Andreia M. Silva

Extracellular Vesicles: Immunomodulatory messengers in the context of tissue repair/regeneration.

&NA; Inflammation is a complex and highly regulated biological process, crucial for a variety of functions in the human body, from host response against infectious agents to initiation of repair/regeneration of injured tissues. In the context of tissue repair, the action of different immune cell populations and their interplay with tissue specific cells, including stem cells, is still being uncovered. Extracellular Vesicles (EV) are small membrane vesicles secreted by cells in a controlled manner, which can act locally and systemically. The ability of EV to influence tissue repair and regeneration has been proposed as a physiologically intelligent and targeted strategy of cell communication. Herein, the role of EV in tissue repair is reviewed, summarising first their contribution to the regulation of immune cell function, and discussing the implications for the resolution of inflammation during repair. Next, the impact of EV on cell proliferation and differentiation, and on extracellular matrix remodelling, key aspects of the subsequent phases of tissue repair, is addressed. Finally, EV‐based therapies are discussed, focusing on the application of naturally produced EV, and the use of EV as delivery vehicles. Graphical Abstract Figure. No caption available.

miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis

Mesenchymal Stromal/Stem Cells (MSC) are currently being explored in diverse clinical applications, including regenerative therapies. Their contribution to regeneration of bone fractures is dependent on their capacity to proliferate, undergo osteogenesis and induce angiogenesis. This study aimed to uncover microRNAs capable of concomitantly regulate these mechanisms. Following microRNA array results, we identified miR-195 and miR-497 as downregulated in human primary MSC under osteogenic differentiation. Overexpression of miR-195 or miR-497 in human primary MSC leads to a decrease in osteogenic differentiation and proliferation rate. Conversely, inhibition of miR-195 increased alkaline phosphatase expression and activity and cells proliferation. Then, miR-195 was used to study MSC capacity to recruit blood vessels in vivo. We provide evidence that the paracrine effect of MSC on angiogenesis is diminishedwhen cells over-express miR-195. VEGF may partially mediate this effect, as its expression and secreted protein levels are reduced by miR-195, while increased by anti-miR-195, in human MSC. Luciferase reporter assays revealed a direct interaction between miR-195 and VEGF 3′-UTR in bone cancer cells. In conclusion, our results suggest that miR-195 regulates important mechanisms for bone regeneration, specifically MSC osteogenic differentiation, proliferation and control of angiogenesis; therefore, it is a potential target for clinical bone regenerative therapies.

Circulating extracellular vesicles: Their role in tissue repair and regeneration

Extracellular vesicles (EVs) have been a growing interest of the scientific community in recent years due to the wide possibilities of their evaluation as biomarkers of disease, and their potential to be used as therapeutic agents or vehicles. EVs that circulate in plasma carry proteins and nucleic acids, potentially to distant locations in the body where they can interfere with several cellular processes. To aid understanding of this rapidly evolving field, circulating EVs, including immune cell-derived ones, are reviewed here. Their cellular origins and described functions are discussed in a perspective of their contribution to regenerative processes. Different techniques for EV engineering and examples of their application are reviewed as a strong future direction of EV research. A summary of important aspects yet to be addressed ties up this review.

Resveratrol as a Natural Anti-Tumor Necrosis Factor-α Molecule: Implications to Dendritic Cells and Their Crosstalk with Mesenchymal Stromal Cells

Dendritic cells (DC) are promising targets for inducing tolerance in inflammatory conditions. Thus, this study aims to investigate the effects of the natural anti-inflammatory molecule resveratrol on human DC at phenotypic and functional levels, including their capacity to recruit mesenchymal stem/stromal cells (MSC). Primary human monocyte-derived DC and bone marrow MSC were used. DC immunophenotyping revealed that small doses of resveratrol (10 µM) reduce cell activation in response to tumor necrosis factor (TNF)-α, significantly decreasing surface expression of CD83 and CD86. Functionally, IL-12/IL-23 secretion induced by TNF-α was significantly reduced by resveratrol, while IL-10 levels increased. Resveratrol also inhibited T cell proliferation, in response to TNF-α-stimulated DC. The underlying mechanism was investigated by Western blot and imaging flow cytometry (ImageStreamX), and likely involves impairment of nuclear translocation of the p65 NF-κB subunit. Importantly, results obtained demonstrate that DC are able to recruit MSC through extracellular matrix components, and that TNF-α impairs DC-mediated recruitment. Matrix metalloproteinases (MMP) produced by both cell populations were visualized by gelatin zymography. Finally, time-lapse microscopy analysis revealed a significant decrease on DC and MSC motility in co-cultures, indicating cell interaction, and TNF-α further decreased MSC motility, while resveratrol recovered it. Thus, the current study points out the potential of resveratrol as a natural anti-TNF-α drug, capable of modulating DC phenotype and function, as well as DC-mediated MSC recruitment.

Fibrinogen scaffolds with immunomodulatory properties promote in vivo bone regeneration

The hypothesis behind this work is that fibrinogen (Fg), classically considered a pro-inflammatory protein, can promote bone repair/regeneration. Injury and biomaterial implantation naturally lead to an inflammatory response, which should be under control, but not necessarily minimized. Herein, porous scaffolds entirely constituted of Fg (Fg-3D) were implanted in a femoral rat bone defect and investigated at two important time points, addressing the bone regenerative process and the local and systemic immune responses, both crucial to elucidate the mechanisms of tissue remodelling. Fg-3D led to early infiltration of granulation tissue (6 days post-implantation), followed by bone defect closure, including periosteum repair (8 weeks post-injury). In the acute inflammatory phase (6 days) local gene expression analysis revealed significant increases of pro-inflammatory cytokines IL-6 and IL-8, when compared with non-operated animals. This correlated with modified proportions of systemic immune cell populations, namely increased T cells and decreased B, NK and NKT lymphocytes and myeloid cell, including the Mac-1+ (CD18+/CD11b+) subpopulation. At 8 weeks, Fg-3D led to decreased plasma levels of IL-1β and increased TGF-β1. Thus, our data supports the hypothesis, establishing a link between bone repair induced by Fg-3D and the immune response. In this sense, Fg-3D scaffolds may be considered immunomodulatory biomaterials.

Systemic delivery of bone marrow mesenchymal stem cells for in situ intervertebral disc regeneration

Cell therapies for intervertebral disc (IVD) regeneration presently rely on transplantation of IVD cells or stem cells directly to the lesion site. Still, the harsh IVD environment, with low irrigation and high mechanical stress, challenges cell administration and survival. In this study, we addressed systemic transplantation of allogeneic bone marrow mesenchymal stem cells (MSCs) intravenously into a rat IVD lesion model, exploring tissue regeneration via cell signaling to the lesion site. MSC transplantation was performed 24 hours after injury, in parallel with dermal fibroblasts as a control; 2 weeks after transplantation, animals were killed. Disc height index and histological grading score indicated less degeneration for the MSC‐transplanted group, with no significant changes in extracellular matrix composition. Remarkably, MSC transplantation resulted in local downregulation of the hypoxia responsive GLUT‐1 and in significantly less herniation, with higher amounts of Pax5+ B lymphocytes and no alterations in CD68+ macrophages within the hernia. The systemic immune response was analyzed in the blood, draining lymph nodes, and spleen by flow cytometry and in the plasma by cytokine array. Results suggest an immunoregulatory effect in the MSC‐transplanted animals compared with control groups, with an increase in MHC class II+ and CD4+ cells, and also upregulation of the cytokines IL‐2, IL‐4, IL‐6, and IL‐10, and downregulation of the cytokines IL‐13 and TNF‐α. Overall, our results indicate a beneficial effect of systemically transplanted MSCs on in situ IVD regeneration and highlight the complex interplay between stromal cells and cells of the immune system in achieving successful tissue regeneration. Stem Cells Translational Medicine 2017;6:1029–1039

miR-195 inhibits macrophages pro-inflammatory profile and impacts the crosstalk with smooth muscle cells

Macrophages are a main component of atherosclerotic plaques. Recent studies suggest that pro-inflammatory M1 macrophages are pro-atherogenic while M2 macrophages promote plaque stability. Moreover, toll-like receptor signalling pathways are implicated in atherosclerotic plaque formation, evolution and regression. We propose microRNAs as key regulators of these processes. In this context, our goal is to promote inflammation resolution using miR-195 to reduce M1-like macrophage polarization and to evaluate the molecular mechanisms underlying such effect, as well as to explore the functional consequences for smooth muscle cell recruitment. Human primary macrophages were differentiated from peripheral blood monocytes and stimulated with LPS or IL-10 to promote M1 or M2c polarization, respectively. miR-195 levels were upregulated in M2c macrophages compared with M1 macrophages. In THP-1 macrophages stimulated with LPS and IFN-γ, results show that TLR2 levels were reduced by miR-195 overexpression compared with scrambled control. In addition, phosphorylated forms of p54 JNK, p46 JNK and p38 MAPK were decreased by miR-195 in macrophages following M1 stimulation. Moreover, miR-195 significantly decreased levels of IL-1β, IL-6 and TNF-α pro-inflammatory cytokines in the supernatants of M1-stimulated macrophage cultures. At the functional level, results from smooth muscle cell recruitment and migration models showed that miR-195 impairs the capacity of M1 macrophages to promote smooth muscle cells migration. In conclusion, miR-195 is involved in macrophage polarization and inhibits TLR2 inflammatory pathway mediators. Moreover, miR-195 impairs the effect of macrophages on smooth muscle cells recruitment capacity and migration profile. Thus, miR-195 might be used as a new potential tool to promote inflammation resolution in cardiovascular research.

Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells

Dendritic cells (DCs) are promising targets for drug delivery, as they can induce immunity or tolerance. The current study aims to examine the potential of using nanostructured lipid carriers (NLC) as delivery systems for human DC by evaluating nanoparticle internalization, cell labeling, and drug activity. NLC were formulated incorporating the fluorochrome fluorescein isothiocyanate (FITC-NLC) or the natural anti-inflammatory molecule resveratrol (rsv-NLC). Primary human DCs were differentiated from peripheral blood monocytes, and the innovative imaging flow cytometry technique was used to examine FITC-NLC internalization. The capacity of rsv-NLC to inhibit DC activation in response to proinflammatory cytokine tumor necrosis factor-α (TNF- α) was investigated by conventional flow cytometry. A combination of imaging and conventional flow cytometry was used to assess NLC cytotoxicity. The results obtained indicate that both NLC formulations were stable over time, with mean diameter <200 nm and highly negative zeta potential (about −30 mV). When DCs were placed in contact with NLC, imaging flow cytometry clearly showed that DCs efficiently internalized FITC-NLC, with nearly 100% of cells internalizing nanoparticles upon 1 hour of incubation. Both immature and mature DCs internalized NLC to high and comparable levels, and without cytotoxicity. Stimulating DC with TNF-α in the presence of rsv-NLC revealed that, using these nanoparticles, very small concentrations of rsv were sufficient to significantly decrease surface expression of activation marker CD83 (5 µM) and major histocompatibility complex-class II molecule human leukocyte antigen – antigen D related (10 µM), both upregulated in response to TNF-α stimulation. Rsv-NLC were compared with free rsv; at 5 µM, rsv-NLC were able to inhibit nuclear factor κ beta phosphorylation and significantly decrease the level of interleukin-12/23, both upregulated in response to TNF-α, while 10 µM free rsv were needed to promote a similar effect. Taken together, the results presented show that NLC are suitable carriers of fluorescent labels or bioactive molecules for human DCs, leading to inflammation modulation.

Integrated Analysis of Biological Samples by Imaging Flow Cytometry.

Instituto de Engenharia Biomédica (INEB). University of Porto, Porto, Portugal b.IMAGE – Bioimaging Center for Biomaterials and Regenerative Therapies, INEB, Porto, Portugal Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Porto, Portugal Instituto de Ciências Biomédicas Abel Salazar (ICBAS). University of Porto. Porto, Portugal Faculdade de Engenharia da Universidade do Porto (FEUP), Porto, Portugal Faculdade de Medicina da Universidade do Porto (FMUP), Porto, Portugal Centre for Cell Biology and Department of Biology, University of Aveiro, Aveiro, Portugal College of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter, Devon, United Kindgom.

Profiling the circulating miRnome reveals a temporal regulation of the bone injury response

Bone injury healing is an orchestrated process that starts with an inflammatory phase followed by repair and remodelling of the bone defect. The initial inflammation is characterized by local changes in immune cell populations and molecular mediators, including microRNAs (miRNAs). However, the systemic response to bone injury remains largely uncharacterized. Thus, this study aimed to profile the changes in the plasma miRnome after bone injury and determine its biological implications. Methods: A rat model of femoral bone defect was used, and animals were evaluated at days 3 and 14 after injury. Non-operated (NO) and sham operated animals were used as controls. Blood and spleen were collected and peripheral blood mononuclear cells (PBMC) and plasma were separated. Plasma miRnome was determined by RT-qPCR array and bioinformatics Ingenuity pathway analysis (IPA) was performed. Proliferation of bone marrow mesenchymal stem/stromal cells (MSC) was evaluated by Ki67 staining and high-throughput cell imaging. Candidate miRNAs were evaluated in splenocytes by RT-qPCR, and proteins found in the IPA analysis were analysed in splenocytes and PBMC by Western blot. Results: Bone injury resulted in timely controlled changes to the miRNA expression profile in plasma. At day 3 there was a major down-regulation of miRNA levels, which was partially recovered by day 14 post-injury. Interestingly, bone injury led to a significant up-regulation of let-7a, let-7d and miR-21 in plasma and splenocytes at day 14 relative to day 3 after bone injury, but not in sham operated animals. IPA predicted that most miRNAs temporally affected were involved in cellular development, proliferation and movement. MSC proliferation was analysed and found significantly increased in response to plasma of animals days 3 and 14 post-injury, but not from NO animals. Moreover, IPA predicted that miRNA processing proteins Ago2 and Dicer were specifically inhibited at day 3 post-injury, with Ago2 becoming activated at day 14. Protein levels of Ago2 and Dicer in splenocytes were increased at day 14 relative to day 3 post-bone injury and NO animals, while in PBMC, levels were reduced at day 3 (albeit Dicer was not significant) and remained low at day 14. Ephrin receptor B6 followed the same tendency as Ago2 and Dicer, while Smad2/3 was significantly decreased in splenocytes from day 14 relative to NO and day 3 post-bone injury animals. Conclusion: Results show a systemic miRNA response to bone injury that is regulated in time and is related to inflammation resolution and the start of bone repair/regeneration, unravelling candidate miRNAs to be used as biomarkers in the monitoring of healthy bone healing and as therapeutic targets for the development of improved bone regeneration therapies.