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 (IGF1, MMP1, TGFB3, PDGFRB, PGF). Senescence-associated secretory phenotype (SASP) modifications after long-term cultivation lead to attenuation of angiogenic potential of ASC.

Multipotent mesenchymal stem/stromal cells (MSCs) are strongly involved in tissue homeostasis mainly through paracrine regulation. Here we examined the influence of simulated microgravity on the angiogenic potential of adipose-derived MSCs (ASCs). The conditioned medium (CM) from RPM-exposed ASCs stimulated the formation of vessel network in ovo, endothelial cell (EC) capillary-like network ex ovo and non-directed EC migration in vitro. These effects were driven by alteration of both angiogenesis-related gene and protein expression. The elevation of angiogenic regulators Serpin E1, Serpin F1, IGFBP, VEGF, IL-8 was detected in ASC-CM after 3D-clinorotation. Besides, transcription of genes encoded growth factors with proangiogenic activity were upregulated including VEGF-с and VEGF-a. These data evidenced that besides direct effect on ECs, microgravity could provoke MSC-mediating specific microenvironment for ECs supporting their functions i.e. proliferation and migration via increased production of IL-8 and VEGF as well as other paracrine factors involved in angiogenesis regulation.

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.

Исследовано влияние трех штаммов пробиотических бактерий: Lactobacillus rhamnosus K32 (L), Bifidobacterium longum GT15 (B), Enterococcus faecium L-3 (E), на уровни экспрессии и содержания ключевых цитокинов при помощи полимеразной цепной реакции с обратной транскрипцией и иммуноферментного анализа соответственно. В работе использованы культура клеток промоноцитарного происхождения (HTP-1) и экспериментальная модель дисбиоза кишечника. В геномах штаммов В и E ранее были выявлены гены, кодирующие бактериоцины, гены компонентов внешней мембраны, пилей и экзополисахаридов, участвующих в модуляции иммунной системы. При исследовании влияния пробиотических штаммов и их супернатантов на экспрессию цитокинов на культуре клеток выявлено увеличение экспрессии TNFα, обусловленное B, E и супернатантом L. Культура В дополнительно вызывала экспрессию IL-8 и IL-10. При коррекции дисбиоза кишечника у крыс (индуцированного метронидазолом и ампициллином) пробиотиками было показано, что нарушение в составе микробиоты (чрезмерный рост Klebsiella spp., низкое содержание Faecalobacterium prausnitzii) оставались у животных после введения L, как в контроле (без введения пробиотиков). В отличие от этих групп у крыс после введения E и B были выявлены: 1) низкие уровни экспрессии провоспалительных цитокинов (IL-8, TNFα, MCP-1) в брыжеечных лимфатических узлах и содержание данных факторов в сыворотке крови; 2) увеличение содержания в сыворотке крови антивоспалительного цитокина TGF-β. В данной работе, при помощи двух взаимодополняющих моделей, выявлены индивидуальные особенности иммуномодулирующих эффектов пробиотических штаммов L. rhamnosus K32, B. longum GT15 (B), E. faecium L-3, оказывающих различное влияние на микробиоту кишечника.

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.