Development and testing of a coculture flow system for studying endothelial and smooth muscle cell interactions under high wall shear stress

Smooth muscle cells (SMCs) have been demonstrated to play a critical role in the formation of intracranial aneurysms (IAs) by undergoing phenotypic modulation to a proinflammatory state. In this state they downregulate contractile proteins, upregulate inflammatory markers, and contribute to vessel wall degradation through secretion of matrix metalloproteinases (MMPs). These changes occur in regions of high wall shear stress (WSS) and positive wall shear stress gradient (WSSG) and an intact endothelium. Although specific mechanisms remain unclear, it is likely that increased blood flow affects the endothelial cells (ECs), inducing a change in signaling that provides the trigger modulating SMCs to an inflammatory state. The objective of this study was to design an in vitro system that delivers physiological WSS as well as pathologically high WSS to cocultures of ECs and SMCs and monocultures of SMCs in order to test the hypothesis that high WSS modulates SMCs to a proinflammatory phenotype through EC signaling. Immunofluorescent staining confirmed the differentiated phenotype of SMCs and ECs at the start of the experiments and qPCR was performed to assess and compare the phenotypic changes of SMCs in coculture and monoculture models under high WSS. This study demonstrated that ECs affect SMC behavior in our cocultures, as is demonstrated by the statistically significant differences in SMC expression between monocultures and cocultures under the same conditions. However, the results of phenotypic assessment under increased WSS did not correlate with the expected results of our hypothesis. The most likely reason for this lack of correlation is the short duration of flow exposure, which is limited to 6 hours due to the limited capability of ECs to remain adhered for longer studies. In order to accurately recreate the inflammatory SMC phenotype observed in intracranial aneurysms, modifications must be made to the system to enhance EC adhesion so that longer studies can be performed. The flow chamber that was built, the qPCR primers designed, and the coculturing techniques established in this study can be used in future studies that will improve the system so that it can be used to perform mechanistic studies.