Advanced Glycated apoA-IV Loses Its Ability to Prevent the LPS-Induced Reduction in Cholesterol Efflux-Related Gene Expression in Macrophages.

We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.

AGE-albumin enhances ABCA1 degradation by ubiquitin-proteasome and lysosomal pathways in macrophages.

BACKGROUND AND AIMS: Advanced glycation end products (AGEs) induce cellular oxidative/endoplasmic reticulum stress and inflammation. We investigated its underlying mechanisms for atherogenesis focusing on regulation of ABCA1 protein decay in macrophages. METHODS: The ABCA1 decay rate was evaluated in macrophages after treatment with LXR agonist and by incubation with control (C) or AGE-albumin concomitant or not with cycloheximide, MG-132, ammonium chloride and calpain inhibitors were utilized to inhibit, respectively, proteasome, lysosome and ABCA1 proteolysis at cell surface. ABCA1 was determined by immunoblot and the protein decay rate calculated along time by the slope of the linear regression. Ubiquitination level was determined in ABCA1 immunoprecipitated from whole cell lysate or bulk cell membrane. AGE effect was also analyzed in THP-1 cells transfected with siRNA-RAGE. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/MS. One-way ANOVA and Student t test were utilized to compare results. RESULTS: CML and PYR-albumin were higher in AGE-albumin as compared to C. AGE-albumin reduced ABCA1 in J774 and THP-1 macrophages (20-30%) and induced a higher ABCA1 ubiquitination and a faster protein decay rate that was dependent on the presence of AGE during the kinetics of measurement in the presence of cycloheximide. Proteasomal inhibition restored and lysosomal inhibition partially recovered ABCA1 in cells treated with AGE-albumin. Calpain inhibition was not able to rescue ABCA1. RAGE knockdown prevented the reduction in ABCA1 elicited by AGE. CONCLUSIONS: AGE-albumin diminishes ABCA1 by accelerating its degradation through the proteasomal and lysosomal systems. This may increase lipid accumulation in macrophages by diminishing cholesterol efflux via RAGE signaling contributing to atherosclerosis in diabetes mellitus.

N-acetylcysteine Counteracts Adipose Tissue Macrophage Infiltration and Insulin Resistance Elicited by Advanced Glycated Albumin in Healthy Rats.

Background: Advanced glycation endproducts elicit inflammation. However, their role in adipocyte macrophage infiltration and in the development of insulin resistance, especially in the absence of the deleterious biochemical pathways that coexist in diabetes mellitus, remains unknown. We investigated the effect of chronic administration of advanced glycated albumin (AGE-albumin) in healthy rats, associated or not with N-acetylcysteine (NAC) treatment, on insulin sensitivity, adipose tissue transcriptome and macrophage infiltration and polarization. Methods: Male Wistar rats were intraperitoneally injected with control (C) or AGE-albumin alone, or, together with NAC in the drinking water. Biochemical parameters, lipid peroxidation, gene expression and protein contents were, respectively, determined by enzymatic techniques, reactive thiobarbituric acid substances, RT-qPCR and immunohistochemistry or immunoblot. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/mass spectrometry (LC-MS/MS) and ELISA. Results: CML and PYR were higher in AGE-albumin as compared to C. Food consumption, body weight, systolic blood pressure, plasma lipids, glucose, hepatic and renal function, adipose tissue relative weight and adipocyte number were similar among groups. In AGE-treated animals, insulin resistance, adipose macrophage infiltration and Col12a1 mRNA were increased with no changes in M1 and M2 phenotypes as compared to C-albumin-treated rats. Total GLUT4 content was reduced by AGE-albumin as compared to C-albumin. NAC improved insulin sensitivity, reduced urine TBARS, adipose macrophage number and Itgam and Mrc mRNA and increased Slc2a4 and Ppara. CD11b, CD206, Ager, Ddost, Cd36, Nfkb1, Il6, Tnf, Adipoq, Retn, Arg, and Il12 expressions were similar among groups. Conclusions: AGE-albumin sensitizes adipose tissue to inflammation due to macrophage infiltration and reduces GLUT4, contributing to insulin resistance in healthy rats. NAC antagonizes AGE-albumin and prevents insulin resistance. Therefore, it may be a useful tool in the prevention of AGE action on insulin resistance and long-term complications of DM.

Glycated albumin induces lipid infiltration in mice aorta independently of DM and RAS local modulation by inducing lipid peroxidation and inflammation.

AIMS: Advanced glycated albumin (AGE-albumin) adversely impairs macrophage lipid homeostasis in vitro, which may be prevented by angiotensin receptor blockers. In vivo studies are inconclusive whether AGE-albumin itself plays important role in early-stage atherogenesis. We aimed at investigating how AGE-albumin by itself drives atherosclerosis development in dyslipidemic non-diabetic mice and if its effects are due to the activation of renin-angiotensin system in the arterial wall and the expression of genes and proteins involved in lipid flux. METHODS AND RESULTS: Murine albumin glycation was induced by incubation with 10mM glycolaldehyde and C-albumin with PBS alone. Twelve-week-old-male apoE knockout mice were submitted to a daily IP injection of control (C) or AGE-albumin (2mg/mL) during 30days with or without losartan (LOS: 100mg/L; C+LOS and AGE+LOS). Aortic arch was removed, and gene expression was determined by RT-PCR and protein content by immunofluorescence. Plasma lipid and glucose levels were similar among groups. Systolic blood pressure was similarly reduced in both groups treated with LOS. In comparison to C-albumin, aortic lipid infiltration was 5.3 times increased by AGE-albumin, which was avoided by LOS. LOS prevented the enhancement induced by AGE-albumin in Ager, Tnf and Cybb mRNA levels but did not reduce Olr1. Nfkb and Agt mRNA levels were unchanged by AGE-albumin. LOS similarly reduced Agtr1a mRNA level in both C and AGE-albumin groups. In AGE-albumin-treated mice, immunofluorescence for carboxymethyl-lysine, 4-hydroxynonenal and RAGE was respectively, 4.8, 2.6 and 1.7 times enhanced in comparison to C-albumin. These increases were all avoided by LOS. CONCLUSIONS: AGE-albumin evokes a pre-stage of atherogenesis in dyslipidemic mice independently of the presence of diabetes mellitus or modulation in the RAS in part by the induction of lipid peroxidation and inflammation.

Transtendon rotator-cuff repair of partial-thickness articular surface tears can lead to medial rotator-cuff failure.

PURPOSE: The purpose of this study was to evaluate clinical and anatomic outcomes of patients following transtendon rotator-cuff repair of partial articular supraspinatus tendon avulsion (PASTA) lesions. PATIENTS AND METHODS: Patients in the senior author's practice who had isolated PASTA lesions treated by transtendon rotator-cuff repair were included (n=8) and retrospectively reviewed. All patients were evaluated preoperatively and at a mean of 21.2 months (±9.7 months) postoperatively using standardized clinical evaluation (physical exam, American Shoulder and Elbow Surgeons, and Simple Shoulder Test). All patients underwent postoperative imaging with a magnetic resonance imaging arthrogram. RESULTS: There was a significant improvement in American Shoulder and Elbow Surgeons (42.7±17.5 to 86.9±25.2) and Simple Shoulder Test (4.6±3.2 to 10.1±3.8) scores from pre- to postoperative, respectively. Postoperative imaging demonstrated full-thickness medial cuff tearing in seven patients, and one patient with a persistent partial articular surface defect. CONCLUSION: Transtendon repair of PASTA lesions may lead to improvements in clinical outcome. However, postoperative imaging demonstrated a high incidence of full-thickness rotator-cuff defects following repair.

Synthesis of stable isotope-labelled monolysyl advanced glycation endproducts.

Advanced Glycation Endproducts (AGEs) are modified amino acids that form on proteins and are known to be implicated in the pathogenesis of diabetes and related diseases. Ready access to synthetic stable isotope-labelled AGEs allows for quantitative mass spectrometry studies to be undertaken, providing key insights into the roles AGEs play in the progression of such diseases. However, the majority of current syntheses of these compounds suffer from poor yields and lengthy procedures and are not suitable for the purposes required here. Here, we report robust syntheses of stable isotope-labelled monolysyl AGEs, N(ε)-(carboxymethyl)lysine, N(ε)-(carboxyethyl)lysine and pyrraline, that provide straightforward access to these compounds for quantitative amino acid analysis. This work will facilitate future investigations with these compounds and lead to a better understanding of the roles they play in diabetes and related diseases.

Telomerase inhibitors from cyanobacteria: isolation and synthesis of sulfoquinovosyl diacylglycerols from Microcystis aeruguinosa PCC 7806.

By using the Telospot assay, 27 different extracts of cyanobacteria were evaluated for telomerase inhibition. All extracts showed varying, but significant activity. We selected Microcystis aeruguinosa PCC 7806 to identify the active compound and a bioassay guided fractionation led us to isolate mixtures of sulfoquinovosyl diacylglycerols (SQDGs), which were identified by 2D NMR and MS/MS experiments. Pure SQDG derivatives were then synthesized. The IC(50) values of pure synthetic sulfoquinovosyl dipalmitoylglycerol and the monopalmitoylated derivative against telomerase were determined to be 17 and 40 µM, respectively. A structure-activity relationship study allowed the identification of compounds with modified lipophilic acyl groups that display improved activity.

Synthesis of monolysyl advanced glycation endproducts and their incorporation into collagen model peptides.

The synthesis of advanced glycation endproducts (AGEs), CML, CEL, and pyrraline and their incorporation into collagen model peptides is reported. AGEs are modified amino acids that form on proteins such as collagen and are thought to play a significant role in the pathogenesis of many diseases, particularly diabetes. The synthesis and incorporation of these compounds into synthetic peptides is a key step in developing model systems with which to investigate AGE-modified proteins.

Surface modifications based on the cyanobacterial siderophore anachelin: from structure to functional biomaterials design.

This review describes the design, synthesis and evaluation of novel catechol based anchors for surface modification. The anachelin chromophore, the catecholate fragment of the siderophore anachelin from the cyanobacterium Anabaena cylindrica, allows for the immobilization of polyethylene glycol (PEG) on titania and glass surfaces thus rendering them protein resistant and antifouling. It is proposed that catecholate siderophores constitute a class of natural products useful for surface modification similar to dihydroxyphenylalanine and dopamine derived compounds found in mussel adhesive proteins. Second-generation dopamine derivatives featuring a quaternary ammonium group were found to be equally efficient in generating antifouling surfaces. The anachelin chromophore, merged via a PEG linker to the glycopeptide antibiotic vancomycin, allowed for the generation of antimicrobial surfaces through an operationally simple dip-and-rinse procedure. This approach offers an option for the prevention of nosocomial infections through antimicrobial implants, catheters and stents. Consequences for the mild generation of functional biomaterials are discussed and novel strategies for the immobilization of complex natural products, proteins and DNA on surfaces are presented.

Phosphinates as new electrophilic partners for cross-coupling reactions.

The use of enol phosphinates as electrophiles for cross-coupling reactions has been explored. Both boronic acids (Suzuki-Miyaura reaction) and stannanes (Stille reaction) couple efficiently with lactam derived phosphinates.

One-pot synthesis of benzothiazolines and napthathiazolines via cascade ortho-lithiation, cyclisation and elimination of N-arylsulfonyl lactams.

ortho-Lithiation of cyclic aryl sulfonamides in the presence of phosphoryl chloride provides a very simple entry to fused polycyclic sultams (benzothiazolines and naphthathiazolines).