High intraluminal pressure promotes vascular inflammation via caveolin-1.

The aetiology and progression of hypertension involves various endogenous systems, such as the renin angiotensin system, the sympathetic nervous system, and endothelial dysfunction. Recent data suggest that vascular inflammation may also play a key role in the pathogenesis of hypertension. This study sought to determine whether high intraluminal pressure results in vascular inflammation. Leukocyte adhesion was assessed in rat carotid arteries exposed to 1 h of high intraluminal pressure. The effect of intraluminal pressure on signaling mechanisms including reactive oxygen species production (ROS), arginase expression, and NFĸB translocation was monitored. 1 h exposure to high intraluminal pressure (120 mmHg) resulted in increased leukocyte adhesion and inflammatory gene expression in rat carotid arteries. High intraluminal pressure also resulted in a downstream signaling cascade of ROS production, arginase expression, and NFĸB translocation. This process was found to be angiotensin II-independent and mediated by the mechanosensor caveolae, as caveolin-1 (Cav1)-deficient endothelial cells and mice were protected from pressure-induced vascular inflammatory signaling and leukocyte adhesion. Cav1 deficiency also resulted in a reduction in pressure-induced glomerular macrophage infiltration in vivo. These findings demonstrate Cav1 is an important mechanosensor in pressure-induced vascular and renal inflammation.

Serum Amyloid A Stimulates Vascular and Renal Dysfunction in Apolipoprotein E-Deficient Mice Fed a Normal Chow Diet.

Elevated serum amyloid A (SAA) levels may promote endothelial dysfunction, which is linked to cardiovascular and renal pathologies. We investigated the effect of SAA on vascular and renal function in apolipoprotein E-deficient (ApoE-/-) mice. Male ApoE-/- mice received vehicle (control), low-level lipopolysaccharide (LPS), or recombinant human SAA by i.p. injection every third day for 2 weeks. Heart, aorta and kidney were harvested between 3 days and 18 weeks after treatment. SAA administration increased vascular cell adhesion molecule (VCAM)-1 expression and circulating monocyte chemotactic protein (MCP)-1 and decreased aortic cyclic guanosine monophosphate (cGMP), consistent with SAA inhibiting nitric oxide bioactivity. In addition, binding of labeled leukocytes to excised aorta increased as monitored using an ex vivo leukocyte adhesion assay. Renal injury was evident 4 weeks after commencement of SAA treatment, manifesting as increased plasma urea, urinary protein, oxidized lipids, urinary kidney injury molecule (KIM)-1 and multiple cytokines and chemokines in kidney tissue, relative to controls. Phosphorylation of nuclear-factor-kappa-beta (NFκB-p-P65), tissue factor (TF), and macrophage recruitment increased in kidneys from ApoE-/- mice 4 weeks after SAA treatment, confirming that SAA elicited a pro-inflammatory and pro-thrombotic phenotype. These data indicate that SAA impairs endothelial and renal function in ApoE-/- mice in the absence of a high-fat diet.

NFκB Inhibition Mitigates Serum Amyloid A-Induced Pro-Atherogenic Responses in Endothelial Cells and Leukocyte Adhesion and Adverse Changes to Endothelium Function in Isolated Aorta.

The acute phase protein serum amyloid A (SAA) is associated with endothelial dysfunction and early-stage atherogenesis. Stimulation of vascular cells with SAA increases gene expression of pro-inflammation cytokines and tissue factor (TF). Activation of the transcription factor, nuclear factor kappa-B (NFκB), may be central to SAA-mediated endothelial cell inflammation, dysfunction and pro-thrombotic responses, while targeting NFκB with a pharmacologic inhibitor, BAY11-7082, may mitigate SAA activity. Human carotid artery endothelial cells (HCtAEC) were pre-incubated (1.5 h) with 10 µM BAY11-7082 or vehicle (control) followed by SAA (10 µg/mL; 4.5 h). Under these conditions gene expression for TF and Tumor Necrosis Factor (TNF) increased in SAA-treated HCtAEC and pre-treatment with BAY11-7082 significantly (TNF) and marginally (TF) reduced mRNA expression. Intracellular TNF and interleukin 6 (IL-6) protein also increased in HCtAEC supplemented with SAA and this expression was inhibited by BAY11-7082. Supplemented BAY11-7082 also significantly decreased SAA-mediated leukocyte adhesion to apolipoprotein E-deficient mouse aorta in ex vivo vascular flow studies. In vascular function studies, isolated aortic rings pre-treated with BAY11-7082 prior to incubation with SAA showed improved endothelium-dependent vasorelaxation and increased vascular cyclic guanosine monophosphate (cGMP) content. Together these data suggest that inhibition of NFκB activation may protect endothelial function by inhibiting the pro-inflammatory and pro-thrombotic activities of SAA.

Nitroxyl (HNO) reduces endothelial and monocyte activation and promotes M2 macrophage polarization.

Nitroxyl anion (HNO) donors are currently being assessed for their therapeutic utility in several cardiovascular disorders including heart failure. Here, we examine their effect on factors that precede atherosclerosis including endothelial cell and monocyte activation, leucocyte adhesion to the endothelium and macrophage polarization. Similar to the NO donor glyceryl trinitrate (GTN), the HNO donors Angeli's salt (AS) and isopropylamine NONOate (IPA/NO) decreased leucocyte adhesion to activated human umbilical vein endothelial cells (HUVECs) and mouse isolated aorta. This reduction in adhesion was accompanied by a reduction in intercellular adhesion molecule-1 (ICAM-1) and the cytokines monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) which was inhibitor of nuclear factor κB (NFκB) α (IκBα)- and subsequently NFκB-dependent. Intriguingly, the effects of AS on leucocyte adhesion, like those on vasodilation, were found to not be susceptible to pharmacological tolerance, unlike those observed with GTN. As well, HNO reduces monocyte activation and promotes polarization of M2 macrophages. Taken together, our data demonstrate that HNO donors can reduce factors that are associated with and which precede atherosclerosis and may thus be useful therapeutically. Furthermore, since the effects of the HNO donors were not subject to tolerance, this confers an additional advantage over NO donors.

High-density lipoprotein inhibits human M1 macrophage polarization through redistribution of caveolin-1.

BACKGROUND AND PURPOSE: Monocyte-derived macrophages are critical in the development of atherosclerosis and can adopt a wide range of functional phenotypes depending on their surrounding milieu. High-density lipoproteins (HDLs) have many cardio-protective properties including potent anti-inflammatory effects. We investigated the effects of HDL on human macrophage phenotype and the mechanisms by which these occur. EXPERIMENTAL APPROACH: Human blood monocytes were differentiated into macrophages in the presence or absence of HDL and were then induced to either an inflammatory macrophage (M1) or anti-inflammatory macrophage (M2) phenotype using LPS and IFN-γ or IL-4, respectively. KEY RESULTS: HDL inhibited the induction of macrophages to an M1-phenotype, as evidenced by a decrease in the expression of M1-specific cell surface markers CD192 and CD64, as well as M1-associated inflammatory genes TNF-α, IL-6 and MCP-1 (CCL2). HDL also inhibited M1 function by reducing the production of ROS. In contrast, HDL had no effect on macrophage induction to the M2-phenotype. Similarly, methyl-ß-cyclodextrin, a non-specific cholesterol acceptor also suppressed the induction of M1 suggesting that cholesterol efflux is important in this process. Furthermore, HDL decreased membrane caveolin-1 in M1 macrophages. We confirmed that caveolin-1 is required for HDL to inhibit M1 induction as bone marrow-derived macrophages from caveolin-1 knockout mice continued to polarize into M1-phenotype despite the presence of HDL. Moreover, HDL decreased ERK1/2 and STAT3 phosphorylation in M1 macrophages. CONCLUSIONS AND IMPLICATIONS: We concluded that HDL reduces the induction of macrophages to the inflammatory M1-phenotype via redistribution of caveolin-1, preventing the activation of ERK1/2 and STAT3.

Nitroxyl: a vasodilator of human vessels that is not susceptible to tolerance.

Pre-clinical studies have identified nitroxyl (HNO), the reduced congener of nitric oxide (NO•), as a potent vasodilator which is resistant to tolerance development. The present study explores the efficacy of HNO in human blood vessels and describes, for the first time, a vasodilator for humans that is not susceptible to tolerance. Human radial arteries and saphenous veins were obtained from patients undergoing coronary artery graft surgery and mounted in organ baths. Repeated vasodilator responses to the HNO donor Angeli's salt (AS) and NO• donor glyceryl trinitrate (GTN) were determined. AS- and GTN-induced concentration-dependent vasorelaxation of both human radial arteries (AS pEC50: 6.5 ± 0.2; -log M) and saphenous veins (pEC50: 6.7 ± 0.1) with similar potency. In human radial arteries, GTN-induced relaxation was reduced by the NO• scavenger hydroxocobalamin (HXC; P<0.05) but was unaffected by the HNO scavenger L-cysteine. Alternately, AS was unaffected by HXC but was reduced by L-cysteine (5-fold shift, P<0.05). The sGC (soluble guanylate cyclase) inhibitor ODQ abolished responses to both AS and GTN in arteries and veins (P<0.05). Inhibition of voltage-dependent potassium channels (Kv channels) with 4-AP also significantly reduced responses to AS (pEC50: 5.5) and GTN, suggesting that the relaxation to both redox congeners is cGMP- and Kv channel-dependent. Critically, a concentration-dependent development of tolerance to GTN (1 and 10 µM; P<0.05), but not to AS, was observed in both saphenous veins and radial arteries. Like GTN, the HNO donor AS causes vasorelaxation of human blood vessels via activation of a cGMP-dependent pathway. Unlike GTN, however, it does not develop tolerance in human blood vessels.

Raised soluble P-selectin moderately accelerates atherosclerotic plaque progression.

Soluble P-selectin (sP-selectin), a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe-/- mice placed on a high fat diet (HFD) were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day) for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe-/- mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe-/- or SM22α-hDTR Apoe-/- mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe-/- HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe-/- HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.

Septic shock: desperately seeking treatment.

Septic shock results from the dysregulation of the innate immune response following infection. Despite major advances in fundamental and clinical research, patients diagnosed with septic shock still have a poor prognostic outcome, with a mortality rate of up to 50%. Indeed, the reasons leading to septic shock are still poorly understood. First postulated 30 years ago, the general view of septic shock as an acute and overwhelming inflammatory response still prevails today. Recently, the fact that numerous clinical trials have failed to demonstrate any positive medical outcomes has caused us to question our fundamental understanding of this condition. New and sophisticated technologies now allow us to accurately profile the various stages and contributory components of the inflammatory response defining septic shock, and many studies now report a more complex inflammatory response, particularly during the early phase of sepsis. In addition, novel experimental approaches, using more clinically relevant animal models, to standardize and stratify research outcomes are now being argued for. In the present review, we discuss the most recent findings in relation to our understanding of the underlying mechanisms involved in septic shock, and highlight the attempts made to improve animal experimental models. We also review recent studies reporting promising results with two vastly different therapeutic approaches influencing the renin-angiotensin system and applying mesenchymal stem cells for clinical intervention.

Interleukin 10 antioxidant effect decreases leukocytes/endothelial interaction induced by tumor necrosis factor α.

Little is known about the endothelial mechanisms involved in the anti-inflammatory effects of interleukin 10 (IL-10). The goal of this study was to evaluate the effects of IL-10 on endothelial oxidative stress and endothelial inflammation induced by tumor necrosis factor α (TNF-α). Production of reactive oxygen species (ROS) in perfused human umbilical vein endothelial cells (HUVECs) was studied by fluorescent microscopy using dichlorodihydrofluorescein diacetate. Tumor necrosis factor α (1 ng/mL) was added to the perfusion medium in the absence and presence of IL-10 (1 ng/mL). The role of phosphatidylinositol 3-kinase (PI3-kinase) was assessed using wortmannin and LY 2940002 (inhibitors of PI3-kinase). Specific inhibition of p110 α and p110 γ/δ PI3-kinase subunits was studied using A66 and TG100-115. As well, levels of ceramide and intercellular adhesion molecule 1 (ICAM-1) expression were measured. Finally, the effect of IL-10 on TNF-α-induced leukocyte/endothelium interaction was examined using an ex vivo perfused vessel model. Interleukin 10 significantly reduced dichlorodihydrofluorescein diacetate fluorescence induced by TNF-α in HUVECs (12.5% ± 3.2% vs. 111.7% ± 21.6% at 60 min). Pretreatment by LY2940002 or wortmannin restored ROS production induced by TNF-α in the presence of IL-10. In HUVECs treated by TNF-α + IL-10, inhibition of p110 α PI3-kinase subunit significantly increased ROS production, whereas p110 γ/δ inhibition did not have a significant effect. Pretreatment with IL-10 significantly decreased TNF-α-induced increased levels of ceramide (TNF-α vs. TNF-α + IL-10: 6,278 ± 1,013 vs. 1,440 ± 130 pmol/mg prot), as well as ICAM-1 expression and leukocyte adhesion (TNF-α vs. TNF-α + IL-10: 26.8 ± 2.6 vs. 6.7 ± 0.4 adherent leukocytes/field at 15 min). Interleukin 10 decreases the level of inflammation induced by TNF-α in endothelial cells by reducing the TNF-α-induced ROS production, ICAM-1 expression, and leukocyte adhesion to the endothelium. The antioxidant effect of IL-10 is mediated through PI3-kinase and is paralleled by a decrease in ceramide synthesis induced by TNF-α.

Caveolin-1 plays a critical role in the differentiation of monocytes into macrophages.

OBJECTIVE: Monocyte to macrophage differentiation is an essential step in atherogenesis. The structure protein of caveolae, caveolin-1, is increased in primary monocytes after its adhesion to endothelium. We explore the hypothesis that caveolin-1 plays a role in monocyte differentiation to macrophages. METHODS AND RESULTS: Both phorbol myristate acetate-induced THP-1 and colony-stimulating factor-induced primary monocyte differentiation was associated with an increase in cellular caveolin-1 expression. Overexpression of caveolin-1 by transfection increased macrophage surface markers and inflammatory genes, whereas caveolin-1 knockdown by small interfering RNA or knockout reduced these. Also, caveolin-1 knockdown inhibited the differentiation-induced nuclear translocation of early growth response 1 (EGR-1) through extracellular signal-regulated kinase phosphorylation, further decreased the binding of EGR-1 to CD115 promoter, thus decreasing EGR-1 transcriptional activity. In functional assays, caveolin-1 inhibited transmigration but promoted phagocytosis in the monocyte-macrophage lineage. Decreasing caveolin-1 inhibited the uptake of modified low-density lipoprotein and reduced cellular lipid content. Finally, we showed that caveolin-1 knockout mice displayed less monocyte differentiation than wild-type mice and that EGR-1 transcription activity was also decreased in these mice because of the inhibition of extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: Caveolin-1 promotes monocyte to macrophage differentiation through the regulation of EGR-1 transcriptional activity, suggesting that phagocytic caveolin-1 may be critical for atherogenesis.

Imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease(2), stroke(3) and heart failure(4). An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species(5). In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers(6) however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration(7-9). When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry(10). Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model(9) which enables real-time analysis of leukocyte-endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter(11) as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.

Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte-endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1-specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-alpha-activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma samples from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin-dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1-mediated rolling and dependent on endothelial P-selectin expression.

An age-related decline in endothelial function is not associated with alterations in L-arginine transport in humans.

OBJECTIVES: Endothelial dysfunction is established in aged individuals; however, the mechanism(s) are not fully elucidated. We have previously identified l-arginine transport as a potential rate-limiting factor in nitric oxide (NO) production in heart-failure patients, characterized with endothelial dysfunction. We therefore aimed to investigate whether the age-related decline in endothelial function is due to reduced transport of the NO precursor, L-arginine. METHODS: Thirty-seven healthy males aged between 19 and 69 were recruited. Throughout 40 min of intra-arterial (i.a.) infusion of [3H]L-arginine (100 nCi/min), venous blood samples were withdrawn for the determination of L-arginine clearance. Venous occlusion plethysmography was then used to record the forearm blood flow responses to i.a. infusions of acetylcholine (ACh; 9.25 and 37 microg/min) and sodium nitroprusside (SNP; 2 and 8 microg/min). RESULTS: While ACh-induced vasodilation decreased with age (37 microg/min; young 15.87 +/- 1.30, middle-aged 9.59 +/- 1.33, older 10.42 +/- 1.12 ml/min per 100 ml tissue; P = 0.001), there was no change in forearm [3H]L-arginine clearance (young 126.08 +/- 19.05, middle-aged 122.47 +/- 20.96, older 126.56 +/- 19.56 ml/min; NS). Further [3H]L-arginine uptake studies in isolated peripheral blood mononuclear cells supported our in vivo findings, demonstrating no difference in [3H]L-arginine transport across the age spectrum. CONCLUSIONS: The present study excludes the hypothesis of impaired L-arginine transport as a potential mechanism for the age-related decline in endothelial function.

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake.

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.

Plasma C-reactive protein, but not protein S, VCAM-1, von Willebrand factor or P-selectin, is associated with endothelium dysfunction in coronary artery disease.

Abnormal endothelium-dependent vasodilatation is well documented in coronary artery disease (CAD), as are significant increases of acute phase inflammatory proteins and other soluble hepatic and endothelium derived proteins. In the current study we investigated whether there is a relationship between endothelium-dependent vasodilatation and the plasma levels of such proteins. Further we examined the association between these proteins, together with age, cholesterol and endothelium function, and participant status (i.e. healthy, smoker, coronary artery disease). Three groups of participants were recruited: healthy controls (n = 20), cigarette smokers (n = 20) and patients with coronary artery disease (n = 20). Forearm vascular dilatation responses to the endothelium-dependent agonist acetylcholine were obtained using venous occlusion plethysmography. Blood was sampled at the time of study for the measurement of the serological proteins described above. Responses to acetylcholine were significantly dampened in patients with CAD when compared with control and smoker groups (forearm blood flow at acetylcholine 37 microg/min): control, smokers, CAD, 12.14 +/- 2.14, 12.35 +/- 09.4, 5.12 +/- 0.81, ANOVA, P < 0.05). Responses to acetylcholine were not different between controls and smokers. C-reactive protein (C-RP) levels (controls, smokers, CAD, 1.40 +/- 0.19, 2.27 +/- 0.71, 4.73 +/- 1.09, P < 0.05) and protein S levels were significantly higher in the CAD group compared with the other two groups. There was a significant inverse correlation between forearm blood flow responses to acetylcholine and C-reactive proteins. Both responses to acetylcholine and C-RP levels demonstrated a significant association with participant status with the r value increasing from 0.405 for responses to acetylcholine to 0.491 for the combination of responses to acetylcholine and C-RP. We conclude that there is an inverse correlation between C-reactive protein, but not protein S, VCAM-1, P-selectin nor von Willebrand factor, levels and abnormal endothelium-dependent vasodilation and further that responses to acetylcholine together with C-RP protein have a strong association with cardiovascular risk and disease.

Impaired acetylcholine-induced release of nitric oxide in the aorta of male aromatase-knockout mice: regulation of nitric oxide production by endogenous sex hormones in males.

The consequences of estrogen deficiency on the cardiovascular system have been widely examined in females. The effects of endogenous estrogen deficiency in males are less clear. The aromatase-knockout (ArKO) mouse lacks a functional Cyp19 gene, which encodes aromatase, and is thus incapable of synthesizing endogenous estrogen. In the present study, we examined the effect of lack of endogenous estrogens on vascular function in aortic rings isolated from male ArKO mice and compared these effects to rings from wild-type (WT) littermates. Full concentration-response curves to norepinephrine, acetylcholine, isoprenaline, and sodium nitroprusside were obtained in the absence and presence of the nitric oxide synthase inhibitor Nomega-nitro-L-arginine in aortic segments set up in isometric myographs. Responses to noradrenaline were not different in aorta from ArKO compared with WT mice. Both Nomega-nitro-L-arginine and endothelium denudation significantly shifted the noradrenaline concentration-response curve to the left; however, this shift was not different in ArKO compared with WT. Responses to the endothelium-dependent vasodilator acetylcholine were significantly blunted in aortic rings from ArKO mice (Emax, 58.2+/-0.9% and 34.0+/-0.5% in wild-type and ArKO, respectively; P<0.05), whereas responses to the endothelium-independent agonist sodium nitroprusside and to the partial endothelium-dependent agonist isoprenaline were not affected. These findings suggest that endogenous estrogen facilitate vasorelaxation in males. This may be via modulating endothelial function rather than vascular smooth muscle cell responsiveness.

L-arginine transport in humans with cortisol-induced hypertension.

A deficient L-arginine-nitric oxide system is implicated in cortisol-induced hypertension. We investigate whether abnormalities in L-arginine uptake contribute to this deficiency. Eight healthy men were recruited. Hydrocortisone acetate (50 mg) was given orally every 6 hours for 24 hours after a 5-day fixed-salt diet (150 mmol/d). Crossover studies were performed 2 weeks apart. Thirty milliliters of blood was obtained for isolation of peripheral blood mononuclear cells after each treatment period. L-arginine uptake was assessed in mononuclear cells incubated with L-arginine (1 to 300 micromol/L), incorporating 100 nmol/L [3H]-l-arginine for a period of 5 minutes at 37 degrees C. Forearm [3H]-L-arginine extraction was calculated after infusion of [3H]-L-arginine into the brachial artery at a rate of 100 nCi/min for 80 minutes. Deep forearm venous samples were collected for determination of L-arginine extraction. Plasma cortisol concentrations were significantly raised during the active phase (323+/-43 to 1082+/-245 mmol/L, P<0.05). Systolic blood pressure was elevated by an average of 7 mm Hg. Neither L-arginine transport into mononuclear cells (placebo vs active, 26.3+/-3.6 vs 29.0+/-2.1 pmol/10 000 cells per 5 minutes, respectively, at an l-arginine concentration of 300 micromol/L) nor L-arginine extraction in the forearm (at 80 minutes, placebo vs active, 1 868 904+/-434 962 vs 2 013 910+/-770 619 disintegrations per minute) was affected by cortisol treatment; ie, that L-arginine uptake is not affected by short-term cortisol treatment. We conclude that cortisol-induced increases in blood pressure are not associated with abnormalities in the l-arginine transport system.