Institute for Molecular Science of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan.
Institute for Molecular Science of Medicine, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan; Biomedical Technology Research Center, Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Matrix Biol. 2020 May;87:77-93. doi: 10.1016/j.matbio.2019.10.006. Epub 2019 Oct 26.
Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix, and is expressed at high levels in tissues during development and remodeling in pathological conditions. Its core protein is cleaved at a region close to the N-terminal end of CSβ domain by several members of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, i.e., ADAMTS-1, 4, 5, 9, 15, and 20. Here, using a CRISPR/Cas9 system, we generated knock-in mice (V1R), which express an ADAMTS cleavage-resistant versican. Some V1R homozygote mice, termed R/R, exhibit syndactyly and organ hemorrhage. In wound healing experiments, R/R wound shows accumulation of versican and activated TGFβ-signaling in the early stage, leading to faster healing than wild type wound. Immunostaining for Ki67, CD31, smooth muscle α-actin, periostin demonstrates higher levels of overall cell proliferation and an increased number of endothelial cells and myofibroblasts. Immunostaining for CD11b and qRT-PCR for macrophage markers revealed increased levels of inflammatory cell infiltration, especially those of M1 macrophages. Cultured R/R dermal fibroblasts revealed increased deposition of versican, type I and III collagens, and hyaluronan, and upregulation of Smad2/3 signaling. Taken together, these results demonstrate that the cleavage site determines versican turnover and that versican plays a central role in the provisional matrix during the wound repair.
蛋白聚糖聚糖是细胞外基质中的一种大型硫酸软骨素/硫酸皮肤素蛋白聚糖，在发育过程中和病理条件下组织的重塑过程中高水平表达。其核心蛋白在靠近 CSβ 结构域 N 端的区域被几种解整合素和金属蛋白酶与血小板反应蛋白基序（ADAMTS）家族成员切割，即 ADAMTS-1、4、5、9、15 和 20。在这里，我们使用 CRISPR/Cas9 系统生成了表达 ADAMTS 切割抗性蛋白聚糖的基因敲入小鼠（V1R）。一些 V1R 纯合子小鼠，称为 R/R，表现出并指和器官出血。在伤口愈合实验中，R/R 伤口显示出在早期积累蛋白聚糖和激活 TGFβ 信号，导致比野生型伤口更快愈合。Ki67、CD31、平滑肌α-肌动蛋白、骨膜蛋白的免疫染色显示出更高水平的整体细胞增殖，以及更多的内皮细胞和肌成纤维细胞。CD11b 的免疫染色和巨噬细胞标志物的 qRT-PCR 显示出炎症细胞浸润水平增加，特别是 M1 巨噬细胞。培养的 R/R 真皮成纤维细胞显示出蛋白聚糖、I 型和 III 型胶原以及透明质酸的沉积增加，以及 Smad2/3 信号的上调。总之，这些结果表明切割位点决定了蛋白聚糖的周转率，并且蛋白聚糖在伤口修复过程中的临时基质中起核心作用。
