Publications by authors named "Hema Viswambharan"

27 Publications

  • Page 1 of 1

Barium yttrium fluoride based upconversion nanoparticles as dual mode image contrast agents.

Mater Sci Eng C Mater Biol Appl 2021 May 6;124:111937. Epub 2021 Feb 6.

School of Chemical and Process Engineering, Faculty of Engineering and Physical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

Dual labeled contrast agents could provide better complementary information for bioimaging than available solely from a single modality. In this paper we investigate the suitability of Yb and Er-doped BaYF upconversion nanoparticles (UCNPs) as both optical and X-ray micro computed tomography (μCT) contrast agents. Stable, aqueous UCNP dispersions were synthesised using a hydrothermal method with the addition of polyethyleneimine (PEI). UCNPs were single crystal and had a truncated cuboidal and/or truncated octahedral morphology, with average particle size of 47 ±9 nm from transmission electron microscopy which was further used to characterize the structure and composition in detail. A zeta potential value of +51 mV was measured for the aqueous nanoparticle dispersions which is beneficial for cell permeability. The outer hydrated PEI layer is also advantageous for the attachment of proteins for targeted delivery in biological systems. The prepared UCNPs were proven to be non-toxic to endothelial cells up to a concentration of 3.5 mg/mL, when assessed using an MTT assay. The particles showed intense green upconversion photoluminescence when excited at a wavelength of 976 nm using a diode laser. Quantitative X-ray μCT contrast imaging confirmed the potential of these UCNPs as X-ray contrast agents and confirming their dual modality for bioimaging.
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http://dx.doi.org/10.1016/j.msec.2021.111937DOI Listing
May 2021

Differential associations of ankle and brachial blood pressures with diabetes and cardiovascular diseases: cross-sectional study.

Sci Rep 2021 Apr 30;11(1):9406. Epub 2021 Apr 30.

NHS England & NHS Improvement (North East and Yorkshire), Quarry Hill, Leeds, LS2 7UE, UK.

Increased brachial systolic blood-pressure (BP) predicts diabetes (T2DM) but is not fully effective. Value of absolute ankle systolic BP for T2DM compared to brachial systolic BP is not known. Our objectives were to assess independent relationships of ankle-systolic BP with T2DM and cardiovascular disease in Europeans and south Asians. Cross-sectional studies of anonymised data from registered adults (n = 1087) at inner city deprived primary care practices. Study includes 63.85% ethnic minority. Systolic BP of the left and right-brachial, posterior-tibial and dorsalis-pedis-arteries measured using a Doppler probe. Regression models' factors were age, sex, ethnicity, body mass index (BMI) and waist height ratio (WHtR). Both brachial and ankle systolic-BP increase with diabetes in Europeans and south Asians. We demonstrated that there was a significant positive independent association of ankle BP with diabetes, regardless of age and sex compared to Brachial. There was stronger negative association of ankle blood pressure with cardiovascular disease, after adjustment for BMI, WHtR and ethnicity. Additionally, we found that ankle BP were significantly associated with cardiovascular disease in south Asians more than the Europeans; right posterior tibial. Ankle systolic BPs are superior to brachial BPs to identify risks of Type 2DM and cardiovascular diseases for enhanced patient care.
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http://dx.doi.org/10.1038/s41598-021-88973-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087686PMC
April 2021

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Am J Physiol Cell Physiol 2020 07 13;319(1):C64-C74. Epub 2020 May 13.

Leeds Institute for Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE and Nox2 (ESMIRO/ApoE/Nox2) were generated and compared with ESMIRO/ApoE/Nox2 littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE/Nox2 mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE/Nox2 mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.
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http://dx.doi.org/10.1152/ajpcell.00389.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468885PMC
July 2020

Piezo1 channel activation mimics high glucose as a stimulator of insulin release.

Sci Rep 2019 11 14;9(1):16876. Epub 2019 Nov 14.

Leeds Institute for Cardiovascular and Metabolic Medicine, Faculty of Medicine and Health, University of Leeds, Leeds, LS2 9JT, United Kingdom.

Glucose and hypotonicity induced cell swelling stimulate insulin release from pancreatic β-cells but the mechanisms are poorly understood. Recently, Piezo1 was identified as a mechanically-activated nonselective Ca permeable cationic channel in a range of mammalian cells. As cell swelling induced insulin release could be through stimulation of Ca permeable stretch activated channels, we hypothesised a role for Piezo1 in cell swelling induced insulin release. Two rat β-cell lines (INS-1 and BRIN-BD11) and freshly-isolated mouse pancreatic islets were studied. Intracellular Ca measurements were performed using the fura-2 Ca indicator dye and ionic current was recorded by whole cell patch-clamp. Piezo1 agonist Yoda1, a competitive antagonist of Yoda1 (Dooku1) and an inactive analogue of Yoda1 (2e) were used as chemical probes. Piezo1 mRNA and insulin secretion were measured by RT-PCR and ELISA respectively. Piezo1 mRNA was detected in both β-cell lines and mouse islets. Yoda1 evoked Ca entry was inhibited by Yoda1 antagonist Dooku1 as well as other Piezo1 inhibitors gadolinium and ruthenium red, and not mimicked by 2e. Yoda1, but not 2e, stimulated Dooku1-sensitive insulin release from β-cells and pancreatic islets. Hypotonicity and high glucose increased intracellular Ca and enhanced Yoda1 Ca influx responses. Yoda1 and hypotonicity induced insulin release were significantly inhibited by Piezo1 specific siRNA. Pancreatic islets from mice with haploinsufficiency of Piezo1 released less insulin upon exposure to Yoda1. The data show that Piezo1 channel agonist induces insulin release from β-cell lines and mouse pancreatic islets suggesting a role for Piezo1 in cell swelling induced insulin release. Hence Piezo1 agonists have the potential to be used as enhancers of insulin release.
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http://dx.doi.org/10.1038/s41598-019-51518-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856185PMC
November 2019

Endothelial Insulin Receptor Restoration Rescues Vascular Function in Male Insulin Receptor Haploinsufficient Mice.

Endocrinology 2018 08;159(8):2917-2925

Leeds Institute of Cardiovascular and Metabolic Medicine, LIGHT Laboratories, The University of Leeds, Leeds, United Kingdom.

Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We investigated whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor knockout (IRKO) mice were crossed with mice expressing a human insulin receptor endothelial cell-specific overexpression (hIRECO) to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO mice in glucose and insulin tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO mice exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild type littermates. These phenotypic changes were associated with increased basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO mice also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling.
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http://dx.doi.org/10.1210/en.2018-00215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047419PMC
August 2018

Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance.

Nat Commun 2017 08 24;8(1):350. Epub 2017 Aug 24.

Schools of Medicine, University of Leeds, Leeds, LS2 9JT, UK.

Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.The mechanisms that regulate the body's response to exercise are poorly understood. Here, Rode et al. show that the mechanically activated cation channel Piezo1 is a molecular sensor of physical exercise in the endothelium that triggers endothelial communication to mesenteric vessel muscle cells, leading to vasoconstriction.
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http://dx.doi.org/10.1038/s41467-017-00429-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571199PMC
August 2017

Endothelial SHIP2 Suppresses Nox2 NADPH Oxidase-Dependent Vascular Oxidative Stress, Endothelial Dysfunction, and Systemic Insulin Resistance.

Diabetes 2017 11 22;66(11):2808-2821. Epub 2017 Aug 22.

Leeds Institute of Cardiovascular and Metabolic Medicine, Leeds Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, U.K.

Shc homology 2-containing inositol 5' phosphatase-2 (SHIP2) is a lipid phosphatase that inhibits insulin signaling downstream of phosphatidylinositol 3-kinase (PI3K); its role in vascular function is poorly understood. To examine its role in endothelial cell (EC) biology, we generated mice with catalytic inactivation of one SHIP2 allele selectively in ECs (ECSHIP2). Hyperinsulinemic-euglycemic clamping studies revealed that ECSHIP2 was resistant to insulin-stimulated glucose uptake in adipose tissue and skeletal muscle compared with littermate controls. ECs from ECSHIP2 mice had increased basal expression and activation of PI3K downstream targets, including Akt and endothelial nitric oxide synthase, although incremental activation by insulin and shear stress was impaired. Insulin-mediated vasodilation was blunted in ECSHIP2 mice, as was aortic nitric oxide bioavailability. Acetylcholine-induced vasodilation was also impaired in ECSHIP2 mice, which was exaggerated in the presence of a superoxide dismutase/catalase mimetic. Superoxide abundance was elevated in ECSHIP2 ECs and was suppressed by PI3K and NADPH oxidase 2 inhibitors. These findings were phenocopied in healthy human ECs after SHIP2 silencing. Our data suggest that endothelial SHIP2 is required to maintain normal systemic glucose homeostasis and prevent oxidative stress-induced endothelial dysfunction.
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http://dx.doi.org/10.2337/db17-0062DOI Listing
November 2017

Response by Viswambharan and Kearney to Letter Regarding Article, "Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop".

Circ Res 2017 02;120(4):e4-e5

Leeds Institute for Cardiovascular and Metabolic Medicine, University of Leeds School of Medicine, University of Leeds, Leeds, United Kingdom.

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http://dx.doi.org/10.1161/CIRCRESAHA.117.310510DOI Listing
February 2017

Insulin-Like Growth Factor Binding Protein 1 Could Improve Glucose Regulation and Insulin Sensitivity Through Its RGD Domain.

Diabetes 2017 02 10;66(2):287-299. Epub 2016 Nov 10.

Division of Cardiovascular and Diabetes Research, Leeds Multidisciplinary Cardiovascular Research Centre, Faculty of Medicine and Health, University of Leeds, Leeds, West Yorkshire, U.K.

Low circulating levels of insulin-like growth factor binding protein 1 (IGFBP-1) are associated with insulin resistance and predict the development of type 2 diabetes. IGFBP-1 can affect cellular functions independently of IGF binding through an Arg-Gly-Asp (RGD) integrin-binding motif. Whether causal mechanisms underlie the favorable association of high IGFBP-1 levels with insulin sensitivity and whether these could be exploited therapeutically remain unexplored. We used recombinant IGFBP-1 and a synthetic RGD-containing hexapeptide in complementary in vitro signaling assays and in vivo metabolic profiling in obese mice to investigate the effects of IGFBP-1 and its RGD domain on insulin sensitivity, insulin secretion, and whole-body glucose regulation. The RGD integrin-binding domain of IGFBP-1, through integrin engagement, focal adhesion kinase, and integrin-linked kinase, enhanced insulin sensitivity and insulin secretion in C2C12 myotubes and INS-1 832/13 pancreatic β-cells. Both acute administration and chronic infusion of an RGD synthetic peptide to obese C57BL/6 mice improved glucose clearance and insulin sensitivity. These favorable effects on metabolic homeostasis suggest that the RGD integrin-binding domain of IGFBP-1 may be a promising candidate for therapeutic development in the field of insulin resistance.
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http://dx.doi.org/10.2337/db16-0997DOI Listing
February 2017

Selective Enhancement of Insulin Sensitivity in the Endothelium In Vivo Reveals a Novel Proatherosclerotic Signaling Loop.

Circ Res 2017 Mar 5;120(5):784-798. Epub 2016 Dec 5.

From the Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, United Kingdom (H.V., N.Y.Y., A.S., H.I., N.H., A.M.N.W., A.S., N.M., S.G., P. Shah, P. Sukumar, K.E.P., P.J.G., J.L., D.J.B., S.B.W., R.M.C., M.T.K.); Division of Medicine, Department of Metabolism & Experimental Therapeutics, University College London, United Kingdom (M.C.G.); and British Heart Foundation Centre of Research Excellence, King's College London, United Kingdom (A.M.S., C.X.C.S.).

Rationale: In the endothelium, insulin stimulates endothelial NO synthase (eNOS) to generate the antiatherosclerotic signaling radical NO. Insulin-resistant type 2 diabetes mellitus is associated with reduced NO availability and accelerated atherosclerosis. The effect of enhancing endothelial insulin sensitivity on NO availability is unclear.

Objective: To answer this question, we generated a mouse with endothelial cell (EC)-specific overexpression of the human insulin receptor (hIRECO) using the Tie2 promoter-enhancer.

Methods And Results: hIRECO demonstrated significant endothelial dysfunction measured by blunted endothelium-dependent vasorelaxation to acetylcholine, which was normalized by a specific Nox2 NADPH oxidase inhibitor. Insulin-stimulated phosphorylation of protein kinase B was increased in hIRECO EC as was Nox2 NADPH oxidase-dependent generation of superoxide, whereas insulin-stimulated and shear stress-stimulated eNOS activations were blunted. Phosphorylation at the inhibitory residue Y657 of eNOS and expression of proline-rich tyrosine kinase 2 that phosphorylates this residue were significantly higher in hIRECO EC. Inhibition of proline-rich tyrosine kinase 2 improved insulin-induced and shear stress-induced eNOS activation in hIRECO EC.

Conclusions: Enhancing insulin sensitivity specifically in EC leads to a paradoxical decline in endothelial function, mediated by increased tyrosine phosphorylation of eNOS and excess Nox2-derived superoxide. Increased EC insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide. Inhibition of proline-rich tyrosine kinase 2 restores insulin-induced and shear stress-induced NO production. This study demonstrates for the first time that increased endothelial insulin sensitivity leads to a proatherosclerotic imbalance between NO and superoxide.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.309678DOI Listing
March 2017

Haploinsufficiency of the insulin-like growth factor-1 receptor enhances endothelial repair and favorably modifies angiogenic progenitor cell phenotype.

Arterioscler Thromb Vasc Biol 2014 Sep 10;34(9):2051-8. Epub 2014 Jul 10.

From the Division of Cardiovascular and Diabetes Research, Leeds Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, United Kingdom.

Objectives: Defective endothelial regeneration predisposes to adverse arterial remodeling and is thought to contribute to cardiovascular disease in type 2 diabetes mellitus. We recently demonstrated that the type 1 insulin-like growth factor receptor (IGF1R) is a negative regulator of insulin sensitivity and nitric oxide bioavailability. In this report, we examined partial deletion of the IGF1R as a potential strategy to enhance endothelial repair.

Approach And Results: We assessed endothelial regeneration after wire injury in mice and abundance and function of angiogenic progenitor cells in mice with haploinsufficiency of the IGF1R (IGF1R(+/-)). Endothelial regeneration after arterial injury was accelerated in IGF1R(+/-) mice. Although the yield of angiogenic progenitor cells was lower in IGF1R(+/-) mice, these angiogenic progenitor cells displayed enhanced adhesion, increased secretion of insulin-like growth factor-1, and enhanced angiogenic capacity. To examine the relevance of IGF1R manipulation to cell-based therapy, we transfused IGF1R(+/-) bone marrow-derived CD117(+) cells into wild-type mice. IGF1R(+/-) cells accelerated endothelial regeneration after arterial injury compared with wild-type cells and did not alter atherosclerotic lesion formation.

Conclusions: Haploinsufficiency of the IGF1R is associated with accelerated endothelial regeneration in vivo and enhanced tube forming and adhesive potential of angiogenic progenitor cells in vitro. Partial deletion of IGF1R in transfused bone marrow-derived CD117(+) cells enhanced their capacity to promote endothelial regeneration without altering atherosclerosis. Our data suggest that manipulation of the IGF1R could be exploited as novel therapeutic approach to enhance repair of the arterial wall after injury.
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http://dx.doi.org/10.1161/ATVBAHA.114.304121DOI Listing
September 2014

Restoring Akt1 activity in outgrowth endothelial cells from South Asian men rescues vascular reparative potential.

Stem Cells 2014 Oct;32(10):2714-23

Leeds Multidisciplinary Cardiovascular Research Centre, LIGHT Laboratories, The University of Leeds, Clarendon Way, Leeds, United Kingdom.

Recent data suggest reduced indices of vascular repair in South Asian men, a group at increased risk of cardiovascular events. Outgrowth endothelial cells (OEC) represent an attractive tool to study vascular repair in humans and may offer potential in cell-based repair therapies. We aimed to define and manipulate potential mechanisms of impaired vascular repair in South Asian (SA) men. In vitro and in vivo assays of vascular repair and angiogenesis were performed using OEC derived from SA men and matched European controls, prior defining potentially causal molecular mechanisms. SA OEC exhibited impaired colony formation, migration, and in vitro angiogenesis, associated with decreased expression of the proangiogenic molecules Akt1 and endothelial nitric oxide synthase (eNOS). Transfusion of European OEC into immunodeficient mice after wire-induced femoral artery injury augmented re-endothelialization, in contrast with SA OEC and vehicle; SA OEC also failed to promote angiogenesis after induction of hind limb ischemia. Expression of constitutively active Akt1 (E17KAkt), but not green fluorescent protein control, in SA OEC increased in vitro angiogenesis, which was abrogated by a NOS antagonist. Moreover, E17KAkt expressing SA OEC promoted re-endothelialization of wire-injured femoral arteries, and perfusion recovery of ischemic limbs, to a magnitude comparable with nonmanipulated European OEC. Silencing Akt1 in European OEC recapitulated the functional deficits noted in SA OEC. Reduced signaling via the Akt/eNOS axis is causally linked with impaired OEC-mediated vascular repair in South Asian men. These data prove the principle of rescuing marked reparative dysfunction in OEC derived from these men.
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http://dx.doi.org/10.1002/stem.1766DOI Listing
October 2014

Endothelium-specific insulin resistance leads to accelerated atherosclerosis in areas with disturbed flow patterns: a role for reactive oxygen species.

Atherosclerosis 2013 Sep 29;230(1):131-9. Epub 2013 Jun 29.

Division of Cardiovascular and Diabetes Research, Leeds Multidisciplinary Cardiovascular Research Centre, University of Leeds, United Kingdom.

Objective: Systemic insulin resistance is associated with a portfolio of risk factors for atherosclerosis development. We sought to determine whether insulin resistance specifically at the level of the endothelium promotes atherosclerosis and to examine the potential involvement of reactive oxygen species.

Methods: We cross-bred mice expressing a dominant negative mutant human insulin receptor specifically in the endothelium (ESMIRO) with ApoE(-/-) mice to examine the effect of endothelium-specific insulin resistance on atherosclerosis.

Results: ApoE(-/-)/ESMIRO mice had similar blood pressure, plasma lipids and whole-body glucose tolerance, but blunted endothelial insulin signalling, in comparison to ApoE(-/-) mice. Atherosclerosis was significantly increased in ApoE(-/-)/ESMIRO mice at the aortic sinus (226 ± 16 versus 149 ± 24 × 10(3) μm(2), P = 0.01) and lesser curvature of the aortic arch (12.4 ± 1.2% versus 9.4 ± 0.9%, P = 0.035). Relaxation to acetylcholine was blunted in aorta from ApoE(-/-)/ESMIRO mice (Emax 65 ± 41% versus 103 ± 6%, P = 0.02) and was restored by the superoxide dismutase mimetic MnTMPyP (Emax 112 ± 15% versus 65 ± 41%, P = 0.048). Basal generation of superoxide was increased 1.55 fold (P = 0.01) in endothelial cells from ApoE(-/-)/ESMIRO mice and was inhibited by the NADPH oxidase inhibitor gp91ds-tat (-12 ± 0.04%, P = 0.04), the NO synthase inhibitor L-NMMA (-8 ± 0.02%, P = 0.001) and the mitochondrial specific inhibitor rotenone (-23 ± 0.04%, P = 0.006).

Conclusions: Insulin resistance specifically at the level of the endothelium leads to acceleration of atherosclerosis in areas with disturbed flow patterns such as the aortic sinus and the lesser curvature of the aorta. We have identified a potential role for increased generation of reactive oxygen species from multiple enzymatic sources in promoting atherosclerosis in this setting.
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http://dx.doi.org/10.1016/j.atherosclerosis.2013.06.017DOI Listing
September 2013

Nox2 NADPH oxidase has a critical role in insulin resistance-related endothelial cell dysfunction.

Diabetes 2013 Jun 24;62(6):2130-4. Epub 2013 Jan 24.

Division of Cardiovascular and Diabetes Research, Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance-induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance-mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance-related vascular disease.
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http://dx.doi.org/10.2337/db12-1294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661635PMC
June 2013

Novel role of the IGF-1 receptor in endothelial function and repair: studies in endothelium-targeted IGF-1 receptor transgenic mice.

Diabetes 2012 Sep 25;61(9):2359-68. Epub 2012 Jun 25.

Division of Cardiovascular and Diabetes Research, Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK.

We recently demonstrated that reducing IGF-1 receptor (IGF-1R) numbers in the endothelium enhances nitric oxide (NO) bioavailability and endothelial cell insulin sensitivity. In the present report, we aimed to examine the effect of increasing IGF-1R on endothelial cell function and repair. To examine the effect of increasing IGF-1R in the endothelium, we generated mice overexpressing human IGF-1R in the endothelium (human IGF-1R endothelium-overexpressing mice [hIGFREO]) under direction of the Tie2 promoter enhancer. hIGFREO aorta had reduced basal NO bioavailability (percent constriction to N(G)-monomethyl-l-arginine [mean (SEM) wild type 106% (30%); hIGFREO 48% (10%)]; P < 0.05). Endothelial cells from hIGFREO had reduced insulin-stimulated endothelial NO synthase activation (mean [SEM] wild type 170% [25%], hIGFREO 58% [3%]; P = 0.04) and insulin-stimulated NO release (mean [SEM] wild type 4,500 AU [1,000], hIGFREO 1,500 AU [700]; P < 0.05). hIGFREO mice had enhanced endothelium regeneration after denuding arterial injury (mean [SEM] percent recovered area, wild type 57% [2%], hIGFREO 47% [5%]; P < 0.05) and enhanced endothelial cell migration in vitro. The IGF-1R, although reducing NO bioavailability, enhances in situ endothelium regeneration. Manipulating IGF-1R in the endothelium may be a useful strategy to treat disorders of vascular growth and repair.
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http://dx.doi.org/10.2337/db11-1494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3425420PMC
September 2012

Increasing circulating IGFBP1 levels improves insulin sensitivity, promotes nitric oxide production, lowers blood pressure, and protects against atherosclerosis.

Diabetes 2012 Apr 22;61(4):915-24. Epub 2012 Feb 22.

Division of Cardiovascular and Diabetes Research, MultidisciplinaryCardiovascular Research Centre, University of Leeds, Leeds, U.K.

Low concentrations of insulin-like growth factor (IGF) binding protein-1 (IGFBP1) are associated with insulin resistance, diabetes, and cardiovascular disease. We investigated whether increasing IGFBP1 levels can prevent the development of these disorders. Metabolic and vascular phenotype were examined in response to human IGFBP1 overexpression in mice with diet-induced obesity, mice heterozygous for deletion of insulin receptors (IR(+/-)), and ApoE(-/-) mice. Direct effects of human (h)IGFBP1 on nitric oxide (NO) generation and cellular signaling were studied in isolated vessels and in human endothelial cells. IGFBP1 circulating levels were markedly suppressed in dietary-induced obese mice. Overexpression of hIGFBP1 in obese mice reduced blood pressure, improved insulin sensitivity, and increased insulin-stimulated NO generation. In nonobese IR(+/-) mice, overexpression of hIGFBP1 reduced blood pressure and improved insulin-stimulated NO generation. hIGFBP1 induced vasodilatation independently of IGF and increased endothelial NO synthase (eNOS) activity in arterial segments ex vivo, while in endothelial cells, hIGFBP1 increased eNOS Ser(1177) phosphorylation via phosphatidylinositol 3-kinase signaling. Finally, in ApoE(-/-) mice, overexpression of hIGFBP1 reduced atherosclerosis. These favorable effects of hIGFBP1 on insulin sensitivity, blood pressure, NO production, and atherosclerosis suggest that increasing IGFBP1 concentration may be a novel approach to prevent cardiovascular disease in the setting of insulin resistance and diabetes.
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http://dx.doi.org/10.2337/db11-0963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314358PMC
April 2012

The insulin-like growth factor-1 receptor is a negative regulator of nitric oxide bioavailability and insulin sensitivity in the endothelium.

Diabetes 2011 Aug 15;60(8):2169-78. Epub 2011 Jun 15.

Division of Cardiovascular and Diabetes Research, Leeds Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds, UK.

Objective: In mice, haploinsufficiency of the IGF-1 receptor (IGF-1R(+/-)), at a whole-body level, increases resistance to inflammation and oxidative stress, but the underlying mechanisms are unclear. We hypothesized that by forming insulin-resistant heterodimers composed of one IGF-1Rαβ and one insulin receptor (IR), IRαβ complex in endothelial cells (ECs), IGF-1R reduces free IR, which reduces EC insulin sensitivity and generation of the antioxidant/anti-inflammatory signaling radical nitric oxide (NO).

Research Design And Methods: Using a number of complementary gene-modified mice with reduced IGF-1R at a whole-body level and specifically in EC, and complementary studies in EC in vitro, we examined the effect of changing IGF-1R/IR stoichiometry on EC insulin sensitivity and NO bioavailability.

Results: IGF-1R(+/-) mice had enhanced insulin-mediated glucose lowering. Aortas from these mice were hypocontractile to phenylephrine (PE) and had increased basal NO generation and augmented insulin-mediated NO release from EC. To dissect EC from whole-body effects we generated mice with EC-specific knockdown of IGF-1R. Aortas from these mice were also hypocontractile to PE and had increased basal NO generation. Whole-body and EC deletion of IGF-1R reduced hybrid receptor formation. By reducing IGF-1R in IR-haploinsufficient mice we reduced hybrid formation, restored insulin-mediated vasorelaxation in aorta, and insulin stimulated NO release in EC. Complementary studies in human umbilical vein EC in which IGF-1R was reduced using siRNA confirmed that reducing IGF-1R has favorable effects on NO bioavailability and EC insulin sensitivity.

Conclusions: These data demonstrate that IGF-1R is a critical negative regulator of insulin sensitivity and NO bioavailability in the endothelium.
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http://dx.doi.org/10.2337/db11-0197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3142083PMC
August 2011

Insulin resistance impairs circulating angiogenic progenitor cell function and delays endothelial regeneration.

Diabetes 2011 Apr 11;60(4):1295-303. Epub 2011 Feb 11.

Division of Cardiovascular and Diabetes Research, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.

Objective: Circulating angiogenic progenitor cells (APCs) participate in endothelial repair after arterial injury. Type 2 diabetes is associated with fewer circulating APCs, APC dysfunction, and impaired endothelial repair. We set out to determine whether insulin resistance adversely affects APCs and endothelial regeneration.

Research Design And Methods: We quantified APCs and assessed APC mobilization and function in mice hemizygous for knockout of the insulin receptor (IRKO) and wild-type (WT) littermate controls. Endothelial regeneration after femoral artery wire injury was also quantified after APC transfusion.

Results: IRKO mice, although glucose tolerant, had fewer circulating Sca-1(+)/Flk-1(+) APCs than WT mice. Culture of mononuclear cells demonstrated that IRKO mice had fewer APCs in peripheral blood, but not in bone marrow or spleen, suggestive of a mobilization defect. Defective vascular endothelial growth factor-stimulated APC mobilization was confirmed in IRKO mice, consistent with reduced endothelial nitric oxide synthase (eNOS) expression in bone marrow and impaired vascular eNOS activity. Paracrine angiogenic activity of APCs from IRKO mice was impaired compared with those from WT animals. Endothelial regeneration of the femoral artery after denuding wire injury was delayed in IRKO mice compared with WT. Transfusion of mononuclear cells from WT mice normalized the impaired endothelial regeneration in IRKO mice. Transfusion of c-kit(+) bone marrow cells from WT mice also restored endothelial regeneration in IRKO mice. However, transfusion of c-kit(+) cells from IRKO mice was less effective at improving endothelial repair.

Conclusions: Insulin resistance impairs APC function and delays endothelial regeneration after arterial injury. These findings support the hypothesis that insulin resistance per se is sufficient to jeopardize endogenous vascular repair. Defective endothelial repair may be normalized by transfusion of APCs from insulin-sensitive animals but not from insulin-resistant animals.
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http://dx.doi.org/10.2337/db10-1080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064103PMC
April 2011

Vascular insulin-like growth factor-I resistance and diet-induced obesity.

Endocrinology 2009 Oct 16;150(10):4575-82. Epub 2009 Jul 16.

Division of Cardiovascular and Diabetes Research, Leeds Multidisciplinary Cardiovascular Research Centre, University of Leeds, Leeds LS2 9JT, United Kingdom.

Obesity and type 2 diabetes mellitus are characterized by insulin resistance, reduced bioavailability of the antiatherosclerotic signaling molecule nitric oxide (NO), and accelerated atherosclerosis. IGF-I, the principal growth-stimulating peptide, which shares many of the effects of insulin, may, like insulin, also be involved in metabolic and vascular homeostasis. We examined the effects of IGF-I on NO bioavailability and the effect of obesity/type 2 diabetes mellitus on IGF-I actions at a whole-body level and in the vasculature. In aortic rings IGF-I blunted phenylephrine-mediated vasoconstriction and relaxed rings preconstricted with phenylephrine, an effect blocked by N(G)-monomethyl L-arginine. IGF-I increased NO synthase activity to an extent similar to that seen with insulin and in-vivo IGF-I led to serine phosphorylation of endothelial NO synthase (eNOS). Mice rendered obese using a high-fat diet were less sensitive to the glucose-lowering effects of insulin and IGF-I. IGF-I increased aortic phospho-eNOS levels in lean mice, an effect that was blunted in obese mice. eNOS activity in aortae of lean mice increased 1.6-fold in response to IGF-I compared with obese mice. IGF-I-mediated vasorelaxation was blunted in obese mice. These data demonstrate that IGF-I increases eNOS phosphorylation in-vivo, increases eNOS activity, and leads to NO-dependent relaxation of conduit vessels. Obesity is associated with resistance to IGF-I at a whole-body level and in the endothelium. Vascular IGF-I resistance may represent a novel therapeutic target to prevent or slow the accelerated vasculopathy seen in humans with obesity or type 2 diabetes mellitus.
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http://dx.doi.org/10.1210/en.2008-1641DOI Listing
October 2009

Endothelial nitric oxide synthase gene transfer inhibits human smooth muscle cell migration via inhibition of Rho A.

J Cardiovasc Pharmacol 2008 Oct;52(4):369-74

Cardiovascular Research, Institute of Physiology, University Zürich, Switzerland.

Smooth muscle cell (SMC) migration contributes to vascular remodeling. Nitric oxide (NO) produced via endothelial NO synthase (eNOS) inhibits SMC migration. This study analyzes signal transduction mechanisms of SMC migration targeted by NO. SMCs were cultured from human saphenous veins, and cell migration was studied using Boyden chambers. PDGF-BB (0.1 to 10 ng/ml) stimulated SMC migration in a concentration-dependent manner, which was inhibited by adenoviral-mediated overexpression of eNOS and by the NO donor diethylentriamine NONOate (DETANO, 10 to 10 mol/L). NO release was enhanced in eNOS-transduced SMCs, and L-NAME blunted the effect of eNOS overexpression on migration. PDGF-BB (10 ng/ml) activated Rho A, which was inhibited by the overexpression of eNOS by DETANO and by 8 bromo-cGMP. The inhibitory effect of DETANO on Rho A activity was prevented by the cGMP-dependant kinase inhibitor. Furthermore, inhibition of Rho A by C3 exoenzyme and inhibition of ROCK by Y-27632 diminished cell migration stimulated by PDGF-BB. Finally, in the cells overexpressing constitutively active ROCK mutant (CAT), DETANO failed to prevent PDGF-BB-induced SMC migration. In conclusion, NO inhibits human SMC migration via blockade of the Rho A pathway.
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http://dx.doi.org/10.1097/FJC.0b013e31818953d0DOI Listing
October 2008

Mutation of the circadian clock gene Per2 alters vascular endothelial function.

Circulation 2007 Apr 2;115(16):2188-95. Epub 2007 Apr 2.

Department of Medicine, Division of Physiology, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland.

Background: The circadian clock regulates biological processes including cardiovascular function and metabolism. In the present study, we investigated the role of the circadian clock gene Period2 (Per2) in endothelial function in a mouse model.

Methods And Results: Compared with the wild-type littermates, mice with Per2 mutation exhibited impaired endothelium-dependent relaxations to acetylcholine in aortic rings suspended in organ chambers. During transition from the inactive to active phase, this response was further increased in the wild-type mice but further decreased in the Per2 mutants. The endothelial dysfunction in the Per2 mutants was also observed with ionomycin, which was improved by the cyclooxygenase inhibitor indomethacin. No changes in the expression of endothelial acetylcholine-M3 receptor or endothelial nitric oxide synthase protein but increased cyclooxygenase-1 (not cyclooxygenase-2) protein levels were observed in the aortas of the Per2 mutants. Compared with Per2 mutants, a greater endothelium-dependent relaxation to ATP was observed in the wild-type mice, which was reduced by indomethacin. In quiescent aortic rings, ATP caused greater endothelium-dependent contractions in the Per2 mutants than in the wild-type mice, contractions that were abolished by indomethacin. The endothelial dysfunction in the Per2 mutant mice is not associated with hypertension or dyslipidemia.

Conclusions: Mutation in the Per2 gene in mice is associated with aortic endothelial dysfunction involving decreased production of NO and vasodilatory prostaglandin(s) and increased release of cyclooxygenase-1-derived vasoconstrictor(s). The results suggest an important role of the Per2 gene in maintenance of normal cardiovascular functions.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.106.653303DOI Listing
April 2007

Sirolimus increases tissue factor expression but not activity in cultured human vascular smooth muscle cells.

BMC Cardiovasc Disord 2005 Jul 15;5:22. Epub 2005 Jul 15.

Vascular Biology, Department of Medicine, Division of Physiology,University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland.

Background: Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).

Methods: SMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 - 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.

Results: Sirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 micromol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.

Conclusion: Although sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.
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http://dx.doi.org/10.1186/1471-2261-5-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190166PMC
July 2005

Endothelial nitric oxide synthase gene transfer restores endothelium-dependent relaxations and attenuates lesion formation in carotid arteries in apolipoprotein E-deficient mice.

Basic Res Cardiol 2005 Mar 24;100(2):102-11. Epub 2004 Nov 24.

Division of Cardiology, University of Lausanne, Faculty of Biology and Medicine CHUV-BH10, Lausanne, Switzerland.

Nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) exert partly opposing effects in vascular biology. NO plays pleiotropic vasoprotective roles including vasodilation and inhibition of platelet aggregation, smooth muscle cell proliferation, and endothelial monocyte adhesion, the last effect being mediated by MCP-1 downregulation. Early stages of arteriosclerosis are associated with reduced NO bioactivity and enhanced MCP-1 expression. We have evaluated adenovirus-mediated gene transfer of human endothelial NO synthase (eNOS) and of a N-terminal deletion (8ND) mutant of the MCP-1 gene that acts as a MCP-1 inhibitor in arteriosclerosis-prone, apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-dependent relaxations were impaired in carotid arteries instilled with a noncoding adenoviral vector but were restored by eNOS gene transfer (p < 0.01). A perivascular collar was placed around the common carotid artery to accelerate lesion formation. eNOS gene transfer reduced lesion surface areas, intima/media ratios, and macrophage contents in the media at 5-week follow-up (p < 0.05). In contrast, 8ND-MCP-1 gene transfer did not prevent lesion formation. In conclusion, eNOS gene transfer restores endothelium-dependent vasodilation and inhibits lesion formation in ApoE(-/-) mouse carotids. Further studies are needed to assess whether vasoprotection is maintained at later disease stages and to evaluate the long-term efficacy of eNOS gene therapy for primary arteriosclerosis.
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http://dx.doi.org/10.1007/s00395-004-0500-9DOI Listing
March 2005

Thrombin stimulates human endothelial arginase enzymatic activity via RhoA/ROCK pathway: implications for atherosclerotic endothelial dysfunction.

Circulation 2004 Dec 29;110(24):3708-14. Epub 2004 Nov 29.

Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Fribourg, Switzerland.

Background: Arginase competes with endothelial nitric oxide synthase (eNOS) for the substrate l-arginine and decreases NO production. This study investigated regulatory mechanisms of arginase activity in endothelial cells and its role in atherosclerosis.

Methods And Results: In human endothelial cells isolated from umbilical veins, thrombin concentration- and time-dependently stimulated arginase enzymatic activity, reaching a 1.9-fold increase (P<0.001) at 1 U/mL for 24 hours. The effect of thrombin was prevented by C3 exoenzyme or the HMG-CoA reductase inhibitor fluvastatin, which inhibit RhoA, or by the ROCK inhibitors Y-27632 and HA-1077. Adenoviral expression of constitutively active RhoA or ROCK mutants enhanced arginase activity (approximately 3-fold, P<0.001), and the effect of active RhoA mutant was inhibited by the ROCK inhibitors. Neither thrombin nor the active RhoA/ROCK mutants affected arginase II protein level, the only isozyme detectable in the cells. Moreover, a significantly higher arginase II activity (1.5-fold, not the protein level) and RhoA protein level (4-fold) were observed in atherosclerotic aortas of apoE-/- compared with wild-type mice. Interestingly, l-arginine (1 mmol/L), despite a significantly higher eNOS expression in aortas of apoE-/- mice, evoked a more pronounced contraction, which was reverted to a greater vasodilation by the arginase inhibitor l-norvaline (20 mmol/L) compared with the wild-type animals (n=5, P<0.001).

Conclusions: Thrombin enhances arginase activity via RhoA/ROCK in human endothelial cells. Higher arginase enzymatic activity is involved in atherosclerotic endothelial dysfunction in apoE-/- mice. Targeting vascular arginase may represent a novel therapeutic possibility for atherosclerosis.
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http://dx.doi.org/10.1161/01.CIR.0000142867.26182.32DOI Listing
December 2004

Reconstituted high-density lipoprotein inhibits thrombin-induced endothelial tissue factor expression through inhibition of RhoA and stimulation of phosphatidylinositol 3-kinase but not Akt/endothelial nitric oxide synthase.

Circ Res 2004 Apr 26;94(7):918-25. Epub 2004 Feb 26.

Department of Medicine, Divisions of Physiology, University of Fribourg, Fribourg, Switzerland.

Endothelial cells express negligible amounts of tissue factor (TF) that can be induced by thrombin, which is important for acute coronary syndromes. Recent research suggests that endothelial TF expression is positively regulated by RhoA and p38mapk, but negatively by Akt/endothelial nitric oxide synthase (eNOS) pathway. High-density lipoprotein (HDL) is atheroprotective and exerts antiatherothrombotic effect. This study investigated the effect of a reconstituted HDL (rHDL) on endothelial TF expression induced by thrombin and the underlying mechanisms. In cultured human umbilical vein and aortic endothelial cells, thrombin (4 U/mL, 4 hours) increased TF protein level, which was reduced by rHDL (0.1 mg/mL, 43% inhibition, n=3 to 7, P<0.01). Activation of RhoA but not p38mapk by thrombin was prevented by rHDL. rHDL stimulated Akt/eNOS pathway. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin or LY294002 abolished the activation of Akt/eNOS and reversed the inhibitory effect of rHDL on TF expression. Adenoviral expression of the active PI3K mutant (p110) reduced TF expression stimulated by thrombin without inhibiting RhoA activation, whereas expression of the active Akt mutant (m/p) further facilitated TF upregulation by thrombin. Moreover, a dominant-negative Akt mutant (KA) reduced thrombin's effect and did not reverse the rHDL's inhibitory effect on TF expression. Inhibition of eNOS by N(omega)-nitro-L-arginine methyl ester (100 micromol/L) did not affect the rHDL's effect. In conclusion, rHDL inhibits thrombin-induced human endothelial TF expression through inhibition of RhoA and activation of PI3K but not Akt/eNOS. These findings implicate a novel mechanism of antiatherothrombotic effects of HDL.
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http://dx.doi.org/10.1161/01.RES.0000124302.20396.B7DOI Listing
April 2004

Enhanced endothelial nitric oxide-synthase activity in mice infected with Trypanosoma brucei.

Int J Parasitol 2003 Sep;33(10):1099-104

Vascular Biology, Institute of Physiology, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland.

Infection of humans with Trypanosoma brucei causes sleeping sickness, which is invariably fatal if left untreated. The course of infection is characterised, among others, by multiple organ damage including cardiovascular dysfunctions such as hypotension and breakdown of the blood-brain barrier. The latter eventually leads to the parasite invasion into central nervous system and ultimately to the death of the patient. Nitric oxide (NO) synthesised from L-arginine via endothelial NO-synthase (eNOS) is involved in the control of vascular tone and permeability. The present study explores the effect of T. brucei infection on the endothelium-dependent in vitro vasomotor response of isolated mouse aortas. Aorta rings were suspended in organ chambers for isometric tension recording. The endothelium-dependent NO-mediated relaxation in response to acetylcholine (10(-9) to 10(-5) M) was markedly enhanced in the infected mice compared to controls (P<0.05), whereas the endothelium-independent vasodilation to an exogenous NO-donor, sodium nitroprusside, was comparable in both groups. Norepinephrine-stimulated contraction was also comparable in the absence or presence of the NO-synthase inhibitor N(omega)-Nitro-L-arginine methyl ester (L-NAME; 10(-4)M) in both groups. The enhanced endothelium-dependent relaxation in the infected mice correlated well with a 3.5-fold increase in eNOS protein level in these aortas as compared to those of control mice (P=0.05). Thus, T. brucei infection enhances eNOS protein expression in the endothelium, causing a pronounced vasodilation. Overproduction of NO in trypanosomiasis may be involved in the observed generalised hypotension and in an increased vascular permeability that facilitates T. brucei invasion into surrounding tissues and its penetration into the central nervous system in later phases of infection.
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http://dx.doi.org/10.1016/s0020-7519(03)00180-2DOI Listing
September 2003

Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells.

Mol Cell Biol 2002 Dec;22(24):8467-77

Vascular Biology, Institute of Physiology, University of Fribourg, CH-1700 Fribourg, Switzerland.

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC139860PMC
http://dx.doi.org/10.1128/MCB.22.24.8467-8477.2002DOI Listing
December 2002