Role of microparticles in atherothrombosis.

Cell activation or apoptosis leads to plasma membrane blebbing and microparticle (MP) release in the extracellular space. MPs are submicron membrane vesicles which express a panel of phospholipids and proteins specific of the cells they are derived from. Exposure of negatively charged phospholipids and tissue factor confers a procoagulant potential to MPs. MPs accumulate in the lipid core of the atherosclertotic plaque and is a major determinant of its thrombogenecity. Elevation of plasma MPs levels, particularly those of endothelial origin, reflects cellular injury and is considered now as a surrogate marker of vascular dysfunction. Thus, MPs can be seen as triggers of a vicious circle for they promote prothrombogenic and pro-inflammatory responses as well as cellular dysfunction within the vascular compartment. A better knowledge of MP composition and biological effects as well as the mechanisms leading to their clearance will probably open new therapeutic approaches in the treatment of atherothrombosis.

Microparticles and type 2 diabetes.

Cell activation or apoptosis leads to plasma membrane blebbing and microparticles (MPs) release in the extracellular space. MPs are submicron membrane vesicles, which harbour a panel of oxidized phospholipids and proteins specific to the cells they derived from. MPs are found in the circulating blood of healthy volunteers. MPs levels are increased in many diseases, including cardiovascular diseases with high thrombotic risk. Exposure of negatively charged phospholipids and tissue factor confers a procoagulant potential to MPs. Elevation of plasma MPs levels, particularly those of endothelial origin, reflects cellular injury and appears now as a surrogate marker of vascular dysfunction. Recent studies demonstrate an elevation of circulating levels of MPs in diabetes. MPs could also be involved in the development of vascular complications in diabetes for they stimulate pro-inflammatory responses in target cells and promote thrombosis, endothelial dysfunction and angiogenesis. Thus, these studies provide new insight in the pathogenesis and treatment of vascular complications of diabetes.

[Circulating endothelial microparticles: a new marker of vascular injury]. / Microparticules endothéliales circulantes: un nouveau marqueur du dysfonctionnement vasculaire.

Cell activation or apoptosis leads to plasma membrane blebbing and microparticles (MPs) release. MPs are submicron membrane vesicles expressing a panel of oxidized phospholipids and proteins specific of the cells they originate from. Exposure of negatively charged phospholipids and tissue factor confers a procoagulant potential to MPs. Increases in plasma MPs levels, particularly those of endothelial origin, reflects cellular injury and appears now as a surrogate marker of vascular dysfunction. MPs are also biologically active and stimulate pro-inflammatory responses in target cells. Thus, MPs can promote a prothrombogenic and pro-inflammatory vicious circle leading to vascular dysfunction. A better understanding of MPs composition, as well as their effects and the mechanisms leading to their clearance will likely open new therapeutic approaches in the treatment and the prognosis of cardiovascular diseases.

Impaired flow-induced dilation in mesenteric resistance arteries from mice lacking vimentin.

The intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

Proangiogenic effect of angiotensin-converting enzyme inhibition is mediated by the bradykinin B(2) receptor pathway.

Recent studies have suggested a proangiogenic effect of angiotensin-converting enzyme (ACE) inhibition. We hypothesized that such a proangiogenic effect of ACE inhibition may be mediated, in part, by bradykinin (BK) B(2)-receptor pathway. This study therefore examined the neovascularization induced by ACE inhibitor treatment in B(2) receptor-deficient mice (B(2)(-/-)) in a model of surgically induced hindlimb ischemia. After artery femoral occlusion, wild-type and B(2)(-/-) mice were treated with or without ACE inhibitor (perindopril, 3 mg/kg/d) for 28 days. Angiogenesis was then quantitated by microangiography, capillary density measurement, and laser Doppler perfusion imaging. The protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were determined by Western blot. In wild-type animals, vessel density and capillary number in the ischemic leg were raised by 1.8- and 1.4-fold, respectively, in mice treated with ACE inhibitor when compared with the nontreated animals (P<0.01). This corresponded to an improved ischemic/nonischemic leg perfusion ratio by 1.5-fold in ACE inhibitor-treated animals when compared with the untreated ones (0.87+/-0.07 versus 0.59+/-0.05, respectively, P<0.01). Activation of the angiogenic process was also associated with a 1.7-fold increase in tissue eNOS protein level in mice treated with ACE inhibitor (P<0.05 versus control) but not with changes in VEGF protein level. Conversely, ACE inhibition did not affect vessel density, blood flow, and eNOS protein level in ischemic hindlimb of B(2)(-/-) mice. Therefore, proangiogenic effect of ACE inhibition is mediated by B(2)-receptor signaling and was associated with upregulation of eNOS content, independently of VEGF expression.

Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice.

- Flow-dependent dilation is a fundamental mechanism by which large arteries ensure appropriate blood supply to tissues. We investigated whether or not the vascular kallikrein-kinin system, especially tissue kallikrein (TK), contributes to flow-dependent dilation by comparing wild-type and TK-knockout mice in which the presence or absence of TK expression was verified. We examined in vitro changes in the outer diameter of perfused carotid arteries from TK(+/+) and TK(-/-) mice. In both groups, exogenous bradykinin caused a similar dilation that was abolished by the B(2) receptor antagonist HOE-140, as well as by the NO synthase inhibitor N:(omega)-nitro-L-arginine methyl ester. However, purified kininogen dilated only TK(+/+) arteries, demonstrating the essential role of TK in the vascular formation of kinins. In TK(+/+) arteries, increasing intraluminal flow caused a larger endothelium-dependent dilation than that seen in TK(-/-). In both strains the flow response was mediated by NO and by endothelium-derived hyperpolarizing factor, whereas in TK(-/-) vasoconstrictor prostanoids participated as well. HOE-140 impaired flow-dependent dilation in TK(+/+) arteries while showing no effect in TK(-/-). This compound reduced the flow response in TK(+/+) arteries to a level similar to that in TK(-/-). After NO synthase inhibition, HOE-140 no longer affected the response of TK(+/+). Impaired flow-dependent dilation was also observed in arteries from knockout mice lacking bradykinin B(2) receptors as compared with wild-type animals. This study demonstrates the active contribution of the vascular kallikrein-kinin system to one-third of the flow-dependent dilation response via activation of B(2) receptors coupled to endothelial NO release.

Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction.

BACKGROUND: Shed membrane microparticles circulate in the peripheral blood of nonischemic (NI) patients and patients with myocardial infarction (MI). We investigated whether or not these microparticles would affect endothelium-dependent responses. METHODS AND RESULTS: Rat aortic rings with endothelium were exposed for 24 hours to circulating microparticles isolated from 7 patients with NI syndromes and 19 patients with acute MI. Endothelium-dependent relaxations to acetylcholine were not affected by high concentrations of microparticles from NI patients (P=0.80). However, significant impairment was observed in preparations exposed to microparticles from patients with MI at low and high concentrations, corresponding to 0.7-fold and 2-fold circulating plasma levels (P=0.05 and 0.001, respectively). Impairment was not affected by diclofenac (P=0.47), nor by the cell-permeable superoxide dismutase mimetic Mn(III)tetra(4-benzoic acid) porphyrin chloride (P=0.33), but it was abolished by endothelium removal or by N(omega)monomethyl-L-arginine. Relaxations to the calcium ionophore ionomycin were decreased in rings exposed to microparticles from MI patients (P=0.05 and 0.009 for low and high concentrations, respectively), but microparticles from NI patients had no effect (P=0.81). Finally, high concentrations of microparticles from MI patients affected neither endothelium-independent relaxation to sodium nitroprusside (P=0.59) nor expression of the endothelial nitric oxide synthase (P=0.43). CONCLUSIONS: Circulating microparticles from patients with MI selectively impair the endothelial nitric oxide transduction pathway and, therefore, could contribute to the general vasomotor dysfunction observed after MI, even in angiographically normal arteries.

Endothelial dysfunction and collagen accumulation: two independent factors for restenosis and constrictive remodeling after experimental angioplasty.

BACKGROUND: Constrictive remodeling plays a prominent role in restenosis after balloon angioplasty, but its regulation remains unclear. Because endothelial dysfunction and changes in extracellular matrix have been reported after angioplasty, this study was designed to simultaneously evaluate endothelial function and collagen and elastin changes after restenosis and arterial remodeling. METHODS AND RESULTS: Atherosclerosis was induced in femoral arteries of 22 New Zealand White rabbits by air-desiccation and a high-cholesterol diet. One month later, angioplasty was performed. Histomorphometry and in vitro assessment of endothelial function were performed 4 weeks after angioplasty. Restenosis correlated with constrictive remodeling (r=0.60, P=0.01) but not with neointimal growth (r=-0.06, P=0.79). Restenosis correlated with an impaired relaxation to acetylcholine (ACh; r=0.61, P=0.02) but not with the response to the endothelium-independent vasodilator sodium nitroprusside (r=-0.25, P=0.40). Restenosis correlated positively with collagen accumulation (r=0.69, P=0.004) and inversely with elastin density (r=-0.48, P=0.05). Relaxations to ACh were significantly more decreased in arteries with constrictive remodeling than in those with enlargement remodeling (3.7+/-7.9% versus 35.5+/-15.0%, P=0.04). Neointimal collagen density was significantly higher in arteries with constrictive remodeling than in those with enlargement remodeling (34.5+/-4.5% versus 18.2+/-4.7%, P=0.03). Endothelial function and collagen and elastin density were independent predictors of restenosis in the study. CONCLUSIONS: These results demonstrate that the severity of restenosis after angioplasty correlated with both defective endothelium-dependent relaxation and increased collagen density.

The combined 5-HT2 receptor and calcium channel inhibitor LU49938 restores endothelium dependent responses in the regenerated endothelium of the porcine coronary artery.

OBJECTIVE: The aim was to evaluate the effect of the combined 5-hydroxytryptamine-2 (5-HT2) receptor antagonist and calcium channel inhibitor LU49938 ((2S)-5-[N-methyl-N-(n-hexyl)] amino-2-isopropyl-2(3.4.5-trimethoxyphenyl)-valeronitrilhydrochloride ) on the endothelium dependent responsiveness of porcine coronary arteries with native and regenerated endothelium. METHODS: Male Yorkshire pigs were assigned randomly to one of four groups: (1) controls; (2) pigs receiving LU49938 daily (5 mg.kg-1) for four weeks; (3) pigs undergoing balloon de-endothelialisation of the left anterior descending coronary artery; and (4) pigs undergoing balloon de-endothelialisation and receiving LU49938 daily. At four weeks, quantitative coronary angiography, organ chamber experiments, and morphometric studies of the tissues were performed. RESULTS: Treatment with LU49938 did not affect the endothelium dependent responses in native porcine coronary arteries. Intracoronary injection of serotonin caused significantly greater coronary vasoconstriction in group 3 compared with group 4. The cross sectional area of the intima and media of previously de-endothelialised left anterior descending coronary artery increased significantly in group 3, but not in group 4. In arteries with regenerated endothelium, augmented endothelium dependent contractions were noted not only in response to serotonin, but also in response to platelets, noradrenaline, and endothelin-1. The endothelium dependent relaxations to platelets, serotonin, and UK14304 were impaired in the regenerated endothelium, but not those to adenosine diphosphate and SIN-1. However, following four weeks of treatment with LU49938, the pertussis toxin sensitive endothelium dependent responses were restored. The augmented endothelium dependent contraction to endothelin-1 was not altered by the treatment. CONCLUSIONS: Chronic treatment with LU49938 restores endothelium dependent, pertussis toxin sensitive, G protein mediated responses in the regenerated endothelium of the porcine coronary artery, and inhibits the intimal thickening following arterial injury.

Interleukin-2 causes endothelium-dependent contractions to arachidonic acid.

The present experiments were designed to investigate the effect of interleukin-2 on the response to arachidonic acid in rings with and without endothelium from Wistar-Kyoto (WKY) and spontaneously hypertensive rat (SHR) aortas. In control rings, arachidonic acid induced contractions of WKY aorta that were not different between preparations with and without endothelium. Incubation with interleukin-2 (10 units/mL) for 6 or 18 hours augmented the response to arachidonic acid in rings with, but not in those without, endothelium from WKY rat aortas. In the WKY aorta, both the endothelium-dependent and endothelium-independent contractions to arachidonic acid observed after incubation with interleukin-2 were abolished by indomethacin and ridogrel (a thromboxane-endoperoxide receptor antagonist and a thromboxane synthase inhibitor) but were not affected by dazoxiben (a thromboxane synthase inhibitor). Interleukin-2 did not augment the vascular reactivity of WKY aortic smooth muscle to activation of the thromboxane-endoperoxide receptor with U46619. In aortas from SHRs, arachidonic acid evoked endothelium-dependent contraction; interleukin-2 did not modify the response to arachidonic acid in preparations with and without endothelium. These data demonstrate that 1) endothelium-dependent contractions to arachidonic acid are observed in SHR but not in WKY rat aortas; 2) interleukin-2 induces endothelium-dependent contractions to arachidonic acid in the WKY aorta that are mediated by an augmented release of a metabolite of cyclooxygenase, different from thromboxane A2 but activating thromboxane-endoperoxide receptors; and 3) interleukin-2 does not affect the endothelium-dependent and endothelium-independent response to arachidonic acid in the SHR aorta.

Endothelial AT1-mediated release of nitric oxide decreases angiotensin II contractions in rat carotid artery.

The purpose of this study was to examine whether angiotensin II (Ang II) stimulates the release of endothelium-derived nitric oxide, which then impairs the contractions of vascular smooth muscle caused by the peptide, and to determine the receptor subtypes mediating these responses. Experiments were performed on isolated rings of rat carotid artery either incubated in the presence of phosphodiesterase inhibitor for the measurement of intracellular levels of cGMP or suspended in organ chambers for recording of changes in isometric force. Ang II (10(-7) mol/L) caused a twofold increase in intracellular cGMP level in preparations with but not in those without endothelium. The presence of endothelium impaired the contractions evoked by the peptide and caused approximately 50% inhibition of the maximal response to Ang II (3 x 10(-8) mol/L); pD2 values for Ang II were 8.9 +/- 0.1 and 9.6 +/- 0.2 in rings with and without endothelium, respectively. In rings with endothelium the contractions to Ang II were augmented by nitro-L-arginine (an inhibitor to nitric oxide synthase) but not indomethacin (an inhibitor of cyclooxygenase), to reach a response comparable to that of preparations without endothelium. In rings without endothelium losartan (a preferential angiotensin type 1 receptor antagonist) displayed competitive antagonism toward Ang II (pA2 = 9.5); PD 123319 (a preferential angiotensin type 2 receptor antagonist; up to 10(-7) mol/L) did not affect the response to the peptide. Losartan (3 x 10(-9) mol/L) but not PD 123319 (10(-7) mol/L) impaired the endothelium-dependent component of the response to the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-derived contracting factors.

The endothelium not only mediates relaxation but is a source of contracting factors. Endothelium-dependent contractions are elicited by physical and chemical stimuli (i.e., hypoxia, pressure, and stretch) and autacoids, local and circulating hormones. The mechanism of endothelium-dependent contractions to hypoxia involves withdrawal of nitric oxide. The endothelial cyclooxygenase pathway can produce thromboxane A2, prostaglandin H2, and superoxide anions. The peptide endothelin is a potent contracting factor; its production is stimulated by vasopressor hormones, platelet-derived factors, coagulation products, and cytokines, whereas endothelium-derived nitric oxide, prostacyclin, and a smooth muscle cell-derived inhibitory factor reduce endothelin production. In hypertension, the release of cyclooxygenase-dependent endothelium-derived contracting factors to stretch, acetylcholine, and platelet-derived products is augmented. Vascular endothelin production in hypertension remains controversial but appears mostly normal; it is augmented in the presence of vascular disease or renal insufficiency. The endothelium-dependent inhibition of endothelin-induced contractions is reduced in hypertension while the reactivity of vascular smooth muscle may be normal, increased, or reduced. The potentiating effects of low concentrations of endothelin on contractions to norepinephrine are augmented with aging and hypertension. In atherosclerosis, the production of the cyclooxygenase-dependent endothelium-derived contracting factors and endothelin is enhanced. Thus, endothelium-derived contracting factors can profoundly affect vascular tone and counteract relaxing factors produced within the endothelium. In hypertension and atherosclerosis, the role of contracting factors appears to become more dominant, leading to an imbalance of endothelium-dependent vascular regulation.

Endothelin stimulated by angiotensin II augments contractility of spontaneously hypertensive rat resistance arteries.

In cultured endothelial cells, endothelin is produced after stimulation with angiotensin II. The effects of angiotensin II and endothelin-1 on vascular sensitivity to norepinephrine were studied in perfused rat mesenteric resistance arteries. Expression of endothelin messenger RNA (mRNA) was determined in endothelial cells obtained from the mesenteric circulation. Perfusion (5 hours) of the arteries with angiotensin II (10(-7) M) potentiated contractions in arteries with endothelium induced by norepinephrine in spontaneously hypertensive rats but not Wistar-Kyoto rats. The potentiation was inhibited by phosphoramidon and an endothelin antibody. Short-term stimulation (1 hour) with angiotensin II did not cause the potentiation. Stimulation with angiotensin I (10(-7) M; 5 hours) caused a potentiation prevented by captopril. In endothelial cells collected from the mesenteric arterial bed of spontaneously hypertensive rats, endothelin-specific mRNA was constitutively expressed, and the level of endothelin transcripts was increased by angiotensin II (10(-7) M). Threshold concentrations of exogenous endothelin-1 potentiated contractions induced by norepinephrine in arteries with and without endothelium of spontaneously hypertensive rats but not Wistar-Kyoto rats. Thus, angiotensin II stimulates the endothelial production of endothelin in situ and therapy potentiates contractions to norepinephrine in mesenteric resistance arteries of spontaneously hypertensive rats. This suggests that vascular endothelin production acts as an amplifier of the pressor effects of the renin-angiotensin system that may play an important role in hypertension.

Endothelium-derived relaxing factors and converting enzyme inhibition.

Endothelial cells can produce at least 3 substances which cause relaxation of vascular smooth muscle: (1) endothelium-derived nitric oxide (NO, which is secreted not only toward the underlying vascular smooth muscle but also into the blood vessel lumen). NO also has a physiological role at the interface between the endothelial cells and the blood content; in particular, NO inhibits the adhesion of platelets and leukocytes to the endothelium. (2) Endothelium-derived hyperpolarizing factor, presumably a labile metabolite of arachidonic acid formed through the P-450 pathway, which appears to act on smooth muscle by being one of the few physiologic openers of the potassium channels. (3) Prostacyclin, which can be considered as an endothelium-derived relaxing substance, given its vasodilator activity and its primarily endothelial origin. One of the main factors modulating the release of these EDRFs is the shear stress of blood on the arterial wall, which explains why flow-induced vasodilation is endothelium-dependent in the intact organism. The peptide bradykinin is a potent stimulus for EDRF release. The normal lifespan of an adult human endothelial cell is some 30 years, after which aging takes its toll and the cells must be replaced. The regenerated cells lose some of their ability to release EDRF, in particular in response to platelet aggregation and thrombin. Finally, in hypertension and atherosclerosis, a decrease in endothelium-dependent relaxation is obvious in response to a variety of stimuli. All converting enzyme inhibitors tested so far share a potentiating effect on endothelium-dependent relaxation to bradykinin, and augmented local production of bradykinin may help to explain the acute vasodilator properties of these compounds.

Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat.

1. Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). 2. Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of NG-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M1), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3). 3. The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 +/- 0.07 (EC50). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (-log M) 9.48 +/- 0.07, 6.74 +/- 0.22, 6.30 +/- 0.20 and 9.39 +/- 0.22 respectively. The potency of acetylcholine in inducing endothelium-dependent relaxation was 7.82 +/- 0.09 (IC50). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (-log M) 9.15 +/- 0.23 and 9.63 +/- 0.28, respectively. These results suggest that the muscarinic M3 receptor subtype mediates both endothelium-dependent relaxation and contraction to acetylcholine in SHR aorta.

Gi proteins and the response to 5-hydroxytryptamine in porcine cultured endothelial cells with impaired release of EDRF.

1. The receptor-mediated release of endothelium-derived relaxing factor(s) (EDRF) requires the presence of different functional G proteins in endothelial cells. Release of EDRF in response to 5-hydroxytryptamine (5-HT), which involves activation of pertussis toxin-sensitive Gi proteins, is impaired in both regenerated endothelium of the coronary artery following balloon catheterization and in porcine cultured endothelial cells. This study used porcine cultured endothelial cells as a model of regenerated endothelium to determine if the abnormal release of EDRF in response to 5-HT may be associated with the loss of functional pertussis toxin-sensitive Gi proteins. 2. Binding studies on porcine cultured endothelial cells demonstrated specific binding sites for [3H]-5-HT. Scatchard analyses revealed a single binding site for [3H]-5-HT with Kd of 7.2 +/- 3.5 nM and maximal binding (Bmax) of 121.4 +/- 51.3 fmol mg-1 protein. Binding of [3H]-5-HT was displaced by methiothepin (5-HT1 and 5-HT2 antagonist; Ki = 6.2 +/- 1.2 nM), but not by ketanserin (preferential 5-HT2 antagonist). 3. Gi alpha 1 protein was expressed in cultured but not in native endothelial cells. Gi alpha 2 and Gi alpha 3 proteins were expressed to significant levels in porcine native and cultured endothelial cells, as detected by Northern and Western blot analysis. 4. In membranes from cultured endothelial cells, two bands of 40 and 41 kDa, which corresponded to the Gi alpha 2 and the combination of Gi alpha 3-Gi alpha 1 proteins, respectively, were ADP-ribosylated by pertussis toxin. The labelling intensity was Gi alpha 2>Gi alpha 3-Gi alpha l and the amount of ADP-ribosylation was not different between porcine native and cultured endothelial cells. Stimulation of the cultured cells with 5-HT (3 x 10-6 M; 4 min) decreased significantly further ADP-ribosylation of Gi alpha 2 by pertussis toxin, but not that of Gi alpha 3 and/or Gi alpha l.5. The present results suggest that porcine endothelial cell culture may lead to the abnormal expression of Gi alpha l protein and that the dysfunctional release of EDRF from cultured porcine endothelial cells in response to 5-HT is not associated with the loss of Gi alpha proteins or the absence of 5-HT binding sites.

Cyclo-oxygenase-1 and -2 contribution to endothelial dysfunction in ageing.

Experiments were designed to investigate the role of cyclo-oxygenase isoforms in endothelial dysfunction in ageing. Aortic rings with endothelium of aged and young (24 vs 4 month-old) Wistar rats, were mounted in organ chambers for the recording of changes in isometric tension. In young rats, acetylcholine (ACh) caused a complete relaxation which was not affected by indomethacin (0.3 microM), NS-398 (a preferential COX-2 inhibitor; 1 microM), SQ-29548 (a thromboxane-receptor antagonist; 1 microM), nor valeryl-salicylate (VAS, a preferential inhibitor of COX-1; 3 mM). In aged rats, ACh caused a biphasic response characterized by a first phase of relaxation (0.01 - 1 microM ACh), followed by a contraction (3 - 100 microM ACh). Indomethacin, NS-398 and SQ-29548, but not VAS, augmented the first phase. Indomethacin, VAS, NS-398 and SQ-29548 decreased the contractions to high ACh concentrations. Then, the sensitivity to thromboxane receptor activation was investigated with U-46619. The results show comparable EC(50) values in young and aged rats. In aged rats, the ACh-stimulated release of prostacyclin, prostaglandin F(2alpha) and thromboxane A(2) was decreased by either indomethacin, NS-398, VAS or endothelium removal. However, in young animals, the ACh-stimulated release of prostacyclin and prostaglandin F(2alpha) were smaller than in older animals and remained unaffected by NS-398. Aortic endothelial cells from aged - but not young - rats express COX-2 isoform, while COX-1 labelling was observed in endothelial cells from both young and aged rats. These data demonstrate the active contribution of COX-1 and -2 in endothelial dysfunction associated with ageing.

Inhibition of the angiotensin converting enzyme by perindoprilat and release of nitric oxide.

Experiments were designed to investigate the mechanism underlying the endothelium-dependent relaxations to perindoprilat, a converting enzyme inhibitor, in canine coronary arteries previously exposed to bradykinin. Rings suspended in organ chambers were exposed to bradykinin for 3 min and washed extensively for 150 min. In rings previously exposed to the peptide, bradykinin induced relaxations which were augmented in the presence of perindoprilat; this response was not affected by indomethacin, but nitro-L-arginine induced a rightward shift of the relaxation to the peptide without affecting its maximal effect. In canine coronary arteries previously exposed to the peptide, perindoprilat caused endothelium-dependent relaxations (IC50 = 7.83), which had been observed previously at concentrations where the converting enzyme inhibitor did not augment the response to bradykinin. Carboxypeptidase B, but not aprotinin, impaired the relaxation to perindoprilat, suggesting a contribution of bradykinin. The relaxation to perindoprilat was not affected by the B1 antagonist Leu8-des-Arg9-bradykinin. However, the bradykinin B2 antagonist HOE-140 displayed a noncompetitive antagonism against the response to perindoprilat. The response to the converting enzyme inhibitor was not affected by indomethacin but was impaired significantly by nitro-L-arginine. The present findings suggest that in canine coronary arteries previously exposed to bradykinin, the relaxation to perindoprilat is mediated mainly by endothelium-derived nitric oxide. In addition, the response to perindoprilat may be due to factors other than just protection of bound bradykinin from degradation.

Endothelium-dependent responses in hypertension.

The endothelium controls the tone of the underlying vascular smooth muscle by releasing relaxing factors. These include prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). In certain types of hypertension, endothelium-dependent relaxations are curtailed, because of a reduced production and/or action of endothelium-derived NO and EDHF. In the spontaneously hypertensive rat, endothelium-dependent relaxations are reduced, because of the endothelium-dependent production of vasoconstrictor prostanoids (endoperoxides and, in some cases, thromboxane A2). These prostanoids may be produced in the vascular smooth muscle cells rather than in the endothelium. The endothelial dysfunction observed in hypertensive blood vessels is likely to be a consequence rather than a cause of the disease process.

The hemoregulatory peptide N-acetyl-ser-asp-lys-pro impairs angiotensin I-induced contractions in rat aorta.

The hemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is degraded by ACE. This study was designed to examine the effect of Ac-SDKP on the contractions to angiotensin I. Experiments were performed on rat aortic rings with endothelium exposed to nitro-L-arginine. Ac-SDKP (10 and 100 microM) significantly augmented angiotensin I ED20 (from 2.0+/-0.4 to 4.2+/-1.0 and 5.0+/-0.9 nM) and ED50 (from 4.3+/-0.7 to 8.6+/-1.0 and 10.7+/-1.3 nM, respectively), but did not alter its maximal response. The contractions to angiotensin II were not affected by Ac-SDKP. No degradation of exogenous Ac-SDKP nor detectable release of endogenous Ac-SDKP were observed in the incubation medium. These results suggest that Ac-SDKP impairs angiotensin I response by inhibiting ACE and subsequent angiotensin II formation.