Nasser Rizk

12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase

The purpose of the current study was to evaluate the effect of 12/15- lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2Akita mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.

iTRAQ-based quantitative protein expression profiling and MRM verification of markers in type 2 diabetes.

The pathogenesis of Type 2 diabetes mellitus (T2DM) is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are noninformative with respect to progression of the disease to other associated pathologies. Thus, predicting the risk and development of T2DM-related complications, such as cardiovascular disease, remains a major challenge. We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of nondiabetic control subjects (n = 76) and patients diagnosed with T2DM (n = 106). In this case-control study, the samples were randomly divided as training and validation data sets. In the first step, iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western blot and QPCR analysis. Functional pathway analysis identified perturbations in lipid and small molecule metabolism as well as pathways that lead to disruption of glucose homeostasis and blood coagulation. These putative biomarkers may be clinically useful for subset stratification of T2DM patients as well as for the development of novel therapeutics targeting the specific pathology.

Adiponectin changes in HCV-Genotype 4: relation to liver histology and response to treatment

Summary.  Recently, attention has been focussed on adiponectin and its changes in different types of chronic liver disease. Its relation to hepatic fibrosis and insulin resistance in post‐hepatitis liver disease is not clear. The aim of this study was to clarify the adiponectin changes in genotype 4 hepatitis C virus (HCV)‐infected patient in relation to liver histology and insulin resistance, and its usefulness as a predictor of hepatic fibrosis and response to treatment. Total adiponectin and its high molecular weight (HMW) form as well as insulin levels were studied in 92 chronic HCV, genotype 4 and 66 healthy control volunteers. Neither total adiponectin (r = 0.101, P = 0.220) nor HMW adiponectin (r = 0.081, P = 0.328) correlated with viral load. Total and not HMW adiponectin was significantly correlated with hepatic fibrosis and inflammation (r = 0.267, P = 0.002, r = 0.278, P < 0.001, respectively).In addition, total adiponectin (r = 0.224, P = 0.002) and HMW adiponectin (r = 0.266, P < 0.0006) significantly correlated with insulin resistance. As fibrosis did not correlate with insulin resistance (r = 0.081, P = 0.204), the correlation between total adiponectin and fibrosis was not mediated by insulin resistance. Multivariable regression analysis, (including pretreatment cases and controls) revealed that total adiponectin was significantly associated with gender, being lower among male subjects (X2 = 13.04, P = 0.0001). The multivariable regression model supported the lack of association between insulin resistance and total adiponectin levels (X2 = 1.88, P = 0.171), while non cirrhotics had significantly lower total adiponectin levels than cirrhotics (X2 = 10.90, P = 0.004) and lower level of inflammation significantly lower total adiponectin levels than more severe inflammation (X2 = 8.95, P = 0.003). Total or HMW adiponectin did not yield receiver operating characteristic (ROC) curves with area under the curve (AUC) >75%, thus the cutoff points have poor sensitivity/specificity as predictors of fibrosis. However, as a predictor of end‐of‐treatment response, the ROC curve of adiponectin index gave yield an AUC = 81.4%. We can conclude that total adiponectin level, in HCV genotype 4 patients, increases with progression of hepatic fibrosis regardless of insulin resistance. Its high molecular form does not have such correlation. The adiponectin changes are not related to viral load, insulin resistance or other demographic data suggesting that this change is histologically related. In spite of this, no adiponectin cutoff level had reasonable sensitivity/specificity for predicting hepatic fibrosis stage, while this may be used as a predictor for antiviral response possibly reflecting improvement in hepatic inflammation post treatment.

Association of adiponectin gene polymorphism (+T45G) with acute coronary syndrome and circulating adiponectin levels.

We investigated the association of adiponectin gene polymorphisms (+T45G and +G276T) and adiponectin levels with acute coronary syndrome (ACS) among Arabs in Qatar. A case–control study was performed in 142 Arab patients with ACS and 122 controls. Genotypes were determined using TaqMan real-time polymerase chain reaction assay. The TT, TG, and GG genotype frequencies of the T45G variant were significantly different among cases and controls (P = .023) but not significant for G276T genotypic frequencies. It was found that only the +45G allele was significantly associated with 3-fold increased risk of ACS (odds ratio = 2.77; 1.03-6.96; P = .043) among patients, using the genetic recessive model. Carriers of GG alleles had significantly lower adiponectin levels compared to TT/TG carriers of T45G in patients with ACS. The present study suggests that only T45G single-nucleotide polymorphism in the adiponectin gene is associated with higher odds for ACS events and has an effect on serum adiponectin levels among Arab populations.

Interleukin-28 and hepatitis C virus genotype-4: Treatment-induced clearance and liver fibrosis

AIM To investigate the association between interleukin-28B (IL28B) genotype and response to treatment and hepatic fibrosis in patients with hepatitis C virus (HCV) genotype 4. METHODS Two hundred and one HCV-genotype 4 patients were included. All patients were treated with Peginterferon alph2a/Ribavirin for 48 wk. End of treatment response (ETR) was defined as loss of detectable serum HCV RNA at the end of treatment. Sustained viral response (SVR) was defined as loss of detectable serum HCV RNA at the end of 24 wk follow up. Genotyping of IL28B rs12979860 was performed using the TaqMan assay. We used logistic regression to estimate the adjusted odds ratio (aOR) and 95%CI. RESULTS The study included 201 HCV-genotype 4 patients. The majority of patients were men (89.6%), with a median age of 47 years, inter-quartile range (40-51). Approximately 62.5% of patients had ETR, and 49.6% had SVR. Individuals who achieved SVR were more likely to be younger (χ(2) = 4.91, P = 0.027), and less likely to have fibrosis (χ(2) = 15.54, P < 0.0001), or inflammation (χ(2) = 7.58, P = 0.006). The genotype distribution of rs12979860 was 36.2%, 49.0% and 14.8% for genotypes CC, CT, and TT, respectively. In these participants, rs12979860 genotype distribution did not differ by gender (P = 0.466), pretreatment viral load (P = 0.600), inflammation (P = 0.435), or fibrosis (P = 0.291). The frequencies of IL28B rs12979860 genotypes were TT (14.8%), CT (49.0%), and CC (36.2%). Compared to rs12979860 genotype TT, aORs (95%CI) for ETR and SVR were: CC genotype, [17.55 (5.34-57.69) and 5.92 (2.09-16.76), respectively]; CT genotype, [5.15 (1.80-14.78) and 2.48 (0.94-6.52), respectively]. In the current study, the patients who did not achieve ETR or SVR had a lower prevalence of rs12979860 CC (17.4% and 23.3%, respectively) than individuals who had ETR or SVR (47.9% and 47.2%, respectively). Individuals with rs12979860 CC genotype had approximately 6 times the odds of SVR compared to individuals with TT genotype (aOR = 5.92; 95%CI: 2.09-16.76). Similarly, patients with CT genotype had SVR more often than patients with TT genotype (aOR = 2.48; 95%CI: 0.94-6.52). Carrying at least one copy of the C allele (genotypes CT and CC) had almost 8 times the probability of ETR compared to those with genotype rs12979860 TT (aOR = 7.87; 95%CI: 2.84-21.82), and approximately 3 times the odds of SVR compared to those with genotype rs12979860 TT (aOR = 3.46; 95%CI: 1.37-8.74). In addition, data were consistent with a significant gene-dose relationship (aOR = 4.05/allele; 95%CI: 2.27-7.22). The association between rs12979860 genotype and SVR was similar among those who achieved and those who did not achieve SVR. CONCLUSION In HCV-genotype 4 patients, rs12979860 is a sensitive predictor of viral clearance, independent of viral load, age, gender or fibrosis, with no similar relation to severity of fibrosis.

Elevated serum leptin levels in patients with acute myocardial infarction; correlation with coronary angiographic and echocardiographic findings

BackgroundTo assess the relationship between serial serum leptin levels in patients with acute myocardial infarction (AMI) who received thrombolysis and the degree of coronary atherosclerosis, coronary reperfusion, echocardiographic findings, and clinical outcome. 51 consecutive patients presenting with AMI were studied. Clinical characteristics including age, sex, body mass index (BMI) and cardiovascular risk factors were recorded. Serial serum leptin levels at the time of admission and subsequently at 0, 6, 12, 24, 36, 60 hours afterwards were obtained. Coronary angiography was performed in 34 patients; the relation between serum leptin levels and evidence of coronary reperfusion as well as the extent of coronary atherosclerosis according to the coronary artery surgery study classification (CASS) were evaluated. Echocardiographic evaluation was performed in all patients. 36 matched patients were enrolled as control group who had serum leptin level 9.4 ± 6.5 ng/ml.ResultsThe patients mean age was 50.5 ± 10.6 years. There were 47 males and 3 females. 37.1% were diabetics, 23.5% were hypertensive, 21.6% were dyslipidemic and 22.7% were obese (BMI ≥ 30). Leptin concentrations (ng/ml) increased and peaked at the 4th sample (36 hrs) after admission (mean ± SD) sample (1) =9.55 ± 7.4, sample (2) =12.9 ± 8.4, sample (3) =13.8 ± 10.4, sample (4) =18.9 ± 18.1, sample (5) =11.4 ± 6.5, sample (6) =10.8 ± 8.9 ng/ml. There was a significant correlation between serum leptin and BMI (r = 0.342; p = 0.03). Leptin levels correlated significantly to creatine kinase level on the second day (r = 0.43, p ≤ 0.01). Significant correlation of mean serum leptin with the ejection fraction (P < 0.05) was found. No difference in timing of peak serum leptin between patients who achieved coronary reperfusion vs. those who did not (p = 0.8). There was a trend for an increase in the mean serum leptin levels with increasing number of diseased vessels. There was no correlation between serum leptin levels and outcome neither during the hospitalization nor at 9 months follow up.ConclusionSerum leptin levels increase after myocardial infarction. Serum leptin level may be a predictor of the left ventricular ejection fraction and the degree of atherosclerosis but not of coronary reperfusion.

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase.

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2−/− mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.

Total and high molecular weight adiponectin in patients with coronary artery disease.

Background Serum adiponectin inversely correlates with the extent of coronary artery disease (CAD). Objectives To investigate the clinical significance of measuring high molecular weight (HMW) adiponectin in addition to total adiponectin from different samples (peripheral veins and ostia of coronary arteries) in patients with CAD. Methods We studied 134 patients; 57 with acute coronary syndrome (ACS), 44 with stable angina and 33 healthy patients. Total and HMW adiponectin were measured in the coronary ostia and peripheral veins simultaneously. Venous levels of lipid profile, C-reactive protein, tumour necrosis factor-α, interleukin-6 and insulin were measured. Results Mean levels of HMW adiponectin in the coronary ostia were significantly correlated with the venous levels of total (r = 0.33, P = 0.002) and HMW (r = 0.37, P = 0.002) adiponectin. Mean levels of total and HMW adiponectin were lower in CAD patients versus controls (6.9 ± 0.5 versus 7.9 ± 0.7 μg/ml, P = 0.3 and 1.9 ± 0.2 versus 3.1 ± 0.3 μg/ml, P = 0.003). In the coronary ostia, levels of HMW adiponectin were higher in ACS than those with stable angina (1.1 ± 0.1 versus 0.8 ± 0.1 μg/ml, P = 0.2). In patients with CAD, levels of ostial adiponectin (total and HMW) were significantly lower in diabetic than non-diabetic patients (6.5 ± 0.8 versus 9.4 ± 1.1 μg/ml, P = 0.04 and 0.8 ± 0.12 versus 1.2 ± 0.1 μg/ml, P = 0.03). Mean levels of venous adiponectin (total and HMW) were non-significantly lower in diabetic patients (5.9 ± 0.7 versus 7.7 ± 0.7, P = 0.12 and 1.8 ± 0.4 versus 1.9 ± 0.1, P = 0.8). Conclusion Measurement of HMW adiponectin and its ratio to total adiponectin may be a better marker for CAD than total adiponectin levels. Ostial levels of adiponectin in ACS may indicate re-distribution of adiponectin molecules towards the acute lesions. The low HMW adiponectin levels in diabetes mellitus may in part explain the worse outcome of CAD in diabetics.

Hypothalamic expression of neuropeptide-Y in the New Zealand obese mouse

OBJECTIVE: Increased levels of hypothalamic neuropeptide-Y (NPY) are thought to contribute to the manifestation of the obese phenotype. The aim of this study was to characterize the interactions between leptin, insulin and NPY in the pathogenesis of polygenic obesity.DESIGN AND METHODS: A polygenic obese animal model, the New Zealand obese mouse (NZO) and its age-matched control, was used to assess the hypothalamic mRNA expression of NPY, the insulin receptor (IR) and the leptin receptor (Ob-Rb), by semiquantitative polymerase chain reaction. Experiments were performed early (at eight weeks old) and late (at 40 weeks old) in the life of these animals.RESULTS: Serum glucose was significantly elevated in obese mice. Serum insulin levels were not different between obese and lean mice, whereas serum leptin levels were significantly elevated in obese mice and increased continuously during life [reaching extremely high values at 40 weeks (41.5±4.1 vs 3.4±0.25 ng/ml for obese and lean, respectively). The hypothalamic expression of NPY mRNA was significantly higher in NZO mice compared to controls at both eight weeks (2.3-fold) and 40 weeks (1.9-fold), respectively, whereas expression of IR and Ob-Rb remained unaffected.CONCLUSION: Increased hypothalamic expression of NPY due to leptin resistance, may be involved in the development of polygenic obesity. Unchanged Ob-Rb expression suggests that either a defective hypothalamic uptake or defects in Ob-Rb signalling underly this process.

Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids

We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.