E. RIP kinase 1 signaling in atherosclerosis

Professor Manolis Pasparakis

Rationale and aims

Atherosclerosis remains the number one cause for morbidity and mortality in western countries. The development and growth of atherosclerotic plaques is the result of a chronic non-resolving inflammatory response involving the activation of endothelial cells to express cytokines, chemokines, and adhesion molecules, thereby resulting in the recruitment and entrapment of monocytes in the subendothelial space. Here, monocytes mature into macrophages that take up lipids to become foam cells. The death of lipid-laden macrophages is believed to critically affect the progression of atherosclerosis. The aim of this project is to experimentally address the role of apoptosis and n

Current state of research and own preliminary work

Figure 1. Schematic depiction of the pathways and key molecules regulating apoptosis and necroptosis downstream of death receptors and Toll-like receptors.

Apoptosis of foam cells within the atherosclerotic plaque, combined with impaired efferocytosis resulting in secondary necrosis has been recognized as an important process fueling inflammation. While apoptosis has been studied since many years and was until recently believed to be the only pathway of regulated cell death, recent research has revealed the existence of regulated necrotic cell death pathways that are controlled by distinct biochemical signaling cascades. Necroptosis is a type of regulated necrotic cell death that is induced by the kinase RIPK3 and its substrate pseudokinase MLKL. RIPK1 is an upstream kinase that has an important role in inducing both FADD-Caspase-8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis (Figure 1). Recent studies have suggested that RIPK3-dependent necroptosis of macrophages contributes to the growth and progression of atherosclerotic plaques in mice. However, since RIPK3 can also induce apoptosis and in addition exhibits cell death-independent proinflammatory functions, the role of necroptosis in atherosclerosis remains poorly understood.

Experimental approach and work program

To experimentally evaluate the role of apoptosis and necroptosis in atherogenesis, we will use genetic mouse models to inhibit specifically necroptosis (MLKL knockout) or both apoptosis and necroptosis (FADD-RIPK3 double knockout) to address the in vivo role of macrophage death in the development and progression of atherosclerotic plaques. In addition, we will also use mice expressing a kinase inactive mutant RIPK1D138N to determine the role of RIPK1 kinase activity mediated cell death in the development and progression of atherosclerotic plaques. For these studies, we will use bone marrow reconstitution in the LDLR knockout mouse model of atherosclerosis. Specifically, 8-week-old LDLR knockout mice will be lethally irradiated (1000 Rad) and will be reconstituted with intravenous injection of bone marrow cells obtained from the different genetic mouse models (RIPK1D138N, Mlkl-/-, Ripk3-/-, FADDMy-KO Ripk3-/- mice). The bone marrow reconstituted mice will be treated with broad spectrum antibiotics in the drinking water for 4 weeks until efficient reconstitution of their hematopoietic system is confirmed by FACS analysis of blood cells. Subsequently, the mice will be fed a high cholesterol diet for 10 weeks to promote the development of atherosclerotic plaques. The levels of cholesterol and triglycerides in the serum of the mice will be assessed before and after the high cholesterol diet feeding. At the and of the 10 week high cholesterol feeding period, the development and progression of atherosclerosis in these mice will be assessed using methodologies that are well established in our lab. Specifically, the aortas of the mice will be dissected and used for en face staining of lipids with Sudan IV followed by morphometric analysis and quantification of plaque formation. Aortic arches will be dissected and will be used for RNA preparation and subsequent qRT-PCR analysis of proinflammatory cytokine and chemokine as well as adhesion molecule expression. The hearts of the high cholesterol diet-fed mice will be dissected and frozen in OCT and consecutive 7 μm cryosections of the heart in the atrioventricular valve region will be collected and stained with toluidine blue, followed by morphometric analysis of atherosclerotic plaque size. Sections will also be immunostained for macrophages as well as for dead cells (apoptotic, necroptotic). Collectively, these studies will provide novel insights into the underlying molecular and cellular mechanisms by which inflammatory and cell death signaling downstream of death and toll-like receptors regulate the progression of atherosclerosis.

Potential future therapeutic implications

This project bears the potential to identify targetable proteins as important contributors to the progression of atherosclerotic plaques. Specifically, our studies aiming to address the role of RIPK1 kinase activity in the pathogenesis of atherosclerosis may identify RIPK1 as a therapeutic target in this disease. Considering that RIPK1 inhibitors are currently in phase II clinical trials for the treatment of different inflammatory diseases, our studies have strong therapeutic potential.

Added value through collaborations within the CCRC

Here, we assess the functional contribution of apoptotic and necroptotic cell death in vascular pathology using mouse models of atherosclerosis. Given the focus on monocytes and macrophages, other groups focusing on this cell species (Rosenkranz, Rudolph, Baldus) will benefit from close collaboration with our group. Since RIP kinase and TLR are redox sensitive, other enzyme systems expressed in monocytes and macrophages are also attractive to study, e.g. MPO (Baldus) or pharmacological interventions such as nitrated fatty acids (Rudolph). Core facilities will help to visualize and quantify plaque morphology and composition (histopathology; A. Quaas / R. Büttner), and to perform proteomics-based analyses (C. Freese / M. Krüger). Additionally, in vivo imaging of cell death may be facilitated by molecular imaging (B. Neumaier).

General research interests

The Pasparakis group focuses on studying the role of inflammation in disease pathogenesis. Inflammatory responses are regulated by intracellular signalling cascades such as the NF-κB and MAP kinase pathways that are activated downstream of cytokine receptors (e.g. TNFR, IL-1R) or innate immune receptors (e.g. Toll-like receptors, NOD-like receptors). The NF-κB pathway functions in essentially all mammalian cell types and is activated in response to injury, infection, inflammation and other stressful conditions requiring rapid reprogramming of gene expression. Genes regulated by NF-κB include cytokines and chemokines, adhesion molecules, regulators of cell survival, proliferation and apoptosis, acute phase proteins and proteins important for protection from oxidative stress. Due to its central role in regulating cellular responses to inflammation and injury, the NF-κB pathway is implicated in the pathogenesis of inflammatory diseases and cancer. To study the function of NF-κB and MAP kinase pathways in the pathogenesis of inflammatory diseases in vivo the group focuses on using recombinase-assisted (Cre/loxP) conditional gene targeting in mice, allowing studying gene function in the adult organism in a spatially and temporally controlled manner. Using conditional gene targeting the group studies the role of NF-κB and MAP kinase signalling in mouse models of inflammatory diseases such as atherosclerosis, inflammatory

Manolis Pasparakis’ profile

Manolis Pasparakis is Professor at the Institute for Genetics at the Faculty of Mathematics and Natural Sciences at the University of Cologne and leads the Mouse Genetics and Inflammation Laboratory. He received his Ph.D. in Biology from the University of Athens and did his postdoctoral training in the group of Professor Klaus Rajewsky in the Department of Immunology at Institute for Genetics in Cologne. He became a group leader at the European Molecular Biology Laboratory (EMBL) Mouse Biology Unit in Monterotondo, where he worked from 2002 to 2005. In 2005, he joined the Institute for Genetics as a professor. He is member of the executive board and coordinator of Research Area E on “Inflammation in Aging-associated Diseases” of the Cologne Cluster of Excellence on Cellular Stress Responses in Ageing-Associated Disesases.