Fibrosis is a pathological process characterized by persistent fibroblast activation and excessive ECM accumulation. Aortic carboxypeptidase-like protein (ACLP), a secreted ECM protein that binds fibrillar collagen, is up-regulated in fibrotic tissues and promotes fibroblast differentiation through canonical TGFβ receptor signaling. We hypothesized that when presented within the collagen matrix, ACLP would engage integrin-dependent mechanical signaling pathways that contribute to fibrogenic activation. Using 10T1/2 mouse mesenchymal progenitor cells, we identify a previously unrecognized mechanism through which collagen-bound ACLP induces fibrogenic activation via β1 integrin–mediated signaling. Collagen-bound ACLP induced rapid cell spreading, increased β1 integrin activation, and promoted focal adhesion maturation. These adhesion events triggered activation of the GTPases RhoA and Rac1, accompanied by enhanced F-actin assembly and nuclear accumulation of myocardin-related transcription factor A, a key regulator of fibrogenic gene expression. Transcriptomic profiling revealed enrichment of focal adhesion, ECM–receptor interaction, and actin cytoskeletal pathways downstream of collagen-bound ACLP, which was conserved in primary adipose-derived stromal cells. Together, these findings establish collagen-bound ACLP as a matrix-derived cue that links ECM composition to integrin-dependent fibrogenic activation. The RNA-sequencing data that support the findings of this study are publicly available on Sequencing Read Archive (SRA) under BioProject [PRJNA1368594][1] and processed data in the Gene Expression Omnibus (GEO) repository under accession numbers [GSE312027][2] (10T1/2 cells) and [GSE324887][3] (primary stromal cells). [1]: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1368594 [2]: /lookup/external-ref?link_type=NCBIGEO&access_num=GSE312027&atom=%2Flsa%2F9%2F6%2Fe202503600.atom [3]: /lookup/external-ref?link_type=NCBIGEO&access_num=GSE324887&atom=%2Flsa%2F9%2F6%2Fe202503600.atom
Extracellular matrix (ECM) mechanical properties regulate tissue homeostasis and disease progression, with persistent ECM stiffening serving as a hallmark of fibrosis; yet, the early transition from healthy to diseased tissue remains poorly understood. Dynamic three-dimensional (3D) tissue models that capture early-stage stiffening are needed to investigate cellular responses during disease initiation. This work presents an innovative platform for studying cell responses in 3D environments undergoing active matrix stiffening. A bioinspired synthetic ECM incorporates collagen-mimetic peptides and employs sequential, non-terminal strain-promoted azide–alkyne cycloaddition (SPAAC) reactions to enable controlled increases in matrix stiffness over physiologically relevant timescales. Alternating polymer incubations produce a 2.5-fold increase in storage modulus over 72 hours, modeling the mechanical transition from healthy to early-stage fibrotic lung tissue. Live-cell reporter fibroblasts enable real-time monitoring of alpha-smooth muscle actin (αSMA) expression, revealing significant upregulation during matrix stiffening that remains transient and difficult to detect via traditional endpoint assays. Active stiffening also modulates fibroblast motility, transiently increasing migration speed while persistently enhancing directional persistence. Complementary computational reaction-diffusion modeling provides mechanistic insight into modulus gradient formation and reaction kinetics. This versatile toolbox enables investigation of early mechanobiological responses to matrix stiffening and may aid identification of markers of fibrotic disease onset. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, DP2-HL152424, P30 GM110758-02 U.S. National Science Foundation, https://ror.org/021nxhr62, DMR-2011824
Rxiv mechanobio
