Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.     

Modulation of Mesenchymal Stem Cells for Enhanced Therapeutic Utility in Ischemic Vascular Diseases

Mesenchymal stem cells are multipotent stem cells isolated from various tissue sources, including but not limited to bone marrow, adipose, umbilical cord, and Wharton Jelly. Although cell-mediated mechanisms have been reported, the therapeutic effect of MSCs is now recognized to be primarily mediated via paracrine effects through the secretion of bioactive molecules, known as the “secretome”. The regenerative benefit of the secretome has been attributed to trophic factors and cytokines that play neuroprotective, anti-angiogenic/pro-angiogenic, anti-inflammatory, and immune-modulatory roles. The advancement of autologous MSCs therapy can be hindered when introduced back into a hostile/disease environment. Barriers include impaired endogenous MSCs function, limited post-transplantation cell viability, and altered immune-modulatory efficiency. Although secretome-based therapeutics have gained popularity, many translational hurdles, including the heterogeneity of MSCs, limited proliferation potential, and the complex nature of the secretome, have impeded the progress. This review will discuss the experimental and clinical impact of restoring the functional capabilities of MSCs prior to transplantation and the progress in secretome therapies involving extracellular vesicles. Modulation and utilization of MSCs–secretome are most likely to serve as an effective strategy for promoting their ultimate success as therapeutic modulators.     

Cell-free Stem Cell-Derived Extract Formulation for Regenerative Medicine Applications

Stem cells for regenerative medicine purposes offer therapeutic benefits, but disadvantages are still ill defined. The benefit of stem cells may be attributed to their secretion of growth factors (GFs), cytokines (CKs), and extracellular vesicles (EVs), including exosomes. We present a novel cell-free stem cell-derived extract (CCM), formulated from human progenitor endothelial stem cells (hPESCs), characterized for biologically active factors using ELISA, nanoparticle tracking analysis and single particle interferometric reflectance imaging sensing. The effect on fibroblast proliferation and ability to induce stem cell migration was analyzed using Alamar Blue proliferation and Transwell migration assays, respectively. GFs including IGFBP 1, 2, 3, and 6, insulin, growth hormone, PDGF-AA, TGF-α, TGF-β1, VEGF, and the anti-inflammatory cytokine, IL-1RA were detected. Membrane enclosed particles within exosome size range and expressing exosome tetraspanins CD81 and CD9 were identified. CCM significantly increased cell proliferation and induced stem cell migration. Analysis of CCM revealed presence of GFs, CKs, and EVs, including exosomes. The presence of multiple factors including exosomes within one formulation, the ability to promote cell proliferation and induce stem cell migration may reduce inflammation and pain, and augment tissue repair.     

Tumor-Derived Small Extracellular Vesicles Induce Pro-Inflammatory Cytokine Expression and PD-L1 Regulation in M0 Macrophages via IL-6/STAT3 and TLR4 Signaling Pathways

Tumor-associated macrophages play a key role in promoting tumor progression by exerting an immunosuppressive phenotype associated with the expression of programmed cell death ligand 1 (PD-L1). It is well known that tumor-derived small extracellular vesicles (SEVs) affect the tumor microenvironment, influencing TAM behavior. The present study aimed to examine the effect of SEVs derived from colon cancer and multiple myeloma cells on macrophage functions. Non-polarized macrophages (M0) differentiated from THP-1 cells were co-cultured with SEVs derived from a colorectal cancer (CRC) cell line, SW480, and a multiple myeloma (MM) cell line, MM1.S. The expression of PD-L1, interleukin-6 (IL-6), and other inflammatory cytokines as well as of the underlying molecular mechanisms were evaluated. Our results indicate that SEVs can significantly upregulate the expressions of PD-L1 and IL-6 at both the mRNA and protein levels and can activate the STAT3 signaling pathway. Furthermore, we identified the TLR4/NF-kB pathway as a convergent mechanism for SEV-mediated PD-L1 expression. Overall, these preliminary data suggest that SEVs contribute to the formation of an immunosuppressive microenvironment.     

Recent Insights into Human Endometrial Peptidases in Blastocyst Implantation via Shedding of Microvesicles

Blastocyst implantation involves multiple interactions with numerous molecules expressed in endometrial epithelial cells (EECs) during the implantation window; however, there is limited information regarding the molecular mechanism underlying the crosstalk. In blastocysts, fibronectin plays a major role in the adhesion of various types of cells by binding to extracellular matrix proteins via the Arg-Gly-Asp (RGD) motif. In EECs, RGD-recognizing integrins are important bridging receptors for fibronectin, whereas the non-RGD binding of fibronectin includes interactions with dipeptidyl peptidase IV (DPPIV)/cluster of differentiation (CD) 26. Fibronectin may also bind to aminopeptidase N (APN)/CD13, and in the endometrium, these peptidases are present in plasma membranes and lysosomal membranes. Blastocyst implantation is accompanied by lysosome exocytosis, which transports various peptidases and nutrients into the endometrial cavity to facilitate blastocyst implantation. Both DPPIV and APN are released into the uterine cavity via shedding of microvesicles (MVs) from EECs. Recently, extracellular vesicles derived from endometrial cells have been proposed to act on trophectoderm cells to promote implantation. MVs are also secreted from embryonal stem cells and may play an active role in implantation. Thus, crosstalk between the blastocyst and endometrium via extracellular vesicles is a new insight into the fundamental molecular basis of blastocyst implantation.     

Extracellular Vesicles Released from Stem Cells as a New Therapeutic Strategy for Primary and Secondary Glomerulonephritis

Current treatment of primary and secondary glomerulopathies is hampered by many limits and a significant proportion of these disorders still evolves towards end-stage renal disease. A possible answer to this unmet challenge could be represented by therapies with stem cells, which include a variety of progenitor cell types derived from embryonic or adult tissues. Stem cell self-renewal and multi-lineage differentiation ability explain their potential to protect and regenerate injured cells, including kidney tubular cells, podocytes and endothelial cells. In addition, a broad spectrum of anti-inflammatory and immunomodulatory actions appears to interfere with the pathogenic mechanisms of glomerulonephritis. Of note, mesenchymal stromal cells have been particularly investigated as therapy for Lupus Nephritis and Diabetic Nephropathy, whereas initial evidence suggest their beneficial effects in primary glomerulopathies such as IgA nephritis. Extracellular vesicles mediate a complex intercellular communication network, shuttling proteins, nucleic acids and other bioactive molecules from origin to target cells to modulate their functions. Stem cell-derived extracellular vesicles recapitulate beneficial cytoprotective, reparative and immunomodulatory properties of parental cells and are increasingly recognized as a cell-free alternative to stem cell-based therapies for different diseases including glomerulonephritis, also considering the low risk for potential adverse effects such as maldifferentiation and tumorigenesis. We herein summarize the renoprotective potential of therapies with stem cells and extracellular vesicles derived from progenitor cells in glomerulonephritis, with a focus on their different mechanisms of actions. Technological progress and growing knowledge are paving the way for wider clinical application of regenerative medicine to primary and secondary glomerulonephritis: this multi-level, pleiotropic therapy may open new scenarios overcoming the limits and side effects of traditional treatments, although the promising results of experimental models need to be confirmed in the clinical setting.     

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.     

Context Dependent Sulf1/Sulf2 Functional Divergence in Endothelial Cell Activity

Signalling activities are tightly regulated to control cellular responses. Heparan sulfate proteoglycans (HSPGs) at the cell membrane and extracellular matrix regulate ligand availability and interaction with a range of key receptors. SULF1 and SULF2 enzymes modify HSPG sulfation by removing 6-O sulfates to regulate cell signalling but are considered functionally identical. Our in vitro mRNA and protein analyses of two diverse human endothelial cell lines, however, highlight their markedly distinct regulatory roles of maintaining specific HSPG sulfation patterns through feedback regulation of HS 6-O transferase (HS6ST) activities and highly divergent roles in vascular endothelial growth factor (VEGF) and Transforming growth factor β (TGFβ) cell signalling activities. Unlike Sulf2, Sulf1 over-expression in dermal microvascular HMec1 cells promotes TGFβ and VEGF cell signalling by simultaneously upregulating HS6ST1 activity. In contrast, Sulf1 over-expression in venous ea926 cells has the opposite effect as it attenuates both TGFβ and VEGF signalling while Sulf2 over-expression maintains the control phenotype. Exposure of these cells to VEGF-A, TGFβ1, and their inhibitors further highlights their endothelial cell type-specific responses and integral growth factor interactions to regulate cell signalling and selective feedback regulation of HSPG sulfation that additionally exploits alternative Sulf2 RNA-splicing to regulate net VEGF-A and TGFβ cell signalling activities.     

Functional Interplay between Methyltransferases and Inflammasomes in Inflammatory Responses and Diseases

An inflammasome is an intracellular protein complex that is activated in response to a pathogenic infection and cellular damage. It triggers inflammatory responses by promoting inflammatory cell death (called pyroptosis) and the secretion of pro-inflammatory cytokines, interleukin (IL)-1β and IL-18. Many types of inflammasomes have been identified and demonstrated to play a central role in inducing inflammatory responses, leading to the onset and progression of numerous inflammatory diseases. Methylation is a biological process by which methyl groups are transferred from methyl donors to proteins, nucleic acids, and other cellular molecules. Methylation plays critical roles in various biological functions by modulating gene expression, protein activity, protein localization, and molecular stability, and aberrant regulation of methylation causes deleterious outcomes in various human diseases. Methylation is a key determinant of inflammatory responses and diseases. This review highlights the current understanding of the functional relationship between inflammasome regulation and methylation of cellular molecules in inflammatory responses and diseases.     

Activation and Function of NLRP3 Inflammasome in Bone and Joint-Related Diseases

Inflammation is a pivotal response to a variety of stimuli, and inflammatory molecules such as cytokines have central roles in the pathogenesis of various diseases, including bone and joint diseases. Proinflammatory cytokines are mainly produced by immune cells and mediate inflammatory and innate immune responses. Additionally, proinflammatory cytokines accelerate bone resorption and cartilage destruction, resulting in the destruction of bone and joint tissues. Thus, proinflammatory cytokines are involved in regulating the pathogenesis of bone and joint diseases. Interleukin (IL)-1 is a representative inflammatory cytokine that strongly promotes bone and cartilage destruction, and elucidating the regulation of IL-1 will advance our understanding of the onset and progression of bone and joint diseases. IL-1 has two isoforms, IL-1α and IL-1β. Both isoforms signal through the same IL-1 receptor type 1, but the activation mechanisms are completely different. In particular, IL-1β is tightly regulated by protein complexes termed inflammasomes. Recent research using innovative technologies has led to a series of discoveries about inflammasomes. This review highlights the current understanding of the activation and function of the NLRP3 (NOD-like receptor family, pyrin domain-containing 3) inflammasome in bone and joint diseases.     

Roles and Applications of Red Blood Cell-Derived Extracellular Vesicles in Health and Diseases

Red blood cell-derived extracellular vesicles (RBCEVs) are vesicles naturally produced by red blood cells and play multiple roles such as acting as cell-to-cell communication messengers in both normal physiological and diseased states. RBCEVs are highly promising delivery vehicles for therapeutic agents such as biomolecules and nucleic acids as they are easy to source, safe, and versatile. RBCEVs autonomously target the liver and pass the blood–brain barrier into the brain, which is highly valuable for the treatment of liver and brain diseases. RBCEVs can be modified by various functional units, including various functional molecules and nanoparticles, to improve their active targeting capabilities for tumors or other sites. Moreover, the RBCEV level is significantly shifted in many diseased states; hence, they can also serve as important biomarkers for disease diagnoses. It is clear that RBCEVs have considerable potential in multiple medical applications. In this review, we briefly introduce the biological roles of RBCEVs, presented interesting advances in RBCEV applications, and discuss several challenges that need to be addressed for their clinical translation.     

Microglia Development and Maturation and Its Implications for Induction of Microglia-Like Cells from Human iPSCs

Microglia are resident immune cells of the central nervous system and play critical roles during the development, homeostasis, and pathologies of the brain. Originated from yolk sac erythromyeloid progenitors, microglia immigrate into the embryonic brain parenchyma to undergo final postnatal differentiation and maturation driven by distinct chemokines, cytokines, and growth factors. Among them, TGFβ1 is an important regulator of microglial functions, mediating homeostasis, anti-inflammation, and triggering the expression of microglial homeostatic signature genes. Since microglia studies are mainly based on rodent cells and the isolation of homeostatic microglia from human tissue is challenging, human-induced pluripotent stem cells have been successfully differentiated into microglia-like cells recently. However, employed differentiation protocols strongly vary regarding used cytokines and growth factors, culture conditions, time span, and cell yield. Moreover, the incomplete differentiation of human microglia can hamper the similarity to primary human microglia and dramatically influence the outcome of follow-up studies with these differentiated cells. This review summarizes the current knowledge of the molecular mechanisms driving rodent microglia differentiation in vivo, further compares published differentiation protocols, and highlights the potential of TGFβ as an essential maturation factor.     

Extracellular Environment-Controlled Angiogenesis,and Potential Application for Peripheral Nerve Regeneration

Endothelial cells acquire different phenotypes to establish functional vascular networks. Vascular endothelial growth factor (VEGF) signaling induces endothelial proliferation, migration, and survival to regulate vascular development, which leads to the construction of a vascular plexuses with a regular morphology. The spatiotemporal localization of angiogenic factors and the extracellular matrix play fundamental roles in ensuring the proper regulation of angiogenesis. This review article highlights how and what kinds of extracellular environmental molecules regulate angiogenesis. Close interactions between the vascular and neural systems involve shared molecular mechanisms to coordinate developmental and regenerative processes. This review article focuses on current knowledge about the roles of angiogenesis in peripheral nerve regeneration and the latest therapeutic strategies for the treatment of peripheral nerve injury.     

辅助性T细胞17与相关疾病发生的研究进展

辅助性T细胞17(T helper cells 17,Th17)是新近发现的一种辅助性T细胞,该类细胞在机体的抗微生物免疫中起非常重要的作用,也与很多自身免疫性疾病的发生有一定的关系,如过敏、糖尿病、类风湿性关节炎等。Th17细胞是与Th1及Th2细胞不同的T细胞亚类,可分泌IL-17A、IL-17F、IL-21、IL-22等细胞因子。本文就Th17细胞的基本生物学功能、与感染性疾病及自身免疫性疾病发生的相关性作一综述。     

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.     

Plasticity of Intestinal Epithelium: Stem Cell Niches and Regulatory Signals

The discovery of Lgr5+ intestinal stem cells (ISCs) triggered a breakthrough in the field of ISC research. Lgr5+ ISCs maintain the homeostasis of the intestinal epithelium in the steady state, while these cells are susceptible to epithelial damage induced by chemicals, pathogens, or irradiation. During the regeneration process of the intestinal epithelium, more quiescent +4 stem cells and short-lived transit-amplifying (TA) progenitor cells residing above Lgr5+ ISCs undergo dedifferentiation and act as stem-like cells. In addition, several recent reports have shown that a subset of terminally differentiated cells, including Paneth cells, tuft cells, or enteroendocrine cells, may also have some degree of plasticity in specific situations. The function of ISCs is maintained by the neighboring stem cell niches, which strictly regulate the key signal pathways in ISCs. In addition, various inflammatory cytokines play critical roles in intestinal regeneration and stem cell functions following epithelial injury. Here, we summarize the current understanding of ISCs and their niches, review recent findings regarding cellular plasticity and its regulatory mechanism, and discuss how inflammatory cytokines contribute to epithelial regeneration.     

Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials.     

Cellular Crosstalk between Endothelial and Smooth Muscle Cells in Vascular Wall Remodeling

Pathological vascular wall remodeling refers to the structural and functional changes of the vessel wall that occur in response to injury that eventually leads to cardiovascular disease (CVD). Vessel wall are composed of two major primary cells types, endothelial cells (EC) and vascular smooth muscle cells (VSMCs). The physiological communications between these two cell types (EC–VSMCs) are crucial in the development of the vasculature and in the homeostasis of mature vessels. Moreover, aberrant EC–VSMCs communication has been associated to the promotor of various disease states including vascular wall remodeling. Paracrine regulations by bioactive molecules, communication via direct contact (junctions) or information transfer via extracellular vesicles or extracellular matrix are main crosstalk mechanisms. Identification of the nature of this EC–VSMCs crosstalk may offer strategies to develop new insights for prevention and treatment of disease that curse with vascular remodeling. Here, we will review the molecular mechanisms underlying the interplay between EC and VSMCs. Additionally, we highlight the potential applicable methodologies of the co-culture systems to identify cellular and molecular mechanisms involved in pathological vascular wall remodeling, opening questions about the future research directions.     

Role of Versican, Hyaluronan and CD44 in Ovarian Cancer Metastasis

There is increasing evidence to suggest that extracellular matrix (ECM) components play an active role in tumor progression and are an important determinant for the growth and progression of solid tumors. Tumor cells interfere with the normal programming of ECM biosynthesis and can extensively modify the structure and composition of the matrix. In ovarian cancer alterations in the extracellular environment are critical for tumor initiation and progression and intra-peritoneal dissemination. ECM molecules including versican and hyaluronan (HA) which interacts with the HA receptor, CD44, have been shown to play critical roles in ovarian cancer metastasis. This review focuses on versican, HA, and CD44 and their potential as therapeutic targets for ovarian cancer.