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Divergent cell fates and functions between skeletal and soft tissue CD146+ pericytes

ABSTRACT: Human pericytes demonstrate multilineage differentiation potential, and their descendants participate in tissue homeostasis and repair. Increasing evidence from developmental biology and tissue engineering suggest that regional specification by tissue of origin exists among human pericytes.Here, we sought to define the differentiation of CD146+ human pericytes from skeletal and soft tissue sources. Uncultured CD146+CD31-CD45- pericytes were derived by fluorescent activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146+CD31-CD45- cells retained canonical features of pericytes, including cell surface marker expression, multilineage differentiation potential, and paracrine induced tubulogenesis. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis, while soft tissue pericytes did not in vitro or in vivo. Microarray analysis demonstrated enrichment in CXCR4 and BMP signaling among periosteal pericytes in comparison to their soft tissue pericyte counterparts. Signaling pathway manipulation of CXCR4 among soft tissue pericytes led to an increase in osteoblastogenesis and bone formation. In sum, skeletal and soft tissue pericytes differ in their relative lineage differentiation potential and ability to form bone. Plasticity exists, however, and manipulation of CXCR4 signaling pathway may ‘coax’ a soft tissue pericyte toward an osteoblastogenic cell fate.

ORGANISM(S): Homo sapiens

PROVIDER: GSE125545 | GEO | 2020/06/22

REPOSITORIES: GEO

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Project description: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.

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Divergent cell fates and functions between skeletal and soft tissue CD146+ pericytes

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