Proteomic approach attracts considerable attention in the study of physiological and pathological changes. Several recent reviews describe the effect of microgravity on the animal and human proteomes [40,41,42,43,44]. Over the past few years, mass spectrometry has become the method of choice for the characterization of ECM composition [45,46] and has been shown to offer new bioinformatic approaches of translating data from the putative biomarkers to the elucidation of new therapeutic targets [47,48,49]. The experimental strategies, new bioinformatic tools, and methods for matrix isolation have been described for the research on the ECM composition and mechanisms of degradation/renewal [50,51]. The new MatrisomeDB version contains selected proteomic data from 17 studies with ECM from 15 various tissues and includes 847 human ECM proteoforms and over 350,000 peptide-to-spectrum matches [52].

The proteomic data on matrisome components are considered to be of diagnostic and prognostic values in clinical studies. A comparison of core ECM and ECM-associated molecules’ data from the human carotid endarterectomy samples demonstrated the differences in the proteome and gene expression in symptomatic and asymptomatic atherosclerotic patients, including MMP-9, chitinase 3-like-1, calcium binding protein S100 A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein [53]. Proteomic analysis revealed the loss of aggrecan and several small leucine-rich proteoglycans, with a compensatory increase in collagen I during ECM remodeling in varicose veins, though there were no significant alterations of gene expression. These data suppose that the remodeling process associated with venous hypertension mainly occurs at the translation level, rather than at the transcription one. [47].

Investigation of the effects of spaceflights and ground-based simulations on the proteomics of matrisome in healthy subjects is of great interest. Several experimental modes that involve healthy volunteers are used to simulate the effects of certain spaceflight factors. These include various types of immersion and head-down tilt bed rest (HDT BR).

After 21 days of “dry” immersion, the altered levels of a number of proteins were detected in plasma with chromatography–mass spectrometric analysis. The identification of overrepresented processes, as well as processes and biological pathways was performed using the GO databases (biological processes, pathways, and KEGG). The significantly changed proteins were annotated as involved in ECM remodeling (alpha, beta, fibrinogen gamma chains), fibronectin, transtyretin), vitronectin and the cell morphogenesis regulation (alipoprotein A-I, prothrombin, alpha, beta, gamma chains of fibrinogen, fibronectin [54,55]. Hypokinesia is accompanied “dry’ immersion as well, and probably causes a protease/counter protease imbalance, which may be responsible for the ECM remodeling activation. Besides, after 21 day “dry” immersion a decrease in ECM proteoglycans, lumican and COMP, was also detected. COMP, as a cartilage structural protein, plays an important role in the ECM stabilization due to the interactions with collagen fibrils and other matrix components [56].

The blood COMP level is sensitive to physiological stress. The study of 14 day HDT BR demonstrated that the joint cartilage thickness was reduced during the experiment, followed by a decrease in the COMP level [57]. Liu et al. [58] reported the chondrocytes’ ability to respond to stress in the extracellular environment (possibly both mechanical and shear stress), which resulted in an altered expression of matrix proteins. Therefore, a decrease in the blood COMP level during “dry” immersion may reflect a reduced metabolic activity of cartilage matrix proteins in response to the lack of mechanical stimuli.

Based on the proteomic data of blood and urine samples collected in HDT BR and “dry” immersion of similar durations (21 days), GO pathways analysis was performed for proteins with significantly changed concentrations. It was demonstrated that ECM remodeling was the most significant process among them (in particular, collagen degradation) [59]. At day 21 of HDT BR, a significant decrease was observed in the levels of collagen I and XV alpha-chains, and cathepsin D that are associated with degradation processes and ECM collagen fibril assembly.

According to the “dry” immersion and HDT BR proteomic data comparison, at day 21, proteins involved in the ECM organization and metabolism were detected: endorepellin, nidogen-1, tenascin X, and vitronectin. Bioinformatic resources confirm that proteins endorepellin and nidogen-1 are primarily involved in the ECM degradation. The analysis demonstrated that the proteins that changed their blood levels under HDT BR have the catalytic activity functions (transferase and hydrolase activities). These findings provide information about the ECM structures’ involvement in the response to a reduced support load.

Urine, blood, and exhaled air condensate are the minimally invasive biological material samples available for proteomic research in astronauts. We believe that blood samples are the most preferable ones for the study of ECM components among the above liquids. da Silveira et al. have applied a multidisciplinary systemic biology analytical approach to determine transcriptomic, proteomic, metabolomic, and epigenetic responses to spaceflight [60]. Multi-omics datasets obtained from the analysis of an astronaut’s biological samples after a one-year space mission showed a significant enrichment of biological processes closely related to the functions of ECM.

Blood samples obtained from the Russian cosmonauts were examined by various proteomic mass spectrometry-based methods. Changes in the serum protein composition, including full-size proteins and the isoforms, fragments, metabolites, and peptides, after long-term spaceflights were characterized by direct mass spectrometry profiling after serum pre-fractioning using MB WCX magnetic particles. After a spaceflight, the peak areas of “acute phase” proteins, lipid metabolism, proteolytic enzymes and their inhibitors were shown to alter [61].

With semi-quantitative label free panoramic method, it was demonstrated that among 419 various proteins in cosmonauts’ blood plasma 17 proteins were significantly increased, while two significantly decreased after a prolonged space flight in comparison with pre-flight levels. In most cases, these proteins do not return to pre-flight baselines by day 7 after spaceflight. They are involved in the blood clotting system, ECM remodeling, and immune processes [62].

The quantitative changes in the cosmonauts’ blood proteome found using targeted MRM method with a panel of stable [¹³C]/[¹⁵N] isotope-labeled proteotypic peptides indicated that almost all proteins with the concentrations reacting to space flight can be combined into a network of interactions between the processes of regulation of protease activity, innate immunity, lipid metabolism, coagulation cascades, and ECM metabolism [43]. Latridis et al. suggest that these reactions may be triggered by extracellular signaling pathways of mechanotransduction [63]. Part of the group of functionally ECM-associated proteins detected in the samples at day 1 after landing in a reduced concentration, were found to return to baseline after 7 days, but another part of proteins retained significantly reduced concentrations [61,62]. The ANDCell program made it possible to identify biological processes involving ECM proteins that are modified by spaceflight factors (Table 2).

As evidenced from Table 2, the reorganization of the matrix structure due to MMPs, the ERK pathway regulation, cell–matrix adhesion, secretion, and assembly of ECM structures are the principal biological processes affected by spaceflight.

Proteomic analysis of urine samples from Russian cosmonauts after six-month missions detected 20 of the 256 proteins with altered levels [43,64]. However, the concentrations of most of them were returned to pre-flight levels within 7 days of the post-flight rehabilitation. At day 1 upon landing, seven proteins involved in ECM remodeling were identified among the significantly changing proteins. According to their functions in the physiological processes, they belong to the proteins involved in the musculoskeletal system metabolism. Thus, the level of osteodifferentiation and bone mineralization associated osteopontin was increased at day 1 upon landing and did not return to baseline after a further 7 days. Interestingly, several proteins not detected in urine samples prior to flight were increased acutely in post-flight: alpha-1-antichymotrypsin, N-acetyl glycosamine-6-sulfatase, cystatin-M, collagen alpha-1(I) chain, and vitronectin, granulin, and LDH beta chain. The above proteins are known to belong to the group of ECM-associated biological processes [64].

Therefore, the secretion and assembly of the matrisome components, its remodeling activity, as well as the ECM/cells associations are affected by spaceflight, primarily by the reduction of mechanical stress under microgravity.^{[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64]}