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Monaco group inflammation in atherosclerosis

What is the best way to target inflammation in atherosclerosis?

The concept of a role of inflammation in the pathogenesis of atherosclerosis was regarded with skepticism in Cardiology, even if eminent groups had already data supporting an immune basis for atherosclerosis. The 90s were exciting times during which several clinical and experimental findings started proving atherosclerosis as an inflammatory disease. A line of evidence, to which we contributed, was the identification of a cytokine-dependent systemic inflammatory response in patients with acute coronary syndromes.

Now atherosclerosis is recognized as an inflammatory disease process that stems from the involvement of the adaptive and innate immune response and is characterized by the upregulation of pro-inflammatory cytokines. In particular the discovery of the receptors involved in innate immune recognition had a significant role in furthering our understanding of atherosclerosis and sensing of lipoproteins.

Innate immune receptors are strong inducers of cytokine upregulation and have a strong impact on the development of atherosclerosis. In atherosclerosis innate immune cells such as monocytes-macrophages, dendritic cells and mast cells are heavily represented in the atherosclerotic plaque. Our team demonstrated that the dysregulated production of pro-inflammatory cytokines in human plaques is dependent on innate immune signalling initiated by the extracellular membrane – bound Toll-like Receptor (TLR)-2 and, to a lesser extent TLR4. Finally, I recently demonstrated that the endosomal Toll-like receptor-3 may instead mediate atheroprotection.

A novel model of human atherosclerosis

Traditional approaches to the study of human atherosclerosis are based on immunohistochemistry, which does not allow functional studies. To overcome this limitation, I established a unique model system to study signalling pathways in cells isolated from human atherosclerotic tissue, onwards referred to as “atheroma cell culture”. Important was the choice of tissue. Fresh surgical tissue rather than autopsy material was fundamental for the collection of viable cells. Size was also crucial in order to maximise cell numbers. Carotid endarterectomy specimens were the best solution. Common misgivings against the use of cultured cells to model disease include the widespread belief that culture conditions and passaging may alter the cell phenotype, rendering observations remote from the disease settings. Hence, I wanted to avoid passaging cells and to study the cells within a short period from their isolation. In the atheroma cell culture, carotid endarterectomy specimens undergo enzymatic dissociation to obtain a single cell suspension. The cells are not passaged nor expanded nor sorted by cell types, but cultured as a mixed population. I carried out extensive analyses to characterise the mixture and its viability and I found that the mixture consist of viable cells representative of the major cell types resident in the human atherosclerotic plaques, (macrophages, smooth muscle cells and T lymphocytes). The macrophage (as detected by CD68 expression) is the most abundant cell type, amounting to approximately 50% of total cell numbers in almost all donors studied so far. Like RA synovial cells, atheroma cells display a significant production of a variety of cytokines, chemokines, tissue factor and matrix metalloproteinases (MMPs) in absence of extrinsic stimulation, as compared to unstimulated human primary cells in culture. Genetic targeting of numerous inflammatory mediators in mouse models of atherosclerosis prevents the development of atherosclerosis. However, mechanistic insights have yet to be translated in humans, due to the lack of experimental tools to test efficacy in human atherosclerosis in the preclinical phase. The “human atheroma cell culture” solves this problem due to its advantage over traditional approaches to the study of human atherosclerosis: the feasibility of functional studies, whereby cells are teased out of the tissue and the role of individual genes can be dissected out using antibodies, soluble receptors, small molecule compounds, or gene transfer.

Besides this model we utilise a combination of experimental models of atherosclerosis based on hypercholesterolemia and arterial injury.

CURRENT WORK

1.  Pattern recognition receptor (PRR) signalling in atherosclerosis

Using the atheroma cell culture, I demonstrated that the production of pro-thrombotic and pro-inflammatory mediators in human atheroma depends on the activation of nuclear factor κB (NFκB) signalling, and in particular of the canonical pathway led by the IκB kinase (IKK) -2/β The NFκB pathway is a major “hub” of inflammatory intracellular signalling, and it is induced by numerous pro-inflammatory mediators. The following question was to identify the upstream rate-limiting step for such cytokine and chemokine production. Blockade of TLR-2 markedly reduced the production of cytokines, chemokines and MMPs. Similar effects were observed with blockade of MyD88, but not IL-1 nor TLR-4 nor TRAM. Our data suggest that, in human atherosclerosis, TLR-2 and MyD88 play a predominant role in NFκB activation, and in the production of inflammatory mediators, and even matrix degrading enzymes. Surprisingly, the TLR-4/TRAM was dispensable for the production of pro-inflammatory cytokines, yet was involved in the production of MMP-1 and -3.

Our observation may have therapeutic implications on the future treatment of cardiovascular disease, defining TLR-2 as a potential therapeutic target. Moreover, it furnishes leads for the identification of TLR ligands in human atherosclerosis. Further exploration is directed towards the identification of PRR agonists involved in the pathogenesis of atherosclerosis and their usage of associated receptros and coreceptors,

2.   Atheroprotective PRR signalling

Finally, I recently demonstrated that not all TLRs are pro-atherogenic. Some can be, instead, atheroprotective. Using human and murine systems, we have investigated for the first time the consequence of TLR3 signaling in vascular disease. We compared the responses of human atheroma-derived smooth muscle cells (AthSMC) and control aortic smooth muscle cells (AoSMC) to various TLR ligands. AthSMC exhibited a specific increase in TLR3 expression and TLR3-dependent functional responses. Intriguingly, exposure to dsRNA in vitro and in vivo induced increased expression of both pro- and anti-inflammatory genes in vascular cells and tissues. Therefore, we sought to assess the contribution of TLR3 signalling in vivo in mechanical and hypercholesterolemia induced arterial injury. Surprisingly, neointima formation in a perivascular collar-induced injury model was reduced by the systemic administration of the dsRNA analogue Poly(I:C) in a TLR3-dependent manner. Furthermore, genetic deletion of TLR3 dramatically enhanced the development of elastic lamina damage after collar-induced injury. Accordingly, deficiency of TLR3 accelerated the onset of atherosclerosis in hypercholesterolemic ApoE-/- mice. Collectively, our data describe for the first time a protective role for TLR signalling in the vessel wall. We are now exploring the role of other PRRs and associated molecules that may have protective function in atherosclerosis by downregulating innate immune responses.