Engineering molecular reporters to investigate effects of shear stress upon endothelial cells

The endothelial monolayer and its associated basement membrane compose a non-thrombogenic surface and selective barrier between the blood and the vessel wall. Loss of normal endothelial integrity, with effects that range from increased permeability to the endothelial disruption, is thought to be an important contributor to development and complication of atherosclerotic lesions ultimately causing life-threatening conditions such as stroke or myocardial infarction. Understand the processes that lead to such outcomes is essential to fashioning effective clinical therapies. Atherosclerotic lesions occur in areas of disturbed flow, thus we investigated the hypothesis that exposure of endothelium to pulsatile flow enhances expression of enzymes which degrade the endothelial basement membrane, called matrix metalloproteinases. Indeed, we found that flow increased production of MMP-9 by endothelial cells. To obtain further insight into this novel regulatory function of flow, we set out to identify flow-responsive regions of the MMP-9 gene promoter. However, endothelial cells are notoriously difficult to transfect effectively, thus we chose to use a new approach in which we engineered promoter reporters into retroviruses, which can efficiently infect and integrate into murine cells. For this promoter deletion analysis, we created six different recombinant retroviruses containing a reporter gene (luciferase) being driven by increasing lengths of the MMP-9 promoter. These constructs infected and integrated into murine endothelial cells with 99+% efficiency. Promoter analysis of infected cells determined that the region between -153 and -93 (relative to the transcriptional start site) was necessary for transcriptional activity and regulation of the MMP-9 gene. We also found that pulsatile shear stress (15 dynes/cm2) increases MMP-9 transcription 2-3 fold, as compared to cells maintained in static culture. In this project, we therefore devised and engineered novel retroviral reporters, and we already demonstrated their utility in conditions previously difficult to tackle, an approach that can become a valuable tool for researchers in the field of tissue engineering. Specifically, through their use, we were able to identify a novel potential regulatory element in the promoter of MMP-9, which could have important consequences for the understanding of MMP role in vascular pathology and could lead to the development of therapeutic interventions.