Roles of Type V Collagen in the Mechanotransduction and Immunoresponses of Fibroblasts

Introduction: Fibroblasts play an important function in the production of collagenous extracellular matrix, and at the same time, are highly adaptive and responsive to various biomechanical and biochemical cues [1]. During wound healing, fibroblasts receive a plethora of stimuli from macrophages, dictating how the fibroblasts respond. M1 macrophages induce a proinflammatory response, leading to matrix degradation and collagen remodeling [2], while M2 macrophages induce a pro-regenerative response, resulting in a myofibroblastic transition and excessive collagen I deposition [1, 3]. In the case of a fibrotic matrix, the shift of fibroblasts into myofibroblasts perpetuates a cycle of scar formation [1, 3]. Type V collagen, a minor regulatory collagen, is canonically known to regulate collagen I fibril assembly and size [4], suggesting it could be a key player in this process. Indeed, in patients with classic Ehlers-Danlos syndrome (cEDS), caused by mutations in the COL5A1 and/or COL5A2 genes, a major symptom is delayed wound healing and formation of atrophic scars [4]. Understanding how collagen V plays a role in fibroblast mechanosensing could advance our knowledge of the fibroblast-matrix crosstalk in fibrosis. This study aims to determine how the haploinsufficiency of collagen V alters fibroblast response to external immunomodulatory and mechanical stimuli.

Materials and

Methods: Mouse embryonic fibroblasts (MEFs) were harvested from the dermis of E18.5 wild-type (WT) or Col5a1+/− mice. Extracted dermis was minced using a razor blade and allowed to adhere to cell culture dishes. Further digestion was performed by treating with collagenase type IV at 5 mg/ml in cDMEM media containing 10%FBS and 1% Penn/Strep/AmphB for 2-3 days. After treatment, non-adherent cells and tissue will be resuspended in fresh cDMEM and was replated. Media were changed every 2 days until cells were confluent. Polyacrylamide (PAAm) (Advanced Biomatrix) gels with 2kPa (soft) and 64kPa (stiff) moduli were used as the primary culturing substrate. cDMEM supplemented with 50 ug/ml ascorbic acid along either TNF-α (20ng/ml), TGF-β (10ng/ml), or a non-drug control was administered to the cells for 48 hours. Here, TNF-α and TGF-β were used to simulate M1-macrophage induced proinflammatory response and M2-macrophage induced remodeling response, respectively . To generate cell derived matrices (CDMs), tissue culture dishes coated with 0.2% bovine skin gelatin were used to culture cells at 4.5×105 cells/dish. After the culture, we performed immunofluorescence staining for collagen V (AB7046, Abcam), phalloidin for F-actin cytoskeleton (A12379, Thermo Fisher), paxillin (5H11, Thermo Fisher) and DAPI for nuclei (62248, Thermo Fisher). Following a 10-day culture to allow for matrix formation, AFM-based nanoindentation was performed using a microspherical tip (R ≈ 5 μm, k ≈ 0.03 N/m) in PBS to quantify matrix indentation modulus, Eind, following our established procedures [5, 6].

Results, Conclusions, and Discussions: Results and

Discussion: The reduced collagen V expression in Col5a1+/− cells relative to the WT control was validate at the protein level by decreased collagen V staining (Fig. 1a), and at the transcription level by qPCR (Fig. 1b). When examining morphological changes of Col5a1+/− versus WT cells under treatments, we observed that WT cells exhibited morphological changes in response to immunological cues, including increased aspect ratio under TNF-α stimulation, as well as increased cell spreading and expression of stress fibers under TGF- β stimulation. In addition, in WT cells, treatment of TNF-α resulted in decreased FAK staining (Fig. 2, magenta), illustrating altered mechanotransduction. In contrast, Col5a1+/− cells did not demonstrate these changes (Fig. 2). Furthermore, comparing the soft versus stiff substrates, we did not note salient changes in cell morphology or FAK distribution response to the substrate stiffness for either cell type, with or without the treatment of TNF-α or TGF-β (Fig. 2). Applying AFM-nanoindentation, we observed Col5a1+/− cells produced a CDM with higher modulus compared to that of WT under TGF- β stimulation. However, such contrast was absent for TNF-α-treated or untreated groups. Together, these results support that loss of collagen V could have a significant impact on the assembly of fibroblast-synthesized matrix, and fibroblast response to biomechanical and inflammatory cues. Our ongoing studies aim to uncover downstream mechanobiological signaling pathways regulated by collagen V and its interactions with fibroblasts, including its modulation on integrin expressions.

Conclusion: This study shows that collagen V impacts the immunomodulatory and mechanoresponses of fibroblasts. Results provided the basis for improving fibroblast-mediated wound healing by targeting collagen V activities.

References: [1] Hannan+2018. [2] Chou+1996. [3] Witherel+2021. [4] Wenstrup+ 2004. [5] Chery+ 2020. [6] Li, Q+2017.

Acknowledgements (Optional): This work was supported by NSF CAREER CMMI-1751898 and the U.S. Department of Education’s Graduate Assistance in Areas of National Need (GAANN) Program.