Integrin activation on circulating inflammatory monocytes: Biomarkers of cardiovascular disease risk

Coronary artery disease (CAD) results in the narrowing of the small blood vessels that supply oxygen to the heart and is the major cause of morbidity and mortality in the U.S. Since CAD is largely asymptomatic, clinicians rely on biomarkers in the blood (e.g., low- and high-density lipoprotein cholesterol, C-reactive protein, and triglyceride levels) to determine cardiovascular risk and to guide therapy for an individual patient. However, these conventional biomarkers lack in specificity to inform on vascular health, as they fail to identify >20% of subjects at risk for future complications resulting from coronary blockage. Atherosclerosis underlying CAD is dependent on the recruitment of circulating monocytes to inflamed endothelium expressing vascular cell adhesion molecule-1 (VCAM-1). Therefore, the objectives of this work were: 1) develop unique assays to assess the functional phenotype of circulating monocytes during inflammation associated with atherogenesis, 2) characterize the relationship between activated monocytes and cardiovascular risk, and 3) elucidate the mechanism through which activated blood monocytes recruit to inflamed endothelium.;To address the objectives of this dissertation, we fabricated a microfluidic based lab-on-a-chip to quantify the adhesion efficiency of monocytes sheared in whole blood on VCAM-1. The lab-chip device, combined with fluorescent antibody labeling against CD14 and CD16, allowed us to characterize the recruitment efficiency of monocyte subsets, in particular the inflammatory CD14 ++CD16+ (Mon2). We found that Mon2 adhered with 7-fold higher efficiency than other subsets and capture was dependent on integrins CD11c/CD18 and very late antigen-4 (VLA-4). To characterize the relationship between capture of inflammatory Mon2 and cardiovascular risk, we studied a range of subjects that included otherwise healthy individuals with elevated serum triglyceride levels to patients presenting with acute myocardial infarction resulting from coronary disease. We found that Mon2 from hypertriglyceridemic subjects exhibited increased CD11c expression and capture on VCAM-1 compared to subjects with normal serum triglyceride levels. CD11c expression and capture efficiency was further elevated in patients with known CAD suggesting a hierarchal relationship between activation of Mon2 and cardiovascular disease risk. Upregulation of CD11c in blood was attributable to Mon2 uptake of triglyceride rich lipoproteins and subsequent signaling via a phospholipase C (PLC)-dependent mechanism to increase CD11c expression, VLA-4 function, and integrin co-clustering within focal adhesive sites on VCAM-1. Using a unique antibody that allosterically binds CD11c and induces a high affinity conformation, we found that outside-in activation of CD11c on the membrane directly activated VLA-4 adhesion via phospho-spleen tyrosine kinase (pSyk) and paxillin within focal adhesion complexes. In summary, this work reveals that CD11c is an inducible integrin on inflammatory Mon2, whose expression correlates with monocyte inflammatory state in subjects at risk for atherogenesis and in patients with CAD. Therefore CD11c on Mon2 is a biomarker of cardiovascular risk and a potential target for drug therapy to ameliorate the recruitment of inflammatory monocytes during atherogenesis.