Belay Tesfamariam

Elevated glucose promotes generation of endothelium-derived vasoconstrictor prostanoids in rabbit aorta.

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.

Inhibition of adrenergic vasoconstriction by endothelial cell shear stress.

Isolated perfused rabbit carotid arteries were used to determine the effects of endotbelial cell shear stress on the response to adrenergk 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 ± 10-6 M), a goanylate 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 bfoassay ring. These results suggest that shear stress on endothelial cells modulates adrenergk vasoconstriction by augmenting release of endothelial cell-derived vasodilators.

Aldose reductase inhibition restores endothelial cell function in diabetic rabbit aorta

A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from al-loxan-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.

Differential effects of pravastatin, simvastatin, and atorvastatin on Ca2+ release and vascular reactivity

The direct effects of the cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitors, on vascular smooth muscle responsiveness were examined by incubation of isolated aorta from normocholesterolemic rats with simvastatin, atorvastatin, or pravastatin. The smooth muscle contractions caused by phenylephrine were progressively inhibited with increasing concentrations of simvastatin. Similarly, atorvastatin at the higher concentration caused decreased responses to phenylephrine. In contrast, incubation with pravastatin had no significant effect at all concentrations studied. In Ca2+-free buffer, the transient contraction caused by phenylephrine, which results from intracellular release of Ca2+, also was inhibited by simvastatin and atorvastatin but not by pravastatin. In cultured rat aortic smooth muscle cells loaded with fura-2, increases in intracellular free-Ca2+ concentration ([Ca2+]i) induced by angiotensin II were markedly inhibited in cells incubated with simvastatin and atorvastatin but not pravastatin. The inhibitory effects of simvastatin and atorvastatin were reversed by mevalonate. These findings demonstrate that inhibition of HMG CoA reductase by using simvastatin and atorvastatin, but not pravastatin, has effects on vascular smooth muscle cell responsiveness that involve alteration of Ca2+ homeostasis through a mevalonate-dependent pathway.

15-Hydroxyeicosatetraenoic acid and diabetic endothelial dysfunction in rabbit aorta

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.

Drug Release Kinetics From Stent Device-Based Delivery Systems

In an effort to overcome the limitations of balloon-expandible intravascular metal stent-induced neointimal formation, drug-coated stent devices have been developed. The stent platform allows the local delivery of drugs to an injury site, thereby reducing the amount of drug exposure to the systemic circulation and other organs. The drug carrier matrix allows the release of the drug in a diffusion-controlled manner over an extended time period after the stent implant. The drugs are chosen such that the complex cascade of events that occurs after stent implantation that leads to smooth muscle cell proliferation and migration towards the intima are inhibited. The success of an antirestenotic drug therapy from a drug-coated stent is dependent, at least partially, on the extent of drug elution from the stent, the duration and rate of release, and accumulation of drug in the arterial wall in such a way that it covers the initiation and progression of vessel wall remodeling. The local vascular drug concentrations achieved are directly correlated with the biological effects and local vascular toxicity, and there is therefore a challenge in finding an optimum dose of drug to be delivered to tissues (ie, one that has the desired therapeutic effect without local adverse effects). There is increased focus on optimization of various factors that affect drug release from the stent system, including the physicochemical properties of the drugs, carrier vehicle formulation, and profile of elution kinetics. This review highlights the various factors involved in drug release kinetics, local vascular toxicity, carrier vehicle matrix, tissue deposition, and distribution through the arterial wall from stent-based drug delivery systems.

Augmented adrenergic contractions of carotid arteries from cholesterol-fed rabbits due to endothelial cell dysfunction.

Summary: Transmural electrical stimulation was used to elicit frequency-dependent adrenergic neurogenic contractions in isolated carotid arteries from cholesterol-fed and control rabbits. In rings with endothelium, responses to adrenergic nerve stimulation were significantly greater in arteries from cholesterol-fed as compared with those from control rabbits. Responses to adrenergic nerve stimulation of rings without endothelium were not different between the two groups. Methylene blue, a guanylate cyclase inhibitor, increased contractions of rings with endothelium and abolished the difference between the responses of arteries from cholesterol-fed and control rabbits. Methylene blue had no significant effect on arteries without endothelium. The overflow of endogenous norepinephrine (NE) caused by transmural electrical stimulation was not different between segments of arteries from cholesterol-fed and control rabbits. In control rabbits, exogenously applied NE contracted arteries with endothelium less than arteries without endothelium, whereas in cholesterol-fed rabbits the contractions caused by NE were not different between arteries with and without endothelium. Acetylcholine-induced relaxations were not different between rings with endothelium from cholesterol-fed and control rabbits. These results suggest that hypercholesterolemia selectively impairs the inhibitory influence of the endothelium on adrenergic contractions.

Paclitaxel potentiates inflammatory cytokine-induced prothrombotic molecules in endothelial 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-α (TNF-α), is known to cause severe endothelial dysfunction and accelerate atherosclerotic lesion progression. The interaction of TNF-α 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-α increased TF. Cotreatment of paclitaxel with TNF-α markedly augmented the release of TF. TNF-α induced release of plasminogen activator inhibitor but no synergism occurred with paclitaxel. Treatment of endothelial cells with paclitaxel and TNF-α reduced expression of thrombomodulin and protein C receptor. Tissue factor pathway inhibitor expression was reduced by prolonged treatment with either paclitaxel or TNF-α. The adhesion molecule, CD62 E, was induced by TNF-α; however, CD31, CD62 P, and CD106 were not affected by paclitaxel and TNF-α. Apoptosis was not observed with cotreatment of endothelial cells with paclitaxel and TNF-α. CD59-positive microparticles were released in response to TNF-α, but the release was not augmented by paclitaxel. Paclitaxel and TNF-α increased the nitrotyrosination of proteins. These findings indicate that paclitaxel enhances TNF-α-induced release of TF, and downregulated thrombomodulin, increased protein nitration, which may subsequently favor prothrombotic intimal surface.

Diabetes Mellitus and the Vascular Endothelium

The endothelium plays an important role in the function of blood vessels. Under normal circumstances substances released from the endothelium, such as endothelium-derived factor(s), prostaglandins, and peptides, modulate the tone of vascular smooth muscle, prevent platelet aggregation and thrombosis in the blood vessel lumen, and may modulate vessel growth. It is well known that vasomotor, thrombotic, and growth control mechanisms are abnormal in blood vessels from humans and animals with atherosclerosis. It is also recognized that the progression and complications of atherosclerosis are increased in patients with diabetes. This chapter will review recent evidence that diabetes and hyperglycemia cause specific abnormalities in the vasomotor function of the endothelium. Because these vasomotor abnormalities result from the abnormal release of substances from the endothelium that may play a role in thrombosis and growth, an understanding of their mechanism of action may provide new insights to the basis of vascular complications in diabetes mellitus.

Enhanced adrenergic neurotransmission in diabetic rabbit carotid artery.

OBJECTIVE The aim was to examine the effects of diabetes mellitus on adrenergic neurotransmission and smooth muscle responsiveness in the densely innervated carotid artery from six-week alloxan diabetic rabbits. METHODS Rings of carotid arteries were isolated from normal and diabetic rabbits and isometric tension was measured in response to stimulation of adrenergic nerves, alpha adrenoceptors, and activation by calcium. RESULTS Basal content and stimulated overflow of endogenous noradrenaline were reduced by approximately 25% in arteries from diabetic as compared to normal rabbits. In contrast, responses to endogenous noradrenaline released from adrenergic nerves by electrical stimulation or tyramine displacement were not different between arteries from normal and diabetic groups. Neuronal uptake blockade using cocaine caused a significantly smaller leftward shift in the contractions produced by electrical stimulation and exogenously applied noradrenaline in arteries from diabetic rabbits. The tonic, but not phasic, contractions caused by phenylephrine were larger in arteries from diabetic rabbits. Calcium-induced contractions caused by readdition of calcium to a calcium-free medium containing potassium (15 mmol.litre-1) were also significantly larger in arteries from diabetic rabbits. BAY K 8644, a calcium channel activator, caused an increase in calcium induced contractions and abolished the difference between the two groups. CONCLUSIONS Although neurogenic contractions of diabetic carotid artery are normal, there is inefficient or reduced neuronal uptake as well as increased activity of calcium channels in the smooth muscle which increase contractions to alpha adrenoceptor agonists.