Extracellular matrix stiffness regulates microvascular stability by controlling endothelial paracrine signaling to determine pericyte fate
Background:  The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions.  Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young's modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results:  Primary microvascular pericytes differentiated into myofibroblasts (NG2+aSMA+) on stiff ECM, even with the TGFß signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+aSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+aSMA-/low) and activated (NG2+aSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin ß1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation.  Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling. Overall design: to investigate the effect of stiffness and TGFßinhibitor A83-01 on microvessels and pericyte-myofibroblast differentiation