Intravital microscopy reveals endothelial dysfunction in resistance arterioles in Angiotensin II-induced hypertension.

It is known that hypertension is associated with endothelial dysfunction and that Angiotensin II (Ang II) is a key player in the pathogenesis of hypertension. We aimed to elucidate whether endothelial dysfunction is a specific feature of Ang II-mediated hypertension or a common finding of hypertension, independently of underlying etiology. We studied endothelial-dependent vasorelaxation in precapillary resistance arterioles and in various large-caliber conductance arteries in wild-type mice with Ang II-dependent hypertension (2-kidney 1-clip (2K1C) model) or Ang II-independent (volume overload) hypertension (1-kidney 1-clip model (1K1C)). Normotensive sham mice were used as controls. Aortic mechanical properties were also evaluated. Intravital microscopy of precapillary arterioles revealed a significantly impaired endothelium-dependent vasorelaxation in 2K1C mice compared with sham mice, as quantified by the ratio of acetylcholine (ACh)-induced over S-nitroso-N-acetyl-D,L-penicillamine (SNAP)-induced vasorelaxation (2K1C: 0.49±0.12 vs. sham: 0.87±0.11, P=0.018). In contrast, the ACh/SNAP ratio in volume-overload hypertension 1K1C mice was not significantly different from sham mice, indicating no specific endothelial dysfunction (1K1C: 0.77±0.27 vs. sham: 0.87±0.11, P=0.138). Mechanical aortic wall properties and endothelium-dependent vasorelaxation, assessed ex vivo in rings of large-caliber conductance (abdominal and thoracic aorta, carotid and femoral arteries), were not different between 2K1C, 1K1C and sham mice. Endothelial dysfunction is an early feature of Ang II- but not volume-overload-mediated hypertension. This occurs exclusively at the level of precapillary arterioles and not in conduit arteries. Our findings, if confirmed in clinical studies, will provide a better understanding of the pathophysiological mechanisms of hypertension.

Fracture risk and zoledronic acid therapy in men with osteoporosis.

BACKGROUND: Fractures in men are a major health issue, and data on the antifracture efficacy of therapies for osteoporosis in men are limited. We studied the effect of zoledronic acid on fracture risk among men with osteoporosis. METHODS: In this multicenter, double-blind, placebo-controlled trial, we randomly assigned 1199 men with primary or hypogonadism-associated osteoporosis who were 50 to 85 years of age to receive an intravenous infusion of zoledronic acid (5 mg) or placebo at baseline and at 12 months. Participants received daily calcium and vitamin D supplementation. The primary end point was the proportion of participants with one or more new morphometric vertebral fractures over a period of 24 months. RESULTS: The rate of any new morphometric vertebral fracture was 1.6% in the zoledronic acid group and 4.9% in the placebo group over the 24-month period, representing a 67% risk reduction with zoledronic acid (relative risk, 0.33; 95% confidence interval, 0.16 to 0.70; P=0.002). As compared with men who received placebo, men who received zoledronic acid had fewer moderate-to-severe vertebral fractures (P=0.03) and less height loss (P=0.002). Fewer participants who received zoledronic acid had clinical vertebral or nonvertebral fractures, although this difference did not reach significance because of the small number of fractures. Bone mineral density was higher and bone-turnover markers were lower in the men who received zoledronic acid (P<0.05 for both comparisons). Results were similar in men with low serum levels of total testosterone. The zoledronic acid and placebo groups did not differ significantly with respect to the incidence of death (2.6% and 2.9%, respectively) or serious adverse events (25.3% and 25.2%). CONCLUSIONS: Zoledronic acid treatment was associated with a significantly reduced risk of vertebral fracture among men with osteoporosis. (Funded by Novartis Pharma; ClinicalTrials.gov number, NCT00439647.).

APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

PPAR-mediated activity of phthalates: A link to the obesity epidemic?

The endocrine disruption hypothesis asserts that exposure to small amounts of some chemicals in the environment may interfere with the endocrine system and lead to harmful effects in wildlife and humans. Many of these chemicals may interact with members of the nuclear receptor superfamily. Peroxisome proliferator-activated receptors (PPARs) are such candidate members, which interact with many different endogenous and exogenous lipophilic compounds. More particularly, the roles of PPARs in lipid and carbohydrate metabolism raise the question of their activation by a sub-class of pollutants, tentatively named "metabolic disrupters". Phthalates are abundant environmental micro-pollutants in Europe and North America and may belong to this class. Mono-ethyl-hexyl-phthalate (MEHP), a metabolite of the widespread plasticizer di-ethyl-hexyl-phthalate (DEHP), has been found in exposed organisms and interacts with all three PPARs. A thorough analysis of its interactions with PPARgamma identified MEHP as a selective PPARgamma modulator, and thus a possible contributor to the obesity epidemic.

Impact of genomic methylation on radiation sensitivity of colorectal carcinoma.

PURPOSE: To investigate the influence of demethylation with 5-aza-cytidine (AZA) on radiation sensitivity and to define the intrinsic radiation sensitivity of methylation deficient colorectal carcinoma cells. METHODS AND MATERIALS: Radiation sensitizing effects of AZA were investigated in four colorectal carcinoma cell lines (HCT116, SW480, L174 T, Co115), defining influence of AZA on proliferation, clonogenic survival, and cell cycling with or without ionizing radiation. The methylation status for cancer or DNA damage response-related genes silenced by promoter methylation was determined. The effect of deletion of the potential target genes (DNMT1, DNMT3b, and double mutants) on radiation sensitivity was analyzed. RESULTS: AZA showed radiation sensitizing properties at >or=1 micromol/l, a concentration that does not interfere with the cell cycle by itself, in all four tested cell lines with a sensitivity-enhancing ratio (SER) of 1.6 to 2.1 (confidence interval [CI] 0.9-3.3). AZA successfully demethylated promoters of p16 and hMLH1, genes associated with ionizing radiation response. Prolonged exposure to low-dose AZA resulted in sustained radiosensitivity if associated with persistent genomic hypomethylation after recovery from AZA. Compared with maternal HCT116 cells, DNMT3b-defcient deficient cells were more sensitive to radiation with a SER of 2.0 (CI 0.9-2.1; p = 0.03), and DNMT3b/DNMT1-/- double-deficient cells showed a SER of 1.6 (CI 0.5-2.7; p = 0.09). CONCLUSIONS: AZA-induced genomic hypomethylation results in enhanced radiation sensitivity in colorectal carcinoma. The mediators leading to sensitization remain unknown. Defining the specific factors associated with radiation sensitization after genomic demethylation may open the way to better targeting for the purpose of radiation sensitization.

Addiction and arousal: the hypocretin connection

The hypocretins, also known as orexins, are two neuropeptides now commonly described as critical components to maintain and regulate the stability of arousal. Several lines of evidence have raised the hypothesis that hypocretin-producing neurons are part of the circuitries that mediate the hypothalamic response to acute stress. Intracerebral administration of hypocretin leads to a dose-related reinstatement of drug and food seeking behaviors. Furthermore, stress-induced reinstatement can be blocked with hypocretin receptor 1 antagonism. These results, together with recent data showing that hypocretin is critically involved in cocaine sensitization through the recruitment of NMDA receptors in the ventral tegmental area, strongly suggest that activation of hypocretin neurons play a critical role in the development of the addiction process. The activity of hypocretin neurons may affect addictive behavior by contributing to brain sensitization or by modulating the brain reward system. Hypocretinergic cells, in coordination with brain stress systems may lead to a vulnerable state that facilitates the resumption of drug seeking behavior. Hence, the hypocretinergic system is a new drug target that may be used to prevent relapse of drug seeking

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester.

OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.

Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation.

Omega-3 fatty acids (ω-3 FAs) have potential anti-inflammatory activity in a variety of inflammatory human diseases, but the mechanisms remain poorly understood. Here we show that stimulation of macrophages with ω-3 FAs, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and other family members, abolished NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1β secretion. In addition, G protein-coupled receptor 120 (GPR120) and GPR40 and their downstream scaffold protein β-arrestin-2 were shown to be involved in inflammasome inhibition induced by ω-3 FAs. Importantly, ω-3 FAs also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder in a high-fat-diet-induced type 2 diabetes model. Our results reveal a mechanism through which ω-3 FAs repress inflammation and prevent inflammation-driven diseases and suggest the potential clinical use of ω-3 FAs in gout, autoinflammatory syndromes, or other NLRP3 inflammasome-driven inflammatory diseases.

Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts.

Antigen-specific T-cell activation implicates a redistribution of plasma membrane-bound molecules in lipid rafts, such as the coreceptors CD8 and CD4, the Src kinases Lek and Fyn, and the linker for activation of T cells (LAT), that results in the formation of signaling complexes. These molecules partition in lipid rafts because of palmitoylation of cytoplasmic, membrane proximal cysteines, which is essential for their functional integrity in T-cell activation. Here, we show that exogenous dipalmitoyl-phosphatidylethanolamine (DPPE), but not the related unsaturated dioleoyl-phosphatidylethanolamine (DOPE), partitions in lipid rafts. DPPE inhibits activation of CD8(+) T lymphocytes by sensitized syngeneic antigen-presenting cells or specific major histocompatibility complex (MHC) peptide tetramers, as indicated by esterase release and intracellular calcium mobilization. Cytotoxic, T lymphocyte (CTL)-target cell conjugate formation is not affected by DPPE, indicating that engagement of the T-cell receptor by its cognate ligand is intact in lipid-treated cells. In contrast to other agents known to block raft-dependent signaling, DPPE efficiently inhibits the MHC peptide-induced recruitment of palmitoylated signaling molecules to lipid rafts and CTL activation without affecting cell viability or lipid raft integrity.

Immunocytochemical and pharmacological characterization of metabotropic glutamate receptors of the vestibular end organs in the frog.

Using immunocytochemistry and multiunit recording of afferent activity of the whole vestibular nerve, we investigated the role of metabotropic glutamate receptors (mGluR) in the afferent neurotransmission in the frog semicircular canals (SCC). Group I (mGluR1alpha) and group II (mGluR2/3) mGluR immunoreactivities were distributed to the vestibular ganglion neurons, and this can be attributed to a postsynaptic locus of metabotropic regulation of rapid excitatory transmission. The effects of group I/II mGluR agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine (MCPG) on resting and chemically induced afferent activity were studied. ACPD (10-100 microM) enhanced the resting discharge frequency. MCPG (5-100 microM) led to a concentration-dependent decrease of both resting activity and ACPD-induced responses. If the discharge frequency had previously been restored by L-glutamate (L-Glu) in high-Mg2+ solution, ACPD elicited a transient increase in the firing rate in the afferent nerve suggesting that ACPD acts on postsynaptic receptors. The L-Glu agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA), were tested during application of ACPD. AMPA- and NMDA-induced responses were higher in the presence than absence of ACPD, implicating mGluR in the modulation of ionotropic glutamate receptors. These results indicate that activation of mGluR potentiates AMPA and NMDA responses through a postsynaptic interaction. We conclude that ACPD may exert modulating postsynaptic effects on vestibular afferents and that this process is activity-dependent.

Determinants of sustained viral suppression in HIV-infected patients with self-reported poor adherence to antiretroviral therapy.

BACKGROUND: Good adherence to antiretroviral therapy (ART) is critical for successful HIV treatment. However, some patients remain virologically suppressed despite suboptimal adherence. We hypothesized that this could result from host genetic factors influencing drug levels. METHODS: Eligible individuals were Caucasians treated with efavirenz (EFV) and/or boosted lopinavir (LPV/r) with self-reported poor adherence, defined as missing doses of ART at least weekly for more than 6 months. Participants were genotyped for single nucleotide polymorphisms (SNPs) in candidate genes previously reported to decrease EFV (rs3745274, rs35303484, rs35979566 in CYP2B6) and LPV/r clearance (rs4149056 in SLCO1B1, rs6945984 in CYP3A, rs717620 in ABCC2). Viral suppression was defined as having HIV-1 RNA <400 copies/ml throughout the study period. RESULTS: From January 2003 until May 2009, 37 individuals on EFV (28 suppressed and 9 not suppressed) and 69 on LPV/r (38 suppressed and 31 not suppressed) were eligible. The poor adherence period was a median of 32 weeks with 18.9% of EFV and 20.3% of LPV/r patients reporting missed doses on a daily basis. The tested SNPs were not determinant for viral suppression. Reporting missing >1 dose/week was associated with a lower probability of viral suppression compared to missing 1 dose/week (EFV: odds ratio (OR) 0.11, 95% confidence interval (CI): 0.01-0.99; LPV/r: OR 0.29, 95% CI: 0.09-0.94). In both groups, the probability of remaining suppressed increased with the duration of continuous suppression prior to the poor adherence period (EFV: OR 3.40, 95% CI: 0.62-18.75; LPV/r: OR 5.65, 95% CI: 1.82-17.56). CONCLUSIONS: The investigated genetic variants did not play a significant role in the sustained viral suppression of individuals with suboptimal adherence. Risk of failure decreased with longer duration of viral suppression in this population.

15-hydroxyprostaglandin dehydrogenase is down-regulated in gastric cancer.

PURPOSE: We have investigated the expression and regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer. EXPERIMENTAL DESIGN: Clinical gastric adenocarcinoma samples were analyzed by immunohistochemistry and quantitative real-time PCR for protein and mRNA expression of 15-PGDH and for methylation status of 15-PGDH promoter. The effects of interleukin-1beta (IL-1beta) and epigenetic mechanisms on 15-PGDH regulation were assessed in gastric cancer cell lines. RESULTS: In a gastric cancer cell line with a very low 15-PGDH expression (TMK-1), the 15-PGDH promoter was methylated and treatment with a demethylating agent 5-aza-2'-deoxycytidine restored 15-PGDH expression. In a cell line with a relatively high basal level of 15-PGDH (MKN-28), IL-1beta repressed expression of 15-PGDH mRNA and protein. This effect of IL-1beta was at least in part attributed to inhibition of 15-PGDH promoter activity. SiRNA-mediated knockdown of 15-PGDH resulted in strong increase of prostaglandin E(2) production in MKN-28 cells and increased cell growth of these cells by 31% in anchorage-independent conditions. In clinical gastric adenocarcinoma specimens, 15-PGDH mRNA levels were 5-fold lower in gastric cancer samples when compared with paired nonneoplastic tissues (n = 26) and 15-PGDH protein was lost in 65% of gastric adenocarcinomas (n = 210). CONCLUSIONS: 15-PGDH is down-regulated in gastric cancer, which could potentially lead to accelerated tumor progression. Importantly, our data indicate that a proinflammatory cytokine linked to gastric carcinogenesis, IL-1beta, suppresses 15-PGDH expression at least partially by inhibiting promoter activity of the 15-PGDH gene.

The orphan receptor CRF2-4 is an essential subunit of the interleukin 10 receptor.

The orphan receptor CRF2-4 is a member of the class II cytokine receptor family (CRF2), which includes the interferon receptors, the interleukin (IL) 10 receptor, and tissue factor. CRFB4, the gene encoding CRF2-4, is located within a gene cluster on human chromosome 21 that comprises three interferon receptor subunits. To elucidate the role of CRF2-4, we disrupted the CRFB4 gene in mice by means of homologous recombination. Mice lacking CRF2-4 show no overt abnormalities, grow normally, and are fertile. CRF2-4 deficient cells are normally responsive to type I and type II interferons, but lack responsiveness to IL-10. By approximately 12 wk of age, the majority of mutant mice raised in a conventional facility developed a chronic colitis and splenomegaly. Thus, CRFB4 mutant mice recapitulate the phenotype of IL-10-deficient mice. These findings suggest that CRF2-4 is essential for IL-10-mediated effects and is a subunit of the IL-10 receptor.