Maria Assunta Potenza

Endothelial Dysfunction in Diabetes: From Mechanisms to Therapeutic Targets

Micro- and macrovascular complications are major causes of disability and death in patients with diabetes mellitus. Functional impairment of endothelial activity precedes the development of morphological alterations during the progression of diabetes. This endothelial dysfunction results from reduced bioavailability of the vasodilator nitric oxide (NO), mainly due to accelerated NO degradation by reactive oxygen species (ROS). Although hyperglycemia, insulin resistance, hyperinsulinemia and dyslipidemia independently contribute to endothelial dysfunction via several distinct mechanisms, increased oxidative stress seems to be the first alteration triggering several others. Mechanisms proposed to explain glucose- and lipid-induced vascular alterations in diabetes include accelerated formation of advanced glycation end-products (AGEs), protein kinase C activation, inflammatory signaling and oxidative stress. Insulin resistance with impaired PI 3-kinase effects decreases insulin mediated production of NO and reduces vasodilation, capillary recruitment and antioxidant properties of endothelium. Compensatory hyperinsulinemia enhances activation of intact MAP-kinase pathways and contributes to pro-atherogenic events by increasing secretion of endothelin-1 (ET-1), stimulating expression of adhesion molecules such as VCAM-1 and E-selectin, and inducing production of ROS. Conventional therapies to reduce hyperglycemia, dyslipidemia and insulin resistance may effectively improve endothelial function and delay the onset of vascular complications. Novel therapeutic approaches designed to inhibit AGEs formation, reduce PKC activation, decrease inflammatory signals and restore the ox/redox balance of endothelium may be predicted to ameliorate vascular function in diabetic state. This review summarizes the current knowledge on the most important mechanisms involved in endothelial dysfunction during diabetes. In addition, novel therapeutic strategies that may result from recently identified targets are also described.

Epigallocatechin Gallate, a Green Tea Polyphenol, Mediates NO-dependent Vasodilation Using Signaling Pathways in Vascular Endothelium Requiring Reactive Oxygen Species and Fyn

Green tea consumption is associated with reduced cardiovascular mortality in some epidemiological studies. Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, mimics metabolic actions of insulin to inhibit gluconeogenesis in hepatocytes. Because signaling pathways regulating metabolic and vasodilator actions of insulin are shared in common, we hypothesized that EGCG may also have vasodilator actions to stimulate production of nitric oxide (NO) from endothelial cells. Acute intra-arterial administration of EGCG to mesenteric vascular beds isolated ex vivo from WKY rats caused dose-dependent vasorelaxation. This was inhibitable by l-NAME (NO synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or PP2 (Src family kinase inhibitor). Treatment of bovine aortic endothelial cells (BAEC) with EGCG (50 μm) acutely stimulated production of NO (assessed with NO-specific fluorescent dye DAF-2) that was inhibitable by l-NAME, wortmannin, or PP2. Stimulation of BAEC with EGCG also resulted in dose- and time-dependent phosphorylation of eNOS that was inhibitable by wortmannin or PP2 (but not by MEK inhibitor PD98059). Specific knockdown of Fyn (but not Src) with small interfering RNA inhibited both EGCG-stimulated phosphorylation of Akt and eNOS as well as production of NO in BAEC. Treatment of BAEC with EGCG generated intracellular H2O2 (assessed with H2O2-specific fluorescent dye CM-H2DCF-DA), whereas treatment with N-acetylcysteine inhibited EGCG-stimulated phosphorylation of Fyn, Akt, and eNOS. We conclude that EGCG has endothelial-dependent vasodilator actions mediated by intracellular signaling pathways requiring reactive oxygen species and Fyn that lead to activation of phosphatidylinositol 3-kinase, Akt, and eNOS. This mechanism may explain, in part, beneficial vascular and metabolic health effects of green tea consumption.

Endothelin-1: The Yin and Yang on Vascular Function

Endothelin-1 (ET-1) is a vasoconstrictor secreted by endothelial cells, which acts as the natural counterpart of the vasodilator nitric oxide (NO). ET-1 contributes to vascular tone and regulates cell proliferation through activation of ETA and ETB receptors. Physical factors such as shear stress, or stimuli including thrombin, epinephrine, angiotensin II, growth factors, cytokines and free radicals enhance secretion of ET-1. By contrast, mediators like nitric oxide (NO), cyclic GMP, atrial natriuretic peptide, and prostacyclin reduce the release of endogenous ET-1. Thus, under normal conditions, the effects of the ET-1 are carefully regulated through inhibition or stimulation of ET-1 release from endothelium. Endothelial dysfunction is one of the earliest landmarks of vascular abnormalities. Altered function of endothelium may result from absolute decrease in bioavailability of NO as well as from relative augment in ET-1 synthesis, release or activity. Imbalance in the production of vasodilator and vasoconstrictor agents may contribute to the onset of hemodynamic disorders. Since dysregulation of the endothelin system is important in the pathogenesis of several cardiovascular diseases, the ETA and ETB receptors are attractive therapeutic targets for disorders associated with elevated ET-1 levels. ET receptor antagonists may be regarded as disease-modifying agents thanks to their ability to preserve endothelial integrity when the endothelin system is overactive. This review summarizes the current knowledge on the role of ET-1 in experimental hypertension and describes recent findings on the involvement of MAPK signalling pathways in ET-1 release in hypertension associated with insulin resistance. Moreover, therapeutic applications of ET-1 receptor blockers are also discussed.

Vascular actions of insulin with implications for endothelial dysfunction

Hemodynamic actions of insulin depend largely on the hormones ability to stimulate synthesis and release of endothelial mediators, whose balanced activity ensures dynamic control of vascular function. Nitric oxide (NO), endothelin-1 (ET-1), and reactive oxygen species (ROS) are important examples of endothelial mediators with opposing properties on vascular tone, hemostatic processes, and vascular permeability. Reduced NO bioavailability, resulting from either insufficient production or increased degradation of NO, characterizes endothelial dysfunction. In turn, endothelial dysfunction predicts vascular complications of metabolic and hemodynamic disorders. In the cardiovascular system, insulin stimulates the production and release of NO, ET-1, and ROS via activation of distinct intracellular signaling pathways. Under insulin-resistant conditions, increased insulin concentrations and/or impaired insulin-signaling pathways in the vasculature may contribute to imbalance in secretion of endothelial mediators that promote pathogenesis of vascular abnormalities. This short review describes signaling pathways involved in insulin-stimulated release of NO, ROS, and ET-1 and suggests possible molecular mechanisms by which abnormal insulin signaling may contribute to endothelial dysfunction.

Treatment of spontaneously hypertensive rats with rosiglitazone and/or enalapril restores balance between vasodilator and vasoconstrictor actions of insulin with simultaneous improvement in hypertension and insulin resistance.

Spontaneously hypertensive rats (SHRs) exhibit endothelial dysfunction and insulin resistance. Reciprocal relationships between endothelial dysfunction and insulin resistance may contribute to hypertension by causing imbalanced regulation of endothelial-derived vasodilators (e.g., nitric oxide) and vasoconstrictors (e.g., endothelin-1 [ET-1]). Treatment of SHRs with rosiglitazone (insulin sensitizer) and/or enalapril (ACE inhibitor) may simultaneously improve hypertension, insulin resistance, and endothelial dysfunction by rebalancing insulin-stimulated production of vasoactive mediators. When compared with WKY control rats, 12-week-old vehicle-treated SHRs were hypertensive, overweight, and insulin resistant, with elevated fasting levels of insulin and ET-1 and reduced serum adiponectin levels. In mesenteric vascular beds (MVBs) isolated from vehicle-treated SHRs and preconstricted with norepinephrine (NE) ex vivo, vasodilator responses to insulin were significantly impaired, whereas the ability of insulin to oppose vasoconstrictor actions of NE was absent (versus WKY controls). Three-week treatment of SHRs with rosiglitazone and/or enalapril significantly reduced blood pressure, insulin resistance, fasting insulin, and ET-1 levels and increased adiponectin levels to values comparable with those observed in vehicle-treated WKY controls. By restoring phosphatidylinositol 3-kinase–dependent effects, rosiglitazone and/or enalapril therapy of SHRs also significantly improved vasodilator responses to insulin in MVB preconstricted with NE ex vivo. Taken together, our data provide strong support for the existence of reciprocal relationships between endothelial dysfunction and insulin resistance that may be relevant for developing novel therapeutic strategies for the metabolic syndrome.

Treatment of spontaneously hypertensive rats with rosiglitazone ameliorates cardiovascular pathophysiology via antioxidant mechanisms in the vasculature

Oxidative stress contributes to cardiovascular complications of diabetes, in part, by reducing the bioavailability of nitric oxide (NO). We investigated the mechanisms whereby the insulin sensitizer rosiglitazone may ameliorate oxidative stress in the vasculature of spontaneously hypertensive rats (SHR). Nine-week-old SHR were treated by gavage for 7 wk with rosiglitazone (5 mg x kg(-1) x day(-1)) or vehicle control. Treatment of SHR with rosiglitazone lowered systolic blood pressure, reduced fasting plasma insulin and asymmetrical dimethylarginine, and increased insulin sensitivity (when compared with vehicle treatment). In vessel homogenates and serum from rosiglitazone-treated SHR, SOD activity was enhanced, while 8-iso-PGF(2alpha) (lipid peroxidation product) was reduced (when compared with samples from vehicle-treated SHR). Moreover, expression of p22phox (catalytic subunit of NADPH oxidase) as well as nitrotyrosine and superoxide content were all reduced in the aortas of rosiglitazone-treated SHR. In mesenteric vascular beds (MVB) isolated ex vivo from rosiglitazone-treated SHR, NO-dependent vasodilator actions of insulin were improved when compared with MVB from vehicle-treated SHR. Acute pretreatment of MVB from vehicle-treated SHR with apocynin (NADPH oxidase inhibitor) enhanced vasodilator actions of insulin (results comparable to those in MVB from rosiglitazone-treated SHR). In Langendorff heart preparations from rosiglitazone-treated SHR, ischemia/reperfusion injury caused infarcts 40% smaller than in hearts from vehicle-treated SHR. Acute pretreatment of hearts from vehicle-treated SHR with apocynin produced similar results. Finally, rosiglitazone treatment of endothelial cells in primary culture reduced superoxide induced by insulin-resistant conditions. We conclude that rosiglitazone therapy in SHR increases SOD activity and decreases p22phox expression in the vasculature to reduce oxidant stress leading to an improved cardiovascular phenotype.

Functional characterization of endothelin receptors in hypertensive resistance vessels.

OBJECTIVE The physiological and pathophysiological functions of endothelin-1 in modulating the regional blood flow of normal and spontaneously hypertensive rats (SHR) were studied in the perfused mesenteric vascular bed, a useful model for investigating resistance vessels. DESIGN AND METHODS We used 12-week-old SHR and Wistar-Kyoto (WKY) rats. Endothelin A (ETA) receptor responsiveness was evaluated by endothelin-1 (0.2-2 mumol/l) concentration-response curves, and repeated in the presence of indomethacin and the ETA and endothelin B (ETB) receptor antagonists BQ-485 and BQ-788, respectively. ETB receptor responsiveness was tested by sarafotoxin S6c concentration-response curves, obtained in the noradrenaline-precontracted mesenteric vascular bed, and repeated after treatment with BQ-788 and after endothelial denudation. RESULTS In both groups, endothelin-1 induced concentration-dependent contraction; SHR exhibited a markedly increased maximal effect compared with WKY rats (P < 0.01). BQ-485 produced a shift to the right for endothelin-1 concentration-response curves in both groups, with a higher pA2 (negative common logarithm of the antagonist that produces an agonist dose ratio of 2) value in SHR than in WKY rats (P < 0.01). The increase in the maximal effect produced by endothelin-1 in SHR was prevented by indomethacin, which also induced a significant increase in the endothelin-1 concentration producing the half-maximal response (EC50) in SHR (P < 0.05). Sarafotoxin S6c produced an ETB-dependent endothelium-mediated relaxant effect in WKY rats, which was not observed in SHR. CONCLUSIONS The higher vasoconstriction induced by endothelin-1 in SHR may be related to a greater number of available ETA receptors, due to the presence of an ETA receptor subtype. This mechanism may be linked to the production of prostanoids that add to the direct endothelin-1-evoked vasoconstriction. These results, together with the lack of relaxation in response to sarafotoxin S6c in SHR, suggest that an imbalance in the endothelin-1 ability to induce both contraction and relaxation is present in SHR with sustained hypertension, manifesting as a greater contractile effect evoked in this strain.

Idiopathic chronic constipation: tachykinins as cotransmitters in colonic contraction

Background Tachykinins (TKs) have been shown to be involved in the excitatory enteric motor pathway. This study aimed to examine the direct and nerve‐mediated effect of specific NK1, NK2 and NK3 receptor agonists and antagonists in colonic preparations from control subjects and patients with idiopathic chronic constipation (ICC).

Cardiovascular Complications in Diabetes: Lessons from Animal Models

Micro- and macro-vascular complications are the leading causes of morbidity and mortality in type 1 and type 2 diabetic patients. Despite the vast clinical experience linking diabetic metabolic abnormalities to cardiovascular lesions, the molecular basis of individual susceptibility to diabetic cardiovascular injury is still largely unknown. Significant advances in this area may come from studies on suitable animal models. Although no animal model can accurately reproduce the human disease, experimental studies in animals have the great advantage to eliminate factors such as ethnicity, economic and geographic variables, drug interactions, diet, gender and age differences that importantly limit clinical studies. Indeed, appropriate animal models have provided important information on genetic and environmental risks of diabetes, and helped to dissect molecular mechanisms underlying the development, progression and therapeutic control of this disease. Unfortunately, none of the diabetic models presently available fully mimics the human syndrome. Therefore, the current knowledge on the pathogenesis of cardiovascular complications relies on the evaluation of distinct phenotypes from various diabetic models. In addition to strains prone to diabetes, this disease can be induced by surgical, pharmacological or genetic manipulation in several animal species. Rodents are the most used, although some studies are still performed in larger animals as rabbits, cats, pigs or monkeys. Far from being exhaustive, this work should serve as a general overview of the most relevant clues provided by major species and models for the overall comprehension of cardiovascular complications in type 1 and type 2 diabetes.

In-vivo administration of CLC-K kidney chloride channels inhibitors increases water diuresis in rats: a new drug target for hypertension?

Objective The human kidney-specific chloride channels ClC-Ka (rodent ClC-K1) and ClC-Kb (rodent ClC-K2) are important determinants of renal function, participating to urine concentration and blood pressure regulation mechanisms. Here we tested the hypothesis that these chloride channels could represent new drug targets for inducing diuretic and antihypertensive effects. Methods To this purpose, the CLC-K blockers benzofuran derivatives MT-189 and RT-93 (10, 50, 100 mg/kg), were acutely administered by gavage in Wistar rats, and pharmacodynamic and pharmacokinetic parameters determined by functional, bioanalytical, biochemical and molecular biology assays. Results Plasma concentration values for MT-189 and RT-93 were indicative of good bioavailability. Both MT-189 and RT-93 dose-dependently increased urine volume without affecting electrolyte balance. A comparable reduction of SBP was observed in rats after MT-189, RT-93 or furosemide administration. Benzofuran derivatives treatment did not affect kidney CLC-K mRNA level or inner medulla osmolality, whereas a significant vasopressin-independent down-regulation of aquaporin water channel type 2 was observed at protein and transcriptional levels. In rats treated with benzofuran derivatives, the observed polyuria was mainly water diuresis; this finding indirectly supports a cross-talk between chloride and water transport in nephron. Moreover, preliminary in-vitro evaluation of the drugs capability to cross the blood-inner ear barrier suggests that these compounds have a limited ability to induce potential auditory side effects. Conclusion CLC-K blockers may represent a new class of drugs for the treatment of conditions associated with expanded extracellular volume, with a hopeful high therapeutic potential for hypertensive patients carrying ClC-K gain-of-function polymorphisms.