Human Cell-Derived Extracellular Matrix Modulates Endothelial Cell Morphology and Metabolism in Response to Fluid Shear Stress.

Endothelial cells integrate biochemical cues from the extracellular matrix (ECM) with mechanical cues from blood flow to regulate vascular function, yet the combined influence of native human ECM complexity and physiologic fluid shear stress remains poorly understood. Here, we developed a custom 3D-printed cone-and-plate rheometer compatible with compliant, heterogeneous, and cell-derived substrates, enabling the application of controlled laminar shear stress to the human cell-derived matrix (hCDM) and hydrogels without structural disruption. Using this platform, we investigated endothelial cell responses to variations in ECM composition and fibrillar microstructure under controlled laminar shear stress. As a demonstration of this platform, we show that hCDM, which contains some proteins found in the intimal basement membrane, pre-aligns endothelial cells and constrains their morphological response to shear stress, in contrast to the robust flow-induced alignment observed on fibronectin. Transcriptomic profiling revealed substrate-dependent differences in mechanotransduction signaling under flow, including differential regulation of integrin expression and significant upregulation of genes in the SREBP-associated cholesterol metabolism pathway. Consistent with these transcriptional trends, endothelial cells cultured on hCDM exhibited increased lipid droplet accumulation under flow. Pre-alignment of hCDM fibrils further decoupled matrix orientation from the flow direction, demonstrating that engineered control of fibrillar architecture can modulate alignment, junctional organization, and metabolic response. Together, these findings establish hCDM as a biologically rich and mechanobiologically active substrate for vascular studies and introduce a versatile rheometer platform that expands experimental access to physiologic shear environments on compliant, ECM-derived materials.