Supplementary Material for: Cadherin Expression is Regulated by Mechanical Phenotypes of Fibroblasts in the Perivascular Matrix

The influence of mechanical forces generated by stromal cells in the perivascular matrix is thought to be a key regulator in controlling blood vessel growth. Cadherins are mechanosensors that facilitate and maintain cell-cell interactions and blood vessel integrity, but little is known about how stromal cells regulate cadherin signaling in the vasculature. Our objective was to investigate the relationship between mechanical phenotypes of stromal cells with cadherin expression in 3D tissue engineering models of vascular growth.

Stromal cell lines were subjected to a bead displacement assay to track matrix distortions and characterize mechanical phenotypes in 3D microtissue models. These cells included human ventricular cardiac (NHCF), dermal (NHDF), lung (NHLF), breast cancer-associated (CAF), and normal breast fibroblasts (NBF). Cells were embedded in a fibrin matrix (10mg/mL) with fluorescent tracker beads; images were collected every 30min. We also studied endothelial cells (ECs) in co-culture with mechanically-active or -inactive stromal cells and quantified N-Cad, OB-Cad, and VE-Cad expression using immunofluorescence.

Bead displacement studies identified mechanically-active stromal cells (CAFs, NHCFs, NHDFs) that generate matrix distortions and mechanically-inactive cells (NHLFs, NBFs). Compared to ECs only, CAFs + ECs as well as NBFs + ECs in 3D co-culture significantly decreased expression of VE-Cad; in addition, the Pearson’s Correlation Coefficient for N-Cad and VE-Cad showed a strong correlation (>0.7), suggesting cadherin colocalization. Using a microtissue model, we demonstrated that mechanical phenotypes associated with increased matrix deformations correspond to enhanced angiogenic growth. The results could suggest a mechanism to control tight junction regulation in developing vascular beds for tissue engineering scaffolds or understanding vascular growth during developmental processes. Our studies provide novel data for how mechanical phenotype of stromal cells in combination with secreted factor profiles is related to cadherin regulation, localization, and vascularization potential in 3D microtissue models.

血管周围基质中基质细胞产生的机械力，被认为是调控血管生长的关键因素。钙粘蛋白（Cadherins）是一类机械感受器，能够介导并维持细胞间相互作用以及血管完整性，但目前对于基质细胞如何在脉管系统中调控钙粘蛋白信号通路仍知之甚少。本研究旨在探究血管生长三维组织工程模型中，基质细胞机械表型与钙粘蛋白表达之间的关联。

本研究采用微球位移实验，对三维微组织模型中的基质细胞系进行基质畸变追踪与机械表型表征。所使用的细胞系包括人心室心肌细胞（NHCF）、皮肤成纤维细胞（NHDF）、肺成纤维细胞（NHLF）、乳腺癌相关成纤维细胞（CAF）以及正常乳腺成纤维细胞（NBF）。将细胞包埋于含荧光追踪微球的纤维蛋白基质（浓度为10mg/mL）中，每隔30分钟采集一次图像。我们还将内皮细胞（ECs）与具有机械活性或无机械活性的基质细胞进行共培养，并采用免疫荧光法定量检测N-钙粘蛋白（N-Cad）、OB-钙粘蛋白（OB-Cad）以及血管内皮钙粘蛋白（VE-Cad）的表达水平。

微球位移实验结果显示，具备基质畸变产生能力的基质细胞为机械活性型（包括CAFs、NHCFs与NHDFs），而无此能力的则为机械非活性型（包括NHLFs与NBFs）。与单纯培养的内皮细胞相比，三维共培养体系中CAFs与内皮细胞共组、NBFs与内皮细胞共组的VE-Cad表达水平均显著下调；此外，N-Cad与VE-Cad的皮尔逊相关系数显示二者存在强相关性（>0.7），提示钙粘蛋白存在共定位现象。借助微组织模型，我们证实与基质形变增强相关的机械表型，可对应促血管生成生长效应。本研究结果可为组织工程支架中发育中血管床的紧密连接调控，或是解析发育过程中的血管生长机制提供新的思路。本研究为三维微组织模型中，基质细胞机械表型与分泌因子谱共同如何调控钙粘蛋白的表达、定位以及血管化潜能提供了全新的数据支撑。