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1.  Deamidated Lipocalin‐2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice 
Background
Lipocalin‐2 is a proinflammatory adipokine upregulated in obese humans and animals. A pathogenic role of lipocalin‐2 in hypertension has been suggested. Mice lacking lipocalin‐2 are protected from dietary obesity‐induced cardiovascular dysfunctions. Administration of lipocalin‐2 causes abnormal vasodilator responses in mice on a high‐fat diet (HFD).
Methods and Results
Wild‐type and lipocalin‐2 knockout mice were fed with standard chow or HFD. Immunoassays were performed for evaluating the circulating and tissue contents of lipocalin‐2. The relaxation and contraction of arteries were studied using a wire myograph. Blood pressure was monitored with implantable radio telemetry. Dietary obesity promoted the accumulation of lipocalin‐2 protein in blood and arteries. Deficiency of this adipokine protected mice from dietary obesity‐induced elevation of blood pressure. Mass spectrometry analysis revealed that human and murine lipocalin‐2 were modified by polyamination. Polyaminated lipocalin‐2 was rapidly cleared from the circulation. Adipose tissue was a major site for lipocalin‐2 deamidation. The circulating levels and the arterial accumulation of deamidated lipocalin‐2 were significantly enhanced by treatment with linoleic acid (18:2n−6), which bound to lipocalin‐2 with high affinity and prevented its interactions with matrix metalloproteinase 9 (MMP9). Combined administration of linoleic acid with lipocalin‐2 caused vascular inflammation and endothelial dysfunction and raised the blood pressure of mice receiving standard chow. A human lipocalin‐2 mutant with cysteine 87 replaced by alanine (C87A) contained less polyamines and exhibited a reduced capacity to form heterodimeric complexes with MMP9. After treatment, C87A remained in the circulation for a prolonged period of time and evoked endothelial dysfunction in the absence of linoleic acid.
Conclusions
Polyamination facilitates the clearance of lipocalin‐2, whereas the accumulation of deamidated lipocalin‐2 in arteries causes vascular inflammation, endothelial dysfunction, and hypertension.
doi:10.1161/JAHA.114.000837
PMCID: PMC4187505  PMID: 24721803
adipokine; endothelial dysfunction; inflammation; lipotoxicity; obesity
2.  New Insights in the Contribution of Voltage-Gated Nav Channels to Rat Aorta Contraction 
PLoS ONE  2009;4(10):e7360.
Background
Despite increasing evidence for the presence of voltage-gated Na+ channels (Nav) isoforms and measurements of Nav channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Nav channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Nav channels in the control of rat aortic contraction.
Methodology/Principal Findings
Nav channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Nav channels and smooth muscle α-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Nav transcripts: Nav1.2, Nav1.3, and Nav1.5. Only the Nav1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Nav channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Nav channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2–10 mM), which induced moderate membrane depolarization (e.g., from −55.9±1.4 mV to −45.9±1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 µM) and blocked by TTX (1 µM). KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX.
Conclusions/Significance
These results define a new role for Nav channels in arterial physiology, and suggest that the TTX-sensitive Nav1.2 isoform, together with the Na+/Ca2+ exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization.
doi:10.1371/journal.pone.0007360
PMCID: PMC2752992  PMID: 19809503
3.  Consequences of reduced production of NO on vascular reactivity of porcine coronary arteries after angioplasty: importance of EDHF 
British Journal of Pharmacology  2002;136(8):1153-1161.
The consequences of the reduced production of nitric oxide (NO) by cells from regenerated endothelium were investigated by measuring membrane potential of smooth muscle cells (SMCs), isometric tension and cyclic nucleotides content in porcine coronary arteries with intimal thickening, four weeks following angioplasty.Under basal conditions, SMCs of coronary arteries with regenerated endothelium were depolarized by 10 mV. This depolarization was associated with 82% decreased level of cGMP without alteration in cAMP.Sodium nitroprusside (SNP, 1 μM) repolarized SMCs of the previously denuded coronary arteries. This repolarization was abolished by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) and not suppressed by glibenclamide (10 μM), iberiotoxin (IbTX, 100 nM) and the combination of charybdotoxin (ChTX, 40 nM) plus apamin (100 nM).Four-aminopyridine (4-AP, 1-5 mM) generated spontaneous rhythmic activities only in coronary arteries with regenerated endothelium which were abolished by SNP. Nevertheless, 4-AP did not suppress the repolarization induced by SNP.In vascular segments with regenerated endothelium, contracted with prostaglandin F2α (PGF2α), relaxation to bradykinin (BK, 30 nM) was unaltered despite a reduced production of cGMP (−70%). Indomethacin (10 μM) plus Nω-nitro-L-arginine (L-NA, 30 μM) reduced relaxation (−12% and −35% for native and regenerated endothelium, respectively) but did not abolish it.The hyperpolarizations induced by BK were not altered by the presence of indomethacin and L-NA and were unchanged in segments with regenerated endothelium.These data are consistent with a contribution of impairment in NO production to the depolarization of SMCs. Nevertheless, EDHF responses to BK are sufficient to maintain a normal relaxation after angioplasty.
doi:10.1038/sj.bjp.0704828
PMCID: PMC1573455  PMID: 12163348
Regenerated endothelium; endothelium-derived hyperpolarizing factor; nitric oxide; cyclic nucleotides; bradykinin
4.  Potassium ions and endothelium-derived hyperpolarizing factor in guinea-pig carotid and porcine coronary arteries 
British Journal of Pharmacology  1999;127(1):27-34.
Experiments were designed to determine in two arteries (the guinea-pig carotid and the porcine coronary arteries) whether or not the endothelium-derived hyperpolarizing factor (EDHF) can be identified as potassium ions, and to determine whether or not the inwardly rectifying potassium current and the Na+/K+ pump are involved in the hyperpolarization mediated by EDHF.The membrane potential of vascular smooth muscle cells was recorded with intracellular microelectrodes in the presence of Nω-L-nitro-arginine (L-NA) and indomethacin.In vascular smooth muscle cells of guinea-pig carotid and porcine coronary arteries, acetylcholine and bradykinin induced endothelium-dependent hyperpolarizations (−18±1 mV, n=39 and −19±1 mV, n=7, respectively). The hyperpolarizations were not affected significantly by ouabain (1 μM), barium chloride (up to 100 μM) or the combination of ouabain plus barium.In both arteries, increasing extracellular potassium concentration by 5 or 10 mM induced either depolarization or in a very few cases small hyperpolarizations which never exceeded 2 mV.In isolated smooth muscle cells of the guinea-pig carotid artery, patch-clamp experiments shows that only 20% of the vascular smooth muscle cells expressed inwardly rectifying potassium channels. The current density recorded was low (0.5±0.1 pA pF−1, n=8).These results indicate that, in two different vascular preparations, barium sensitive-inwardly rectifying potassium conductance and the ouabain sensitive-Na+/K+ pump are not involved in the EDHF-mediated hyperpolarization. Furthermore, potassium did not mimic the effect of EDHF pointing out that potassium and EDHF are not the same entity in those arteries.
doi:10.1038/sj.bjp.0702493
PMCID: PMC1565980  PMID: 10369452
Potassium; endothelium-derived hyperpolarizing factor; EDHF; endothelium; vascular smooth muscle cells; inwardly rectifying potassium current; Na+/K+ pump; ouabain; barium
5.  Morphological heterogeneity with normal expression but altered function of G proteins in porcine cultured regenerated coronary endothelial cells 
British Journal of Pharmacology  1997;122(6):999-1008.
Experiments were designed to investigate whether the pertussis toxin-dependent endothelial dysfunction following balloon injury is due to a reduced expression or an insufficient function of G-proteins.Endothelium-dependent responses of porcine coronary arteries were examined in vitro by use of conventional organ chambers. Morphological analysis was performed by isolating and culturing the endothelial cells from these arteries. The expression of Gi-proteins in regenerated endothelial cells was measured by Western blots and immunolabelling. The function of G-proteins was assessed by measuring the GTPase activity of cultured endothelial cells.Eight days following denudation, endothelial regrowth was confirmed by histological examination and by demonstrating the presence of endothelium-dependent relaxations to bradykinin and 5-hydroxytryptamine (5-HT). In primary culture, the regenerated endothelial cells displayed a ‘cobblestone' pattern as seen with native endothelial cells.Twenty eight days after denudation, the endothelium-dependent relaxations induced by 5-HT were impaired, but those to bradykinin were maintained. However, the latter were reduced when endothelium-dependent hyperpolarization was prevented.Twenty eight days after denudation, multinucleated giant cells were present in the regenerated but not in the native cultured endothelial cell populations. These regenerated endothelial cells incorporated less tritiated thymidine than native endothelial cells.The intensities of the bands on the immunoblot of the regenerated endothelial cells, when several antibodies against Giα1/α2/α3 were used, were the same as those obtained in native endothelial cells. The immunolabelling with the same antibodies was similar between the giant cells and the regenerated endothelial cells of normal size. The hydrolysis of GTP was lower in regenerated than in native endothelial cell membranes.In conclusion, endothelium-dependent relaxations mediated by Gi-proteins are impaired in balloon denuded coronary arteries. This dysfunction following regeneration cannot be explained by a reduced expression of Gi proteins but rather reflects an abnormal function of the G-proteins in the regenerated endothelium.
doi:10.1038/sj.bjp.0701475
PMCID: PMC1565034  PMID: 9401761
Regenerated endothelium; endothelial dysfunction; Gi-protein; pertussis toxin; bradykinin; NaF; 5-hydroxytryptamine

Results 1-5 (5)