Kosta Theodorou

Myeloid A Disintegrin and Metalloproteinase Domain 10 Deficiency Modulates Atherosclerotic Plaque Composition by Shifting the Balance from Inflammation toward Fibrosis

A disintegrin and metalloproteinase domain 10 (ADAM10) is a metalloprotease involved in cleavage of various cell surface molecules, such as adhesion molecules, chemokines, and growth factor receptors. Although we have previously shown an association of ADAM10 expression with atherosclerotic plaque progression, a causal role of ADAM10 in atherosclerosis has not been investigated. Bone marrow from conditional knockout mice lacking Adam10 in the myeloid lineage or from littermate controls was transplanted into lethally irradiated low density lipoprotein receptor Ldlr(-/-) mice on an atherogenic diet. Myeloid Adam10 deficiency did not affect plaque size, but it increased plaque collagen content. Matrix metalloproteinase 9 and 13 expression and matrix metalloproteinase 2 gelatinase activity were significantly impaired in Adam10-deficient macrophages, whereas their capacity to stimulate collagen production was unchanged. Furthermore, relative macrophage content in advanced atherosclerotic lesions was decreased. In vitro, Adam10-deficient macrophages showed reduced migration toward monocyte chemoattractant protein-1 and transmigration through collagen. In addition, Adam10-deficient macrophages displayed increased anti-inflammatory phenotype with elevated IL-10, and reduced production of proinflammatory tumor necrosis factor, IL-12, and nitric oxide in response to lipopolysaccharide. These data suggest a critical role of Adam10 for leukocyte recruitment, inflammatory mediator production, and extracellular matrix degradation. Thereby, myeloid ADAM10 may play a causal role in modulating atherosclerotic plaque stability.

Fine Tuning Cell Migration by a Disintegrin and Metalloproteinases

Cell migration is an instrumental process involved in organ development, tissue homeostasis, and various physiological processes and also in numerous pathologies. Both basic cell migration and migration towards chemotactic stimulus consist of changes in cell polarity and cytoskeletal rearrangement, cell detachment from, invasion through, and reattachment to their neighboring cells, and numerous interactions with the extracellular matrix. The different steps of immune cell, tissue cell, or cancer cell migration are tightly coordinated in time and place by growth factors, cytokines/chemokines, adhesion molecules, and receptors for these ligands. This review describes how a disintegrin and metalloproteinases interfere with several steps of cell migration, either by proteolytic cleavage of such molecules or by functions independent of proteolytic activity.

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE−/− mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3+ T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3+ T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.

HDL and macrophages: explaining the clinical failures and advancing HDL-based therapeutics in cardiovascular diseases?

Atherosclerosis, a chronic inflammatory disease affecting mediumand large-sized arteries, is the main underlying cause of cardiovascular syndromes such as myocardial infarction and stroke [1]. Atherosclerosis is commonly viewed as a chronic inflammatory disease, driven by (modified) lipids. This rendered lipid status a major target for intervention in or even reversal of this disease [2]. One of the most effective strategies appeared to be lowering of plasma cholesterol levels, in particular lowdensity lipoprotein (LDL) cholesterol levels by statins and more recently by pro-protein convertase subtilisin/kexin type 9 (PCSK9) inhibitors [2]. With the growing insight that highdensity lipoproteins (HDL), unlike LDL, protects against cardiovascular diseases (CVDs), an increasing number of studies focus on elevating HDL as therapeutic target. The physiological role of HDL is to transport cholesterol from the periphery (e.g. the vessel wall) back to the liver for degradation. Therefore, elevated HDL levels should lead to attenuated atherosclerosis. However, as summarized in the following, the outcome of several clinical trial studies investigating the therapeutic effects of HDL-elevating drugs was disappointing which fostered a still ongoing lively debate. In this editorial we will focus on several recent publications which fuelled this debate regarding the potency of HDL as therapeutic target for atherosclerosis. Already more than 50 years ago, the Framingham Heart Study was the first study to provide compelling evidence of an inverse relationship between HDL and the risk of a cardiovascular event [3]. This rooted the concept that HDL, ‘the good cholesterol,’ could potentially protect against atherosclerosisrelated diseases (HDL hypothesis). During the 1990s this notion was confirmed in several animal studies. For example, Badimon et al. observed a regression of atherosclerotic lesions after infusion of cholesterol-fed rabbits with HDL isolated from human plasma [4]. Moreover, Rubin et al. could show that transgenic overexpression of human apoA-I in mice, resulting in high circulating HDL levels, inhibited early atherogenesis [5]. Based on the aforementioned epidemiological and experimental data, all pointing to beneficial effects of HDL in CVD, major efforts were made to develop HDL-elevating therapies. Besides the lifestyle interventions, like increased physical activity, a lot of pharmacological strategies, such as niacin, CETP inhibitors, PPAR agonists (fibrates), and direct HDL/apoA-I mimetics, have been developed and tested in human clinical trials. Surprisingly, several of these trials had to be discontinued due to unforeseen, detrimental side-effects or did not show the expected beneficial outcome on CVD risk. The HDL hypothesis recently suffered another blow with the study of Keene et al. [6]. Their meta-analysis of 39 clinical trials aiming to raise plasma HDL levels showed that niacin, fibrates, and CETP inhibitors fail to reduce all-cause mortality or even the incidence of cardiovascular events like myocardial infarction or stroke. Furthermore, recent Mendelian randomization analyses by Voight et al. showed that polymorphisms exclusively altering HDL levels actually did not affect the risk of myocardial infarctions [7]. After decades of research the originally described association of HDL and cardiovascular risk, the actual basis of the HDL hypothesis, thus seems to be not so straightforward as initially expected. The recent discrepant findings of studies focusing on vascular cell-type-specific effects of HDL may offer some enlightenments. Clearly, HDL exerts anti-inflammatory effects on endothelial cells and smooth muscle cells (SMCs), evidenced by strongly reduced secretion of critical cytokines/chemokines [8,9]. Recent studies on the effects of HDL on macrophages reported rather conflicting results. However, the use of different cell states (naïve or lipid-loaded cells), HDL preparations and concentrations, or readout models may at least partly explain these observed discrepancies. Suzuki et al. [10] reported inhibitory (anti-inflammatory) effects of HDL on the type I interferon response in acetylated LDL-loaded foam cells, whereas all other studies used nonloaded macrophages. In support, anti-inflammatory effects were also observed by De Nardo et al. [11]. However, in their study a soybean phospholipid reconstituted HDL compound (CSL-111) was used at supraphysiological concentrations, twoto threefold higher than in humans. As several soybean components have already been shown to be anti-inflammatory in macrophages, conclusions on specific HDL effects should be taken with caution. In addition, this study reported antiinflammatory effects of native HDL, although these experiments were performed in TLR4 mutant C3H/HeJ mice, which display a defective response to bacterial endotoxins like LPS. In contrast, we recently showed clear pro-inflammatory effects of HDL on

Whole body and hematopoietic ADAM8 deficiency does not influence advanced atherosclerotic lesion development, despite its association with human plaque progression

Although A Disintegrin And Metalloproteinase 8 (ADAM8) is not crucial for tissue development and homeostasis, it has been implicated in various inflammatory diseases by regulating processes like immune cell recruitment and activation. ADAM8 expression has been associated with human atherosclerosis development and myocardial infarction, however a causal role of ADAM8 in atherosclerosis has not been investigated thus far. In this study, we examined the expression of ADAM8 in early and progressed human atherosclerotic lesions, in which ADAM8 was significantly upregulated in vulnerable lesions. In addition, ADAM8 expression was most prominent in the shoulder region of human atherosclerotic lesions, characterized by the abundance of foam cells. In mice, Adam8 was highly expressed in circulating neutrophils and in macrophages. Moreover, ADAM8 deficient mouse macrophages displayed reduced secretion of inflammatory mediators. Remarkably, however, neither hematopoietic nor whole-body ADAM8 deficiency in mice affected atherosclerotic lesion size. Additionally, except for an increase in granulocyte content in plaques of ADAM8 deficient mice, lesion morphology was unaffected. Taken together, whole body and hematopoietic ADAM8 does not contribute to advanced atherosclerotic plaque development, at least in female mice, although its expression might still be valuable as a diagnostic/prognostic biomarker to distinguish between stable and unstable lesions.