Jean-Paul Vilaine

Role of gap junctions and EETs in endothelium-dependent hyperpolarization of porcine coronary artery

The effects of endothelium‐derived hyperpolarizing factor (EDHF: elicited using substance P or bradykinin) were compared with those of 11,12‐EET in pig coronary artery. Smooth muscle cells were usually impaled with microelectrodes through the adventitial surface. Substance P (100u2003nM) and 11,12‐EET (11,12‐epoxyeicosatrienoic acid; 3u2003μM) hyperpolarized endothelial cells in intact arteries. These actions were unaffected by 100u2003nM iberiotoxin but were abolished by charybdotoxin plus apamin (each 100u2003nM). Substance P (100u2003nM) and bradykinin (30u2003nM) hyperpolarized intact artery smooth muscle; Substance P had no effect after endothelium removal. 11,12‐EET hyperpolarized de‐endothelialized vessels by 12.6±0.3u2003mV, an effect abolished by 100u2003nM iberiotoxin. 11,12‐EET hyperpolarized intact arteries by 18.6±0.8u2003mV, an action reduced by iberiotoxin, which was ineffective against substance P. Hyperpolarizations to 11,12‐EET and substance P were partially inhibited by 100u2003nM charybdotoxin and abolished by further addition of 100u2003nM apamin. 30u2003μM barium plus 500u2003nM ouabain depolarized intact artery smooth muscle but responses to substance P and bradykinin were unchanged. 500u2003μM gap 27 markedly reduced hyperpolarizations to substance P and bradykinin which were abolished in the additional presence of barium plus ouabain. Substance P‐induced hyperpolarizations of smooth muscle cells immediately below the internal elastic lamina were unaffected by gap 27, even in the presence of barium plus ouabain. In pig coronary artery, 11,12‐EET is not EDHF. Smooth muscle hyperpolarizations attributed to ‘EDHF’ are initiated by endothelial cell hyperpolarization involving charybdotoxin‐ (but not iberiotoxin) and apamin‐sensitive K+ channels. This may spread electrotonically via myoendothelial gap junctions but the involvement of an unknown endothelial factor cannot be excluded.

Potassium ions and endothelium‐derived hyperpolarizing factor in guinea‐pig carotid and porcine coronary arteries

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±1u2003mV, n=39 and −19±1u2003mV, n=7, respectively). The hyperpolarizations were not affected significantly by ouabain (1u2003μM), barium chloride (up to 100u2003μM) or the combination of ouabain plus barium. In both arteries, increasing extracellular potassium concentration by 5 or 10u2003mM induced either depolarization or in a very few cases small hyperpolarizations which never exceeded 2u2003mV. 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±0.1u2003pAu2003pF−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.

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries.

1 The purpose of these experiments was to determine whether or not the endothelium‐dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo‐oxygenase) can be attributed to the production of an endogenous cannabinoid. 2 Membrane potential was recorded in the guinea‐pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3 In the rat mesenteric artery, the cannabinoid receptor antagonist, SRu2003141716 (1u2003μM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium‐dependent hyperpolarization induced by acetylcholine (1u2003μM) (17.3±1.8u2003mV, n=4 and 17.8±2.6u2003mV, n=4, in control and presence of SRu2003141716, respectively). Anandamide (30u2003μM) induced a hyperpolarization of the smooth muscle cells (12.6±1.4u2003mV, n=13 and 2.0±3.0u2003mV, n=6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SRu2003141716 (1u2003μM). HU‐210 (30u2003μM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30u2003μM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4 In the rat mesenteric artery, the endothelium‐dependent hyperpolarization induced by acetylcholine (1u2003μM) (19.0±1.7u2003mV, n=6) was not altered by glibenclamide (1u2003μM; 17.7±2.3u2003mV, n=3). However, the combination of charybdotoxin (0.1u2003μM) plus apamin (0.5u2003μM) abolished the acetylcholine‐induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7±2.7u2003mV, n=3). The hyperpolarization induced by anandamide (30u2003μM) (12.6±1.4u2003mV, n=13) was significantly inhibited by glibenclamide (4.0±0.4u2003mV, n=4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3±2.3u2003mV, n=4). 5 In the guinea‐pig carotid artery, acetylcholine (1u2003μM) evoked endothelium‐dependent hyperpolarization (18.8±0.7u2003mV, n=15). SRu2003141716 (10u2003nM to 10u2003μM), caused a direct, concentration‐dependent hyperpolarization (up to 10u2003mV at 10u2003μM) and a significant inhibition of the acetylcholine‐induced hyperpolarization. Anandamide (0.1 to 3u2003μM) did not influence the membrane potential. At a concentration of 30u2003μM, the cannabinoid agonist induced a non‐reproducible hyperpolarization (5.6±1.3u2003mV, n=10) with a slow onset. SRu2003141716 (1u2003μM) did not affect the hyperpolarization induced by 30u2003μM anandamide (5.3±1.5u2003mV, n=3). 6 In the porcine coronary artery, anandamide up to 30u2003μM did not hyperpolarize or relax the smooth muscle cells. The endothelium‐dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SRu2003141716 (1u2003μM). 7 These results indicate that the endothelium‐dependent hyperpolarizations, observed in the guinea‐pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.

Hypercholesterolemia Increases Coronary Endothelial Dysfunction, Lipid Content, and Accelerated Atherosclerosis After Heart Transplantation

Hyperlipidemia may increase endothelial damage and promote accelerated atherogenesis in graft coronary vasculopathy. To study the effects of hypercholesterolemia on coronary endothelial dysfunction, intimal hyperplasia, and lipid content, a porcine model of heterotopic heart transplantation, allowing nonacute rejection without immunosuppressive drugs, was used. A high cholesterol diet was fed to donor and recipient swine 1 month before and after transplantation. The endothelial function of coronary arteries of native and transplanted hearts from cholesterol-fed animals was studied in organ chambers 30 days after implantation and compared with endothelial function in arteries from animals fed a normal diet. The total serum cholesterol increased 3-fold in donors and recipients. Endothelium-dependent relaxations to serotonin, to the alpha(2)-adrenergic agonist UK14,304, and to the direct G-protein activator sodium fluoride were decreased significantly in allografted hearts compared with native hearts from both groups. Relaxations to the calcium ionophore A23187 and bradykinin were decreased significantly in allografts from animals fed the high cholesterol diet. The prevalence of intimal hyperplasia was significantly increased in coronary arteries from hypercholesterolemic swine. There was a significant increase in the lipid content of allograft arteries of hypercholesterolemic recipients. Hypercholesterolemia causes a general coronary endothelial dysfunction, increases the prevalence of intimal hyperplasia, and augments the incorporation of lipids in the vascular wall after heart transplantation. Hyperlipidemia accelerates graft coronary atherosclerosis through its effects on the endothelium.

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.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen--A novel biomarker of atherosclerotic plaque remodeling.

OBJECTIVEnDegradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine.nnnDESIGN AND METHODSnA monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice.nnnRESULTSnThe lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice.nnnCONCLUSIONnWe have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.

Cells Derived from Regenerated Endothelium of the Porcine Coronary Artery Contain More Oxidized Forms of Apolipoprotein-B-100 without a Modification in the Uptake of Oxidized LDL

Increased accumulation of lipoproteins and cholesterol within cells from regenerated endothelium may be responsible for their reported dysfunction. This study compared the presence and uptake of oxidized forms of low-density lipoprotein (LDL) in cells derived from native and regenerated endothelium. Four weeks after balloon denudation, primary cultures of native and regenerated endothelial cells were prepared from porcine coronary arteries. Regenerated endothelium stained more strongly using an antibody against oxidized lipoproteins. The increase in oxidized forms of apolipoprotein-B-100 exhibited by cells from regenerated endothelium was not due to an increase in extracellular-induced oxidation of native LDL, measured as the production of thiobarbituric-acid-reactive substances, being identical in both cell types. Intracellular cholesterol and cholesterol ester content were unchanged in regenerated cells. Using flow cytometry, accumulation of oxidized LDL was investigated further by quantifying the uptake of a mildly oxidized preparation of 1,1’-dioctadecyl-3,3,3’,3-tetramethyl-indocarbocyanine perchlorate-labelled LDL. The parameters of uptake, EC50 and Emax, were not different between cells from native and regenerated endothelium suggesting that the number of LOX-1 receptors was identical in the two cell types. Moreover, a negative correlation between the increased uptake of acetylated LDL and decreased cGMP production in response to bradykinin was observed in cells from regenerated endothelium. Thus, the increased incorporation of modified LDL and their intracellular oxidation could be responsible for the alteration in NO production. The presence of oxidized forms of LDL may be a marker of endothelium regeneration and could be involved in the endothelial dysfunction of pig coronary arteries 4 weeks after balloon denudation.

Monohydroperoxidized fatty acids but not 4-hydroxynonenal induced acute cardiac cell damage

Unsaturated fatty acids constitutive of cardiac membranal lipid matrix are one of the primary targets for reactive oxygen species generated during ischemia-reperfusion cycle. Lipid peroxidation is a cascade of intricate reactions involving the successive formations of fatty acids hydroperoxides and aldehydic compounds such as alkenals derived from the oxidative fragmentation of these hydroperoxides. The potential deleterious effects of different classes of lipid peroxidation products on cardiac cells were compared using three in vitro approaches: (i) cardiomyocyte integrity, (ii) electromechanical activity of papillary muscle, and (iii) atrial contractility. The following products of lipid peroxidation were tested: (i) photoperoxidized arachidonic acid pooling hydroperoxidized derivatives and aldehydic compounds, (ii) fatty acids hydroperoxides, and (iii) 4-hydroxynonenal, a characteristic alkenal derived from the oxidative fragmentation of hydroperoxidized n-6 fatty acids. Only fatty acids hydroperoxides induced drastic loss of cellular integrity and severe disturbances in electromechanical activity of cardiomyocytes. 4-hydroxynonenal induced only a slight leak of lactate dehydrogenase at high concentrations and did not modify the electromechanical behavior of cardiac preparations. Under our conditions, monohydroperoxidized fatty acids but not 4-hydroxynonenal induced acute cardiac cell damages. In conclusion, lipid hydroperoxides can be considered both as markers of oxidative injury and relay sources of oxidative stress.

Effect of uncoupling endothelial nitric oxide synthase on calcium homeostasis in aged porcine endothelial cells

AIMSnThe requirement of endothelial NO synthase (NOS3) calcium to produce NO is well described, although the effect of NO on intracellular calcium levels [Ca(2+)](i) is still confusing. Therefore, NO and [Ca(2+)](i) cross-talk were studied in parallel in endothelial cells possessing a functional or a dysfunctional NO pathway.nnnMETHODS AND RESULTSnDysfunctional porcine endothelial cells were obtained either in vitro by successive passages or in vivo from regenerated endothelium 1 month after coronary angioplasty. Activity of NOS3 was characterized by conversion of arginine to citrulline, BH(4) intracellular availability, cGMP, and superoxide anion production. Imaging of the Ca(2+) indicator FURA 2-AM was recorded and sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) pump activity was analysed by (45)Ca(2+) uptake into cells. In endothelial cells with a functional NO pathway, NOS3 inhibition increased [Ca(2+)](i) and, conversely, an NO donor decreased it. In aged cells with an uncoupled NOS3 as shown by the reduced BH(4) level, the increase in superoxide anion and the lower production of cGMP and the decrease in NO bioavailability were linearly correlated with the increase in basal [Ca(2+)](i). Moreover, when stimulated by bradykinin, the calcium response was reduced while its decay was slowed down. These effects on the calcium signalling were abolished in calcium-free buffer and were similarly induced by SERCA inhibitors. In aged cells, NO improved the reduced SERCA activity and tended to normalize the agonist calcium response.nnnCONCLUSIONnIn control endothelial cells, NO exerts a negative feedback on cytosolic Ca(2+) homeostasis. In aged cells, uncoupled NOS3 produced NO that was insufficient to control the [Ca(2+)](i). Consequently, under resting conditions, SERCA activity decreased and [Ca(2+)](i) increased. These alterations were reversible as exogenous NO, in a cGMP-independent way, refilled intracellular calcium stores, reduced calcium influx, and improved the agonist-evoked calcium response. Therefore, prevention of the decrease in NO in dysfunctional endothelium would normalize the calcium-dependent functions.

Protective effect of S12340 on cardiac cells exposed to oxidative stress.

Oxidative stress induced by reactive oxygen species is one aspect of the deleterious mechanisms involved in myocardial post-ischemic reperfusion injury. The antioxidant properties of the new molecule S12340 (8-[3-(3,5-diterbutyl-4-hydroxyphenyl-thio)propyl]-1-oxa-2- oxo-3,8-diazaspiro[4.5]decane) were evaluated using three successive in vitro approaches mimicking the cardiac cell damages induced by reactive oxygen species released into the reperfused myocardium. (i) The effects of S12340 on lipid peroxidation were evaluated using an original cell-free model of non-enzymatic peroxidation of 1.32 mM arachidonic acid induced by reactive oxygen species generated photochemically. S12340 (13.2 microM) inhibited by 29% the rate of oxidative fragmentation of monohydroperoxidized arachidonic acid into aldehydic products. (ii) S12340 (10 microM) inhibited by 96% and 58% the oxidative necrosis of cultured rat cardiomyocytes induced by xanthine oxidase (20 mU/ml) and monohydroperoxidized arachidonic acid (30 microM), respectively. (iii) Superfusion of guinea-pig papillary muscle with monohydroperoxidized arachidonic acid (20 microM) resulted in marked alterations of their electrophysiological and mechanical activities. These modifications, maximal 15-17 min after the addition of lipid hydroperoxide, were completely abolished by S12340 (30 microM).