Cells respond to physical forces in their environment through a process called mechanotransduction. Mechanotransduction molecules on the cell surface recognize physical forces and transmit an internal biochemical signal that can affect cell growth, gene expression, etc. Endothelial cells (ECs), or the cells lining the blood vessels, can sense shear stress induced by the blood flow. In regions of high shear stress, the cells elongate and align with the direction of the flow. However, in regions of low shear stress or disturbed flow, the ECs do not have an elongated and oriented morphology. These regions of low or disturbed flow are susceptible to the formation of atherosclerotic lesions. Therefore the study of mechanotransduction in ECs will aid in our understanding of atherosclerosis and cardiovascular disease. Experiments with ECs exposed to fluid flow or stretched ECs have shown that cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1) is phosphorylated by the protein kinase Fyn. SHP-2, a protein tyrosine phosphatase, propagates the signal along the ERK/MAPK biochemical pathway, eventually altering the EC growth and alignment. It is hypothesized that physical stretching of PECAM-1 unravels the cytoplasmic domain and exposes the region that is phosphorylated. The proposed research will build an understanding of how PECAM-1 responds to physical forces through three aims.
In Aim 1, a construct consisting of the cytoplasmic domain of PECAM-1 will be produced through molecular biology and biotechnology techniques.
In Aim 2, the physical characteristics of the construct will be measured using single molecule force spectroscopy techniques. To perform these measurements, the PECAM-1 construct will be elongated with an atomic force microscope (AFM), and the resultant forces will be measured. Finally, in Aim 3, the PECAM-1 construct will be stretched with the AFM while the signal propagation event will be measured in real time using fluorescence. This will allow the determination of the forces required to perform PECAM-1 mechanotransduction.
Atherosclerotic plaques form in the region of blood vessels that experience disturbed fluid flow. This project will address the manner in which cells recognize physical forces, like fluid flow, and transmit the force as a biochemical signal. The research will focus on platelet endothelial cell adhesion molecule-1 (PECAM-1), a molecule that is involved in mechanical signaling in the cells that line the blood vessels and implicated in the formation of plaques.