Nowadays, paracrine regulation is considered as a major tool of mesenchymal stem cell (MSC) involvement in tissue repair and renewal in adults. Aging results in alteration of tissue homeostasis ...including neovascularization. In this study, we examined the influence of replicative senescence on the angiogenic potential of adipose-derived MSCs (ASCs). Angiogenic activity of conditioned medium (CM) from senescent and "young" ASCs was evaluated in chorioallantoic membrane (CAM) assay in ovo using Japanese quail embryos. Also, the formation of capillary-like tubes by human umbilical vein endothelial cells (HUVECs) in 3D basement membrane matrix ''Matrigel'' and HUVEC migration capacity were analyzed. Multiplex, dot-blot and gene expression analysis were performed to characterize transcription and production of about 100 angiogenesis-associated proteins. The results point to decreased angiogenic potential of senescent ASC secretome in ovo. A number of angiogenesis-associated proteins demonstrated elevation in CM after long-term cultivation. Meanwhile, VEGF (key positive regulator of angiogenesis) did not change transcription level and concentration in CM. Increasing both pro- (FGF-2, uPA, IL-6, IL-8 etc.) and antiangiogenic (IL-4, IP-10, PF4, Activin A, DPPIV etc.) factors was observed. Some proangiogenic genes were downregulated (
). Senescence-associated secretory phenotype (SASP) modifications after long-term cultivation lead to attenuation of angiogenic potential of ASC.
Despite the use of countermeasures (including intense physical activity), cosmonauts and astronauts develop muscle atony and atrophy, cardiovascular system failure, osteopenia, etc. All these ...changes, reminiscent of age-related physiological changes, occur in a healthy person in microgravity quite quickly – within a few months. Adaptation to the lost of gravity leads to the symptoms of aging, which are compensated after returning to Earth. The prospect of interplanetary flights raises the question of gravity thresholds, below which the main physiological systems will decrease their functional potential, similar to aging, and affect life expectancy. An important role in the aging process belongs to the body’s cellular reserve – progenitor cells, which are involved in physiological remodeling and regenerative/reparative processes of all physiological systems. With age, progenitor cell count and their regenerative potential decreases. Moreover, their paracrine profile becomes pro-inflammatory during replicative senescence, disrupting tissue homeostasis. Mesenchymal stem/stromal cells (MSCs) are mechanosensitive, and therefore deprivation of gravitational stimulus causes serious changes in their functional status. The review compares the cellular effects of microgravity and changes developing in senescent cells, including stromal precursors.
Rodent hindlimb unloading (HU) model was developed to elucidate responses/mechanisms of adverse consequences of space weightlessness. Multipotent mesenchymal stromal cells (MMSCs) were isolated from ...rat femur and tibia bone marrows and examined ex vivo after 2 weeks of HU and subsequent 2 weeks of restoration of load (HU + RL). In both bones, decrease of fibroblast colony forming units (CFU-f) after HU with restoration after HU + RL detected. In CFU-f and MMSCs, levels of spontaneous/induced osteocommitment were similar. MMSCs from tibia initially had greater spontaneous mineralization of extracellular matrix but were less sensitive to osteoinduction. There was no recovery of initial levels of mineralization in MMSCs from both bones during HU + RL. After HU, most bone-related genes were downregulated in tibia or femur MMSCs. After HU + RL, the initial level of transcription was restored in femur, while downregulation persisted in tibia MMSCs. Therefore, HU provoked a decrease of osteogenic activity of BM stromal precursors at transcriptomic and functional levels. Despite unidirectionality of changes, the negative effects of HU were more pronounced in stromal precursors from distal limb-tibia. These observations appear to be on demand for elucidation of mechanisms of skeletal disorders in astronauts in prospect of long-term space missions.
The mesenchymal stromal/stem cells (MSCs) are known to secrete pleiotropic paracrine factors, contributing to tissue regeneration. This unique ability makes MSCs promising therapeutic tools for many ...diseases, including even those that were previously untreatable. Thus, the development of preconditioning approaches aimed at enhancing the paracrine function of MSCs attracts great interest. In the present work, we studied how the extracellular matrix, the essential part of the native tissue microenvironment, affects the secretory capacity of MSCs of various origins. The MSC-derived decellularized extracellular matrix (dECM), used as the cell culture substrate, triggered strong upregulation of FGF-2, MMP-1, HGF, GRO-α, GRO-β, CXCL-5, CXCL-6, IL-6, IL-8, G-CSF and MCP-1. Functional in vitro tests revealed that conditioned media derived from MSCs cultured on dECM significantly improved 3T3 fibroblast and HaCaT keratinocyte scratch wound healing, stimulated THP-1 monocyte migration and promoted capillary-like HUVEC-based tube formation compared to conditioned media from MSCs grown on plastic. In addition, we found that FAK inhibition promoted dECM-induced upregulation of paracrine factors, suggesting that this kinase participates in the MSCs' paracrine response to dECM. Together, these findings demonstrate that dECM provides cues that considerably enhance the secretory function of MSCs. Thus, dECM usage as a cell culture substrate alone or in combination with a FAK inhibitor may be viewed as a novel MSC preconditioning technique.
The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ...ECM by osteogenic cells. To elucidate the cellular effects of microgravity, human mesenchymal stromal cells (MSCs) and their osteocommitted progeny were exposed to simulated microgravity (SMG) for 10 days using random positioning machine (RPM). After RPM exposure, an imbalance of MSC collagen/non-collagen ratio at the expense of a decreased level of collagenous proteins was detected. At the same time, the secretion of proteases (cathepsin A, cathepsin D, MMP3) was increased. No significant effects of SMG on the expression of stromal markers and cell adhesion molecules on the MSC surface were noted. Upregulation of COL11A1, CTNND1, TIMP3, and TNC and downregulation of HAS1, ITGA3, ITGB1, LAMA3, MMP1, and MMP11 were detected in RPM exposed MSCs. ECM-associated transcriptomic changes were more pronounced in osteocommitted progeny. Thus, 10 days of SMG provokes a decrease in the collagenous components of ECM, probably due to the decrease in collagen synthesis and activation of proteases. The presented data demonstrate that ECM-associated molecules of both native and osteocommitted MSCs may be involved in bone matrix reorganization during spaceflight.
Muscle and skeleton structures are considered most susceptible to negative factors of spaceflights, namely microgravity. Three-dimensional clinorotation is a ground-based simulation of microgravity. ...It provides an opportunity to elucidate the effects of microgravity at the cellular level. The extracellular matrix (ECM) content, transcriptional profiles of genes encoding ECM and remodelling molecules, and secretory profiles were investigated in a heterotypic primary culture of bone marrow cells after 14 days of 3D clinorotation. Simulated microgravity negatively affected stromal lineage cells, responsible for bone tissue formation. This was evidenced by the reduced ECM volume and stromal cell numbers, including multipotent mesenchymal stromal cells (MSCs). ECM genes encoding proteins responsible for matrix stiffness and cell-ECM contacts were downregulated. In a heterotypic population of bone marrow cells, the upregulation of genes encoding ECM degrading molecules and the formation of a paracrine profile that can stimulate ECM degradation, may be mechanisms of osteodegenerative events that develop in real spaceflight.
The process of aging is intimately linked to alterations at the tissue and cellular levels. Currently, the role of senescent cells in the tissue microenvironment is still being investigated. Despite ...common characteristics, different cell populations undergo distinctive morphofunctional changes during senescence. Mesenchymal stem cells (MSCs) play a pivotal role in maintaining tissue homeostasis. A multitude of studies have examined alterations in the cytokine profile that determine their regulatory function. The extracellular matrix (ECM) of MSCs is a less studied aspect of their biology. It has been shown to modulate the activity of neighboring cells. Therefore, investigating age-related changes in the MSC matrisome is crucial for understanding the mechanisms of tissue niche ageing. This study conducted a broad proteomic analysis of the matrisome of separated fractions of senescent MSCs, including the ECM, conditioned medium (CM), and cell lysate. This is the first time such an analysis has been conducted. It has been established that there is a shift in production towards regulatory molecules and a significant downregulation of the main structural and adhesion proteins of the ECM, particularly collagens, fibulins, and fibrilins. Additionally, a decrease in the levels of cathepsins, galectins, S100 proteins, and other proteins with cytoprotective, anti-inflammatory, and antifibrotic properties has been observed. However, the level of inflammatory proteins and regulators of profibrotic pathways increases. Additionally, there is an upregulation of proteins that can directly cause prosenescent effects on microenvironmental cells (SERPINE1, THBS1, and GDF15). These changes confirm that senescent MSCs can have a negative impact on other cells in the tissue niche, not only through cytokine signals but also through the remodeled ECM.
Multipotent mesenchymal stromal cells are considered as a perspective tool in cell therapy and regenerative medicine. Unfortunately, autologous cell therapy does not always provide positive outcomes ...in elder donors, perhaps as a result of the alterations of stem cell compartments. The mechanisms of stem and progenitor cell senescence and the factors engaged are investigated intensively. In present paper, we elucidated the effects of tissue‐related O2 on morphology, functions, and transcriptomic profile of adipose tissue‐derived stromal cells (ASCs) in replicative senescence in vitro model. Replicatively senescent ASCs at ambient (20%) O2 (12‐21 passages) demonstrated an increased average cell size, granularity, reactive oxygen species level, including anion superoxide, lysosomal compartment activity, and IL‐6 production. Decreased ASC viability and proliferation, as well as the change of more than 10 senescence‐associated gene expression were detected (IGF1, CDKN1C, ID1, CCND1, etc). Long‐term ASC expansion at low O2 (5%) revoked in part the replicative senescence‐associated alterations.
Ex vivo expansion of hematopoietic progenitors is considered as an attractive tool to increase the number of stem and progenitor cells (HSPCs) for cell therapy. The efficacy of ex vivo expansion is ...strongly depends on the feeder cell activity to mimic hematopoietic microenvironment. Here we demonstrated, that combination of mitomycin C-induced growth arrest and tissue-related O2 (physiological hypoxia) modulated stromal capacity of adipose tissue derived stromal cells (ASCs). Growth arrest did not affect viability, stromal phenotype and multilineage potential of ASCs permanently expanded at tissue-related O2. Meanwhile, the PCR analysis revealed an up-regulation of genes, encoded molecules of cell–cell (ICAM1, HCAM/CD44) and cell–matrix adhesion (ITGs), extracellular matrix production (COLs) and remodeling (MMPs, HAS1) in growth-arrested ASCs at physiological hypoxia in comparison with ambient O2 (20%). The number of ICAM-1 positive ASCs was increased under low O2 as well. These alterations contributed into the ex vivo expansion of cord blood HSPCs providing the preferential production of primitive HSPCs. The number of cobblestone area forming cell (CAFC) colonies was 1.5-fold higher at physiological hypoxia (p < 0.05). CAFCs considered as long-term culture-initiating cells (LTC-IC) known to support long-term hematopoiesis restoration in vivo. The presented data may be applicable in the development of upscale protocols of HSPC expansion.
Microgravity is a principal risk factor hampering human cardiovascular regulation during space flights. Endothelial dysfunction associated with the impaired integrity and uniformity of the monolayer ...represents a potential trigger for vascular damage. We characterized the expression profile of the multi‐step cascade of adhesion molecules (ICAM‐1, VCAM‐1, E‐selectin, VE‐cadherin) in umbilical cord endothelial cells (ECs) after 24 h of exposure to simulated microgravity (SMG), pro‐inflammatory cytokine TNF‐α, and the combination of the two. Random Positioning Machine (RPM)‐mediated SMG was used to mimic microgravity effects. SMG stimulated the expression of E‐selectin, which is known to be involved in slowing leukocyte rolling. Primary ECs displayed heterogeneity with respect to the proportion of ICAM‐1‐positive cells. ECs were divided into two groups: pre‐activated ECs displaying high proportion of ICAM‐1+‐cells (ECs‐1) (greater than 50%) and non‐activated ECs with low proportion of ICAM‐1+‐cells (ECs‐2) (less than 25%). Only non‐activated ECs‐2 responded to SMG by elevating gene transcription and increasing ICAM‐1 and VE‐cadherin expression. This effect was enhanced after cumulative SMG‐TNF‐α exposure. ECs‐1 displayed an unexpected decrease in number of E‐selectin‐ and ICAM‐1‐positive ECs and pronounced up‐regulation of VCAM1 upon activation of inflammation, which was partially abolished by SMG. Thus, non‐activated ECs‐2 are quite resistant to the impacts of microgravity and even exhibited an elevation of the VE‐cadherin gene and protein expression, thus improving the integrity of the endothelial monolayer. Pre‐activation of ECs with inflammatory stimuli may disturb the EC adhesion profile, attenuating its barrier function. These alterations may be among the mechanisms underlying cardiovascular dysregulation in real microgravity conditions.
Non‐activated ECs (low ICAM‐1 expression) are quite resistant to simulated microgravity impact demonstrating even elevation of VE‐cadherin gene and protein expression improving the integrity of endothelial monolayer. Pre‐activation of ECs with inflammatory stimuli could hamper ECs adhesion profile attenuating its barrier function. These alterations may be among of the mechanisms underlying cardiovascular dysregulation in the real microgravity.
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