Poster BB11 - Mechanisms of Aligned Collagen Formation in Human Adipose-Derived Stromal/Stem Cells

Introduction: Tendons transmit muscle force to bone via a collagen fiber network, maintained by tenocytes. Injuries result in disorganized collagen, leading to scar tissue formation. Tendon tissue engineering aims to create aligned collagen matrices with adequate mechanical properties and normal tendon fibroblast-matrix coupling. Mechanisms behind cellular production of aligned collagen are not fully understood but involve microscale cues and cellular forces. Mechanosensitive calcium channel transient receptor potential vanilloid 4 (TRPV4) plays a role in aligned collagen formation in mesenchymal stem cells. TRPV4 regulates aligned collagen formation in mesenchymal stem cells, with its activation enhancing alignment and inhibition preventing it. TRPV4's downstream effectors, including yes-associated protein (YAP), are involved in various cellular processes. YAP, activated by TRPV4 in a cell-dependent manner, is crucial for aligned collagen formation during tendon development. However, the TRPV4-YAP relationship varies with tissue and cell context. The roles of TRPV4 and YAP in engineered tendon development on 2D scaffolds are unknown. The objective of this study was to investigate the relationship between TRPV4, YAP, and aligned collagen formation in human adipose-derived mesenchymal stem cells (hASCs) on 2D scaffolds.

Materials and

Methods: Microphotopatterning: Cell-adhesive microphotopatterns (μPP) (Fig. 1A) were created with a 10x1 macro-boundary aspect ratio (1250 μm x 125 μm) with 2 μm diameter channels spaced 5 μm apart (center-to-center) in glass-bottom dishes (MatTek Corporation). This pattern was chosen because it promotes a tendon-like phenotype (unpublished data) and promotes tendon-like gene expression. Cell Culture: hASCs (IRB exempt) were seeded at 20,000 cells/dish and cultured for 3- 7 days in media with 2% FBS and no exogenous growth factors. Agonists and Antagonists: TRPV4 agonists RN1747 (Tocris 3745) and GSK101 (GSK1016790A; Selleck Chem S8107) and antagonist GSK219 (GSK2193874; Tocris 5106) were used to modulate TRPV4. Verteporfin (Tocris 5305) was used to inhibit YAP. All chemicals (Verteporfin, GSK101, GSK219, RN1747) were solubilized in dimethylsulfoxide (DMSO) and added to cell culture media at 0.1% DMSO v/v, and compared to both an untreated, and a 0.1% DMSO only vehicle control. hASCs were recovered for 24-hours in culture on the microphotopatterns before treatment with agonists or antagonists. Actin/Nuclei: After 7 days culture, hASCs were fixed and stained with (4’,6-diamidino-2-phenylindole) (DAPI) (nuclei) and Alexa Fluor 555 phalloidin (actin), then FIJI was used for analysis. Collagen Alignment: After 7 days culture, hASCs were fixed with 4% paraformaldehyde and stained with Picrosirius red. Images were taken using a light microscope with a graded polarizer and analyzer, and collagen alignment was quantified. Statistics: All data (n=4-7) were evaluated using the appropriate parametric or non-parametric test in JMP Pro 15 with confidence level (α=0.05).

Results, Conclusions, and Discussions: TRPV4 activation via GSK101 or RN1747 led to more robust tissue growth over 7 days in culture, with engineered tissues forming larger areas than untreated control or vehicle (p < 0.05). GSK101 treatment led to an increase in aligned collagen formation (Fig 1B,C). GSK101 treatment also resulted in greater green birefringence in Picrosirius red stains under polarized light, suggesting immature or loosely packed collagen fibers or newly deposited collagen fibers. Conversely, inhibiting TRPV4 with GSK219 reduced aligned collagen (Fig 1B,C) without affecting the total collagen area. Similarly, inhibiting YAP with verteporfin reduced aligned collagen without affecting collagen area at low doses (0.25 μM) (Fig 1B,C), while higher doses of verteporfin (>0.5 μM) prevent a cohesive tissue from forming. Simultaneously activating TRPV4 with GSK101 while inhibiting YAP via verteporfin partially rescued YAP-regulated collagen alignment (Fig. 1B,C). The amount of aligned collagen increased significantly with TRPV4 activation in YAP inhibited samples (p < 0.05) but did not achieve the levels of collagen alignment seen in untreated or vehicle control samples (p < 0.05) (Fig. 1B,C). Inhibiting YAP also reduced actin alignment, which was moderately rescued by TRPV4 activation (p>0.05). However, the TRPV4 activation did not rescue actin alignment back to the level of controls (p>0.05). YAP inhibition also reduced nuclear alignment (p < 0.05) and nuclear aspect ratio (p < 0.05), which was also not rescued by TRPV4 activation.
From other studies, TRPV4 activation with GSK101 led to increased force at focal adhesions in mesenchymal stem cells, where it could potentially bind discoidin domain receptor 1 (DDR1) and interact with non-muscle myosin IIA (NMMIIA) to align actin stress fibers and generate forces to align collagen in the extracellular matrix -- a process regulated by TRPV4 signaling. The effect of GSK101 treatment on DDR1 expression is unknown but knocking out TRPV4 expression led to undetectable levels of DDR1 in mouse gingival fibroblasts. If GSK101 stimulation increases DDR1 expression, it could have a protective effect on YAP degradation via LATS1 inactivation and actin alignment via NMMIIA, which could lead to the rescued actin and collagen alignment seen in hASCs treated simultaneously with GSK101 and verteporfin.

Acknowledgements (Optional): NIH grants AR073882 (DL), AR065764 (DL), and AR079844 (TJ).