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.

Metabolic syndrome is defined as a combination of obesity, elevated triglycerides, decreased high-density lipoproteins, hypertension and insulin resistance. It is at least partially caused by sedentary life style and unhealthy dietary habits and is a major risk factor for development and progression of cardiovascular disease and type 2 diabetes. Growing medical and socioeconomic impact of the metabolic syndrome warrants further active search for novel risk markers and therapeutic targets. Recent experimental and epidemiological studies have demonstrated the multiple roles of the endogenous methylarginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) as wells as the enzymes, which are involved in their catabolism, dimethyarginine dimethylaminohydrolases (DDAHs) and alanine:glyoxylate aminotransferase 2 (AGXT2) in the pathogenesis of metabolic syndrome and its complications. ADMA is thought to exhibit its pathological effects by inhibiting and uncoupling nitric oxide synthases (NOS), while SDMA can inhibit transport of L-arginine. DDAHs, namely DDAH1 and DDAH2, have been thought as the major enzymes metabolizing ADMA to citrulline, while being inactive towards SDMA. Experimental studies with upregulation of DDAH1 in animal models showed that lowering ADMA results in protection against endothelial dysfunction, atherosclerosis, ischemia/reperfusion injury and vascular remodeling, acceleration of angiogenesis in the settings of ischemia and improvement of insulin sensitivity. Unfortunately, all the attempts to upregulate DDAH1 using small drugs have not been successful. The data regarding the role of DDAH2 are contradictory, with some studies showing that it can metabolize ADMA under certain conditions and other studies questioning its enzymatic activity towards ADMA. AGXT2 is a mitochondrial aminotransferase, which can metabolize, among its other substrates, both ADMA and SDMA. It is a large protein with possible allosteric regulatory sites, suggesting that, in contrast to DDAH1, it could be upregulated by small molecules. The role of AGXT2 in different pathophysiological processes involving ADMA and SDMA is poorly understood. It has been recently discovered in the offspring cohort of the Framingham Heart Study participants that a composite compound, consisting of the products of metabolism of ADMA and SDMA by AGXT2 (asymmetric dimethylguanidino valeric acid (ADGV) and symmetric dimethylguanidino valeric acid (SDGV), correspondingly) is an independent biomarker of CT (computed tomography)-defined NAFLD (non-alcoholic fatty liver disease) and a predictor of future diabetes up to 12 years before disease, suggesting that AGXT2 may play a key role in development of metabolic disease and its progression. We and other have recently identified several other metabolically active substrates of AGXT2, such as a marker of cardiovascular and overall mortality homoarginine and a regulator of fatty acid oxidation and browning of adipose tissue beta-amino-isobutyric acid (BAIBA), which further supports the importance of AGXT2 in pathogenesis of cardiovascular and metabolic diseases. The data presented in the current thesis enable answering the two research aims: 1) Identification of the tissue and intracellular expression pattern of human AGXT2 and 2) Testing the hypothesis that ubiquitous transgenic overexpression of AGXT2 protects from ADMA-induced vascular damage in vivo. The first research aim provided a thorough characterization of AGXT2 expression in humans using multiple complimentary techniques and addressed the current discrepancy in the literature with previous demonstration of comparable levels of Agxt2 expression by RT-PCR and Western Blot in the kidneys and liver in mice, and previous reports on detection of predominant Agxt2 expression in the kidneys by Northern Blot and in-situ RNA-hybridization in rats. In our current study we analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. We saw the strongest expression of AGXT2 in the kidney and liver both on the mRNA and protein levels. Our immunohistochemistry stainings showed that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed the intracellular localization of AGXT2 in mitochondria. In the second research aim we investigated whether long-term upregulation of AGXT2 is safe and can protect from ADMA- mediated vascular damage in the setting of DDAH1 deficiency, which is commonly observed in cardiovascular pathologies. We generated AGXT2 transgenic (TG) mice with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of ADGV, the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. The work, included into this thesis demonstrates that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans, where the enzyme is localized in mitochondria. The expression of AGXT2 in the liver is consistent with the proposed role of AGXT2 in development and progression of NAFLD and is consistent with our previous discovery of hepatocyte nuclear factor 4 alpha (HNF4α) as the major regulator of Agxt2 expression in the mouse liver. Chronic upregulation of AGXT2 in mice lowered systemic ADMA levels without any obvious effects on viability, development, growth and fertility, suggesting potential safety of this ADMA-lowering approach. Overexpression of AGXT2 protected from ADMA-induced vascular damage in the highly clinically relevant settings of DDAH1 deficiency, suggesting that the observed vascular damage was indeed caused by ADMA itself, rather than by some ADMA-independent effects of DDAH1 deficiency. The observed protective effects of AGXT2 upregulation are especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful. The current study, therefore, provides the basis for the future screens to identify small molecules, which would upregulate AGXT2 activity.

Homocystein (tHcy) gilt als unabhängiger kardiovaskulärer Risikofaktor und korreliert eng mit einer endothelialen Dysfunktion, welche nichtinvasiv mittels der flussinduzierten Vasodilatation (FMD) messbar ist. Experimentelle Hyperhomocysteinämie ist mit einer reduzierten Bioverfügbarkeit von endothelialen Stickstoffmonoxid (NO) bei gleichzeitig erhöhten Spiegeln des kompetetiven Inhibitors der NO-Biosynthese asymmetrisches Dimethylarginin (ADMA) assoziiert. In-vivo senkt eine Östrogenbehandlung neben tHcy auch die ADMA-Spiegel und verbessert signifikant die Endothelfunktion. Hinsichtlich ihrer Wirkung als selektive Östrogenrezeptormodulatoren wird angenommen, dass Phytoöstrogene, speziell Sojaisoflavone, ähnliche Effekte hervorrufen.<br><br> Innerhalb einer europäischen, multizentrischen, doppelblinden Interventionsstudie an 89 gesunden, postmenopausalen Frauen wurde der Einfluss von Sojaisoflavonen auf den Homocysteinmetabolismus, den Blutdruck und die in-vivo Endothelfunktion untersucht. Die cross-over Studie umfasste zwei achtwöchige Interventionsperioden, die von einer gleichlangen Wash-out-Phase unterbrochen waren. Die Zuteilung zum Isoflavon- (50 mg/d) oder Plazeboregime für die erste Interventionsphase erfolgte randomisiert. Endpunkterhebungen fanden jeweils in den Wochen 0 und 8 der Interventionsperioden statt.<br><br> Die renale Ausscheidung von Genistein, Daidzein und Equol war während der Isoflavonintervention signifikant erhöht (P>0,001). Die Phyoöstrogene hatten weder einen Effekt auf die tHcy-Konzentration (P=0,286), noch auf ADMA, Erythrozytenfolat und Vitamin B-12 (P>0,05) im Plasma. Während die Summe aus Nitrat und Nitrit (NOx), welche die NO-Bioverfügbarkeit reflektiert, im Verlaufe der Plazebobehandlung abfiel, wurde ein leichter Anstieg bei der Isoflavonsupplementation beobachtet (Delta Wo8-Wo0: -2,60 [-8,75; 2,25] vs. 1,00 [-6,65; 7,85] µmol/L P<0,001), was zu einem signifikanten Behandlungseffekt führte. Weiterhin wurde eine positive Korrelation zwischen ADMA und Vitamin B-12 gefunden (R=0,252; P=0,018). Die flussinduzierte Vasodilatation (P=0,716), ein Maß für die Endothelfunktion, blieb durch die Isoflavonbehandlung unbeeinflusst, obwohl sich diese über die Zeit insgesamt verbesserte (P>0,001). Bis auf einen marginalen Anstieg des systolischen Wertes (P=0,032) im Vergleich zur Plazebobehandlung blieb der Blutdruck während der Isoflavonintervention unverändert.<br><br> Im Gegensatz zu Östrogen übten Sojaisoflavone weder einen Einfluss auf die in-vivo Endothelfunktion noch auf die traditionellen und neuen kardiovaskulären Risikofaktoren den Blutdruck, tHcy und ADMA aus. Demzufolge ist der gesundheitliche Nutzen isolierter Isoflavone hinsichtlich einer Prävention hormonmangelbedingter Erkrankungen in gesunden postmenopausalen Frauen fraglich. / Homocysteine (tHcy) is a strong and independent risk factor for cardiovascular disease. Hyperhomocysteinemia contributes to endothelial dysfunction as assessed by flow-mediated vasodilation (FMD). The mechanisms by which homocysteine generates endothelial dysfunction remain incompletely understood although a growing body of data suggests that the bioavailability of nitric oxide (NO) is reduced. The principal competitive inhibitor of endothelial NO-synthase asymmetric dimethylarginine (ADMA) may play a central role in homocysteine related dysfunction as it is derived from homocysteine metabolism. Cardiovascular risk factor modification has suggested beneficial effects of estrogen on endothelial function by lowering homocysteine and ADMA levels. We hypothesize that phytoestrogens particular isoflavones act in a similar manner.<br><br> The effects of soy isoflavones on homocysteine metabolism and endothelial function were investigated within a multi-centre, double blind, cross-over intervention trial in 89 European postmenopausal women. Subjects consumed either fruit cereal bars with or without soy isoflavones (50 mg/d) for 8 weeks each with a 8 weeks washout period in between. Endpoint measurements were during both treatment phases at baseline and weeks 8, respectively. <br><br> Urinary phytoestrogens increased significantly after isoflavone intervention (P<0.001). Isoflavone supplementation did affect neither plasma total homocysteine (P=0.286) nor ADMA, vitamin B-12 or folate (P<0.05). Isoflavones had a favorable effect on NO-metabolism assessed by analysis of NO-metabolites (NOx) nitrite and nitrate. While NOx concentration significantly decreased during placebo there was a slight increase after isoflavone supplementation leading to a significant treatment difference (delta wk8-wk0: -2.60 [-8.75; 2.25] vs. 1.00 [-6.65; 7.85] µmol/L P<0.001). There was no association between total homocysteine and ADMA whereas a positive correlation was found for ADMA and vitamin B-12 (R=0.252; P=0.018). The endothelial function model did not demonstrate any difference between either treatment regime (P=0.716), although endothelial function assessed by flow-mediated vasodilation improved in general (P<0.001). A potential adverse effect was noted, with an elevation in systolic blood pressure (P=0.032) whereas diastolic blood pressure and mean arterial pressure remained unaffected.<br><br> Soy isoflavones did not have beneficial effects on endothelial function as well as on traditional and novel cardiovascular risk factors like plasma homocysteine, blood pressure and ADMA as observed for estrogen treatment. The health benefit of isolated isoflavones in healthy postmenopausal women is questionable.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.