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Extracellular matrix stiffness regulates microvascular stability by controlling endothelial paracrine signaling to determine pericyte fate

ABSTRACT: 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+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) 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 β1 (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.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE237069 | GEO | 2023/07/18

REPOSITORIES: GEO

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Project description:Pericytes have been implicated in regulation of inflammatory, reparative, fibrogenic and angiogenic responses in several different organs and pathologic conditions. Although the adult mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. We used NG2Dsred;PDGFRaEGFP pericyte-fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in a model of myocardial infarction. The transcriptomic profile of pericyte-derived fibroblasts was studied using PCR arrays. The transcriptomic profile of NG2 lineage cells (pericytes) was studied in control and infarcted hearts using single cell RNA-sequencing analysis. The role of TGF-b signaling in regulation of pericyte phenotype in vivo was investigated using pericyte-specific Tgfbr2 knockout mice. In vitro, the effects of TGF-b were studied in cultured human placental pericytes.In normal mouse hearts, NG2 and PDGFRa identified distinct non-overlapping populations of pericytes and fibroblasts respectively. Following myocardial infarction, a population of cells expressing both pericyte and fibroblast markers emerged. These cells expressed large amounts of extracellular matrix (ECM) genes. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes underwent fibroblast conversion. Single cell RNA-seq experiments demonstrated expansion and diversification of pericyte-derived cells in the infarct, associated with emergence of subpopulations exhibiting accentuated matrix gene synthesis. In vitro studies and the profile of pericyte-derived fibroblasts identified TGF-b as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific Tgfbr2 disruption had no significant effects on myofibroblast infiltration and collagen deposition in the infarct. Pericyte-specific TGF-b signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels. These reparative effects of infarct pericytes protected the infarcted heart from dilative remodeling.

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Extracellular matrix stiffness regulates microvascular stability by controlling endothelial paracrine signaling to determine pericyte fate

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