Carmela Nacci

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.

Endothelial dysfunction in mice with streptozotocin-induced type 1 diabetes is opposed by compensatory overexpression of cyclooxygenase-2 in the vasculature.

Cardiovascular complications of diabetes result from endothelial dysfunction secondary to persistent hyperglycemia. We investigated potential compensatory mechanisms in the vasculature that oppose endothelial dysfunction in diabetes. BALB/c mice were treated with streptozotocin (STZ) to induce type 1 diabetes (T1D). In mesenteric vascular beds (MVBs), isolated ex vivo from mice treated with STZ for 1 wk, dose-dependent vasorelaxation to acetylcholine (ACh) or sodium nitroprusside was comparable with that in age-matched control mice (CTRL). By contrast, MVBs from mice treated with STZ for 8 wk had severely impaired vasodilator responses to ACh consistent with endothelial dysfunction. Pretreatment of MVBs from CTRL mice with nitric oxide synthase inhibitor nearly abolished vasodilation to ACh. In MVB from 1-wk STZ-treated mice, vasodilation to ACh was only partially impaired by L-N(omega)-arginine methyl ester. Thus, vasculature of mice with T1D may have compensatory nitric oxide-independent mechanisms to augment vasodilation to ACh and oppose endothelial dysfunction. Indeed, pretreatment of MVBs isolated from 1-wk STZ-treated mice with NS-398 [selective cyclooxygenase (COX)-2 inhibitor] unmasked endothelial dysfunction not evident in CTRL mice pretreated without or with NS-398. Expression of COX-2 in MVBs, aortic endothelial cells, and aortic vascular smooth muscle cells from STZ-treated mice was significantly increased (vs. CTRL). Moreover, concentrations of the COX-2-dependent vasodilator 6-keto-prostaglandin F-1alpha was elevated in conditioned media from aorta of STZ-treated mice. We conclude that endothelial dysfunction in a mouse model of T1D is opposed by compensatory up-regulation of COX-2 expression and activity in the vasculature that may be relevant to developing novel therapeutic strategies for diabetes and its cardiovascular complications.

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.

Molecular and clinical aspects of endothelial dysfunction in diabetes

Diabetic patients have an increased risk for cardiovascular complications with respect to the general population. Micro- and macrovascular complications such as nephropathy, retinopathy, atherosclerosis, and coronary artery disease are usually preceded by endothelial dysfunction, a condition characterized by impaired vasorelaxation resulting from reduced bioavailability of the endothelial mediator nitric oxide (NO). Nitric oxide is among endothelial mediators released by endothelial cells in response to insulin stimulation. Therefore, metabolic abnormalities such as insulin resistance, dyslipidemia, compensatory hyperinsulinemia and overt hyperglycemia may all contribute to impaired NO bioavailability and abnormal vasodilatation in diabetic patients. Each of these alterations may trigger endothelial dysfunction by multiple intracellular mechanisms including accelerated formation of advanced glycolysis end products, activation of protein kinase C, increased pro-inflammatory signaling, and impaired sensitivity of the PI 3-kinase signaling pathways. This review outlines the most important mechanisms by which insulin takes part in physiological regulation of endothelial function. Abnormal insulin signaling in endothelium under diabetic conditions and patho-physiological consequences on cardiovascular homeostasis will also be discussed.

Stress, Neuropsychiatric Disorders and Immunological Effects Exerted by Benzodiazepines

Psychoneuroimmunology is a growing scientific field which deals with the mutual interplay between nervous and immune systems. In this framework, many data have demonstrated that cytokines (CKs) derived from the periphery are able to cross the blood brain barrier and act upon the central nervous system (CNS) [e.g., the hypothalamic-pituitary-adrenal axis (HPAA)], thus regulating several physiological functions (thermoregulation, sleep, appetite) or damaging the nervous tissue, when released in exaggerated amounts. On the other hand, nervous cells, such as astrocytes and microglial cells also generate proinflammatory CKs which may be detrimental for the CNS. The neuromodulating CK network can be triggered by microorganisms and/or their products (i.e. bacterial endotoxins), but also stressful life events may activate the HPAA, thus affecting the immune system function. This review will place emphasis on some clinical conditions, such as phobia and migraine without aura (MWA), characterized by anxiety disorders. Patients affected by these neuropsychiatric alterations exhibit multiple functional deficits of phagocytes and T lymphocytes which allow penetration of various pathogens into the host. This is also supported by the detection of circulating bacterial endotoxins and the evidence of both spontaneous and induced exaggerated release of proinflammatory CKs in phobic and MWA patients. The possible iatrogenic effects of benzodiazepines (BDZ) on the immune system have been evaluated by in vitro and in vivo studies. In this respect, it emerges that diazepam exerts an inhibitory function on the immune system, while alprazolam behaves as an immunoenhancer. The presence of central and/or peripheral BDZ receptors on immune cells seems to be the key mechanism responsible for the immunomodulation exerted by these drugs.

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).

Globular adiponectin counteracts VCAM-1-mediated monocyte adhesion via AdipoR1/NF-κB/COX-2 signaling in human aortic endothelial cells

Adiponectin (Ad) is an insulin-sensitizing adipocytokine with anti-inflammatory and vasoprotective properties. Cleavage of native full-length Ad (fAd) by elastases from activated monocytes generates globular Ad (gAd). Increased gAd levels are observed in the proximity of atherosclerotic lesions, but the physiological meaning of this proteolytic Ad fragment in the cardiovascular system is controversial. We compared molecular and biological properties of fAd and gAd in human aortic endothelial cells (HAEC). In control HAEC, both fAd and gAd acutely stimulated nitric oxide (NO) production by AMPK-dependent pathways. With respect to fAd, gAd more efficiently increased activation of NF-κB signaling pathways, resulting in cyclooxygenase-2 (COX-2) overexpression and COX-2-dependent prostacyclin 2 (PGI(2)) release. In contrast with fAd, gAd also increased p38 MAPK phosphorylation and VCAM-1 expression, ultimately enhancing adhesion of monocytes to endothelial cells. In HAEC lacking AdipoR1 (by siRNA), both activation of NF-κB as well as COX-2 overexpression by gAd were abrogated. Conversely, gAd-mediated p38MAPK activation and VCAM-1 expression were unaffected, and monocyte adhesion was greatly enhanced. In HAEC lacking COX-2 (by siRNA), reduced levels of PGI(2) further increased gAd-dependent monocyte adhesion. Our findings suggest that biological activities of fAd and gAd in endothelium do not completely overlap, with gAd possessing both AdipoR1-dependent ability to stimulate COX-2 expression and AdipoR1-independent effects related to expression of VCAM-1 and adhesion of monocytes to endothelium.

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.

Endothelin-1-receptor-mediated responses in resistance vessels of young and adult spontaneously hypertensive rats.

Objective To assess whether primary changes in endothelin-1 (ET-1) receptor responsiveness or secondary vessel functional modifications could characterize the effects evoked by ET-1 in the mesenteric vascular bed (MVB) of prehypertensive 5-week-old and 12-week-old spontaneously hypertensive rats (SHRs). Design and methods We used male 5-week-old and 12-week-old SHRs and sex- and age-matched Wistar-Kyoto (WKY) rats as controls. ET-1 receptor responsiveness was evaluated by ET-1 (0.04–2 μmol/l) concentration-response curves and repeated with indomethacin and BQ-123 (0.1–0.5 μmol/l), the latter a selective ETA receptor antagonist. ETB receptor responsiveness was tested by sarafotoxin S6c (1–100 nmol/l) and IRL-1620 (0.1–10 nmol/l) concentration-response curves, obtained in the noradrenaline-precontracted MVB. Results At 5 weeks of age, ET-1 induced a similar concentration-dependent contraction in SHRs and WKY rats, with an overlapping BQ-123 pA2 value (negative common logarithm of the antagonist that produces an agonist dose ratio of 2) in the two strains. Indomethacin was ineffective in both groups. Sarafotoxin S6c and IRL-1620 both evoked an ETB-mediated, significant relaxation, only in WKY rats. In 12-week-old SHRs, ET-1 evoked a markedly increased maximal effect compared with the response in WKY rats (P < 0.01); this was prevented by treatment with indomethacin. The BQ-123 pA2 value was higher in SHRs than in WKY rats (P < 0.01). Both sarafotoxin S6c and IRL-1620 evoked a significant concentration-dependent relaxation in WKY rats, which was not detected in SHR preparations. Conclusions Our results could suggest that the different responses evoked by ET-1 in the MVB of SHRs during the onset of hypertension may be related partially to primary alterations in the ET-1 receptorial pattern and partially to the onset of high blood pressure, leading to an impairment in the haemodynamic balance.

Elevated Endothelin-1 (ET-1) Levels May Contribute to Hypoadiponectinemia in Childhood Obesity

CONTEXT Pediatric obesity is associated with endothelial dysfunction and hypoadiponectinemia, but the relationship between these two conditions remains to be fully clarified. Whether enhanced release of endothelin-1 (ET-1) may directly impair adiponectin (Ad) production in obese children is not known. OBJECTIVE The aim of the study was to explore whether and how high circulating levels of ET-1 may contribute to impair Ad production, release, and vascular activity. DESIGN AND PARTICIPANTS Sixty children were included into obese (Ob; n = 30), overweight (OW; n = 11), and lean (n = 19) groups. Total and high-molecular-weight Ad, ET-1, vascular cell adhesion molecule-1, and von Willebrand factor levels were measured in serum samples. Adipocytes were stimulated with exogenous ET-1 or with sera from lean, OW, and Ob, and Ad production and release measured in the absence or in the presence of ETA (BQ-123) and ETB (BQ-788) receptor blockers, p42/44 MAPK inhibitor PD-98059, or c-Jun NH2-terminal protein kinase inhibitor SP-600125. Vasodilation to Ad was evaluated in rat isolated arteries in the absence or in the presence of BQ-123/788. RESULTS Total and high-molecular-weight Ad was significantly decreased and ET-1 levels significantly increased in OW (P < .01) and Ob (P < .001) children. A statistically significant linear regression (P < .01) was found between Ad and ET-1. Exposure of adipocytes to exogenous ET-1 or serum from OW and Ob significantly decreased Ad mRNA and protein levels (P < 0.001). The inhibitory effect of ET-1 on Ad was reverted by BQ-123/788 or PD-98059 but not SP-600125. Ad-mediated vasodilation was further increased in arteries pretreated with BQ-123/788. CONCLUSIONS ET-1-mediated inhibition of Ad synthesis via p42/44 MAPK signaling may provide a possible explanation for hypoadiponectinemia in pediatric obesity and contribute to the development of cardiovascular complications.