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+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.研究背景：周细胞向肌成纤维细胞分化可引发微血管退变、细胞外基质（extracellular matrix, ECM）堆积与组织硬化，此为纤维化疾病的典型病理特征。目前学界尚未明确微血管微环境中周细胞-肌成纤维细胞分化的调控机制。本团队前期研究构建了一种全新的二维共培养平台，用于共培养源自同一组织的微血管内皮细胞（endothelial cells, ECs）与周细胞。本研究旨在探究细胞外基质硬度对微血管内皮细胞、周细胞及其相互作用的调控作用。 实验方法：采用TGM2D培养基培养原代微血管。硬质细胞外基质的制备方式为：将基质溶液置于常规培养皿中孵育1小时，随后经磷酸盐缓冲液（phosphate buffered saline, PBS）清洗；除非另有说明，本研究均采用杨氏模量约6 kPa的软质细胞外基质。以大鼠颅骨制备骨移植物。对细胞开展免疫荧光染色、RNA测序、实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qRT-PCR）、蛋白质印迹（western blotting）及基因敲低实验。 实验结果：在硬质细胞外基质上，即使加入转化生长因子β（transforming growth factor β, TGFβ）信号通路抑制剂A83-01，原代微血管周细胞仍会分化为肌成纤维细胞（NG2+αSMA+）。软质细胞外基质与A83-01可协同维持微血管稳定性，并抑制周细胞向肌成纤维细胞分化（NG2+αSMA-/low）。据此我们定义了两类周细胞亚群：静息周细胞（NG2+αSMA-/low）与活化周细胞（NG2+αSMA+）。体内骨移植实验证实，软质细胞外基质可促进微血管再生并抑制纤维化进程。鉴于整合素是主要的机械感受器，我们基于RNA测序数据筛选整合素家族成员并开展qRT-PCR验证，发现整合素β1（Integrin β1, Itgb1）是受软质细胞外基质与A83-01处理下调的核心亚基。敲低Itgb1可抑制硬质细胞外基质上的肌成纤维细胞分化。值得注意的是，整合素β1磷酸化位点Y783主要定位于硬质细胞外基质上的微血管内皮细胞，该磷酸化修饰可促进内皮细胞分泌包括结缔组织生长因子（connective tissue growth factor, CTGF）在内的旁分泌因子，进而诱导周细胞向肌成纤维细胞分化。敲低CTGF或采用单克隆抗体处理可部分抑制肌成纤维细胞分化，提示纤维化形成过程涉及多条信号通路。 研究结论：微血管内皮细胞通过旁分泌信号通路介导细胞外基质硬度诱导的周细胞-肌成纤维细胞分化。