Zirkulierende endotheliale Vorläuferzellen, kurz EPCs, werden als Biomarker für kardiovaskuläre Erkrankungen genutzt. In einer prospektiven, randomisierten Cross-over-Studie wurden 18 chronische Dialysepatienten während jeweils vier Wochen mit Low-Flux-HD, High-Flux-HD sowie Hämodiafilatration behandelt. EPCs wurden zu Beginn sowie am Ende jedes Intervalls bestimmt. Trotz deutlicher Unterschiede bezüglich der Toxinentfernung im mittelmolekularen Bereich zeigte sich kein Einfluss auf Qualität und Quantität der EPCs / The number and function of endothelial progenitor cells (EPCs) are associated with the cardiovascular risk. In a prospective, randomized, cross-over trial, 18 maintenance dialysis patients were subjected to four weeks of each low-flux HD (LF-HD), high-flux HD (HF-HD), and online predilution hemodiafiltration (HDF). EPCs were determined at baseline and at the end of each four-week period. Despite a clear separation in middle molecular uremic toxin removal, there was no difference found in the effects of LF-HD, HF-HD, and HDF on EPCs

Intaktes Endothel und die ausreichende Funktion der endothelialen NO-Synthase (eNOS) sind Voraussetzungen für gesunde Gefäße. Eine endotheliale Dysfunktion besteht bei Patienten mit kardiovaskulären Risikofaktoren bzw. manifester koronarer Herzerkrankung (KHK). Endotheliale Vorläuferzellen (EPC) sind ein wichtiger Faktor für die Aufrechterhaltung der Homöostase des Endothels. Im klinischen Teil der vorliegenden Arbeit konnte gezeigt werden, dass diese Vorläuferzellen mit zunehmender Schwere der KHK in geringerem Maß im Blut zirkulieren. Die Anzahl der EPC war bei Patienten mit einer koronaren 3-Gefäßerkrankung um 77 % geringer, die Anzahl der kolonie-bildenden Einheiten (CFUs) um 50,3%, jeweils verglichen mit Patienten ohne KHK. Bei diesen Patienten konnte ebenfalls gezeigt werden, dass sich die Konzentration des endogenen eNOS-Inhibitors asymmetrisches Dimethylarginin (ADMA) im Plasma mit zunehmender Schwere der KHK erhöhte (0,47 ± 0,02 μmol/l bei fehlender KHK gegenüber 0,58 ± 0,02 μmol/l bei koronarer 3-Gefäßerkrankung). ADMA ist über eine Hemmung der eNOS an der Entstehung und Aufrechterhaltung einer endothelialen Dysfunktion beteiligt. Über diesen Weg wird vermutlich auch die Funktion der EPC erheblich eingeschränkt. Dies konnten wir anhand der In­vitro­Versuche mit EPC gesunder Spender zeigen. Dabei reduzierte sich unter ADMA-Einfluß die Anzahl der EPC in Kultur, die Anzahl und Größe der CFUs und ihre Funktion bzw. ihre Fähigkeit, sich in gefäßähnliche Strukturen zu integrieren. Eine gleichzeitige Gabe des HMG-CoA-Reduktase-Inhibitors Rosuvastatin wirkte in all diesen In-vitro-Versuchen der hemmenden Wirkung von ADMA entgegen. Die vorliegende Arbeit zeigt erstmals eine inverse Korrelation zwischen ADMA-Spiegeln und der Anzahl und Funktion der EPC. Der negative Einfluss auf EPCs ist vermutlich ein wichtiger Mechanismus, über den ADMA der Entstehung und dem Fortschreiten kardiovaskulärer Erkrankungen Vorschub leistet. / Endothelial progenitor cells play a pivotal role in regeneration of injured endothelium, thereby limiting the formation of atherosclerotic lesions. Reduced numbers of EPCs may affect progression of coronary artery disease. Regulation of EPC mobilization and function is mediated in part by nitric oxide (NO). Endogenous inhibitors of NO synthases, such as ADMA, contribute to endothelial dysfunction and injury. We tested the hypothesis that asymmetric dimethylarginine (ADMA) may be an endogenous inhibitor of endothelial progenitor cells (EPCs). We used flow cytometry and in vitro assays to investigate the relationship between EPC number and function with ADMA plasma levels in patients with stable angina. The plasma concentration of ADMA was related to the severity of coronary artery disease and correlated inversely with the number of circulating CD34+/CD133+ progenitor cells (r = -0.69; p < 0.0001) and endothelial colony forming units (CFUs) (r = -0.75; p < 0.0001). Adjusting for all patient characteristics, we confirmed these findings in multivariate regression analyses. In vitro differentiation of EPCs was repressed by ADMA in a concentration-dependent manner. Compared with untreated cells, ADMA reduced EPC incorporation into endothelial tube-like structures to 27 +/- 11% (p < 0.001). Asymmetric dimethylarginine repressed the formation of CFUs from cultured peripheral blood mononuclear cells to 35 +/- 7% (p < 0.001). Asymmetric dimethylarginine decreased endothelial nitric oxide synthase activity in EPCs to 64 +/- 6% (p < 0.05) when compared with controls. Co-incubation with the hydroxymethyl glutaryl coenzyme A reductase inhibitor rosuvastatin abolished the detrimental effects of ADMA. CONCLUSIONS: Asymmetric dimethylarginine is an endogenous inhibitor of mobilization, differentiation, and function of EPCs. This contributes to the cardiovascular risk in patients with high ADMA levels and may explain low numbers and function of EPCs in patients with coronary artery disease.

No description available.

No description available.

Despite intense research efforts, the repair of large bone defects is still not satisfactory and remains a major challenge in Orthopaedic Surgery. In this context bone tissue engineering has emerged as a promising strategy. However, one of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Thus an active blood vessel network is an essential pre-requisite for scaffold constructs to integrate within existing host tissue. Currently, great efforts are made to address this problem employing transplantation of vascular cells and loading of appropriate biological factors. Endothelial progenitor cells (EPCs) are a heterogeneous subpopulation of bone marrow mononuclear progenitor cells with potential for differentiation to the endothelial lineage and thus vasculogenic capacity. However, clinical studies reported that with the increase of age, increased susceptibility to apoptosis and accelerated senescence may contribute to the numerical and functional impairments observed in EPCs, which may lead to a reduced angiogenic capacity and an increased risk of vascular disease. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. A large body of evidence indicates that in Traditional Chinese Medicine (TCM) a plethora of herbs and herbal extracts are effective in the treatment of vascular diseases such as chronic wounds, diabetic retinopathy and rheumatoid arthritis. Thus, it seems rational to explore these medicinal plants as potential sources of novel angiomodulatory factors. In this thesis we demonstrated that treatment with TCM herbal extracts promote cell growth, cell migration, cell-matrix and capillary-like tube formation of BM-EPCs. Among these TCM extracts, Salidroside (SAL) and Icariin (ICAR) incubation increased VEGF and nitric oxide secretion, which in turn mediated the enhancement of angiogenic differentiation of BM-EPCs. A mechanic evaluation provided evidence that SAL stimulates the phosphorylation of Akt, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K), as well as phosphorylated ERK1/2, which is associated with the cell migration and tube formation. Furthermore, a pilot in vivo study showed that SAL has the potential to enhance bone formation in a murine femoral critical-size bone defects model. Another new finding of the present study is that hydrogen peroxide (H2O2)-induced cytotoxicity is counteracted by TCM extracts. We found that SAL, Salvianolic acid B (SalB) and ICAR significantly abrogated H2O2-induced cell apoptosis, reduced the intracellular level of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) expression, and restored the mitochondrial membrane potential of BM-EPCs. Our data suggest that this protective effect of SalB is mediated by the activation of mTOR, p70S6K, 4EBP1, and by the suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways after H2O2 stress. In addition, the investigation also demonstrates that ICAR owns the ability to inhibit apoptotic and autophagic programmed cell death via restoring the loss of mTOR and attenuation of ATF2 activity upon oxidative stress. Based on the outcomes of the present work, we propose SAL, SalB and ICAR as novel proanigiogenic and cytoprotective therapeutic agents with potential applications in the fields of systemic and site-specific tissue regeneration including ischaemic disease and extended musculoskeletal tissue defects.

Despite intense research efforts, the repair of large bone defects is still not satisfactory and remains a major challenge in Orthopaedic Surgery. In this context bone tissue engineering has emerged as a promising strategy. However, one of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Thus an active blood vessel network is an essential pre-requisite for scaffold constructs to integrate within existing host tissue. Currently, great efforts are made to address this problem employing transplantation of vascular cells and loading of appropriate biological factors. Endothelial progenitor cells (EPCs) are a heterogeneous subpopulation of bone marrow mononuclear progenitor cells with potential for differentiation to the endothelial lineage and thus vasculogenic capacity. However, clinical studies reported that with the increase of age, increased susceptibility to apoptosis and accelerated senescence may contribute to the numerical and functional impairments observed in EPCs, which may lead to a reduced angiogenic capacity and an increased risk of vascular disease. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. A large body of evidence indicates that in Traditional Chinese Medicine (TCM) a plethora of herbs and herbal extracts are effective in the treatment of vascular diseases such as chronic wounds, diabetic retinopathy and rheumatoid arthritis. Thus, it seems rational to explore these medicinal plants as potential sources of novel angiomodulatory factors. In this thesis we demonstrated that treatment with TCM herbal extracts promote cell growth, cell migration, cell-matrix and capillary-like tube formation of BM-EPCs. Among these TCM extracts, Salidroside (SAL) and Icariin (ICAR) incubation increased VEGF and nitric oxide secretion, which in turn mediated the enhancement of angiogenic differentiation of BM-EPCs. A mechanic evaluation provided evidence that SAL stimulates the phosphorylation of Akt, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K), as well as phosphorylated ERK1/2, which is associated with the cell migration and tube formation. Furthermore, a pilot in vivo study showed that SAL has the potential to enhance bone formation in a murine femoral critical-size bone defects model. Another new finding of the present study is that hydrogen peroxide (H2O2)-induced cytotoxicity is counteracted by TCM extracts. We found that SAL, Salvianolic acid B (SalB) and ICAR significantly abrogated H2O2-induced cell apoptosis, reduced the intracellular level of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) expression, and restored the mitochondrial membrane potential of BM-EPCs. Our data suggest that this protective effect of SalB is mediated by the activation of mTOR, p70S6K, 4EBP1, and by the suppression of MKK3/6-p38 MAPK-ATF2 and ERK1/2 signaling pathways after H2O2 stress. In addition, the investigation also demonstrates that ICAR owns the ability to inhibit apoptotic and autophagic programmed cell death via restoring the loss of mTOR and attenuation of ATF2 activity upon oxidative stress. Based on the outcomes of the present work, we propose SAL, SalB and ICAR as novel proanigiogenic and cytoprotective therapeutic agents with potential applications in the fields of systemic and site-specific tissue regeneration including ischaemic disease and extended musculoskeletal tissue defects.

There is convincing evidence in vivo that the vascular homing of endothelial progenitor cells (EPCs) contributes to rapid endothelial regeneration, which could prevent thrombosis and restenosis of cardiovascular devices. To enhance the EPC homing on cardiovascular devices, immobilization of an EPC capture agent (e.g. an anti-CD34 antibody) on the surface of cardiovascular devices is critical. We describe a way of immobilizing anti-CD34 Ab on 316L Stainless Steel (316L SS). For this, surface modification of 316L SS was performed via self-polymerization of dopamine (DA) and covalent grafting of staphylococcal protein A (SPA). On this coating the anti-CD34 Abs were oriented immobilized through their Fc constant region with SPA. In this process, the results of quartz crystal microbalance, X-ray photoelectron spectroscopy and water contact angle studies indicate that DA, SPA and anti-CD34 Ab were successfully immobilized onto the surface step by step. In vitro blood-compatibility tests confirmed that the modified surface induced less pro-coagulant fibrinogen denaturation, less platelet adhesion and lower activation of the adherent platelets. The affinity of EPCs for the modified surface has been demonstrated under flow conditions. This study provides potential applications for cardiovascular implant materials.

Efficient immobilization of the antibody to the substrate is of crucial importance in the development of anti-CD34-based endothelial progenitor cells capturing cardiovascular devices. This should go along with precise control of the antibody orientation by appropriate immobilization technology for retaining antibody activity, like in immunosensors. Recently, great attention was paid to immobilization of anti-CD34 antibody onto substrates by covalent binding, but at random orientation. Here, to investigate the biological effect of antibody orientation, we have prepared two kinds of anti-CD34 antibody coated surfaces, with random immobilization and oriented immobilization. The immunological binding activity (IBA) of the antibody at oriented immobilization was 3.48 times higher than at random immobilization, indicating that the two different surfaces were successfully prepared. The endothelial progenitor cell-capturing capability of oriented antibody-immobilized surface was 1.35 and 1.64 times higher than for the random immobilized surface after seeding for 2 and 12 h under flow condition, respectively. The endothelial progenitor cell-capturing efficiency per antibody by oriented immobilization was 5.16 and 6.26 times higher than for the random after seeding for 2 and 12 h under flow condition, respectively. In addition, the oriented antibody-immobilized surface possessed better blood-compatibility. These results clearly revealed the significance of antibody orientation which could retain its biological effect and may revolutionize the antibody-immobilization protocols used in cardiovascular and other bloodcontacting biomedical devices.

Endothelzell-basierte Therapien vermitteln regenerative Effekte hinsichtlich der Revaskularisierung von ischämischen Geweben. Doch ist die Verfügbarkeit von autologen Endothelzellen aufgrund einer krankheitsbedingt reduzierten Frequenz im peripheren Blut oder einer verminderten Integrität der endogenen Endothelzell-Populationen eingeschränkt. Hingegen ist es möglich, allogene endotheliale Vorläuferzellen aus der Nabelschnur in zelltherapeutisch relevanten Mengen zu isolieren. In der vorliegenden Arbeit wurden zunächst die Eigenschaften allogener humaner Nabelschnur (NS)-abgeleiteter sog. Endothelial Colony-Forming Cells (ECFCs) mit denen von venösen NS-abgeleiteten Endothelzellen verglichen. Aufgrund der nachgewiesenen Immunogenität von allogenen ECFCs wurde eine weiterführende Strategie zur Reduktion dieser immunogenen Eigenschaften durch die Koapplikation mit Mesenchymalen Vorläuferzellen (MSCs) verfolgt. Humane ECFCs wurden mit MSCs desselben Spenders kombiniert und in funktionellen in vitro- und in vivo-Assays untersucht. Dadurch konnte nachgewiesen werden, dass IFNγ-stimulierte ECFC/MSC-Kokulturen eine reduzierte Expression von HLA-Molekülen zeigen. Entsprechend induzierten spezifische CD8+ T-Zellen eine reduzierte Lyse der kokultivierten ECFCs und MSCs. Die Kokultur von ECFCs und MSCs mit allogenen Immunzellen führte zu einer nahezu vollständigen Inhibition der T-Zell-Proliferation. Um die reduzierte Immunogenität von ECFC und MSC in vivo zu verifizieren, wurden die Zellen in immundefiziente Mäuse injiziert, welche nachfolgend mit humanen PBMCs rekonstituiert wurden. So konnte nachgewiesen werden, dass die Koapplikation von ECFCs und MSCs nicht nur die Entstehung von stabilen Gefäßnetzwerken begünstigt, sondern zudem in den Transplantaten zu einer verringerten Immunzell-Infiltration führte. Die Koapplikation von ECFCs mit MSCs könnte daher eine klinische Nutzung dieser allogenen Quelle für die therapeutische Unterstützung der Vaskularisierung ermöglichen. / Endothelial cell-based therapies promote tissue regeneration and vascularization after ischemic damage. The availability of autologous endothelial progenitor cells is restricted in diseased patients, however therapeutically relevant numbers of allogeneic Endothelial Progenitor Cells can be isolated from an umbilical cord (UC). In the present study, the immunogenic properties of these Endothelial Colony Forming Cells (ECFCs) were first compared to human umbilical vein endothelial cells (HUVECs). Both cytokine-treated endothelial cells induced CD4+ and CD8+ T cell proliferation after coculture with allogeneic immune cells. So far, the potential interactions between ECFCs and Mesenchymal Stem/Progenitor Cells (MSCs) concerning their immunological features is poorly understood, but we hypothesize that MSCs might improve the immune compatibility and vessel building characteristics of ECFCs. Therefore, human UC-derived ECFC and MSC cocultures from the same donor were analyzed using various functional in vitro and in vivo assays. Stimulation of these cocultures with IFNγ caused strongly reduced expression levels of HLA-molecules compared to ECFC monocultures. The decreased molecular density on the cocultured ECFCs resulted in reduced cytotoxic CD8+ T cell-mediated lysis. Further, during IFNγ stimulation, the combination of ECFCs with MSCs prevented initiation of allogeneic T cell proliferation. To verify this concept in vivo, ECFCs and MSCs were co-transplanted in a humanized allograft mouse model in immunodeficient mice in order to effectively induce stable microvessels. These experiments demonstrate that when MSCs are co-applied with ECFCs, they not only support the formation of stable blood vessels, but also lead to fewer HLA-DR+ human vascular structures and fewer infiltrating human leukocytes. The data presented indicate that crosstalk between UC-derived ECFCs and MSCs might lower the risk of allogeneic ECFC rejection.