Aldosterone induces endothelial dysfunction in resistance arteries from normotensive and hypertensive rats by increasing thromboxane A(2) and prostacyclin

Background and purpose: The present study was designed to assess whether cyclooxygenase-2 (COX-2) activation is involved in the effects of chronic aldosterone treatment on endothelial function of mesenteric resistance arteries (MRA) from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Experimental approach: Relaxation to acetylcholine was measured in MRA from both untreated and aldosterone-treated strains. Vasomotor responses to prostacyclin and U46619 were also analysed. Release of 6-oxo-prostaglandin (PG)F(1 alpha) and thromboxane B(2) (TxB(2)) was determined by enzyme immunoassay. COX-2 protein expression was measured by western blot. Key results: Aldosterone reduced acetylcholine relaxation in MRA from both strains. In MRA from both aldosterone-treated strains the COX-1/2 or COX-2 inhibitor (indomethacin and NS-398, respectively), Tx2 synthesis inhibitor (furegrelate), prostacyclin synthesis inhibitor (tranylcypromine) or Tx2/PG2 receptor antagonist (SQ 29 548), but not COX-1 inhibitor SC-560, increased acetylcholine relaxation. In untreated rats this response was increased only in SHR. Prostacyclin elicited a biphasic vasomotor response: lower concentrations elicited relaxation, whereas higher concentrations elicited contraction that was reduced by SQ 29 548. Aldosterone increased the acetylcholine-stimulated production of 6-oxo-PGF(1 alpha) and TxB(2) in MRA from both strains. COX-2 expression was higher in both strains of rats treated with aldosterone. Conclusions and implications: Chronic treatment with aldosterone impaired endothelial function in MRA under normotensive and hypertensive conditions by increasing COX-2-derived prostacyclin and thromboxane A(2). As endothelial dysfunction participates in the pathogenesis of many cardiovascular disorders we hypothesize that anti-inflammatory drugs, specifically COX-2 inhibitors, could ameliorate vascular damage in patients with elevated aldosterone production.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1 beta (IL-1 beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alphalbeta (CINC-2 alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process. Copyright (c) 2007 John Wiley & Sons, Ltd.

Oxidative stress and inflammatory mediators contribute to endothelial dysfunction in high-fat diet-induced obesity in mice

Objective We investigated the effects of high-fat diet-induced obesity on vascular proinflammatory factors and oxidative stress on endothelium-dependent relaxation of the aorta. Methods Female Swiss mice were submitted to a high-fat diet for 16 weeks. At the end of the experimental period, we evaluated blood pressure, relaxation in response to acetylcholine in aortic rings in the absence and the presence of the superoxide anion scavenger, superoxide dismutase (SOD, 150 U/ml), and the nuclear factor (NF)-kappa B inhibitor, sodium salicylate (5 mmol/l). Aortic protein expression of endothelial nitric oxide synthase, Cu/Zn-SOD, NF-kappa B, I kappa B-alpha, and proinflammatory cytokines were also evaluated. Results Obese mice presented higher systolic and diastolic blood pressure than control mice (P<0.05). The relaxation of aortas to acetylcholine, but not to sodium nitroprusside, was significantly decreased in obese mice and was corrected by both SOD and sodium salicylate (P<0.05). The protein expression of endothelial nitric oxide synthase and Cu/Zn-SOD was significantly decreased in aorta from obese mice (P<0.05). Total p65 NF-kappa B subunit protein expression was not affected by obesity, but the protein expression of NF-kappa B inhibitor I kappa B-alpha was lower in aorta from obese mice (P<0.05). There were no significant differences in the interleukin (IL)-1 beta and IL-6 protein expression between groups. In contrast, the expression of TNF-alpha was significantly increased in aortas from obese mice. Conclusion Our resultssuggest that the reducedantioxidant defense and the local NF-kappa B pathway play an important role in the impairment of endothelium-dependent relaxation in aorta from obese mice. J Hypertens 28: 2111-2119 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

GLP-1 Inhibits and Adrenaline Stimulates Glucagon Release by Differential Modulation of N- and L-Type Ca(2+) Channel-Dependent Exocytosis

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 mu M) concentrations of forskolin, respectively. The expression of GLP-1 receptors in a cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on a cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates a cell electrical activity, increases [Ca(2+)] enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP]). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP],.

Lymphocytes transfer [(14)C]-labeled fatty acids to skeletal muscle in culture; modulation by exercise

Previous studies have shown that lipids are transferred from lymphocytes (Ly) to different cell types including macrophages. enterocytes, and pancreatic beta cells in co-culture This study investigated whether [(14)C]-labeled fatty acids (FA) can be transferred from Ly to skeletal muscle (SM), and the effects of exercise on such phenomenon Ly obtained from exercised (EX) and control (C) male Wistar rats were preloaded with the [(14)C]-labeled free FA palmitic (PA), oleic (OA), linoleic (LA), or arachidonic (AA) Radioactively loaded Ly were then co-cultured with SM from the same Ly donor animals Substantial amounts of FA were transferred to SM being the profile PA = OA > AA > LA to the C group. and PA > OA > LA > AA to the EX group These FA were incorporated predominantly as phospholipids (PA = 66 75%: OA = 63 09%, LA = 43 86%, AA - 47 40%) in the C group and (PA = 63 99% OA = 52 72%, LA = 55 99%, AA = 63 40%) in the EX group Also in this group, the remaining radioactivity from AA, LA, and OA acids was mainly incorpoiated in structural and energetic lipids These results support the hypothesis that Ly are able to export lipids to SM in co-culture Furthermore. exercise modulates the lipid transference profile, and its incorporation on SM The overall significance of this phenomenon in vivo remains to be elucidated. Copyright (C) 2010 John Wiley & Sons, Ltd

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK. gene transcription.

Vascular endothelial growth factor (VEGF) and VEGF-receptor expression in placenta of hyperglycemic pregnant women

Hyperglycemia occurs in a variety of conditions such as overt diabetes, gestational diabetes and mild hyperglycemia, all of which are generally defined based on the oral glucose tolerance test and glucose profiles. Whereas diabetes has received considerable attention in recent decades, few studies have examined the mechanisms of mild hyperglycemia and its associated disturbances. Mild gestational hyperglycemia is associated with macrosomia and a high risk of perinatal mortality. Morphologically, the placenta of these women is characterized by an increase in the number of terminal villi and capillaries, presumably as part of a compensatory mechanism to maintain homeostasis at the maternal-fetal interface. In this study, we analised the expression of VEGF and its receptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR) in placentas from mildly hyperglycemic women. This expression was compared with that of normoglycemic women and women with gestational and overt diabetes. Immunohistochemistry revealed strong staining for VEGF and VEGFR-2 in vascular and trophoblastic cells of mildly hyperglycemic women, whereas the staining for VEGFR-1 was discrete and limited to the trophoblast. The pattern of VEGF and VEGF-receptor reactivity in placentas from women with overt diabetes was similar to that of normoglycemic women. In women with gestational diabetes, strong staining for VEGFR-1 was observed in vascular and trophoblastic cells whereas VEGF and VEGFR-2 were detected only in the trophoblast. The expression of these proteins was confirmed by western blotting, which revealed the presence of an additional band of 75 kDa. In the decidual compartment, only extravillous trophoblast reacted with all antibodies. Morphological analysis revealed collagen deposition around large arteries in all groups with altered glycemia. These findings indicate a placental response to altered glycemia that could have important consequences for the fetus. The change in the placental VEGF/VEGFR expression ratio in mild hyperglycemia may favor angiogenesis in placental tissue and could explain the hypercapillarization of villi seen in this gestational disturbance. (C) 2010 Elsevier Ltd. All rights reserved.

Retinoic Acid and VEGF Delay Smooth Muscle Relative to Endothelial Differentiation to Coordinate Inner and Outer Coronary Vessel Wall Morphogenesis

Rationale: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. Objective: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. Methods and Results: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. Conclusion: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels. (Circ Res. 2010;107:204-216.)

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

Differential effects of formaldehyde exposure on the cell influx and vascular permeability in a rat model of allergic lung inflammation

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days. and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB(4). TXB(2), IL-1 beta, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung Vitamins C, E and apocynin reduced the levels of LTB(4) in BAL-cultured cells of the FA and FA/OVA groups, but Increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1) In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability Our data are indicative that the actions of FA involve mechanisms related to endothelium-leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Short-term induction of thrombocytopenia delays periodontal healing in rats with periodontal disease: participation of endostatin and vascular endothelial growth factor

Background and Objective: Platelets contain factors, including VEGF and endostatin, that can modulate the healing process. We evaluated the effects of severe thrombocytopenia on periodontal healing in rats and determined the contribution of VEGF and endostatin to the healing process. Material and Methods: Rats were distributed into three test groups and two control groups. Cotton ligatures were placed at the gingival margin level of the lower first molar in the test groups. Sham-operated rats and rats in one of the periodontitis groups were killed 15 days later. Rats in the remaining two periodontitis groups had the ligatures removed in order to study the spontaneous recovery from the periodontal disease 15 days later, and these rats were treated with rabbit antiplatelet serum, in order to induce thrombocytopenia, or normal rabbit serum. An additional group without ligatures received antiplatet serum in the same period. Results: After ligature removal, rats treated with normal rabbit serum showed reduced myeloperoxidase activity, decreased alveolar bone loss and increased numbers of blood vessels. Thrombocytopenia caused a delay in alveolar bone regeneration, a decrease in the number of vessels and a modest decrease in myeloperoxidase activity. In the rats with periodontitis, serum endostatin concentrations were slightly decreased and serum VEGF remained unchanged compared with sham-operated animals. After ligature removal, a significant VEGF increase and endostatin decrease were observed in the rats treated with normal rabbit serum. Thrombocytopenia led to a dramatic fall in both VEGF and endostatin concentrations. Conclusion: Thrombocytopenia leads to a delay of periodontal healing in the situation of experimental periodontitis, which might be mediated in part by a decrease in the serum concentration of VEGF and endostatin derived from the platelets. However, other factors derived from the platelets may also have contributed to a delay of periodontal healing in the rats with thrombocytopenia.

Micronutrient prenatal supplementation prevents the development of hypertension and vascular endothelial damage induced by intrauterine malnutrition

Aims: The premise that intrauterine malnutrition plays an important role in the development of cardiovascular and renal diseases implies that these disorders can be programmed during fetal life. Here, we analyzed the hypothesis that supplementation with mixed antioxidant vitamins and essential mineral in early life could prevent later elevation of blood pressure and vascular and renal dysfunction associated with intrauterine malnutrition. Main methods: For this, female Wistar rats were randomly divided into three groups on day 1 of pregnancy: control fed standard chow ad libitum; restricted group fed 50% of the ad libitum intake and a restricted plus micronutrient cocktail group treated daily with a combination of micronutrient (selenium, folate, vitamin C and vitamin E) by oral gavage. Key findings: In adult offspring, renal function and glomerular number were impaired by intrauterine malnutrition. and the prenatal micronutrient treatment did not prevent it. However, increased blood pressure and reduced endothelium-dependent vasodilation were prevented by the micronutrient prenatal treatment. Intrauterine malnutrition also led to reduced NO production associated with increased superoxide generation, and these parameters were fully normalized by this prenatal treatment. Significance: Our current findings indicate that programming alterations during fetal life can be prevented by interventions during the prenatal period, and that disturbance in availability of both antioxidant vitamins and mineral may play a crucial role in determining the occurrence of long-term cardiovascular injury. (C) 2009 Elsevier Inc. All rights reserved.

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN] = 17.6 +/- 4 versus 10.7 +/- 2 control; n = 6) and decreased relaxation to acetylcholine (47.6 +/- 6% versus 73.2 +/- 10% control; n = 8) versus arteries from uninephrectomized rats. O- GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units = 3.8 +/- 0.3 versus 2.3 +/- 0.3 control; n = 5). PugNAc (O- GlcNAcase inhibitor; 100 mu mol/L; 24 hours) increased vascular O- GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2 +/- 2 versus 10.7 +/- 3 vehicle; n = 6), and decreased acetylcholine dilation in uninephrectomized (41.4 +/- 6 versus 73.2 +/- 3 vehicle; n = 6) but not in DOCA rats (phenylephrine, 16.5 +/- 3 versus 18.6 +/- 3 vehicle, n = 6; acetylcholine, 44.7 +/- 8 versus 47.6 +/- 7 vehicle, n = 6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P < 0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P < 0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O- GlcNAc transferase, glutamine: fructose-6-phosphate amidotransferase, and O- GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P < 0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O- GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension. (Hypertension. 2009; 53: 166-174.)

The renin-angiotensin system is modulated by swimming training depending on the age of spontaneously hypertensive rats

Aim: To investigate the effects of swimming training on the renin-angiotensin system (RAS) during the development of hypertensive disease. Main methods: Male spontaneously hypertensive rats (SHR) were randomized into: sedentary young (SY), trained young (TV), sedentary adult (SA), and trained adult (TA) groups. Swimming was performed 5 times/wk/8wks. Key findings: Trained young and adult rats showed both decreased systolic and mean blood pressure, and bradycardia after the training protocol. The left ventricular hypertrophy (LVH) was observed only in the TA group (12.7%), but there was no increase on the collagen volume fraction. Regarding the components of the RAS, TV showed lower activity and gene expression of angiotensinogen (AGT) compared to SY. The TA group showed lower activity of circulatory RAS components, such as decreased serum ACE activity and plasma renin activity compared to SA. However, depending on the age, although there were marked differences in the modulation of the RAS by training, both trained groups showed a reduction in circulating angiotensin II levels which may explain the lower blood pressure in both groups after swimming training. Significance: Swimming training regulates the RAS differently in adult and young SHR rats. Decreased local cardiac RAS may have prevented the LVH exercise-induced in the TV group. Both groups decreased serum angiotensin II content, which may, at least in part, contribute to the lowering blood pressure effect of exercise training. (C) 2011 Elsevier Inc. All rights reserved.

Environmental modulation of ethanol-induced locomotor activity: Correlation with neuronal activity in distinct brain regions of adolescent and adult Swiss mice

Drug abuse is a concerning health problem in adults and has been recognized as a major problem in adolescents. induction of immediate-early genes (IEG), such as c-Fos or Egr-1, is used to identify brain areas that become activated in response to various stimuli, including addictive drugs. It is known that the environment can alter the response to drugs of abuse. Accordingly, environmental cues may trigger drug-seeking behavior when the drug is repeatedly administered in a given environment. The goal of this study was first to examine for age differences in context-dependent sensitization and then evaluate IEG expression in different brain regions. For this, groups of mice received i.p. ethanol (2.0 g/kg) or saline in the test apparatus, while other groups received the solutions in the home cage, for 15 days. One week after this treatment phase, mice were challenged with ethanol injection. Acutely, ethanol increased both locomotor activity and IEG expression in different brain regions, indistinctly, in adolescent and adult mice. However, adults exhibited a typical context-dependent behavioral sensitization following repeated ethanol treatment, while adolescent mice presented gradually smaller locomotion across treatment, when ethanol was administered in a paired regimen with environment. Conversely, ethanol-treated adolescents expressed context-independent behavioral sensitization. Overall, repeated ethanol administration desensitized IEG expression in both adolescent and adult mice, but this effect was greatest in the nucleus accumbens and prefrontal cortex of adolescents treated in the context-dependent paradigm. These results suggest developmental differences in the sensitivity to the conditioned and unconditioned locomotor effects of ethanol. (C) 2008 Elsevier B.V. All rights reserved.