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Final Report Summary - MACROPHAGE ITGB3 (Cell autonomous role of beta3 integrin: function in macrophages.)

Atherosclerosis is a chronic inflammation of the arterial wall caused by accumulation of cholesterol. The complex process involves endothelial and smooth muscle cells as well as the recruitment and differentiation of circulating monocytes. Integrins are membrane-bound molecules that are involved in signaling between the cells and the extracellular matrix, thereby influencing intracellular signaling and cytoskeletal stability. We and others have described the in vivo consequences of germline deletion of the αvβ3 heterodimer in inflammation, atherosclerosis and cancer. However, the distinct pathophysiological consequences of its absence for macrophage function especially in development of atherosclerotic lesions and for the process of inflammation remain mostly elusive. We hypothesized that the role of β3 integrin is cell autonomous, i.e. depending on the context of its expression. The main objective of the project was to characterize the role of the β3 integrin in macrophages during inflammatory atherosclerosis.
To study the objectives we employed novel genetically modified mouse models as well as existing Cre/Lox technology for tissue specific deletion of the β3 integrin in macrophages. Tissue culture of primary cells was employed to demonstrate mechanisms for the observed phenotypes in vivo. In addition, gene arrays and proteomics followed by sophisticated bioinformatics analysis was used to construct networks of the underlying pathophysiology. At first we found that tissue specific β3-deficiency in macrophages alone is sufficient to cause increased lesion formation in the aorta of mice on high fat diet on atherosclerosis-prone background suggesting an important atheroprotective role played by this integrin.
β3integrin is expressed during differentiation of macrophages with a peak expression shortly before maturation of cells. Macrophages from β3 deficient mice treated with oxidized LDL in vitro demonstrated a strong cholesterol uptake and increased foam cell formation. We found that β3integrin-deficiency in macrophages caused an early activation of Ras MAPK activity followed by ERK phosphorylation, deficient cholesterol efflux and decreased expression of cholesterol transporter (ABCA1) which is a major regulator of cellular cholesterol. Blocking of ERK pathway reduced foam cell formation of macrophages at least partially by restoring expression of ABCA1. We also demonstrated an important interplay between oxLDL-treated macrophages and vascular smooth muscle cells (VSMC) in vitro. The conditioned media from oxLDL-treated macrophages caused a significant increase in NFKB- but not ERK-phosphorylation in VSMCs. When challenged with bacterial wall compounds those macrophages also displayed increased secretion of inflammatory cytokines. Computational analyses showed the increased activity of an interleukin 1 beta-dependent network of signals responsible for the induced inflammatory activity. As a third phenotypic feature, we found that macrophages from that β3integrin-deficient mice show an impaired ability to migrate.
Taken together, our results show that macrophage β3 integrin is an important signaling molecule for the cellular activation during atherosclerosis and inflammation. With regards to the common “response to retention hypothesis” we speculate that that β3integrin deficient macrophages leave the blood stream through the endothelial layer when attracted by adhesion molecules such as VCAM and ICAM. Within the lesion, they take up oxidized lipoproteins such as oxidized LDL. The oxLDL uptake is facilitated through increased expression of scavenger receptors like CD36 and decreased ABCA-1 mediated efflux. The cells start producing inflammatory cytokines. Due to their impaired migratory capacity the cells are unable to leave the subendothelial space and form foam cells which are the substrate of atherosclerotic lesions.
Our results next prompted us to extend our research to investigate the role of β3integrin-deficiency in allergic diseases. We specifically addressed the role of that β3integrin in mast cells and in mast cell mediated diseases. We found that that β3integrin is robustly expressed in mast cells during and after their differentiation from bone marrow. The cells lacking that β3integrin were smaller and had a migratory deficit. Population based research suggest that people carrying certain polymorphisms in the β3integrin display a greater susceptibility to allergies and allergic inflammation. The underlying mechanism for these observations are subject to our further investigations.
The establishment of research collaborations fostered research on the adhesion molecule β3 integrin in Europe. Collaborations with the University of Norwich, UK, led to the discovery of novel aspects of β3 integrin in endothelial cells. This refers to the novel findings characterizing neoangiogenesis, but also to β3 integrin function within the adhesome. Mass spectrometry experiments showed that β3 integrin is part of the endothelial adhesome. Depletion of β3-integrin led to changes in microtubule behaviour that control their migration. β3 integrin regulates microtubule stability in endothelial cells. β3 integrin is considered a key anti-angiogenic target for cancer treatment due to its expression on neovasculature, but whether it is pro- or anti-angiogenic depends on the context in which it is expressed. We have made the same observation in the context of inflammation. In primary macrophages we found the β3 integrin to be anti-inflammatory while tumor associated macrophages may promote tumor growth through predominant M2 polarization of the macrophages in their interaction with the epithelial cells.
In another collaborative effort we showed that lungs of mice infected with influenza were massively invaded by thrombin-dependent protease-receptor 4 (PAR4-) activated platelets causing an aggravated cytokine storm, chemotaxis of inflammation, acute lung injury and death. β3 integrin-deficient mice were protected from influenza infection-mediated lung pathologies. The observation that β3 integrin-mediated platelet inhibition has protecting effects in influenza-dependent pneumonitis may have important consequences on current influenza management. Our preliminary results showed that other platelet inhibiting drugs works as well in this direction in mice. Available drugs such as clopidogrel or aspirin may also be tested in that respect as immediate impact of the study.
In summary, our results support the hypothesis that the role of β3 integrin in health and disease is cell-autonomous and context dependent. It confirms that observation that germline deletion and pharmacological inhibition may produce very differential findings. Further understanding of the cell-autonomous role of β3 integrin is necessary to understand unwanted off-target effects when systemic antagonists are tested for different indications. The idea of amplifying the β3 integrin-signalling awaits further investigations.

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