Publications by authors named "Paul M Vanhoutte"

268 Publications

TNFα induces endothelial dysfunction in rheumatoid arthritis via LOX-1 and arginase 2: reversal by monoclonal TNFα antibodies.

Cardiovasc Res 2021 Jan 23. Epub 2021 Jan 23.

Center for Molecular Cardiology, Schlieren Campus, University of Zurich, Switzerland.

Aims: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting joints and blood vessels. Despite low levels of low-density lipoprotein cholesterol (LDL-C), RA patients exhibit endothelial dysfunction and are at increased risk of death from cardiovascular (CV) complications, but the molecular mechanism of action is unknown.We aimed in the present study to identify the molecular mechanism of endothelial dysfunction in a mouse model of RA and in patients with RA.

Methods And Results: Endothelium-dependent relaxations to acetylcholine were reduced in aortae of two TNFα transgenic mouse lines with either mild (Tg3647) or severe (Tg197) forms of RA in a time- and severity-dependent fashion as assessed by organ chamber myograph. In Tg197, TNFα plasma levels were associated with severe endothelial dysfunction. LOX-1 receptor was markedly upregulated leading to increased vascular oxLDL uptake and NFκB-mediated enhanced Arg2 expression via direct binding to its promoter resulting in reduced NO bioavailability and vascular cGMP levels as shown by ELISA and chromatin immunoprecipitation. Anti-TNFα treatment with infliximab normalized endothelial function together with LOX-1 and Arg2 serum levels in mice. In RA patients, soluble LOX-1 serum levels were also markedly increased and closely related to serum levels of C-reactive protein. Similarly, ARG2 serum levels were increased. Similarly, anti-TNFα treatment restored LOX-1 and ARG2 serum levels in RA patients.

Conclusions: Increased TNFα levels not only contribute to RA, but also to endothelial dysfunction by increasing vascular oxLDL content and activation of the LOX-1/NFκB/Arg2 pathway leading to reduced NO bioavailability and decreased cGMP levels. Anti-TNFα treatment improved both articular symptoms and endothelial function by reducing LOX-1, vascular oxLDL and Arg2 levels.
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http://dx.doi.org/10.1093/cvr/cvab005DOI Listing
January 2021

Natriuretic peptides relax human intrarenal arteries through natriuretic peptide receptor type-A recapitulated by soluble guanylyl cyclase agonists.

Acta Physiol (Oxf) 2021 03 5;231(3):e13565. Epub 2020 Nov 5.

Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.

Aim: Natriuretic peptides, BNP and ANP increase renal blood flow in experimental animals. The signalling pathway in human kidney vasculature is unknown. It was hypothesized that BNP and ANP cause endothelium-independent relaxation of human intrarenal arteries by vascular natriuretic peptide receptor-A, but not -B and -C, which is mimicked by agonists of soluble guanylyl cyclase sGC.

Methods: Human (n = 54, diameter: 665 ± 29 µm 95% CI) and control murine intrarenal arteries (n = 83, diameter 300 ± 6 µm 95% CI) were dissected and used for force recording by four-channel wire myography. Arterial segments were pre-contracted, then subjected to increasing concentrations of BNP, ANP, phosphodiesterase 5-inhibitor sildenafil, sGC-activator BAY 60-2770 and -stimulator BAY 41-2272. Endothelial nitric oxide synthase (eNOS) dependence was examined by use of L-NAME and eNOS knockout respectively. Molecular targets (NPR A-C, sGC, phosphodiesterase-5 and neprilysin) were mapped by PCR, immunohistochemistry and RNAscope.

Results: BNP, ANP, sildenafil, sGC-activation and -stimulation caused concentration-dependent relaxation of human and murine intrarenal arteries. BNP responses were independent of eNOS and were not potentiated by low concentration of phosphodiesterase-5-inhibitor, sGC-stimulator or NPR-C blocker. PCR showed NPR-A and C, phosphodiesterase-5, neprilysin and sGC mRNA in renal arteries. NPR-A mRNA and protein was observed in vascular smooth muscle and endothelial cells in arteries, podocytes, Bowmans capsule and vasa recta. NPR-C was observed in tubules, glomeruli and vasculature.

Conclusion: Activation of transmembrane NPR-A and soluble guanylyl cyclase relax human preglomerular arteries similarly to phosphodiestase-5 inhibition. The human renal arterial bed relaxes in response to cGMP pathway.
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http://dx.doi.org/10.1111/apha.13565DOI Listing
March 2021

Major histocompatibility complexes are up-regulated in glomerular endothelial cells via activation of c-Jun N-terminal kinase in 5/6 nephrectomy mice.

Br J Pharmacol 2020 11 30;177(22):5131-5147. Epub 2020 Sep 30.

Shanghai Key Laboratory of Organ Transplantation, Fudan University, Shanghai, China.

Background And Purpose: This study aims to explore the mechanism underlying the up-regulation of major histocompatibility complex (MHC) proteins in glomerular endothelial cells in 5/6 nephrectomy mice.

Experimental Approach: C57/BL6 mice were randomly allocated to sham-operated (2K) and 5/6 nephrectomy (5/6Nx) groups. Mouse splenic lymphocytes, from either syngeneic or allogeneic background, were injected into 5/6Nx mice after total body irradiation. Human glomerular endothelial cells (HGECs) were cultured for experiments in vitro. Western blots, PCR, immunohistochemical and fluorescent staining were used, along with assays of tissue cytokines, lymphocyte migration and renal function.

Key Results: Four weeks after nephrectomy, expression of both mRNA and protein of MHC II, CD80, and CD86 were increased in 5/6Nx glomerular endothelial cells. After total body irradiation, 5/6Nx mice injected with lymphocytes from Balb/c mice, but not those from C57/BL6 mice, exhibited increased creatinine levels, indicating that allograft lymphocyte transfer impaired renal function. In HGECs, the protein levels of MHC and MHC Class II transactivator (CIITA) were increased by stimulation with TNF-α or IFN-γ, which promoted human lymphocytes movement. These increases were reduced by JNK inhibitors. In the 5/6Nx mice, JNK inhibition down-regulated MHC II protein in glomerular endothelial cells, suggesting that JNK signalling participates in the regulation of MHC II protein.

Conclusion And Implications: Chronic inflammation in mice subjected to nephrectomy induces the up-regulation of MHC molecules in glomerular endothelial cells. This up-regulation is reduced by inhibition of JNK signalling.
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http://dx.doi.org/10.1111/bph.15237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589013PMC
November 2020

The NO-donor MPC-1011 stimulates angiogenesis and arteriogenesis and improves hindlimb ischemia via a cGMP-dependent pathway involving VEGF and SDF-1α.

Atherosclerosis 2020 07 30;304:30-38. Epub 2020 May 30.

Center for Molecular Cardiology, Schlieren Campus, University of Zurich, Zurich, Switzerland. Electronic address:

Background And Aims: Peripheral arterial disease (PAD) is an important cause of morbidity and mortality with little effective medical treatment currently available. Nitric oxide (NO) is crucially involved in organ perfusion, tissue protection and angiogenesis.

Methods: We hypothesized that a novel NO-donor, MPC-1011, might elicit vasodilation, angiogenesis and arteriogenesis and in turn improve limb perfusion, in a hindlimb ischemia model. Hindlimb ischemia was induced by femoral artery ligation in Sprague-Dawley rats, which were randomized to receive either placebo, MPC-1011, cilostazol or both, up to 28 days. Limb blood flow was assessed by laser Doppler imaging.

Results: After femoral artery occlusion, limb perfusion in rats receiving MPC-1011 alone or in combination with cilostazol was increased throughout the treatment regimen. Capillary density and the number of arterioles was increased only with MPC-1011. MPC-1011 improved vascular remodeling by increasing luminal diameter in the ischemic limb. Moreover, MPC-1011 stimulated the release of proangiogenic cytokines, including VEGF, SDF1α and increased tissue cGMP levels, reduced platelet activation and aggregation, potentiated proliferation and migration of endothelial cells which was blunted in the presence of soluble guanylyl cyclase inhibitor LY83583. In MPC-1011-treated rats, Lin/CD31/CXCR4 cells were increased by 92.0% and Lin/VEGFR2/CXCR4 cells by 76.8% as compared to placebo.

Conclusions: Here we show that the NO donor, MPC-1011, is a specific promoter of angiogenesis and arteriogenesis in a hindlimb ischemia model in an NO-cGMP-VEGF- dependent manner. This sets the basis to evaluate and confirm the efficacy of such therapy in a clinical setting in patients with PAD and impaired limb perfusion.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.05.012DOI Listing
July 2020

Adipocyte fatty acid-binding protein exacerbates cerebral ischaemia injury by disrupting the blood-brain barrier.

Eur Heart J 2020 09;41(33):3169-3180

State Key Laboratory of Pharmaceutical Biotechnology, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.

Aims: Adipocyte fatty acid-binding protein (A-FABP) is an adipokine implicating in various metabolic diseases. Elevated circulating levels of A-FABP correlate positively with poor prognosis in ischaemic stroke (IS) patients. No information is available concerning the role of A-FABP in the pathogenesis of IS. Experiments were designed to determine whether or not A-FABP mediates blood-brain barrier (BBB) disruption, and if so, to explore the molecular mechanisms underlying this deleterious effects.

Methods And Results: Circulating A-FABP and its cerebral expression were increased in mice after middle cerebral artery occlusion. Genetic deletion and pharmacological inhibition of A-FABP alleviated cerebral ischaemia injury with reduced infarction volume, cerebral oedema, neurological deficits, and neuronal apoptosis; BBB disruption was attenuated and accompanied by reduced degradation of tight junction proteins and induction of matrix metalloproteinases-9 (MMP-9). In patients with acute IS, elevated circulating A-FABP levels positively correlated with those of MMP-9 and cerebral infarct volume. Mechanistically, ischaemia-induced elevation of A-FABP selectively in peripheral blood monocyte-derived macrophages and cerebral resident microglia promoted MMP-9 transactivation by potentiating JNK/c-Jun signalling, enhancing degradation of tight junction proteins and BBB leakage. The detrimental effects of A-FABP were prevented by pharmacological inhibition of MMP-9.

Conclusion: A-FABP is a key mediator of cerebral ischaemia injury promoting MMP-9-mediated BBB disruption. Inhibition of A-FABP is a potential strategy to improve IS outcome.
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http://dx.doi.org/10.1093/eurheartj/ehaa207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556749PMC
September 2020

Deficiency of T-type voltage-gated calcium channels results in attenuated weight gain and improved endothelium-dependent dilatation of resistance vessels induced by a high-fat diet in mice.

J Physiol Biochem 2020 Feb 3;76(1):135-145. Epub 2020 Feb 3.

Institute of Molecular Medicine, Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark.

The deletion of T-type Ca3.1 channels may reduce high-fat diet (HFD)-induced weight gain, which correlates positively with obesity and endothelial dysfunction. Therefore, experiments were designed to study the involvement of T-type Ca3.1 channels in HFD-induced endothelial dysfunction in mice. Wildtype (WT) and Ca3.1 mice were fed either a normal diet (ND) or an HFD for 8 weeks. Body composition was assessed, and thoracic aortae and mesenteric arteries were harvested for myography to assess endothelium-dependent responses. Changes in intracellular calcium were measured by fluorescence imaging, and behavior was assessed with the open-field test. Ca3.1 mice had attenuated HFD-induced weight gain and lower total fat mass compared with WT mice. Ca3.1 mice on an HFD had reduced plasma cholesterol levels compared with WT mice on the same diet. Increased feeding efficiency, independent of food intake, was observed in WT mice on an HFD compared with an ND, but no difference in feeding efficiency between diets was observed for Ca3.1 mice. Nitric oxide-dependent dilatation was increased in mesenteric arteries of Ca3.1 mice compared with WT mice on an HFD, with no difference observed in aortae. No differences in mouse locomotor activity were observed between the experimental groups. Mice on an HFD lacking T-type channels have reduced weight gain, lower total cholesterol levels, and increased dilatation of resistance vessels compared with WT mice on an HFD, suggesting that Ca3.1 deletion protects against endothelial dysfunction in resistance vessels but not in large conduit vessels.
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http://dx.doi.org/10.1007/s13105-020-00728-2DOI Listing
February 2020

Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Thromb Haemost 2020 Jan 13;120(1):168-180. Epub 2019 Dec 13.

Center for Molecular Cardiology, Schlieren Campus, University of Zurich, Zurich, Switzerland.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac messenger ribonucleic acid expression was reduced while increased. Cardiac-specific JunD overexpressing mice ( ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.
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http://dx.doi.org/10.1055/s-0039-3400299DOI Listing
January 2020

Piezo Ion Channels in Cardiovascular Mechanobiology.

Trends Pharmacol Sci 2019 12 5;40(12):956-970. Epub 2019 Nov 5.

Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Institut de Pharmacologie Moléculaire et Cellulaire, Labex ICST, Valbonne, France. Electronic address:

Mechanotransduction has a key role in vascular development, physiology, and disease states. Piezo1 is a mechanosensitive (MS) nonselective cationic channel that occurs in endothelial and vascular smooth muscle cells. It is activated by shear stress associated with increases in local blood flow, as well as by cell membrane stretch upon elevation of blood pressure. Here, we briefly review the pharmacological modulators of Piezo and discuss current understanding of the role of Piezo1 in vascular mechanobiology and associated clinical disorders, such as atherosclerosis and hypertension. Ultimately, we believe that this research will help identify novel therapeutic strategies for the treatment of vascular diseases.
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http://dx.doi.org/10.1016/j.tips.2019.10.002DOI Listing
December 2019

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues.

J Vis Exp 2019 06 3(148). Epub 2019 Jun 3.

The State Key Laboratory of Pharmaceutical Biotechnology and the Department of Pharmacology and Pharmacy, University of Hong Kong;

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.
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http://dx.doi.org/10.3791/59688DOI Listing
June 2019

Acute activation of endothelial AMPK surprisingly inhibits endothelium-dependent hyperpolarization-like relaxations in rat mesenteric arteries.

Br J Pharmacol 2019 08 4;176(16):2905-2921. Epub 2019 Jul 4.

Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.

Background And Purpose: Endothelium-dependent hyperpolarizations (EDHs) contribute to the regulation of peripheral resistance. They are initiated through opening of endothelial calcium-activated potassium channels (K ); the potassium ions released then diffuse to the underlying smooth muscle cells, causing hyperpolarization and thus relaxation. The present study aimed to examine whether or not AMPK modulates EDH-like relaxations in rat mesenteric arteries.

Experimental Approach: Arterial rings were isolated for isometric tension recording. AMPK activity and protein level were measured by ELISA and western blotting respectively.

Key Results: The AMPK activator, AICAR, reduced ACh-induced EDH-like relaxations and increased AMPK activity in preparations with endothelium; these responses were prevented by compound C, an AMPK inhibitor. AICAR inhibited relaxations induced by SKA-31 (opener of endothelial K ) but did not affect potassium-induced, hyperpolarization-attributable relaxations or increase AMPK activity in preparations without endothelium. A769662, another AMPK activator, not only caused a similar inhibition of relaxations to ACh and SKA-31 in preparations with endothelium but also inhibited hyperpolarization-attributable relaxations and augmented AMPK activity in rings without endothelium. Protein levels of total AMPKα, AMPKα1, or AMPKβ1/2 were comparable between preparations with and without endothelium.

Conclusions And Implications: Activation of endothelial AMPK, by either AICAR or A769662, acutely inhibits EDH-like relaxations of rat mesenteric arteries. Furthermore, A769662 inhibits signalling downstream of smooth muscle hyperpolarization. In view of the major blunting effect of AMPK activation on EDH-like relaxations, caution should be applied when administering therapeutic agents that activate AMPK in patients with endothelial dysfunction characterized by reduced production and/or bioavailability of NO.
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http://dx.doi.org/10.1111/bph.14716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6637042PMC
August 2019

Endothelial muscarinic M -receptors: A Σ-target?

Authors:
Paul M Vanhoutte

Acta Physiol (Oxf) 2019 05 29;226(1):e13273. Epub 2019 Mar 29.

State Key Laboratory of Biopharmaceutical Technologies, Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China.

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http://dx.doi.org/10.1111/apha.13273DOI Listing
May 2019

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Am J Physiol Renal Physiol 2018 12 3;315(6):F1670-F1682. Epub 2018 Oct 3.

Cardiovascular and Renal Research, University of Southern Denmark , Odense , Denmark.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.
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http://dx.doi.org/10.1152/ajprenal.00391.2018DOI Listing
December 2018

The acute blood pressure-lowering effect of amiloride is independent of endothelial ENaC and eNOS in humans and mice.

Acta Physiol (Oxf) 2019 01 11;225(1):e13189. Epub 2018 Oct 11.

Department of Cardiovascular and Renal Research, Institute for Molecular Medicine, University of Southern Denmark, Odense C, Denmark.

Aims: The epithelial sodium channel (ENaC) is expressed in cultured endothelial cells and inhibitory coupling to eNOS activity has been proposed. The present study tested the hypothesis that ENaC blockers increase systemic NO-products and lower blood pressure in patients and mice, depending on eNOS.

Methods: NO-products and cGMP were measured in diabetes patient urine and plasma samples before and after amiloride treatment (20-40 mg for two days, plasma n = 22, urine n = 12 and 5-10 mg for eight weeks, plasma n = 52, urine n = 55). Indwelling catheters were implanted in the femoral artery and vein in mice for continuous arterial blood pressure and heart rate recordings and infusion.

Results: Treatment with amiloride for two days increased plasma and urine NO-products, while plasma cGMP decreased and urinary cGMP was unchanged in patient samples. Eight weeks of treatment with amiloride did not alter NO-products and cGMP. In mice, amiloride boli of 5, 50, and 500 µg/kg lowered heart rate and arterial blood pressure significantly and acutely. Benzamil had no effect on pressure and raised heart rate. In hypertensive eNOS and L-NAME-treated mice, amiloride lowered blood pressure significantly. L-NAME infusion significantly decreased NO-products in plasma; amiloride and eNOS-deletion had no effect. An acetylcholine bolus resulted in acute blood pressure drop that was attenuated in eNOS and L-NAME mice. ENaC subunit expressions were not detected consistently in human and mouse arteries and endothelial cells.

Conclusion: Amiloride has an acute hypotensive action not dependent on ENaC and eNOS and likely related to the heart.
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http://dx.doi.org/10.1111/apha.13189DOI Listing
January 2019

Lipocalin-2 derived from adipose tissue mediates aldosterone-induced renal injury.

JCI Insight 2018 09 6;3(17). Epub 2018 Sep 6.

The State Key Laboratory of Pharmaceutical Biotechnology and.

Lipocalin-2 is not only a sensitive biomarker, but it also contributes to the pathogenesis of renal injuries. The present study demonstrates that adipose tissue-derived lipocalin-2 plays a critical role in causing both chronic and acute renal injuries. Four-week treatment with aldosterone and high salt after uninephrectomy (ANS) significantly increased both circulating and urinary lipocalin-2, and it induced glomerular and tubular injuries in kidneys of WT mice. Despite increased renal expression of lcn2 and urinary excretion of lipocalin-2, mice with selective deletion of lcn2 alleles in adipose tissue (Adipo-LKO) are protected from ANS- or aldosterone-induced renal injuries. By contrast, selective deletion of lcn2 alleles in kidney did not prevent aldosterone- or ANS-induced renal injuries. Transplantation of fat pads from WT donors increased the sensitivity of mice with complete deletion of Lcn2 alleles (LKO) to aldosterone-induced renal injuries. Aldosterone promoted the urinary excretion of a human lipocalin-2 variant, R81E, in turn causing renal injuries in LKO mice. Chronic treatment with R81E triggered significant renal injuries in LKO, resembling those observed in WT mice following ANS challenge. Taken in conjunction, the present results demonstrate that lipocalin-2 derived from adipose tissue causes acute and chronic renal injuries, largely independent of local lcn2 expression in kidney.
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http://dx.doi.org/10.1172/jci.insight.120196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171815PMC
September 2018

Endothelial SIRT1 prevents age-induced impairment of vasodilator responses by enhancing the expression and activity of soluble guanylyl cyclase in smooth muscle cells.

Cardiovasc Res 2019 03;115(3):678-690

State Key Laboratory of Pharmaceutical Biotechnology and Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, LKS Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China.

Aims: Aged arteries are characterized by attenuated vasodilator and enhanced vasoconstrictor responses, which contribute to the development of diseases such as arterial hypertension, atherosclerosis, and heart failure. SIRT1 is a longevity regulator exerting protective functions against vascular ageing, although the underlying mechanisms remain largely unknown. This study was designed to elucidate the signalling pathways involved in endothelial SIRT1-mediated vasodilator responses in the arteries of young and old mice. In particular, the contributions of nitric oxide (NO), endothelial NO synthase (eNOS), cyclooxygenase (COX), and/or soluble guanylyl cyclase (sGC) were examined.

Methods And Results: Wild type (WT) or eNOS knockout (eKO) mice were cross-bred with those overexpressing human SIRT1 selectively in the vascular endothelium (EC-SIRT1). Arteries were collected from the four groups of mice (WT, EC-SIRT1, eKO, and eKO-SIRT1) to measure isometric relaxations/contractions in response to various pharmacological agents. Reduction of NO bioavailability, hyper-activation of COX signalling, and down-regulation of sGC collectively contributed to the decreased vasodilator and increased vasoconstrictor responses in arteries of old WT mice. Overexpression of endothelial SIRT1 did not block the reduction in NO bioavailability but attenuated the hyper-activation of COX-2, thus protecting mice from age-induced vasoconstrictor responses in arteries of EC-SIRT1 mice. Deficiency of eNOS did not affect endothelial SIRT1-mediated anti-contractile activities in arteries of eKO-SIRT1 mice. Mechanistic studies revealed that overexpression of endothelial SIRT1 enhanced Notch signalling to up-regulate sGCβ1 in smooth muscle cells. Increased expression and activity of sGC prevented age-induced hyper-activation of COX-2 as well as the conversion of endothelium-dependent relaxations to contractions in arteries of EC-SIRT1 mice.

Conclusion: Age-induced down-regulation of sGC and up-regulation of COX-2 in arteries are at least partly attributable to the loss-of-endothelial SIRT1 function. Enhancing the endothelial expression and function of SIRT1 prevents early vascular ageing and maintains vasodilator responses, thus representing promising drug targets for cardiovascular diseases.
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http://dx.doi.org/10.1093/cvr/cvy212DOI Listing
March 2019

Low but not high frequency of intermittent hypoxia suppresses endothelium-dependent, oxidative stress-mediated contractions in carotid arteries of obese mice.

J Appl Physiol (1985) 2018 11 9;125(5):1384-1395. Epub 2018 Aug 9.

Division of Respiratory Medicine, Department of Medicine, University of Hong Kong , China.

Obstructive sleep apnea is characterized by intermittent hypoxia (IH) during sleep and predisposes to endothelial dysfunction. Obesity is a major risk factor for the occurrence of sleep apnea. The present study compared the functional impact of low- (IH10; 10 hypoxic events/h) and high-frequency (IH60; 60 hypoxic events/h) IH for 4 wk on endothelial function in male C57BL/6 mice with or without high-fat (HF) diet-induced obesity. Mean arterial blood pressure (tail cuff method) was increased in obese mice after IH60 exposure, i.e., HF + IH60 group. The serum levels of the oxidative stress marker malondialdehyde were augmented in lean IH60 and HF groups, with a further increase in HF + IH60 but a reduction in HF + IH10 mice compared with the HF group. Vascular responsiveness was assessed as changes in isometric tension in isolated arteries. Relaxations to the endothelium-dependent vasodilator acetylcholine were impaired in HF + IH60 aortae. Endothelium-dependent contractions (EDC; response to acetylcholine in the presence of the nitric oxide synthase inhibitor l-NAME) in carotid arteries were augmented in the HF group, but this HF-induced augmentation was suppressed by low-frequency IH exposure. The addition of apocynin (antioxidant) reduced EDC in HF and HF + IH60 groups but not in HF + IH10 group. In conclusion, these findings suggest that exposure of obese mice to mild IH exerts preconditioning-like suppression of endothelium-dependent and oxidative stress-mediated contractions. When IH severity increases, this suppression diminishes and endothelial dysfunction accelerates. NEW & NOTEWORTHY The present study demonstrates, for the first time, that low-frequency intermittent hypoxia may exert a preconditioning-like suppression of oxidative stress-induced endothelium-dependent contractions in mice with diet-induced obesity. This relative suppression was diminished as intermittent hypoxia became more severe, and a deleterious effect on endothelial function emerged.
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http://dx.doi.org/10.1152/japplphysiol.00224.2018DOI Listing
November 2018

Activation of NQO-1 mediates the augmented contractions of isolated arteries due to biased activity of soluble guanylyl cyclase in their smooth muscle.

Naunyn Schmiedebergs Arch Pharmacol 2018 11 30;391(11):1221-1235. Epub 2018 Jul 30.

Department of Pharmacology and Pharmacy and State Key Laboratory for Pharmaceutical Biotechnology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 2/F Laboratory Block, Li Ka Shing Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, SAR, China.

Earlier studies on isolated arteries demonstrated that the para-quinone thymoquinone, like acute hypoxia, induces augmentation of contractions, depending on biased activity of soluble guanylyl cyclase (sGC), generating inosine-3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine-3',5'-cyclic monophosphate (cyclic GMP). NAD(P)H:quinone oxidoreductase 1 (NQO-1), the enzyme responsible for biotransformation of quinones into hydroquinones, was examined for its involvement in these endothelium-dependent augmentations, establishing a link between the metabolism of quinones by NQO-1 and biased sGC activity. Isolated arteries of Sprague-Dawley rats (aortae and mesenteric arteries) and farm pigs (coronary arteries) were studied for measurement of changes in tension and collected to measure NQO-1 activity or its protein level. β-lapachone, an ortho-quinone and hence substrate of NQO-1, increased the activity of the enzyme and augmented contractions in arteries with endothelium. This augmentation was inhibited by endothelium removal and inhibitors of endothelial NO synthase (eNOS), sGC, or NQO-1; in preparations without endothelium or treated with an eNOS inhibitor, it was restored by the NO donor DETA NONOate and by ITP and cyclic IMP, revealing biased sGC activity as the underlying mechanism, as with thymoquinone. Hydroquinone, the end product of quinone metabolism by NQO-1, augmented contractions depending on sGC activation but in an endothelium-independent manner. In coronary arteries, repeated acute hypoxia caused similar augmentations as those to quinones that were inhibited by the NQO-1 inhibitor dicoumarol. Augmentations of contraction observed with different naturally occurring quinones and with acute hypoxia are initiated by quinone metabolism by NQO-1, in turn interfering with the NO/biased sGC pathway, suggesting a possibly detrimental role of this enzyme in ischemic cardiovascular disorders.
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http://dx.doi.org/10.1007/s00210-018-1548-7DOI Listing
November 2018

No Protective Effect of Constitutive Activation of AMPK in Endothelial Cells on Vascular Function in Aged Obese Mice but Augmented α1-Adrenergic Contractions in Renal Arteries Reversible by Weight Loss.

J Vasc Res 2018 11;55(4):189-202. Epub 2018 Jul 11.

Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

Background: Aging, obesity, and diabetes favor vascular dysfunction. Endothelial activation of adenosine monophosphate-activated protein kinase (AMPK) has protective effects in diabetes.

Methods: Mice with constitutive endothelial activation of AMPK (CA-AMPK) were given a high fat diet to induce obesity or kept on standard chow as lean controls for up to 2 years. A subset of obese animals was changed to standard chow after 30 weeks of high fat feeding. En-dothelium-dependent and endothelium-independent responses were examined by isometric tension recording.

Results And Conclusion: Endothelium-dependent nitric oxide (NO)- and apamin plus charybdotoxin-sensitive relaxations were preserved and similar between aortic or renal arterial preparations of lean and obese CA-AMPK mice and their wild-type littermates. Despite comparable release of vasoconstrictor prostanoids, cyclooxygenase-dependent contractions were enhanced during NO synthase inhibition in carotid arterial rings of obese CA-AMPK mice. Contractions to the α1-adrenoceptor agonist phenylephrine were augmented in renal arteries of obese animals, a genotype-independent phenomenon reversible by weight loss. These data indicate a higher α1-adrenergic reactivity in renal arteries of aged mice with obesity. The current results highlight the potential of weight loss to alleviate vascular dysfunction. However, endothelial activation of the AMPK pathway in obesity may not be sufficient to prevent vascular dysfunction without lifestyle changes.
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http://dx.doi.org/10.1159/000489959DOI Listing
August 2019

EP4 emerges as a novel regulator of bile acid synthesis and its activation protects against hypercholesterolemia.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 09 8;1863(9):1029-1040. Epub 2018 Jun 8.

Department of Pharmacology and Pharmacy and State Key Laboratory of Pharmaceutical Biotechnology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. Electronic address:

Prostaglandin E receptor subtype 4 (EP4) knockout mice develops spontaneous hypercholesterolemia but the detailed mechanisms by which EP4 affects cholesterol homeostasis remains unexplored. We sought to determine the cause of hypercholesterolemia in EP4 knockout mice, focusing on the role of EP4 in regulating the synthesis and elimination of cholesterol. Deficiency of EP4 significantly decreased total bile acid levels in the liver by 26.2% and the fecal bile acid content by 27.6% as compared to wild type littermates, indicating that the absence of EP4 decreased hepatic bile acid synthesis and their subsequent excretion in stools. EP4 deficiency negatively regulate bile acid synthesis through repression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK)-mediated cholesterol 7α-hydroxylase (CYP7A1) expression and that the hypercholesterolemia in EP4 knockout mice is due to a defect in cholesterol conversion into bile acids. Deficiency of EP4 also increased de novo cholesterol synthesis and altered cholesterol fluxes in and out of the liver. Treating high fat diet-challenged mice with the pharmacological EP4 agonist, CAY10580 (200 μg/kg body weight/day i.p) for three weeks effectively prevented diet-induced hypercholesterolemia, enhanced endogenous bile acid synthesis and their fecal excretion. In summary, EP4 plays a critical role in maintaining cholesterol homeostasis by regulating the synthesis and elimination of bile acids. Activation of EP4 serves as an effective novel strategy to promote cholesterol disposal in the forms of bile acids in order to lower plasma cholesterol levels.
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http://dx.doi.org/10.1016/j.bbalip.2018.06.003DOI Listing
September 2018

Apolipoprotein E favours the blunting by high-fat diet of prostacyclin receptor activation in the mouse aorta.

Br J Pharmacol 2018 09 22;175(17):3453-3469. Epub 2018 Jul 22.

Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, SAR, China.

Background And Purpose: NO-mediated, endothelium-dependent relaxations of isolated arteries are blunted by ageing and high-fat diets, as well as by apolipoprotein E deletion. The present study was designed to test the hypothesis that apolipoprotein E deletion impairs endothelium-dependent responses to prostacyclin (IP) receptor activation.

Experimental Approach: Five-week-old ApoE and ApoE mice were fed normal chow or high-fat diet for 29 weeks. The aortae were isolated for the measurements of isometric tension in Halpern-Mulvany myographs. Levels of proteins were assessed by Western blotting and immunofluorescence, and cyclic nucleotide levels by elisa.

Key Results: The IP receptor agonist, iloprost, induced endothelium-, NO-synthase- and IP-dependent relaxations in aortae of young ApoE mice. High-fat diet favoured activation of thromboxane receptors by iloprost, causing contraction. Apolipoprotein E was present in aortae of ApoE mice, especially in endothelium. Its presence was augmented by high-fat diet. Its deletion potentiated iloprost-induced relaxations in aortae of young mice and prevented the blunting of this response by high-fat diet. Levels of cAMP were higher, but those of cGMP were lower in the aorta of ApoE than in ApoE mice of the same age. The levels of IP receptor protein were not different between ApoE and ApoE mice.

Conclusions And Implications: Iloprost induced an endothelium-dependent relaxation in the aorta of young healthy mice which involved both the cGMP and cAMP pathways. This response was blunted by prolonged exposure to a high-fat diet. Apolipoprotein E deletion potentiated relaxations to IP receptor activation, independently of age and diet.
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http://dx.doi.org/10.1111/bph.14386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086984PMC
September 2018

Circulating MicroRNAs in Young Patients with Acute Coronary Syndrome.

Int J Mol Sci 2018 May 15;19(5). Epub 2018 May 15.

Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Circulating microRNAs (miRNAs) hold great potential as novel diagnostic markers for acute coronary syndrome (ACS). This study sought to identify plasma miRNAs that are differentially expressed in young ACS patients (mean age of 38.5 ± 4.3 years) and evaluate their diagnostic potentials. Small RNA sequencing (sRNA-seq) was used to profile plasma miRNAs. Discriminatory power of the miRNAs was determined using receiver operating characteristic (ROC) analysis. Thirteen up-regulated and 16 down-regulated miRNAs were identified in young ACS patients. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation showed miR-183-5p was significantly up-regulated (8-fold) in ACS patients with non-ST-segment elevated myocardial infarction (NSTEMI) whereas miR-134-5p, miR-15a-5p, and let-7i-5p were significantly down-regulated (5-fold, 7-fold and 3.5-fold, respectively) in patients with ST-segment elevated myocardial infarction (STEMI), compared to the healthy controls. MiR-183-5p had a high discriminatory power to differentiate NSTEMI patients from healthy controls (area under the curve (AUC) of ROC = 0.917). The discriminatory power for STEMI patients was highest with let-7i-5p (AUC = 0.833) followed by miR-134-5p and miR-15a-5p and this further improved (AUC = 0.935) with the three miRNAs combination. Plasma miR-183-5p, miR-134-5p, miR-15a-5p and let-7i-5p are deregulated in STEMI and NSTEMI and could be potentially used to discriminate the two ACS forms.
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http://dx.doi.org/10.3390/ijms19051467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983847PMC
May 2018

Nitric Oxide: From Good to Bad.

Authors:
Paul M Vanhoutte

Ann Vasc Dis 2018 Mar;11(1):41-51

State Key Laboratory of Pharmaceutical Biotechnology and Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, HKSAR, China.

This essay summarizes a lecture presented on October 19th, 2017, during the 58th Annual Meeting of the Japanese College of Angiology in Nagoya, Japan. The lecture summarizes several instances where the absence of relaxations of isolated blood vessels in response to endothelium-dependent vasodilator agonists, which cause activation of endothelial nitric oxide synthase (eNOS) and consequent production of endothelium-derived nitric oxide (NO) and stimulation of soluble guanylyl cyclase (sGC) in underlying vascular smooth muscle, or hypoxia are curtailed or reversed to endothelium-dependent contractions. Chosen examples include selective dysfunction of eNOS activation in regenerated endothelial cells, unresponsiveness of vascular smooth muscle cells to NO during subarachnoid hemorrhage, and biased activation of sGC in vascular smooth muscle cells during acute exposure to hypoxia. The main message of this essay is that absence, blunting, or reversal of endothelium-dependent relaxations in response to vasodilator agonists cannot necessarily be interpreted as a sign of endothelial dysfunction. (This is a review article based on the invited lecture of the 58th Annual Meeting of Japanese College of Angiology.).
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http://dx.doi.org/10.3400/avd.ra.17-00134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882356PMC
March 2018

Deletion of Rap1 disrupts redox balance and impairs endothelium-dependent relaxations.

J Mol Cell Cardiol 2018 02 23;115:1-9. Epub 2017 Dec 23.

Department of Pharmacology and Pharmacy, State Key Laboratory of Pharmaceutical Biotechnology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China; School of Biomedical Science, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China. Electronic address:

Aims: Repressor activator protein 1 (Rap1) is conventionally known as a static structural component of the telomere, but recent evidence indicates that it exerts functions within and outside the nucleus taking part in metabolic regulation and promoting inflammatory responses. The present study investigated whether or not Rap1 deletion affects oxidative stress and nitric oxide (NO) bioavailability in the vascular wall, thus modulating endothelial function.

Methods And Results: Vascular responsiveness was studied in wire myographs in aortae from Rap1 wildtype and knockout mice. Deletion of Rap1 impaired endothelium-dependent relaxations elicited by acetylcholine. Rap1 deficiency did not affect the activation of endothelial NO synthase or the sensitivity of vascular smooth muscle to NO donors. The blunted acetylcholine-mediated relaxations in Rap1 deficient aortae were restored with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors, apocynin or VAS2870. Rap1 deletion lowered cellular thiol-redox status and diminished activities of thiol-redox enzymes, thioredoxin 1 and glutaredoxin 1.

Conclusions: The capacity of thioredoxin 1 and glutaredoxin 1 to reduce intra-protein disulfide bridges is weakened in Rap1 deficient mice, resulting in hyper-activation of NADPH oxidase and greater reactive oxygen species generation. The high oxidative stress in Rap1 deficient mice is implicated with greater oxidative breakdown of NO, explaining the blunted acetylcholine-mediated relaxations in this animal. These findings imply that Rap1 plays an unanticipated role in regulating the fate of NO (a pivotal determinant of vascular homeostasis) and thus identify a new physiological importance of the telomere-associated protein.
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http://dx.doi.org/10.1016/j.yjmcc.2017.12.009DOI Listing
February 2018

Paeonol Attenuates LPS-Induced Endothelial Dysfunction and Apoptosis by Inhibiting BMP4 and TLR4 Signaling Simultaneously but Independently.

J Pharmacol Exp Ther 2018 03 19;364(3):420-432. Epub 2017 Dec 19.

Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia (K.W.C., Y.S.L., D.M., M.R.M.); and State Key Laboratory of Pharmaceutical Biotechnology, Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, People's Republic of China (P.M.V.)

Inflammatory injury of the endothelium leads to apoptosis and endothelial dysfunction. The current study explored the effect and mechanisms of paeonol in inflammation-induced apoptosis and endothelial dysfunction induced by lipopolysaccharides (LPSs). The effects of paeonol on LPS-induced inflammatory injury were assessed by Western blotting, flow cytometry and reactive oxygen species (ROS) measurement in human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice. Vascular reactivity of isolated mouse aortae was examined using wire myographs. The exposure of HUVECs to LPS increased the protein presence of Toll-like receptor 4 (TLR4), bone morphogenic protein 4 (BMP4), BMP receptor type 1A, nicotinamide adenine dinucleotide phosphate oxidase subunit 2, mitogen-activated protein kinase (MAPK), inducible nitric oxide synthase (iNOS), and cleaved caspase 3, as well as decreased it in phosphorylated endothelial nitric oxide synthase; these effects were prevented by treatment with paeonol. Similarly, cotreatment with paeonol reversed BMP4-induced apoptosis in HUVECs. Relaxation in response to the endothelium-dependent vasodilator acetylcholine were impaired in mouse aortae after exposure to LPSs; this endothelial dysfunction was reversed by cotreatment with paeonol, noggin (a BMP4 inhibitor), TAK242 (TLR4 antagonist), apocynin (an ROS scavenger), MAPK inhibitors, and AG (an iNOS inhibitor). BMP4 small interfering RNAs (siRNAs) abolished LPS-induced upregulation of BMP4 and cleaved caspase 3 protein, but not in cells treated with TLR4 siRNA and vice versa. The silencing of TLR4 and BMP4 abolished the inhibitory effects of paeonol on LPS-induced activation of cleaved caspase 3. The present results demonstrate that paeonol reduces LPS-induced endothelial dysfunction and apoptosis by inhibiting TLR4 and BMP4 signaling independently.
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http://dx.doi.org/10.1124/jpet.117.245217DOI Listing
March 2018

L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

Anesthesiology 2018 03;128(3):564-573

From the Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China; Applied Science and Environmental Studies, School of Science and Technology, The Open University of Hong Kong, Kowloon, Hong Kong Special Administrative Region, China (E.S.W.W.); and the State Key Laboratory of Pharmaceutical Biotechnologies, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China (S.W.S.L., P.M.V.).

Background: The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine.

Methods: Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 μM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed.

Results: Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4).

Conclusions: These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.
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http://dx.doi.org/10.1097/ALN.0000000000002032DOI Listing
March 2018

Measuring non-polyaminated lipocalin-2 for cardiometabolic risk assessment.

ESC Heart Fail 2017 11 27;4(4):563-575. Epub 2017 Jun 27.

State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, Hong Kong, China.

Aims: Lipocalin-2 is a pro-inflammatory molecule characterized by a highly diversified pattern of expression and structure-functional relationships. In vivo, this molecule exists as multiple variants due to post-translational modifications and/or protein-protein interactions. Lipocalin-2 is modified by polyamination, which enhances the clearance of this protein from the circulation and prevents its excessive accumulation in tissues. On the other hand, animal studies suggest that non-polyaminated lipocalin-2 (npLcn2) plays a causal role in the pathogenesis of obesity-associated medical complications. The present study examined the presence of npLcn2 in samples from healthy volunteers or patients with cardiac abnormalities and evaluated npLcn2 as a biomarker for cardiometabolic risk assessment.

Methods And Results: Immunoassays were developed to quantify npLcn2 in blood and urine samples collected from 100 volunteers (59 men and 41 women), or venous plasma and pericardial fluid samples obtained from 37 cardiothoracic surgery patients. In healthy volunteers, npLcn2 levels in serum are significantly higher in obese and overweight than in lean subjects. After adjustment for age, gender, smoking, and body mass index (BMI), serum npLcn2 levels are positively correlated with heart rate, circulating triglycerides, high-sensitivity C-reactive protein (hsCRP), and creatinine in plasma. The npLcn2 levels in urine are significantly increased in subjects with metabolic syndrome and positively correlated with BMI, heart rate, circulating triglycerides, and urinary aldosterone. In cardiothoracic surgery patients, the circulating concentrations of npLcn2 are higher (more than two-fold) than those of healthy volunteers and positively correlated with the accumulation of this protein in the pericardial fluid. Heart failure patients exhibit excessive expression and distribution of npLcn2 in mesothelial cells and adipocytes of the parietal pericardium, which are significantly correlated with the elevated plasma levels of npLcn2, total cholesterol, and creatinine.

Conclusions: Quantitative and qualitative evaluation of npLcn2 in human biofluid samples and tissue samples can be applied for risk assessment of healthy individuals and disease management of patients with obesity-related cardiometabolic and renal complications.
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http://dx.doi.org/10.1002/ehf2.12183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695172PMC
November 2017

Inhibition of Vascular c-Jun N-Terminal Kinase 2 Improves Obesity-Induced Endothelial Dysfunction After Roux-en-Y Gastric Bypass.

J Am Heart Assoc 2017 Nov 14;6(11). Epub 2017 Nov 14.

Center for Molecular Cardiology, University of Zurich, Switzerland

Background: Roux-en-Y gastric bypass (RYGB) reduces obesity-associated comorbidities and cardiovascular mortality. RYGB improves endothelial dysfunction, reducing c-Jun N-terminal kinase (JNK) vascular phosphorylation. JNK activation links obesity with insulin resistance and endothelial dysfunction. Herein, we examined whether JNK1 or JNK2 mediates obesity-induced endothelial dysfunction and if pharmacological JNK inhibition can mimic RYGB vascular benefits.

Methods And Results: After 7 weeks of a high-fat high-cholesterol diet, obese rats underwent RYGB or sham surgery; sham-operated ad libitum-fed rats received, for 8 days, either the control peptide D-TAT or the JNK peptide inhibitor D-JNKi-1 (20 mg/kg per day subcutaneous). JNK peptide inhibitor D-JNKi-1 treatment improved endothelial vasorelaxation in response to insulin and glucagon-like peptide-1, as observed after RYGB. Obesity increased aortic phosphorylation of JNK2, but not of JNK1. RYGB and JNK peptide inhibitor D-JNKi-1 treatment blunted aortic JNK2 phosphorylation via activation of glucagon-like peptide-1-mediated signaling. The inhibitory phosphorylation of insulin receptor substrate-1 was reduced, whereas the protein kinase B/endothelial NO synthase pathway was increased and oxidative stress was decreased, resulting in improved vascular NO bioavailability.

Conclusions: Decreased aortic JNK2 phosphorylation after RYGB rapidly improves obesity-induced endothelial dysfunction. Pharmacological JNK inhibition mimics the endothelial protective effects of RYGB. These findings highlight the therapeutic potential of novel strategies targeting vascular JNK2 against the severe cardiovascular disease associated with obesity.
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http://dx.doi.org/10.1161/JAHA.117.006441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721746PMC
November 2017

Deletion of T-type calcium channels Ca3.1 or Ca3.2 attenuates endothelial dysfunction in aging mice.

Pflugers Arch 2018 02 29;470(2):355-365. Epub 2017 Oct 29.

Cardiovascular and Renal Research, University of Southern Denmark, J. B. Winsløwsvej 21, 3., 5000, Odense C, Denmark.

Impairment of endothelial function with aging is accompanied by reduced nitric oxide (NO) production. T-type Ca3.1 channels augment nitric oxide and co-localize with eNOS. Therefore, the hypothesis was that T-type channels contribute to the endothelial dysfunction of aging. Endothelial function was determined in mesenteric arteries (perfusion) and aortae (isometric contraction) of young and old wild-type (WT), Ca3.1, and Ca3.2 knockout mice. NO production was measured by fluorescence imaging in mesenteric arteries. With age, endothelium-dependent subsequent dilatation (following depolarization with KCl) of mesenteric arteries was diminished in the arteries of WT mice, unchanged in Ca3.2 preparations but increased in those of Ca3.1 mice. NO synthase inhibition abolished the subsequent dilatation in mesenteric arteries and acetylcholine-induced relaxations in aortae. NO levels were significantly reduced in mesenteric arteries of old compared to young WT mice. In Ca3.1 and Ca3.2 preparations, NO levels increased significantly with age. Relaxations to acetylcholine were significantly smaller in the aortae of old compared to young WT mice, while such responses were comparable in preparations of young and old Ca3.1 and Ca3.2 mice. The expression of Ca3.1 was significantly reduced in aortae from aged compared to young WT mice. The level of phosphorylated eNOS was significantly increased in aortae from aged Ca3.1 mice. In conclusion, T-type calcium channel-deficient mice develop less age-dependent endothelial dysfunction. Changes in NO levels are involved in this phenomenon in WT and Ca3.1 mice. These findings suggest that T-type channels play an important role in age-induced endothelial dysfunction.
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http://dx.doi.org/10.1007/s00424-017-2068-xDOI Listing
February 2018