Mesangiogenic Progenitor Cells Derived from One Novel CD64brightCD31brightCD14neg Population in Human Adult Bone Marrow

Mesenchymal Stromal Cells (MSCs) have been the object of extensive research for decades, due to their intrinsic clinical value. Nonetheless, the unambiguous identification of a unique in vivo MSC progenitor is still lacking, and the hypothesis that these multipotent cells could possibly arise from different in vivo precursors has been gaining consensus in the last years. We identified a novel multipotent cell population in human adult bone marrow that we firstly named Mesodermal Progenitor Cells (MPCs) for the ability to differentiate toward the mesenchymal lineage while still retaining angiogenic potential. Despite extensive characterization, MPCs positioning within the differentiation pathway and whether they can be ascribed as possible distinctive progenitor of the MSC lineage is still unclear. Here we describe the ex vivo isolation of one novel bone marrow sub-population (Pop#8) with the ability to generate MPCs. Multicolor flow cytometry in combination with either FACS or MACS cell sorting were applied to characterize Pop#8 as CD64brightCD31brightCD14neg. We defined Pop#8 properties in culture, including the potential of Pop#8-derived MPCs to differentiate into MSCs. Gene expression data were suggestive of Pop#8 in vivo involvement in HSC niche constitution/maintenance. Pop#8 resulted over three logs more frequent than other putative MSC progenitors, corroborating the idea that most of the controversies regarding culture expanded MSCs could be the consequence of different culture conditions which select or promote particular sub-populations of precursors

ED-B fibronectin expression is a marker of epithelial-mesenchymal transition in translational oncology

Fibronectin is a component of the extracellular matrix that links collagen fibers to integrins on the cell's surface. The splicing isoforms, containing the ED-B domain, are not expressed in adult tissues but only in tumor stroma or during embryonic development. Fibroblasts and endothelial cells express ED-B fibronectin during angiogenesis. Also cancer cells can synthetize ED-B fibronectin, but its function in tumor growth needs to be further elucidated. We evaluated the expression of ED-B fibronectin in prostate cancer cell lines: PC3 and DU145. Using TGF-β, we induced epithelial to mesenchymal transition in culture and observed an increase of ED-B fibronectin expression. Thereafter, we evaluated the expression of ED-B fibronectin in multipotent mesangiogenic progenitor cells, and in mesenchymal stromal cells. The expression of ED-B fibronectin was much higher in mesenchymal than prostate cancer cells even after the epithelial to mesenchymal transition. Epithelial to mesenchymal transition is a key step for tumor progression contributing to the metastatic spread. Therefore, circulating cancer cells could seed into the metastatic niche taking advantage from the ED-B fibronectin that secrete their own

The effect of high glucose on the inhibitory action of C21, a selective AT2R agonist, of LPS-stimulated tissue factor expression in human mononuclear cells

Background: Intimate links connect tissue factor (TF), the principal initiator of the clotting cascade, to inflammation, a cross-talk amplified by locally generated Angiotensin (AT) II, the effector arm of the Renin Angiotensin System (RAS). C21, a selective AT2R agonist, downregulates the transcriptional expression of TF in LPS-activated peripheral blood mononuclear cell(PBMC)s implying the existence of ATII type 2 receptor (AT2R)s whose stimulation attenuates inflammation-mediated procoagulant responses. High glucose, by activating key signalling pathways and increasing the cellular content of RAS components, augments TF expression and potentiates the inhibitory effect of AT1R antagonists. It is unknown, however, the impact of that stimulus on AT2R-mediated TF inhibition, an information useful to understand more precisely the role of that signal transduction pathway in the inflammation-mediated coagulation process. TF antigen (ELISA), procoagulant activity (PCA, 1-stage clotting assay) and TF-mRNA (real-time polymerase chain reaction) were assessed in PBMCs activated by LPS, a pro-inflammatory and procoagulant stimulus, exposed to either normal (N) or HG concentrations (5.5 and 50 mM respectively). Results: HG upregulated TF expression, an effect abolished by BAY 11-7082, a NFκB inhibitor. C21 inhibited LPS-stimulated PCA, TFAg and mRNA to an extent independent of glucose concentration but the response to Olmesartan, an AT1R antagonist, was quite evidently potentiated by HG. Conclusions: HG stimulates LPS-induced TF expression through mechanisms completely dependent upon NFkB activation. Both AT2R-stimulation and AT1R-blockade downregulate inflammation-mediated procoagulant response in PBMCs but HG impacts differently on the two different signal transduction pathway

Constitutive Expression of Pluripotency-Associated Genes in Mesodermal Progenitor Cells (MPCs)

Background: We recently characterized a progenitor of mesodermal lineage (MPCs) from the human bone marrow of adults or umbilical cord blood. These cells are progenitors able to differentiate toward mesenchymal, endothelial and cardiomyogenic lineages. Here we present an extensive molecular characterization of MPCs, from bone marrow samples, including 39 genes involved in stem cell machinery, differentiation and cell cycle regulation. Methodology/Principal Findings: MPCs are cytofluorimetrically characterized and quantitative RT-PCR was performed to evaluate the gene expression profile, comparing it with MSCs and hESCs lines. Immunofluorescence and dot-blot analysis confirm qRT-PCR data. MPCs exhibit an increased expression of OCT4, NANOG, SALL4, FBX15, SPP1 and to a lesser extent c-MYC and KLF4, but lack LIN28 and SOX2. MPCs highly express SOX15. Conclusions/Significance: MPCs express many pluripotency-associated genes and show a peculiar Oct-4 molecular circuit. Understanding this unique molecular mechanism could lead to identifying MPCs as feasible, long telomeres, target cells for reprogramming with no up-regulation of the p53 pathway. Furthermore MPCs are easily and inexpensively harvested fro

Non enzymatic upregulation of tissue factor expression by gamma-glutamyl transferase in human peripheral blood mononuclear cells

Background Besides maintaining intracellular glutathione stores, gamma-glutamyltransferase(GGT) generates reactive oxygen species and activates NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the principal initiator of the clotting cascade. Thus, GGT might be involved in TF-mediated coagulation processes, an assumption untested insofar. Methods Experiments were run with either equine, enzymatically active GGT or human recombinant (hr) GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation. TF Procoagulant Activity (PCA, one-stage clotting assay), TF antigen(ELISA) and TFmRNA(real-time PCR) were assessed in unpooled human peripheral blood mononuclear cell(PBMC) suspensions obtained from healthy donors through discontinuous Ficoll/Hystopaque density gradient. Results Equine GGT increased PCA, an effect insensitive to GGT inhibition by acivicin suggesting mechanisms independent of its enzymatic activity, a possibility confirmed by the maintained stimulation in response to hrGGT, an enzymatically inactive molecule. Endotoxin(LPS) contamination of GGT preparations was excluded by heat inactivation studies and direct determination(LAL method) of LPS concentrations <0.1 ng/mL practically devoid of procoagulant effect. Inhibition by anti-GGT antibodies corroborated that conclusion. Upregulation by hrGGT of TF antigen and mRNA and its downregulation by BAY-11-7082, a NFkB inhibitor, and N-acetyl-L-cysteine, an antioxidant, was consistent with a NFkB-driven, redox-sensitive transcriptional site of action. Conclusions GGT upregulates TF expression independent of its enzymatic activity, a cytokine-like behaviour mediated by NFκB activation, a mechanism contributing to promote acute thrombotic events, a possibility in need, however, of further evaluation

A Proteomic Approach Identified TFEB as a Key Player in the Protective Action of Novel CB2R Bitopic Ligand FD22a against the Deleterious Effects Induced by β-Amyloid in Glial Cells

Neurodegenerative diseases (NDDs) are progressive multifactorial disorders of the nervous system sharing common pathogenic features, including intracellular misfolded protein aggregation, mitochondrial deficit, and inflammation. Taking into consideration the multifaceted nature of NDDs, development of multitarget-directed ligands (MTDLs) has evolved as an attractive therapeutic strategy. Compounds that target the cannabinoid receptor type II (CB2R) are rapidly emerging as novel effective MTDLs against common NDDs, such as Alzheimer's disease (AD). We recently developed the first CB2R bitopic/dualsteric ligand, namely FD22a, which revealed the ability to induce neuroprotection with fewer side effects. To explore the potential of FD22a as a multitarget drug for the treatment of NDDs, we investigated here its ability to prevent the toxic effect of β-amyloid (Aβ25-35 peptide) on human cellular models of neurodegeneration, such as microglia (HMC3) and glioblastoma (U87-MG) cell lines. Our results displayed that FD22a efficiently prevented Aβ25-35 cytotoxic and proinflammatory effects in both cell lines and counteracted β-amyloid-induced depression of autophagy in U87-MG cells. Notably, a quantitative proteomic analysis of U87-MG cells revealed that FD22a was able to potently stimulate the autophagy-lysosomal pathway (ALP) by activating its master transcriptional regulator TFEB, ultimately increasing the potential of this novel CB2R bitopic/dualsteric ligand as a multitarget drug for the treatment of NDDs

Metabolic reprogramming by 3-Iodothyronamine (T1AM): a new perspective to reverse obesity through co-regulation of sirtuin 4 and 6 expression

Obesity is a complex disease associated with environmental and genetic factors. 3-Iodothyronamine (T1AM) has revealed great potential as an effective weight loss drug. We used metabolomics and associated transcriptional gene and protein expression analysis to investigate the tissue specific metabolic reprogramming effects of subchronic T1AM treatment at two pharmacological daily doses (10 and 25 mg/kg) on targeted metabolic pathways. Multi-analytical results indicated that T1AM at 25 mg/kg can act as a novel master regulator of both glucose and lipid metabolism in mice through sirtuin-mediated pathways. In liver, we observed an increased gene and protein expression of Sirt6 (a master gene regulator of glucose) and Gck (glucose kinase) and a decreased expression of Sirt4 (a negative regulator of fatty acids oxidation (FAO)), whereas in white adipose tissue only Sirt6 was increased. Metabolomics analysis supported physiological changes at both doses with most increases in FAO, glycolysis indicators and the mitochondrial substrate, at the highest dose of T1AM. Together our results suggest that T1AM acts through sirtuin-mediated pathways to metabolically reprogram fatty acid and glucose metabolism possibly through small molecules signaling. Our novel mechanistic findings indicate that T1AM has a great potential as a drug for the treatment of obesity and possibly diabetes

A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in Vero E6 cells

: We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells

Mesodermal Progenitor Cells (MPCs) Differentiate into Mesenchymal Stromal Cells (MSCs) by Activation of Wnt5/Calmodulin Signalling Pathway

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4+CD105+CD90(neg) phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4(neg)CD105+CD90(bright) and MSCA-1+), in the primary cultures, resulted lower than 2%.We demonstrate that MPCs differentiate to MSCs through an SSEA-4+CD105+CD90(bright) early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4(neg)). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ =  0.5 µM, p<0.01), while endothelial differentiation was unaffected.The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required

Specific integrin expression is associated with podosome-like structures on mesodermal progenitor cells.

Mesenchymal stromal cells (MSCs) are a heterogeneous cell population capable of differentiating toward several cell lines in vitro and, possibly, in vivo. Within cultured MSCs, we identified and purified a precursor cell population [mesodermal progenitor cells (MPCs)] retaining robust proliferation potential and ability to differentiate into endothelial or mesenchymal cells. MPC-derived MSCs retain the ability to further differentiate into osteoblasts, cartilage, or fat cells. Here we further characterized MPCs and MSCs by evaluating expression of integrins and adhesion molecules showing their ability to assemble the molecular machinery involved in endothelium adhesion. MPCs were shown to interact with activated and nonactivated endothelium, whereas MSCs exhibited activation of focal adhesion complexes, higher cell motility, and reduced or absent adhesiveness onto endothelial cells, suggesting a matrix remodeling vocation. We also reported a consistent expression of CXCR4 on the MPC cell surface, suggesting that the different phenotypic behavior could be related to specific functions of the cell in each differentiation stage