GM-CSF-activated STAT5A regulates macrophage functions and inflammation in atherosclerosis.

Introduction: Inhibition of STAT5 was recently reported to reduce murine atherosclerosis. However, the role of STAT5 isoforms, and more in particular STAT5A in macrophages in the context of human atherosclerosis remains unknown. Methods and results: Here, we demonstrate reciprocal expression regulation of STAT5A and STAT5B in human atherosclerotic lesions. The former was highly upregulated in ruptured over stable plaque and correlated with macrophage presence, a finding that was corroborated by the high chromosomal accessibility of STAT5A but not B gene in plaque macrophages. Phosphorylated STAT5 correlated with macrophages confirming its activation status. As macrophage STAT5 is activated by GM-CSF, we studied the effects of its silencing in GM-CSF differentiated human macrophages. STAT5A knockdown blunted the immune response, phagocytosis, cholesterol metabolism, and augmented apoptosis terms on transcriptional levels. These changes could partially be confirmed at functional level, with significant increases in apoptosis and decreases in lipid uptake and IL-6, IL-8, and TNFa cytokine secretion after STAT5A knockdown. Finally, inhibition of general and isoform A specific STAT5 significantly reduced the secretion of TNFa, IL-8 and IL-10 in ex vivo tissue slices of advanced human atherosclerotic plaques. Discussion: In summary, we identify STAT5A as an important determinant of macrophage functions and inflammation in the context of atherosclerosis and show its promise as therapeutic target in human atherosclerotic plaque inflammation.

Asymmetrically Substituted m-Terphenyl Phosphates Inhibit the Transcription Factor STAT5a.

We recently presented Stafia-1 as the first chemical entity that inhibits the transcription factor STAT5a with selectivity over the highly homologous STAT5b. Stafia-1, which was identified from a series of symmetrically substituted m-terphenyl phosphates, binds to the interface between the SH2 domain and the linker domain of STAT5a. Here, we outline a synthetic strategy for the synthesis of asymmetrically substituted m-terphenyl phosphates, which can be tailored to address their asymmetric STAT5a binding site in a more specific manner. The asymmetrically substituted m-terphenyl phosphate with the highest activity against STAT5a was converted to a phosphatase-stable monofluoromethylene phosphonate. The synthetic methodology and activity analysis described here provide first insights into the structure-activity relationships of m-terphenyl phosphates for use as selective STAT5a inhibitors.

Stafia-1: a STAT5a-Selective Inhibitor Developed via Docking-Based Screening of in Silico O-Phosphorylated Fragments.

We present a new approach for the identification of inhibitors of phosphorylation-dependent protein-protein interaction domains, in which phenolic fragments are adapted by in silico O-phosphorylation before docking-based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product-derived phenolic fragments, which were virtually O-phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia-1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia-1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small-molecule inhibitor.

Free oxysterols and bile acids including conjugates - Simultaneous quantification in human plasma and cerebrospinal fluid by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI(+)-MS/MS) assay was developed and qualified for analyzing 35 analytes of the cholesterol metabolism, including free cholesterol, 17 free, non-esterified oxysterols and 17 free and conjugated bile acids in plasma and cerebrospinal fluid. As internal standards, 25 commercially available stable deuterium-labeled analogs of the analytes were used. Pre-analytical investigations included stability tests of analyte concentrations affected by different anticoagulation additives: lithium heparin-, citrate-, EDTA-K3-stabilized plasma and serum, and the stability in EDTA whole blood at RT. This LC-ESI(+)-MS/MS method was successfully applied for the analysis of paired serum/cerebrospinal fluid samples of patients with and without blood-brain barrier disturbance, as well as of 100 plasma samples of a LIFE-Adult study sub-cohort. A fast and simple sample preparation including protein precipitation and on-line solid-phase extraction was developed. As little as 55 µL of human plasma/serum or cerebrospinal fluid were needed for the analysis. It was possible to separate isomeric oxysterols and bile acids within 23 min using a C18 core-shell column. The assay is capable of quantifying in a linear range of 0.8-250 ng mL-1 for free hydroxycholesterols, 0.2-10 ng mL-1 for dihydroxycholesterols, 0.2-500 ng mL-1 for bile acids and 16-2000 µg mL-1 for cholesterol with acceptable accuracy and precision. In cerebrospinal fluid one free oxysterols, five free and five conjugated bile acids could be quantified. No significant differences between patients with and without blood-brain barrier disturbance were obtained. In the LIFE-Adult sub-cohort two free oxysterols, four free and seven conjugated bile acids could be quantified in EDTA plasma. Men showed significantly higher concentrations of 26-OHC than women (p = 0.035). Furthermore, in women lower levels of cholic acid, glycocholic acid, glycodeoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, glycoursodeoxycholic acid, glycolithocholic acid and higher levels of taurocholic acid, taurochenodeoxycholic acid, ursodeoxycholic acid/hyodeoxycholic acid were quantified.