Role of H(2)O(2) in hypertension, renin-angiotensin system activation and renal medullary disfunction caused by angiotensin II

BACKGROUND AND PURPOSE

Activation of the intrarenal renin-angiotensin system (RAS) and increased renal medullary hydrogen peroxide (H₂O₂) contribute to hypertension. We examined whether H₂O₂ mediated hypertension and intrarenal RAS activation induced by angiotensin II (Ang II).

EXPERIMENTAL APPROACH

Ang II (200 ng·kg⁻¹·min⁻¹) or saline were infused in Sprague Dawley rats from day 0 to day 14. Polyethylene glycol (PEG)-catalase (10 000 U·kg⁻¹·day⁻¹) was given to Ang II-treated rats, from day 7 to day 14. Systolic blood pressure was measured throughout the study. H₂O₂, angiotensin AT₁ receptor and Nox4 expression and nuclear factor-κB (NF-κB) activation were evaluated in the kidney. Plasma and urinary H₂O₂ and angiotensinogen were also measured.

KEY RESULTS

Ang II increased H₂O₂, AT₁ receptor and Nox4 expression and NF-κB activation in the renal medulla, but not in the cortex. Ang II raised plasma and urinary H₂O₂ levels, increased urinary angiotensinogen but reduced plasma angiotensinogen. PEG-catalase had a short-term antihypertensive effect and transiently suppressed urinary angiotensinogen. PEG-catalase decreased renal medullary expression of AT₁ receptors and Nox4 in Ang II-infused rats. Renal medullary NF-κB activation was correlated with local H₂O₂ levels and urinary angiotensinogen excretion. Loss of antihypertensive efficacy was associated with an eightfold increase of plasma angiotensinogen.

CONCLUSIONS AND IMPLICATIONS

The renal medulla is a major target for Ang II-induced redox dysfunction. H₂O₂ appears to be the key mediator enhancing intrarenal RAS activation and decreasing systemic RAS activity. The specific control of renal medullary H₂O₂ levels may provide future grounds for the treatment of hypertension.     

Chronic administration of BMS309403 improves endothelial function in apolipoprotein E-deficient mice and in cultured human endothelial cells

BACKGROUND AND PURPOSE

Adipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE^−/−) mice and in cultured human endothelial cells.

EXPERIMENTAL APPROACH

A-FABP was measured in aortae of ApoE^−/−mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortae

KEY RESULTS

A-FABP was expressed in aortic endothelium of ApoE^−/− mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective α₂-adrenoceptor agonist) and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE^−/− mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403.

CONCLUSIONS AND IMPLICATIONS

Elevated expression of A-FABP in endothelial cells contributes to their dysfunction both in vivo and in vitro.     

Protective effects of prostaglandin E1 on human umbilical vein endothelial cell injury induced by hydrogen peroxide

Aim:

To investigate the protective effects of prostaglandin E₁ (PGE₁) against H₂O₂-induced oxidative damage on human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were pretreated with PGE₁ (0.25, 0.50, and 1.00 μmol/L) for 24 h and exposed to H₂O₂ (200 μmol/L) for 12 h, and cell viability was measured by the MTT assay. LDH, NO, SOD, GSH-Px, MDA, ROS, and apoptotic percentage were determined. eNOS expression was measured by Western blotting and real-time PCR.

Results:

PGE₁ (0.25−1.00 μmol/L) was able to markedly restore the viability of HUVECs under oxidative stress, and scavenged intracellular reactive oxygen species induced by H₂O₂. PGE₁ also suppressed the production of lipid peroxides, such as MDA, restored the activities of endogenous antioxidants including SOD and GSH-Px, and inhibited cell apoptosis. In addition, PGE₁ significantly increased NO content, eNOS protein, and mRNA expression.

Conclusion:

PGE₁ effectively protected endothelial cells against oxidative stress induced by H₂O₂, an activity that might depend on the up-regulation of NO expression.     

Telmisartan, ramipril and their combination improve endothelial function in different tissues in a murine model of cholesterol-induced atherosclerosis

BACKGROUND AND PURPOSE

Erectile dysfunction correlates with cardiovascular disease and its common risk factors due to the development of endothelial dysfunction. Positive effects on endothelial and erectile function have been described for substances inhibiting the renin-angiotensin-system. Here, we investigated in an atherosclerosis model, whether telmisartan (angiotensin receptor blocker) and ramipril (angiotensin converting enzyme inhibitor) are equivalent or the combination of both is superior in improving endothelial function in the aorta and the corpus cavernosum and in reducing atherosclerosis.

EXPERIMENTAL APPROACH

Wild-type (WT, C57/B6) and apolipoprotein-E-deficient (ApoE^−/−) mice were treated with a cholesterol-rich diet for 8 weeks. ApoE^−/− mice were supplemented with either telmisartan (20 mg·kg⁻¹·day⁻¹), ramipril (2.5 mg·kg⁻¹·day⁻¹) or the combination thereof.

KEY RESULTS

Systolic blood pressure significantly decreased in treatment groups (P < 0.001), with significantly smaller reduction under ramipril monotherapy (P < 0.05). Endothelial function (assessed by pharmacological stimulation of aortic rings and corpus cavernosum in organ bath chambers) was impaired in ApoE^−/− mice compared to WT animals, which was improved by all three treatments to a comparable extent (P < 0.05). Atherosclerotic lesion size in the ascending aorta and aortic sinus (P < 0.001), the amount of lipid peroxides in cavernosal and aortic tissue (P < 0.05) and free radical load (dihydroethidium-stain) (P < 0.05) were enhanced in untreated ApoE^−/− mice in comparison to WT animals and were significantly reduced by either treatment. In penile tissue, expression of eNOS could be restored by renin-angiotensin-aldosterone system blockade.

CONCLUSIONS AND IMPLICATIONS

Telmisartan and ramipril significantly improved endothelial function of aortic and cavernosal tissues in ApoE^−/− via reduction of oxidative stress. Combination of both agents does not enhance beneficial effects significantly.     

β3-Adrenoceptor activation attenuates atherosclerotic plaque formation in ApoE?/? mice through lowering blood lipids and glucose

Aim:

To examine the effects of β₃-adrenoceptor (β₃-AR) activation on atherosclerotic plaque development in ApoE^−/− mice.

Methods:

Thirty six week-old male ApoE^−/− mice on a high-fat diet were treated with atorvastatin (10 mg·kg^-1·d^-1, po), BRL37344 (β₃-AR agonist, 1.65 or 3.30 μg/kg, ip, twice a week) or SR52390A (β₃-AR antagonist, 50 μg/kg, ip, twice a week) for 12 weeks. Wild-type C57BL/6J mice receiving a normal diet were taken as healthy controls. At the end of the treatments, serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-high density lipoprotein cholesterol (nHDL-C), glucose and insulin were measured. The thoracic aortas were dissected out, the area of atherosclerotic plaques and extent of fibrosis in the plaques were examined using HE and Masson''s trichome staining, respectively.

Results:

Compared to wild-type mice, ApoE^−/− mice fed on a high-fat diet exhibited prominent hyperlipidemia and insulin resistance, associated with large area of atherosclerotic plaques and great extent of fibrosis in aortas. Atorvastatin significantly decreased the serum levels of TC and nHDL-C, and reduced the plaque area and collagen content in aortas. BRL37344 significantly decreased the serum levels of TG, TC, nHDL-C, glucose and insulin, and increased HDL-C and the insulin sensitivity, and dose-dependently reduced the plaque area and collagen content in aortas. SR52390A treatment did not affect any parameters studied.

Conclusion:

The β₃-AR agonist impedes the progression of atherosclerosis in ApoE^−/− mice, through improvement of the lipid and glucose profiles.     

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

Aim:

To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.

Methods:

Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na⁺/H⁺ exchanger 1 (NHE1) and calpain, intracellular free Ca²⁺level ([Ca²⁺]_i), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE^−/−) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.

Results:

LPS treatment increased NHE1 activity and [Ca²⁺]_i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca²⁺-dependent protease calpain. Amiloride (1−10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca²⁺]_i. and calpain activity. In the presence of the Ca²⁺ chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE^−/− mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.

Conclusion:

LPS stimulates NHE1 activity, increases [Ca²⁺]_i, and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.     

A role for neuropeptide Y in the gender-specific gastrointestinal, corticosterone and feeding responses to stress

BACKGROUND AND PURPOSE

Exposure to an acute stress inhibits gastric emptying and stimulates colonic transit via central neuropeptide Y (NPY) pathways; however, peripheral involvement is uncertain. The anxiogenic phenotype of NPY^−/− mice is gender-dependent, raising the possibility that stress-induced gastrointestinal (GI) responses are female-dominant through NPY. The aim of this study was to determine GI transit rates, corticosterone levels and food intake after acute restraint (AR) or novel environment (NE) stress in male and female NPY^−/− and WT mice.

EXPERIMENTAL APPROACH

Upper gastrointestinal transit (UGIT) (established 30 min after oral gavage) and corticosterone levels were determined under basal or restrained conditions (30 min) and after treatment i.p. with Y₁ antagonist BIBO3304 or Y₂ antagonist BIIE0246. Faecal pellet output (FPO) was established after AR and treatment i.p. with NPY in the NE, as were colonic bead expulsion rates.

KEY RESULTS

UGIT and FPO were similar in unrestrained male and female mice. NPY^−/− females displayed significantly slower UGIT than NPY^−/− males after AR, but both genders displayed significantly higher FPO and reduced food intake relative to WT counterparts. Peripheral NPY treatment increased bead expulsion time in WT mice. AR male NPY^−/− mice had higher levels of corticosterone than male WT mice; whilst in AR WT mice, after peripheral Y₁ and Y₂ receptor antagonism in males, and Y₂ antagonism in females, corticosterone was significantly elevated.

CONCLUSIONS AND IMPLICATIONS

NPY possesses a role in the gender-dependent susceptibility to stress-induced GI responses. Furthermore, NPY inhibits GI motility through Y₂ receptors and corticosterone release via peripheral Y₁ and Y₂ receptors.     

Simvastatin suppresses vascular inflammation and atherosclerosis in ApoE?/? mice by downregulating the HMGB1-RAGE axis

Aim:

High mobility group box protein 1 (HMGB1) and receptor for the advanced glycation end product (RAGE) play pivotal roles in vascular inflammation and atherosclerosis. The aim of this study was to determine whether the HMGB1-RAGE axis was involved in the actions of simvastatin on vascular inflammation and atherosclerosis in ApoE^−/− mice.

Methods:

Five-week old ApoE^−/− mice and wild-type C57BL/6 mice were fed a Western diet. At 8 weeks of age, ApoE^−/− mice were administered simvastatin (50 mg·kg⁻¹·d⁻¹) or vehicle by gavage, and the wild-type mice were treated with vehicle. The mice were sacrificed at 11 weeks of age, and the atherosclerotic lesions in aortic sinus were assessed with Oil Red O staining. Macrophage migration was determined with scanning EM and immunohistochemistry. Human umbilical vein endothelial cells (HUVECs) were used for in vitro study. Western blots were used to quantify the protein expression of HMGB1, RAGE, vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1).

Results:

Vehicle-treated ApoE^−/− mice exhibited significant increases in aortic inflammation and atherosclerosis as well as enhanced expression of HMGB1, RAGE, VCAM-1, and MCP-1 in aortic tissues as compared to the wild-type mice. Furthermore, serum total cholesterol, triglyceride and LDL levels were markedly increased, while serum HDL level was decreased in vehicle-treated ApoE^−/− mice. Administration with simvastatin in ApoE^−/− mice markedly attenuated the vascular inflammation and atherosclerotic lesion area, and decreased the aortic expression of HMGB1, RAGE, VCAM-1, and MCP-1. However, simvastatin did not affect the abnormal levels of serum total cholesterol, triglyceride, LDL and HDL in ApoE^−/− mice. Exposure of HUVECs to HMGB1 (100 ng/mL) markedly increased the expression of HMGB1, RAGE and VCAM-1, whereas pretreatment of the cells with simvastatin (10 μmol/L) blocked the HMGB1-caused changes.

Conclusion:

Simvastatin inhibits vascular inflammation and atherosclerosis in ApoE^−/− mice, which may be mediated through downregulation of the HMGB1-RAGE axis.     

Amplified NO/cGMP-mediated relaxation and ryanodine receptor-to-BKCa channel signalling in corpus cavernosum smooth muscle from phospholamban knockout mice

BACKGROUND AND PURPOSE

Relaxation of corpus cavernosum smooth muscle (CCSM) is induced by NO. NO promotes the formation of cGMP, which activates cGMP-dependent protein kinase I (PKGI). The large conductance calcium-activated potassium (BK_Ca) channel is regarded as a major target of NO/cGMP signalling; however, the mechanism of BK_Ca activation remains unclear. The aim of the present study was to determine whether sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ release from the SR via ryanodine receptors (RyRs) is important for BK_Ca channel activation in response to NO/cGMP.

EXPERIMENTAL APPROACH

In vitro myography was performed on CCSM strips from wild-type and PLB knockout (PLB^−/−) mice to evaluate contraction and relaxation in response to pharmacological agents and electrical field stimulation (EFS).

KEY RESULTS

In CCSM strips from PLB^−/− mice, a model of increased SR Ca²⁺ load, contractile force in response to EFS or phenylephrine (PE) was increased by nearly 100%. EFS of strips precontracted with PE induced transient relaxation in CCSM, an effect that was significantly larger in PLB^−/− strips. Likewise, the relaxation of PE-induced contraction in response to SNP and cGMP was greater in PLB^−/−, as demonstrated by a shift in the concentration–response curve towards lower concentrations. Blocking RyRs and BK_Ca channels diminished the induced relaxations and eliminated the difference between wild-type and PLB^−/−.

CONCLUSIONS AND IMPLICATIONS

NO/cGMP activates BK_Ca channels through RyR-mediated Ca²⁺ release. This signalling pathway is responsible for approximately 40% of the NO/cGMP effects and is amplified by increased SR Ca²⁺ concentrations.     

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

BACKGROUND AND PURPOSE

One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O₂^•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.

EXPERIMENTAL APPROACH

HUVECs were incubated with ox-LDL with/without pyridoxine, N^G-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O₂^•− was measured using lucigenin chemiluminescence, and O₂^•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.

KEY RESULTS

Ox-LDL significantly increased O₂^•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O₂^•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.

CONCLUSIONS AND IMPLICATIONS

Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.     

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

Background and purpose:

P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Experimental approach:

mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE^–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE^–/– mice.

Key results:

P2Y₆ receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y₂ receptor expression remained unchanged. Expression of P2Y₁ or P2Y₄ receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y₆ mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y₆-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y₆ receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y₆-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE^–/– mice with suramin or PPADS (50 and 25 mg·kg⁻¹·day⁻¹ respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.

Conclusions and implications:

These results suggest involvement of nucleotide receptors, particularly P2Y₆ receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y₆ receptor-deficient mice.     

Oxidative species increase arginase activity in endothelial cells through the RhoA/Rho kinase pathway

BACKGROUND AND PURPOSE

NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available l-arginine, reducing NO and increasing superoxide (O₂^.−) production via NOS uncoupling. Elevated O₂^.− combines with NO to form peroxynitrite (ONOO^-), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide-induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase (ROCK) pathway.

EXPERIMENTAL APPROACH

Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO^- generator (SIN-1) or H₂O₂. Pretreatment with inhibitors of Rho kinase (Y-27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation.

KEY RESULTS

Exposure to SIN-1 (25 µM, 24 h) or H₂O₂ (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Gö6976 or siRNA to p115-Rho GEF. There was an early activation of p115-Rho GEF (SIN-1, 2 h; H₂O₂, 1 h) and Rho A (SIN-1, 4 h; H₂O₂, 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN-1 and H₂O₂ also reduced NOS activity, which was blocked by pretreatment with p115-RhoGEF siRNA.

CONCLUSIONS AND IMPLICATIONS

Our data indicate that the oxidative species ONOO^- and H₂O₂ increase arginase activity/expression through PKC-mediated activation of RhoA/Rho kinase pathway.     

Chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice

BACKGROUND AND PURPOSE

ApolipoproteinE-deficient [apoE (−/−)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (−/−) mice.

EXPERIMENTAL APPROACH

ApoE (−/−) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg⁻¹·h⁻¹) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg⁻¹·h⁻¹) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys.

KEY RESULTS

Ang-(1-7)-treated apoE (−/−) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (−/−) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (−/−) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased.

CONCLUSION AND IMPLICATIONS

Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.     

The dual PPARα/γ agonist aleglitazar increases the number and function of endothelial progenitor cells: implications for vascular function and atherogenesis

Background and Purpose

Aleglitazar is a dual PPARα/γ agonist but little is known about its effects on vascular function and atherogenesis. Hence, we characterized its effects on circulating angiogenic cells (CAC), neoangiogenesis, endothelial function, arteriogenesis and atherosclerosis in mice.

Experimental Approach

C57Bl/6 wild-type (WT, normal chow), endothelial NOS (eNOS)^−/− (normal chow) and ApoE^−/− (Western-type diet) mice were treated with aleglitazar (10 mg·kg⁻¹·day⁻¹, i.p.) or vehicle.

Key Results

Aleglitazar enhanced expression of PPARα and PPARγ target genes, normalized glucose tolerance and potently reduced hepatic fat in ApoE^−/− mice. In WT mice, but not in eNOS^−/−, aleglitazar up-regulated Sca-1/VEGFR2-positive CAC in the blood and bone marrow and up-regulated diLDL/lectin-positive CAC. Aleglitazar augmented CAC migration and enhanced neoangiogenesis. In ApoE^−/− mice, aleglitazar up-regulated CAC number and function, reduced markers of vascular inflammation and potently improved perfusion restoration after hindlimb ischaemia and aortic endothelium-dependent vasodilatation. This was associated with markedly reduced formation of atherosclerotic plaques. In human cultured CAC from healthy donors and patients with coronary artery disease with or without diabetes mellitus, aleglitazar increased migration and colony-forming units in a concentration-dependent manner. Furthermore, oxidative stress-induced CAC apoptosis and expression of p53 were reduced, while telomerase activity and expression of phospho-eNOS and phospho-Akt were elevated. Comparative agonist and inhibitor experiments revealed that aleglitazar''s effects on CAC migration and colony-forming units were mediated by both PPARα and PPARγ signalling and required Akt.

Conclusions and Implications

Aleglitazar augments the number, function and survival of CAC, which correlates with improved vascular function, enhanced arteriogenesis and prevention of atherosclerosis in mice.     

Increased endothelin-1 vasoconstriction in mesenteric resistance arteries after superior mesenteric ischaemia-reperfusion

BACKGROUND AND PURPOSE

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone. We aimed to evaluate the influence of superior mesenteric artery (SMA) ischaemia-reperfusion (I/R) on mesenteric resistance artery vasomotor function and the mechanism involved in the changes in vascular responses to ET-1.

EXPERIMENTAL APPROACH

SMA from male Sprague-Dawley rats was occluded (90 min) and following reperfusion (24 h), mesenteric resistance arteries were dissected. Vascular reactivity was studied using wire myography. Protein and mRNA expression, superoxide anion (O₂^•−) production and ET-1 plasma concentration were evaluated by immunofluorescence, real-time quantitative PCR, ethidium fluorescence and elisa, respectively.

KEY RESULTS

I/R increased ET-1 plasma concentration, ET-1-mediated vasoconstriction and ET_B mRNA expression, and down-regulated ET_A mRNA expression. Immunofluorescence confirmed mRNA results and revealed an increase in ET_B receptors in the mesenteric resistance artery media layer after I/R. Therefore, the ET_B receptor agonist sarafotoxin-6 induced a contraction that was inhibited by the ET_B receptor antagonist BQ788 only in vessels, with and without endothelium, from I/R rats. Furthermore, BQ788 potentiated ET-1 vasoconstriction only in sham rats. Endothelium removal in rings from I/R rats unmasked the inhibition of ET-1 vasoconstriction by BQ788. Endothelium removal, N^ω-nitro-L-arginine methyl ester and superoxide dismutase abolished the differences in ET-1 vasoconstriction between sham and I/R rats. We also found that I/R down-regulates endothelial NOS mRNA expression and concomitantly enhanced O₂^•− production by increasing NADPH oxidase 1 (NOX-1) and p^47phox mRNA.

CONCLUSIONS AND IMPLICATIONS

Mesenteric I/R potentiated the ET-1-mediated vasoconstriction by a mechanism that involves up-regulation of muscular ET_B receptors and decrease in NO bioavailability.     

Loss of multidrug and toxin extrusion 1 (MATE1) is associated with metformin-induced lactic acidosis

BACKGROUNDS AND PURPOSE

Lactic acidosis is a fatal adverse effect of metformin, but the risk factor remains unclear. Multidrug and toxin extrusion 1 (MATE1) is expressed in the luminal membrane of the kidney and liver. MATE1 was revealed to be responsible for the tubular and biliary secretion of metformin. Therefore, some MATE polymorphisms, that cause it to function abnormally, are hypothesized to induce lactic acidosis. The purpose of this study is to clarify the association between MATE dysfunction and metformin-induced lactic acidosis.

EXPERIMENTAL APPROACH

Blood lactate, pH and bicarbonate ion (HCO₃^-) levels were evaluated during continuous administration of 3 mg·mL⁻¹ metformin in drinking water using Mate1 knockout (−/−), heterozygous (+/−) and wild-type (+/+) mice. To determine the tissue accumulation of metformin, mice were given 400 mg·kg⁻¹ metformin orally. Furthermore, blood lactate data were obtained from diabetic patients given metformin.

KEY RESULTS

Seven days after metformin administration in drinking water, significantly higher blood lactate, lower pH and HCO₃^- levels were observed in Mate1^−/− mice, but not in Mate1^+/− mice. The blood lactate levels were not affected in patients with the heterozygous MATE variant (MATE1-L125F, MATE1-G64D, MATE2-K-G211V). Sixty minutes after metformin administration (400 mg·kg⁻¹, p.o.) the hepatic concentration of metformin was markedly higher in Mate1^−/− mice than in Mate1^+/+ mice.

CONCLUSION AND IMPLICATIONS

MATE1 dysfunction caused a marked elevation in the metformin concentration in the liver and led to lactic acidosis, suggesting that the homozygous MATE1 variant could be one of the risk factors for metformin-induced lactic acidosis.     

Apolipoprotein A-I inhibits LPS-induced atherosclerosis in ApoE?/? mice possibly via activated STAT3-mediated upregulation of tristetraprolin

Aim:

To investigate the effects of the major component of high-density lipoprotein apolipoprotein A-I (apoA-I) on the development of atherosclerosis in LPS-challenged ApoE^−/− mice and the underlying mechanisms.

Methods:

Male ApoE-KO mice were daily injected with LPS (25 μg, sc) or PBS for 4 weeks. The LPS-challenged mice were intravenously injected with rAAV-apoA-I-GFP or rAAV-GFP. After the animals were killed, blood, livers and aortas were collected for biochemical and histological analyses. For ex vivo experiments, the abdominal cavity macrophages were harvested from each treatment group of mice, and cultured with autologous serum, then treated with LPS.

Results:

Chronic administration of LPS in ApoE^−/− mice significantly increased the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), increased infiltration of inflammatory cells, and enhanced the development of atherosclerosis. In LPS-challenged mice injected with rAAV-apoA-I-GFP, viral particles and human apoA-I were detected in the livers, total plasma human apoA-I levels were grammatically increased; HDL-cholesterol level was significantly increased, TG and TC were slightly increased. Furthermore, overexpression of apoA-I significantly suppressed the expression of proinflammatory cytokines, reduced the infiltration of inflammatory cells, and decreased the extent of atherosclerotic lesions. Moreover, overexpression of apoA-I significantly increased the expression of the cytokine mRNA-destabilizing protein tristetraprolin (TTP), and phosphorylation of JAK2 and STAT3 in aortas. In ex vivo mouse macrophages, the serum from mice overexpressing apoA-I significantly increased the expression of TTP, accompanied by accelerated decay of mRNAs of the inflammatory cytokines.

Conclusion:

ApoA-I potently suppresses LPS-induced atherosclerosis by inhibiting the inflammatory response possibly via activation of STAT3 and upregulation of TTP.     

Hydrogen peroxide affects contractile activity and anti-oxidant enzymes in rat uterus

Background and purpose:

The effects of hydrogen peroxide (H₂O₂) on uterine smooth muscle are not well studied. We have investigated the effect and the mechanism of action of exogenous hydrogen peroxide on rat uteri contractile activity [spontaneous and calcium ion (Ca²⁺)-induced] and the effect of such treatment on anti-oxidative enzyme activities.

Experimental approach:

Uteri were isolated from virgin Wistar rats and suspended in an organ bath. Uteri were allowed to contract spontaneously or in the presence of Ca²⁺ (6 mM) and treated with H₂O₂ (2 µM–3 mM) over 2 h. Anti-oxidative enzyme activities (manganese superoxide dismutase-MnSOD, copper-zinc superoxide dismutase-CuZnSOD, catalase-CAT, glutathione peroxidase-GSHPx and glutathione reductase-GR) in H₂O₂-treated uteri were compared with those in uteri immediately frozen after isolation or undergoing spontaneous or Ca²⁺-induced contractions, without treatment with H₂O₂. The effect of inhibitors (propranolol, methylene blue, L-NAME, tetraethylamonium, glibenclamide and 4-aminopyridine) on H₂O₂-mediated relaxation was explored.

Key results:

H₂O₂ caused concentration-dependent relaxation of both spontaneous and Ca²⁺-induced uterine contractions. After H₂O₂ treatment, GSHPx and MnSOD activities were increased, while CuZnSOD and GR (In Ca²⁺-induced rat uteri) were decreased. N^ω-nitro-L-arginine methyl ester antagonized the effect of H₂O₂ on Ca²⁺-induced contractions. H₂O₂-induced relaxation was not affected by propranolol, potentiated by methylene blue and antagonized by tetraethylamonium, 4-aminopyridine and glibenclamide, with the last compound being the least effective.

Conclusions and implications:

H₂O₂ induced dose-dependent relaxation of isolated rat uteri mainly via changes in voltage-dependent potassium channels. Decreasing generation of reactive oxygen species by stimulation of anti-oxidative pathways may lead to new approaches to the management of dysfunctional uteri.