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. 2024 May 14;25(10):5332.
doi: 10.3390/ijms25105332.

Senescence-Associated Alterations in Matrisome of Mesenchymal Stem Cells

Diana Matveeva  1 , Daria Kashirina  1 , Mariia Ezdakova  1 , Irina Larina  1 , Ludmila Buravkova  1 , Andrey Ratushnyy  1
Affiliations

Senescence-Associated Alterations in Matrisome of Mesenchymal Stem Cells

Diana Matveeva et al. Int J Mol Sci. .

Abstract

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.

Keywords: extracellular matrix (ECM); matrisome; mesenchymal stem cells (MSCs); senescence; senescence-associated secretory phenotype (SASP).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Senescence markers of MmC-treated MSCs: (a) cell monolayer morphology, and light microscopy; (b) forward scattering (FSCA) and side scattering (SSC-A) to illustrate size and cytoplasm vacuolization of MSCs, respectively, and flow cytometry; (c) autofluorescence intensity, and flow cytometry; (d) cell viability, annexin/propidium iodide (Ann/PI) staining, and flow cytometry; (e) histochemical evaluation of senescence-associated-β-galactosidase (SA-β-gal) activity in MSCs; and (f) colony-forming unit assay, and light microscopy. Data are shown as mean ± SD; n ≥ 4, * p < 0.05. MmC—mitomycin C, MSCs—mesenchymal stem cells.
Figure 2
Figure 2
Characteristics of ECM proteins produced by young (MmC−) and senescent (MmC+) MSCs: (a) histological identification of collagenous (Sirius Red) and non-collagenous (Fast Green) proteins and their semiquantitative spectrophotometric determination, bar—50 µm; (b) immunocytochemical detection of ECM proteins (ECM proteins—green, DAPI—blue), representative microphotographs, bar—20 µm; (c) the content of pro-collagen 1α1, detected by enzyme immunoassay, in the conditioned medium of young and senescent MSCs; data are presented as M ± SD, p ≤ 0.05, n = 3; (d) relative gene expression (2−ΔΔCt) in MmC+ versus MmC− MSCs encoding ECM proteins: type I collagen (COL1A1), fibronectin (FN1), osteonectin (SPARC), metalloproteinases (MMP1, MMP2), tissue inhibitor of metalloproteinases (TIMP3), and urokinase plasminogen activator (PLAU); data normalized to the expression of HPRT1, RPLP0; * p ≤ 0.05, n ≥ 3.
Figure 3
Figure 3
Quantitative distribution of MSC proteins identified by mass spectrometry in samples of young (MmC−), senescent (MmC+), and both cell types (MmC−/MmC+) by functional groups of the matrisome.
Figure 4
Figure 4
Venn diagram analysis. Graph show common and specific proteins among dcECM obtained from intact (MmC−) and senescent (MmC+) MSCs analyzed by mass spectrometry.
Figure 5
Figure 5
Gene Ontology functional enrichment analysis of the biological processes: (a) unique and upregulated matrisome proteins in MmC− dcECM samples; (b) unique and upregulated matrisome proteins in MmC+ dcECM samples. dcECM—decellularized extracellular matrix.
Figure 6
Figure 6
Matrisome proteins identified in dcECM, cell lysate, or conditioned medium of MmC− and MmC+ MSCs. The heat map reflects the direction of change in the relative content of proteins in MmC+ compared to MmC− samples.
Figure 7
Figure 7
Matrisome analysis of senescent MSCs revealed the main patterns of changes in the produced ECM. A shift in production towards regulatory molecules and significant downregulation of the main structural and adhesion molecules of the ECM have been established. Such changes can contribute to the deterioration of cell function, the activation of sensory phenotype, and the development of tissue pathologies.
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