We hypothesize that different subsets of myeloid cells have distinct and overlapping functions in atherosclerosis. Here, we propose to test which subsets are responsible for inflammatory cytokine production, antigen presentation, and foam cell formation, and how these processes are modulated by platelet factor 4 (Pf4, also known as CXCL4). Macrophage accumulation initiates the formation of fatty streaks, which eventually develop into atherosclerotic lesions. Macrophages are also critically involved in vulnerable plaque, whose rupture triggers major adverse cardiovascular events (MACE). Macrophages and DCs are antigen- presenting cells (APCs) that interact with T cells, amplifying inflammation. CD4 T cell interaction with APCs is evident in multiphoton microscopy: the T cells pause when they form an immunological synapse. CXCL4, the product of the Pf4 gene, is pro-atherogenic and drastically alters macrophage phenotype. To investigate APC functions, foam cell formation, antigen presentation and the impact of Pf4 on these processes, we propose to combine newly developed multiphoton live cell imaging of the aortic wall with flow cytometry, immunofluorescence and histology.
Specific aim 1 is to test which subsets of myeloid cells become foam cells in the aortic wall, using aortic wall flow cytometry and multiphoton live cell imaging in GFP-tagged Apoe-/- mice. We have generated LysMGFP Apoe-/-, Cx3cr1GFP Apoe-/- and CD11cYFP Apoe-/- mice. In each of these mouse strains, different subsets of myeloid cells are green (or yellow) fluorescent.
Specific aim 2 is to test the effects of T cell interactios with macrophages and dendritic cells on atherosclerosis. T cells derived from OTII transgenic mice express a monoclonal TCR that recognizes an ovalbumin peptide. Polyclonal T cells, some of which are specific to "atherosclerosis antigens", are isolated from dsRed Apoe-/- mice. These T cells are transferred to aortas of LysMGFP Apoe-/-, Cx3cr1GFP Apoe-/- and CD11cYFP Apoe-/- mice to test their interactions with APCs, which are expected to induce T cell proliferation (BrdU), polarization (interferon-?, IL-17A) and foam cell formation (oxLDL uptake).
Specific aim 3 is to investigate the effect of platelet factor 4 on foam cell formation an function. PF4 is a strong modulator of macrophage phenotype, promoting the M4 phenotype in vitro. Pf4-/-Apoe-/- mice have been reported to develop smaller atherosclerotic lesions. To address possible mechanisms, we propose to cross Pf4-/-Apoe-/- mice with our fluorescent reporter mice to visualize changes in myeloid subset composition, phenotype and behavior in these mice. When this research is complete, we will know which macrophage subset(s) become foam cells, which interact with T cells in a productive immune response to trigger re-stimulation, proliferation and cytokine production, and how PF4 influences both processes. PF4 or other molecules investigated here may emerge as targets for prevention or therapy of atherosclerosis and its complications.

Public Health Relevance

Atherosclerosis is the disease underlying heart attacks and strokes. Both are triggered by acute blood clotting (thrombosis) in an artery with vulnerable atherosclerotic plaque. "Vulnerable" means that the plaque can rupture and trigger these catastrophic events. Vulnerable plaques are full of macrophages, derived from white blood cells, creating an inflammatory environment. These macrophages can engorge lipids to become foam cells and they can stimulate the immune system. This proposal uses advanced microscopy that can image deep in the tissue to investigate how subsets of macrophages regulate inflammation, lipid accumulation and activation of the immune system. Some of the molecules investigated could become targets for future drug development.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL115232-01
Application #
8346057
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Hasan, Ahmed AK
Project Start
2012-07-01
Project End
2017-05-31
Budget Start
2012-07-01
Budget End
2013-05-31
Support Year
1
Fiscal Year
2012
Total Cost
$566,124
Indirect Cost
$199,897
Name
La Jolla Institute
Department
Type
DUNS #
603880287
City
La Jolla
State
CA
Country
United States
Zip Code
92037