Endothelial Metabolism Research Articles

Endothelial cell metabolism and implications for cancer therapy

Tumour tissue is characterised by fluctuating oxygen concentrations, decreased nutrient supply, and acidic pH. The primarily glycolytic metabolism of tumour cells contributes to this, with increased glucose consumption and increased lactate secretion. Endothelial cells are particularly challenged when recruited towards the tumour metabolic environment. They are required to proliferate and form functional networks in order to establish continuous blood flow. Considering that deregulated metabolism is an emerging hallmark of cancer and target of tumour therapy, it is of importance to incorporate the current knowledge about how the tumour metabolic environment, as a therapy target, can affect endothelial cell metabolism and the angiogenic response. Recent studies have shown differences in metabolic pathways in endothelial cells compared with other normal or tumour tissues. Therefore, we have reviewed relevant literature on endothelial metabolism and the response to angiogenic activation in conditions of metabolic stress.

The influence of high glucose on the aerobic metabolism of endothelial EA.hy926 cells

The endothelium is considered to be relatively independent of the mitochondrial energy supply. The goals of this study were to examine mitochondrial respiratory functions in endothelial cells and isolated mitochondria and to assess the influence of chronic high glucose exposure on the aerobic metabolism of these cells. A procedure to isolate of bioenergetically active endothelial mitochondria was elaborated. Human umbilical vein endothelial cells (EA.hy926 line) were grown in medium containing either 5.5 or 25 mM glucose. The respiratory response to elevated glucose was observed in cells grown in 25 mM glucose for at least 6 days or longer. In EA.hy926 cells, growth in high glucose induced considerably lower mitochondrial respiration with glycolytic fuels, less pronounced with glutamine, and higher respiration with palmitate. The Crabtree effect was observed in both types of cells. High glucose conditions produced elevated levels of cellular Q10, increased ROS generation, increased hexokinase I, lactate dehydrogenase, acyl-CoA dehydrogenase, uncoupling protein 2 (UCP2), and superoxide dismutase 2 expression, and decreased E3-binding protein of pyruvate dehydrogenase expression. In isolated mitochondria, hyperglycaemia induced an increase in the oxidation of palmitoylcarnitine and glycerol-3-phosphate (lipid-derived fuels) and a decrease in the oxidation of pyruvate (a mitochondrial fuel); in addition, increased UCP2 activity was observed. Our results demonstrate that primarily glycolytic endothelial cells possess highly active mitochondria with a functioning energy-dissipating pathway (UCP2). High-glucose exposure induces a shift of the endothelial aerobic metabolism towards the oxidation of lipids and amino acids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-012-1156-1) contains supplementary material, which is available to authorized users.

Endothelial Nitric Oxide Synthase Expression in Postmenopausal Women: A Sex-Specific Risk Factor in Coronary Surgery

After coronary artery bypass graft surgery, older women have less favorable clinical outcome and lower conduit patency compared with men. This less favorable outcome can be in part ascribed to impaired endothelium-derived nitric oxide (eNOS) production. This study evaluated endothelial nitric oxide synthase expression in internal mammary artery from postmenopausal women undergoing coronary artery bypass graft surgery. Internal mammary artery segments were obtained from 20 postmenopausal woman and 20 matched male patients. Twenty more segments from younger patients were used as controls. Expression of eNOS messenger RNA in internal mammary artery endothelial cells were evaluated by polymerase chain reaction and real-time quantitative reverse transcription polymerase chain reaction. The eNOS protein level was assayed by Western blot. Vascular dynamics of specimens were evaluated by organ chamber methodology. In postmenopausal women, the band of messenger RNA for eNOS was reduced by 37.4% and by 25.2%, respectively, compared with matched men and the control group (62.6%±4.8% versus 74.8%±5.3%, p<0.001). In comparison with the control group lane, the eNOS protein immunoreactive band was 44.2% decreased in postmenopausal women and 34.5% decreased in matched men, and was significantly decreased in postmenopausal women as compared with matched men (55.8%±4.6% versus 65.5%±5.2%, p<0.001). Nitric oxide-mediated vasomotor dynamics were consistent with reduced eNOS production. Internal mammary artery endothelial cells from women after menopause undergoing coronary surgery have impaired expression of messenger RNA for eNOS and reduced eNOS levels. Reduced bioactivity of nitric oxide translates into impaired endothelial metabolism that could contribute to worse surgical outcome.

Endothelial Lipase Is Localized to Follicular Epithelial Cells in the Thyroid Gland and Is Moderately Expressed in Adipocytes

Endothelial lipase (EL), a member of the triglyceride lipase gene family, has been shown to be a key player in HDL metabolism. Northern blots revealed that EL was highly expressed in endothelium, thyroid, lung, placenta, liver, and testis. In liver and adrenal gland, EL protein was localized with vascular endothelial cells but not parenchymal cells. EL was shown to be upregulated in tissues such as atherosclerotic plaque where it was located in macrophages, endothelial cells, and medial smooth muscle cells. The purpose of this study was to investigate the cellular localization of EL in thyroid and other tissues where EL is known to be expressed. Besides its presence in vascular endothelial and smooth muscle cells, EL protein was detected in the epithelial cells that line the follicles within the thyroid gland. EL-specific immunostaining was also found near the cell surface as well as in the cytoplasm of adipocytes. Using immunoblots, EL expression was confirmed in cultured human omental and subcutaneous adipocytes. EL expression, however, was not found in preadipocytes. These findings suggest that EL plays a role in thyroid and adipocyte biology in addition to its well-known role in endothelial function and HDL metabolism.

Chronic Inhibition of cGMP Phosphodiesterase 5A Improves Diabetic Cardiomyopathy

Background— cGMP phosphodiesterase type 5 protein is upregulated in myocardial hypertrophy. However, it has never been ascertained whether phosphodiesterase type 5 inhibition exerts an antiremodeling effect in nonischemic heart disease in humans. We explored the cardioreparative properties of a selective phosphodiesterase type 5 inhibitor, sildenafil, in a model of diabetic cardiomyopathy. Methods and Results— Fifty-nine diabetic men (60.3±7.4 years) with cardiac magnetic resonance imaging consistent with nonischemic, nonfailing diabetic cardiomyopathy (reduced circumferential strain [σ], −12.6±3.1; increased left ventricular [LV] torsion [θ], 18.4±4.6°; and increased ratio of LV mass to volume, 2.1±0.5 g/mL) were randomized to receive sildenafil or placebo (100 mg/d). At baseline, the metabolic indices were correlated with torsion, strain, N-terminal pro–B-type natriuretic peptide, vascular endothelial growth factor, monocyte chemotactic protein-1, and blood pressure. After 3 months, sildenafil produced a significant improvement compared with placebo in LV torsion (Δθ: sildenafil, −3.89±3.11° versus placebo, 2.13±2.35°; P &lt;0.001) and strain (Δσ: sildenafil, −3.30±1.86 versus placebo, 1.22±1.84; P &lt;0.001). Sildenafil-induced improvement of LV contraction was accompanied by consistent changes in chamber geometry and performance, with a 6.5±11 improvement in mass-to-volume ratio over placebo ( P =0.021). Monocyte chemotactic protein-1 and transforming growth factor-β were the only markers affected by active treatment (Δmonocyte chemotactic protein-1: −75.30±159.28 pg/mL, P =0.032; Δtransforming growth factor-β: 5.26±9.67 ng/mL, P =0.009). No changes were found in endothelial function, afterload, or metabolism. Conclusions— The early features of diabetic cardiomyopathy are LV concentric hypertrophy associated with altered myocardial contraction dynamics. Chronic phosphodiesterase type 5 inhibition, at this stage, has an antiremodeling effect, resulting in improved cardiac kinetics and circulating markers. This effect is independent of any other vasodilatory or endothelial effects and is apparently exerted through a direct intramyocardial action. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00692237.

Abstract 2332: The glucose analog 2-DG selectively inhibits AKT and ERK in endothelial cells via interference of N-linked glycosylation and induces endothelial cell apoptosis in vitro and in vivo.

Abstract BACKGROUND: As every step in tumor angiogenesis is an energy consuming process, targeting endothelial (EC) glucose metabolism is a rational strategy for angiogenesis inhibition. We recently showed that interference with endothelial glucose metabolism by the glucose analog 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis in vitro and in vivo, at concentrations lower than those required to induce tumor cell cytotoxicity. The objectives of the current study are to further understand the basis for EC sensitivity to 2-DG in vitro and in vivo. METHODS: Time dependent effects of 2-DG were studied on EC tube formation at 4, 8 and 18 hrs. Apoptosis was assessed by the TUNEL assay at 24, 48 and 72 hours. The effects of low antiangiogenic concentrations (0.6 mM) of 2-DG on ERK, AKT and mTOR pathways were assessed in endothelial (HUVEC) and cancer (MDA-MB-231, HT-29, 786-0) cells by western blot. The in vivo effects of intraocular 2-DG administration on newly forming (CD-105) and total (lectin) tumor vessels were evaluated in the LHBETATAG retinoblastoma model. In vivo EC apoptosis after 2-DG treatment was assessed by TUNEL/CD31 staining of treated tumors. RESULTS: Inhibition of HUVEC capillary formation by 2-DG occurred as early as 8 hours after treatment, and the effects were clear at 18 hours compared to controls. EC apoptosis was non-significantly increased at 24 hours, and was prominent at 48 and 72 hours. Antiangiogenic 2-DG concentrations (0.6 mM) significantly inhibited phosphorylation of Akt473, S6 and Erk at 24 hours. On the other hand, 2-DG did not affect these pathways in MDA-MB-231 or HT-29 cells, while they occurred at a lower level in 786-0 cells. Mannose successfully reversed AKT, S6 and ERK inhibition only in HUVECs, but not in 786-0 cells, suggesting that the EC selective inhibition of ERK and AKT are mediated by inhibition of N-linked glycosylation, while other mechanisms mediate the effects in cancer cells. Importantly, these effects were 2-DG specific, as other glucose analogs (FDG) and the glycolytic inhibitor oxamate did not significantly affect ERK or AKT. Intraocular 2-DG administration decreased tumor microvasculature, and importantly, induced EC apoptosis, as demonstrated by TUNEL+/CD 31 co-localization. CONCLUSIONS: Our data indicate that in angiogenic ECs, 2-DG's inhibition of N-linked glycosylation targets critical proliferation and survival pathways. 2-DG mediated Inhibition of AKT and ERK pathways may explain the significant endothelial sensitivity cytotoxic and pro-apoptotic effects of 2-DG in vitro and in vivo, and further support targeting EC glucose metabolism as a viable strategy for angiogenesis inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2332. doi:1538-7445.AM2012-2332

Changes on the expression of ectopic inhibitor protein subunit of f1-f0 atp synthase (if1) in osteoarthritis

Purpose: Osteoarthritis (OA) is a disease characterized by the cartilage degradation, and involves the entire joint, including the subchondral bone, synovial membrane, menisci and periarticular structures. The inflammation and angiogenesis occurs at the osteochondral junction as well as within the osteoarthritic synovium providing a potential mechanism for the genesis of pain in knee OA. Menisci play a role in the OA disease while is injured and there is a strong association between meniscal damage/degenerative tears and cartilage loss with pathological changes extracellular matrix degeneration with secretion of cytokines and growth factors. A few studies have investigated on meniscal cells in the process of OA and angiogenesis. Previous studies have demonstrated the ectopic localization of mitochondrial F1-F0 ATP synthase as a target for anti-angiogenesis intervention. It has been shown that β subunits of ATP synthase bind angiostatin on cell surface of endothelial cells (EC) and mediate down-regulation of endothelial cell proliferation and migration. Some data suggest that angiostatin inhibits vascularization by suppression of endothelial ATP metabolism regulating vascular physiology. Angiostatin blocks IF1 subunit binding at the same binding site on ATP synthase and abolishes its ability to conserve ATP. We demonstrated the distribution of IF1 subunit on OA knee joint and evaluate the regulation of the ectopic expression of IF1 by IL-1β and TNF-α. Methods: The identification of the IF1 subunit of ATP synthase was using flow cytometry and confocal microscopy using rabbit polyclonal antibodies on cultured human menisci cells. The distribution was detected by immunofluorescence using the same antibodies. Results: The fluorescence microscopy results showed a superficial cartilage and meniscus marker distribution of IF1 in control whereas the IF1 signal was localized on subchondral bone and hypertrofic chondrocytes in OA samples. Human menisci cells were cultivated after digestion with collagenase 2 and incubated with polyclonal antibody against IF1 subunit to show a plasma membrane location in control and different distribution on OA samples. Histograms of flow cytometry show changes on the IF1 membrane expression under TNF-α and IL-1β. There is low expression of ectopic IF-1 under IL-1β effect. Conclusions: We showed that the distribution of the IF1 subunit of ATP synthase on joint knee change under OA and this expression is regulated by IL-1β and TNF-α.

CLINICAL SIGNIFICANCE OF INSULIN RESISTANCE SYNDROME IN DEVELOPMENT OF ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH PRIMARY GOUT

Endothelial function has been studied in 175 males with primary gout. It has been established that incidence of endothelial dysfunction in patients with primary gout was associated with the clinical course and was marked most of all in patients who had a chronic gout. The correlations found between the parameters of endothelial dysfunction and impaired carbohydrate metabolism suggest close link and coordination between the above processes which contribute greatly to development and progress of atherosclerosis in such patients.

Period2 gene mutant mice show compromised insulin-mediated endothelial nitric oxide release and altered glucose homeostasis

Period2 (Per2) is an important component of the circadian clock. Mutation of this gene is associated with vascular endothelial dysfunction and altered glucose metabolism. The aim of this study is to further characterize whole body glucose homeostasis and endothelial nitric oxide (NO) production in response to insulin in the mPer2Brdm1 mice. We show that mPer2Brdm1 mice exhibit compromised insulin receptor activation and Akt signaling in various tissues including liver, fat, heart, and aortas with a tissue-specific heterogeneous diurnal pattern, and decreased insulin-stimulated NO release in the aortas in both active and inactive phases of the animals. As compared to wild type (WT) mice, the mPer2Brdm1 mice reveal hyperinsulinemia, hypoglycemia with lower fasting hepatic glycogen content and glycogen synthase level, no difference in glucose tolerance and insulin tolerance. The mPer2Brdm1 mice do not show increased predisposition to obesity either on normal chow or high fat diet compared to WT controls. Thus, mice with Per2 gene mutation show altered glucose homeostasis and compromised insulin-stimulated NO release, independently of obesity.

Individualized therapy of HHT driven by network analysis of metabolomic profiles

BackgroundHereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease with a varying range of phenotypes involving abnormal vasculature primarily manifested as arteriovenous malformations in various organs, including the nose, brain, liver, and lungs. The varied presentation and involvement of different organ systems makes the choice of potential treatment medications difficult.ResultsA patient with a mixed-clinical presentation and presumed diagnosis of HHT, severe exertional dyspnea, and diffuse pulmonary shunting at the microscopic level presented for treatment. We sought to analyze her metabolomic plasma profile to assist with pharmacologic treatment selection. Fasting serum samples from 5 individuals (4 healthy and 1 with HHT) were metabolomically profiled.A global metabolic network reconstruction, Recon 1, was used to help guide the choice of medication via analysis of the differential metabolism between the patient and healthy controls using metabolomic data. Flux Balance Analysis highlighted changes in metabolic pathway activity, notably in nitric oxide synthase (NOS), which suggested a potential link between changes in vascular endothelial function and metabolism. This finding supported the use of an already approved medication, bevacizumab (Avastin). Following 2 months of treatment, the patient's metabolic profile shifted, becoming more similar to the control subject profiles, suggesting that the treatment was addressing at least part of the pathophysiological state.ConclusionsIn this 'individualized case study' of personalized medicine, we carry out untargeted metabolomic profiling of a patient and healthy controls. Rather than filtering the data down to a single value, these data are analyzed in the context of a network model of metabolism, in order to simulate the biochemical phenotypic differences between healthy and disease states; the results then guide the therapy. This presents one approach to achieving the goals of individualized medicine through Systems Biology and causal models analysis.

Combination of nitric oxide stimulation with high-dose 18F-FDG promotes apoptosis and enhances radiation therapy of endothelial cells

High-dose (18)F-FDG can provide targeted nuclear therapy of cancer. Endothelial cell injury is a key determinant of tumor response to radiotherapy. Here, we tested the hypothesis that activation of endothelial cell glycolytic metabolism with nitric oxide can enhance the therapeutic effect of high-dose (18)F-FDG. Calf pulmonary artery endothelial (CPAE) cells were treated with graded doses of (18)F-FDG. Glycolysis was stimulated by 24 h of exposure to the nitric oxide donor, sodium nitroprusside (SNP). Cell viability was assessed by MTT and clonogenic assays. Apoptosis was evaluated by ELISA of cytosolic DNA fragments and Western blots of cleaved caspase-3. SNP stimulation (0.1 and 1 mM) augmented CPAE cell (18)F-FDG uptake to 2.6- and 4.6-fold of controls without adverse effects. Treatment with 333 μCi/ml (18)F-FDG alone reduced viable cell number to 35.4% of controls by Day 3. Combining 0.1 mM SNP stimulation significantly enhanced the killing effect, reducing cell numbers to 19.2% and 39.2% of controls by 333 and 167 μCi/ml of (18)F-FDG, respectively. (18)F-FDG also suppressed clonogenic survival to 80.8% and 43.2% of controls by 83 and 167 μCi/ml, which was again intensified by SNP to 59.7% and 21.1% of controls. The cytotoxic effect of (18)F-FDG was attributed to induction of apoptosis as shown by increased cytosolic fragmented DNA and cleaved caspase-3 levels (26.4% and 30.7% increases by 167 μCi/ml). Combining SNP stimulation significantly increased both of these levels to 1.8-fold of control cells. High-dose (18)F-FDG combined with nitric oxide-stimulated glycolysis is an effective method to inhibit endothelial cell survival and promote apoptosis. These results suggest a potential role of this strategy for targeted radiotherapy of angiogenic vasculature.

Induction of Oxidative Stress and Human Leukocyte/Endothelial Cell Interactions in Polycystic Ovary Syndrome Patients with Insulin Resistance

Insulin resistance is a feature of polycystic ovary syndrome (PCOS) and is related to mitochondrial and endothelial function. We tested whether hyperandrogenic insulin-resistant women with PCOS, who have an increased risk of vascular disease, display impaired leukocyte-endothelium interactions, and mitochondrial dysfunction. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 43 lean reproductive-age women with PCOS and 39 controls subjects. We evaluated anthropometric and metabolic parameters, adhesion molecules, and interactions between leukocytes and human umbilical vein endothelial cells. Mitochondrial function was studied by assessing mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels (GSH), and the oxidized glutathione (GSSG)/GSH ratio in polymorphonuclear cells. Impairment of mitochondrial function was observed in the PCOS patients, evident in a decrease in oxygen consumption, an increase in reactive oxygen species production, a decrease in the GSH/GSSG ratio and GSH levels, and an undermining of the membrane potential. PCOS was related to a decrease in polymorphonuclear cell rolling velocity and an increase in rolling flux and adhesion. Increases in IL-6 and TNFα and adhesion molecules (vascular cell adhesion molecule-1 and E-selectin) were also observed. This study supports the hypothesis of an association between insulin resistance and an impaired endothelial and mitochondrial oxidative metabolism. The evidence obtained shows that the inflammatory state related to insulin resistance in PCOS induces a leukocyte-endothelium interaction. These findings may explain the increased risk of vascular disease in women with PCOS.

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism

Kinin B1 and B2 receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B1 (B1−/−) and B2 receptor (B2−/−) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B1−/− and B2−/− in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B1−/− and B2−/−. In contrast, in the presence of l-arginine, Ca2+ and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B1−/− and B2−/−. The present study demonstrated that targeted deletion of B1 or B2 receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B1−/− and B2−/− vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.

Preclinical Evaluation Methods for Screening of Anti-Atherosclerotic Drugs: An Overview

Atherosclerosis is a disease of large and medium-sized muscular arteries and is characterized by endothelial dysfunction, vascular inflammation, and the buildup of lipids, cholesterol, calcium, and cellular debris within the intima of the vessel wall. This buildup results in plaque formation, vascular remodeling, acute and chronic luminal obstruction, abnormalities of blood flow and diminished oxygen supply to target organs. Vasomotor function, the thrombogenicity of the blood vessel wall, the state of activation of the coagulation cascade, the fibrinolytic system, smooth muscle cell migration and proliferation, and cellular inflammation are complex and interrelated biological processes that contribute to atherogenesis and the clinical manifestations of atherosclerosis. Elevated serum levels of low-density lipoprotein cholesterol overwhelm the antioxidant properties of the healthy endothelium and result in abnormal endothelial metabolism of this lipid moiety. Oxidized low-density lipoprotein is capable of a wide range of toxic effects and cell/vessel wall dysfunctions that are characteristically and consistently associated with the development of atherosclerosis. Detail study of atherosclerosis can be done by using various animal models. Animals different species mainly use for screening methods are mice, rats, rabbits, squil, hamsters, guinea pig. Various animal models are hyperlipidemic model, hypercholestermic model, hypolipidemic model, hereditary hypercholestermic model hereditary hyper lipidemic model, transgenic model. These models are used to observed effect of drug on diseased animal and find out various drugs for treatment of atherosclerosis disease.

Effects of obstructive sleep apnea on serum brain-derived neurotrophic factor protein, cortisol, and lipid levels

Obstructive sleep apnea (OSA) is a sleep-disordered breathing leading to vascular endothelial cells dysfunction, cognitive impairment, and abnormal lipid metabolism. serum brain-derived neurotrophic factor (BDNF) protein, cortisol, and lipid levels in OSA were investigated. All middle-aged subjects including healthy individuals without signs and symptoms of apnea-hypopnea and ear nose throat (ENT) outpatients were randomly recruited and screened by overnight polysomnogram (PSG). Apnea-hypopnea index (AHI) was used as a criteria to determine subjects to enroll in this program. According to AHI, they were separated into control and OSA groups. A group of 39 OSA patients (AHI ≥ 10 events/h) and 24 control subjects (AHI < 5 events/h) were selected. Serum BDNF protein was analyzed by enzyme-linked immunosorbent assay (ELISA) from venous blood collection at 8:00 a.m. following PSG. Serum cortisol was assayed by enzyme-chemiluminescense immuno assay (ECLIA). Serum lipid profile levels were determined by enzymatic colorimetric and homogeneous method. Characteristics of OSA patients and control groups including gender, age, AHI, body weight, height, and BMI showed significant differences. Serum BDNF protein, cortisol, triglyceride, and total cholesterol levels in OSA and control groups were not significantly different. High density lipoprotein-cholesterol (HDL-c) in OSA was significantly lower than that of control (p = 0.008) while low density lipoprotein-cholesterol (LDL-c) was significantly higher than that of control (p = 0.04). OSA had no significant effect on serum BDNF, cortisol, triglyceride, or total cholesterol levels while LDL-c and HDL-c levels in OSA patients compared to control were significantly different at p = 0.04, and p = 0.008, respectively.

Pattern of P450 expression at the human blood–brain barrier: Roles of epileptic condition and laminar flow

P450 enzymes (CYPs) play a major role in hepatic drug metabolism. It is unclear whether these enzymes are functionally expressed by the diseased human blood-brain barrier (BBB) and are involved in local drug metabolism or response. We have evaluated the cerebrovascular CYP expression and function, hypothesizing possible implication in drug-resistant epilepsy. CYP P450 transcript levels were assessed by cDNA microarray in primary endothelial cultures established from a cohort of brain resections (n = 12, drug-resistant epilepsy EPI-EC and aneurism domes ANE-EC). A human brain endothelial cell line (HBMEC) and non-brain endothelial cell line (HUVEC) were used as controls. The effect of exposure to shear stress on CYP expression was evaluated. Results were confirmed by Western blot and immunohistochemistry on brain specimens. Endothelial drug metabolism was assessed by high performance liquid chromatography (HPLC-UV). cDNA microarray showed the presence of CYP enzymes in isolated human primary brain endothelial cells. Using EPI-EC and HBMEC we found that CYP mRNA levels were significantly affected by exposure to shear stress. CYP3A4 protein was overexpressed in EPI-EC (290 ± 30%) compared to HBMEC and further upregulated by shear stress exposure. CYP3A4 was increased in the vascular compartment at regions of reactive gliosis in the drug-resistant epileptic brain. Metabolism of carbamazepine was significantly elevated in EPI-EC compared to HBMEC. These results support the hypothesis of local drug metabolism at the diseased BBB. The direct association between BBB CYP enzymes and the drug-resistant phenotype needs to be further investigated.

Estradiol-17β Stimulates Specific Receptor and Endogenous Nitric Oxide-Dependent Dynamic Endothelial Protein S-Nitrosylation: Analysis of Endothelial Nitrosyl-Proteome

Covalent adduction of a nitrosyl group to cysteines [S-nitrosylation (S-NO)] is emerging as a key route for nitric oxide (NO) to directly modulate protein functions. Here, we studied the effects of estrogens on endothelial protein S-NO and analyzed the nitrosyl-proteomes by biotin/CyDye switch technique combined with two-dimensional fluorescence difference gel electrophoresis and identified nitrosoproteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Estradiol-17beta (E2) rapidly stimulated protein S-NO in human umbilical vein endothelial cells, maximizing within 10- to 30-min post-E2 (10 nm) exposure. E2-BSA also rapidly stimulated protein S-NO. Both E2 and E2-BSA-induced protein S-NO was blocked by ICI 182,780 and N-nitro-l-arginine-methylester. Human umbilical vein endothelial cells expressed estrogen receptor (ER)alpha and ERbeta; both seemed to be required for E2 stimulation of protein S-NO because: 1) neither ERalpha or ERbeta agonist alone, but their combination, stimulated protein S-NO; and 2) either ERalpha or ERbeta antagonist blocked E2-induced protein S-NO. Numerous nitrosoproteins (spots) were observed on two-dimensional fluorescence difference gel. One hundred spots of interest were picked up; 58 were identified and, of which 15 were novel nitrosoproteins, 28 were up-regulated, 11 were decreased, and the rest were unchanged by E2. Pathway analysis suggested that nitrosoproteins are involved in regulating various endothelial functions, including apoptosis, cell structure and metabolism, redox homeostasis, etc. Thus, estrogens stimulate dynamic endothelial protein S-NO via mechanisms linked to specific ERs possibly on the plasma membrane and endogenous NO. These findings signify a critical next step for the understanding of the biological targets of enhanced NO production by estrogens.

Regulation of arginine transport and metabolism by Protein Kinase Cα in endothelial cells: stimulation of CAT2 transporters and arginase activity

Endothelial metabolism of arginine plays a key role in vascular homeostasis. While it is documented that the availability of extracellular arginine is critical for nitric oxide synthesis by eNOS, little is known about the relationships existing between arginine transport and the activity of arginase, the enzyme responsible for the production of ornithine and urea. The present study aims to characterize the role of PKC in the regulation of arginine transport and metabolism by human umbilical vein (HUVEC) and aortic (HAEC) endothelial cells. The results obtained demonstrate that the activation of PKCα by phorbol esters or thymeleatoxin causes a transient increase of arginine transport through system y+, referable to the induction of SLC7A2 mRNAs and to the increased expression of CAT2 transporters. PKCα-dependent stimulation of arginine transport requires the activation of MEK/ERK1/2 cascade, which leads to the stimulation of AP-1 and to the consequent induction of CAT2 expression. In parallel, PKCα activation also increases arginase expression and activity and promotes eNOS phosphorylation, resulting in decreased NO production. It is concluded that the activation of PKCα stimulates arginine entry in human endothelial cells and shifts the metabolism of the cationic amino acid from NO synthesis to arginase-dependent production of ornithine and urea. This metabolic deviation may contribute to the endothelial dysfunction associated with conditions of PKC overactivity.

Role of Dimethylarginine Dimethylaminohydrolases in the Regulation of Endothelial Nitric Oxide Production

Reduced NO is a hallmark of endothelial dysfunction, and among the mechanisms for impaired NO synthesis is the accumulation of the endogenous nitric-oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Free ADMA is actively metabolized by the intracellular enzyme dimethylarginine dimethylaminohydrolase (DDAH), which catalyzes the conversion of ADMA to citrulline. Decreased DDAH expression/activity is evident in disease states associated with endothelial dysfunction and is believed to be the mechanism responsible for increased methylarginines and subsequent ADMA-mediated endothelial nitric-oxide synthase impairment. Two isoforms of DDAH have been identified; however, it is presently unclear which is responsible for endothelial ADMA metabolism and NO regulation. The current study investigated the effects of both DDAH-1 and DDAH-2 in the regulation of methylarginines and endothelial NO generation. Results demonstrated that overexpression of DDAH-1 and DDAH-2 increased endothelial NO by 24 and 18%, respectively. Moreover, small interfering RNA-mediated down-regulation of DDAH-1 and DDAH-2 reduced NO bioavailability by 27 and 57%, respectively. The reduction in NO production following DDAH-1 gene silencing was associated with a 48% reduction in l-Arg/ADMA and was partially restored with l-Arg supplementation. In contrast, l-Arg/ADMA was unchanged in the DDAH-2-silenced cells, and l-Arg supplementation had no effect on NO. These results clearly demonstrate that DDAH-1 and DDAH-2 manifest their effects through different mechanisms, the former of which is largely ADMA-dependent and the latter ADMA-independent. Overall, the present study demonstrates an important regulatory role for DDAH in the maintenance of endothelial function and identifies this pathway as a potential target for treating diseases associated with decreased NO bioavailability.

Hyperglycemia attenuates angiogenic capability of survivin in endothelial cells

Survivin, an anti-apoptotic protein, can be induced by hypoxia and contributes to angiogenic activity in endothelial cells. To determine the potential mechanism of survivin in endothelial dysfunction caused by hyperglycemia in diabetes, we evaluated the role of survivin in hyperglycemia and its effect on endothelial homeostasis. We demonstrated that an increase of d-glucose was sufficient to down-regulate survivin expression, impacting survivin's angiogenic role in endothelial cells. We additionally showed that survivin expression was increased in response to hypoxia yet this reaction was mitigated when the endothelial cells were in hyperglycemic conditions prior to hypoxia. Hyperglycemia also affected survivin-related proliferation and migration of endothelial cells and increased the number of apoptotic cells. In the ischemic porcine myocardium, the expression of survivin was induced. Moreover, survivin expression in the aorta, myocardium, and isolated endothelial cells was attenuated in a porcine model of diabetes in comparison to non-diabetes, which correlated negatively with the levels of fasting blood sugars and positively with territory perfusion. These results demonstrate that hyperglycemia critically alters survivin expression in vitro and in vivo, which leads to attenuation of angiogenic activity and impacts endothelial metabolism.