Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, Medical College, Qingdao University, China (Y.Y., Y. Leng, X.S., J.M., L.M., L.Y., Y.Z., J.L., D.W.).
School of Basic Medicine, Qingdao University, China (Y.Y., Y. Leng, X.S., L.M., L.Y., Y.Z.).
Arterioscler Thromb Vasc Biol. 2023 Oct;43(10):1887-1899. doi: 10.1161/ATVBAHA.123.319119. Epub 2023 Aug 31.
The differentiation of pericytes into myofibroblasts causes microvascular degeneration, ECM (extracellular matrix) 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 2-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.
Primary microvessels were cultured in the TGM2D medium (tubular microvascular growth medium on 2-dimensional substrates). Stiff ECM was prepared by incubating ECM solution in regular culture dishes for 1 hour followed by PBS wash. Soft ECM with Young modulus of ≈6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, RT-qPCR (real-time quantitative polymerase chain reaction), Western blotting, and knockdown experiments were performed on the cells.
Primary microvascular pericytes differentiated into myofibroblasts (NG2αSMA) on stiff ECM, even with the TGFβ (transforming growth factor beta) signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2αSMA). We thus defined 2 pericyte subpopulations: primary (NG2αSMA) and activated (NG2αSMA) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As integrins are the major mechanosensor, we performed RT-qPCR screening of integrin family members and found Itgb1 (integrin β1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down 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 (connective tissue growth factor), to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation.
ECM stiffness and TGFβ signaling cooperatively regulate microvascular stability and pericyte-myofibroblast differentiation. Stiff ECM promotes EC ITGB1 phosphorylation (Y783) and CTGF secretion, which induces pericyte-myofibroblast differentiation.
周细胞向肌成纤维细胞的分化导致微血管退化、细胞外基质（ECM）积累和组织僵硬，这是纤维化疾病的特征。周细胞在微血管环境中的分化是如何调节的尚不清楚。我们之前的研究建立了一种新的 2 维平台，用于共培养来自同一组织的微血管内皮细胞（ECs）和周细胞。本研究探讨了 ECM 硬度如何调节微血管 ECs、周细胞及其相互作用。
原代微血管在 TGM2D 培养基（管状微血管生长培养基在 2 维基质上）中培养。通过将 ECM 溶液在普通培养皿中孵育 1 小时，然后用 PBS 洗涤来制备刚性 ECM。除非另有说明，否则使用杨氏模量约为 6 kPa 的软 ECM。骨移植物从大鼠颅骨中制备。对细胞进行免疫染色、RNA 测序、实时定量聚合酶链反应（RT-qPCR）、Western 印迹和敲低实验。
在刚性 ECM 上，即使存在 TGFβ（转化生长因子β）信号抑制剂 A83-01，原代微血管周细胞也分化为肌成纤维细胞（NG2αSMA）。软 ECM 和 A83-01 协同维持微血管稳定，同时抑制周细胞-肌成纤维细胞分化（NG2αSMA）。因此，我们定义了 2 种周细胞亚群：原代（NG2αSMA）和激活（NG2αSMA）周细胞。软 ECM 促进了体内骨移植物移植中的微血管再生和抑制纤维化。由于整合素是主要的机械感受器，我们对整合素家族成员进行了 RT-qPCR 筛选，发现 Itgb1（整合素β1）是软 ECM 和 A83-01 处理下调的主要亚基。敲低抑制了刚性 ECM 上的肌成纤维细胞分化。有趣的是，在刚性 ECM 上，ITGB1 磷酸化（Y783）主要位于微血管 ECs 上，这促进了 EC 分泌旁分泌因子，包括 CTGF（结缔组织生长因子），从而诱导周细胞-肌成纤维细胞分化。CTGF 敲低或单克隆抗体治疗部分减少了肌成纤维细胞分化，这表明纤维化形成涉及多种途径。
ECM 硬度和 TGFβ 信号协同调节微血管稳定性和周细胞-肌成纤维细胞分化。刚性 ECM 促进 EC ITGB1 磷酸化（Y783）和 CTGF 分泌，从而诱导周细胞-肌成纤维细胞分化。
