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.

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.

Currently, there is a growing focus on aging and age-related diseases. The processes of aging are based on cell senescence, which results in changes in intercellular communications and pathological alterations in tissues. In the present study, we investigate the influence of senescent mesenchymal stem cells (MSCs) on endothelial cells (ECs). In order to induce senescence in MSCs, we employed a method of stress-induced senescence utilizing mitomycin C (MmC). Subsequent experiments involved the interaction of ECs with MSCs in a coculture or the treatment of ECs with the secretome of senescent MSCs. After 48 h, we assessed the EC state. Our findings revealed that direct interaction led to a decrease in EC proliferation and migratory activity of the coculture. Furthermore, there was an increase in the activity of the lysosomal compartment, as well as an upregulation of the genes P21, IL6, IL8, ITGA1, and ITGB1. Treatment of ECs with the “senescent” secretome resulted in less pronounced effects, although a decrease in proliferation and an increase in ICAM-1 expression were observed. The maintenance of high levels of typical “senescent” cytokines and growth factors after 48 h suggests that the addition of the “senescent” secretome may have a prolonged effect on the cells. It is noteworthy that in samples treated with the “senescent” secretome, the level of PDGF-AA was higher, which may explain some of the pro-regenerative effects of senescent cells. Therefore, the detected changes may underlie both the negative and positive effects of senescence. The findings provide insight into the effects of cell senescence in vitro, where many of the organism’s regulatory mechanisms are absent.

Mesenchymal stromal cells (MSCs) are considered a valuable tool for cell therapy. After systemic administration, the outcome of MSCs and endothelial cells (ECs) interactions strongly depend on the local microenvironment and tissue O₂ levels in particular. In vitro analysis of EC effects on MSC regenerative potential in co-culture was performed after short-term interaction at “physiological” hypoxia (5% O₂) and acute hypoxic stress (0.1% O₂). At 5% O₂, MSCs retained stromal phenotype and CFU-f numbers, osteogenic RUNX2 was upregulated. A shift in the expression of adhesion molecules, and an increase in transcription/synthesis of IL-6, IL-8 contributed to facilitation of directed migration of MSCs. In the presence of MSCs, manifestations of oxidative stress in ECs were attenuated, and a decrease in adhesion of PBMCs to TNF-α-activated ECs was observed. Under 0.1% O₂, reciprocal effects of ECs and MSCs were similar to those at 5% O₂. Meanwhile, upregulation of RUNX2 was canceled, IL-6 decreased, and IL-8 significantly increased. “Protective” effects of MSCs on TNF-α-ECs were less pronounced, manifested as NOS3 downregulation and intracellular NO elevation. Therefore, interaction with ECs at “physiological” hypoxia enhanced pro-regenerative capacities of MSCs including migration and anti-inflammatory modulation of ECs. Under acute hypoxic stress, the stimulating effects of ECs on MSCs and the “protective” potential of MSCs towards TNF-α-ECs were attenuated.

Mesenchymal stromal cells (MSCs) are promising therapeutic agents, largely due to their capacity for self-renewal, differentiation, and immunomodulation. Importantly, these beneficial effects are frequently mediated by the MSC secretome, which contains factors with anti-inflammatory, anti-apoptotic, and pro-regenerative properties. However, cellular senescence can impair these critical functions. To identify senescence-associated changes in the MSC secretome that may regulate aging and intercellular communication, we performed a mass spectrometry-based proteomic analysis of the conditioned medium from MSCs undergoing stress-induced senescence. Our analysis confirmed the upregulation of established aging markers, such as IL-6, PAI-1, and IGFBP7. Furthermore, we identified a significant increase in lesser-known senescence-associated secretory phenotype (SASP) components, including INHBA—a known inhibitor of proliferation—and DKK3, which blocks stromal cell pluripotency. Pathway analysis revealed that stress-induced senescence broadly affected proteins involved in glycolysis, immune response, hemostasis, and the regulation of cell death and the cell cycle. These alterations are likely to negatively impact the MSC microenvironment. Interestingly, the cellular response to senescence was dualistic. Alongside detrimental SASP factors, we observed an increase in protective proteins such as annexins (ANXA1, ANXA2), antioxidants (TXN, PRDX1, PRDX6), and the heat shock protein HSPB1, which collectively defend neighboring cells from inflammation and oxidative stress. These findings underscore the complex etiology of cellular senescence and the paradoxical nature of the SASP. The obtained data also emphasize the necessity of comprehensive proteomic profiling of the MSC secretome across different aging models to harness the full therapeutic potential of MSCs and their secretomes for regenerative medicine.