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TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

ABSTRACT: Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca²⁺) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca²⁺ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca²⁺-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca²⁺ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca²⁺ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca²⁺ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

SUBMITTER: Gilchrist CL

PROVIDER: S-EPMC6369759 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

Gilchrist Christopher L CL Leddy Holly A HA Kaye Laurel L Case Natasha D ND Rothenberg Katheryn E KE Little Dianne D Liedtke Wolfgang W Hoffman Brenton D BD Guilak Farshid F

Proceedings of the National Academy of Sciences of the United States of America 20190123 6

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca<sup>2+</sup>) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca<sup>2+</sup> osc ...[more]

PMID: 30674675

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Project description:Unlabelled: Mesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin-8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies.SignificanceThe present study investigated and characterized select aspects of the human mesenchymal stem cell (hMSC) secretome and assessed its in vitro and in vivo biological bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. The present study provided the first evidence of a shift of the hMSC cytokine signature induced by oxygen tension, particularly near anoxia (0.1% O2). Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine, could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest in using conditioned media in therapeutic modalities.

Dataset Information

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

Publications

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension.

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