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2nd Waddensymposium Vascular regeneration - from biology to clinical use - June 28 – July 1, 2009, Grand Hotel Opduin, Texel, the Netherlands nd 2 Waddensymposium Vascular regeneration - from biology to clinical use - This symposium is organized in collaboration with: www.waddensymposium.eu Location: Grand Hotel Opduin, Texel www.opduin.nl Hereby the final program for our second “Waddensymposium” organized by the department of Immunohematology and Blood Transfusion of the LUMC. Location: Grand Hotel Opduin Ruijslaan 22 1796 AD De Koog (Texel) The Netherlands Department of Immunohematology and Blood Transfusion Symposium secretariat: Amber Günthardt Mail: [email protected] Phone: +31 71 526 38 27 cell phone: +31.6.53527185 Organizing committee: Wim Fibbe Paul Quax Peter van den Elsen Jaap Jan Zwaginga cell phone +31.6.50655116 Sunday 28th of June Final program 28th of June till 1st of July 2009 18.00 Arrival Hotel Opduin, Texel 20.00 Dinner Monday 29th of June 07.30 – 08.15 Breakfast 08.15 – 08.30 Welcome, symposium in perspective: Willem Fibbe, Leiden [The Netherlands] Vascular remodeling Chair: Jaap Jan Zwaginga 08.30 – 09.05 Erik Biessen, Maastricht [The Netherlands] “Role of mast cells in plaque instability “ 09.05 – 09.40 Robert Kleemann, Leiden [The Netherlands] “Pharmacological modulation of atherosclerotic plaques: effects on lesion progression and regression” 09.40 – 10.15 Peter van den Elsen, Leiden [The Netherlands] “Epigenetics and vascular disease” 10.15 – 10.45 Quick abstracts: R. Wierda, J. Karper, H. Roelofs, M. Ewing and L. Seghers 10.45 – 11.00 Coffee break 11.00 – 11.25 Christian Weber, Aachen [Germany] “Chemokines in atherosclerotic vascular remodeling“ 11.25 – 12.30 Abstracts Marco de Ruiter, Leiden [The Netherlands] “Maternal Transmission of Risk for Atherosclerosis” Karolina Janeczek, Twente [The Netherlands] “Mesenchymal Stem Cells as Source for Endothelial Progenitors” Diana Ploeger, Groningen [The Netherlands] “Modulated monocyte adherence: implications for peripheral vascular disease” Margreet de Vries, Leiden [The Netherlands] “Timp-1 modulates vein graft thickening and intra-plaque hemorrhages” 12.30 – 13.15 Lunch 13.15 – 18.00 Social event 18.00 – 20.00 Diner Chair: Anton Jan van Zonneveld 20.00 – 21.30 Douglas Losordo, Chicago [USA] “Intramyocardial autologous CD34 cell therapy for refractory angina” Tuesday 30th of June Biology of arteriogenesis and neovascularization Chair: Peter van den Elsen 08.30 – 09.05 Paul Quax, Leiden [The Netherlands] “NK cells and their role in modulating arteriogenesis” 09.05 – 09.40 Mervin Yoder, Indianapolis [USA] “Circulating cord blood endothelial colony forming cells (ECFC) can be specified to an arterial endothelial gene expression fate in vitro” 09.40 – 10.15 Victor van Hinsbergh, Amsterdam [The Netherlands] “Hypoxia and angiogenesis” 10.15 – 10.45 Coffee break 10.45 – 11.20 Marco Harmsen, Groningen [The Netherlands] “Stem cells in angiogenesis” 11.20 – 11.55 Imo Hoefer, Utrecht [The Netherlands] “Modulation of arteriogenic response by monocytes” 11.55 – 12.30 Mat Daemen, Maastricht [The Netherlands] “Mechanisms of Plaque Instability: role of circulating cells” 12.30 – 13.15 Lunch 13.15 – 18.00 Social event 18.00 – 20.00 Diner Chair: Wim Fibbe 20.00 – 21.30 Toyoaki Murohara, Kumamoto [Japan] “Implantation of Adipose-Derived Regenerative Cells Enhances Ischemia-induced Angiogenesis” Wednesday 1st of July Therapeutic vascularization Chair: Paul Quax 08.30 – 09.05 Anton Jan van Zonneveld, Leiden [The Netherlands] “Mechanisms in endothelial maintenance and repair” 09.05 – 09.40 Jamie Case, Indianapolis [USA] “Application of Poly-Chromatic Flow Cytometry Analysis and Cell Sorting Techniques to Identify Novel Subsets of Rare Circulating Cells with Angiogenic Potential” 09.40 – 10.15 Alain Tedgui, Paris [France] “Immune-regulatory Pathways in Atherosclerosis” 10.15 – 11.15 Abstracts Sacha Geutskens, Leiden [The Netherlands] “Monocytes as cellular therapy for vascular regeneration” Marie-Jose Goumans, Leiden [The Netherlands] “CXCR4/ CD26 modulated recruitment of HHT-1 mononuclear cells to the ischemic heart” Eric van der Veer, Leiden [The Netherlands] “The mRNA-binding protein Quaking is essential for vascular smooth muscle cell function” 11.15 – 11.35 Coffee break 11.35 – 13.00 Position paper 13.00 – 13.30 Lunch END OF MEETING ABSTRACTS Role of mast cells in plaque instability MONDAY Erik A.L. Biessen, Saskia C..A. de Jager , Theo J.C. Van Berkel, Ilze Bot Manifest atherosclerosis may present as myocardial infarction and stroke and is generally caused by thrombotic plaque rupture. The processes underlying plaque rupture are only poorly understood, but may amongst others involve a fulminant inflammation. Pathology studies indicated the increased presence of mast cells, important inflammatory effector cells in allergy responses, in (peri)vascular tissue during plaque progression, suggestive of a causal role in disease progression. Recent experimental data in mouse models for atherosclerosis have shown that mast cells and derived mediators can profoundly impact plaque progression and reduce plaque stability. In this presentation, we will discuss recent advances in experimental research on mast cells in atherosclerosis and the therapeutic potential of modulation of mast cell function in cardiovascular disease. We will delineate the multitethered effects of acute and chronic focal mast cell activation, discuss the relevant players in these processes and speculate on the endogenous triggers of mast cell mobilization and activation in the adventitia. MONDAY Pharmacological modulation of atherosclerotic plaques: effects on lesion progression and regression Robert Kleemann Atherosclerosis was previously thought to be a disease primarily involving lipid accumulation in the arterial wall. Current concepts of the disease include involvement of the immune system and chronic inflammation as crucial factors in all stages of the atherosclerotic process: the initiation of endothelial dysfunction, fatty streak formation and lesion progression and complication. This central role of inflammation and immunity in atherogenesis suggests that anti-inflammatory therapies might have a beneficial role in management of the disease. Because lipid lowering clearly causes inflammation associated with atherosclerosis to diminish, it is difficult to assess whether pharmaceuticals used in clinical practice have direct anti-inflammatory effects (ie, independent of their cholesterollowering effects). Furthermore, the set-up of many animal studies studying intervention in the inflammatory component of atherogenesis also deviates from the clinical norm because intervention is started simultaneously with onset of the experimentally controlled disease (ie long before first lesions are formed). This translational discrepancy is mainly due to the lack of appropriate (drug-sensitive) animal models that allow studying regression. Using a humanized and drug-sensitive animal model, ApoE3Leiden mouse, evidence is provided for an additional health benefit by quenching the inflammatory component of disease. Newly developed experimental conditions enabling the study of lesion regression reveal that not all compounds that reduce lesion progression are also effective in promoting lesion regression. Epigenetics and vascular disease MONDAY Peter J. van den Elsen Recent insights into the pathogenesis of atherosclerosis reveal the importance of chronic inflammation in the initiation and progression of vascular remodelling. It is now widely appreciated that in addition to macrophages also NK, NKT and T cells play a critical role in disease pathogenesis. Notably the unusual expression of molecules that play an important role in immune regulation found on vascular wall components is thought to contribute to the ongoing inflammatory process. It has become increasingly clear over the past years that epigenetic mechanisms play an essential and fundamental role in the transcriptional control of genes. These epigenetic mechanisms act to change the accessibility of chromatin by modification of DNA and by modifications of histones. Epigenetic regulators, such as those involved in histone acetylation and methylation modifications, are increasingly being recognized as direct or indirect components in the regulation of expression of vascular, immune and other tissue-specific genes. Deviations from these tightly controlled epigenetic mechanisms therefore have a direct impact on the cellular portrait of expressed genes and contribute to disease pathogenesis. Moreover, epigenetic modifications are influenced by environmental factors and accumulate in time. Epigenetic processes are reversible and therefore provide excellent targets for therapy. A small number of drugs directed against epigenetic processes are already FDA approved. The basis of epigenetic regulation and the contribution thereof in inflammatory processes, which contribute to the initiation and progression of vascular remodelling, will be discussed. Epigenetics and Atherosclerosis MONDAY Rutger J. Wierda, I.M. Rietveld, P.H. Quax, H.C. de Boer, J.H. Lindeman and P.J. van den Elsen Over the past decades it has become increasingly clear that gene regulation by epigenetic mechanisms – such as histone methylation, histone acetylation and DNA methylation – plays an important role in complex diseases – such as atherosclerosis – and inflammation. Epigenetic processes modulate gene expression without modifying the actual genetic code, whilst it can be inherited over multiple cell divisions. Epigenetics also provides an attractive explanation how diet, environment and lifestyle may contribute to disease development. In principle, epigenetics explains how external factors can impose aberrant gene expression patterns in an individual life time and even transgenerationally. Epigenetic processes are reversible by nature and can be modulated by small molecule inhibitors which target the enzymatic activities critical for histone methylation, histone acetylation and DNA methylation. Because multiple inhibitors are already FDA approved for usage in cancer therapy, this provides an excellent opportunity for therapeutic intervention in vascular diseases.Using immunohistochemical staining of atherosclerotic plaques in various stages of disease development we hope to identify epigenetic factors which are involved in disease pathogenesis. Subsequently, we will indentify the genes which are under the control of the disease-associated epigenetic factors by chromatin immunoprecipitation (ChIP) and massive parallel sequencing. Using cell cultures of Human Umbilical Vein Endothelial Cells (HUVECs) and vascular Smooth Muscle Cells (vSMCs) we will investigate the influence of small molecule inhibitors on the expression patterns of the identified genes. Preliminary data using IHC-P show the existence of differential histone methylation modifications revealing the dynamic nature of these epigenetic processes in the vascular wall. Furthermore, by using bisulfite sequencing and ChIP it was revealed that several chemokine receptor genes display differential DNA methylation and histone methylation patterns between HUVECs and SMCs which explains the observed differential expression patterns of these genes upon stimulation with various inflammatory mediators. MONDAY TLR4 is involved in vein graft remodelling and can serve as a local therapeutic target. J.C Karper, M.R de Vries, P.H.A Quax Despite that the survival of auto-transplanted veins for revascularization is limited by remodelling processes such as neointimal formation and atherotrombotic evolution, veins are still widely used for bypass surgery. This is due to their easier accessibility than arterial conduits. Toll like receptors are located on most of the cells in the vessel wall and are capable of recognizing exogenous as well as endogenous ligands that are considered to be involved in vascular remodelling. We hypothesize that TLR4 is an important mediator in the initiation and development of vein graft disease. To explore this potential role of TLR4, a venous bypass procedure was performed in Balb/c mice and in C3H/Hej mice. The latter lacks proper TLR4 signalling. After 28 days the vein graft was removed and analysed. C3H/Hej mice had 48% less vessel wall thickening than the Balb/c controls. To increase venous graft survival, we hypothesize to interfere in the local TLR4 expression of the vein graft in ApoE3Leiden mice using gene silencing approaches. These mice, when fed a high cholesterolemic diet, are well known to develop massive vein graft disease due to neointimal formation and accelerated atherosclerosis. Out of 5 lentiviral based constructs, all containing a different short hairpin RNA vector against TLR4 for silencing TLR4 expression in vivo the most potent vector for TLR4 protein knockdown was selected using infection of CHO cells expressing murine TLR4 and subsequent measurement of TLR4/MD2 complex protein expression by FACS analysis. This vector was used to infect vein graft segments using a perivascular delivery procedure. At sacrifice at 28 days after surgery, the combination of local lenti-shTLR4 delivery and subsequent TLR4 knockdown led to a 38% reduction of vessel wall thickening in the vein graft segment and thus a superior graft patency. This reduction of hyperplasia in vein graft remodelling after local interfering in the TLR4 pathway indicates the potential of TLR4 as a local therapeutic target. MSCs for vascular repair MONDAY Helene Roelofs Multipotent stromal cells (MSC) are characterized by their capacity to differentiate into bone, fat and cartilage and they are able to moderate immune responses and to enhance engraftment. These characteristics have rendered them interesting candidates for various new cell therapeutic approaches. We have already obtained good results with MSCs derived bone marrow (BM-MSC) for treatment of graft versus host disease and engraftment enhancement in clinical transplantation settings. MSCs have been found in a variety of tissues and can also be isolated from more easily accessible adipose tissue (AT-MSC). Upon characterization of MSCs from this interesting alternative source, we detected a CD34+ cell subset, that was not present in BM-MSC populations. Recently, it was reported that pericytes (CD34+/CD31/CD144-) from the adipose stromal-vascular fraction coexpress mesenchymal markers (CD10, CD13, and CD90) and are able to enhance the stability of endothelial networks and produce angiogenic factors (VEGF, HGF, bFGF) and inflammatory factors (IL-6 and -8 and MCP-1 and -2). We are currently investigating how the AT-MSC-specific CD34+ subpopulation is related to these pericytes and whether this subpopulation can be developed into a cell therapeutic product with improved properties for vascular repair. MONDAY Therapeutic Effects of Annexin A5 on Accelerated Atherosclerosis in Vein Grafts Mark M Ewing, M.R. de Vries, M. Nordzell, K. Pettersson, J.W. Jukema, P.H.A. Quax Rationale Venous bypass grafts can fail due to the occurrence of thrombosis, vein graft thickening and accelerated atherosclerosis. Annexin A5 has antithrombotic, anti-inflammatory and antiapoptotic properties, making annexin A5 a promising therapeutic agent against accelerated atherosclerosis. The aim of this study is to assess the therapeutic effects of annexin A5 on post-interventional vascular remodeling, including inflammation and accelerated atherosclerosis, in murine vein grafts. Methods & Results Post-interventional vascular remodeling was induced by either femoral artery cuff placement or by grafting of a venous segment in the common carotid artery of APOE*3-Leiden transgenic mice, fed a cholesterol-rich diet. Mice were treated daily by intraperitoneal injections with (1 mg/kg/day) recombinant human annexin A5 or vehicle only. After three days, the presence of inflammatory cells within the lesions of the femoral arteries was evaluated. Vein graft thickening and cellular composition of the vascular wall were analyzed after 28 days. Three days after femoral cuff placement adhesion and infiltration of leukocytes and macrophages was retarded in recombinant human annexin A5 treated mice by 51 and 71%, also the expression of TNF α and MCP-1in the arterial wall was significantly reduced by respectively 31% and 43%. Treatment with annexin A5 led to a significant reduction of vein graft thickening of 48% and a reduced number of leukocytes in the vein graft wall of 46%. Annexin A5 treatment resulted in stabilization of a rupture-prone phenotype of the lesions by showing a trend to reduced macrophage content, an increase in smooth muscle cell number and collagen deposition, a decrease in fibrin/fibrinogen deposition, and a decreased number of apoptotic cells. Furthermore, plaque hemorrhage features such as leaky vessels and dissections were also decreased in the annexin A5 treatment group. Conclusion Daily treatment with annexin A5 resulted in reduction of inflammation, thrombosis and accelerated atherosclerosis in murine vein grafts, thus a more stabile plaque phenotype. Clinical Relevance Annexin A5 is currently already used safely in patients as a diagnostic tool to detect atherosclerosis non-invasively and possible clinical applications include systemic administration and local delivery through drug-eluting stents. This study showed pronounced effects of annexin A5 in vivo on accelerated atherosclerosis and this provides a perspective for further research into therapeutic application of annexin A5 in cardiovascular disease. MONDAY C57BL/6 NK cell gene complex crucially involved in general vascular remodelling Leonard Seghers, V. van Weel, J. van Bergen, R. Toes, A. Hellingman, M. de Vries, V. van Hinsbergh, P. Quax Vascular remodelling is key in disease processes such as Peripheral Arterial Disease (PAD), restenosis and vein graft disease. Over years, evidence is accumulating that the immune system plays an important role in vascular remodelling. Inflammatory cells as monocytes, CD4+ and CD8+ T cells were already proven to be modulators of vascular remodelling. Recently, NK cells were indicated to contribute to vascular remodelling in processes of revascularisation and atherosclerotic lesion development. Here we further wished to address the role of NK cells in vascular remodelling in general. Interestingly, C57BL/6 and BALB/c mice are different with respect to remodelling; C57BL/6 mice display profound remodelling, whereas BALB/c mice display much less vascular remodelling. In addition, these two mouse strains are different in their NK gene complex (NKC) alleles, which are encoding for activating and inhibitory NK cell receptors. Subsequently, these two mouse strains and a third congenic BALB/c mouse strain expressing the C57BL/6 NKC alleles, BALB.B6CMV1r (CMV1r), were used in two different animal models of vascular remodelling, the mouse model for restenosis and the vein graft mouse model respectively. At first, contribution of NK cells in neointimal thickening as a response to vascular injury upon nonconstrictive cuff placement around the femoral artery was assessed. To do so, C57BL/6 mice were depleted for NK cells using a NK1.1 antibody, control mice received a matched isotype control. Mice depleted for NK cells showed significantly less formation of neointima when compared to control mice, indicating a contributory role for NK cells in the process of neointima formation. Given the differences in vascular remodelling between C57BL/6 and BALB/c mice and the differences in NK cell receptor repertoire, C57BL/6, BALB/c mice and the congenic BALB/c mouse strain CMV1r received a non-constrictive cuff around their femoral arteries. Surprisingly, congenic CMV1r showed progressive formation of neointima, comparative to C57BL/6 and significantly different from BALB/c mice that displayed very little formation of neointima. To verify whether NK cell contribution could be applied to vascular remodelling processes in general, the vein graft mouse model was performed in the aforementioned three mouse strains. In concert with our previous observations, CMV1r mice responded similar to C57BL/6 mice by displaying intimal hyperplasia in the venous bypass graft. BALB/c mice showed significantly less hyperplasia when compared to both CMV1r and C57BL/6 mice. Importantly, CMV1r displayed inflammatory infiltrate similar to that of the C57BL/6 mice, whereas BALB/c mice nearly show inflammatory cell influx into the vessel wall. In summary, we showed that NK cells play an important role in the induction of neointima formation. This was shown by inhibited formation of neointima when NK cells were absent. Subsequently, it was demonstrated that the expression of the C57BL/6 NK cell receptor repertoire leads to more profound vascular remodelling, i.e. formation of neointima and vein graft thickening by intimal hyperplasia. Furthermore, the observed differences in inflammatory infiltrate between the three mouse strains strongly suggest that the C57BL/6 NK receptor repertoire might be involved in the initiation of an immune response that is associated with profound vascular remodelling. Taken together, these data provide the first clue to involvement of C57BL/6 NK gene complex in vascular remodelling in general. Maternal Transmission of Risk for Atherosclerosis MONDAY Marco C De Ruiter In the last 20 years increasing amount of epidemiological and pathological evidence has become available illustrating the relationship between an adverse in-utero environment and increased risk of vascular disease in the offspring. The fetal origins hypothesis of Barker states that adaptation to unfavorable aspects of the maternal environment is beneficial to the developing embryo in utero. However, when the adult environment differs from the fetal environment, these adaptations may lead to an increased risk for cardiovascular disease. Question is how the adverse maternal environment can be translated into an increased atherosusceptibility of the offspring. We have developed an apolipoprotein E heterozygous knockout mouse model showing that maternal hypercholesterolemia is associated with endothelial damage in the fetal vasculature. After birth, neither difference in morphology, in intima/media thickness nor in lipid profile could be demonstrated between the maternally exposed and non-exposed groups. The maternally exposed group, however, develops substantial intimal hyperplasia in response to reduced shear stress levels and/or an atherogenic diet. This indicates that athero-susceptibility can be imprinted in fetuses with arteries that do not as yet manifest atherosclerotic disease. Global gene expression analysis using microarrays of adult non-compromised arteries demonstrates upregulation of genes involved in immune pathways and fatty acid metabolism in the offspring from hypercholesterolemic mothers. These changes coincide with specific local changes in histon modifcations in the fetal endothelial and the smooth muscle cells. We conclude that maternal apoE-deficiency affects chromatin modification marks in specific cells of the vasculature of the offspring in a non-uniform manner and that these changes are associated with an alteration in athero-susceptibility. Our observations also imply that epigenetic marks are variable within specific cells of the same tissue and that global analyses of epigenetic marks will thus underestimate the complexity of the process. Mesenchymal Stem Cells as Source for Endothelial Progenitors MONDAY Karolina Janeczek, N. Groen, H. Fernandes, N. Rivron, J. Rouwkema, C. van Blitterswijjk and J. de Boer Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from the bone marrow, expanded in the culture dish and differentiated into various cell types. Therefore MSCs are increasingly used in regenerative medicine as a source of cells for restoring wornout or damaged tissues such as cartilage, cardiac muscle and bone. The issue that remains to be improved in current implantation strategies is maintaining cell survival after implantation. The key parameter in this problem is the supply of nutrients and oxygen with diffusion as rate limiting factor. To maintain cell survival in big grafts, a vascular structure needs to be introduced within the graft which can rapidly hook up to the host’s blood vessels upon implantation. Such attempts were already successfully performed, however only with clinically irrelevant endothelial cell lines. We investigated the potential application of MSCs as a source for endothelial progenitors that can be used to cerate vascular network within grafts. We cultured cells on surfaces covered with extracellular matrix proteins (Matrigel, collagen, fibronectin) to differentiate MSCs into endothelial progenitors. We used both primary and immortalized MSC clones and utilized human umbilical vein endothelial cells (HUVEC) as positive control. We examined the optimal culture conditions to gain the highest possible differentiation efficiency. We investigated three basic characteristics of the examined cells: their ability to form capillary-like structures (CSLs), ability to take up acetylated LDL and finally expression of markers typical for endothelial cells such as CD31. We were able to obtain CSLs with similar efficiency in both cell lines when cells were cultured on Matrigel. These structures were not observed when MSCs were grown on tissue culture plastic, collagen or fibronectin. The fact that iMSCs reacted in a similar way as hMSCs allowed us to use the former in further research without depending on fresh hMSCs of different donors. The crucial parameters in culture condition were cell seeding density (minimum 12000 cells per cm2) and medium components (MSCs created CSLs in both basic and EGM-2 medium but expressed CD31 only when cultured in EGM-2 medium). Some of the cells we obtained took up AcLDL. We are currently investigating which medium component is crucial in such differentiation and what are the signaling pathways responsible for it. To confirm our results we will use siRNA to interfere with selected signaling pathways. We will select MSC-derived CD31+ cells by cell sorting and further examine their nature by investigating the expression of other endothelial markers. We will also perform several functional assays associated with endothelial cells in order to determine further potential use of obtained cells. Our results confirmed the ability of MSCs to differentiate into cells that can possibly be used to create a vessel network in grafts before implantation. Furthermore, MSC-derived CD31+ cells can be tested in other clinical application such as blood vessel tissue engineering or treatment of stent-inflicted vascular damage. MONDAY Specific extracellular matrix and serum components decrease monocyte adherence: implications for monocyte plasticity and function in peripheral vascular disease Diana Ploeger, M.J.A.van Luyn, M.C.Harmsen Introduction: Peripheral vascular disease (PVD) is a condition in which the blood vessels, in particular arteries, are damaged or dysfunctional, often as the result of atherosclerosis. PVD associates with limited perfusion of the extremities and may severely handicap patients. Beside stenting and bypass grafting, novel cell-based therapies employing so-called endothelial progenitor cells (EPC) are promising in animal models for arteriogenesis. Peripheral blood mononuclear cells contain different types of EPC of which the CD14positive monocytes are most abundant (~10%). In vitro and in vivo and under angiogenic conditions these monocytes differentiate into EPC that secrete a host of pro-angiogenic proteinaceous factors such as VEGF-A and FGF-2. Remarkably, the monocytes in PVD patients do not warrant adequate vascular maintenance and repair because of the chronic nature of this disease. In PVD, the endothelial monolayer is compromised, exposing the extracellular matrix (ECM) to leukocytes. We hypothesized that part of the presumed EPC dysfunction relates to a disturbed adherence of monocytes to the ECM and the subsequent cell fate. We initiated our studies by investigating the in vitro interaction of the CD14+ monocytes of healthy donors to selected ECM components. Materials and Methods: CD14+ monocytes were isolated from 0.5L buffy coats using immunomagnetic bead isolation. To determine the influence of the ECM on the plasticity and cell function of CD14+ monocytes, we optimized ECM coating procedures first. Thereafter, cell seeding densities and the influence of culture medium compounds (e.g. FCS) on cell adhesion were determined in time. Results: For all ECM components tested, a stable coating was present within one hour incubation. Cross- linking of ECM components with glutaraldehyde resulted in a decreased coating efficiency, compared to their non-cross linked controls. The coating density of ECM components strongly influenced adherence of the CD14+ monocytes. CD14+ monocytes were capable of efficient binding to low coating densities of Collagen I but lost their adhesive capacity to high coating densities of Collagen I. Adherence of CD14+ monocytes to ECM is influenced by culture medium compounds, such as FCS, which caused a 40-fold reduction in adherence. Conclusion & Perspective: In conclusion, we show that adherence of CD14+ monocytes is influenced by different ECM components and common culture medium compounds. Cross-linking of ECM coatings, as preformed in most cell adhesion studies, interferes with cell adhesion and may thus influence plasticity and subsequent function of cells. Here, we have devised an optimal protocol which will be used to study the influence of ECM components on cell plasticity and function in an in vitro model for arteriogenesis. MONDAY Overexpression of Timp-1 Modulates Plasma Cytokine Levels which Positively Affect Vein Graft Thickening and Spontaneous Intra-plaque Hemorrhages in Vein grafts Margreet R de Vries, J. Wouter Jukema, Paul H.A. Quax Introduction MMPs and their inhibitors Timps are known for their modulation of the extracellular matrix and are thought to be key players in plaque stability. MMPs can also activate or degradate cytokines, chemokines, interleukins and growth factors. Smooth muscle cell proliferation, apoptosis and inflammation can be stimulated by Timp-1 in a MMP-dependent and MMPindependent manner. Methods One day prior to the vein graft procedure pCDNA3.1Timp-1 and Luciferase plasmids were introduced via electroporation into the calf muscles of the mice. A venous segment (caval vein) was placed as an interposition in the carotid artery in ApoE3Leiden mice fed a high cholesterol diet for 3 weeks. 28 days after engraftment the grafts were harvested and analysed for morphologic changes and intraplaque hemorrhage.. At day 7 and day 28 plasma samples were collected for a 23-plex cytokine assay. Results In these vein graft lesions, accelerated atherosclerosis with accumulation of lipid loaded foam cells was observed. This accelerated atherosclerosis progresses in time and results in a significant increase in vein graft thickening with foam cell rich lesions, calcification and necrosis within 28 days after surgery. More importantly, in these atherosclerotic lesions features of intraplaque hemorrhage due to dissection, leaky vessels and erosion were observed. In plasmasamples of the Timp-1 group (n=4) at 7 days, both pro-inflammatory and anti-inflammatory cytokines as IL-1α, IL-1β, GM-CSF, KC, TNFα, Rantes, MIP1α, IFNγ, IL2, IL3, IL-10 and IL-12 were significantly elevated compared to the Luciferase plasmasamples. After 28 days only Rantes and Eotaxin were significantly higher in the Timp-1 group. At this timepoint Timp-1 overexpression resulted in a 47 % reduction of vessel wall thickening compared to the control group. More importantly, only one of the Timp-1 treated animals (n=13) showed intraplaque hemorrhage, whereas 66% of the control animals (10 out of 15) displayed features of intraplaque hemorrhage. Conclusion Overexpression of Timp-1 results in pronounced expression of pro- and anti-inflammatory cytokines after 7 days which almost all declined to comparable levels as the Luciferase group at day 28 except for Rantes. After 28 days reduced vessel wall thickening but more importantly reduced intraplaque hemorrhage could be detected in the Timp-1 group. Targeting the Microvasculature for Ischemic Tissue Repair MONDAY Douglas W. Losordo As the population ages and the acute mortality from cardiovascular disease decreases, a large population of patients is emerging who have symptomatic chronic ischemic vascular disease, many of whom remain severely symptomatic despite exhausting conventional medical therapy and mechanical revascularization. In addition, mounting evidence suggests that microvascular insufficiency plays a significant role in the pathophysiology of ischemia. At the present time, there are no therapies that directly address the needs of this patient population. Pre-clinical and early clinical data indicate that a variety of growth factors and stem/progenitor cells may be employed therapeutically for repair of ischemic tissue. Preclinical studies documented the potential therapeutic potency of endothelial progenitor cells, both as cultured and freshly isolated cells. Early phase clinical trials using a variety of approaches have been completed providing data of feasibility, safety and bioactivity. Later phase trials are under way. Accordingly, the goal of ischemic tissue repair appears feasible and is being approached in human clinical trials. We performed a phase II randomized controlled trial of autologous CD34 cell therapy in 167 patients with refractory, class 3 and 4 angina who were not candidates for revascularization. The results of this study will be presented. The evolution of the strategy of ischemic tissue repair will require an ongoing dialogue between clinicians, scientists, regulators and industry to take full advantage of advances in our understanding of the biology of these processes and their appropriate application to patients. NK cells and their role in modulating arteriogenesis TUESDAY Leonard Seghers, Paul Quax The involvement of inflammatory cells in the induction of neovascularisation and arteriogenesis (collateral formation) is well established. It has been suggested for several years that monocytes are involved in the regulation of arteriogenesis. Moreover, we and others have demonstrated a key role for CD4+ T-cells and NK cells in arteriogenesis using depletion studies and mice deficient in e.g. MHC class II, or NK cells. The exact mechanism how these cells affect arteriogenesis is not completely understood. However, it is tempting to suggest that the NK-cells might function as cytokine factories that upon activation start to produce cytokines, chemokines and growth factors. Therefore we hypothesize that the NK cell effector function in arteriogenesis relies on certain specific NK cell receptors. The fact that NK cell receptors play a role in arteriogenesis is firstly confirmed by the observation that mice lacking MHC class I molecule expression, necessary for NK cell interactions with the host, show impaired arteriogenesis. To map out the possible candidate receptors, we made use of the difference in NK cell receptor repertoire between Balb/C and C57B6, which are also known to be two strains of mice that are their opposites in their arteriogenic response. A congenic Balb/C mouse strain which is carrying the C57B6 NK cell receptor repertoire shows a rescue of the poor responding Balb/C (max. 50% flow recovery after 28 days) phenotype towards the rapid arteriogenic response seen in C57B6 mice (100% flow recovery in 14 days). This difference might be caused by the presence of the activating B6 NK cell receptor Ly49H, which is normally absent in Balb/C mice. To verify the involvement of this candidate receptor in arteriogenesis, its natural ligand the CMV protein m157 was overexpressed by non-viral electroporation mediated intramuscular gene transfer one day prior to femoral artery ligation. Blood flow recovery was monitored up to 28 days showing a significantly impaired arteriogenic response in mice overexpressing m157 as compared to control mice which were overexpressing Luciferase (p<0.01). These results show that, induction of hyposensitivity of NK cells via in vivo systemic exposure of the Ly49H NK receptor to its specific ligand m157, leads to impaired arteriogenesis. This indicates that the Ly49H receptor most likely plays a key role in modulation of arteriogenesis. This might provide an interesting pathway for modulating the arteriogenic response by modulation of the immune response, and therefore has impact for cell therapy approaches to induce revascularization. EPC plasticity and function in angiogenesis and inflammation TUESDAY Martin C. Harmsen Endothelial progenitor cells (EPC) from bone marrow or peripheral blood have been celebrated for their capacity to augment therapeutic neovascularisation over the past decade. The phenotype of cultured EPC is dictated by culture time, growth factors and extracellular matrix substratum. The appropriate choice of culture conditions may present a relatively easy accessible model for the in vivo behavior of circulating EPC. In general, monocytes (CD14+ fraction of MNC) emerge as rapidly proliferating colonies of spindle-shaped cell in MNC cultures, hence their name: early EPC (eEPC). These eEPC are shortlived, and harbor a marker profile that would qualify these cells as endothelial-like monocytes. More importantly, eEPC can transiently engraft vascular lesions and secrete a plethora of pro-angiogenic growth factors, which renders eECP promising candidates for therapeutic angiogenesis. On the other hand, endothelial colony forming cells (ECFC) emerge from CD34+ MNC after prolonged culture as genuine stem cells with a typical endothelial phenotype and limited secretome. In vivo ECFC contribute to neovascularisation. Unfortunately, ECFC appear refractory to culturing from adult blood. The concerted action of eEPC and ECFC warrants (pre)clinical studies to assess future cellbased therapy to improve perfusion in patients with PVD or CVD. We set out to dissect the role of circulating EPC, i.e. CD34+ and CD14+ MNC, in ischemic tissue repair. The subcutaneous in vivo administration of human CD34+ EPC embedded in Matrigel plugs, showed these hCD34+ EPC to augment influx of host (murine) inflammatory monocytic cells. Simultaneously, the presence of hCD34+ EPC enhanced host angiogenesis. The hCD34+ appeared to generate a pro-angiogenic niche through secretion of related growth factors including IL-8, MCP-1 (monocyte attraction) and VEGF, FGF-2, and HGF (angiogenesis). Remarkably, in vitro the presence of CD34+ EPC upregulated the proliferation of CD14+ EPC in a paracrine fashion through secretion of HGF. Moreover, CD34+ and CD14+ mutually interacted through secretion of HGF, VEGF, FGF-1, IL-8 and MCP-1 among others. In the rodent subcutaneous Matrigelplug model, the inclusion of these five paracrine factors effected influx of inflammatory cells and vascularisation as efficient as the combination of CD34+/CD14+ cells. Thus, the local microenvironment in vascular lesions or tissue lesions can be adjusted to a regenerative state by administration of therapeutic cells or their paracrine mediators. However, the ‘adjustable’ microenvironment by itself will also affect administered cells. Transforming growth factor-beta is one the prominent mediators that is present in vascular pathologies. In atherosclerosis, TGF can induce adverse dedifferentiation of vascular endothelial cells, known as EnMT or endothelial to mesenchymal transdifferentiation. We have shown that ECFC are also prone to TGF-driven EnMT. In vitro, after EnMT ECFC feature typical smooth muscle cells characteristics such as contractibility, secretion of ECM components and increase of thrombogenicity. Thus, in vivo administered ECFC may adopt a phenotype after EnMT that augments vascular remodeling during revascularization therapy. Modulation of the arteriogenic response by monocytes TUESDAY Imo Hoefer The growth of pre-existent arteriolar connections into functional collateral arteries is a multistep process involving mechanical, cellular and chemical factors. Nowadays, the pivotal role of changes in shear stress within the collateral vessels in the initiation of arteriogenesis is widely accepted. This primary event is followed by the adhesion and migration of inflammatory cells into the collateral vessel wall and the perivascular space. Within this space, these cells facilitate the remodeling and growth of the vasculature by providing growth factors and proteases. Monocytes are known to be actively involved in vascular growth since the late 1960s, but it took several decades until arteriogenesis was sought to be stimulated by attracting circulating monocytes to the growing collateral network. This can be achieved by a variety of cytokines, MCP-1 being the strongest single arteriogenic factor. However, due to the mechanistic similarity between arteriogenesis and atherogenesis, a non-selective attraction of inflammatory cells poses the risk of worsening the underlying atherosclerotic disease. Recent evidence suggests differential effects of monocyte/ macrophage subsets on vessel growth and tissue preservation. These insights and the ability to specifically target these subsets might provide new approaches to positively modulate the arteriogenic response by monocytes combined with neutral or even beneficial effects on atherosclerotic plaques TUESDAY Implantation of Adipose-Derived Regenerative Cells Enhances Ischemia-induced Angiogenesis Toyoaki Murohara Therapeutic angiogenesis using autologous stem/progenitor cells represents a novel strategy for severe ischemic diseases. Recent reports indicated that adipose tissues could supply adipose-derived regenerative cells (ADRCs) or adipose-derived stem cells (ADSCs). Accordingly, we examined whether implantation of ADRCs would augment ischemiainduced angiogenesis using a mouse model. Subcutaneous adipose tissue was obtained from the inguinal fat pad of C57BL/6J mice, and ADRCs were isolated using standard methods including collagenase digestion followed by centrifuge. Flow cytometry revealed that ADRCs expressed Sca-1 but not other lineage markers or endothelial markers. ADRCs expressed stromal cell-derived factor 1 (SDF-1) proteins. Hind limb ischemia was induced and culture-expanded ADRCs, PBS or mature adipocytes (MAs) as control cells were injected into the ischemic muscles. At 3 wks, the ADRC group had a greater laser Doppler blood perfusion index and a higher capillary density compared to the controls. Interestingly the MA group showed suppressed blood flow recovery and capillary density compared to the control group. Implantation of ADRCs increased circulating endothelial progenitor cells (EPCs) as judged by flow cytometry and culture assay of early outgrowth EPCs. SDF-1 mRNA abundance at ischemic tissues and serum SDF-1 protein levels were greater in the ADRC group than in the control group. Finally, intraperitoneal injection of an anti-SDF-1 neutralizing monoclonal antibody reduced the number of circulating EPCs and overall therapeutic efficacies of ADRCs. In conclusion, adipose tissue would be a valuable source for cell-based therapeutic angiogenesis. Moreover, chemokine SDF-1 may play a pivotal role in the ADRCs-mediated angiogenesis at least in part by facilitating mobilization of EPCs. Mechanisms in endothelial maintenance and repair WEDNESDAY Anton Jan van Zonneveld. Disruption of the integrity of the endothelial monolayer will lead to the immediate establishment of an injury-specific micro-environment, that includes aggregated platelets and platelet secretion products, deposited fibrin and other injury-associated components, such as growth factors and chemokines. Circulating stem- and progenitor cells can contribute to vascular regeneration after an injury. The contribution of these stem cells to vascular regeneration depends on the actual homing to the injury and the differentiation towards a more endothelial cell phenotype. The factors that lead to homing of these cells and subsequent differentiation, are not known. We developed models to characterize the injury-associated factors in stem cell-mediated vascular repair under ex vivo conditions, such as flow and the presence of an injury-specific micro-environment. This allows the detailed assessment of the initial steps that take place when vascular progenitor cells (CD34+ cells purified from umbilical cord blood) home to sites of vascular injury.Following homing, the injury-associated micro-environment mediates differentiation of CD34+ progenitors toward a more endothelial cell phenotype. As it has become apparent that microRNAs play a key role in cellular differentiation and vascular development, we study the role of endothelial microRNAs in endothelial differentiation and dedifferentiation. To that end, we perform microRNA profiling and set up the technology to assess the function of microRNAs in vitro and in vivo. WEDNESDAY Application of Poly-Chromatic Flow Cytometry Analysis and Cell Sorting Techniques to Identify Novel Subsets of Rare Circulating Cells with Angiogenic Potential Jamie Case Assay for circulating blood cell subsets that comprise the endothelial progenitor cell (EPC) pool in human peripheral blood by flow cytometry is increasingly used as a biomarker to determine cardiovascular disease risk and tumor angiogenesis since EPCs function in vasculogenesis and angiogenesis. Despite analytical advances in poly-chromatic flow cytometry (PFC), conventional flow cytometric approaches are exclusively utilized to enumerate and isolate EPCs, which has led to varied results as a biomarker in clinical studies, potential cellular misidentification, and thus a lack of a plausible biological explanation for how purported EPCs function in vascular repair. Using a novel protocol for PFC data acquisition and analysis, we discovered that purported circulating endothelial cells (CECs) and progenitors previously identified by a conventional flow cytometry approach, are not endothelial cells, but largely microvesicles and in vivo engrafting hematopoietic stem cells and myeloblasts, respectively. Further, accurate application of PFC identifies a rare circulating endothelial colony forming cell with proliferative potential. Thus, application of optimal PFC technology challenges existing paradigms of previously reported CECs and EPCs and provides a robust methodological platform for future definitive studies of these cells in human clinical studies of vascular disease. Immune-regulatory Pathways in Atherosclerosis WEDNESDAY Alain Tedgui Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive immuno-inflammatory mechanisms are involved. Inflammation is central at all stages of atherosclerosis. It is implicated in the formation of early fatty streaks, when the endothelium is activated and expresses chemokines and adhesion molecules leading to monocyte/lymphocyte recruitment and infiltration into the subendothelium. It also acts at the onset of adverse clinical vascular events, when activated cells within the plaque secrete matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap, leading to rupture and thrombus formation. Cells involved in the atherosclerotic process secrete and are activated by pro-inflammatory cytokines. Recent advances in our understanding of the mechanisms of atherosclerosis provided evidence that the immunoinflammatory response in atherosclerosis is modulated by regulatory pathways involving the two anti-inflammatory cytokines IL-10 and TGF, which play a critical role in counterbalancing the effects of pro-inflammatory cytokines. Interestingly, IL-10 and TGF- are also the two cytokines that mediate the immune regulatory functions of a sub-population of T cells, named regulatory T (Treg) cells. We recently demonstrated that natural CD4+CD25+ Treg cells play an important role in the control of atherosclerosis in apoE-/- mice. In addition, administration of ovalbumin-specific Tr1 cells, with its cognate antigen, to apoE-/- mice, can induce a marked suppression of Th1 (and Th2)-mediated responses with increased IL-10 production by stimulated peripheral T cells. Tr1 responses are associated with reduction in the accumulation of inflammatory macrophages and T lymphocytes in lesions, as well as partial inhibition of plaque development. Modulation of the peripheral immune response is achievable by transfer of regulatory T cells. It is therefore believed that atherosclerosis results from an imbalance between pathogenic T cells, either Th1 or Th2, producing proatherogenic mediators, and Treg cells with immunosuppressive properties, and that promotion, expansion or exogenous administration of Treg cells, ideally specific of plaquederived antigens, might limit disease development and progression. Monocytes as cellular therapy for vascular regeneration WEDNESDAY Sacha B. Geutskens, A.W. Hellingman, R.T. van Beem, L. Seghers, M.R. de Vries, AJ van Zonneveld, P.J. van den Elsen, C.E. van der Schoot, P.H.A. Quax and JJ Zwaginga Monocytes are multipotent hematopoietic progenitors that develop into immune cells, such as macrophages and myeloid dendritic cells, when stimulated accordingly. We recently found that CD14+ monocytes show a pro-angiogenic phenotype upon culture with CD4+ Tlymphocytes. Pro-angiogenic monocyte differentiation was characterized by the formation of CFU-Hill colonies and the increased production of pro-angiogenic factors such as VEGF, PDGF, TNF, TGF- and MCP-1. Initial monocyte-T-cell interactions were TCR-MHC class II-mediated followed by the induction of pro-angiogenic monocyte differentiation via medium-soluble factors. We now observe that transfusion of human monocytes that were stimulated with activated CD4+ T-cell-conditioned medium significantly improve bloodflow recovery after induction of ischemia in the hind limb of nude mice, as compared to transfusion with unstimulated monocytes or PBS. Using -actin labelling we found that more collaterals formed in the adductor muscles of mice transplanted with stimulated monocytes, as well as in mice transplanted with unstimulated monocytes as compared to PBS-treated mice. However, a significantly increased collateral size was only observed in mice transplanted with stimulated monocytes, which is in agreement with the improved hind limb perfusion observed. Thus, in addition to classic immune cell differentiation monocytes develop into cells that efficiently promote collateral artery formation (arteriogenesis) upon receiving the appropriate stimulation. These results support a role for alternative T-cellmonocyte mediated responses in vascular repair and render the pro-angiogenic monocyte an interesting candidate for the development of a regenerative cellular therapy that promotes adult re-vascularization in patients with arterial obstructive diseases. WEDNESDAY Impaired recruitment of HHT-1 mononuclear cells to the ischemic heart is due to an altered CXCR4/CD26 balance S Post, A Smits, A vdn Broek, J Sluijter, I Hoefer, B Janssen, R Snijder, J Mager, G Pasterkamp, C Mummery, P Doevendans, Marie-José Goumans Aim: Mononuclear cells (MNCs) from patients with hereditary hemorrhagic telangiectasia type 1 (HHT1), a genetic disorder caused by mutations in endoglin, show a reduced ability to home to infarcted mouse myocardium. Stromal-cell derived factor-1α (SDF-1α) and its receptor CXCR4 are crucial for homing and negatively influenced by CD26. The aim of this study was to gain insight into the impaired homing of HHT1-MNCs. Methods: CXCR4 and CD26 expression on MNCs was determined by flow cytometry. Transwell migration to SDF-1α was used to analyze in vitro migration. Experimentally induced myocardial infarction in mice, followed by tail vein injection of MNCs, was used to study homing in vivo. Results: Although HHT1-MNCs express elevated levels of CXCR4, this was counterbalanced by high levels of CD26, resulting in decreased migration towards a SDF-1α gradient in vitro. Their migration was enhanced by inhibiting CD26 with Diprotin A. Furthermore, while MNCs from healthy controls responded to TGFβ stimulation by increasing CXCR4 and lowering CD26 expression levels, HHT1-MNCs did not react as efficiently. In particular, CD26 expression remained high. Interestingly, homing of HHT1MNCs to the infarcted region of the murine heart was restored by pre-incubating the HHT1MNCs with Diprotin A before injection into the tail vein. Conclusions: We show that impaired homing of HHT1-MNCs is caused by an impaired ability of the cells to respond to SDF-1α. Our results suggest that modulating CD26 levels using inhibitors like Diprotin A can restore homing in cases where increased CD26 contributes to the underlying pathological mechanism. WEDNESDAY The mRNA-binding protein Quaking is essential for proper vascular smooth muscle cell function Eric van der Veer, A.O. Kraaijeveld, M.R. de Vries, F. Segers, D. Pons, M. Doop, S. Richard, P. Quax, AJ van Zonneveld, J.W. Jukema and E.A.L. Biessen The mRNA-binding protein Quaking (QKI) has a well established role in the nervous system during myelination. Recently, Quaking has also been implicated in embryonic vascular development, where it plays a vital role in regulating vascular cell differentiation, such as pericytes and vascular smooth muscle cells (VSMC). However, a role in adulthood remains elusive. We hypothesized that decreased QKI expression could trigger aberrant VSMC migration and proliferation, a frequent complication of percutaneous coronary intervention (PCI) that increases restenotic risk. To establish a causal involvement of QKI in vascular remodelling we focused on the consequences of decreased QKI expression on VSMC phenotype and functionality. For this, we utilized primary VSMCs derived from aortas of Qk viable mutant (Qkv) mice versus wild type (WT) littermates. VSMCs derived from Qkv mice were characterized by a flattened, enlarged morphology with prominent stress-fibers as opposed to WT cells which displayed a healthy phenotype. In keeping with this finding, Qk v VSMC displayed a decreased capacity to deal with oxidative and serum withdrawal-induced stress. Moreover, Qkv VSMCs displayed significantly decreased transwell cellular migration (WT 116 vs. Qkv 40 migrated cells; N=5; P<0.001), cellular proliferation (WT 2,149 vs. Qkv 607 DPI; N=25; P<0.001), and extracellular matrix production (WT 36 vs. Qkv 18 µm/ml; N=6; P<0.05). To gain insight into whether the levels of quaking expression could impact restenotic risk, we screened the qki gene for single nucleotide polymorphisms (SNPs) that could influence proper VSMC functioning. For this, we assessed the GENetic DEterminants of Restenosis (GENDER) project, a multicenter, prospective study design that enrolled 3,104 consecutive patients after successful PCI. We set out to characterize the role of QKI in instent restenosis by analysis for association of 12 SNPs throughout the qki gene and the risk of restenosis. As many as seven SNPs were found to significantly associate with the risk for target vessel revascularization (TVR). The strongest associations were found close to the transcription start site (2786 T/C: HR: 2.3, 95%CI: 1.3-3.9, P=0.002) and in intron 3 (57896 A/G: HR: 0.7, 95%CI: 0.6-0.9, P=0.005 and 65752 A/G: HR: 1.5, 95%CI: 1.1-1.9, P=0.006). Ten percent of patients carried the 65752G risk allele but lacked the protective 57896G allele. Importantly, these patients were at high risk to develop restenosis (HR: 1.8, 95%CI: 1.3-2.5, p<0.001). In conclusion, we have identified QKI as a central regulator of VMSC function, with decreased expression of QKI leading to VSMC dysfunction. Furthermore, these studies reveal a strong link between QKI and the risk for in-stent restenosis after PCI, suggesting that Qk may serve as a marker for TVR risk prediction and a therapeutic target. The natural history of aortic atherosclerosis: a systematic histopathological evaluation R.A.van Dijk, R. Virmani, J.H. von der Thüsen, A.F. Schaapherder, J.H.N. Lindeman Risk factor profiles for the different vascular beds (i.e. coronary, carotid, peripheral and aortic) are remarkably different, suggesting that atherosclerosis is a heterogeneous disorder. Little is known about the morphologic progression of atherosclerosis in the aorta, one of the primary predilection sites of atherosclerosis. Methods: A systematic analysis was performed in 260 consecutive peri-renal aortic patches (stained with Movat Pentachrome and H&E) collected during organ transplantation (mean donor age 46.5 (range 5-76) years; 54% ♂; mean BMI 24.9; 40% smokers; 20% hypertensive). Plaque morphology was classified according to the modified AHA classification scheme proposed by Virmani et al in 2000. Immunostaining against CD68 was used to identify the distribution of intimal macrophages and monocytes in several predefined locations among various plaque types and fibrous cap thickness. Results: There was significant intimal thickening (p<0.002) and medial thinning (p<0.001) with advancing age. The incidence of atherosclerotic plaques in the abdominal aorta correlated with age (r=0.640, p=0.01). During the first three decades of life adaptive intimal thickening and intimal xanthomas were the predominant lesions. In contrast, the fourth, fifth and sixth decades hallmarked more complicated plaques of pathological intimal thickening, early and late fibroatheromas (EFAs and LFAs), thin-cap FAs (TCFAs; cap thickness <155 µm), ruptured plaques (PRs), healed rupture and fibrotic calcified plaques. The mean percentage of lesional macrophages increased significantly from LFAs to TCFAs (5% to 17 %; p<0,007). Macrophage infiltration of the fibrous cap was negatively correlated with fibrous cap thickness (p<0.006); TCFAs and PRs (caps <100μm) contained significantly more macrophages (19%) compared with caps 101-300μm (6%) and >300μm (2%). Macrophages in shoulder regions were highest in early and late FAs (~45%) followed by TCFAs (27%) and PR (20%). Further, intimal vasa vasorum were mostly seen adjacent to the necrotic core of advanced atherosclerotic plaques and remained confined to the intimomedial border despite marked thickening of the intima. Conclusion: This study shows that aortic atherosclerosis starts early in life. Gross plaque morphologies of the peri-renal abdominal aorta are similar to coronary atherosclerosis yet indications were found for site specific differences in macrophage distribution and neovascularization. PARTICIPANTS Dr. Douwe E. Atsma Leiden University Medical Center Department of Cardiology Albinusdreef 2, building 1, C5-P P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Prof. Erik A.L. Biessen University of Maastricht Department of Pathology Cardiovasculair Research Instituut Maastricht P. Debeyelaan 25 6229 HX Maastricht [email protected] Jamie Case, Ph.D. Indiana University School of Medicine Assistant Research Professor of Pediatrics 1044 West Walnut Street, R4-440 Indianapolis, IN, 46202-5525, United States [email protected] Prof. Mat J.A.P. Daemen University of Maastricht Department of Medicine Section Pathology, room 5.76 PO Box 616 6200 MD Maastricht [email protected] Prof. Peter J. van den Elsen Leiden University Medical Center Department of Immunohematology and Blood Transfusion Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Drs. Mark Ewing Leiden University Medical Center Department of Cardiology Albinusdreef 2, building 1, C5-P P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Prof. Wim E. Fibbe Leiden University Medical Center Department of Immunohematology and Blood Transfusion Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Sacha Geutskens Leiden University Medical Center Department of Immunohematology and Blood Transfusion Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Marie-José Goumans Leiden University Medical Center Department of Molecular Cell Biology Albinusdreef 2, building 1 P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Marco C. Harmsen University Medical Centre Groningen Department of Pathology and Medical Biology Hanzeplein 1 9713 GZ Groningen The Netherlands [email protected] Prof. Victor W.M. van Hinsbergh VU University Medical Center Laboratory for Physiology Institute for Cardiovascular Research Van der Boechorststraat 7 1081 BT Amsterdam The Netherlands [email protected] Drs. Karolina Janeczek University of Twente Department of Tissue Regeneration P.O. Box 271 7500 AE Enschede The Netherlands [email protected] Dr. Imo E. Hoefer University Medical Center Utrecht Experimental Cardiology, G02.523 Heidelberglaan 100 3584 CX Utrecht The Netherlands [email protected] Drs. Jacco Karper Leiden University Medical Center Department of Vascular Surgery, C11-15 Albinusdreef 2, building 1 P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Robert Kleemann Dr. Jan Lindeman Leiden University Medical Center Department of Surgery PO Box 9600, K6-R 2300 RC Leiden The Netherlands [email protected] TNO-Pharma, Gaubius-Laboratory Department of Vascular and Metabolic Diseases PO Box 2215 2301 CE Leiden The Netherlands robert.kleemann@tno Dr. Douglas W. Losordo Feinberg Cardiovascular Research Institute Department of Medicine 303 E Chicago Avenue, Tarry 14-725 Chicago, IL 60611 United States [email protected] Prof. Toyoaki Murohara University Graduate School of Medicine Department of Cardiology, Nagoya, 65 Tsurumai, Showa-ku, Nagoya 466-8550 Japan [email protected] Ing. Diana Ploeger University Medical Center Groningen Department of Pathology & Medical Biology Stem Cell and Tissue Engineering Research Group Hanzeplein 1 (EA11) 9713 GZ Groningen The Netherlands [email protected] Prof. Paul H.A. Quax Leiden University Medical Center Department of Surgery PO Box 9600, K6-R 2300 RC Leiden The Netherlands [email protected] Prof. Ton J. Rabelink Leiden University Medical Center Department of Nephrology Albinusdreef 2, building 1, C3-P P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Marco de Ruiter Leiden University Medical Center Department of Anatomy and Embryology Albinusdreef 2, building 1 P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Helene Roelofs Leiden University Medical Center Department of Immunohematology and Blood Transfusion Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Drs. Leonard Seghers Leiden University Medical Center Department of Surgery, K-6 Albinusdreef 2, building 1 P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Prof. Alain Tedgui PARCC-INSERM 56 rue Leblanc 75010 Paris France [email protected] Ir. Rutger Wierda Leiden University Medical Center Department of Immunohematology and Blood Transfusion Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Mervin C. Yoder M.D. IUSOM 1044 West Walnut, R4-402E Indianapolis, IN 46202 United States [email protected] Ing. Margreet de Vries Leiden University Medical Center Department of Surgery Albinusdreef 2, building 1 P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Prof. D.C. (Christian) Weber Institute for Molecular Cardiovascular Research IMCAR Pauwelsstraße 30 D-52074 Aachen Germany [email protected] Prof. Anton Jan van Zonneveld Leiden University Medical Center Department of Nephrology Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected] Dr. Jaap Jan Zwaginga Leiden University Medical Center Department of IHB Albinusdreef 2, building 1, E3-Q P.O. Box 9600 2300 RC Leiden The Netherlands [email protected]