Virginia S. Lemos

The endothelium-dependent vasodilator effect of the nonpeptide Ang(1-7) mimic AVE 0991 is abolished in the aorta of mas-knockout mice.

Recently, we demonstrated that the endothelium-dependent vasodilator effect of angiotensin(1-7) in the mouse aorta is abolished by genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene. To circumvent the limitations posed by the possible metabolism of Ang(1-7) in this vessel, in this work we studied the mechanism underlying the vasorelaxant effect of AVE 0991, a nonpeptide mimic of the effects of Ang(1-7), using wild-type and Mas-deficient mice. Ang(1-7) and AVE 0991 induced an equipotent concentration-dependent vasodilator effect in aortic rings from wild-type mice that was dependent on the presence of endothelium. The vasodilator effect of Ang(1-7) and AVE 0991 was completely blocked by 2 specific Ang(1-7) receptor antagonists, A-779 and D-Pro7-Ang(1-7), and by inhibition of NO synthase with L-NAME. Moreover, in aortic rings from Mas-deficient mice, the vasodilator effect of both Ang(1-7) and AVE 0991 was abolished. In contrast, the vasodilator effect of acetylcholine and substance P were preserved in Mas-null mice. In addition, the vasoconstriction effect induced by Ang II was slightly increased, and the vasodilation induced by the AT2 agonist CGP 42112A was not altered in Mas-deficient mice. Our results show that Ang(1-7) and AVE 0991 produced an NO-dependent vasodilator effect in the mouse aorta that is mediated by the G protein-coupled receptor Mas.

Evidence for a new angiotensin-(1–7) receptor subtype in the aorta of Sprague–Dawley rats ☆

We have recently described, in the mouse aorta, the vasodilator effect of angiotensin-(1-7) (Ang-(1-7)) was mediated by activation of the Mas Ang-(1-7) receptor and that A-779 and D-Pro7-Ang-(1-7) act as Mas receptor antagonists. In this work we show pharmacological evidence for the existence of a different Ang-(1-7) receptor subtype mediating the vasodilator effect of Ang-(1-7) in the aorta from Sprague-Dawley (SD) rats. Ang-(1-7) induced an endothelium-dependent vasodilator effect in aortic rings from SD rats which was inhibited by removal of the endothelium and by L-NAME (100 microM) but not by indomethacin (10 microM). The Ang-(1-7) receptor antagonist D-Pro7-Ang-(1-7) (0.1 microM) abolished the vasodilator effect of the peptide. However, the other specific Ang-(1-7) receptor antagonist, A-779 in concentrations up to 10 microM, did not affect vasodilation induced by Ang-(1-7). The Ang II AT1 and AT2 receptors antagonists CV11974 (0.01 microM) and PD123319 (1 microM), respectively, the bradykinin B2 receptor antagonist HOE 140 (1 microM) and the inhibitor of ACE captopril (10 microM) did not change the effect of Ang-(1-7). Our results show that in the aorta of SD rats, the vasodilator effect of Ang-(1-7) is dependent on endothelium-derived nitric oxide. This effect is mediated by the activation of Ang-(1-7) receptors sensitive to D-Pro7-Ang-(1-7), but not to A-779, which suggests the existence of a different Ang-(1-7) receptor subtype.

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.

Characterization of a New Selective Antagonist for Angiotensin-(1–7), d-Pro7-Angiotensin-(1–7)

Abstract—Angiotensin-(1–7) [Ang-(1–7)] has biological actions that can often be distinguished from those of angiotensin II (Ang II). Recent studies indicate that the effects of Ang-(1–7) are mediated by specific receptor(s). We now report the partial characterization of a new antagonist selective for Ang-(1–7), d-Pro7-Ang-(1–7). d-Pro7-Ang-(1–7) (50 pmol) inhibited the hypertensive effect induced by microinjection of Ang-(1–7) [4±1 vs 21±2 mm Hg, 25 pmol Ang-(1–7) alone] into the rostral ventrolateral medulla without changing the effect of Ang II (16±2.5 vs 19±2.5 mm Hg after 25 pmol Ang II alone). At 10−7 mol/L concentration, it completely blocked the endothelium-dependent vasorelaxation produced by Ang-(1–7) (10−10 to 10−6 mol/L) in the mouse aorta. The antidiuresis produced by Ang-(1–7) (40 pmol/100 g body weight) in water-loaded rats was also blocked by its analog [1 &mgr;g/100 g body weight; 3.08±0.8 vs 1.27±0.33 mL in Ang-(1–7)–treated rats]. d-Pro7-Ang-(1–7) at a molar ratio of 40:1 did not change the hypotensive effect of bradykinin. Moreover, d-Pro7-Ang-(1–7) did not affect the dipsogenic effect produced by intracerebroventricular administration of Ang II (11.4±1.15 vs 8.8±1.2 mL/h after Ang II) and did not show any demonstrable angiotensin-converting enzyme inhibitory activity in assays with the synthetic substrate Hip-His-Leu and rat plasma as a source of enzyme. Autoradiography studies with 125I–Ang-(1–7) in mouse kidney slices showed that d-Pro7-Ang-(1–7) competed for the binding of Ang-(1–7) to the cortical supramedullary region. In Chinese hamster ovary cells stably transfected with the AT1 receptor subtype, d-Pro7-Ang-(1–7) did not compete for the specific binding of 125I–Ang-II in concentrations up to 10−6 mol/L. There was also no significant displacement of Ang II binding to angiotensin type 2 receptors in membrane preparations of adrenal medulla. These data indicate that d-Pro7-Ang-(1–7) is a selective antagonist for Ang-(1–7), which can be useful to clarify the functional role of this heptapeptide.

Relative contribution of eNOS and nNOS to endothelium-dependent vasodilation in the mouse aorta.

In large vessels, endothelium-dependent vasodilation is mainly attributed to endothelial nitric oxide synthase (eNOS)-derived NO production. However, we have recently shown that neuronal nitric oxide synthase (nNOS)-derived H(2)O(2) is also an endothelium-dependent relaxing factor in the mouse aorta. The relative contribution of nNOS/eNOS, H(2)O(2)/NO remains to be characterized. This work was undertaken to determine the relative contribution of NO versus H(2)O(2), and eNOS versus nNOS to endothelium-dependent vasodilation in the mouse aorta. We used carbon microsensors placed next to the lumen of the vessels to simultaneously measure NO, H(2)O(2) and vascular tone. Acetylcholine produced a concentration-dependent increase in NO and H(2)O(2) production with a good coefficient of linearity with acetylcholine-induced relaxation (R(2)=0.93 and 0.96 for NO and H(2)O(2), respectively). L-NAME, a non-selective inhibitor of nitric oxide synthase, abolished NO and H(2)O(2) production, and impaired vasodilation. Selective pharmacological inhibition of nNOS with L-Arg(NO2)-L-Dbu-NH(2) 2TFA and specific knock-down of nNOS abrogated H(2)O(2) and decreased by half acetylcholine-induced vasodilation. Catalase, which specifically decomposes H(2)O(2), did not interfere with NO, but impaired H(2)O(2) and decreased vasodilation to the same level as those obtained with nNOS inhibition or knocking down. Specific knocking down of eNOS had no effect on H(2)O(2) production but greatly reduced NO and decreased vasodilation to levels similar to those found with nNOS inhibition. In eNOS knocked-down mice, pharmacological nNOS inhibition dramatically reduced H(2)O(2) production and further reduced the residual acetylcholine-induced vasodilation. It is concluded that nNOS/eNOS and H(2)O(2)/NO both contribute in a significant way to relaxation in the mouse aorta.

Alterations in Calcium Stores in Aortic Myocytes From Spontaneously Hypertensive Rats

The aim of the present work was to further characterize intracellular calcium stores released by angiotensin II (Ang II) in spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) vascular smooth muscle cells (VSMCs) and to study their alterations associated with proliferation. Intracellular Ca2+ concentration was monitored by image analysis in aortic myocytes loaded with fura 2. In the presence of extracellular Ca2+, sensitivity to Ang II in proliferating VSMCs was not different in the two strains, but it increased 10-fold in confluent VSMCs from SHR-compared with those from WKY. In Ca(2)+-free medium, Ca2+ release induced by thapsigargin (10 mumol/L) was significantly greater (about twofold) in SHR than WKY, in both proliferating and confluent cultures, with responses during proliferation being 0.7-fold smaller. Responses to Ang II were abolished after exposure of the cells to thapsigargin. In proliferating cultures, ryanodine (10 mumol/L) did not modify the rises in intracellular Ca2+ concentration induced by Ang II in VSMCs from both strains. Conversely, in confluent cultures, ryanodine reduced Ang II (100 nmol/L)-induced Ca2+ release to the same level as in proliferating cultures, and it suppressed the difference between SHR and WKY. These results show that the ryanodine-sensitive Ca2+ release induced by Ang II is enhanced in VSMCs from SHR at confluence and is impaired during proliferation. Thus, they suggest that differences in Ca2+(-)induced Ca2+ release from the sarcoplasmic reticulum may participate in increased responsiveness of VSMCs to Ang II in SHR and in phenotypic modulation of vascular myocytes during proliferation.

Phosphoinositide-3 Kinase γ Activity Contributes to Sepsis and Organ Damage by Altering Neutrophil Recruitment

RATIONALE Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase γ (PI3Kγ) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. OBJECTIVES This study aimed to determine the specific role of the kinase activity of PI3Kγ in the pathogenesis of sepsis and multiorgan damage. METHODS PI3Kγ wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation-induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3Kγ inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. MEASUREMENTS AND MAIN RESULTS Both genetic and pharmaceutical PI3Kγ kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3Kγ pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. CONCLUSIONS This study establishes PI3Kγ as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Decreased production of neuronal NOS-derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis

BACKGROUND AND PURPOSE Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)‐derived H2O2 is an important endothelium‐derived relaxant factor in the mouse aorta. The role of H2O2 and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS‐derived H2O2 contributes to the impaired vasodilatation in apolipoprotein E‐deficient mice (ApoE−/−).

Endothelium dysfunction in LDL receptor knockout mice: a role for H2O2.

In this study, the role of endogenous H2O2 as an endothelium‐dependent relaxant factor was characterised in aortas from C57BL/6J and LDL receptor‐deficient mice (LDLR−/−). Aortic rings from LDLR−/− mice showed impaired endothelium‐dependent relaxation to acetylcholine (ACh; 0.001–100 μM) and to the Ca2+ ionophore A23187 (0.001–3 μM) compared with aortic rings from control mice. Endothelium‐independent relaxation produced by the NO donor, 3‐morpholino‐sydnonimine (SIN‐1) was not different between strains. Pretreatment of vessels with L‐NNA (100 μM) or L‐NNA (100 μM) plus L‐NAME (300 μM) plus haemoglobin (10 μM) markedly decreased, but did not abolish the relaxation to ACh in control mice. In the aortas from LDLR−/− mice treated with L‐NNA (100 μM), ACh induced a contractile effect. Catalase (800 and 2400 U ml−1) shifted to the right the endothelium‐dependent relaxation to ACh in aortas from control but not from LDLR−/− mice. Aminotriazole (50 mM), which inhibits catalase, abolished its effect on control mice. Treatment of vessels with L‐NNA and catalase abolished vasorelaxation induced by ACh. Indomethacin (10 μM) did not modify the concentration–response curve to ACh. Superoxide dismutase (300 U ml−1) did not change ACh‐induced relaxation in both strains. Exogenous H2O2 produced a concentration‐dependent relaxation in endothelium‐denuded aortic rings, which was not different between strains. It is concluded that H2O2 greatly contributes to relaxation to ACh in aorta from control mice. Endothelial‐dependent relaxation to ACh is impaired in LDLR−/− mice. Reduced biosynthesis or increased inactivation of H2O2 is the possible mechanism responsible for endothelial dysfunction in aortas of atherosclerosis‐susceptible LDLR−/− mice.

Angiotensin‐(1‐7) is involved in the endothelium‐dependent modulation of phenylephrine‐induced contraction in the aorta of mRen‐2 transgenic rats

The contribution of the local vascular production of angiotensin‐(1‐7) [Ang‐(1‐7)] to the control of α‐adrenergic‐induced contractions in the aorta of Sprague‐Dawley (SD) and TGR(mRen‐2)27 [mRen‐2] rats was studied. In mRen‐2 rats, contractile responses to phenylephrine were diminished as compared to control SD rats in endothelium containing but not in endothelium‐denuded vessels. L‐NAME increased contractile responses to phenylephrine in mRen‐2 rats and, after nitric oxide synthase blockade, responses to phenylephrine became comparable in both strains. Inhibition of angiotensin‐converting enzyme (ACE) by captopril potentiated contractile responses in mRen‐2 rats and diminished contractile responses in SD rats, both effects being dependent on the presence of a functional endothelium. The effect of captopril in mRen‐2 rats was abolished in vessels pre‐incubated with Ang‐(1‐7). Blockade of Ang‐(1‐7) and bradykinin (BK) receptors by A‐779 and HOE 140 respectively, increased phenylephrine‐induced contraction in mRen‐2, but not in SD rats. This effect was seen only in endothelium‐containing vessels. Angiotensin II AT1 and AT2 receptor blockade by CV 11974 and PD 123319 did not affect the contractile responses to phenylephrine in aortas of transgenic animals but diminished the response in SD rats. This effect was only seen in the presence of a functional endothelium. It is concluded that the decreased contractile responses to phenylephrine in aortas of mRen‐2 rats was dependent on an intact endothelium, the local release and action of Ang‐(1‐7) and bradykinin.