Flow Cytometer analysis of monocyte activation as a biomarker for risk of atherosclerosis.

Fatuma-Ayaan Rinderknecht Gregory FosterDr. Scott Simon

Purpose of the StudyCardio vascular disease associated with

atherosclerosis affects more than 1/3 of Americans. Atherosclerosis is a condition where plaque starts to form on the walls of the arteries. This plaque often limits blood flow and lead to more serious heart conditions. Current biomarkers of atherosclerosis are only able to identify 50% of diseased population. A biomarker is anything that can be used as an indicator of a particular disease. The purpose of this study is to find if high fructose corn syrup can be another biomarker of atherosclerosis. These findings could lead to better prevention, detection and treatment of atherosclerosis.

Study Design24 hour study looking at TGRL and monocyte activation to establish baselineBlood is drawn every hour and analyzed

Individual is sent home with case of high fructose corn syrup drink to consume with every meal for 2 weeks.

Individual returns after 2 weeks and 24 hour study is repeated looking at TGRL and monocytes.

Atherosclerosis and Monocytes

Atherosclerosis is a condition in which an artery wall thickens as a result of the accumulation of fatty materials such as cholesterol. The inflammatory response in the walls of the arteries is caused largely by the accumulation of macrophage white blood cells and promoted by low density lipopreteins without removal of fats from the macrophages by function high density lipoproteins. Monocytes are a type of white blood cell that is part of the immune system. Monocytes replenish macrophages and respond to inflammation signals. Monocytes are often tied to atherosclerosis because when the endothelial walls of the arteries become inflammed, monocytes are recruited to that site, where the bind to the wall and become part of the plaque.

Figure 1. Monocyte Adhesion to Arterial Wall

Dr. Simon’s LabDr. Simon’s lab focuses on white blood cells, like

monocytes and their role in cardiovascular disease. Monocytes are the primary inflammatory cell type that infiltrates early atherosclerotic plaques. Their recruitment into plaques drives disease progression. The lab takes the blood samples from the patients before and after their high fructose drink and analyzes the role of the fructose in monocyte activity using flow cytommetry. Monocytes take up circulating triglyceride rich lipoproteins (TGRL), increase expression of adhesion receptors (CD11c, CD11b, VLA-4, etc). These are receptors on the monocyte cells that allow the monocyte arrest on endothelium. This is one of the early events in plaque formation.

My Work in Flow Cytometry

While working in Dr. Simon’s lab, I focused on operating and helping analyze some data obtained by the flow cytometer. The flow cytometer is a machine that counts microscopic particles by suspending them in a fluid and passing them by an electronic detection apparatus and scatters light off of the internal components of the cell. Using different antibodies/flourophors for the monocytes, such as CD11b and Cd11c, which are used as labels, I can use the flow cytometer to distinguish monocyte populations in the blood samples of the patients.

Working at the Flow Cytometer

How to Analyze DataThe data generated by flow ctymotery is usually in

form of a histogram or two-dimensional dot plot on a lograthmic scale. In analyzing monocyte populations, I used a two dimensional dot plot that graphed forward scatter (FSC) and side scatter (SSC). FSC correlates with cell volume, and SSC has to do with the inner complexity of the cell. I first “gate” the monocytes on the graph, which seperates it from other populations based on flourescent intensity. Because different fluorescent dyes' emission spectra overlap, signals at the detectors have to be compensated electronically as well as computationally. Using software programs such as FlowJo and CellQuest Pro, I was able to compensate the data and ready it for further analysis.

Fluorescence-activated cell sorting

Monocytes

Summary and ResultsThis study was done to link the consuming of high

fructose corn syrup to atherosclerosisMonocytes adhesion to the walls of the arteries is

associated with plaque build-up in atherosclerosisUsing flow cytometry, blood samples from people who

had high amounts of high fructose corn syrup in their diet were tested for increased monocyte adhesion to endothelial walls of the arteries

It was found that increased high fructose corn syrup leads to increased monocyte expression and triglyceride levels and could be a biomarker of atherosclerosis

TGRL and CD11c increase following 2 week high fructose corn syrup