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Vascular scaffold implantation induces injury to the intimal layer and causes discontinuity of the regenerated endothelial monolayer, compromising barrier integrity, increasing permeability, and allowing the transmigration of leukocytes and lipoproteins into the subendothelial space. Mechanical vascular wall stretching triggers Ras homolog family member A (RhoA)/Rho kinase-mediated actomyosin contractility and destabilization of adherens junctions, leading to endothelial barrier dysfunction. Assembly of intercellular adhesion and actin cytoskeletal organization of interendothelial junctions are controlled by downregulation of RhoA guanosine triphosphatase (GTPase)-mediated barrier-disruptive activity and upregulation of repressor-activator protein 1 (Rap1) and Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase-mediated cytoskeletal reorganization, leading to endothelial barrier stabilization. This review highlights the involvement of Rho GTPases in the disruption of endothelial barrier integrity following vascular scaffold implantation and the targeting of downstream Rho-associated protein kinases, which signal the network to restore endothelial barrier integrity and stability., (Published by Elsevier Ltd.)

Coronary artery intervention using device implants significantly reduce the risk of restenosis and the need for revascularization but are associated with endothelial denudation and impaired function. This may be due to incomplete endothelial recovery as a result of intimal injury, presence of polymer and/or high antiproliferative drug accumulation in the intima. The permanent presence of a metal prosthesis or polymer may impair the proliferation of resident endothelial cells to cover empty areas. Attention has focused on the robust replenishment of endothelial monolayer by recruitment of circulating endothelial progenitor cells derived from the bone marrow to areas of endothelial injury. The balance between endothelial damage and repair is critical for the maintenance of intimal integrity, function, and prevention of thrombotic complications. This review will discuss on the aftereffects of intravascular device implants on endothelial injury and the pathways involved in endothelial repair and regeneration with an emphasis on endothelial progenitor cells.

To overcome the limitations of balloon expandible metal stent-induced neointimal smooth muscle cell proliferation, drug-coated stent devices have been developed. Drug eluting stents release high concentrations of antiproliferative agents, such as paclitaxel, to reduce neointimal hyperplasia. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), is known to cause severe endothelial dysfunction and accelerate atherosclerotic lesion progression. The interaction of TNF-alpha and paclitaxel on the release of prothrombotic molecules was examined in endothelial cells. Treatment of endothelial cells with paclitaxel had no direct effect on tissue factor (TF) expression, but TNF-alpha increased TF. Cotreatment of paclitaxel with TNF-alpha markedly augmented the release of TF. TNF-alpha induced release of plasminogen activator inhibitor but no synergism occurred with paclitaxel. Treatment of endothelial cells with paclitaxel and TNF-alpha reduced expression of thrombomodulin and protein C receptor. Tissue factor pathway inhibitor expression was reduced by prolonged treatment with either paclitaxel or TNF-alpha. The adhesion molecule, CD62 E, was induced by TNF-alpha; however, CD31, CD62 P, and CD106 were not affected by paclitaxel and TNF-alpha. Apoptosis was not observed with cotreatment of endothelial cells with paclitaxel and TNF-alpha. CD59-positive microparticles were released in response to TNF-alpha, but the release was not augmented by paclitaxel. Paclitaxel and TNF-alpha increased the nitrotyrosination of proteins. These findings indicate that paclitaxel enhances TNF-alpha-induced release of TF, and downregulated thrombomodulin, increased protein nitration, which may subsequently favor prothrombotic intimal surface.

Endothelial cell dysfunction is considered to be an early event which subsequently leads to vascular wall disorders. Ultrastructural studies indicate that the endothelial cell changes involve membrane damage, increased permeability, swelling and necrosis. The endothelial cell loss of function could be as a result of changes in hemodynamic forces (shear and/or hoop stress), direct drug-induced cytotoxicity, mechanical device implant-induced injury and/or immune-mediated mechanisms. Drugs may perturb endothelial cell integrity by directly triggering inflammatory signaling cascades, enhancing expression of cellular adhesion molecules, activation of cytotoxic T cells and/or autoantibodies directed against endothelial cell membranes. Local release of inflammatory cytokines and chemokines activate endothelial cells to upregulate soluble adhesion molecules, activate neutrophils and generate reactive oxygen species which serve to amplify the initial inflammation leading to dysregulated apoptosis, secondary necrosis and overt vascular injury lesions. Considering the role of the endothelium in the initiation and propagation of vascular wall injury, there is a need for the discovery of validated biomarkers to serve as a predictor of activation of inflammatory cascades in the development of vascular injury. This article reviews some aspects of the multifaceted mechanisms that lead to the initial endothelial cell disruption and subsequent vascular wall injury.

Vascular endothelial dysfunction is a complex phenomenon that is caused by an imbalance of vasodilator and vasoconstrictor factors that regulate the equilibrium-maintaining vascular tone. In the early phase of hypercholesterolemia, endothelial dysfunction precedes vascular wall lesions. One of the earliest recognizable benefits of treatment with HMG-CoA reductase inhibitors (statins) is the normalization of endothelium-dependent relaxation in hypercholesterolemia; this effect occurs before significant lowering of serum cholesterol levels. Recent insights into cellular mechanisms indicate that statins promote vasorelaxation by upregulating the expression of endothelial nitric oxide (NO) synthase, activating the phosphatidylinositide 3-kinase/Akt pathway, inhibiting superoxide anion generation and endothelin synthesis, and by anti-inflammatory effects. These effects appear to be linked to the inhibition of geranylgeranylation of small G proteins such as Rho and Rac GTPases. In this regard, statins preserve endothelial function through the improvement of NO bioavailability and the reduction of oxidative stress, thereby shifting the balance from vasoconstriction to vasodilation. This review highlights the various mechanisms underlying the vasculoprotective effects of statins, independent of their effects on cholesterol lowering.

HMGCoA reductase inhibitors (statins) can have effects outside the target tissue, liver, including serious side-effects such as rhabdomyolysis as well as beneficial pleiotrophic effects. One such effect is upregulation of endothelial nitric oxide synthase (e-NOS) which generally leads to vasorelaxation. However, changing the balance between localized NO and O(2-) fluxes can also lead to oxidant stress and cellular injury through formation of reactive secondary oxidants such as peroxynitrite. We compared different statins for e-NOS subcellular localization, formation of pro-oxidants, and endothelial-dependent vascular function. Vascular relaxation in aortas of statin-dosed rats was inhibited with simvastatin (sevenfold higher EC50 for acetyl-choline induced relaxation) and atorvastatin (twofold increase) but not pravastatin. Ex vivo oxidation of the fluorescent redox probe dihydrorhodamine-123 (DHR-123) was increased in aortas from simvastatin treated rats, indicating increased reactive nitrogen and oxygen species. Human aortic endothelial cells incubated with simvastatin exhibited up to threefold higher intracellular oxidation of DHR-123 along with a twofold increase in total e-NOS protein. The elevated e-NOS was found in the Golgi/mitochondrial fraction and not in the plasma membrane, and using immunofluorescence greater e-NOS was observed proximal to Golgi and cytoskeletal structures and away from plasma membrane in simvastatin-treated cells. The data suggest that the action of lipophilic statins in endothelium can shift e-NOS localization towards intracellular domains, thereby increasing the encounter with metabolically generated O(2-) to produce peroxynitrite and related oxidants. Thus, under some conditions the direct action of lipophilic HMGCoA reductase inhibitors may unbalance NO and O(2-) fluxes and promote oxidant stress, compromising potentially beneficial vascular effects of e-NOS upregulation and increasing the potential for damage to muscle and other tissues.

This study was designed to examine the impairment of endothelium-dependent relaxation in spontaneously hypertensive rats (SHR), to determine whether endothelial cell function is normalized by in vivo treatment with a thromboxane A2-prostaglandin endoperoxide (TP)-receptor blocker, and to establish whether endothelial dysfunction contributes to the elevated blood pressure. In isolated aortic rings from SHR, endothelium-dependent relaxations caused by acetylcholine, adenosine diphosphate, and alpha-thrombin were markedly impaired compared with those from Wistar-Kyoto (WKY) normotensive rats. Arachidonic acid-induced contractions were significantly enhanced in aorta from SHR. In contrast, relaxations caused by direct smooth muscle vasodilators, nitroprusside and cromakalim, and contractions caused by U-46619 were not different between SHR and WKY rats. Treatment of SHR with the oral TP-receptor antagonist, ifetroban, at 20 and 50 mg.kg-1.day-1 fully restored endothelium-dependent relaxation toward normal. However, ifetroban produced no effect on blood pressure in SHR. In vitro incubation of aortic rings from SHR with ifetroban also normalized relaxations to acetylcholine but had no effect in aorta from WKY. In contrast, the thromboxane A synthase inhibitor, dazoxiben, only partially improved abnormal acetylcholine-induced relaxations in aorta from SHR. The results demonstrate that endothelial cell dysfunction in hypertension can be restored to normal by selective TP-receptor blockade. Furthermore, endothelial cell dysfunction and TP-receptor activation may not significantly contribute to elevated systemic blood pressure in SHR.

We examined the effects of diabetes on eicosanoid metabolism and endothelium-dependent relaxation in isolated aorta from alloxan-induced diabetic rabbits and that from normal rabbits incubated in increased concentrations (44 mM) of glucose in vitro for 6 h. Immunoreactive 15-hydroxyeicosatetraenoic acid (HETE) was assayed in the incubation media of isolated aortic segments. Basal and acetylcholine (ACh)-stimulated release of 15-HETE was significantly greater in aorta of diabetic animals as compared with those of normal rabbits. Incubation of aortic segments from normal rabbits in increased concentrations of glucose caused a significant increase in basal and ACh-stimulated release of 15-HETE; and the release was significantly greater in aortic segments with endothelium than in segments without endothelium. Basal and ACh-stimulated release of 15-HETE was inhibited by indomethacin, a cyclooxygenase inhibitor. 15-HETE caused contractions of aortic rings that were inhibited by the prostaglandin H2 (PGH2) thromboxane A2 (TXA2) receptor blocker SQ-29548, but not by the TXA2 synthase inhibitor carbethoxyhexyl imidazole or indomethacin. Treatment of aortic rings with subthreshold concentrations of 15-HETE impaired ACh-induced relaxation; this was prevented by treatment with SQ-29548. Thus, abnormal release of endothelium-derived 15-HETE may play a role in endothelial cell dysfunction and increased vasoconstriction in diabetes by a mechanism that involves interaction with PGH2/TXA2 receptors.

Diabetes is characterized by hyperglycemia, a relative lack of insulin, and an inclination to vascular disease and neuropathy. The link between diabetes and vascular disease is not understood, but autonomic dysfunction could partly account for alterations in reactivity of diabetic blood vessels to neurotransmitters and circulating hormones. Changes in local control of vascular tone, such as imbalance in production of relaxing and contracting factors by the endothelium, may be related to the initiation and maintenance of abnormal vascular reactivity characteristically seen in diabetic vascular complications. The emphasis is to discuss functional changes of blood vessel adrenergic neuroeffector mechanisms and endothelial cell dysfunction, together with the complex interrelationship of cyclooxygenase catalysis, protein kinase C activity, sodium-potassium ATPase activity, and flux through the polyol pathway. This review focuses on the common mechanisms by which hyperglycemia causes changes in vascular function.

Several studies have shown impairment of endothelium-dependent relaxations as well as increased release of vasoconstrictor prostanoids in arteries from diabetic animals and humans. This impairment is restored towards normal by prostaglandin (PG) H2/thromboxane A2 receptor blockade or superoxide dismutase, indicating that the PGH2 and/or superoxide anion (O2-.) generated contributes to the abnormality. Of particular note is that PGH2 impairs endothelium-dependent relaxations and causes contractions by a mechanism that involves generation of O2-. in the endothelium. The effects of elevated glucose are exacerbated by increased aldose reductase activity leading to depletion of NADPH and generation of reactive oxidants. Because NADPH is required for generation of nitric oxide from L-arginine, the depletion of NADPH leads to reduced nitric oxide formation. In a manner similar to that observed with elevated glucose, oxygen-derived free radicals or activation of protein kinase C also cause impairment of endothelium-dependent relaxations, smooth muscle contractions, and release constrictor prostanoids, indicating that a common mechanism for the impairment of endothelial cell function may be operative in diabetes. In this review the cumulative effects of oxidative stress on diabetic endothelial cell dysfunction, together with the complex interrelationship of cyclooxygenase catalysis, protein kinase C activity, and flux through the polyol pathway, are considered.

Objective: Studies of aortas from hypertensive and diabetic rats and rabbits have demonstrated impairment of endothelium-dependent relaxations, which were associated with increased release of endothelium-derived thromboxane A2 (TXA2). This implicates enhanced release of TXA2 or its precursor prostanoid, prostaglandin endoperoxide (PGH2), or both, as factors mediating the endothelial cell dysfunction., Methods: The interaction of vasoconstrictor prostanoids (PGH2, PGF2 alpha and U-46619, a stable thromboxane-receptor agonist) and oxygen-derived free radicals with the release of nitric oxide was examined in isolated aortas from Sprague-Dawley rats., Results: Exogenously applied PGH2 or U-46619 caused concentration-dependent contractions of aortic rings, these contractions being blocked by the newly developed, potent and selective PGH2- and TXA2-receptor antagonist BMS-180291, but not by inhibition of TXA synthase or cyclo-oxygenase (using dazoxiben and indomethacin, respectively). In aortic rings contracted submaximally with phenylephrine, brief exposure to a subthreshold concentration of PGH2 caused impairment of acetylcholine- and ADP-induced, but not of nitroprusside-induced, relaxations. The impairment was restored towards normal by BMS-180291 or by superoxide dismutase (SOD), a superoxide anion scavenger, but not by dazoxiben or indomethacin. In contrast, treatment of aortic rings with U-46619 or PGF2 alpha did not impair the relaxations. Oxygen-derived free radicals generated by xanthine oxidase caused contractions and impaired acetylcholine relaxations which were reversed by SOD but not by BMS-180291., Conclusions: These findings indicate that activation of PGH2 receptors causes contractions and selective impairment of endothelium-dependent relaxations by a mechanism involving generation of oxygen-derived free radicals in the endothelium.

The mechanisms by which the serine protease, alpha-thrombin, mediates relaxations were examined in isolated dog and pig coronary arteries and dog saphenous veins. In rings of coronary arteries and saphenous veins contracted submaximally with prostaglandin F2 alpha or U46619, alpha-thrombin (0.1-10 nM) caused relaxations that were abolished by treatment with N omega-nitro-L-arginine (L-NNA) or removal of the endothelium, indicating that the relaxations were mediated by endothelium-derived nitric oxide. These relaxations were blocked by the thrombin active site inhibitor, MD-805, indicating the requirement of thrombin's catalytic site to induce the relaxations. The thrombin exosite inhibitor, BMS-180742, decreased the sensitivity to alpha-thrombin without altering maximal relaxations. Indomethacin, a cyclooxygenase inhibitor, had no inhibitory effect on the relaxations caused by alpha-thrombin, indicating that the relaxations were not mediated by cyclooxygenase products. Similar to alpha-thrombin, the thrombin receptor activating peptide (human sequence: SFLLRNP, 1-100 microM) caused relaxations in pig coronary artery and dog saphenous vein but not in dog coronary artery. These relaxations were blocked by L-NNA but not by indomethacin. The results indicate that alpha-thrombin induces endothelium-dependent relaxations by a novel signaling mechanism that involves proteolytic cleavage of the thrombin receptor to expose a new amino terminus that functions as a "tethered peptide ligand" to activate thrombin receptors on the endothelial cells and release nitric oxide.

A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from alloxan-induced diabetic rabbits. Isolated aorta of diabetic rabbits, contracted submaximally with phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine or adenosine diphosphate compared to those from normal rabbits. Basal and acetylcholine-stimulated levels of cyclic GMP and the relaxations in response to an endothelium-independent vasodilator, sodium nitroprusside, were not significantly different between diabetic and normal rabbits, indicating that nitric oxide release and action on the vascular smooth muscle were unchanged. The release of thromboxane A2 from diabetic vessels was increased, as previously demonstrated. Treatment with an aldose reductase inhibitor, zopolrestat, normalized the elevated red blood cell sorbitol levels in diabetic rabbits. Zopolrestat also restored the abnormal acetylcholine- and adenosine diphosphate-induced relaxations of the aorta. The aldose reductase inhibitor had no effect on the levels of cyclic GMP or on the increased release of thromboxane A2 in diabetic aorta. These findings suggest that increased activity of the aldose reductase pathway in hyperglycemia is responsible for the abnormal endothelium-dependent relaxation in diabetic blood vessels. Significant alterations in endothelial production of neither nitric oxide nor vasoconstrictor prostanoids could be directly implicated in the improvement caused by the drug, suggesting another mechanism of action.

A possible relationship between aldose reductase activity and myo-inositol levels and endothelium-dependent relaxations was examined in isolated rabbit aorta incubated with elevated concentrations of glucose (44 mM) for 6 hr to mimic hyperglycemic conditions. Rings of aorta incubated in elevated glucose and contracted submaximally by phenylephrine showed significantly decreased endothelium-dependent relaxations induced by acetylcholine compared with aorta incubated in control (5.5 or 11 mM) glucose. Acetylcholine-induced relaxations of aorta incubated in hyperosmotic mannitol (44 mM) were not different from those incubated in control glucose. Treatment with two structurally unrelated aldose reductase inhibitors, sorbinil or zopolrestat, or supplementation with myo-inositol, prevented the abnormal acetylcholine relaxations of aortic rings caused by elevated glucose. No effects of sorbinil, zopolrestat or myo-inositol were observed on the response to acetylcholine of aorta incubated in control glucose. Neither sorbinil nor myo-inositol affected the increase in release of vasoconstrictor prostanoids caused by elevated glucose. These findings suggest that sorbitol accumulation and myo-inositol depletion contribute to the abnormal endothelial cell function caused by exposure to elevated glucose. The increased release of vasoactive prostanoids is either independent of, or possibly contributes to, the abnormal aldose reductase activity and/or myo-inositol depletion in intact blood vessels exposed to elevated concentrations of glucose.

The presence of the endothelium reduced the sensitivity of isolated rabbit carotid artery to endogenous norepinephrine released by electrical stimulation of adrenergic nerves or displaced by tyramine and to exogenously applied norepinephrine, phenylephrine and UK 14304. The maximal contractions induced by the selective alpha 2-agonist UK 14304 were much more profoundly depressed in arteries with endothelium than those induced by the nonselective alpha-adrenoceptor agonist norepinephrine or by the selective alpha 1-agonist. LY 83583, a cyclic-guanosine-monophosphate (GMP)-lowering agent, abolished the endothelium-dependent depression of tone induced by the agonists and converted the sensitivity of arteries with endothelium to that of endothelium-denuded preparations. M & B 22948, a selective cyclic GMP phosphodiesterase inhibitor, significantly inhibited contractions caused by electrical stimulation of adrenergic nerves, tyramine, norepinephrine and UK 14304 in rings with, but not in those without, endothelium. Yohimbine, an alpha 2-adrenoceptor antagonist, increased contractions caused by UK 14304 in rings with endothelium only, but had no significant effect on the contractions caused by exogenously applied norepinephrine or phenylephrine. In the presence of prazosin, an alpha 1-blocker, UK 14304 caused minimal relaxation (about 20%) in rings with endothelium only which were inhibited by yohimbine, suggesting a minor role of direct endothelial cell alpha 2-mediated release of relaxing factors. The over-flow of endogenous norepinephrine caused by electrical stimulation was not affected by treatment with LY 83583 or M & B 22948, suggesting that altering cyclic GMP levels has no major role in prejunctional modulation of norepinephrine release. These findings support the notion that intrinsic levels of cyclic GMP may act as a regulator of adrenergic neurotransmission due primarily to endothelium-derived relaxing factor which is released basally, and to a lesser extent by an activation of endothelial cell alpha 2-adrenoceptors.

Impaired endothelium-dependent relaxation occurs in diabetic rabbit aorta and normal aorta exposed to elevated concentrations of glucose and is prevented by cyclooxygenase inhibitors. The role of free radicals in the endothelial cell impairment was examined with free radical scavengers and in aortas from rabbits fed with probucol (1% wt/wt, a lipid-soluble antioxidant). Rings of aorta suspended for measurement of isometric tension were incubated for 6 h in control (5.5 mM) or elevated (44 mM) glucose. Impairment of endothelium-dependent relaxation to acetylcholine caused by exposure to elevated glucose was prevented by superoxide dismutase, catalase, deferoxamine, or allopurinol and did not occur in aortas from probucol-fed rabbits. Similarly, impairment of acetylcholine relaxations in aortas from alloxan-induced diabetic rabbits was restored to normal by superoxide dismutase. Oxygen-derived free radicals generated by xanthine oxidase also caused impaired acetylcholine relaxations. Exposure of aortic segments to elevated glucose or to xanthine oxidase caused a significant increase in release of immunoreactive prostanoids. These data indicate that the endothelial cell dysfunction caused by elevated glucose is mediated by free radicals that are likely generated through the increased cyclooxygenase catalysis occurring in the endothelium. Treatment with antioxidants protects against impaired endothelium-dependent relaxations caused by elevated glucose.

The vasoconstrictor actions of prostaglandin (PG) endoperoxide, PGH2, were examined in isolated rabbit aortic rings suspended for measurement of isometric tension. In aortic rings with an intact endothelium, PGH2 caused concentration-dependent contractions which were blocked by SQ 29548 (a PGH2-thromboxane A2 receptor blocker) or superoxide dismutase (a superoxide anion scavenger) but not by carbethoxyhexyl imidazole (a thromboxane A2 synthase inhibitor) or catalase (a hydrogen peroxide scavenger). In contrast U 46619, a thromboxane A2 mimic, caused contractions, which were blocked by SQ 29548 but not by superoxide dismutase. PGH2 caused significantly greater contractions in aortic rings without endothelium or in those with intact endothelium treated with NG-nitro-L-arginine, a nitric oxide inhibitor; these contractions were inhibited by SQ 29548 but not by superoxide dismutase. In aortic rings with endothelium contracted with phenylephrine, a subthreshold concentration of PGH2, but not U 46619, impaired relaxations to acetylcholine; the inhibition was prevented by treatment with SQ 29548 or superoxide dismutase, indicating that the abnormality of endothelial cell function was specific for PGH2. These observations indicate that PGH2 causes contractions and inhibits endothelium-dependent relaxation by a mechanism involving formation of superoxide anion, which interacts with endothelium-derived nitric oxide.

A possible relationship between protein kinase C activation and impaired receptor-mediated endothelium-dependent relaxation in diabetes mellitus was examined in isolated aorta from normal rabbit exposed to elevated glucose. Aorta treated for 10 min with 4-phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, showed decreased relaxations to the endothelium-dependent vasodilator, acetylcholine, similar to normal aorta exposed to elevated glucose (22 and 44 mM) for 6 h. Relaxations to the receptor-independent endothelium-dependent vasodilator, A23187, and those caused by the direct smooth muscle vasodilator, sodium nitroprusside, were unaffected by treatment with PMA or exposure to elevated glucose. Indomethacin increased relaxations to acetylcholine of aorta treated with PMA indicating a role for vasoconstrictor prostanoids. PMA caused a significant increase in basal and acetylcholine-stimulated release of vasoconstrictor prostanoids including thromboxane A2 from aortic segments with, but not without endothelium. Protein kinase C inhibitors, H-7 or sphingosine, restored the abnormal acetylcholine-induced relaxations as well as suppressed the abnormal release of prostanoids in aorta exposed to elevated glucose. These findings suggest that the dysfunction of receptor-mediated endothelium-dependent relaxation associated with exposure to elevated glucose is due to increased production of vasoconstrictor prostanoids by the endothelium as a consequence of protein kinase C activation.

The effects of glucose on endothelium-dependent responses and vasoactive prostanoid production were determined by incubating isolated rabbit aortae in control (5.5 or 11 mM) or elevated (44 mM) glucose for 6 h to mimic euglycemic and hyperglycemic conditions. Rings of aortae incubated in elevated glucose, contracted submaximally by phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine compared with the aortae incubated in control glucose. Treatment with indomethacin, a cyclooxygenase inhibitor, or SQ29548, a prostaglandin H2/thromboxane A2 receptor antagonist, restored acetylcholine relaxations of rings in elevated glucose to normal, while these agents had no effect on the relaxation of rings incubated in control glucose. Aortae incubated with mannose (44 mM) as a hyperosmotic control relaxed to acetylcholine normally. The relaxations in response to A23187 and sodium nitroprusside were not different between rings exposed to control and elevated glucose. Radioimmunoassay measurements showed a significant increase in acetylcholine-stimulated release of thromboxane A2 and prostaglandin F2 alpha in aortae with, but not without endothelium incubated with elevated, but not with control glucose. Thus a possible mechanism for endothelium dysfunction in diabetes mellitus is the hyperglycemia-induced increased generation of endothelium-derived vasoconstrictor prostanoids.

Transmural electrical stimulation of isolated ring segments of the rabbit carotid artery caused frequency-dependent contractions; these were blocked by tetrodotoxin or prazosin. Mechanical or chemical removal of the endothelium markedly augmented responses to electrical stimulation. Inhibition of norepinephrine uptake and metabolism with cocaine, hydrocortisone, and pargyline increased contractions in rings with endothelium more than those without endothelium, but responses remained greater in rings denuded of endothelium. Methylene blue, an inhibitor of guanylate cyclase, enhanced responses to electrical stimulation of rings with intact endothelium only. Combined inhibition of guanylate cyclase and norepinephrine disposition increased the contractions and abolished the difference between the responses of rings with and without endothelium. In a perfusion-cascade system, the perfusate of donor segments with endothelium relaxed a bioassay ring without endothelium. Electrical stimulation of the segment caused no further relaxation of the bioassay ring. However, contractions caused by electrically stimulating the bioassay ring were depressed during superfusion with the perfusate of segments with, but not without, endothelium, indicating that vasodilators spontaneously released from the endothelium inhibit responses to nerve stimulation. These observations suggest that inhibition by the endothelium of the response to adrenergic nerve stimulation results from 1) spontaneous release of endothelium-derived vasodilators and 2) disposition of norepinephrine by the endothelial cells.

The endothelium-dependent and presumed endothelium-independent vasodilators acetylcholine and sodium nitroprusside, respectively, were used to characterize relaxation responses of mesenteric resistance arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY). Vessels were preconstricted using concentrations of norepinephrine or 5-hydroxytryptamine, which reduced their diameters by 50 to 60%. Relaxation responses to acetylcholine (10(-8) - 10(-7) M) were significantly smaller (p less than 0.05) in vessel segments from SHRSP, but the maximal relaxations at higher concentrations were the same in both strains. However, SHRSP vessels relaxed to a greater extent than did those of the WKY at all concentrations of sodium nitroprusside. Endothelium removal significantly enhanced sodium nitroprusside-induced dilations in both rat strains, and the dilations were significantly greater in segments from SHRSP in the concentration range of 3 X 10(-8) to 10(-6) M. The decreased relaxation to acetylcholine in resistance arteries from adult hypertensive rats compared with those from the normotensive strain suggests that functional alterations in the endothelium may play a role in hypertensive disease.

Isolated perfused rabbit carotid arteries were used to determine the effects of endothelial cell shear stress on the response to adrenergic nerve stimulation. Arterial segments with and without endothelium were cannulated and perfused with physiological salt solution. Adrenergic nerves were activated by transmural electrical field stimulation. Neurogenic vasoconstriction was significantly greater in segments without endothelium when compared with that of segments with endothelium. In segments with endothelium only, vasoconstriction was depressed when shear stress was increased by increasing the viscosity of the perfusate with dextran. Perfusion with methylene blue (2 X 10(-6) M), a guanylate cyclase inhibitor, increased vasoconstriction in segments with endothelium only. In the presence of methylene blue, vasoconstriction was no longer different between segments with and without endothelium, and perfusion with dextran had no effect. In a perfusion-cascade system, perfusion with dextran of donor segments with but not without endothelium caused further relaxation of a contracted bioassay ring. These results suggest that shear stress on endothelial cells modulates adrenergic vasoconstriction by augmenting release of endothelial cell-derived vasodilators.

The influence of the endothelium on smooth muscle tone and the response of the pig right coronary artery to norepinephrine (NE) was studied. Isolated rings of artery with and without endothelium were stretched in the presence of nitroprusside to a tension previously determined to be optimal for contraction. During wash out of the nitroprusside, rings without endothelium spontaneously generated tone representing 24% of the contraction caused by potassium (120 mM); in rings with endothelium no significant spontaneous tone was observed. Relaxations were caused by NE in rings with endothelium contracted with prostaglandin F2 alpha (PGF2 alpha). In rings without endothelium, NE relaxed spontaneously generated tone as well as that produced by PGF2 alpha. Independent of the mode or degree of contraction, rings with endothelium were more sensitive to NE than rings without endothelium. The difference in sensitivity to NE between rings with and without endothelium was likely due to endothelial cell alpha-2 adrenoceptors, inasmuch as the difference was abolished by rauwolscine. In the presence of propranolol and prazosin, endothelium-dependent relaxations were observed which were also inhibited by rauwolscine. Nevertheless, beta adrenoceptors are the predominant mediator of the relaxation to NE of pig coronary smooth muscle, because propranolol caused a greater shift to the right of the relaxation induced by NE compared to that caused by endothelium removal. Accordingly, under resting conditions, NE caused contractions only in the presence of propranolol. These contractions were attenuated by prazosin or rauwolscine, but blocked only by a combination of both alpha adrenoceptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of calcium in the inhibition by the endothelium of adrenergic neurotransmission was studied in isolated rabbit carotid artery. Contractions induced by transmural electrical field stimulation (0.5-8 Hz), norepinephrine (10(-8)-3 X 10(-5) M), potassium depolarization (15-30 mM), or by readdition of calcium (0.15-2.4 mM) to a calcium-free medium containing potassium (15 mM) were significantly smaller in rings with compared with rings without endothelium. The voltage-dependent calcium channel activator, BAY K 8644 (10(-6) M), increased contractions to all contractile stimuli in rings with more than in rings without endothelium and thereby abolished the inhibitory influence of the endothelium. The inhibition of neurogenic contractions by the endothelium was also, in part, prejunctional, as indicated by decreased overflow of endogenous norepinephrine from superfused segments with compared with segments without endothelium evoked by electrical stimulation (2 Hz) or by reinfusion of calcium (2.5 mM) to calcium-free medium containing potassium (80 mM). BAY K 8644 (10(-6) M) enhanced the overflow of norepinephrine evoked by electrical stimulation or calcium from segments with more than from segments without endothelium and abolished the difference. Thus the endothelium inhibits activation by extracellular calcium of adrenergic nerves and vascular smooth muscle. The action of the endothelium is overcome by BAY K 8644, suggesting that voltage-dependent calcium channels are important in the inhibitory role of the endothelium in both adrenergic nerves and smooth muscle cells.

Endothelium-dependent relaxations and vasoactive prostanoid production caused by acetylcholine were determined in the aortas of rabbits with diabetes mellitus induced by alloxan. Aortas of diabetic rabbits, contracted submaximally by phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine compared with the aortas of normal rabbits. Indomethacin, a cyclooxygenase inhibitor, and SQ 29548, a prostaglandin H2-thromboxane A2 (PGH2-TxA2) receptor antagonist, normalized the sensitivity of diabetic aortas to acetylcholine, whereas these agents had no effect on the response of normal aortas. The relaxations in response to a nonreceptor-mediated endothelium-dependent vasodilator, A23187, and an endothelium-independent vasodilator, sodium nitroprusside, were not different between normal and diabetic aortas. Acetylcholine also caused contractions of resting aortic rings with endothelium from diabetic, but not normal rabbits; these contractions were inhibited by indomethacin. Synthesis of TxA2, measured as immunoreactive TxB2, was significantly increased in diabetic aortic segments only when the endothelium was present. These results suggest that in the diabetic state, the endothelium releases a major vasoconstrictor cyclooxygenase product that either directly counteracts the relaxation caused by or selectively interferes with the release of endothelium-derived relaxing factor(s) induced by cholinergic receptor stimulation. The vasoconstrictor is most likely TxA2 or possibly its precursor, PGH2.