Eleonora Cianci

Wnt Signaling Behaves as a “Master Regulator” in the Osteogenic and Adipogenic Commitment of Human Amniotic Fluid Mesenchymal Stem Cells

AbstractHuman amniotic fluid mesenchymal stem cells (huAFMSCs) are emerging as a promising therapeutic option in regenerative medicine. Here, we characterized huAFMSC phenotype and multipotentiality. When cultured in osteogenic medium, huAFMSC displayed a significant increase in: Alkaline Phosphatase (ALP) activity and mRNA expression, Alizarin Red S staining and Runx2 mRNA expression; whereas maintaining these cells in an adipogenic culture medium gave a time-dependent increase in PPARγ and FABP4 mRNA expression, glycerol-3-phosphate dehydrogenase (GPDH) activity and positivity to Oil Red Oil staining. These results confirm that huAFMSCs can differentiate toward osteogenic and adipogenic phenotypes. The canonical Wnt/ßcatenin signaling pathway appears to trigger huAFMSC osteoblastogenesis, since during early phases of osteogenic differentiation, the expression of Dishevelled-2 (Dvl-2), of the non-phosphorylated form of ß-catenin, and the phosphorylation of glycogen synthase kinase-3ß (GSK3ß) at serine 9 were upregulated. On the contrary, during adipogenic differentiation Dvl-2 expression decreased, whereas that of ß-catenin remained unchanged. This was associated with a late increase in GSK3ß phosphorylation. Consistent with this scenario, huAFMSCs exposure to Dickkopf-1, a selective inhibitor of the Wnt signaling, abolished Runx2 and ALP mRNA upregulation during huAFMSC osteogenic differentiation, whereas it enhanced FABP4 expression in adipocyte-differentiating cells. Taken together, these results unravel novel molecular determinants of huAFMSC commitment towards osteoblastogenesis, which may represent potential targets for directing the differentiation of these cells and improving their use in regenerative medicine. FigureSchematic representation of Wnt pathway involved in the osteogenic and adipogenic differentiation of huAFMSCs. Our paper demonstrates that osteogenic commitment of these cells is linked to the stimulation of Wnt signal leading to the final transcriptional activation of early osteogenic markers such as RUNX-2 and ALP, mediated by β-catenin. DKK1 is a secreted Wnt antagonist that may be used as a drug to inhibit Wnt signal. In contrast, adipogenic commitment involves early inhibition of Wnt pathway leading to ubiquitination/degradation of β-catenin. This results in the transcription of PPARγ and FABP4, considered as the main initiators of adipogenesis. APC, adenomatous polyposis coli; βcat, β-catenin; CK1, casein kinase 1; DKK1, dickkopf 1; Dvl, Dishevelled; GSK3β, glycogen synthase kinase 3β; LRP5/6, low density lipoprotein receptor-related protein 5/6

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.

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

Lipoxin (LX) A4 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 immuno‐precipitation 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. LXA4 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 LXA4‐FPR2/ALX axis in human disease —Simiele, F., Recchiuti, A., Mattoscio, D., De Luca, A., Cianci, E., Franchi, S., Gatta, V., Parolari, A., Werba, J. P., Camera, M., Favaloro, B., Romano, M. Transcriptional regulation of the human FPR2/ALX gene: Evidence of a heritable genetic variant that impairs promoter activity. FASEB J. 26, 1323‐1333 (2012). www.fasebj.org

Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins

Unresolved inflammation and tissue destruction are underlying mechanisms of periodontitis, which is linked to dysregulated polymorphonuclear neutrophil (PMN) functions. Lipoxin A4 (LXA4) is a specialized proresolving lipid mediator (SPM) that dampens excessive inflammation, promotes resolution, and protects from leukocyte‐mediated tissue damage. Human periodontal ligament stem cells (hPDLSCs) represent key players during tissue regeneration and may contribute to resolution of inflammation; thus, they may represent a promising tool in regenerative dentistry. In the present study, we investigated the actions of hPDLSCs on PMN apoptosis and antimicrobial functions, and determined the impact of LXA4 on hPDLSCs. hPDLSCs significantly reduced apoptosis and stimulated microbicidal activity of human PMNs, via both cell‐cell interactions and paracrine mechanisms. Lipid mediator metabololipidomics analysis demonstrated that hPDLSCs biosynthesize SPMs, including resolvin D1, D2, D5, and D6; protectin D1; maresins; and LXB4; as well as prostaglandins D2, E2, and F2α. LXA4 significantly enhanced proliferation, migration, and wound healing capacity of hPDLSCs through the activation of its cognate receptor ALX/FPR2, expressed on hPDLSCs. Together, these results demonstrate that hPDLSCs modulate PMN functions, and provide the first evidence that stem cells generate SPM and that the LXA4‐ALX/FPR2 axis regulates regenerative functions of hPDLSCs by a novel receptor‐mediated mechanism.

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. FASEB J. 28, 3090–3102 (2014). www.fasebj.org

Diabetes Mellitus During Pregnancy Interferes with the BiologicalCharacteristics of Wharton's Jelly Mesenchymal Stem Cells

Recent research indicates that the origin of obesity and related metabolic disorders is not only caused by genetic and risk factors in adult life (unbalanced diet, insufficient physical activity) but also may be influenced by the perinatal environment. In addition, studies in animal models suggest that the mesenchymal stem cell commitment into pre-adipocytes can already occur during fetal development and perinatal life. Since the number of pre-adipocytes and mature adipocytes is lower in normal subjects than in obese subjects, changes in the prenatal maturational process may play a role in the pathogenesis of obesity and metabolic-associated diseases. Hyperglycemia during pregnancy is related to an increased risk of obesity, early onset of metabolic syndrome and type 2 diabetes in the offspring. For this reason it would be useful to investigate how the perinatal environment may affect fetal mesenchymal stem cells, especially in deregulated gestational diabetes, where the fetal environment is modified in terms of hormone levels and nutrition. Therefore, we have compared Wharton’s jelly mesenchymal stem cells (WJ-MSC) obtained from umbilical cord of both healthy and diabetic mothers, in order to better understand the mechanisms involved in metabolic diseases in offspring of diabetic mothers. Results indicate that WJ-MSC from diabetic mothers display, in contrast to cells from healthy mothers, a higher ability to differentiate towards the adipogenic lineage. This suggests that the diabetic uterine environment may be responsible for a “pre-commitment” that could give rise in the post natal life to an alteration of adipocyte production upon an incorrect diet style, which in turn would produce obesity.

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 upregulated 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. FASEB J. 31, 1856–1866 (2017). www.fasebj.org

Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.

Immobilization and delivery of biologically active Lipoxin A4 using electrospinning technology.

Lipoxin (LX)A4 is a lipoxygenase-formed arachidonic acid metabolite with potent anti-inflammatory, pro-resolution properties. Its therapeutic efficacy has been largely demonstrated in a variety of cellular, preclinical and clinical models. Among these, periodontal disease, where LXA4 promotes tissue repair, also by modulating functions of human periodontal ligament stem cells (hPDLSCs). As medicated biomembranes may be particularly useful in clinical settings, where local stimulation of tissue repair is needed, we used electrospinning to embed LXA4 in membranes made of poly(ethylene oxide) (PEO) and poly(d,l-lactide) (PDLLA). These membranes were fully characterized by scanning electron microscopy, differential scanning calorimetry and biocompatibility with hPDLSCs. Here, we report that LXA4 is retained in these membranes and that ∼15-20% of the total LXA4 amount added to the reaction can be eluted from the membranes using an aqueous buffered medium. The eluted LXA4 fully retained its capability to stimulate hPDLSC proliferation. A similar effect was obtained by adding directly the LXA4-containing membranes to cells. These results demonstrate for the first time that LXA4 can be incorporated into biomembranes, which may be useful to combat local inflammation and promote tissue repair in selected clinical settings.

Lipoxins, Resolvins, and the Resolution of Inflammation

Resolution of acute inflammation is an active process, where endogenous specialized pro-resolving mediators (SPM), derived from polyunsaturated fatty acids via lipoxygenase (LO)-driven biochemical pathways, have pivotal roles in “turning off” pro-inflammatory signals, prompting timely resolution. SPM potent and stereospecific bioactions are of considerable interest in immunopharmacology since unresolved inflammation represents a key pathogenetic mechanism of several widespread diseases.