Angiogenesis array data for “Identification of a pro-angiogenic functional role for FSP1-positive fibroblast subtype in wound healing”

Abstract

Fibrosis accompanying wound healing can drive the failure of many different organs. Activated fibroblasts are the principal determinants of post-injury pathological fibrosis along with physiological repair, making them a difficult therapeutic target. Although activated fibroblasts are phenotypically heterogeneous, they are not recognized as distinct functional entities. Using mice that express GFP under the FSP1 or αSMA promoter, we characterized two non-overlapping fibroblast subtypes from mouse hearts after myocardial infarction. Here, we report the identification of FSP1-GFP+ cells as a non-pericyte, non-hematopoietic fibroblast subpopulation with a predominant pro-angiogenic role, characterized by in vitro phenotypic/cellular/ultrastructural studies and in vivo granulation tissue formation assays combined with transcriptomics and proteomics. This work identifies a fibroblast subtype that is functionally distinct from the pro-fibrotic αSMA-expressing myofibroblast subtype. Our study has the potential to shift our focus towards viewing fibroblasts as molecularly and functionally heterogeneous and provides a paradigm to approach treatment for organ fibrosis.

Angiogenesis protein array

The Proteome Profiler Mouse Angiogenesis Array Kit (# ARY015; R & D) was used to assess the relative levels of 53 mouse angiogenesis-related proteins in 300 µg of FSP1+ and αSMA+ cell lysates according to manufacturer’s instructions. Angiogenesis Array is a membrane-based sandwich immunoassay. The cells were cultured for 72 h in 0.5% FBS prior to protein collection. Briefly, a cocktail of biotinylated detection antibodies was mixed with cell lysates. The array membrane was then treated with the cell lysate and antibody mixture overnight at 4 °C, washed, and then incubated with streptavidin-HRP. The array membrane is spotted with capture antibodies to specific target proteins. The captured proteins were visualized using chemiluminescence.

### 摘要
伴随伤口愈合过程发生的纤维化，可引发多种器官功能衰竭。活化成纤维细胞既是损伤后生理性修复的核心参与者，也是病理性纤维化的主要决定因素，这使其成为难以攻克的治疗靶点。尽管活化成纤维细胞在表型上存在异质性，但目前尚未将其作为功能独立的亚群进行区分。本研究利用在成纤维细胞特异性蛋白1（FSP1）或α平滑肌肌动蛋白（αSMA）启动子调控下表达绿色荧光蛋白（GFP）的小鼠，对心肌梗死模型小鼠心脏中的两种非重叠成纤维细胞亚群进行了系统表征。本研究通过体外表型、细胞及超微结构分析，体内肉芽组织形成实验，并结合转录组学与蛋白质组学技术，鉴定出FSP1-GFP阳性细胞为一类非周细胞、非造血起源的成纤维细胞亚群，其核心功能为促血管生成。本研究鉴定出一类功能与促纤维化型表达αSMA的肌成纤维细胞亚群截然不同的成纤维细胞亚群。本研究有望推动学界将成纤维细胞视作分子与功能层面均具有异质性的群体，并为器官纤维化的治疗研究提供全新范式。

### 血管生成蛋白阵列实验
本研究采用蛋白质组谱分析小鼠血管生成阵列试剂盒（货号ARY015；R&D公司），依照产品说明书，对300μg的FSP1阳性与αSMA阳性细胞裂解液中53种小鼠血管生成相关蛋白的相对表达水平进行定量检测。该血管生成阵列属于基于膜载体的夹心免疫检测技术。在收集蛋白前，将细胞置于含0.5%胎牛血清（FBS）的培养基中培养72小时。简要实验流程如下：将生物素标记的检测抗体混合液与细胞裂解液充分混匀；随后将阵列膜与该裂解液-抗体混合液置于4℃孵育过夜，经洗涤后再与链霉亲和素-辣根过氧化物酶（HRP）孵育。阵列膜上预点有针对各特定靶蛋白的捕获抗体；结合的靶蛋白可通过化学发光法实现可视化检测。