Molecular properties prediction, anticancer and anti-inflammatory activities of some pyrimido[1,2-b]pyridazin-2-one derivatives.

Introduction: The anticancer and anti-inflammatory activities of a novel series of eleven pyrimido[1,2-b]pyridazin-2-one analogues substituted at position 7 were assessed in the current study. Methods: The physicochemical characteristics were studied using MolSoft software. The antiproliferative activity was investigated by MTT cell viability assay, and cell cycle analysis elucidated the antiproliferative mechanism of action. Western blot analysis examined the expression levels of key pro-apoptotic (Bax, p53) and pro-survival (Bcl-2) proteins. The anti-inflammatory activity was assessed by measuring the production levels of nitric oxide in RAW264.7 cells, and the expression levels of COX-2 enzyme in LPS-activated THP-1 cells. In addition, the gene expression of various pro-inflammatory cytokines (IL-6, IL-8, IL-1ß, TNF-α) and chemokines (CCL2, CXCL1, CXCL2, CXCL3) was assessed by RT-qPCR. Results: Compound 1 bearing a chlorine substituent displayed the highest cytotoxic activity against HCT-116 and MCF-7 cancer cells where IC50 values of 49.35 ± 2.685 and 69.32 ± 3.186 µM, respectively, were achieved. Compound 1 increased the expression of pro-apoptotic proteins p53 and Bax while reducing the expression of pro-survival protein Bcl-2. Cell cycle analysis revealed that compound 1 arrested cell cycle at the G0/G1 phase. Anti-inflammatory assessments revealed that compound 1 displayed the strongest inhibitory activity on NO production with IC50 of 29.94 ± 2.24 µM, and down-regulated the expression of COX-2. Compound 1 also induced a statistically significant decrease in the gene expression of various cytokines and chemokines. Conclusion: These findings showed that the pyrimidine derivative 1 displayed potent anti-inflammatory and anticancer properties in vitro, and can be selected as a lead compound for further investigation.

Effect of very long-term storage and multiple freeze and thaw cycles on 11-dehydro-thromboxane-B2 and 8-iso-prostaglandin F2α, levels in human urine samples by validated enzyme immunoassays.

Biological samples are often frozen and stored for years and/or thawed multiple times, thus assessing their stability on long-term storage and repeated freeze-thaw cycles is crucial. The study aims were to assess:-the long-term stability of two major enzymatic and non-enzymatic metabolites of arachidonic acid, i.e. urinary 11-dehydro-thromboxane-(Tx) B2, 8-iso-prostaglandin (PG)F2α, and creatinine in frozen urine samples;-the effect of multiple freeze-thaw cycles. Seven-hundred and three urine samples measured in previously-published studies, stored at -40 °C, and measured for a second time for 11-dehydro-TxB2 (n = 677) and/or 8-iso-PGF2α (n = 114) and/or creatinine (n = 610) were stable over 10 years and the 2 measurements were highly correlated (all rho = 0.99, P < 0.0001). Urine samples underwent 10 sequential freeze-thaw cycles, with and without the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (10 mM); urinary 11-dehydro-TxB2 and creatinine were stable across all cycles (11-dehydro-TxB2: 100.4 ± 21%; creatinine: 101 ± 7% of baseline at cycle ten; n = 17), while 8-iso-PGF2α significantly increased by cycle 6 (151 ± 22% of baseline at cycle ten, n = 17, P < 0.05) together with hydrogen peroxide only in the absence of antioxidant. Arachidonic acid metabolites and creatinine appear stable in human urines stored at -40 °C over 10 years. Multiple freeze-thaw cycles increase urinary 8-iso-PGF2α in urine samples without antioxidants. These data are relevant for studies using urine samples stored over long-term and/or undergoing multiple freezing-thawing.

Thromboxane biosynthesis and future events in diabetes: the ASCEND trial.

BACKGROUND AND AIMS: Thromboxane (TX) A2, released by activated platelets, plays an important role in atherothrombosis. Urinary 11-dehydro-TXB2 (U-TXM), a stable metabolite reflecting the whole-body TXA2 biosynthesis, is reduced by ∼70% by daily low-dose aspirin. The U-TXM represents a non-invasive biomarker of in vivo platelet activation and is enhanced in patients with diabetes. This study assessed whether U-TXM is associated with the risk of future serious vascular events or revascularizations (SVE-R), major bleeding, or cancer in patients with diabetes. METHODS: The U-TXM was measured pre-randomization to aspirin or placebo in 5948 people with type 1 or 2 diabetes and no cardiovascular disease, in the ASCEND trial. Associations between log U-TXM and SVE-R (n = 618), major bleed (n = 206), and cancer (n = 700) during 6.6 years of follow-up were investigated by Cox regression; comparisons of these associations with the effects of randomization to aspirin were made. RESULTS: Higher U-TXM was associated with older age, female sex, current smoking, type 2 diabetes, higher body size, urinary albumin/creatinine ratio of ≥3 mg/mmol, and higher estimated glomerular filtration rate. After adjustment for these, U-TXM was marginally statistically significantly associated with SVE-R and major bleed but not cancer [hazard ratios per 1 SD higher log U-TXM (95% confidence interval): 1.09 (1.00-1.18), 1.16 (1.01-1.34), and 1.06 (0.98-1.14)]. The hazard ratio was similar to that implied by the clinical effects of randomization to aspirin for SVE-R but not for major bleed. CONCLUSIONS: The U-TXM was log-linearly independently associated with SVE-R in diabetes. This is consistent with the involvement of platelet TXA2 in diabetic atherothrombosis.

Lestaurtinib (CEP-701) reduces the duration of limbic status epilepticus in periadolescent rats.

BACKGROUND: The timely abortion of status epilepticus (SE) is essential to avoid brain damage and long-term neurodevelopmental sequalae. However, available anti-seizure treatments fail to abort SE in 30% of children. Given the role of the tropomyosin-related kinase B (TrkB) receptor in hyperexcitability, we investigated if TrkB blockade with lestaurtinib (CEP-701) enhances the response of SE to a standard treatment protocol and reduces SE-related brain injury. METHODS: SE was induced with intra-amygdalar kainic acid in postnatal day 45 rats under continuous electroencephalogram (EEG). Fifteen min post-SE onset, rats received intraperitoneal (i.p.) CEP-701 (KCEP group) or its vehicle (KV group). Controls received CEP-701 or its vehicle following intra-amygdalar saline. All groups received two i.p. doses of diazepam, followed by i.p. levetiracetam at 15 min intervals post-SE onset. Hippocampal TrkB dimer to monomer ratios were assessed by immunoblot 24 hr post-SE, along with neuronal densities and glial fibrillary acid protein (GFAP) levels. RESULTS: SE duration was 50% shorter in the KCEP group compared to KV (p < 0.05). Compared to controls, SE induced a 1.5-fold increase in TrkB dimerization in KV rats (p < 0.05), but not in KCEP rats which were comparable to controls (p > 0.05). The KCEP group had lower GFAP levels than KV (p < 0.05), and both were higher than controls (p < 0.05). KCEP and KV rats had comparable hippocampal neuronal densities (p > 0.05), and both were lower than controls (p < 0.05). CONCLUSIONS: Given its established human safety, CEP-701 is a promising adjuvant drug for the timely abortion of SE and the attenuation of SE-related brain injury.

Monoacylglycerol lipase reprograms hepatocytes and macrophages to promote liver regeneration.

Background & Aims: Liver regeneration is a repair process in which metabolic reprogramming of parenchymal and inflammatory cells plays a major role. Monoacylglycerol lipase (MAGL) is an ubiquitous enzyme at the crossroad between lipid metabolism and inflammation. It converts monoacylglycerols into free fatty acids and metabolises 2-arachidonoylglycerol into arachidonic acid, being thus the major source of pro-inflammatory prostaglandins in the liver. In this study, we investigated the role of MAGL in liver regeneration. Methods: Hepatocyte proliferation was studied in vitro in hepatoma cell lines and ex vivo in precision-cut human liver slices. Liver regeneration was investigated in mice treated with a pharmacological MAGL inhibitor, MJN110, as well as in animals globally invalidated for MAGL (MAGL-/-) and specifically invalidated in hepatocytes (MAGLHep-/-) or myeloid cells (MAGLMye-/-). Two models of liver regeneration were used: acute toxic carbon tetrachloride injection and two-thirds partial hepatectomy. MAGLMye-/- liver macrophages profiling was analysed by RNA sequencing. A rescue experiment was performed by in vivo administration of interferon receptor antibody in MAGLMye-/- mice. Results: Precision-cut human liver slices from patients with chronic liver disease and human hepatocyte cell lines exposed to MJN110 showed reduced hepatocyte proliferation. Mice with global invalidation or mice treated with MJN110 showed blunted liver regeneration. Moreover, mice with specific deletion of MAGL in either hepatocytes or myeloid cells displayed delayed liver regeneration. Mechanistically, MAGLHep-/- mice showed reduced liver eicosanoid production, in particular prostaglandin E2 that negatively impacts on hepatocyte proliferation. MAGL inhibition in macrophages resulted in the induction of the type I interferon pathway. Importantly, neutralising the type I interferon pathway restored liver regeneration of MAGLMye-/- mice. Conclusions: Our data demonstrate that MAGL promotes liver regeneration by hepatocyte and macrophage reprogramming. Impact and Implications: By using human liver samples and mouse models of global or specific cell type invalidation, we show that the monoacylglycerol pathway plays an essential role in liver regeneration. We unveil the mechanisms by which MAGL expressed in both hepatocytes and macrophages impacts the liver regeneration process, via eicosanoid production by hepatocytes and the modulation of the macrophage interferon pathway profile that restrains hepatocyte proliferation.

Fluorinated Benzofuran and Dihydrobenzofuran as Anti-Inflammatory and Potential Anticancer Agents.

Benzofuran and 2,3-dihydrobenzofuran scaffolds are heterocycles of high value in medicinal chemistry and drug synthesis. Targeting inflammation in cancer associated with chronic inflammation is a promising therapy. In the present study, we investigated the anti-inflammatory effects of fluorinated benzofuran and dihydrobenzofuran derivatives in macrophages and in the air pouch model of inflammation, as well as their anticancer effects in the human colorectal adenocarcinoma cell line HCT116. Six of the nine compounds suppressed lipopolysaccharide-stimulated inflammation by inhibiting the expression of cyclooxygenase-2 and nitric oxide synthase 2 and decreased the secretion of the tested inflammatory mediators. Their IC50 values ranged from 1.2 to 9.04 µM for interleukin-6; from 1.5 to 19.3 µM for Chemokine (C-C) Ligand 2; from 2.4 to 5.2 µM for nitric oxide; and from 1.1 to 20.5 µM for prostaglandin E2. Three novel synthesized benzofuran compounds significantly inhibited cyclooxygenase activity. Most of these compounds showed anti-inflammatory effects in the zymosan-induced air pouch model. Because inflammation may lead to tumorigenesis, we tested the effects of these compounds on the proliferation and apoptosis of HCT116. Two compounds with difluorine, bromine, and ester or carboxylic acid groups inhibited the proliferation by approximately 70%. Inhibition of the expression of the antiapoptotic protein Bcl-2 and concentration-dependent cleavage of PARP-1, as well as DNA fragmentation by approximately 80%, were described. Analysis of the structure-activity relationship suggested that the biological effects of benzofuran derivatives are enhanced in the presence of fluorine, bromine, hydroxyl, and/or carboxyl groups. In conclusion, the designed fluorinated benzofuran and dihydrobenzofuran derivatives are efficient anti-inflammatory agents, with a promising anticancer effect and a combinatory treatment in inflammation and tumorigenesis in cancer microenvironments.

Platelet thromboxane inhibition by low-dose aspirin in polycythemia vera: Ex vivo and in vivo measurements and in silico simulation.

Low-dose aspirin is currently recommended for patients with polycythemia vera (PV), a myeloproliferative neoplasm with increased risk of arterial and venous thromboses. Based on aspirin pharmacodynamics in essential thrombocythemia, a twice-daily regimen is recommended for patients with PV deemed at particularly high thrombotic risk. We investigated the effects of low-dose aspirin on platelet cyclooxygenase activity and in vivo platelet activation in 49 patients with PV, as assessed by serum thromboxane (TX) B2 and urinary TXA2 /TXB2 metabolite (TXM) measurements, respectively. A previously described pharmacokinetic-pharmacodynamic in silico model was used to simulate the degree of platelet TXA2 inhibition by once-daily (q.d.) and twice-daily (b.i.d.) aspirin, and to predict the effect of missing an aspirin dose during q.d. and b.i.d. regimens. Serum TXB2 averaged 8.2 (1.6-54.7) ng/ml and significantly correlated with the platelet count (γ = 0.39) and urinary TXM (γ = 0.52) in multivariable analysis. One-third of aspirin-treated patients with PV displayed less-than-maximal platelet TXB2 inhibition, and were characterized by significantly higher platelet counts and platelet-count corrected serum TXB2 than those with adequate inhibition. Eight patients with PV were sampled again after 12 ± 4 months, and had reproducible serum TXB2 and urinary TXM values. The in silico model predicted complete inhibition of platelet-derived TXB2 by b.i.d. aspirin, a prediction verified in a patient with PV with the highest TXB2 value while on aspirin q.d. and treated short-term with a b.i.d. regimen. In conclusion, one in three patients with PV on low-dose aspirin display less-than-maximal inhibition of platelet TXA2 production. Serum TXB2 measurement can be a valuable option to guide precision dosing of antiplatelet therapy in patients with PV.

The effect of clopidogrel and aspirin on the severity of traumatic brain injury in a rat model.

Traumatic Brain Injury (TBI) is one of the leading causes of death and disability worldwide. Aspirin (ASA) and clopidogrel (CLOP) are antiplatelet agents that inhibit platelet aggregation. They are implicated in worsening the intracerebral haemorrhage (ICH) risk post-TBI. However, antiplatelet drugs may also exert a neuroprotective effect post-injury. We determined the impact of ASA and CLOP treatment, alone or in combination, on ICH and brain damage in an experimental rat TBI model. We assessed changes in platelet aggregation and measured serum thromboxane by enzyme immune assay. We also explored a panel of brain damage and apoptosis biomarkers by immunoblotting. Rats were treated with ASA and/or CLOP for 48 h prior to TBI and sacrificed 48 h post-injury. In rats treated with antiplatelet agents prior to TBI, platelet aggregation was completely inhibited, and serum thromboxane was significantly decreased, compared to the TBI group without treatment. TBI increases UCHL-1 and GFAP, but decreases hexokinase expression compared to the non-injured controls. All groups treated with antiplatelet drugs prior to TBI had decreased UCH-L1 and GFAP serum levels compared to the TBI untreated group. Furthermore, the ASA and CLOP single treatments increased the hexokinase serum levels. We confirmed that αII-spectrin cleavage increased post-TBI, with the highest cleavage detected in CLOP-treated rats. Aspirin and/or CLOP treatment prior to TBI is a double-edged sword that exerts a dual effect post-injury. On one hand, ASA and CLOP single treatments increase the post-TBI ICH risk, with a further detrimental effect from the ASA + CLOP treatment. On the other hand, ASA and/or CLOP treatments are neuroprotective and result in a favourable profile of TBI injury markers. The ICH risk and the neuroprotection benefits from antiplatelet therapy should be weighed against each other to ameliorate the management of TBI patients.

Modulation of Neuro-Inflammatory Signals in Microglia by Plasma Prekallikrein and Neuronal Cell Debris.

Microglia, the resident phagocytes of the central nervous system and one of the key modulators of the innate immune system, have been shown to play a major role in brain insults. Upon activation in response to neuroinflammation, microglia promote the release of inflammatory mediators as well as promote phagocytosis. Plasma prekallikrein (PKall) has been recently implicated as a mediator of neuroinflammation; nevertheless, its role in mediating microglial activation has not been investigated yet. In the current study, we evaluate the mechanisms through which PKall contributes to microglial activation and release of inflammatory cytokines assessing PKall-related receptors and their dynamics. Murine N9-microglial cells were exposed to PKall (2.5 ng/ml), lipopolysaccharide (100 ng/ml), bradykinin (BK, 0.1 µM), and neuronal cell debris (16.5 µg protein/ml). Gene expression of bradykinin 2 receptor (B2KR), protease-activated receptor 2 (PAR-2), along with cytokines and fibrotic mediators were studied. Bioinformatic analysis was conducted to correlate altered protein changes with microglial activation. To assess receptor dynamics, HOE-140 (1 µM) and GB-83 (2 µM) were used to antagonize the B2KR and PAR-2 receptors, respectively. Also, the role of autophagy in modulating microglial response was evaluated. Data from our work indicate that PKall, LPS, BK, and neuronal cell debris resulted in the activation of microglia and enhanced expression/secretion of inflammatory mediators. Elevated increase in inflammatory mediators was attenuated in the presence of HOE-140 and GB-83, implicating the engagement of these receptors in the activation process coupled with an increase in the expression of B2KR and PAR-2. Finally, the inhibition of autophagy significantly enhanced the release of the cytokine IL-6 which were validated via bioinformatics analysis demonstrating the role of PKall in systematic and brain inflammatory processes. Taken together, we demonstrated that PKall can modulate microglial activation via the engagement of PAR-2 and B2KR where PKall acts as a neuromodulator of inflammatory processes.

Plasma Kallikrein as a Modulator of Liver Injury/Remodeling.

The occurrence and persistence of hepatic injury which arises from cell death and inflammation result in liver disease. The processes that lead to liver injury progression and resolution are still not fully delineated. The plasma kallikrein-kinin system (PKKS) has been shown to play diverse functions in coagulation, tissue injury, and inflammation, but its role in liver injury has not been defined yet. In this study, we have characterized the role of the PKKS at various stages of liver injury in mice, as well as the direct effects of plasma kallikrein on human hepatocellular carcinoma cell line (HepG2). Histological, immunohistochemical, and gene expression analyses were utilized to assess cell injury on inflammatory and fibrotic factors. Acute liver injury triggered by carbon tetrachloride (CCl4) injection resulted in significant upregulation of the plasma kallikrein gene (Klkb1) and was highly associated with the high mobility group box 1 gene, the marker of cell death (r = 0.75, p < 0.0005, n = 7). In addition, increased protein expression of plasma kallikrein was observed as clusters around necrotic areas. Plasma kallikrein treatment significantly increased the proliferation of CCl4-induced HepG2 cells and induced a significant increase in the gene expression of the thrombin receptor (protease activated receptor-1), interleukin 1 beta, and lectin-galactose binding soluble 3 (galectin-3) (p < 0.05, n = 4). Temporal variations in the stages of liver fibrosis were associated with an increase in the mRNA levels of bradykinin receptors: beta 1 and 2 genes (p < 0.05; n = 3-10). In conclusion, these findings indicate that plasma kallikrein may play diverse roles in liver injury, inflammation, and fibrosis, and suggest that plasma kallikrein may be a target for intervention in the states of liver injury.

Cyclooxygenase-2 Glycosylation Is Affected by Peroxynitrite in Endothelial Cells: Impact on Enzyme Activity and Degradation.

The exposure of human endothelial cells to 3-morpholinosydnonimine (SIN-1) induced the expression of cyclooxygenase-2 (COX-2) in a dose- and time-dependent manner. Interestingly, after a prolonged incubation (>8 h) several proteoforms were visualized by Western blot, corresponding to different states of glycosylation of the protein. This effect was specific for SIN-1 that generates peroxynitrite and it was not detected with other nitric oxide-donors. Metabolic labeling experiments using 35S or cycloheximide suggested that the formation of hypoglycosylated COX-2 was dependent on de novo synthesis of the protein rather than the deglycosylation of the native protein. Moreover, SIN-1 reduced the activity of the hexokinase, the enzyme responsible for the first step of glycolysis. The hypoglycosylated COX-2 induced by SIN-1 showed a reduced capacity to generate prostaglandins and the activity was only partially recovered after immunoprecipitation. Finally, hypoglycosylated COX-2 showed a more rapid rate of degradation compared to COX-2 induced by IL-1α and an alteration in the localization with an accumulation mainly detected in the nuclear membrane. Our results have important implication to understand the effect of peroxynitrite on COX-2 expression and activity, and they may help to identify new pharmacological tools direct to increase COX-2 degradation or to inhibit its activity.

Modulation of proteomic and inflammatory signals by Bradykinin in podocytes.

Podocyte damage is one of the hallmarks of diabetic nephropathy leading to proteinuria and kidney damage. The underlying mechanisms of podocyte injury are not well defined. Bradykinin (BK) was shown to contribute to diabetic kidney disease. Here, we evaluated the temporal changes in proteome profile and inflammatory signals of podocytes in response to BK (10-7M). Protein profile was evaluated by liquid chromatography mass Spectrometry (LC-MS/MS) analysis. Proteome profile analysis of podocytes treated with BK (10-7M) for 3 and 6 h, revealed 61 proteins that were differentially altered compared to unstimulated control podocytes. Pathway enrichment analysis suggested inhibition of cell death pathways, engagement of cytoskeletal elements and activation of inflammatory pathways. One of the inflammatory proteins that was identified to be induced by BK treatment is Prostaglandin (PG) H Synthase-2 (Cyclooxygenase-2, COX-2). In addition, BK significantly induced the production and release of PGE2 and this effect was inhibited by both COX-2 and MEK Kinase inhibitors, demonstrating that the production of PGE2 by BK is mediated via COX-2 and MAPK-dependent mechanisms. These findings provide a global understanding of the effector modulated proteome in response to BK and also reveal BK as an important modulator of inflammation and a potential player in podocyte injury.

Pathophysiology of Thrombosis in Peripheral Artery Disease.

Under physiological conditions, peripheral arteries release endogenous vascular-protective and antithrombotic agents. Endothelial cells actively synthesize vasoactive mediators, which regulate vascular tone and platelet reactivity thus preventing thrombosis. Atherosclerosis disrupts homeostasis and favours thrombosis by triggering pro-thrombotic responses in the vessels, platelet activation, aggregation as well as vasoconstriction, phenomena that ultimately lead to symptomatic lumen restriction or complete occlusion. In the present review, we will discuss the homeostatic role of arterial vessels in releasing vascular-protective agents, such as nitric oxide and prostacyclin, the role of pro- and anti-thrombotic vascular receptors as well as the contribution of circulating platelets and coagulation factors in triggering the pro-thrombotic response(s). We will discuss the pathological consequences of disrupting the protective pathways in the arteries and the pharmacological interventions along these pathways.

Monoacylglycerol Lipase Inhibition Protects From Liver Injury in Mouse Models of Sclerosing Cholangitis.

BACKGROUND AND AIMS: Monoacylglycerol lipase (MGL) is the last enzymatic step in triglyceride degradation, hydrolyzing monoglycerides into glycerol and fatty acids (FAs) and converting 2-arachidonoylglycerol into arachidonic acid, thus providing ligands for nuclear receptors as key regulators of hepatic bile acid (BA)/lipid metabolism and inflammation. We aimed to explore the role of MGL in the development of cholestatic liver and bile duct injury in mouse models of sclerosing cholangitis, a disease so far lacking effective pharmacological therapy. APPROACH AND RESULTS: To this aim we analyzed the effects of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding to induce sclerosing cholangitis in wild-type (WT) and knockout (MGL-/- ) mice and tested pharmacological inhibition with JZL184 in the multidrug resistance protein 2 knockout (Mdr2-/- ) mouse model of sclerosing cholangitis. Cholestatic liver injury and fibrosis were assessed by serum biochemistry, liver histology, gene expression, and western blot characterization of BA and FA synthesis/transport. Moreover, intestinal FAs and fecal microbiome were analyzed. Transfection and silencing were performed in Caco2 cells. MGL-/- mice were protected from DDC-induced biliary fibrosis and inflammation with reduced serum liver enzymes and increased FA/BA metabolism and ß-oxidation. Notably, pharmacological (JZL184) inhibition of MGL ameliorated cholestatic injury in DDC-fed WT mice and protected Mdr2-/- mice from spontaneous liver injury, with improved liver enzymes, inflammation, and biliary fibrosis. In vitro experiments confirmed that silencing of MGL decreases prostaglandin E2 accumulation in the intestine and up-regulates peroxisome proliferator-activated receptors alpha and gamma activity, thus reducing inflammation. CONCLUSIONS: Collectively, our study unravels MGL as a metabolic target, demonstrating that MGL inhibition may be considered as potential therapy for sclerosing cholangitis.

Tobacco cigarette smoking exacerbates aortic calcification in an early stage of myocardial infarction in a female mouse model.

Despite increased social awareness, marketing restraints, tobacco taxation, and available smoking cessation rehab programs, active and passive smoking remain a worldwide challenging epidemic and a key risk factor for cardiovascular diseases development. Although cardiovascular (CV) protection is more pronounced in women than in men due to estrogenic effects, tobacco cigarette smoking exposure seems to alter this protection by modulating estrogen actions via undefined mechanisms. Premenopausal cigarette smoking women are at higher risk of adverse CV effects than non-smokers. In this study, we investigated the impact of cigarette smoking on early CV injury after myocardial infarction (MI) in non-menopausal female mice. Aortic arch calcification, fibrosis, reactive oxygen species, and gene expression of inflammatory and calcification genes were exaggerated in mice exposed to cigarette smoke (CS). These findings suggest that aortic injury following MI, characterized by vascular smooth muscle cells transdifferentiation, calcification, inflammation, and collagen deposition but not cardiac dysfunction is exacerbated with CS exposure. The novel findings of this study highlight the importance of aortic injury on short and long-term prognosis in CS-exposed MI females. Linking those findings to estrogen alteration is probable and entails investigation.

gem-Difluorobisarylic derivatives: design, synthesis and anti-inflammatory effect.

INTRODUCTION: New fluorinated diaryl ethers and bisarylic ketones were designed and evaluated for their anti-inflammatory effects in primary macrophages. METHODS: The synthesis of the designed molecules started from easily accessible and versatile gem-difluoro propargylic derivatives. The desired aromatic systems were obtained using Diels-Alder/aromatization sequences and this was followed by Pd-catalyzed coupling reactions and, when required, final functionalization steps. Both direct inhibitory effects on cyclooxygenase-1 or -2 activities, protein expression of cyclooxygenase-2 and nitric oxide synthase-II and the production of prostaglandin E2, the pro-inflammatory nitric oxide and interleukin-6 were evaluated in primary murine bone marrow-derived macrophages in response to lipopolysaccharide. Docking of the designed molecules in cyclooxygenase-1 or -2 was performed. RESULTS: Only fluorinated compounds exerted anti-inflammatory activities by lowering the secretion of interleukin-6, nitric oxide, and prostaglandin E2, and decreasing the protein expression of inducible nitric oxide synthase and cyclooxygenase-2 in mouse primary macrophages exposed to lipopolysaccharide, as well as cyclooxygenase activity for some inhibitors with different efficiencies depending on the R-groups. Docking observation suggested an inhibitory role of cyclooxygenase-1 or -2 for compounds A3, A4 and A5 in addition to their capacity to inhibit nitrite, interleukin-6, and nitric oxide synthase-II and cyclooxygenase-2 expression. CONCLUSION: The new fluorinated diaryl ethers and bisarylic ketones have anti-inflammatory effects in macrophages. These fluorinated compounds have improved potential anti-inflammatory properties due to the fluorine residues in the bioactive molecules.

Heteromerization fingerprints between bradykinin B2 and thromboxane TP receptors in native cells.

Bradykinin (BK) and thromboxane-A2 (TX-A2) are two vasoactive mediators that modulate vascular tone and inflammation via binding to their cognate "class A" G-protein coupled receptors (GPCRs), BK-B2 receptors (B2R) and TX-prostanoid receptors (TP), respectively. Both BK and TX-A2 lead to ERK1/2-mediated vascular smooth muscle cell (VSMC) proliferation and/or hypertrophy. While each of B2R and TP could form functional dimers with various GPCRs, the likelihood that B2R-TP heteromerization could contribute to their co-regulation has never been investigated. The main objective of this study was to investigate the mode of B2R and TP interaction in VSMC, and its possible impact on downstream signaling. Our findings revealed synergistically activated ERK1/2 following co-stimulation of rat VSMC with a subthreshold dose of BK and effective doses of the TP stable agonist, IBOP, possibly involving biased agonist signaling. Single detection of each of B2R and TP in VSMC, using in-situ proximity ligation assay (PLA), provided evidence of the constitutive expression of nuclear and extranuclear B2R and TP. Moreover, inspection of B2R-TP PLA signals in VSMC revealed agonist-modulated nuclear and extranuclear proximity between B2R and TP, whose quantification varied substantially following single versus dual agonist stimulations. B2R-TP interaction was further verified by the findings of co-immunoprecipitation (co-IP) analysis of VSMC lysates. To our knowledge, this is the first study that provides evidence supporting the existence of B2R-TP heteromerization fingerprints in primary cultured VSMC.

Lack of monoacylglycerol lipase prevents hepatic steatosis by favoring lipid storage in adipose tissue and intestinal malabsorption.

Monoacylglycerol lipase (MGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. To examine the role of MGL in hepatic steatosis, WT and MGL KO (MGL-/-) mice were challenged with a Western diet (WD) over 12 weeks. Lipid metabolism, inflammation, and fibrosis were assessed by serum biochemistry, histology, and gene-expression profiling of liver and adipose depots. Intestinal fat absorption was measured by gas chromatography. Primary adipocyte and 3T3-L1 cells were analyzed by flow cytometry and Western blot. Human hepatocytes were treated with MGL inhibitor JZL184. The absence of MGL protected mice from hepatic steatosis by repressing key lipogenic enzymes in liver (Srebp1c, Pparγ2, and diacylglycerol O-acyltransferase 1), while promoting FA oxidation. Liver inflammation was diminished in MGL-/- mice fed a WD, as evidenced by diminished epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 (F4/80) staining and C-C motif chemokine ligand 2 gene expression, whereas fibrosis remained unchanged. Absence of MGL promoted fat storage in gonadal white adipose tissue (gWAT) with increased lipogenesis and unchanged lipolysis, diminished inflammation in gWAT, and subcutaneous AT. Intestinal fat malabsorption prevented ectopic lipid accumulation in livers of MGL-/- mice fed a WD. In vitro experiments demonstrated increased adipocyte size/lipid content driven by PPARγ. In conclusion, our data uncover that MGL deletion improves some aspects of nonalcoholic fatty liver disease by promoting lipid storage in gWAT and fat malabsorption.

Heme oxygenase-1-Dependent anti-inflammatory effects of atorvastatin in zymosan-injected subcutaneous air pouch in mice.

Statins exert pleiotropic and beneficial anti-inflammatory and antioxidant effects. We have previously reported that macrophages treated with statins increased the expression of heme oxygenase-1 (HO-1), an inducible anti-inflammatory and cytoprotective stress protein, responsible for the degradation of heme. In the present study, we investigated the effects of atorvastatin on inflammation in mice and analyzed its mechanism of action in vivo. Air pouches were established in 8 week-old female C57BL/6J mice. Atorvastatin (5 mg/kg, i.p.) and/or tin protoporphyrin IX (SnPPIX), a heme oxygenase inhibitor (12 mg/kg, i.p.), were administered for 10 days. Zymosan, a cell wall component of Saccharomyces cerevisiae, was injected in the air pouch to trigger inflammation. Cell number and levels of inflammatory markers were determined in exudates collected from the pouch 24 hours post zymosan injection by flow cytometry, ELISA and quantitative PCR. Analysis of the mice treated with atorvastatin alone displayed increased expression of HO-1, arginase-1, C-type lectin domain containing 7A, and mannose receptor C-type 1 in the cells of the exudate of the air pouch. Flow cytometry analysis revealed an increase in monocyte/macrophage cells expressing HO-1 and in leukocytes expressing MRC-1 in response to atorvastatin. Mice treated with atorvastatin showed a significant reduction in cell influx in response to zymosan, and in the expression of proinflammatory cytokines and chemokines such as interleukin-1α, monocyte chemoattractant protein-1 and prostaglandin E2. Co-treatment of mice with atorvastatin and tin protoporphyrin IX (SnPPIX), an inhibitor of heme oxygenase, reversed the inhibitory effect of statin on cell influx and proinflammatory markers, suggesting a protective role of HO-1. Flow cytometry analysis of air pouch cell contents revealed prevalence of neutrophils and to a lesser extent of monocytes/macrophages with no significant effect of atorvastatin treatment on the modification of their relative proportion. These findings identify HO-1 as a target for the therapeutic actions of atorvastatin and highlight its potential role as an in vivo anti-inflammatory agent.

Inhibition of monoacylglycerol lipase, an anti-inflammatory and antifibrogenic strategy in the liver.

OBJECTIVE: Sustained inflammation originating from macrophages is a driving force of fibrosis progression and resolution. Monoacylglycerol lipase (MAGL) is the rate-limiting enzyme in the degradation of monoacylglycerols. It is a proinflammatory enzyme that metabolises 2-arachidonoylglycerol, an endocannabinoid receptor ligand, into arachidonic acid. Here, we investigated the impact of MAGL on inflammation and fibrosis during chronic liver injury. DESIGN: C57BL/6J mice and mice with global invalidation of MAGL (MAGL -/- ), or myeloid-specific deletion of either MAGL (MAGLMye-/-), ATG5 (ATGMye-/-) or CB2 (CB2Mye-/-), were used. Fibrosis was induced by repeated carbon tetrachloride (CCl4) injections or bile duct ligation (BDL). Studies were performed on peritoneal or bone marrow-derived macrophages and Kupffer cells. RESULTS: MAGL -/- or MAGLMye-/- mice exposed to CCl4 or subjected to BDL were more resistant to inflammation and fibrosis than wild-type counterparts. Therapeutic intervention with MJN110, an MAGL inhibitor, reduced hepatic macrophage number and inflammatory gene expression and slowed down fibrosis progression. MAGL inhibitors also accelerated fibrosis regression and increased Ly-6Clow macrophage number. Antifibrogenic effects exclusively relied on MAGL inhibition in macrophages, since MJN110 treatment of MAGLMye-/- BDL mice did not further decrease liver fibrosis. Cultured macrophages exposed to MJN110 or from MAGLMye-/- mice displayed reduced cytokine secretion. These effects were independent of the cannabinoid receptor 2, as they were preserved in CB2Mye-/- mice. They relied on macrophage autophagy, since anti-inflammatory and antifibrogenic effects of MJN110 were lost in ATG5Mye-/- BDL mice, and were associated with increased autophagic flux and autophagosome biosynthesis in macrophages when MAGL was pharmacologically or genetically inhibited. CONCLUSION: MAGL is an immunometabolic target in the liver. MAGL inhibitors may show promising antifibrogenic effects during chronic liver injury.