Vascular NO Production Research Articles

Angiotensin II type 1 receptor is involved in flow-induced vasomotor responses of isolated middle cerebral arteries: role of oxidative stress.

This study aimed to determine the mechanosensing role of angiotensin II type 1 receptor (AT1R) in flow-induced dilation (FID) and oxidative stress production in middle cerebral arteries (MCA) of Sprague-Dawley rats. Eleven-week old, healthy male Sprague-Dawley rats on a standard diet were given the AT1R blocker losartan (1 mg/mL) in drinking water (losartan group) or tap water (control group) ad libitum for 7 days. Blockade of AT1R attenuated FID and acetylcholine-induced dilation was compared with control group. Nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) and cyclooxygenase inhibitor indomethacin (Indo) significantly reduced FID in control group. The attenuated FID in losartan group was further reduced by Indo only at Δ100 mmHg, whereas l-NAME had no effect. In losartan group, Tempol (a superoxide scavenger) restored dilatation, whereas Tempol + l-NAME together significantly reduced FID compared with restored dilatation with Tempol alone. Direct fluorescence measurements of NO and reactive oxygen species (ROS) production in MCA, in no-flow conditions revealed significantly reduced vascular NO levels with AT1R blockade compared with control group, whereas in flow condition increased the NO and ROS production in losartan group and had no effect in the control group. In losartan group, Tempol decreased ROS production in both no-flow and flow conditions. AT1R blockade elicited increased serum concentrations of ANG II, 8-iso-PGF2α, and TBARS, and decreased antioxidant enzyme activity (SOD and CAT). These results suggest that in small isolated cerebral arteries: 1) AT1 receptor maintains dilations in physiological conditions; 2) AT1R blockade leads to increased vascular and systemic oxidative stress, which underlies impaired FID.NEW & NOTEWORTHY The AT1R blockade impaired the endothelium-dependent, both flow- and acetylcholine-induced dilations of MCA by decreasing vascular NO production and increasing the level of vascular and systemic oxidative stress, whereas it mildly influenced the vascular wall inflammatory phenotype, but had no effect on the systemic inflammatory response. Our data provide functional and molecular evidence for an important role of AT1 receptor activation in physiological conditions, suggesting that AT1 receptors have multiple biological functions.

Impaired Dilator Reactivity of Middle Cerebral Arteries in Goto‐Kakizaki Rats with Type II Diabetes Mellitus

Type II diabetes mellitus (T2DM) is a highly prevalent disease and is associated with the development of cerebrovascular disease. The objective of the study was to determine whether alterations to the regulation of cerebral vascular tone exist in a non‐obese model of T2DM and to determine the mechanistic contributors to changes in this response. Middle cerebral arteries (MCA) from 17–18‐wk‐old male Wistar Kyoto (WKY) and Goto‐Kakizaki (GK) were isolated and cannulated with glass micropipettes. Vascular reactivity was assessed in response to challenge with acetylcholine (ACh), sodium nitroprusside (SNP), reductions in Po2, 5‐HT, and increased intraluminal pressure. Vessels were treated with the competitive nitric oxide synthase (NOS) inhibitor L‐NAME, the cyclooxygenase inhibitor indomethacin, and the cell‐permeable superoxide dismutase (SOD) mimetic TEMPOL to support interpretation of mechanistic contributors to changes in vessel reactivity. Similar measurements of vascular reactivity to methacholine (MCh), phenylephrine, SNP, and reductions in PO2 were measured in aortic ring preparations, along with measurements of vascular NO production and metabolites of arachidonic acid. There was an impairment of vessel dilation within the MCA and aorta of GK in response to ACh/MCh and hypoxia but not SNP. Decreased vascular NO release and ratio of PGI2/TxA2 were measured within the aorta in response to hypoxia, and increasing MCh concentration. No change was seen in the constrictor response to either 5‐HT or phenylephrine within the MCA or aorta respectively or in the myogenic activation of the MCA. Treatment with L‐NAME severely blunted the dilator reactivity of both the MCA and aorta, while TEMPOL showed mild improvements in GK, and indomethacin yielded improved dilator reactivity to MCh in GK but blunted it in WKY. These results suggest that the vasodilator reactivity of the MCA and aorta of GK in response to endothelium‐dependent dilator stimuli is impaired vs. WKY. This may be due to decreased NO bioavailability and altered arachidonic acid metabolism as a result of a pro‐oxidant pro‐inflammatory condition present in T2DM. The lack of change to the constrictor response of MCA and aorta in GK suggests a lack of structural remodeling as a result of T2DM at this age.Support or Funding InformationAmerican Heart Association and National Institutes of Health (USA); Canadian Institutes for Health Research and Natural Sciences and Engineering Research Council (Canada)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chemerin/CMKLR1 axis influences pulmonary arterial pressure and the NO signaling pathway

Pulmonary arterial hypertension (PAH) is a clinical condition associated with sustained elevations in pulmonary arterial pressure, leading to right ventricular failure. The adipokines leptin and adiponectin have shown respectively deleterious and protective effects in animal models of PAH. Chemerin is a novel adipokine implicated in blood pressure regulation and recently associated with atherosclerosis, essential hypertension and obesity-induced hypertension. In this context, we aimed to determine whether the chemerin/CMKLR1 axis could influence pulmonary arterial pressure and the severity of pulmonary hypertension. Age-matched wild-type (WT) and CMKLR1 knockout (KO) mice were used. We localized chemerin and the chemokine-like receptor-1 (CMKLR1) in lung sections, pulmonary artery and thoracic aorta. WT and KO mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks ( N = 6 per group). Right ventricular systolic pressure (RVSP) was measured with a micro-tip catheter. Segments of pulmonary artery and thoracic aorta were incubated, immediately after sacrifice, with recombinant mouse chemerin (10–8 mol/L, 1 hour) or vehicle, then stimulated or not with acetylcholine (ACH) (10–4 mol/L, 5 min) to measure NO production in supernatants by determination of nitrites. RVSP was higher in KO than in WT mice in normoxia (26.6 ± 0.4 vs. 22.2 ± 1 mmHg, P = 0.001) and in hypoxia (37.8 ± 0.5 vs. 31.4 ± 1.2 mmHg, P = 0.001). Hypoxia reduced NO production in WT and KO mice. In WT, ACH-induced NO production was abolished by chemerin pre-incubation. In normoxia and hypoxia, basal NO production was lower in KO than WT mice and ACH-stimulated NO production was totally inhibited in KO mice. These observations provide support for a role of the chemerin/CMKLR1 axis as regulator of pulmonary vascular homeostasis and as a disease-modifier in the context of pulmonary hypertension by the negative modulation of pulmonary vascular NO production.

Magnesium lithospermate B ameliorates microcirculation perfusion in rats by promoting vascular NO production via activating the PI3K/AKT pathway.

Microcirculation morphologically refers to the blood flow in vessels of less than 150 μm in diameter, including arterioles, capillaries and venules, which provides nutrients and removes metabolic byproducts within tissues. Microcirculation dysfunction is involved in the pathological progress of many diseases, such as obesity, hypertension, and insulin resistance. In this study we investigated the effects of magnesium lithospermate B (MLB), an active compound of the traditional Chinese medicine Slavia miltiorrhiza, on the microcirculation dysfunction in rats and the underlying molecular mechanisms. The effects of MLB on microcirculation were assessed in vivo by measuring the hindlimb blood perfusion in dextran-induced microcirculation dysfunction rats and mesentery blood flow in anesthetized rats. We demonstrated that administration of MLB restored the impaired rat hindlimb blood flow and promoted the mesenteric micoperfusion in vivo. We further revealed in these two animal models that MLB treatment significantly increased the production of total nitrite in vascular tissues (mesentery, aorta, and heart), which was confirmed in human microvascular endothelial cells (HMEC-1) treated with MLB in vitro. Moreover, we showed that MLB treatment significantly increased the phosphorylation of endothelium nitric oxide synthase (eNOS) via inducing AKT phosphorylation in vivo and in vitro. Co-administration of the eNOS inhibitor L-NAME (20 mg/kg) abolished the protective effects of MLB against dextran-induced microcirculation dysfunction in rats, whereas pretreatment with PI3K inhibitor LY294002 (10 μM) prevented eNOS activation in MLB-treated HMEC-1 cells. Our results suggest that MLB can restore the microcirculation dysfunction via activating eNOS, and in turn enhancing the vascular nitric oxide production, which is medicated by MLB-caused activation of the PI3K/AKT pathway.

VASCULAR NO AND SUPROXIDE PRODUCTION IS INFLUENCED BY HIGH DIETARY SALT INTAKE IN TFF3−/− MICE

Objective: Arachidonic acid metabolism and products are shown to affect vascular reactivity mechanism. TFF3−/− mice have protective phenotype of favorable ratio of ω6/ω-3 fatty acids and modified metabolism of arachidonic acid. However, the role of TFF3 peptide in hypertension and vascular function is still underinvestigated. The aim of this study was to asses the effect of high salt diet on flow-induced endothelial NO and superoxide production in carotid arteries of wild type mice of mixed background C57Bl6/Sv/129 (WT) and TFF3−/− knock out mice with the same background. Design and method: Eleven weeks old male TFF3−/− or WT mice were divided in 2 groups (N = 4 per group): low salt (LS) and high salt (HS; 4% NaCl for 7 days). Following dietary protocol, mice were anaesthetized with ketamine (75 mg/kg) and midazolam (2.5 mg/kg). Carotid arteries were isolated and cannulated on pressure myograph with or without flow (at delta 80 mmHg), in the absence/presence of the NOS inhibitor L-NAME. NO production was determined by DAF-2DA to DAF-2T conversion fluorescence assay. To assess the production of superoxide radicals, the carotid arteries were loaded with dihydroethidine (DHE, 20 μM). All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by institutional Ethical Committee. p < 0.05 was significant. Results: Basal NO production in no-flow condition was similar among groups. L-NAME blocked the production of NO in all groups. Flow-induced NO production was decreased in HS-WT group compared to no-flow conditions in HS-WT group, and compared to LS-WT group and LS-TFF3−/− group (p < 0.05). There were no changes in endothelial NO production in TFF3−/− mice. Superoxide production was increased in LS-TFF3−/− no-flow group compared to LS-WT in no-flow condition Flow-induced superoxide production was increased in both HS-WT and HS-TFF3−/− groups compared to corresponding LS groups. Conclusions: HS intake decreases flow-induced vascular NO production in WT mice probably due to increased oxidative stress. However, HS intake does not affect NO production in TFF3−/− mice eventhough superoxide levels are increased in basal levels and in flow conditions, suggesting protective role of this phenotype on NO bioavailability.

Xyloketal B exerts antihypertensive effect in renovascular hypertensive rats via the NO-sGC-cGMP pathway and calcium signaling.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. (No 2508). We previously showed that Xyl-B promoted endothelial NO release and protected against atherosclerosis through the Akt/eNOS pathway. Vascular NO production regulates vasoconstriction in central and peripheral arteries and plays an important role in blood pressure control. In this study, we examined whether Xyl-B exerted an antihypertensive effect in a hypertensive rat model, and further explored the possible mechanisms underlying its antihypertensive action. Administration of Xyl-B (20 mg·kg-1·d-1, ip, for 12 weeks) significantly decreased the systolic and diastolic blood pressure in a two-kidney, two-clip (2K2C) renovascular hypertensive rats. In endothelium-intact and endothelium-denuded thoracic aortic rings, pretreatment with Xyl-B (20 μmol/L) significantly suppressed phenylephrine (Phe)-induced contractions, suggesting that its vasorelaxant effect was attributed to both endothelial-dependent and endothelial-independent mechanisms. We used SNP, methylene blue (MB, guanylate cyclase inhibitor) and indomethacin (IMC, cyclooxygenase inhibitor) to examine which endothelial pathway was involved, and found that MB, but not IMC, reversed the inhibitory effects of Xyl-B on Phe-induced vasocontraction. Moreover, Xyl-B increased the endothelial NO bioactivity and smooth muscle cGMP level, revealing that the NO-sGC-cGMP pathway, rather than PGI2, mediated the anti-hypertensive effect of Xyl-B. We further showed that Xyl-B significantly attenuated KCl-induced Ca2+ entry in smooth muscle cells in vitro, which was supposed to be mediated by voltage-dependent Ca2+ channels (VDCCs), and reduced ryanodine-induced aortic contractions, which may be associated with store-operated Ca2+ entry (SOCE). Taken together, these findings demonstrate that Xyl-B exerts significant antihypertensive effects not only through the endothelial NO-sGC-cGMP pathway but also through smooth muscle calcium signaling, including VDCCs and SOCE.

Abstract 196: DDAH1 Knockout Exacerbates Hypoxia-induced Pulmonary Arterial Hypertension In Mice

Pulmonary arterial hypertension (PAH) is a progressive disease with a very poor prognosis. Recent studies have demonstrated that PAH is associated with diminished nitric oxide bioavailability, increased levels of endogenous nitric oxide synthase (NOS) inhibitor ADMA, and decreased lung dimethylarginine dimethylaminohydrolase (DDAH) activity. We have demonstrated that DDAH1 is essential for degradation of endogenous NOS inhibitor ADMA, and is important for optimal vascular endothelial NO production. However, it is not clear whether decreased DDAH activity and ADMA accumulation exacerbates development or progression of PAH and right ventricular hypertrophy. In addition, the impact of cardiomyocyte specific DDAH1 dysfunction on right ventricular hypertrophy is unknown. Using global DDAH1 gene deficient mice, we demonstrate that chronic ADMA accumulation by global DDAH1 gene deletion did not cause spontaneous PAH under control conditions, but significantly exacerbated chronic hypoxia-induced PAH, as indicated by significantly increased right ventricular (RV) pressure, more RV hypertrophy, and enhanced pulmonary vascular remodeling in DDAH1 deficient mice as compared to wild type mice. Chronic hypoxia resulted in reduced lung DDAH activity and increased circulating ADMA content in wild type mice. Cardiomyocyte specific DDAH1 gene deletion did not exacerbate hypoxia-induced increases in RV pressure or lung vascular remodeling, but significantly exacerbated hypoxia-induced RV hypertrophy in comparison to wild type littermates, indicating DDAH1 distributed in cardiomyocytes protect the heart against RV hypertrophy independent to pressure overload. Collectively, our data indicate that lung DDAH1 plays a critical role in protection against hypoxia-induced PAH and right ventricular hypertrophy.

Endothelial nitric oxide synthase activation through obacunone-dependent arginase inhibition restored impaired endothelial function in ApoE-null mice

Endothelial arginase constrains the activity of endothelial nitric oxide synthase (eNOS) by substrate depletion and reduces nitric oxide bioavailability. During the screening course of arginase inhibitor, we found obacunone as an arginase inhibitor. We tested the hypothesis that obacunone regulates vascular endothelial NO production. Obacunone incubation inhibited arginase I and II activities in liver and kidney lysates, respectively, in dose-dependent manner. Obacunone reciprocally increased nitrite/nitrate (NOx) production in HUVECs. In isolated aortic rings, obacunone increased intracellular l-arginine concentration and enhanced eNOS coupling, leading to increased NO and decreased superoxide production, with no changes in protein expression. Vasoconstriction response to U46619 was attenuated in obacunone-treated aortic vessels compared to that in untreated vessels. Endothelium-dependent vasorelaxant response to acetylcholine was significantly increased in obacunone-treated vessels and was modulated by the NO-dependent signaling cascade. The dose-dependent vasorelaxant response to Ach was reduced in the aortic vessels of ApoE−/− mice fed a high-cholesterol diet. Obacunone incubation increased vasorelaxation to the level of a WT mouse, although the endothelium-independent response to sodium nitroprusside was identical among the groups. Therefore, obacunone may help treat cardiovascular diseases derived from endothelial dysfunction and may be useful for designing pharmaceutical compounds.

Age-related alterations in endothelial function of femoral artery in young SHR and WKY rats.

The present study was designed to evaluate the effects of vascular aging in juvenescence on endothelial function in femoral arteries and to assess differences between normotensive and hypertensive rats. The aim of the study was to determine if age affected nitric oxide- (NO-) mediated relaxations in normotensive and hypertensive rats. Juvenile (7-week-old) and young adult (22-week-old) male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were used in this study. Femoral artery (FA) reactivity was determined by wire myograph and NO synthase activity by conversion of [3H]-L-arginine. During juvenescence systolic blood pressure (tail-cuff) increased significantly only in SHR, while NO synthesis decreased significantly in both strains. Endothelium-dependent relaxations to acetylcholine were reduced in the FA of SHR compared to age-matched WKY at both ages, yet these parameters were unchanged in adult rats compared with juvenile animals. The NO-dependent component of vasorelaxation was markedly reduced, whereas the NO-independent component was increased in adult compared to juvenile rats in both strains. The endothelial dysfunction in SHR at both ages was associated with reduction of NO-independent mechanisms. In conclusion, aging in early periods of life was associated with reduction of vascular NO production and bioavailability in both strains investigated. This reduction was however fully compensated by accentuation of NO-independent mechanisms.

Increased Nitric Oxide Bioavailability in Adult GRK2 Hemizygous Mice Protects Against Angiotensin II–Induced Hypertension

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous serine/threonine protein kinase able to phosphorylate and desensitize the active form of several G protein-coupled receptors. Given the lack of selective inhibitors for GRK2, we investigated the effects elicited by GRK2 inhibition in vascular responses using global adult hemizygous mice (GRK2(+/-)). The vasodilator responses to acetylcholine or isoproterenol were increased in aortas and mesenteric resistance arteries from GRK2(+/-) mice compared with wild-type (WT) littermates. After angiotensin II (AngII) infusion, GRK2(+/-) mice were partially protected against hypertension, vascular remodeling, and mechanical alterations, even when resting basal blood pressures were not significantly different. AngII infusion also (1) increased GRK2 levels in WT but not in GRK2(+/-) vessels; (2) increased vasoconstrictor responses to phenylephrine in WT but not in GRK2(+/-) mice; and (3) decreased vasodilator responses to acetylcholine and vascular pAkt and eNOS levels more in WT than in GRK2(+/-) animals. Vascular NO production and the modulation of vasoconstrictor responses by endothelial-derived NO remained enhanced in GRK2(+/-) mice infused with AngII. Thus, GRK2(+/-) mice are resistant to the development of vascular remodeling and mechanical alterations, endothelial dysfunction, increased vasoconstrictor responses, and hypertension induced by AngII at least partially through the preservation of NO bioavailability. In conclusion, our results describe an important role for GRK2 in systemic hypertension and further establish that an inhibition of GRK2 could be a beneficial treatment for this condition.

Abstract 186: ACE2 Activation Protects Corpus Cavernosum Impairment Induced by Hypercholesterolemia

Erectile dysfunction (ED) is multicausal disorder strictly associated with cardiovascular diseases. ACE2 is a key enzyme of the renin angiotensin system which plays an essential role in the cardiovascular system; however, the function of this enzyme in erectile tissues has never been investigated. Here we examined the expression of ACE2 in erectile tissues and its protective actions against hypercholesterolemic-induced corpus cavernosum injures. ACE2 expression was evaluated by immunostaining and western blot. Apolipoprotein E knockout (ApoE-/-) mice fed with Western-type diet for 11 weeks were treated with DIZE, an ACE2 activator compound, or vehicle during the last 3 weeks. Collagen and reactive oxygen species (ROS) content within the corpus cavernosum (CC) were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of nNOS, eNOS, NADPH subunits (p67-phox and p22-phox) was assessed by western blot. Functional studies in CC strips were performed in an isometric organ bath. ACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, this enzyme was downregulated in penis from hypercholesterolemic mice. The treatment with DIZE reduced ROS production and NADPH oxidase expression in penis from ApoE-/- mice. Furthermore, DIZE treatment elevated vascular NO production (untreated-ApoE-/-: 11.3±1.1 vs. treated-ApoE-/-: 18.7±4.5, mol/L of serum NO3-) and augmented the expression of nNOS and eNOS in the penis. The NO-dependent relaxation induced by acetylcholine (ACh) was significantly increased in CC strip from DIZE-treated ApoE-/- mice (wild type 53.7%; untreated-ApoE-/- 39.3%; treated-ApoE-/- 52.1%, percentage of relaxation at 1μM of ACh). Additionally, DIZE treatment decreased collagen content within the CC. Of note, these beneficial actions of DIZE on the CC were not associated with changes in atherosclerotic plaque deposition or serum lipid profile. In summary, ACE2 activation attenuated oxidative stress, augmented NO bioavailability and decreased collagen deposition in CC of hypercholesterolemic mice. These results demonstrate that ACE2 is expressed and functional in erectile tissue, suggesting that this enzyme might have significant therapeutic benefits against ED.

Ceacam1 deletion causes vascular alterations in large vessels

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulin's stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Generation of a conditional allele for the mouse endothelial nitric oxide synthase gene

Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here, we have generated a floxed eNOS allele in which exons 9-12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre-deleted alleles displayed truncated eNOS protein, lack of vascular NO production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scarring. These findings demonstrate that the floxed allele is normal and it can be converted to a non-functional eNOS allele through Cre recombination. This mouse will allow time- and cell-specific eNOS deletion.

Sistema Neuregulina1/ErbB: importância no controlo da função cardíaca.

The family of Neuregulins (NRG), growth factors like epidermal growth factor, is known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. This family comprises four members, being NRG1 the most largely studied, particularly at the cardiovascular level. The biological effects of NRG1 in the adult heart are mediated by the tyrosine kinase receptors ErbB. In the adult heart, NRG1 is expressed by cells of the endocardial endothelium and the cardiac microvascular endothelium, and the receptors ErbB2/ErbB4 are expressed by ventricular cardiomyocytes and are located in T-tubule system and intercalated disks in close proximity to the system components of excitation-contraction coupling. The importance of the NRG/ErbB signaling axis at the cardiovascular level became evident after discovering that patients treated with trastuzumab (inhibitory antibody against ErbB2, used in the treatment of breast cancer) can develop ventricular dysfunction and have higher risk of cardiomyopathy when co-administered with anthracyclines. Subsequent studies in vitro and in vivo have clarified the effects and the respective signaling pathways associated with the NRG/ErbB system in the adult heart. Some cardiovascular functions of the NRG1/ErbB system have been described at the vascular (stimulation of angiogenesis and ateroprotector effect) and myocardium level (negative inotropic effect) as well as effect on the survival, cell growth and organization of the cardiomyocytes (myofibrillar organization and cell-to-cell contact between cardiomyocytes). Furthermore, the interaction of this system with other neurohumoral mediators has been studied. Thus, there seems to be a physiological role in modulating the sympathovagal balance and an interaction with endothelin-1 signaling. All these effects result from the activation of different intracellular signaling cascades, as a consequence of the binding of NRG1 to ErbB receptors. Some cardiac signaling pathways identified until now include molecules such as MEK / Erk 1/2, phosphatidylinositol 3-kinase/ Akt, focal adhesion kinase, Gab (Grb-2-associated binder) family, vascular endothelial growth factor and NO production by endothelial nitric oxide synthase. Thus, the aim of this paper was to make an up-to-date review of existing information on NRG1/ErbB signaling axis, with particular focus on its cardiovascular effects.

Implant‐induced intraperitoneal inflammatory angiogenesis is attenuated by fluvastatin

1. Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors, exert anti-inflammatory, anti-oxidant and anti-angiogenic effects. These effects are associated with downregulation of pro-inflammatory/pro-angiogenic molecules and upregulation of endothelial nitric oxide synthase (e-NOS) expression/nitric oxide (NO) production. 2. Using the murine sponge model to induce chronic intraperitoneal inflammatory response, we evaluated the inflammatory components, angiogenic and NO production of the fibrovascular tissue, and their modulation by fluvastatin. 3. Our results showed that fluvastatin (0.6 and 6 mg/kg per day) inhibited haemoglobin (Hb) content 4.9±0.4 (n=15; control) vs 2.2±0.2 (n=6; fluvastatin 0.6) and 1.8±0.2 (n=6; fluvastatin 6.0) and the number of vessels in the treated group when compared with the control group. The inflammatory component, as assessed by myeloperoxidase and N-acetyl-β-d-glucosaminidase activities and by the pro-inflammatory cytokines, tumour necrosis factor-α (TNF-α) and Monocyte chemotactic protein-1 (MCP-1)/CCL2/JE levels, was also decreased by the compound. In the treated group, inhibition of both enzyme activities was 54% and 57%, respectively. The levels of the cytokines (TNF-α and CCL2/JE) intra-implant were decreased relative to the control. In these implants, fluvastatin was also able to increase NO production, as detected with an NO-sensitive electrode. 4. The inhibitory function of fluvastatin on key components of intraperitoneal inflammatory angiogenesis shown in the present study is clearly associated with the modulatory effects of this statin on vascular endothelial growth factor, TNF-α and NO production.

Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice

Apolipoprotein (apo) O is a newly discovered apolipoprotein preferentially contained within HDL; however, currently, no data are available on the (patho)physiological effects of apoO. Therefore, the present study assessed the impact of apoO overexpression on (i) plasma lipids and lipoproteins as well as on (ii) HDL functionality. Human apoO was overexpressed by means of recombinant adenovirus (AdhapoO) in human apoA-I transgenic mice, a humanized mouse model of HDL metabolism. AdhapoO substantially increased apoO in plasma and within HDL. However, plasma triglycerides, phospholipids, total cholesterol and HDL cholesterol did not change. HDL size distribution, lipid composition and the apoA-I and the apoO distribution over the different HDL fractions separated by FPLC remained unaltered. Furthermore, enrichment of HDL with apoO did not impact on HDL functionality assessed in four independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells, (ii) protection against LDL oxidation, (iii) anti-inflammatory activity on endothelial cells, and (iv) induction of vasodilation in isolated aortic rings ex vivo as a measure of stimulating vascular NO production. These results demonstrate that although overexpression of apoO results in a substantial enrichment of HDL particles with this novel apolipoprotein, apoO does not impact the plasma lipoprotein profile or HDL functionality.

New labdane-type diterpenes and antitubercular constituents from Hedychium coronarium

Hedychium coronarium Koenig (Zingiberaceae) is a perennial rhizomatous herb, distributed in India, Malaysia, Vietnam, southern China, and Taiwan [1]. Various labdane-type diterpenes are widely distributed in plants of the genus Hedychium. Many of these compounds exhibit cytotoxic, anti-inflammatory, and hepatoprotective activities and show inhibitory effects on the increase in vascular permeability, NO production, and iNOS induction. Two new labdane-type diterpenes, hedychicoronarin A (1) and hedychicoronarin B (2), and ten known compounds (3-12) have been isolated and identified from the rhizome of Hedychium coronarium. The structures of new compounds 1 and 2 were determined through spectral analyses including extensive 2D NMR data. Among the isolated compounds, (+)-coronarin A (3) and coronarin D methyl ether (4) exhibited antitubercular activities with MICs=80 and 50µg/mL, respectively, against Mycobacterium tuberculosis H37Rv in vitro. The structural elucidation of new compounds 1 and 2 and the antitubercular activities of the isolates will be discussed in this symposium.

M1 PROTEIN FROM STREPTOCOCCUS PYOGENES INDUCES NITRIC OXIDE-MEDIATED VASCULAR HYPORESPONSIVENESS TO PHENYLEPHRINE

Streptococcus pyogenes carrying M1 protein causes the severe and increasingly prevalent streptococcal toxic shock syndrome and necrotizing fasciitis. M1 protein is an important virulence factor of S. pyogenes and induces an inflammatory response in human monocytes. We wanted to investigate if purified M1 protein in solution could induce vascular NO production leading to vasopressor hyporesponsiveness. Rat aortic segments were incubated with M1 protein or LPS in vitro. M1 protein (10 microg mL) and LPS (1 ng mL) to a similar extent induced NO production and hyporesponsiveness to the vasoconstrictor phenylephrine. Immunogold electron microscopy demonstrated that M1 protein binds to Toll-like receptor 2 (TLR2) as well as TLR4 in mouse aorta but only to TLR2 in human omental artery. Incubation with M1 protein caused a reduction in the contractile response to phenylephrine in aortic segments from wild-type and TLR2-knockout but not from TLR4-knockout mice. In conclusion, M1 protein causes vascular NO production leading to hyporesponsiveness to vasopressors via a mechanism involving TLR, but the subtypes may be species dependent. M1 protein could contribute to the circulatory disturbances accompanying severe invasive streptococcal infections.

Therapeutic Targeting of Mitochondrial Superoxide in Hypertension

Superoxide (O2(-) ) has been implicated in the pathogenesis of many human diseases including hypertension; however, commonly used antioxidants have proven ineffective in clinical trials. It is possible that these agents are not adequately delivered to the subcellular sites of superoxide production. Because the mitochondria are important sources of reactive oxygen species, we postulated that mitochondrial targeting of superoxide scavenging would have therapeutic benefit. In this study, we found that the hormone angiotensin (Ang II) increased endothelial mitochondrial superoxide production. Treatment with the mitochondria-targeted antioxidant mitoTEMPO decreased mitochondrial O2(-), inhibited the total cellular O2(-), reduced cellular NADPH oxidase activity, and restored the level of bioavailable NO. These effects were mimicked by overexpressing the mitochondrial MnSOD (SOD2), whereas SOD2 depletion with small interfering RNA increased both basal and Ang II-stimulated cellular O2(-). Treatment of mice in vivo with mitoTEMPO attenuated hypertension when given at the onset of Ang II infusion and decreased blood pressure by 30 mm Hg following establishment of both Ang II-induced and DOCA salt hypertension, whereas a similar dose of nontargeted TEMPOL was not effective. In vivo, mitoTEMPO decreased vascular O2(-), increased vascular NO production and improved endothelial-dependent relaxation. Interestingly, transgenic mice overexpressing mitochondrial SOD2 demonstrated attenuated Ang II-induced hypertension and vascular oxidative stress similar to mice treated with mitoTEMPO. These studies show that mitochondrial O2(-) is important for the development of hypertension and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in this and possibly other diseases.

Cocoa, Chocolate, and Cardiovascular Disease

A significant body of evidence demonstrates that diets rich in fruits and vegetables promote health and attenuate, or delay, the onset of various diseases, including cardiovascular disease, diabetes, certain cancers, and several other age-related degenerative disorders. The concept that moderate chocolate consumption could be part of a healthy diet has gained acceptance in past years based on the health benefits ascribed to selected cocoa components. Specifically, cocoa as a plant and chocolate as food contain a series of chemicals that can interact with cell and tissue components, providing protection against the development and amelioration of pathological conditions. The most relevant effects of cocoa and chocolate have been related to cardiovascular disease. The mechanisms behind these effects are still under investigation. However, the maintenance or restoration of vascular NO production and bioavailability and the antioxidant effects are the mechanisms most consistently supported by experimental data. This review will summarize the most recent research on the cardiovascular effects of cocoa flavanols and related compounds.