Bala Vaishali, Patel Vidhi, Sewell-Loftin Mary Kathryn
Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, Alabama, USA.
O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Cells Tissues Organs. 2024;213(6):446-463. doi: 10.1159/000539319. Epub 2024 May 20.
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 (10 mg/mL) with fluorescent tracker beads; images were collected every 30 min. 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). CAFs, NHCFs, and NHDFs displaced the matrix with an average magnitude of 3.17 ± 0.11 μm, 3.13 ± 0.06 μm, and 2.76 ± 0.05 μm, respectively, while NHLFs and NBFs displaced the matrix with an average of 1.82 ± 0.05 μm and 2.66 ± 0.06 μm in fibrin gels. Compared to ECs only, CAFs + ECs as well as NBFs + ECs in 3D co-culture significantly decreased expression of VE-Cad; in addition, 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.
血管周围基质中的基质细胞产生的机械力被认为是控制血管生长的关键调节因子。钙黏蛋白是机械传感器，可促进和维持细胞间相互作用及血管完整性，但关于基质细胞如何调节脉管系统中的钙黏蛋白信号传导，人们知之甚少。我们的目标是在血管生长的三维组织工程模型中研究基质细胞的机械表型与钙黏蛋白表达之间的关系。
对基质细胞系进行微珠位移试验，以追踪基质变形并在三维微组织模型中表征机械表型。这些细胞包括人心室心肌细胞（NHCF）、真皮细胞（NHDF）、肺细胞（NHLF）、乳腺癌相关细胞（CAF）和正常乳腺成纤维细胞（NBF）。将细胞嵌入含有荧光追踪微珠的纤维蛋白基质（10mg/mL）中；每30分钟收集一次图像。我们还研究了与机械活性或非活性基质细胞共培养的内皮细胞（EC），并使用免疫荧光定量N-钙黏蛋白、OB-钙黏蛋白和VE-钙黏蛋白的表达。
微珠位移研究确定了产生基质变形的机械活性基质细胞（CAF、NHCF、NHDF）和机械非活性细胞（NHLF、NBF）。CAF、NHCF和NHDF使基质位移的平均幅度分别为3.17±0.11μm、3.13±0.06μm和2.76±0.05μm，而NHLF和NBF在纤维蛋白凝胶中使基质位移的平均值分别为1.82±0.05μm和2.66±0.06μm。与仅培养内皮细胞相比，三维共培养中的CAF+EC以及NBF+EC显著降低了VE-钙黏蛋白的表达；此外，N-钙黏蛋白和VE-钙黏蛋白的皮尔逊相关系数显示出强相关性（>0.7），表明钙黏蛋白共定位。使用微组织模型，我们证明与基质变形增加相关的机械表型对应于血管生成生长增强。这些结果可能提示了一种机制，用于控制组织工程支架发育中的血管床紧密连接调节或理解发育过程中的血管生长。
我们的研究为基质细胞的机械表型与分泌因子谱如何与三维微组织模型中的钙黏蛋白调节、定位和血管化潜力相关提供了新数据。
