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Absence of the G protein-coupled receptor G2A in mice promotes monocyte/endothelial interactions in aorta
David T Bolick1, Angela M Whetzel, Marcus Skaflen
1Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.
Abstract:
The G protein-coupled receptor G2A is highly expressed on macrophages and lymphocytes and has been localized to atherosclerotic plaques. We examined the role of G2A in modulating monocyte/endothelial interactions in the vessel wall. We measured adhesion of WEHI 78/24 monocytes to aortas of C57BL/6 (B6) and G2A-deficient (G2A(-/-)) mice using an ex vivo adhesion assay. G2A(-/-) mice had 10-fold elevations in adhesion of monocytes to aortas. Injection of GFP-expressing wild-type macrophages into B6 and G2A(-/-) mice in vivo showed increased macrophage accumulation in the aortic wall of G2A(-/-) mice. We isolated aortic endothelial cells (ECs) from B6 and G2A(-/-) mice and found a 2-fold increase in intercellular adhesion molecule-1 and E-selectin surface expression on G2A(-/-) ECs using flow cytometry. Using ELISA, we found a 3-fold increase in interleukin-6 and monocyte chemoattractant protein-1 production by G2A(-/-) ECs compared with B6 ECs. We found a dramatic increase in nuclear localization of the p65 subunit of nuclear factor kappaB in G2A(-/-) ECs. Transfection of G2A into G2A(-/-) ECs to restore normal expression levels reduced p65 nuclear localization to 35%. Restoration of G2A expression in G2A(-/-) ECs significantly reduced intercellular adhesion molecule-1 and endothelial selectin surface expression and reduced monocyte chemoattractant protein-1 and interleukin-6 production. Restoring G2A to G2A(-/-) ECs reduced monocyte adhesion by 80% compared with G2A(-/-) ECs in a flow chamber assay. Absence of G2A in endothelium results in proinflammatory signaling and increased monocyte/endothelial interactions in the aortic wall. Thus, endothelial G2A expression may aid in prevention of vascular inflammation and atherosclerosis.
Insights
The G2A receptor, absent in endothelial cells, promotes inflammation and monocyte adhesion in blood vessels. Restoring G2A expression reduces these effects, suggesting a role in preventing atherosclerosis.
Area of Science:
- Immunology
- Cardiovascular Biology
- Cell Biology
Background:
- G protein-coupled receptor G2A is expressed on immune cells and found in atherosclerotic plaques.
- Monocyte-endothelial interactions are critical in vascular inflammation and atherosclerosis development.
Purpose of the Study:
- To investigate the role of G2A in regulating monocyte adhesion to the endothelium.
- To determine the impact of G2A deficiency on inflammatory signaling in endothelial cells.
Main Methods:
- Ex vivo monocyte adhesion assays using mouse aortas.
- In vivo studies tracking macrophage accumulation in mouse aortic walls.
- Flow cytometry to assess cell surface marker expression (ICAM-1, E-selectin).
- ELISA for cytokine production (IL-6, MCP-1).
- Nuclear factor-kappaB (NF-κB) p65 subunit localization analysis.
- G2A gene rescue experiments in endothelial cells.
Main Results:
- G2A-deficient mice exhibited a 10-fold increase in monocyte adhesion to aortas and greater macrophage accumulation.
- G2A-deficient endothelial cells showed increased ICAM-1 and E-selectin expression and elevated IL-6 and MCP-1 production.
- Absence of G2A led to increased nuclear localization of NF-κB p65.
- Restoring G2A expression in deficient cells significantly reduced inflammatory markers and monocyte adhesion.
Conclusions:
- Endothelial G2A deficiency promotes a pro-inflammatory state, enhancing monocyte-endothelial interactions.
- G2A signaling in endothelial cells plays a protective role against vascular inflammation.
- Targeting endothelial G2A may offer a therapeutic strategy for preventing atherosclerosis.
