Effect of shear stress on coronary smooth muscle maturation

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Project Summary The broad objective of this proposal is to understand why the developing coronary arteries are consistently formed at specific locations. During development of the coronary arteries, numerous immature coronary strands connect to the aorta and then remodel to form the two mature arteries observed in the adult heart. During this remodeling process, one of the biggest changes applied to the newly connected coronary vasculature is the sudden onset of fluid shear stress, yet the regulatory role of fluid shear stress on the development of the coronary vasculature is completely unknown. This proposal specifically addresses how the onset of blood flow, which causes shear stress, induces signaling pathways that would promote smooth muscle migration, proliferation, and maturation. Aim 1 examines the environment surrounding the aorta during the early stages of smooth muscle recruitment to the coronaries. Low levels of shear stress are predicted to promote expression of ET1 and thus lead to matrix metalloprotease (MMP) activity, yielding an environment that promotes smooth muscle migration. This aim will evaluate ET1, MMP2, and MMP9 expression as well as smooth muscle proliferation in the developing coronary arteries of the chick embryo. In ovo injection of MMP2 and 9 inhibitors will confirm the necessity of these MMPs for smooth muscle migration. Aim 2 examines the effects of increased levels of shear stress on cell signaling, particularly the transcription factor KLF2 and the nitric oxide synthase eNOS. High levels of shear stress are predicted to induce KLF2 and lead to eNOS phosphorylation, which promote smooth muscle maturation. Asymmetrical shear stress would thus selectively mature smooth muscle, explaining the consistent patterning of the coronary arteries. In ovo inhibition of eNOS activity will confirm whether eNOS activity is required for smooth muscle maturation and coronary artery stability. Most of the proposed techniques are easily accessible to undergraduate research students, and the preliminary data were generated by undergraduate students, supporting the feasibility of this project at the University of Portland. Altogether, this proposal sits at the intersection among physical forces, molecular responses, and cellular behavior, with the end objective of understanding the formation of the coronary arteries. By better understanding the normal development of these arteries, the proposed research will support future work to examine how these developmental processes go awry, leading to life-threatening congenital coronary artery anomalies.