+) reuptake in the sarcoplasmic reticulum (SR) in both mouse models. DKO, but not ob/ob, developed increased end-diastolic ventricular stiffness, paralleled by increased left ventricular myocardial fibrosis. Cardiac output was preserved in ob/ob mice by favourable loading conditions, whereas it decreased in DKO mice. CONCLUSIONS: Type II diabetes in mice leads to impaired contractility and relaxation due to disturbed Ca(2+) reuptake in the SR, but only when dyslipidaemia and hypertension are superimposed does vascular-ventricular stiffening increase and left ventricular myocardial fibrosis develop.

A cathepsin D-cleaved 16 kDa form of prolactin mediates postpartum cardiomyopathy.

Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology and exposes women to high risk of mortality after delivery. Here, we show that female mice with a cardiomyocyte-specific deletion of stat3 develop PPCM. In these mice, cardiac cathepsin D (CD) expression and activity is enhanced and associated with the generation of a cleaved antiangiogenic and proapoptotic 16 kDa form of the nursing hormone prolactin. Treatment with bromocriptine, an inhibitor of prolactin secretion, prevents the development of PPCM, whereas forced myocardial generation of 16 kDa prolactin impairs the cardiac capillary network and function, thereby recapitulating the cardiac phenotype of PPCM. Myocardial STAT3 protein levels are reduced and serum levels of activated CD and 16 kDa prolactin are elevated in PPCM patients. Thus, a biologically active derivative of the pregnancy hormone prolactin mediates PPCM, implying that inhibition of prolactin release may represent a novel therapeutic strategy for PPCM.

Decrease of endothelin receptor subtype ETB and release of COX-derived products contribute to endothelial dysfunction of porcine epicardial coronary arteries in left ventricular hypertrophy.

Alterations in the regulation of coronary circulation play a major role in the enhanced susceptibility to ischemic injury of the myocardium in left ventricular hypertrophy (LVH). The present study was designed to assess the role of endothelium-dependent contracting factors and endothelin receptors in the coronary endothelial dysfunction in LVH, occurring 2 months after aortic banding in a swine model. Hemodynamic and morphologic analyses were performed in LVH and control groups. Vascular reactivity studies were performed in rings from control and aortic banding groups to assess the contribution of endothelin (ET-1) receptor subtypes to the contraction induced by ET-1 and IRL-1620 (an ETB receptor agonist), with and without endothelium. The effects of cyclooxygenase (COX)-derived products induced by ET-1, serotonin (5-HT), and bradykinin (BK) were evaluated, with or without indomethacin (a COX antagonist). ET-1 receptor density was assessed by confocal microscopy and Western blot experiments. The wall-to-lumen ratio, determined in digital planimetry, was increased in the LVH group with no significant changes in coronary perfusion pressures. There was a significant increase in contractions to ET-1 in the LVH group, which were reduced by exposure to indomethacin and daltroban (thromboxane A2 [TXA2] receptor antagonist). Relaxations to 5-HT and BK were improved by indomethacin in the LVH group. There was no significant change in ETA receptor density (3.113 +/- 0.389 vs 3.594 +/- 0.314) but a decrease in ETB receptor density (6.435 +/- 0.265 vs 4.588 +/- 0.089; P < 0.001) in the LVH group. The coronary endothelial dysfunction of swine epicardial coronary arteries in LVH secondary to 2 months of aortic banding involves both relaxing and contracting factors. ETA receptors and COX-derived products are preferentially implicated in the increased contractions to ET-1. Strategies aimed at decreasing ET-1 effects with ET-1 antagonists selective for ETA receptors could improve the coronary endothelial dysfunction in LVH.

Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction.

BACKGROUND: In the heart, nitric oxide synthases (NOS) modulate cardiac contraction in an isoform-specific manner, which is critically dependent on their cellular and subcellular localization. Defective NO production by NOS3 (endothelial NOS [eNOS]) in the failing heart may precipitate cardiac failure, which could be reversed by overexpression of NOS3 in the myocardium. METHODS AND RESULTS: We studied the influence of NOS3 in relation to its subcellular localization on the function of cardiomyocytes isolated from transgenic mice overexpressing NOS3 under the alpha-myosin heavy chain promoter (NOS3-TG). Immunoblot analysis demonstrated moderate (5-fold) NOS3 overexpression in cardiomyocytes from NOS3-TG heterozygotes. Caveolar localization of transgenic eNOS was demonstrated by immunofluorescence, coimmunoprecipitation with caveolin-3, sucrose gradient fractionation, and immunogold staining revealed by electron microscopy. Compared with wild-type littermate, contractility of NOS3-TG cardiomyocytes analyzed by videomicroscopy revealed a lower incidence of spontaneous arrhythmic contractions (n=32, P<0.001); an attenuation of the beta-adrenergic positive inotropic response (isoproterenol, 10(-7) mol/L: 62.1+/-7.8% versus 90.8+/-8.0% of maximum Ca2+ response; n=10 to 17; P<0.05); a potentiation of the muscarinic negative chronotropic response (carbamylcholine, 3.10(-8) mol/L: -63.9+/-14% versus -27.7+/-5.6% of basal rate; n=8 to 10; P<0.05), confirmed by telemetry in vivo; and an attenuation of the accentuated antagonism of beta-adrenergically stimulated contraction (-14.6+/-1.5% versus -3.5+/-1.5; n=7 to 11; P<0.05). Cardiomyocyte NOS inhibition reversed all 4 effects (P<0.05). CONCLUSIONS: Moderate overexpression of NOS3, targeted to caveolae in murine cardiomyocytes, potentiates the postsynaptic muscarinic response and attenuates the effect of high concentrations of catecholamines. Cardiomyocyte NOS3 may represent a promising therapeutic target to restore the sympathovagal balance and protect the heart against arrhythmia.

Tetrahydrobiopterin and antioxidants reverse the coronary endothelial dysfunction associated with left ventricular hypertrophy in a porcine model.

OBJECTIVE: Endothelium-dependent G-protein mediated relaxations of epicardial coronary arteries is impaired with left ventricular hypertrophy. The objective of this study was to assess the effect of L-arginine, BH(4) and the combination of two antioxidants, superoxide dismutase and catalase, on endothelium-dependent relaxations in a swine left ventricular hypertrophy model. METHODS: Aortic banding was performed 3 cm above the coronary ostia. Vascular reactivity studies were performed in standard organ chamber experiments to assess the NO pathway in the presence of methyltetrahydropterin (a BH(4) analogue), L-arginine, superoxide dismutase and catalase. RESULTS: There was a statistically significant increase in endothelium-dependent relaxation to serotonin and to bradykinin with methyltetrahydropterin and with superoxide dismutase plus catalase (P<0.05) but not with L-arginine compared to untreated coronary arteries from left ventricular hypertrophy animals. Plasma 3-nitrotyrosine level increased significantly from 918+/-122 to 1844+/-300 microM (P<0.05 vs. control) after 60 days of aortic banding. Endothelial dysfunction was not associated with a reduced expression of endothelial nitric oxide synthase 2 months after pressure overload left ventricular hypertrophy. CONCLUSIONS: Treatment with BH(4) and antioxidants constitutes an interesting approach for the prevention of endothelial dysfunction in epicardial coronary arteries associated with left ventricular hypertrophy.