Osteocyte-derived exosomes inhibit osteoblast activity and induce osteoclast formation

Osteocytes, which are the most abundant cell type in bone, regulate osteoblasts and osteoclasts via both cell–cell interactions and paracrine signaling, and osteocyte-derived exosomes might contribute to this paracrine action. In this study, we investigated the effects of osteocyte-derived exosomes on regulating osteoblasts and osteoclasts and studied the potential mechanism. Materials and Methods: Osteocyte-derived exosomes were extracted and identified. PKH67-labeled exosomes were incubated with MC3T3-E1 cells and RAW264.7 cells, and fluorescence confocal microscopy was used to analyze the uptake of exosomes. ALP stain- ing and TRAP staining were used to analyze osteoblast activity and osteoclast formation. The level of miR-214-3p in exosomes was analyzed by qPCR and the incorporation of FAM-labeled miR-214-3p from exosomes into MC3T3-E1 cells was evaluated. The expressions of ephrinA2 and RANKL in exosomes were studied. Results: Our results demonstrated that osteocyte-derived exosomes might recognize osteoblasts through the ephrinA2 protein; thus, miR-214-3p in exosomes was transferred into osteoblasts to inhibit osteoblast activity. Meanwhile, we found that osteocyte-derived exosomes could be transferred into osteoclasts to induce osteoclast formation by releasing RANKL. Conclusion: These findings suggest that osteocyte-derived exosomes play an important role in the regulation of osteoblast and osteoclast activity, which might occur via miR-214-3p and RANKL.

Effects of PACAP on Schwann Cells: Focus on Nerve Injury

Schwann cells, the most abundant glial cells of the peripheral nervous system, represent the key players able to supply extracellular microenvironment for axonal regrowth and restoration of myelin sheaths on regenerating axons. Following nerve injury, Schwann cells respond adaptively to damage by acquiring a new phenotype. In particular, some of them localize in the distal stump to form the Bungner band, a regeneration track in the distal site of the injured nerve, whereas others produce cytokines involved in recruitment of macrophages infiltrating into the nerve damaged area for axonal and myelin debris clearance. Several neurotrophic factors, including pituitary adenylyl cyclase-activating peptide (PACAP), promote survival and axonal elongation of injured neurons. The present review summarizes the evidence existing in the literature demonstrating the autocrine and/or paracrine action exerted by PACAP to promote remyelination and ameliorate the peripheral nerve inflammatory response following nerve injury.

SDF-1 secreted by mesenchymal stem cells promotes the migration of endothelial progenitor cells via PI3K/Akt pathway

Background: Cell-based therapeutics bring great hope in areas of unmet medical needs. Mesenchymal stem cells (MSCs) has been suggested to facilitate neovascularization mainly by paracrine action, and endothelial progenitor cells (EPCs) can differentiate into mature endothelial cells. Studies have demonstrated that a combination cell therapy that includes MSCs and EPCs has a favorable effect on ischemic limbs. However, the mechanism of combination cell therapy remains unclear. Herein, we investigate whether stromal cell-derived factor (SDF)-1 secreted by MSCs contributes to. Furthermore, we examined whether SDF-1 affects EPC migration via Phosphoinositide 3-Kinases (PI3K)/protein kinase B (termed as Akt) signaling pathway.Methods: First, intramuscular MSC injections were supplemented with intravenous EPC injections in the mouse model of hind limb ischemia. The incorporation of Qdot® 525 labeled-EPC into the vasculature and capillary density was evaluated by CD31 immunohistochemistry and immunofluorescence, respectively. Then, the concentration of SDF-1 secreted by MSCs was detected via quantitative immunoassay. Flow cytometry was performed to quantify CXC chemokine receptor (CXCR) 4-positive EPCs. The effect of MSCs on EPC migration was measured by a transwell system and a tube-like structure formation on Matrigel. The SDF-1 antagonist AMD3100 and the PI3K inhibitor wortmannin were separately used to determine the participation of CXCR4 and PI3K into EPC migration. Finally, western blot assay was performed to detect the effect of SDF-1 secreted by MSCs on Akt phosphorylation in EPCs.Results: The combination delivery of MSCs and EPCs via a “dual-administration” approach enhanced the incorporation of EPCs into the vasculature and increased the capillary density in mouse ischemic hind limb. The SDF-1 concentration secreted by MSCs was 2.61 ng/ml after 48 h. CXCR4-positive EPCs increased after incubation with MSC-conditioned medium (CM). MSCs contributed to EPC migration and tube-like structure formation, both of which were suppressed by AMD3100 and wortmannin. Phospho-Akt induced by MSC-CM was attenuated when EPCs were pretreated with AMD3100 and wortmannin.Conclusions: The paracrine action of MSCs contributes to EPC migration. Furthermore, SDF-1 secreted by MSCs induces EPC migration. The mechanism of this migration is related to the activation of the Akt pathway

Soluble PTX3 of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Attenuates Hyperoxic Lung Injury by Activating Macrophage Polarization in Neonatal Rat Model

Therapeutic treatment of various inflammation-related diseases using mesenchymal stem cells (MSCs) has increased in recent years because of the paracrine action of these cells but shows several limitations. First, MSC-based therapies exhibit varying efficacies; thus, biomarkers should be determined to identify who may benefit from these candidate therapeutic agents. Second, the mechanism underlying the therapeutic effects is poorly understood. To evaluate the effects of human umbilical cord blood-derived MSCs (UCB-MSCs) on macrophages, the macrophage cell line NR8383 stimulated with lipopolysaccharide (LPS) was cocultured by UCB-MSCs. We found that UCB-MSCs mediated changes in macrophage polarization towards M2 from M1 macrophages. To identify the paracrine action underlying the anti-inflammation effect of UCB-MSCs, the secretion of UCB-MSCs exposed to LPS-stimulated NR8383 cells was tested using a biotin label-based 507 antibody array. Among the secreted proteins, we selected pentraxin-related protein PTX3/tumor necrosis factor-inducible gene 14 protein (PTX3) to investigate its association with UCB-MSCs in macrophage polarization. We found that human PTX3 was secreted from UCB-MSCs under inflammation condition and reinforced the M2 macrophage marker via the Dectin-1 receptor by activating MSK1/2 phosphorylation signaling in NR8383 cells. Accordingly, knockdown of PTX3 in UCB-MSCs significantly attenuated their therapeutic effects in a neonatal hyperoxic lung injury resulting in reduced survival, lung alveolarization, M2 marker expression, Dectin-1 levels, anti-inflammatory cytokines, and improved M1 marker expression and inflammatory cytokines compared to control MSC-injected rats. UCB-MSCs show therapeutic potential by controlling macrophage polarization. Interestingly, higher PTX3 levels in UCB-MSCs induced greater improvement in the therapeutic effects than lower PTX3 levels. Collectively, PTX3 is a potential marker with critical paracrine effects for predicting the therapeutic potential of MSC therapy in inflammatory diseases; quality control assessments using PTX3 may be useful for improving the therapeutic effects of UCB-MSCs.

Role of extracellular vesicles in stem cell biology

The extracellular vesicles (EVs) are membrane vesicles carrying proteins, nucleic acids, and bioactive lipids of the cell of origin. These vesicles released within the extracellular space and entering into the circulation may transfer their cargo to neighboring or distant cells and induce phenotypical and functional changes that may be relevant in several physiopathological conditions. In an attempt to define the biological properties of EVs, several investigations have focused on their cargo and on the effects elicited in recipient cells. EVs have been involved in modulation of tumor microenvironment and behavior, as well as in the immune and inflammatory response. In the present review, we address the paracrine action of EVs released by stem cells and their potential involvement in the activation of regenerative programs in injured cells.

Encapsulated mononuclear stem cells: paracrine action for the treatment of acute myocardial infarction

Cell therapy is considered as a treatment option for acute myocardial infarction (AMI). Released molecules by cells paracrine action may promote tissue regeneration. Therefore we used bone-marrow mononuclear cells (BMMNCs) from GFP+ Wistar rats encapsulated in sodium alginate for AMI treatment. Animals were randomly allocated into groups – empty (EC); BMMNC capsules; or sham. AMI was induced by occlusion of left anterior artery and capsules were delivered intrathoracically. Troponin I was measured 24h after AMI and echocardiography was performed at 48h and 7d after AMI. On day 7 animals were euthanized and their hearts were harvested. Tissue levels of TNF-α, IL-6, IL-10, cleaved caspase-3, and catalase were measured. Technical procedures were performed by blinded operators. There was no difference in either heart morphofunctional parameters or biochemical analysis between AMI groups. We conclude that the paracrine effects of BMMNCs lacks efficacy to modulate events associated with AMI in the rat.