Abstract Rationale Mannose receptor C-type 2 (MRC2) is predominantly expressed by stromal cells and serves as a key endocytic receptor for collagen internalization and degradation. MRC2 expression declines with aging and has been implicated in the pathogenesis of pulmonary fibrosis. We previously showed that lung fibroblasts from Mrc2-/- mice exhibit defective collagen uptake, and that these mice have increased baseline lung stiffness without changes in total hydroxyproline content. These observations led us to hypothesize that loss of MRC2 alters matrix collagen architecture. To test this, we used second-harmonic generation (SHG) imaging to quantify the organization of collagen deposited by primary lung fibroblasts isolated from Mrc2-/- and wild-type (WT) mice. Methods Primary lung fibroblasts were isolated from age- and sex-matched Mrc2-/- and WT mice. Cells were treated with ascorbic acid and cultured on chamber slides for 14 days. Collagen structure within the extracellular matrix was assessed by second-harmonic generation (SHG) imaging. The degree of alignment was computed as the extent to which the fibers run in a common direction within the image. Fiber orientation metrics were determined using an innovative Fourier-space fitting technique. Results The degree of alignment, reflecting the uniformity of fiber orientation, was significantly higher in Mrc2-/- derived matrix than in WT, indicating a greater degree of directional organization with the loss of MRC2. In contrast, the fiber orthogonality, representing the extent of cross-oriented (orthogonal) fiber structures, was lower in Mrc2-/- derived matrix. Conclusions These findings show that the collagen-rich matrix deposited by Mrc2-/- fibroblasts is characterized by a quantifiable reduction in the intersecting or lattice-like organization typically seen in WT and a corresponding increase in parallel aligned fibers. These altered collagen fibers may lead to changes in fibroblast behaviors. Future studies will analyze the response of fibroblasts to Mrc2-/- vs WT matrices and will assess if collagen fiber architecture is similarly altered in vivo in Mrc2-/- lungs, both at baseline and during fibrosis. This abstract is funded by: This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Directed Medical Research Programs (CDMRP), Peer Reviewed Medical Research Program (PRMRP) Focused Program Award under Award No. HT9425-24-1-0543. Funding for this project was also provided by the University of Wisconsin School of Medicine and Public Health from the Wisconsin Partnership Program.
Yamamoto et al. (Fri,) studied this question.