Vascular Aspects Thrombosis and Haemostasis

This research program aims to unravel molecular players and mechanisms underlying vice versa transition between the two plaque phenotypes. It focuses on molecular players and mechanisms involved in processes of calcification, blood coagulation and apoptosis. We will exploit this knowledge to identify biomarkers and targets for diagnosis, employing molecular imaging (MI), and therapy, employing concepts of targeted drug delivery and nutrition. The subtheme furthermore aims to translate basic insights into clinical applications.

Apoptosis is a biochemically well-regulated form of cell suicide that is associated with various pathologies including acute myocardial infarction, heart failure, unstable atherosclerotic plaque and cancer. Apoptosis is an attractive cellular process to be measured by MI to support diagnosis and to monitor efficacy of therapy.

Apoptosis, the most abundant and best studied form of cell death, is characterized morphologically by cell shrinkage, nuclear fragmentation, membrane blebbing and apoptotic body formation. At the molecular level apoptosis involves the appearance of phosphatidylserine (PS) in the outer leaflet of the plasma membrane bilayer. Translocation of PS to the outer leaflet results from inhibition of aminophospholipid translocase and a concomitant activation of scramblas.
Cell surface expressed PS triggers surrounding phagocytesto engulf the dying cell through a macropinocytic process. Identified PS receptors on phagocytes mediating engulfment include TIM4, KIM-1, BAI1, stabilin-2 and the disputed PSR. In addition PS opsonins such as MFG-E8 and GAS6 mediate engulfment by binding PS on the apoptotic cell and interacting with phagocyte receptors. Rapid engulfment is considered essential to maintaining homeostasis in the immediate environment of the dying cell. Delayed or absent engulfment is accompanied by a spill of pro-inflammatory compounds into the extracellular space and a persistent exposure of PS to the environment. Cell surface exposed PS catalyzes i) the formation of the prothrombotic and pro-inflammatory enzyme thrombin and ii) the ectopic deposition of calciumhydroxyapatite. In addition impaired engulfment of apoptotic cells causes generation of autoantibodies, enhanced procoagulant activity and increased calcification. Together this supports accelerated progression of atherosclerosis towards unstable phenotype.

Our group discovered annexin A5 (anxA5), a protein that binds in the presence of calcium-ions to cell surface expressed PS with a Kd of less then 10-9M. Annexin A5 is the best studied member of the multigene Annexin family of proteins that have structural and functional similarities. The physiological significances of the individual annexin and the annexin family as a whole are not well understood to date but are believed to involve calcium regulated membrane associated processes. Annexin A5 binds PS with high affinity in a calcium dependent manner. AnxA5 is well-known as a molecular imaging agent to measure apoptosis in vitro and in vivo in animal models and patient. Our group showed that anxA5 not only binds PS on the cellular surface of early apoptotic cells but also induces its internalization by a novel process of endocytosis. Various experiments employing synthetic phospholipid surfaces have shown that anxA5 is a monomer in solution and once bound to PS organizes in trimers and aggregates of trimers driven by homotypic protein-protein interaction. It is hypothesized that anxA5 binds PS, forms a 2D-crystal on the cellular surface and then bends the membrane inwards causing invagination and subsequent closure and intracellular trafficking of the endocytic vesicle. The biological property of internalization has potential value for targeted intracellular delivery of compounds employing anxA5 astargeting vector.

Vascular calcification is recognized as a strong and independent predictor of cardiovascular mortality and morbidity. Clinicopathological studies suggest that superficial nodules of calcium deposition in vascular intima are closely associated with rupture of atherosclerotic plaques. In addition micro calcification in the thin fibrous cap may render the cap vulnerable to rupture. Until recently vascular calcification was regarded a biologically passive process dominated by inorganic interactions between excesses in calcium (Ca2+) and phosphate (P). Currently vascular calcification is appreciated as a complex and actively regulated process involving cells and proteinous catalysts and inhibitors akin to bone formation. The precise cellular and molecular mechanisms underlying ectopic calcium depositions remain, however, largely unknown.
Vitamin K dependent extrahepatic proteins (VKEP) such as Growth Arrest Specific gene-6 protein (GAS6), Matrix Gla Protein (MGP) and periostin play important roles in vascular calcification. Evidence supporting their crucial role arises from experiments with cell cultures, knockout mouse models and animal models employing vitamin K antagonizing diets. GAS6 (growth arrest specific gene-6 protein) inhibits hydroxyapatite induced apoptosis of Smooth Muscle Cells (SMC) through Gas6-Axl receptor tyrosine kinase signaling.MGP, which is abundantly present in Matrix Vesicles, inhibits ectopic calcification through as yet unidentified mechanisms. Structure/function relationship analysis has unveiled MGP’s gamma-carboxylated glutamic acid residues mandatory for its inhibitory activity. Furthermore, MGP binding to BMP-2/4 (Bone Morphogenetic Protein) seems to be essential. MGP-/- mice show strong vascular calcification that can be prevented by local MGP expression in arteries but not by systemic expression yielding high serum MGP levels. These findings suggest that MGP associated with MV in the plaque inhibits calcification. Periostin is a novel member of the VKEP family. Its role in vascular calcification is still unknown. Interestingly, Gas6 stimulates phagocytosis of apoptotic cells by bridging PS and Mer receptor on apoptotic cell and macrophage respectively. These relationships couple vitK-dependent proteins also to progression of atherosclerosis via apoptosis and phagocytosis.