Johan Van de Voorde

Endothelial dysfunction in diabetes

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. The endothelium controls the tone of the underlying vascular smooth muscle through the production of vasodilator mediators. The endothelium‐derived relaxing factors (EDRF) comprise nitric oxide (NO), prostacyclin, and a still elusive endothelium‐derived hyperpolarizing factor (EDHF). Impaired endothelium‐dependent vasodilation has been demonstrated in various vascular beds of different animal models of diabetes and in humans with type 1 and 2 diabetes. Several mechanisms of endothelial dysfunction have been reported, including impaired signal transduction or substrate availibility, impaired release of EDRF, increased destruction of EDRF, enhanced release of endothelium‐derived constricting factors and decreased sensitivity of the vascular smooth muscle to EDRF. The principal mediators of hyperglycaemia‐induced endothelial dysfunction may be activation of protein kinase C, increased activity of the polyol pathway, non‐enzymatic glycation and oxidative stress. Correction of these pathways, as well as administration of ACE inhibitors and folate, has been shown to improve endothelium‐dependent vasodilation in diabetes. Since the mechanisms of endothelial dysfunction appear to differ according to the diabetic model and the vascular bed under study, it is important to select clinically relevant models for future research of endothelial dysfunction.

Regulatory Mechanisms in the Retinal and Choroidal Circulation

The retina receives its nutrients from two separate circulations: retinal and choroidal circulation. This short overview describes the determinants in the regulation of these circulations. Retinal circulation is characterized by a low blood flow while flow in the choroid is high. The choroidal circulation is mainly controlled by sympathetic innervation and is not autoregulated. Retinal circulation lacks autonomic innervation, shows an efficient autoregulation and is mainly influenced by local factors. Local mediators released by endothelial cells and surrounding retinal tissue also have a substantial role in the regulation of retinal circulation.

Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine

Despite their potent vasodilating action in vivo, acetylcholine and histamine often show a vasoconstricting action in vitro. As the endothelium has an important role in the vasodilating effect of acetylcholine, we investigated the possible role of the endothelium in the vasodilating effect of histamine in comparison to acetylcholine. Experiments were done on ring segments of rat thoracic aorta mounted for isometric tension measurements. We demonstrated that relaxation by histamine and acetylcholine of pre-contracted rat aorta segments required the presence of endothelial cells. Acetylcholine acting on muscarinic receptors, and histamine acting on H1-receptors seemed to initiate the production of mediator(s) from the endothelial cells, which leads to relaxation of the vascular smooth muscle cells. This production appeared to be depressed by ETYA and hydroquinone, and under hypoxic conditions.

Evidence Against the Involvement of Cytochrome P450 Metabolites in Endothelium‐Dependent Hyperpolarization of the Rat Main Mesenteric Artery

1 The influence of different inhibitors of cytochrome P450 mono‐oxygenase on the endothelium‐dependent and ‐independent hyperpolarization in the isolated rat main mesenteric artery was investigated. 2 Application of acetylcholine (ACh; 1 μm) for 10 min evoked an endothelium‐dependent peak hyperpolarization of about 18 mV followed by a partial recovery to a level 7 mV more negative than the resting value (‐50.2 ± 0.5 mV). 3 Proadifen (30 μm) completely and reversibly inhibited the ACh‐induced hyperpolarization. Conversely, the imidazole antimycotics clotrimazole (30 μm) and miconazole (100 μm) had less effect on the peak endothelium‐dependent hyperpolarization. The suicide substrate inhibitors 17‐octadecynoic acid (17‐ODYA; 5 μm) and 1‐aminobenzotriazole (1‐ABT; 2 mm) did not significantly influence endothelium‐dependent hyperpolarization. 4 The endothelium‐independent hyperpolarization (16 mV) evoked by levcromakalim (300 nm) was completely inhibited by proadifen as well as by clotrimazole and miconazole but was not affected by 17‐ODYA or 1‐ABT. 5 These results do not support the view that the ACh‐induced endothelium‐dependent hyperpolarization in the rat mesenteric artery is mediated by cytochrome P450 mono‐oxygenase metabolites. Proadifen and imidazole antimycotics impair the activation of ATP‐regulated K+ channels in mesenteric artery cells, rendering non‐specific inhibition of smooth muscle K+ channel activation an alternative explanation for the inhibitory influence of some (but not all) P450 inhibitors on endothelium‐dependent hyperpolarization in this preparation.

Retinal Arterial Tone Is Controlled by a Retinal-Derived Relaxing Factor

The present study provides evidence that retinal tissue may profoundly influence the retinal arterial smooth muscle cell tone by releasing an unknown retinal relaxing factor. Isolated bovine retinal arteries with and without adhering retinal tissue were mounted in a wire myograph for isometric tension recordings. The maximal contraction induced by prostaglandin F2alpha was 0.95+/-0.7 mN (n=6) in the presence and 5.15+/-0.76 mN (n=6) in the absence of adhering retinal tissue. The contractions induced by U-46619, serotonin, and endothelin-1 were similarly blocked in the presence of retinal tissue. The K+ 120 mmol/L-induced contraction was not significantly affected (2.8+/-0.7 mN, n=6, in the presence and 3. 6+/-0.7 mN, n=6, in the absence of retinal tissue). Placing a piece of bovine retinal tissue in the proximity of a contracted (ie, with prostaglandin F2alpha) retinal artery induced a complete relaxation of the retinal vessel, suggesting the involvement of a diffusible chemical vasorelaxant. Also porcine, canine, and ovine retinal tissue completely relaxed the contracted (with prostaglandin F2alpha) bovine retinal artery. Other smooth muscle preparations, including rat mesenteric and renal arteries and rat main bronchi, also relaxed with the application of a piece of bovine retinal tissue. Incubation of bovine retinas in a Krebs-Ringer bicarbonate solution yielded a solution that relaxed isolated precontracted bovine retinal arteries, confirming the involvement of a diffusible chemical messenger. Hexane extraction, heating the solution to 70 degrees C, or treatment with trypsin did not alter the relaxing properties of the incubation solution. The characteristics of the retinal relaxing factor do not correspond with those of nitric oxide, prostanoids, adenosine, acetylcholine, or any other of the known vasoactive neurotransmitters released from the retina. Our results suggest that retinal arterial tone is controlled by a diffusible, hydrophilic, and heat-stable relaxing factor that does not correspond with a known vasoactive molecule formed within the retina.

Endothelium-Derived Hyperpolarizing Factor–Mediated Renal Vasodilatory Response Is Impaired During Acute and Chronic Hyperhomocysteinemia

Background—Endothelial dysfunction is an early event in the development of vascular complications in hyperhomocysteinemia. Endothelial cells release a number of vasodilators, including NO and prostacyclin. Several lines of evidence have indicated the existence of a third vasodilator pathway, mediated by endothelium-derived hyperpolarizing factor (EDHF). EDHF is a major determinant of vascular tone in small resistance vessels. The influence of hyperhomocysteinemia on EDHF is unknown. The present in vivo study evaluates the integrity of the EDHF pathway in the renal microcirculation of rats with acute and chronic hyperhomocysteinemia. Methods and Results—EDHF-mediated vasodilation was evaluated as the renal blood flow (RBF) response to intrarenal acetylcholine during systemic NO synthase and cyclooxygenase inhibition. Acute hyperhomocysteinemia induced by intravenous homocysteine did not affect EDHF-mediated vasodilation. In contrast, intravenous methionine with subsequent hyperhomocysteinemia impaired the EDHF-mediated RBF response. When the methionine infusion was preceded by adenosine periodate oxidized to prevent the cleavage of S-adenosylhomocysteine to homocysteine and adenosine, a similar impairment of EDHF was observed, but with normal homocysteine levels. Animals with chronic hyperhomocysteinemia induced by a high-methionine, low–B vitamin diet during 8 weeks had a severely depressed EDHF-mediated vasodilation compared with those on a standard diet. Endothelium-independent vasodilation to deta-NONOate and pinacidil was not affected in acute and chronic hyperhomocysteinemia, demonstrating intact vascular smooth muscle reactivity. Conclusions—EDHF-dependent responses are impaired in the kidney of hyperhomocysteinemic rats. Because EDHF is a major regulator of vascular function in small vessels, these findings have important implications for the development of microangiopathy in hyperhomocysteinemia.

Protein-Bound Uremic Toxins Stimulate Crosstalk between Leukocytes and Vessel Wall

Leukocyte activation and endothelial damage both contribute to cardiovascular disease, a major cause of morbidity and mortality in CKD. Experimental in vitro data link several protein-bound uremic retention solutes to the modulation of inflammatory stimuli, including endothelium and leukocyte responses and cardiovascular damage, corroborating observational in vivo data. However, the impact of these uremic toxins on the crosstalk between endothelium and leukocytes has not been assessed. This study evaluated the effects of acute and continuous exposure to uremic levels of indoxylsulfate (IS), p-cresylsulfate (pCS), and p-cresylglucuronide (pCG) on the recruitment of circulating leukocytes in the rat peritoneal vascular bed using intravital microscopy. Superfusion with IS induced strong leukocyte adhesion, enhanced extravasation, and interrupted blood flow, whereas pCS caused a rapid increase in leukocyte rolling. Superfusion with pCS and pCG combined caused impaired blood flow and vascular leakage but did not further enhance leukocyte rolling over pCS alone. Intravenous infusion with IS confirmed the superfusion results and caused shedding of heparan sulfate, pointing to disruption of the glycocalyx as the mechanism likely mediating IS-induced flow stagnation. These results provide the first clear in vivo evidence that IS, pCS, and pCG exert proinflammatory effects that contribute to vascular damage by stimulating crosstalk between leukocytes and vessels.

Protective effect of cromakalim and diazoxide, and proulcerogenic effect of glibenclamide on indomethacin-induced gastric injury.

We investigated the influences of the K+ channel opening drugs cromakalim and diazoxide and their blocker, glibenclamide, in indomethacin-induced gastric injury in rats. Cromakalim (0.1 and 0.3 mg/kg) and diazoxide (10 and 30 mg/kg) produced dose-dependent gastroprotection at doses that were also effective on the cardiovascular system. Glibenclamide reversed their gastroprotective effects and aggravated indomethacin-induced gastric damage by its own. Cromakalim (10(-9)-10(-5) M) and diazoxide (10(-9)-10(-4) M) relaxed noradrenaline pre-contracted gastric arteries (94.59+/-1.58% and 93.86+/-2.99%, respectively). Their relaxant effects were inhibited by glibenclamide (10(-5) M) but not by indomethacin (10(-5) M) and LG-nitro-L-arginine (10(-4) M). Cromakalim (0.1 and 0.3 mg/kg) did not change gastric mucosal blood flow but increased the gastric mucosal vascular conductance in anaesthetized rats as measured by the hydrogen gas clearance technique. Indomethacin increased myeloperoxidase activity in the gastric mucosa, an effect which was reversed by cromakalim and diazoxide. Glibenclamide abolished their effects on myeloperoxidase activity and, alone, increased this parameter. Additionally, indomethacin caused infiltration of neutrophils which was reduced by cromakalim and diazoxide in a glibenclamide sensitive manner. The effects of cromakalim and diazoxide on mucosal myeloperoxidase activity, neutrophil infiltration and gastric injury correlated with each other. The effects of diazoxide (30 mg/kg) and glibenclamide (10 mg/kg) on blood glucose level were not correlated with their effects on gastric injury. Taken together, K+ channel opening drugs show misoprostol-like protective effects in indomethacin-induced gastric injury which seems to be related to modulation of neutrophil function.

EDHF and residual NO: different factors

See article by Ge et al. [61] (pages 547–556) in this issue. The endothelium plays a pivotal role in the control of vascular tone and blood pressure. In response to a variety of physiological stimuli, such as bradykinin, acetylcholine, histamine, substance P, shear stress and pulsatile stretch, endothelial cells release vasodilator substances. Those include prostacyclin [1] and endothelium-derived relaxing factor (EDRF) [2]. The latter has been identified as nitric oxide (NO) [3]. This labile compound is continuously synthesized from l-arginine by the nitric oxide synthase (NOS) enzyme constitutively expressed in the endothelial cell. It acts by direct stimulation of the soluble guanylyl cyclase in vascular smooth muscle cells. Since the elucidation of the biosynthesis of NO, l-arginine analogues such as N G-monomethyl-l-arginine (l-NMMA) and N G-nitro-l-arginine (l-NA) and its methyl ester (l-NAME) have been used as inhibitors of NOS to assess the contribution of the NO/cGMP pathway in endothelium-dependent responses. In the presence of inhibitors of both cyclo-oxygenase and NOS, residual endothelium-dependent relaxations were observed in a variety of preparations. Moreover, direct measurements of the smooth muscle membrane potential showed agonist-induced hyperpolarizations which disappeared after removal of the endothelium. Hyperpolarization of the vascular smooth muscle cells is known to cause relaxation by decreasing the open-probability of voltage-dependent calcium channels and by interfering with intracellular calcium release mechanisms. The presence of haemoglobin, which binds and inactivates NO, or of methylene blue, which inhibits guanylyl cyclase and destroys NO, did not inhibit the endothelium-dependent hyperpolarization in the rat aorta and pulmonary artery [4], the rabbit femoral artery [5] or the dog mesenteric artery [6]. Therefore, the existence of another relaxing factor, unrelated to NO and prostanoids, was proposed to contribute to endothelium-mediated relaxations [4,7,8]. This factor … * Corresponding author. Tel.: +32-9-240-3330; fax: +32-9-240-3059 Bert.Vanheel{at}rug.ac.be

Prostanoid-induced contractions are blocked by sulfonylureas

The sulfonylureas glibenclamide and tolbutamide are blockers of ATP-regulated K+ channels. The present study shows that these drugs also block contractions induced by prostaglandin F2 alpha, prostaglandin E2 and the thromboxane A2 mimetic U-46619 on rat aorta. This effect of sulfonylureas is not related to the endothelium since it is also found in endothelium-denuded preparations. The blockade is specific for prostanoids since contractions with norepinephrine, phenylephrine, serotonin, endothelin-1 or K+ (120 mM) are not or much less affected. On the other hand, contraction induced by activation of G-proteins with aluminium tetrafluoride anion (AlF4-) is significantly blocked by the sulfonylureas. Also on rat carotid artery the contraction of prostaglandin F2 alpha is importantly blocked by glibenclamide. It is concluded that the sulfonylureas glibenclamide and tolbutamide exert a specific inhibitory influence on prostanoid-induced contractions. This inhibition might be due to interference at the level of regulatory G-proteins, since the contractions induced by agonists that, like the prostanoids, activate phospholipase C (serotonin, phenylephrine, norepinephrine, endothelin) are not blocked.