Identification of microRNAs 758 and 33b as potential modulators of ABCA1 expression in human atherosclerotic plaques.

BACKGROUND AND AIM: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters A1 and G1 are the main transporters involved in macrophage cholesterol efflux. The understanding of the molecular mechanism(s) of their regulation in atherosclerosis is crucial for potential therapeutic approaches. Preclinical studies support a role for microRNAs in the posttranscriptional regulation of these transporters; however, no evidence is still available on human atherosclerosis. Thus, the aim of this study was to investigate the modulation of the ABCA1 and ABCG1 pathway in human atherosclerotic plaques and microRNA involvement in its modulation. METHODS AND RESULTS: Thirty-one human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy for high-grade (>70%) vessel stenosis, and divided into normocholesterolemic (n = 15) and hypercholesterolemic groups (n = 16) according to the presence/absence of hypercholesterolemia. Both ABCA1 and ABCG1 messenger RNAs (mRNAs) were significantly upregulated in carotid plaques from hypercholesterolemic patients as assessed by real-time polymerase chain reaction (RT-PCR). Despite this result, no difference was found at the protein levels analyzed by Western blot, thus suggesting a strong posttranscriptional modulation. MicroRNA microarray and subsequent validation by RT-PCR showed a significant upregulation of ABCA1-linked miR-758 and miR-33b in plaques from hypercholesterolemic patients. CONCLUSION: We provide evidence of a strong posttranscriptional regulation of ABCA1 and ABCG1 expression in human atherosclerotic plaques from hypercholesterolemic patients. This effect is potentially due to the concomitant increase of miR-33b and miR-758, two well-established regulators of ABCA1 and ABCG1 expression. The identification of miR-33b and miR-758 as putative key regulators of ABCA1 protein expression within human atherosclerotic plaques provides further data for the realization of new anti-atherosclerotic drugs with specific targets based on anti-miRNA technologies.

Care manager to control cardiovascular risk factors in primary care: the Raffaello cluster randomized trial.

BACKGROUND AND AIM: This cluster randomized trial evaluated the efficacy of a disease and care management (D&CM) model in cardiovascular (CVD) prevention in primary care. METHODS AND RESULTS: Eligible subjects had ≥ 1 among: blood pressure ≥ 140/90 mmHg; glycated hemoglobin ≥ 7%; LDL-cholesterol ≥ 160 or ≥ 100 mg/dL (primary or secondary prevention, respectively); BMI ≥ 30; current smoking. The D&CM intervention included a teamwork including nurses as care managers for the implementation of tailored care plans. Control group was allocated to usual-care. The main outcome was the proportion of subjects achieving recommended clinical targets for ≥ 1 of uncontrolled CVD risk factors at 12-month. During 2008-2009 we enrolled 920 subjects in the Abruzzo/Marche regions, Italy. Following the exclusion of L'Aquila due to 2009 earthquake, final analyses included 762 subjects. The primary outcome was achieved by 39.1% (95%CI: 34.2-44.2) and 25.2% (95%CI: 20.9-29.9) of subjects in the intervention and usual-care group, respectively (p < 0.001). The D&CM intervention significantly increased the proportion of subjects who achieved clinical targets for both diabetes and hypertension, with no differences in hypercholesterolemia, smoking status and obesity. CONCLUSIONS: The D&CM intervention was effective in controlling cardiovascular risk factors, in particular hypertension and diabetes. Numbers needed to treat were small. Such intervention may deserve further consideration in clinical practice. REGISTRATION NUMBER: ACTRN12611000813987.

Lower incidence of macrovascular complications in patients on insulin glargine versus those on basal human insulins: a population-based cohort study in Italy.

BACKGROUND AND AIM: The aim of this study was to compare the use of insulin glargine and intermediate/long-acting human insulin (HI) in relation to the incidence of complications in diabetic patients. METHODS AND RESULTS: A population-based cohort study was conducted using administrative data from four local health authorities in the Abruzzo Region (900,000 inhabitants). Diabetic patients without macrovascular diseases and treated with either intermediate/long-acting HI or glargine were followed for 3-years; the incidence of diabetic (macrovascular, microvascular and metabolic) complications was ascertained by hospital discharge claims and estimated using Cox proportional hazard models. Propensity score (PS) matching was also used to adjust for significant differences in the baseline characteristics between the two groups. RESULTS: Overall, 1921 diabetic patients were included: 744 intermediate/long-acting HI and 1177 glargine users. During the 3-year follow-up, 209 (28.1%) incident events of any diabetic complication occurred in the intermediate/long-acting HI and 159 (13.5%) in the glargine group. After adjustment for covariates, glargine users had an HR (95% CI) of 0.57 (0.44-0.74) for any diabetic complication and HRs of 0.61 (0.44-0.84), 0.58 (0.33-1.04) and 0.35 (0.18-0.70) for macrovascular, microvascular and metabolic complications, respectively, compared to intermediate/long-acting HI users. PS analyses supported these findings. CONCLUSIONS: The use of glargine is associated with a lower risk of macrovascular complications compared with traditional basal insulins. However, limitations inherent to the study design including the short length of observation and the lack of data on metabolic control or diabetes duration, do not allow us to consider this association as a proof of causality.

Efficacy of a nurse-led email reminder program for cardiovascular prevention risk reduction in hypertensive patients: a randomized controlled trial.

BACKGROUND: Many strategies have been evaluated to improve the prevention and control of cardiovascular (CVD) risk factors. Nursing telephonic and tele-counseling individualized lifestyle educational programs have been found to improve blood pressure control and adherence to lifestyle recommendation. This study tested the efficacy of a nurse-led reminder program through email (NRP-e) to improve CVD risk factors among hypertensive adults. METHODS: All participants received usual CVD prevention and a guideline-based educational program. Subjects in the NRP-e group also received weekly email alerts and phone calls from a nurse care manager for 6 months. Emails contained a reminder program on the need for adherence with a healthy lifestyle based upon current guidelines. Follow-up visits were scheduled at 1, 3 and 6 months after enrollment; randomization was made centrally and blood samples were evaluated into a single laboratory. RESULTS: The final sample consisted of 98 (control) and 100 (NRP-e) subjects (mean age 59.0 ± 14.5 years; 51.0% males). After 6 months, the following CVD risk factors significantly improved in both groups: body mass index, alcohol and fruit consumption, cigarette smoking, adherence to therapy hours, systolic and diastolic blood pressure, fasting blood glucose, low-density lipoproteins (LDL) and total cholesterol, triglycerides, and physical activity. In the NRP-e group, however, the prevalence of several behaviors or conditions at risk decreased significantly more than in the control group: obesity (-16%), low fruit consumption (-24%), uncontrolled hypertension (-61%), LDL (-56%), and total cholesterol (-40%). CONCLUSIONS: The NRP-e improved a range of CVD risk factors. The program had low costs, required only an average of <20 min per day in addition to normal practice, and may deserve further evaluation for the inclusion among existing care management approaches.

Pitfalls of contrast-enhanced ultrasound (CEUS) in the diagnosis of splenic sarcoidosis.

By observing the real-time behavior of focal liver lesions at three vascular phases (arterial, portal-venous, and late), contrast-enhanced ultrasound (CEUS) has been successfully applied to differentiate benign from malignant hepatic nodules. In recent years, numerous studies highlighted the usefulness of CEUS also for other applications such as abdominal trauma, renal, pancreatic, thyroid, and inflammatory bowel diseases, supporting its role even in differentiating benign from malignant splenic nodules. Therefore, the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) recently updated the guidelines for the use of ultrasound contrast agents in clinical practice, pointing out the indication to characterize splenic parenchymal inhomogeneity or suspected lesions found on conventional ultrasound (BUS). We describe the case of a patient with a history of colon cancer and finding, at BUS and CEUS, of hypoechoic lesions with a highly suggestive pattern for metastases, subsequently histologically proved to be splenic localizations of a benign and multisystemic granulomatous disease such as sarcoidosis. We therefore reviewed the current literature focusing on the role of CEUS in differentiating benign from malignant splenic lesions, emphasizing on the lack of data and numerical shortage of sarcoidosis derived-lesions in the available studies. We conclude that sarcoidosis remains a diagnosis of exclusion and new studies are needed before defining precise indications of CEUS in these patients.

Immunopathogenesis of sarcoidosis and risk of malignancy: a lost truth?

The hypothesis of a relationship between sarcoidosis and malignancy was firstly formulated in 1972 by Brincker. He documented an association of sarcoid reactions or sarcoidosis with 19 lymphomas and associated malignancies. Based on various epidemiological studies, for more than 20 years sarcoidosis has been considered as a condition at increased risk for cancer, particularly lymphoproliferative disorders. The existence of a sarcoidosis-lymphoma syndrome was therefore proposed, highlighting, as a potential mechanism, the uncontrolled lymphocyte proliferation and mitotic activity. A reduced ability to eliminate an antigen and chronic inflammation have been suggested as triggering events. Leading to a reduced tumor immune surveillance, a diminished myeloid dendritic cells (mDC) function, despite up-regulated co-stimulatory and maturation markers, was also raised as potential mechanism. However, some subsequent studies have questioned the presence of a close association between the two entities and have explained those previously published as the result of selection bias and misclassification. Recently, a Swedish population-based cohort study documented a significant overall excess incidence of cancer among sarcoidosis patients, especially those with multiple hospitalizations or admission in older age, emphasizing again a potential neoplastic risk. Therefore, currently, whether these patients have an increased risk of developing malignant lesions is still debated. Larger and unbiased studies are needed before drawing definite conclusions.

MicroRNAs and atherosclerosis: new actors for an old movie.

To date, cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. MicroRNAs (miRNAs) are endogenous, short, non-coding RNA sequences able to regulate gene expression principally at the post-transcriptional level. Initially, they were thought to be involved only in developmental timing of worms. Their involvement in human biology was recently discovered and many studies have been performed to demonstrate the role of miRNA in human cancer. Since the first observation in 2005 of their implication in cardiac biology, many studies have demonstrated their role in the genetic modulation of cardiovascular development and in cardiovascular diseases such as cardial remodeling and heart failure, cardiac arrhythmias, cardiac ischaemia, cardiac fibrosis, atherosclerosis and stroke. Thus, the aim of this review is to describe the role of miRNA in atherosclerosis development and evolution and to individuate their role as potential therapeutic target.

Formulation and characterization of polyphenol-loaded lipid nanocapsules.

The purpose of this study was to design and characterize two flavonoid-loaded lipid nanocapsules (LNC) by applying the phase inversion process, and to enhance their apparent solubility and/or the stability. The flavonoid-loaded LNC were characterized by particle size, encapsulation efficiency, drug leakage rates, stability and spectroscopic studies. It was observed that quercetin-loaded LNC30 (3%) and LNC60 (2%) carried a particle size of 30.3 and 55.1 nm, respectively and significant higher entrapment efficiency. Encapsulation of quercetin (QC) in LNC enabled us to increase its apparent aqueous solubility by a factor of 100. And in view of calculations and results, it seems most probable that QC is arranged at this LNC interface between the oil phase and the hydrophilic polyethylene glycol moieties of the surfactant. In addition, colloidal suspensions proved to be stable in term of encapsulation for at least 10 weeks and QC was not oxidised. With simple chemical modification of (-)-epigallocatechin-3-gallate or (-)-EGCG, it was possible to reach very high encapsulation rates (95%). Thus we obtained stable colloidal suspensions of (-)-EGCG in water over 4 weeks while free (-)-EGCG solubilised in water exhibited 100% degradation within 4h. The initial problems (solubility and stability) of these flavonoids were resolved thanks to drug-loaded LNC.

Effects of a phytosterol-enriched dairy product on lipids, sterols and 8-isoprostane in hypercholesterolemic patients: a multicenter Italian study.

BACKGROUND AND AIMS: Plant sterols, added to several food sources, lower serum cholesterol concentrations. Plant sterol-induced cholesterol lowering is paralleled by a mild decrease in plasma levels of the antioxidant beta-carotene, the amount of this decrease being considered clinically non-significant. Whether the effect on lipid profile of daily consumption of plant sterol-enriched low-fat fermented milk (FM) is paralleled by a concomitant variation in a reliable marker of the oxidative burden like plasma isoprostane levels is unresolved. METHODS AND RESULTS: The effect of plant sterol consumption on plasma lipid and isoprostane levels of hypercholesterolemic patients was evaluated in a multicenter, randomized double blind study. Hypercholesterolemic patients consumed a FM daily for 6 weeks. Subjects were randomized to receive either 1.6g of plant sterol-enriched FM (n=60) or control FM product (n=56). After 6 weeks of plant sterol-enriched FM consumption, LDL cholesterol was reduced from 166.2+/-2.0 to 147.4+/-2.8 mg/dL (p=0.01). A significant reduction was observed for total cholesterol (from 263.5+/-2.6 to 231.0+/-3.2mg/dL, p=0.01). There was greater LDL cholesterol lowering among hypercholesterolemic patients with higher LDL cholesterol at baseline. We found a reduction of plasma 8-isoprostane in patients taking plant sterol-enriched FM (from 43.07+/-1.78 to 38.04+/-1.14 pg/ml, p=0.018) but not in patients taking the control product (from 42.56+/-2.12 to 43.19+/-2.0 pg/ml, p=NS). Campesterol and beta-sitosterol levels were not influenced by phytosterol consumption. CONCLUSIONS: Daily consumption of low-fat plant sterol dairy product favourably changes lipid profile by reducing LDL-cholesterol, and may also have an anti-oxidative effect through a reduction of plasma isoprostanes.

Increased concentrations of soluble CD40 ligand may help to identify type 1 diabetic adolescents and young adults at risk for developing persistent microalbuminuria.

BACKGROUND: Recent studies on the pathogenesis of diabetic complications have demonstrated the important role of a number of aberrantly expressed molecules acting together in the development of early diabetic microvascular complications. Soluble CD40 ligand (sCD40L) is supposed to be one of the most likely candidates for both retinopathy and nephropathy. METHODS: In January 1989, sCD40L was measured in 340 normoalbuminuric diabetic adolescents and young adults. Participants were examined at baseline and biannually thereafter. sCD40L was measured every 2 years during a 16-year follow-up period. sCD40L was also measured in parents. RESULTS: Over 16 years, 32 out of 340 patients developed persistent microalbuminuria; no patient developed overt diabetic nephropathy. The risk of developing microalbuminuria was higher in children with increased sCD40L at the beginning of the study (using 6 ng/mL as the arbitrary cut-off value) (group A) compared with those with normal sCD40L (group B). Sex did not influence predictive value, sensitivity, or specificity. sCD40L was not significantly correlated with duration of diabetes. The percentage of offspring with both parents having sCD40L above the mean values was significantly higher in group A than in group B. The odds ratio (OR) for the occurrence of microalbuminuria after adjustment for confounding variables in patients with elevated baseline sCD40L was 4.2 (95% CI, 2.1-10.7). CONCLUSIONS: Persistently increased sCD40L levels from the onset of diabetes might help to identify those normotensive and normoalbuminuric young patients at increased risk of developing incipient nephropathy later in life.

COX-2 expression in atherosclerosis: the good, the bad or the ugly?

Cyclooxygenase (COX) is the rate limiting enzyme catalyzing the conversion of arachidonic acid into prostanoids, lipid mediators critically implicated in a variety of physiological and pathophysiological processes, including inflammation, vascular and renal homeostasis, and immune responses. Since the early 1990s it has been appreciated that two isoforms of COX exist, referred to as COX-1 and COX-2. Although structurally homologous, COX-1 and COX-2 are regulated by two independent and quite different systems and have different functional roles. In the setting of acute ischemic syndromes it has been recognized that COX pathway plays an important role; however, whereas the function of platelet COX-1 in acute ischemic diseases is firmly established, the role of COX-2 in atherothrombosis remains controversial. The complex role of COX-2 in this setting is also confirmed by the unexpected cardiovascular side effects of long-term treatment with COX-2 inhibitors. In this article, we review the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque vulnerability, the effects of the variable expression of upstream and downstream enzymes in the prostanoid biosynthesis on COX-2 expression and inhibition.

Multivariate curve resolution of rapid-scan FTIR difference spectra of quinone photoreduction in bacterial photosynthetic membranes.

Photosynthetic reaction centres and membranes are systems of particular interest and are often taken as models to investigate the molecular mechanisms of selected bioenergetic reactions. In this work, a multivariate curve resolution by alternating least squares procedure is detailed for resolution of time-resolved difference FTIR spectra probing the evolution of quinone reduction in photosynthetic membranes from Rhodobacter sphaeroides under photoexcitation. For this purpose, different data sets were acquired in the same time range and spectroscopic domain under slightly different experimental conditions. To enable resolution and provide meaningful results the different data sets were arranged in an augmented matrix. This strategy enabled recovery of three different species despite rank-deficiency conditions. It also results in better definition (identity and evolution) of the contributions. From the resolved spectra, the species have been attributed to: 1. the formation of ubiquinol, more precisely the disappearance of Q/appearance of QH(2); 2. conformational change of the protein in the surrounding biological medium; 3. oxidation of diaminodurene, a redox mediator. Because, moreover, results obtained from augmented data sets strategies enable quantitative and qualitative interpretation of concentration profiles, other effects, for example the consequence of repeated light excitation of the same sample, choice of illumination power, or the number of spectra accumulated could be compared and discussed.

Pharmacological modulation of plaque instability.

Atherosclerotic plaque rupture is promoted by metalloproteinase (MMP)-2 and MMP-9, enzymes that degrade the fibrous cap leading to plaque erosion. MMP biosynthesis is mediated by prostaglandin (PG)E2, the product of cyclooxygenase (COX)-2/inducible PGE synthase (mPGES) activity. We have recently reported the overexpression of COX-2/mPGES-1 in vulnerable plaques as a basis of MMP-mediated plaque instability. Hypercholesterolemia and hypertension are two important risk factors for atherosclerosis. Recent trial showed that statins and AT1 receptor blockers significantly reduce the incidence of cardiovascular events in humans. Since anti-inflammatory effects have been reported in association to therapy with statins or AT1 receptor blockers, in two different studies we hypothesized that these drugs can stabilize atherosclerotic plaques through modulation of COX-2/mPGES-1-dependent MMP biosynthesis. Our data demonstrated the stabilizing effect of atherosclerotic plaques by simvastatin or irbesartan, that is due, at least in part, to the reduction of inflammatory burden and suppression of PGE2-dependent metalloproteinases release.

Novel determinants of plaque instability.

Arachidonic acid metabolism plays an important role in acute ischemic syndromes affecting the coronary or cerebrovascular territory, as reflected by biochemical measurements of eicosanoid biosynthesis and the results of inhibitor trials in these settings. Two cyclooxygenase (COX)-isozymes have been characterized, COX-1 and COX-2, that differ in terms of regulatory mechanisms of expression, tissue distribution, substrate specificity, preferential coupling to upstream and downstream enzymes and susceptibility to inhibition by the extremely heterogeneous class of COX-inhibitors. While the role of platelet COX-1 in acute coronary syndromes and ischemic stroke is firmly established through approximately 20 years of thromboxane metabolite measurements and aspirin trials, the role of COX-2 expression and inhibition in atherothrombosis is substantially uncertain, because the enzyme was first characterized in 1991 and selective COX-2 inhibitors became commercially available only in 1998. In this review, we discuss the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque 'vulnerability,' and the clinical consequences of COX-2 inhibition. Recent studies from our group suggest that variable expression of upstream and downstream enzymes in the prostanoid biosynthetic cascade may represent important determinants of the functional consequences of COX-2 expression and inhibition in different clinical settings.

Enhanced soluble CD40 ligand contributes to endothelial cell dysfunction in vitro and monocyte activation in patients with diabetes mellitus: effect of improved metabolic control.

AIMS/HYPOTHESIS: Inflammation plays a pathogenic role in the development of accelerated atherosclerosis in diabetes. Soluble CD40 ligand (sCD40L) is enhanced in diabetes; however, the molecular mechanisms linking sCD40L to accelerated atherosclerosis in diabetes are still unclear. We tested the hypothesis that sCD40L may be involved in the vascular complications in diabetes and exerts its effect by triggering inflammatory reactions on mononuclear and endothelial cells (ECs). METHODS: We studied 70 patients, 40 with type 2 and 30 with type 1 diabetes, with a history or physical examination negative for cardiovascular disease, and 40 non-diabetic and 30 healthy subjects, matched with the type 2 and type 1 diabetic patients, respectively. Plasma and serum sCD40L, and plasma soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, E-selectin and monocyte chemo-attractant protein-1 (MCP-1) were measured. Adhesion molecules and MCP-1 release, the ability to repair an injury in ECs, and O2- generation in monocytes were analysed in vitro after stimulation with serum from patients or controls. RESULTS: Type 2 and type 1 diabetic patients had significantly higher sCD40L levels than controls. Furthermore, high sCD40L was associated with in vitro adhesion molecules and MCP-1 release, impaired migration in ECs and enhanced O2- generation in monocytes. Improved metabolic control was associated with a reduction of plasma sCD40L by 37.5% in 12 type 1 diabetic patients. Furthermore, elevated sCD40L in diabetic patients was significantly correlated with HbA1c levels. CONCLUSIONS/INTERPRETATION: Upregulation of sCD40L as a consequence of persistent hyperglycaemia in diabetic patients results in EC activation and monocyte recruitment to the arterial wall, possibly contributing to accelerated atherosclerosis development in diabetes.

Relationship between reduced BCL-2 expression in circulating mononuclear cells and early nephropathy in type 1 diabetes.

Microalbuminuria is the earliest clinical evidence of diabetic nephropathy, but the mechanisms linking hyperglycemia and kidney complications are not clear. The aim of this study was to evaluate whether enhanced oxidative stress in patients with microalbuminuria can contribute to diabetic nephropathy development through downregulation of the antiapoptotic gene Bcl-2 that promotes in turn a pro-inflammatory status. We studied 30 patients with type 1 diabetes (15 with and 15 without microalbuminuria) compared to 15 matched healthy controls. Plasma oxidant status, and expression of Bcl-2, activated NF-kB, inducible Nitric Oxide synthase (iNOS), and monocyte chemoattractant protein (MCP)-1 in circulating monocytes were evaluated at baseline and after 8-week oral vitamin E treatment (600 mg b.i.d.). Bcl-2 expression was significantly reduced in microalbuminuric diabetic patients as a consequence of increased oxidant burden secondary to persistent hyperglycemia. Bcl-2 down-regulation was associated with enhanced expression of NF-kB, iNOS and MCP-1, and showed a strong correlation with the albumin excretion rate. Low Bcl-2 expression and high inflammatory status were normalized by vitamin E both in vivo and in vitro. Our study showed that Bcl-2 down-regulation in diabetic patients with poor glycemic control results in the activation of the NF-kB pathway leading to the development of nephropathy. Vitamin E might provide a novel form of therapy for prevention of nephropathy in diabetic patients in which an acceptable glycemic control is difficult to achieve despite insulin therapy.

Factors associated with apoptosis in symptomatic and asymptomatic carotid atherosclerotic plaques.

The aim of this study was to investigate the differences that are present between apoptosis in symptomatic (with symptoms of cerebral ischemic attack) and asymptomatic carotid atherosclerotic plaques. The apoptotic process in macrophages and smooth muscle cells was evaluated. Cellular markers and products of immune cells in symptomatic and asymptomatic atherosclerotic plaque and endoarterectomy specimen were analyzed by immunohistochemistry. No statistically significant differences were present regarding the mean SMC actin-positive area. Using double staining of alpha-smooth muscle actin and TUNEL techniques, the number of smooth muscle cells in apoptosis was statistically higher in symptomatic plaque as compared with asymptomatic plaque. Statistically significant differences (p=0.009) were also found in the CD45-positive cells in the inflammatory infiltrate. The CD68-positive macrophages showed statistically significant differences (p=0.0001). Similarly, the double staining with CD68 and TUNEL revealed that apoptotic macrophages were mainly present in asymptomatic plaques rather than symptomatic plaques. Statistically significant differences (p<0.001) were found in the Bcl-2 expression, with higher values in asymptomatic plaques. Our data showed that the increase of the inflammatory cells contributes to plaque instability and that death due to apoptosis of smooth muscle cells in symptomatic plaques could contribute to their destabilization and explains their tendency to fracture.

The role of nitric oxide in the development of diabetic angiopathy.

Diabetic angiopathy is the main cause of morbidity and mortality in patients with diabetes mellitus. Clinical manifestations and pathophysiological mechanisms of diabetic angiopathy can be traced back to the development of endothelial cell dysfunction with alterations in the eNOS/NO system production or availability as the primum movens in its natural history. Hyperglycemia per se or through the accumulation of AGEs, increased oxidative stress, leading to NOS uncoupling and NO-quenching by excess superoxide and peroxynitrite, and individual genetic background are thought to be responsible for this NO metabolism imbalance. The complex interplay of these mechanisms results in a perturbation of the physiological properties of NO in the maintenance of endothelial homeostasis, such as vasodilation, anticoagulation, leukocyte adhesion, smooth muscle cell proliferation, and antioxidant capacity. Hence, abnormality in NO availability results in generalized accelerated atherosclerosis, hyperfiltration, glomerulosclerosis, tubulointerstitial fibrosis and progressive decline in glomerular filtration rate, and apoptosis and neovascularization in the retina. Indeed, the parallel development of nephropathy, retinopathy, and macroangiopathy may be considered as manifestations of endothelial dysfunction at distinct vascular sites. Given this scenario, intervention targeting any of the pathways involved in the NOS/NO system cascade may prove potential therapeutic targets in the prevention of long-term diabetic complications.

Resveratrol and quercetin down-regulate tissue factor expression by human stimulated vascular cells.

BACKGROUND: Epidemiological studies have shown that consumption of wine reduces the risk of coronary heart disease. Resveratrol and quercetin, two polyphenolic compounds found in grapes and red wine, have been shown to contribute to this protection by exerting several biological properties which could be associated with cardioprotection. Tissue factor (TF), the cellular receptor that initiates blood coagulation, plays a primary role both in hemostasis following tissue injury and in the pathogenesis of atherosclerosis which predisposes to thrombosis. OBJECTIVES: We investigated the role of resveratrol and quercetin on TF expression by endothelial and mononuclear cells (MN). METHODS: Confluent human umbilical vein endothelial cells and MN collected from healthy donors were stimulated with bacterial lipopolysaccharide, interleukin-1beta or tumor necrosis factor-alpha after incubation with increasing concentrations of resveratrol or quercetin. RESULTS: In both cell types, TF activity induced by any agonist was significantly reduced by resveratrol or quercetin in a dose-dependent fashion. Northern blot analysis indicated that resveratrol and quercetin strongly reduce TF mRNA in both cell types. The inhibition of TF mRNA originated from a reduction in nuclear binding activity of the transacting factor c-Rel/p65, which was induced by the agonists and measured by electromobility shift assay. Western blot analysis revealed that the diminished c-Rel/p65 activity was dependent upon inhibition of degradation of the c-Rel/p65 inhibitory protein IkappaBalpha. CONCLUSIONS: These results provide a molecular basis which could help explain the protective activity of red wine against cardiovascular disease.