Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

The proresolution lipid mediator lipoxin (LX)A4 bestows protective bioactions on endothelial cells. We examined the impact of LXA4 on transcellular endothelial signaling via microRNA (miR)-containing microvesicles. We report LXA4 inhibition of MV release by TNF-α-treated HUVECs, associated with the down-regulation of 18 miR in endothelial microvesicles (EMVs) and the up-regulation of miR-126-5p, both in HUVECs and in EMVs. LXA4 up-regulated miR-126-5p by ∼5-fold in HUVECs and promoted a release of microvesicles (LXA4-EMVs) that enhanced miR-126-5p by ∼7-fold in recipient HUVECs. In these cells, LXA4-EMVs abrogated the up-regulation of VCAM-1, induced in recipient HUVECs by EMVs released by untreated or TNF-α-treated HUVECs. LXA4-EMVs also reduced by ∼40% the expression of SPRED1, which we validated as an miR-126-5p target, whereas they stimulated monolayer repair in an in vitro wound assay. This effect was lost when the EMVs were depleted of miR-126-5p. These results provide evidence that changes in miR expression and microvesicle packaging and transfer represent a mechanism of action of LXA4, which may be relevant in vascular biology and inflammation.-Codagnone, M., Recchiuti, A., Lanuti, P., Pierdomenico, A. M., Cianci, E., Patruno, S., Mari, V. C., Simiele, F., Di Tomo, P., Pandolfi, A., Romano, M. Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

Epigenetic regulation of the formyl peptide receptor 2 gene.

Lipoxin (LX) A4, a main stop signal of inflammation, exerts potent bioactions by activating a specific G protein-coupled receptor, termed formyl peptide receptor 2 and recently renamed ALX/FPR2. Knowledge of the regulatory mechanisms that drive ALX/FPR2 gene expression is key for the development of innovative anti-inflammatory pharmacology. Here, we examined chromatin patterns of the ALX/FPR2 gene. We report that in MDA-MB231 breast cancer cells, the ALX/FPR2 gene undergoes epigenetic silencing characterized by low acetylation at lysine 27 and trimethylation at lysine 4, associated with high methylation at lysine 27 of histone 3. This pattern, which is consistent with transcriptionally inaccessible chromatin leading to low ALX/FPR2 mRNA and protein expression, is reversed in polymorphonuclear leukocytes that express high ALX/FPR2 levels. Activation of p300 histone acetyltransferase and inhibition of DNA methyltransferase restored chromatin accessibility and significantly increased ALX/FPR2 mRNA transcription and protein levels in MDA-MB231 cells, as well as in pulmonary artery endothelial cells. In both cells types, changes in the histone acetylation/methylation status enhanced ALX/FPR2 signaling in response to LXA4. Collectively, these results uncover unappreciated epigenetic regulation of ALX/FPR2 expression that can be exploited for innovative approaches to inflammatory disorders.

MicroRNA-181b regulates ALX/FPR2 receptor expression and proresolution signaling in human macrophages.

Regulatory mechanisms of ALX/FPR2, the lipoxin A4 receptor, expression have considerable relevance in inflammation resolution. Because microRNAs (miRs) are emerging as key players in inflammation resolution, here we examined microRNA-mediated regulation of ALX/FPR2 (lipoxin A4 receptor/formyl peptide receptor 2) expression. By matching data from bioinformatic algorithms, we found 27 miRs predicted to bind the 3'-UTR of ALX/FPR2. Among these, we selected miR-181b because of its link with inflammation. Using a luciferase reporter system, we assessed miR-181b binding to ALX/FPR2 3'-UTR. Consistent with this, miR-181b overexpression in human macrophages significantly down-regulated ALX/FPR2 protein levels (-25%), whereas miR-181b knockdown gave a significant increase in ALX/FPR2 (+60%). miR-181b levels decreased during monocyte to macrophage differentiation (-50%), whereas ALX/FPR2 expression increased significantly (+60%). miR-181b overexpression blunted lipoxin A4 (0.1-10 nm)- and resolvin D1 (0.01-10 nm)-stimulated phagocytic activity of macrophages. These results unravel novel regulatory mechanisms of ALX/FPR2 expression and ligand-evoked macrophages proresolution responses mediated by miR-181b, thus uncovering novel components of the endogenous inflammation resolution circuits.

Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.

Resolvin D1 (RvD1; 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) is an endogenous immunoresolvent that regulates acute inflammation and orchestrates resolution. Here, we investigated anti-inflammatory and proresolving actions of RvD1 after oral administration. RvD1 rapidly accumulated in the mouse plasma after oral delivery and dose-dependently (1-100 ng/mouse) reduced leukocyte infiltration in zymosan A-induced acute peritonitis. Using mathematical resolution indices, RvD1 reduced Ψmax by ∼50%, shortened the resolution interval by 3 h, and significantly reduced total leukocyte (by ∼30-45%) and polymorphonuclear neutrophil (by ∼40-55%) accumulation when administered at the peak of peritonitis. RvD1 also improved course and outcome of severe peritonitis, shifting it toward resolution. In peritoneal macrophages (MΦs) from the resolution phase of peritonitis, RvD1 down-regulated (by 2- to 3-fold) select genes that control gene transcription, namely coactivator-associated arginine methyltransferase 1 (CARM1), and downstream genes, such as colony-stimulating factor 3, intercellular adhesion molecule 1, and monocyte inflammatory protein 2, which promote neutrophil infiltration and reduce MΦ phagocytosis. Congruently, CARM1 knockdown in human and murine MΦs induced a proresolving phenotype, recapitulating in vivo actions of RvD1. These results establish novel properties of RvD1 and demonstrate that RvD1 modifies the transcription control machinery in MΦs, as part of its mechanisms of action during the resolution of acute inflammation.-Recchiuti, A., Codagnone, M., Pierdomenico, A. M., Rossi, C., Mari, V. C., Cianci, E., Simiele, F., Gatta, V., Romano, M. Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.

Transcriptional regulation of the human FPR2/ALX gene: evidence of a heritable genetic variant that impairs promoter activity.

Lipoxin (LX) A(4,) a main endogenous stop-signal of inflammation, activates the G-protein-coupled receptor FPR2/ALX, which triggers potent anti-inflammatory signaling in vivo. Thus, the regulation of FPR2/ALX expression may have pathophysiological and therapeutic relevance. Here, we mapped a nucleotide sequence with strong FPR2/ALX promoter activity. Chromatin immunoprecipitation revealed specificity protein 1 (Sp1) binding to the core promoter. Site-directed mutagenesis of the Sp1 cis-acting element and Sp1 overexpression established that this transcription factor is key for maximal promoter activity, which is instead suppressed by DNA methylation. LXA(4) enhanced FPR2/ALX promoter activity (+74%) and mRNA expression (+87.5%) in MDA-MB231 cells. A single nucleotide mutation (A/G) was detected in the core promoter of one subject with history of cardiovascular disease and of his two daughters. This mutation reduced by ∼35-90% the promoter activity in vitro. Moreover, neutrophils from individuals carrying the A/G variant displayed ∼10- and 3-fold reduction in FPR2/ALX mRNA and protein, respectively, compared with cells from their relatives or healthy volunteers expressing the wild-type allele. These results uncover FPR2/ALX transcriptional regulation and provide the first evidence of mutations that affect FPR2/ALX transcription, thus opening new opportunities for the understanding of the LXA(4)-FPR2/ALX axis in human disease.

Persistence and adherence to androgen deprivation therapy in men with prostate cancer: an administrative database study.

BACKGROUND: The aim of this study was to assess adherence to and persistence with androgen deprivation therapy (ADT) in a large cohort of prostate cancer (PCa) patients selected from an administrative database, with special attention to elderly patients. METHODS: Patients treated with LHRH analogues, LHRH antagonists, the novel androgen antagonist enzalutamide, and the CYP17 inhibitor abiraterone were included spanning the years 2011-2017. Descriptive statistics were used to analyze persistence and adherence in older patients stratified by age (46-55, 56-65, 66-75, 76-85, and >85 years). The effect of persistence duration on overall survival in super-elderly patients was analyzed by the Kaplan-Meier method, together with the influence of multiple prescriptions on overall survival. RESULTS: A total of 1160 male patients were treated with ADT. Of these, 1075 were given LHRH analogues, 80 LHRH antagonists, 14 novel androgen antagonists, and 109 the CYP17 inhibitor. Median adherence values were 0.93, 0.97, 0.95, and 0.99 respectively. The highest persistence was recorded for LHRH analogues/antagonists (24 months), followed enzalutamide and abiraterone (8 months). A total of 107 patients (9.2%) were classified as super-elderly (age range 85-97 years). Median persistence and OS in this group were 13 months and 29 months, respectively. The adherence was 0.92. Overall survival was significantly associated with additional prescriptions for other conditions-indications (P=0.0047) but not with differences in adherence rates (P=0.98). CONCLUSIONS: Our data showed high adherence and persistence rates in men on ADT. The overall survival in the super-elderly is not influenced by persistence and/or adherence but rather by coprescriptions.

microRNA-181b is increased in cystic fibrosis cells and impairs lipoxin A4 receptor-dependent mechanisms of inflammation resolution and antimicrobial defense.

The involvement of microRNA (miR) in cystic fibrosis (CF) pathobiology is rapidly emerging. We previously documented that miR-181b controls the expression of the ALX/FPR2 receptor, which is recognized by the endogenous proresolution ligand, lipoxin (LX)A4. Here, we examined whether the miR-181b-ALX/FPR2 circuit was altered in CF. We examined human airways epithelial cells, normal (16HBE14o-), carrying the ΔF508 mutation (CFBE41o-) or corrected for this mutation (CFBE41o-/CEP-CFTR wt 6.2 kb), as well as monocyte-derived macrophages (MΦs) from CF patients. CFBE41o- cells exhibited higher miR-181b and reduced ALX/FPR2 levels compared to 16HBE14o- and CFBE41o-/CEP-CFTR wt 6.2 kb cells. An anti-mir-181b significantly enhanced ALX/FPR2 expression (+ 60%) as well as LXA4-induced increase in transepithelial electric resistance (+ 25%) in CFBE41o- cells. MΦs from CF patients also displayed increased miR-181b (+ 100%) and lower ALX/FPR2 levels (- 20%) compared to healthy cells. An anti-mir-181b enhanced ALX/FPR2 expression (+ 40%) and normalized receptor-dependent LXA4-induced phagocytosis of fluorescent-labeled zymosan particles as well as of Pseudomonas aeruginosa by CF-MΦs. These results provide the first evidence that miR-181b is overexpressed in CF cells, impairing some mechanisms of the ALX/FPR2-dependent pathway of inflammation resolution. Thus, targeting miR-181b may represent a strategy to enhance anti-inflammatory and anti-microbial defense mechanisms in CF.

Lipoxins and aspirin-triggered lipoxins in resolution of inflammation.

The resolution of the inflammatory response is highly regulated by the timely biosynthesis of a number of endogenous lipid mediators. Among these, lipoxins (LX) and their 15-epimers, aspirin triggered lipoxins (ATL) derived by the lipoxygenase (LO) route of arachidonic acid metabolism. In particular, they are formed and released by cells expressing 5-, 12- and 15-LO such as leukocytes, platelets, vascular endothelium and epithelium, alone or during transcellular interactions. ATL biosynthesis requires cyclooxygenase-2 acetylation by aspirin. LX and ATL exert potent bioactions on leukocytes, vascular and epithelial cells to stop inflammation and promote resolution. They have shown to be beneficial in a broad spectrum of preclinical models of disease as well as in some clinical trials. Counter-regulatory signaling by LXA4 and 15-epi-LXA4 follows the activation of a G protein-coupled receptor, termed ALX/FPR2, which is emerging as a key anti-inflammatory receptor.

Role of Hydroxamate-Based Histone Deacetylase Inhibitors (Hb-HDACIs) in the Treatment of Solid Malignancies.

Hydroxamate-based histone deacetylase inhibitors (Hb-HDACIs), such as vorinostat, belinostat and panobinostat, have been previously shown to have a wide range of activity in hematologic malignancies such as cutaneous T-cell lymphoma and multiple myeloma. Recent data show that they synergize with a variety of cytotoxic and molecular targeted agents in many different solid tumors, including breast, prostate, pancreatic, lung and ovarian cancer. Hb-HDACIs have a quite good toxicity profile and are now being tested in phase I and II clinical trials in solid tumors with promising results in selected neoplasms, such as hepatocarcinoma. This review will focus on their clinical activity and safety in patients with advanced solid neoplasms.