Funding

American Heart Association Grant

American Heart Association

 Summary. Atherosclerosis is caused by the recruitment of monocytes into the artery walls, followed by their transformation into foam cells and subsequently the formation of fatty streaks. The selective recruitment of monocytes by vascular endothelium during atherogenesis involves multiple types of cell adhesion molecules. The initial attachment of circulating monocytes to the endothelium is mediated by the interaction of P-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1). Subsequent intercellular interaction by a second pair of cell adhesion molecules, Very Late Antigen-4 (VLA-4) and Vascular Cell Adhesion Moleule-1 (VCAM-1), permits the monocytes to roll along and eventually adhere firmly to the endothelium. Although much is known about the adhesion molecules involved in the recruitment of monocytes, the underlying mechanisms that permit a cell to resist the shear force of the blood stream are poorly understood. Our research in cell adhesion focuses on understanding how cell adhesion molecules contribute to the selective recruitment of monocytes into the arteries. It is based on the application of advanced biophysical approaches such as the Atomic Force Microscope (AFM) to probe the intrinsic properties of the individual adhesion complexes and the interplay between the adhesion complexes. In this application, we propose to elaborate on the mechanisms of cell adhesion by: 1) investigating the temporal and spatial dependences of the mechanical force generated by the interaction between monocyte and endothelial cells and 2) evaluating potential mechanisms for avidity modulation of integrin-mediated adhesion. The proposed research will provide an improved quantitative understanding of the mechanisms of leukocyte adhesion at the molecular level that would be of great relevance to atherosclerosis and in the design of inhibitors that modulate the trafficking of cells.

SPECIFIC AIMS

Atherosclerosis is caused by the recruitment of monocytes into the artery walls, followed by their transformation into foam cells and the subsequent formation of fatty streaks. The selective recruitment of monocytes by the vascular endothelium during atherogenesis involves the interaction of multiple types of cell adhesion molecules, including selectins, integrins, and members of the immunoglobulin superfamily. The long term goal of the proposed research is to understand how the biophysical properties of these adhesion molecules contribute to the selective recruitment of monocytes into the arteries. An improved understanding of the mechanisms involved in molecular adhesion is of great relevance to vascular science and in the design of inhibitors that modulate the trafficking of monocytes. The specific aims of this application are:

Aim 1: To determine the temporal and spatial dependence of the mechanical force generated by the interaction between monocyte and endothelial cell by atomic force microscopy (AFM).

The initial attachment of monocytes to the endothelium has been attributed to binding of P-selectin to its ligand P-selectin Glycoprotein Ligand-1 (PSGL-1). This interaction is subsequently stabilized by the binding of Very Late Antigen-4 (VLA-4) to its ligand, Vascular Cell Adhesion Moleule-1 (VCAM-1). The main goal of this aim is to determine how the adhesive force resulting from these interaction changes with the duration of monocyte-endothelium contact. The proposed research will determine if there are preferred sites on an endothelial monolayer for initial monocyte attachment and whether there is a force gradient generated by the monocyte-endothelial cell interaction that dictate the movement of the monocyte to the site of transmigration. We will achieve these objectives using the atomic force microscope (AFM), which allows us to regulate precisely the area of contact between the cells, the duration of the contact, and location of monocyte-endothelial layer contact. Moreover, the AFM has the sensitivity to detect the forces generated by a single adhesion complex.

Aim 2: To evaluate potential mechanisms for avidity modulation of integrin-mediated cell adhesion.

As a follow up to Aim 1, the proposed experiments in this aim will determine the relative contributions of different mechanisms for avidity modulation of VLA-4/VCAM-1 interaction. Potential mechanisms for avidity modulation of integrin-ligand interaction include change in cell compliance upon cell activation, a structural rearrangement of VLA 4 and/or a lateral redistribution of VLA 4, and the expression of a multiple integrin binding site in different isoforms of VCAM-1. The relative contributions of these mechanisms will be assessed using a combination of biophysical and biochemical methods including AFM, reflection interference microscopy (RICM) and chemical crosslinking methods.