Project area III

P08: Role of ceramide-rich macrodomains in regulating ABC transporters post ischemia

• Prof. Dr. Dirk Hermann

Department of Neurology, E-Mail: dirk.hermann@uk-essen.de

• Prof. Dr. Zvi Fuks

Memorial-Sloan-Kettering Cancer Center, NY, USA,
E-Mail: fuksz@mskcc.org

• Prof. Dr. Richard Kolesnick

Memorial-Sloan-Kettering Cancer Center, NY, USA,
E-Mail: r-kolesnick@ski.mskcc.org

This project tests the hypothesis that post ischemia-reperfusion (I/R) alterations in the configuration of ATP-binding cassette (ABC) transporters in brain plasma membranes, and resultant changes in neurovascular function, are regulated by acid sphingomyelinase (Asm)/ceramide. The neurovascular unit significantly dysfunctions in I/R. ABCB1 at the luminal endothelial membrane increases, enhancing extrusion of pharmacologic compounds from brain, while abluminal ABCC1 decreases, reducing drug transport from blood into brain. Recent evidence indicates low density lipoprotein (LDL)-related protein-8 (LRP8; also: apolipoprotein-E [ApoE] receptor-2) ligation regulates these ABC changes by repressing c-Jun signaling. Since I/R injury activates Asm to generate the second messenger ceramide that coalesces into ceramide-rich macrodomains (CRMs) in the plasma membrane, in which ApoE preferentially integrates, engagement of the Asm/ceramide machinery in neurovascular dysfunction would offer attractive therapeutic targets.

P09: Sphingosine-1-Phosphate (S1P) in arterial remodeling and aneurysm formation

• Dr. Petra Keul

Institute of Pathophysiology, E-Mail: petra.keul@uni-due.de

• Prof. Dr. Timothy Hla

Weill Cornell Medical College, NY, USA, E-Mail: thi2002@med.cornell.edu

S1P has numerous functions in the vasculature in general and vascular smooth muscle cells (VSMC) in particular, but its role in the pathological remodeling process after arterial injury in vivo is largely unknown. The most important clinical manifestations of acute or chronic arterial injury are restenosis after angioplasty and aortic aneurysms. The underlying process involves proliferation and migration of VSMC along with vascular inflammation, and causally depends on the excessive activation of matrix metalloproteinases (MMPs), proteolytic enzymes that degrade the extracellular matrix (ECM) of the vessel. The most deleterious clinical consequence of MMP-dependent pathological remodeling of an artery is the formation of aortic aneurysms that progressively expand and ultimately rupture with extremely high mortality (1). Both our published and preliminary studies have revealed a causal interaction between S1P, S1P receptors and MMPs: we have shown that S1P3 protects, while pharmacological S1P elevation exacerbates pathological remodeling and aneurysm formation after carotid artery ligation, and have identified S1P to inhibit TNFalpha-induced MMP-9 activation via S1P2. MMPs and S1P receptors are also known to be regulated both in the injured carotid and aortic aneurysms. Here, we will characterize the S1P receptors and the mechanisms by which they regulate MMP-dependent arterial remodeling and will pursue their relevance for aneurysm formation, progression and rupture in an angiotensin II-induced aneurysm model. We will use genetic and pharmacologic tools to influence individual S1P receptors relevant to MMP regulation and will employ them as strategies to inhibit and stabilize aneurysms. This may have immediate relevance for the clinic as there is no current consensus treatment of aneurysms besides extensive stenting and high-risk emergency surgery, and since the first S1P analogue FTY720 (Gilenya®) has already entered the clinic for relapsing multiple sclerosis making it rapidly available for potential vascular applications.

P10: Sphingosine-1-phosphate (S1P) as a causal factor for impaired function of high-density lipoproteins (HDL) in patients with coronary artery disease (CAD)

• Prof. Dr. Bodo Levkau

Institute of Pathophysiology, E-Mail: bodo.levkau@uni-due.de

• Prof. Dr. Timothy Hla

Weill Cornell Medical College, NY, USA, E-Mail: tih2002@med.cornell.edu

S1P and ceramide regulate the molecular ‘rheostat’ that integrates stress and survival signals in the cell and consecutively translates them into life or death decisions. Extracellular S1P transmits signals through five cognate G protein-coupled receptors but does not exist in a ‘free’, soluble form because of its amphipathic structure. Instead, it associates with specific physiological carriers such as HDL. High plasma levels of HDL have been well known to protect against cardiovascular diseases, but only recently we and others have attributed several of the beneficial HDL functions to their S1P content (1). We have also found that HDL from patients with coronary artery disease (CAD) contain less S1P than healthy individuals (3). As patients with CAD are also known to suffer from impaired, dysfunctional HDL (2), we have hypothesized here that the reduced S1P within their HDL contributes to the impaired HDL function. Recently, we have identified the binding partner for S1P in HDL to be apolipoprotein M (ApoM) (4). As several post-translational modifications of HDL apolipoproteins as well as changes in HDL composition and subclasses have been observed in CAD (2), we will determine whether they are responsible for reduced HDL-S1P content. The overall goals of this project are to identify the molecular HDL apolipoprotein alterations that underlie the reduced HDL-S1P content in CAD patients, to test how this reduction may result in impaired S1P-dependent HDL functions and to design approaches to prevent or possibly even restore S1P content and possibly S1P-dependent HDL function in CAD patients. Pharmacological interventions aimed at increasing the bioactive S1P content in HDL to enhance HDL functionality and thus their protective effects may be of clinical benefit in CAD and could constitute a novel therapeutic paradigm in cardiovascular medicine.

P11: Trafficking of sphingosine-1-phosphate (S1P) between cells and high-density lipoproteins as crucial component of S1P signalling and lipid transfer

• Prof. Dr. Bodo Levkau

Institute of Pathophysiology, E-Mail: bodo.levkau@uni-due.de

• Prof. Dr. Anant Menon

Weill Cornell Medical College, NY, USA, E-Mail: akm2003@med.cornell.edu

High-density lipoproteins (HDL) are the most potent endogenous factors that protect against atherosclerosis-related cardiovascular diseases by taking up cholesterol from peripheral tissues and eliminating it via billiary excretion. Although several atheroprotective functions of HDL have been attributed to their S1P content (1, 2) and the majority of plasma S1P is contained in HDL, the mechanisms regulating its uptake, localization and transport are not well understood. Neither has the mode of S1P delivery from HDL to cellular S1P receptors for the purpose of signaling that, indisputably, takes place in vivo has been tackled yet. In its first part, this proposal addresses fundamental biochemical, biophysical and biostructural questions concerning the mechanisms of S1P incorporation into and its off-loading from HDL, respectively. In particular, we will examine whether there is a functional connection between S1P translocation from cells to HDL and the efflux of cholesterol to HDL via ABC-type transporters A1 and G1 and the genuine HDL receptor scavenger receptor type B1 (Srb1), respectively. In its second part, the proposal addresses the role of Srb1 in the delivery of HDL-associated S1P to S1P receptors for signaling. The project also considers where S1P is localized within the HDL particle as this pertains directly to its bioavailability. The insights will help design pharmacological strategies to enhance S1P-mediated HDL signaling in particular and beneficial S1P-attributed cardiovascular effects of HDL in general.