Morphological and Quantitative Evidence for Altered Mesenchymal Stem Cell Remodeling of Collagen in an Oxidative Environment—Peculiar Effect of Epigallocatechin-3-Gallate

Abstract

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin-3-gallate (EGCG), attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labelled with FITC before being oxidized according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometric analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells, over the cells, and central (nuclear/perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC and is accompanied by a substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometric analysis showed that oxidized FITC-collagen tends to accumulate beneath cells and around cell nuclei, suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometric analysis also revealed that both processes are supported by EGCG pretreatment.

Early Stages of Ex Vivo Collagen Glycation Disrupt the Cellular Interaction and Its Remodelling by Mesenchymal Stem Cells—Morphological and Biochemical Evidence

“…Therefore, we decided to deepen the study by quantifying (1) the cells-driven enzymatic degradation of collagen and (2) its direct proteolysis by exogenously adding collagenase in a cell-free system. To analyse glycated FITC collagen, we used the previously described approach based on the proteolytic de-quenching of fluorescent conjugated probes (FRET effect) [23,25]. As evident from the Results section, glycation significantly altered both the cell-driven (Figure 5) and exogenous collagenase-driven (Figure 6) enzymatic remodelling of collagen.…”

“…It has to be noted that this quantitative approach was successfully applied in our previous study [23,25], but here we plotted the photometric signal in the presence of cells against the corresponding signal from absolutely identical controls (also with cells), but using non-labelled protein. According to the calculated values of Δ RPU presented in Figure 5, the glycation significantly reduces the typical ADMSC de-quenching effect (left column), again going along with the extent of glycation, 1 day or 5 days (middle and left columns).…”

Early Stages of Ex Vivo Collagen Glycation Disrupt the Cellular Interaction and Its Remodeling by Mesenchymal Stem Cells—Morphological and Biochemical Evidence

“…However, the system is hardly that simple from a physiological point of view, since the recruitment of stem cells to the sites of injury is also supported by the local release of signaling molecules and growth factors [ 49 ] that promote their tropism and potentially influence the downstream expression of adhesion molecules [ 50 ]. Hence, to be more relevant, we added serum during the incubation of cells for over 2 h (for 5 and 24 h) to optimize their functionality during the anticipated remodeling of collagen, a phenomenon of primary interest [ 51 ].…”