Die koronare Herzkrankheit ist derzeit die häufigste Todesursache auf der Welt. Die Prävention der Arteriosklerose und ihrer klinischen Manifestationen wie der koronaren Herzkrankheit sind wichtige Ziele. Das Gen des endothelialen leukozytären Adhäsionsmoleküls-1 (ELAM-1,E-Selektin) sollte auf DNA-Polymorphismen untersucht werden, um einen möglichen genetischen Hintergrund von zellulären Interaktionen, die in den arteriosklerotischen Prozeß involviert sind, zu untersuchen. Eine signifikant höhere Mutationsrate (P=0,039) wurde bei 41 Patienten mit 40 Jahren oder jünger mit einer angiographisch nachgewiesenen schweren koronaren Arteriosklerose, im Vergleich zu 51 Patienten zwischen 40 und 50 Jahren, gefunden. Assoziationsanalysen zum Vergleich der Häufigkeiten des DNA-Polymorphismus in Abhängigkeit von den Risikofaktoren (männliches Geschlecht, Myokardinfarkt in der Eigenanamnese, positive Familienanamnese, Zigarettenrauchen, Hyperlipidämie (Hypercholesterinämie, Hypertriglyzeridämie), niedriges HDL-Cholesterin, Diabetes mellitus, Adipositas und arterielle Hypertonie) zeigten nur für die Hypertriglyzeridämie und die positive Familienanamnese eine statistisch signifikante Assoziation. Diese Daten lassen den Schluß zu, daß der DNA-Polymorphismus des endothelialen leukozytären Adhäsionsmoleküls-1 mit einer frühzeitigen schweren koronaren Arteriosklerose assoziiert ist. Der gefundene Polymorphismus ist ein zusätzlicher Risikofaktor bei einer positiven Familienanmnese. / Coronary heart disease is the major cause of death in the world. Prevention of atherosclerosis and its clinical manifestations such as coronary heart disease are fundamental goals. To contribute to the analysis of the genetic background of atherosclerosis especially endothelial dysfunction we searched for DNA polymorphisms in the endothelial-leukocyte adhesion molecule-1 (ELAM-1,E-selectin). A significantly higher mutation frequency (P=0,039) was observed in 41 patients aged 40 years or less with angiographically proven severe coronary atherosclerosis compared with 51 patients aged between 40 and 50 years. Association studies with risk factors for coronary heart disease (male sex, myocardial infarction, positive family history, cigarette smoking, hyperlipidaemia (hypercholesterolaemia, hypertriglyceridaemia), low HDL cholesterol, diabetes mellitus, obesity and hypertension) showed associations only for hypertriglyceridaemia and a positive family history. These data suggest that the polymorphisms in the ELAM-1 are associated with a higher risk for premature severe coronary atherosclerosis. DNA polymorphism in the ELAM-1 gene is an additional coronary risk factor if a positive family history exists.

<p>UVOD: Procesi koji obuhvataju endotelnu disfunkciju, oksidativni stres, hroničnu inflamaciju, hiperaktivnost i aktivaciju trombocita te naru&scaron;avanje ravnoteže procesa koagulacije i fibrinolize od najranijih faza razvoja dijabetes melitusa tip 2 (T2DM) promovi&scaron;u aterogenezu i nastanak aterotromboznih komplikacija. Kompleksan terapijski pristup u T2DM ima za cilj ne samo uspostavljanje glikoregulacije, korekciju brojnih metaboličkih poremećaja i modifikaciju pridruženih faktora rizika za nastanak ateroskleroze već i primenu antitrombocitne terapije u cilju primarne ili sekundarne prevencije aterotromboznih komplikacija. Uprkos primenjenoj antiagregacionoj terapiji, deo bolesnika doživi rekurentne aterotrombozne atake. Bolesnici sa T2DM se izdvajaju kao grupa sa posebnim rizikom za recidivantne aterotromboze &scaron;to može biti uslovljeno rezistencijom na primenjenu antitrombocitnu terapiju. Praćenje efekata antitrombocitne terapije i blagovremeno identifikovanje rezistentnih bolesnika ima za cilj optimizaciju primenjene antitrombocitne terapije &scaron;to može biti od izuzetnog kliničkog značaja u smislu sprečavanja progresije aterotromboznog procesa. CILJ: Proceniti i uporediti nivoe biomarkera, pokazatelja endotelne aktivacije, aktivacije i agregabilnosti trombocita u bolesnika sa bole&scaron;ću arterijskih krvnih sudova u tipu 2 dijabetes melitusa u odnosu na njihove vrednosti u zdravoj populaciji. Uporediti efikasnost primenjene antitrombocitne terapije tienopiridinima u bolesnika sa tipom 2 dijabetes melitusa i bole&scaron;ću arterijskih krvnih sudova u odnosu na efikasnost ove terapije u nedijabetičnoj populaciji bolesnika sa bole&scaron;ću arterijskih krvnih sudova. MATERIJAL I METODE: U ispitivanje je uključeno 100 ispitanika oba pola, starosti od 33 do 70 godina života, kod kojih je prethodno utvrđeno postojanje neke od kliničkih manifestacija bolesti arterijskih krvnih sudova (IBS, CVB, PAB) koji kao antitrombocitnu terapiju uzimaju tienopiridinski preparat, klopidogrel. Od toga, 50 uključenih ispitanika imalo je dijagnozu dijabetes melitus tipa 2, a 50 su bili bolesnici bez dijabetesa. Kontrolnu grupu je činilo 30 klinički i biohemijski zdravih ispitanika, nepu&scaron;ača koji su prema polnoj i dobnoj strukturi odgovarali ispitivanim grupama bolesnika. Svim ispitanicima su urađena antropometrijska merenja, laboratorijska analiza uzoraka krvi na automatizovanim analizatorima sa određivanjem parametara metabolizma glukoze, lipida, parametera inflamacije, KKS, parmetara koagulacije i trombocitnih pokazatelja. Određivanje serumske koncentracije sE&ndash;selektina i sP-selektina je vr&scaron;eno ELISA tehnikom (R&amp;D Systems, Inc. Minneapolis, USA). Plazmatska koncentracija vWFAg-a određivana je imunoturbidimetrijskom metodom na koagulacionom analizatoru Siemens Healthcare Diagnostics, Nemačka. Agregabilnost trombocita je određivana impedantnom agregometrijom (Multiple Electrode Aggregometry - MEA) na Multiplate analizatoru, Dynabyte, Minhen, Nemačka. Bazalna agregabilnost trombocita procenjivana je TRAP testom, rezidualna agregabilnost trombocita pod terapijom klopidogrela ADP testom, rezidualna agregabilnost trombocita pod terapijom aspirina, ASPI testom. Individualni odgovor na primenjenu antiagregacionu terapiju je procenjivan i na osnovu procenta sniženja bazalne agregabilnosti trombocita (%SAT) nakon primenjene antiagregacione terapije &scaron;to je izračunato sledećim formulama: procena antiagregacionog efekta klopidogrela:%SATadp =100 x (1-ADP/TRAP) i procena antiagregacionog efekta aspirina:%SATaspi =100 x (1-ASPI/TRAP). REZULTATI: Nivo sE-slektina je bio signifikantno vi&scaron;i u bolesnika sa T2DM u odnosu na bolesnike bez dijabetesa (45,1&plusmn;18,1vs.31,8&plusmn;10,5ng/ml; p&lt;0,001) i kontrolnu grupu zdravih ispitanika (45,1&plusmn;18,1vs.27,2&plusmn;11,2ng/ml; p&lt;0,001). Plazmatski nivo vWF Ag, bio je statistički značajno vi&scaron;i u bolesnika sa T2DM u odnosu na grupu ispitanika bez dijabetesa (172&plusmn;75,2vs. 146&plusmn;40,6%; p=0,045), kao i u odnosu na kontrolnu grupu zdravih (172&plusmn;75,2vs.130&plusmn;33,8%; p=0,007). Nivo sPselektina bio je statistički značajno vi&scaron;i kod bolesnika s T2DM u odnosu na ispitanike u grupi dijabetesa (95,2&plusmn;31,8vs.84,0&plusmn;21,8 ng/ml; p=0,042) i kontrolnoj grupi (95,2&plusmn;31,8vs.76,7&plusmn;16,2ng/ml; p=0,004). Uočeno je da je %rP statistički bio značajno vi&scaron;i u grupi dijabetičara u odnosu na grupu ispitanika bez dijabetesa (3,47&plusmn;1,30vs.2,30&plusmn;1,30%; p&lt;0,001) i kontrolnu grupu zdravih (3,47&plusmn;1,30vs.2,29&plusmn;1,23%; p&lt;0,001). Bolesnici sa T2DM imali su statistički značajno vi&scaron;e vrednosti ADP testa (70,3&plusmn;22,0vs.56,9&plusmn;19,7U; p=0,002) u odnosu na bolesnike bez dijabetesa, a uočen je i značajno niži stepen procenta sniženja bazalne agregabilnosti, %SATadp, u dijabetičara u odnosu na ispitanike bez dijabetesa (31,6&plusmn;12,4vs. 48,6&plusmn;12,6 %; p&lt;0,001). U grupi ispitanika sa T2DM vrednost TRAP testa statistički značajno pozitivno koreli&scaron;e sa brojem neutrofila (r=0,349;p= 0,013) i NLR-om (r=0,472;p=0,001), a multivarijantnom linearnom regresionom analizom dokazana je nezavisna povezanost TRAP testa i fibrinogena (B=9,61;p=0,009). Takođe, u istoj ispitivanoj grupi postoji pozitivna povezanost ADP testa sa HOMAIR (r=0,319;p=0,024), NLR-om (r=0,515;p&lt;0,001), hsCRP-om (r=0,356;p=0,011), kao i sa %rP (r=0,302;p=0,049). Multivarijantnom linearnom regresionom analizom dokazana je nezavisna povezanost ADP testa i ITM (B=1,43;p=0,043). %SATadp u bolesnika sa T2DM negativno je korelisao sa ITM (r= -0,381;p=0,006), OS (r= - 0,387;p=0,006), HOMA-IR (r= -0,349;p=0,013), hsCRP-om (r= -0,288; p=0,043), %rP (r= -0,302;p=0,049), sE-selektinom (r= -0,369; p=0,008) i sP-selektinom (r= - 0,374;p=0,007). U grupi dijabetičara, postoji pozitivna povezanost %rP sa ITM (r=0,365;p= 0,016), OS (r=0,435;p=0,004), HOMA-IR (r=0,409;p=0,006), hsCRP (r=0,374;p=0,014), sP-selektinom (r=0,341;p=0,025) i vWFAg-om (r=0,348;p=0,022). Takođe, sE-selektin pozitivno koreli&scaron;e sa ITM (r=0,380;p =0,006), OS (r=0,380; p=0,007), HOMA-IR (r=0,339;p=0,016), hsCRP-om (r=0,351;p=0,013), a sP-selektin sa ITM (r=0,312;p=0,027), OS (r=0,395;p=0,005), HOMA-IR (r=0,286;p=0,044), hsCRP-om (r=0,369; p=0,008) i nivoom sE &ndash; selektina (r=0,560;p &lt;0,001). Evaluirajući odgovor na terapiju klopidogrelom u podgrupama bolesnika sa dijabetesom, napravljenim prema kvartilnoj distribuciji nivoa ADP-a, tj. stepenu rezidualne agregabilnosti trombocita u toku terapije klopidogrelom, uočeno je da ukupna bazalna agregabilnost trombocita procenjena TRAP testom statistički značajno raste od prvog do četvrtog kvartila (76,50 &plusmn;19,91 vs. 94,54&plusmn;16,67 vs. 112,00&plusmn;10,22 vs. 128,92&plusmn;15,69U;p&lt;0,001), dok se %SATadp od prvog do četvrtog kvartila značajno smanjivao (40,44&plusmn;13,33 vs. 31,20&plusmn;11,82 vs. 33,16&plusmn;7,03 vs. 21,53&plusmn;10,16%). ZAKLJUČAK: Koncentracije cirkuli&scaron;ućih biomarkera endotelne aktivacije, sE &ndash; selektina i vWF Ag-a, solubilnog biomarkera trombocitne aktivacije, sP &ndash; selektina, kao i procenat retikulisanih trombocita, %rP, markera trombocitnog prometa, značajno su povi&scaron;ene kod bolesnika sa bole&scaron;ću arterijskih krvnih sudova u tipu 2 dijabetes melitusa u odnosu na njihove koncentracije kod zdravih ispitanika i bolesnika bez dijabetesa. Bolesnici sa T2DM imali su znatno vi&scaron;i stepen rezistencije na antitrombocitnu terapiju klopidogrelom u odnosu na bolesnike bez dijabetesa, procenjene stepenom rezidualne agregabilnosti trombocita, ADP test, kao i procentom sniženja ukupne bazalne agregabilnosti trombocita, %SATadp, metodom impedantne agregometrije, a &scaron;to je uslovilo i trend učestalijeg ponavljanja ishemijskih ataka u odnosu na bolesnike bez dijabetesa. Međusobna povezanost ispitivanih biomarkera endotelne i trombocitne aktivacije (sE &ndash; selektina, vWF Aga, sP &ndash; selektina), kao i markera prometa trombocita (%rP) sa metaboličko inflamatornim parametrima i sa indikatorima odgovora na antiagregacionu terapiju, može ukazivati na to da nepovoljan metabolički milje dijabetičara može biti jedan od doprinosnih faktora lo&scaron;em odgovoru na antitrombocitnu terapiju klopidogrelom.</p> / <p>INTRODUCTION: Processes involving endothelial dysfunction, oxidative stress, chronic inflammation, platelet activation and the imbalance between coagulation and fibrinolysis promote atherogenesis and atherothrombotic complications at early stage of diabetes mellitus type 2 (T2DM). The complex therapeutic approach in T2DM aims not only to reestablish glycemic control and to correct a number of metabolic disorders, but also to achieve primary or secondary prevention of atherothrombotic complications. Despite the applied antiplatelet therapy, some patients experience recurrent atherothrombotic attacks. Patients with T2DM are the group at particular risk for recurrent atherothrombosis, which can be caused by antiplatelet therapy resistance. Monitoring the effectiveness of antiplatelet therapy and identification of resistant patients aims to optimize the applied antiplatelet therapy, which can be of great clinical significance in terms of preventing progression of atherotrombotic processes. AIM: Evaluate and compare the levels of biomarkers, indicators of endothelial activation, platelet activation and aggregability in patients with arterial vascular disease in type 2 diabetes mellitus compared to their values in a healthy population. Compare the effectiveness of applied antiplatelet therapy with thienopyridines in patients with type 2 diabetes mellitus and arterial vascular disease compared to the efficacy of this therapy in nondiabetic population of patients with arterial vascular disease. MATERIAL AND METHODS: The study included 100 patients, 33 to 70 years of age, with previously established existence of some of the clinical manifestations of arterial vascular disease (CAD, CVD, PAD), taking thienopyridine antiplatelet therapy with clopidogrel. 50 patients was previously diagnosed with diabetes mellitus type 2 and 50 were nondiabetic patients. Control group included 30 age and sex matched healthy participants, non-smokers. All subjects underwent anthropometric measurements and laboratory analysis of blood samples on automated analyzers with determining the parameters of glucose metabolism, lipids, inflammation parameters, complete blood count, coagulation and platelet parameters. Serum concentrations of sEselectin and sP-selectin were determined by ELISA (R&amp;D Systems, Inc., Minneapolis, USA). vWFAg was determined by immunoturbidimetry on coagulometer Siemens Healthcare Diagnostics, Germany. Platelet aggregability was determined by impedance aggregometry (Multiple Electrode Aggregometry - MEA) on Multiplate analyzer, Dynabyte, Munich, Germany. Basal platelet aggregability was estimated by TRAP test, residual platelet aggregability during clopidogrel treatment was estimated by ADP test and during aspirin treatement by ASPI test. Individual response to antiplatelet therapy was estimated by the percentage of decrease in basal platelet aggregability (%DPA) obtained after antiplatelet therapy, calculated bypresented formulas: %DPAadp =100 x (1-ADP/TRAP)and %DPAaspi =100 x (1- ASPI/TRAP). RESULTS: Concentration of sE-selectin was significantly higher in patients with T2DM in order to non-diabetic patients (45,1&plusmn;18,1vs.31,8&plusmn;10,5ng/ml;p&lt;0,001) and healthy control group (45,1&plusmn;18,1vs.27,2&plusmn;11,2ng/ml; p&lt;0,001). vWF Ag was significantly higher in diabetic patients than in non-diabetics (172&plusmn;75,2vs. 146&plusmn;40,6%; p=0,045) and healthy controls (172&plusmn;75,2vs.130&plusmn;33,8%; p=0,007). sP-selectin was also significantly higher in patients with T2DM than in non-diabetics (95,2&plusmn;31,8vs.84,0&plusmn;21,8 ng/ml; p=0,042) and healthy controls (95,2&plusmn;31,8vs.76,7&plusmn;16,2ng/ml; p=0,004). %rP was significantly higher in group of patients with T2DM than in nondiabetic patients (3,47&plusmn;1,30vs.2,30&plusmn;1,30%; p&lt;0,001) and healthy control group (3,47&plusmn;1,30vs.2,29&plusmn;1,23%; p&lt;0,001). T2DM patients had statistically higher values of ADP test (70,3&plusmn;22,0vs.56,9&plusmn;19,7U; p=0,002) compared to patients without diabetes, and significantly lower %DPAadp (31,6&plusmn;12,4vs. 48,6&plusmn;12,6 %; p&lt;0,001). In T2DM group of patients, level of TRAP test correlated positively with number of white blood cells (r=0,349;p= 0,013) and NLR (r=0,472;p=0,001), and multivariant linear regression analisys showed significant independent association of TRAP test with fibrinogen (B=9,61;p=0,009). Statistically significant positive correlation of ADP test with HOMA-IR (r=0,319;p=0,024), NLR (r=0,515;p&lt;0,001), hsCRP (r=0,356;p=0,011) and %rP (r=0,302;p=0,049) was observed in patients with T2DM. Multivariant linear regression analisys showed significant independent association of ADP test with BMI (B=1,43;p=0,043). %DPAadp negatively correlated with BMI (r=-0,381;p=0,006), WC (r= - 0,387;p=0,006), HOMA-IR (r= -0,349;p=0,013), hsCRP (r= -0,288; p=0,043), %rP (r= -0,302;p=0,049), sE-selectin (r= -0,369; p=0,008) and sP-selectin (r= -0,374;p=0,007) in diabetic patients. Significant positive correlation of %rP with BMI (r=0,365;p= 0,016), WC (r=0,435;p=0,004), HOMA-IR (r=0,409;p=0,006), hsCRP (r=0,374;p=0,014), sP-selectin (r=0,341;p=0,025) and vWFAg (r=0,348;p=0,022) was found in diabetics. Also, sE-selectin positively correlated with BMI (r=0,380;p =0,006), WC (r=0,380; p=0,007), HOMA-IR (r=0,339;p=0,016), hsCRP(r=0,351;p=0,013), and sPselectin correlated positively with BMI (r=0,312;p=0,027), WC (r=0,395;p=0,005), HOMA-IR (r=0,286;p=0,044), hsCRP (r=0,369; p=0,008) and sE &ndash; selectin (r=0,560;p &lt;0,001). Evaluating the response to clopidogrel therapy in subgrpoups of diabetic patients accoarding the quartile distribution of ADP test (clopidogrel on-treatment platelet reactivity), it is found that total basal aggregability estimated by TRAP test significantly increased from the first to the fourth quartile (76,50 &plusmn;19,91 vs. 94,54&plusmn;16,67 vs. 112,00&plusmn;10,22 vs. 128,92&plusmn;15,69U;p&lt;0,001) while %DPAadp decreased (40,44&plusmn;13,33 vs. 31,20&plusmn;11,82 vs. 33,16&plusmn;7,03 vs. 21,53&plusmn;10,16%). CONCLUSION: Concentration of circulating biomarkers of endothelial activation, sE-selectin and vWF Ag, soluble marker of platelet activation, sP &ndash; selectin, as well as percentage of reticulated platelets, %rP, marker of platelet turnover, were significantly higher in patients with arterial vascular disease in T2DM compared to healthy controls and non-diabetics. Patients with T2DM had significantly higher degree of resistance to antiplatelet therapy with clopidogrel compared to non diabetics, estimated by ADP test, as well as with %DPAadp, what caused more frequent recurrent ischemic attacks compared to nondiabetic patients. Correlation of biomarkers of endothelial and platelet activation (sE &ndash; selectin, vWF Ag, sP &ndash; selectin) and markers of platelet turnover (%rP) with metabolic profile indicators and poor antiplatelet therapy response suggest that altered metabolic profile can be one of contributing factors of poor antiplatelet response in diabetic patients.</p>

Das Ziel dieser Arbeit war die Untersuchung der Rollgeschwindigkeiten von Leukozyten auf humanen kardialen mikrovaskulären Endothelzellen (HCMEC) und humanen umbilikalen venösen Endothelzellen (HUVEC). Die Endothelzellen wurden aus explantierten humanen Herzen sowie aus menschlichen Nabelschnüren unmittelbar postpartal gewonnen. Unter definierten Bedingungen wurden die in einer Flusskammer gemessenen Geschwindigkeiten von L-Selektin exprimierenden Nalm6-IF4 Zellen auf unstimulierten Endothelzellen mit denen auf stimulierten Endothelzellen verglichen. Die langsamere Geschwindigkeit der Leukozyten auf stimulierten Endothelzellen weist darauf hin, dass L-Selektin Liganden auf humanen kardialen mikrovaskulären Endothelzellen erst nach Stimulation exprimiert werden. Die beobachtete Geschwindigkeitsreduktion der Leukozyten ist jedoch von dem in der Literatur beschriebenen Selektin vermittelten Rollen zu unterscheiden. In den Versuchen mit Tunicamycin wurde gezeigt, dass N-glykosidisch gebundene Zucker kritische Bestandteile der Liganden für ihre Interaktion mit L-Selektin sind. Unter den gleichen Versuchsbedingungen wurde auch der Einfluss E-Selektin vermittelter Interaktionen auf die Geschwindigkeit der HL60 Zellen untersucht. Neben dem typischen Rollen wurde hier ebenfalls eine Selektin abhängige Geschwindigkeitsreduktion gesehen. / The aim of the present study was to investigate selectin mediated rolling velocities of leucocytes on human cardiac microvascular endothelial cells (HCMEC) and human umbilical vein endothelial cells (HUVEC). HCMEC were gained from explanted human hearts and HUVEC from umbilical cords immediately postpartum. Flow velocities of L-Selectin expressing Nalm6-IF4 cells on quiesent endothelial cells were compared to those on stimulated endothelial cells. Stimulation of endothelial cells with TNF led to significantly slower velocities of Nalm6-IF4 cells indicating that HCMEC express L-Selectin ligands only after stimulation. The observed reduction of flow velocities differs from rolling of leucocytes described in the literature. Experiments with tunicamycin showed that N-glycosylated carbohydtrate moieties are needed for proper function of L-Selectin ligands. E-Selectin mediated interactions between HL60 cells and endothelial cells were studied under the same conditions. Besides the typical rolling, a selectin mediated reduction of flow velocity was observed.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

We examined the effect of cyclosporin A, tacrolimus, sirolimus and everolimus on the cell growth, viability, proliferation, expression of cellular adhesion molecules (CAM) and leukocyte (PBMC) binding of human macrovascular (coronary artery, saphenous vein) and microvascular endothelial cells (EC). Tacrolimus did not affect EC integrity, growth or expression of CAM. Exclusively, EC from the coronary arteries showed a reduced cellular growth (about 30%) under cyclosporin A and tacrolimus treatment. In contrast, treatment with mTOR inhibitors reduced EC proliferative activity by about 40%, independently of the EC origin. No induction of apoptosis (caspase-3/7 activity) or cytotoxicity (MTS test) was observed. Long-term treatment with high concentrations of sirolimus and everolimus did not enhance the expression of CAM. Stimulation with tumor necrosis factor significantly increased the expression of CAM, independently of the drugs used. None of the mTOR inhibitors influenced the tumor necrosis factor-induced expression of CAM, whereas adhesion of PBMC increased significantly, as described by other papers. In summary, neither calcineurin inhibitors nor mTOR inhibitors activate human micro- and macrovascular EC. Therefore, the investigated drugs are unlikely to contribute to EC activation during transplant-associated vasculopathy. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

<p>UVOD: Gojaznost je hronično, multifaktorijalno i kompleksno oboljenje povezano sa povećanim rizikom od aterosklerotskih kardiovaskularnih bolesti (KVB). Disfunkcija vaskularnog endotela predstavlja rani događaj u patofiziolo&scaron;kom kontinuumu aterosklerotskog procesa, a produženo izlaganje vaskularnog endotela faktorima rizika za aterosklerozu udruženim sa gojaznosti (insulinska rezistencija, dislipidemija, proinflamatorno/protrombozno stanje), može doprineti procesima aktivacije/disfunkcije endotela i progresiji ateroskleroze u supkliničku, odnosno kliničku formu bolesti. CILJ: Uporediti koncentracije solubilne forme adhezionih molekula &ndash; intracelularnog adhezivnog molekula &ndash;1 (sICAM&ndash;1) i E selektina (sE&ndash;selektin), između ispitanika sa hiperalimentacionim tipom gojaznosti i normalno uhranjenih zdravih ispitanika, kao i utvrditi eventualno postojanje razlika u koncentraciji sICAM&ndash;1 i sE&ndash;selektina između ispitanika kod kojih je merenjem debljine kompleksa intima medija karotidne arterije (IMK) uočen supklinički stadijum ateroskleroze i ispitanika koji imaju normalnu debljinu IMK. Ispitati povezanost parametara telesne kompozicije (ukupne masne mase tela i masne mase abdominalnih depoa), cirkuli&scaron;ućih koncentracija biomarkera disfunkcije vaskularnog endotela (sICAM&ndash;1 i sE&ndash;selektina) i IMK kod ispitanika sa hiperalimentacionim tipom gojaznosti. MATERIJAL I METODE: U istraživanje je uključeno 60 ispitanika sa hiperalimentacionim tipom gojaznosti bez pridruženih komorbiditeta i 30 zdravih normalno uhranjenih učesnika usklađenih sa ispitanicima po godinama života i polu koji su činili kontrolnu grupu. Svim ispitanicima su urađena antropometrijska merenja, analiza komponenata telesne kompozicije (bioelektrična impedansna analiza, Tanita Body Composition Analyzer BC &ndash; 418 MA III), laboratorijska analiza uzoraka krvi na automatizovanim analizatorskim sistemima sa određivanjem parametara metabolizma glukoze (bazalno i 2 h u toku oralnog glukoza tolerans testa), lipida i lipoproteina, inflamacije i homocisteina. Određivanje serumske koncentracije sICAM&ndash;1 i sE&ndash;selektina je vr&scaron;eno ELISA tehnikom (R&amp;D Systems, Inc. Minneapolis, USA). Vrednosti IMK&ndash;a su određivane pomoću karotidnog dupleks ultrazvuka (Aloka SSD&ndash;650 US system, Tokyo), a na osnovu izmerenih (IMK) i normalno očekivanih vrednosti IMK za svakog ispitanika je izračunavan IMK Z&ndash;skor. Supklinički stadijum ateroskleroze je definisan kao vrednost IMK Z&ndash;skora veća od 1 (&scaron;to odgovara vrednosti IMK većoj od 95 percentila normalno očekivane vrednosti u odnosu na pol i godine života ispitanika). REZULTATI: Ispitanici sa hiperalimentacionim tipom gojaznosti su imali statistički značajno vi&scaron;e vrednosti medijane serumske koncentracije sE&ndash;selektina u poređenju sa medijanom serumske koncetracije sE&ndash;selektina učesnika u kontrolnoj grupi (36,2 (33,21&ndash;43.7) vs. 25,14 (23,1&ndash;29,48) ng/mL, P=0,00). Gojazni ispitanici III stepena gojaznosti su imali statistički značajno vi&scaron;u medijanu serumske koncenracije sE&ndash;selektina u odnosu na medijanu sE&ndash;selektina u ispitanika I stepena gojaznosti (41,5 (36,58&ndash;49,48) vs. 34,34 (22,49&ndash;36,62) ng/mL, P=0,00), odnosno medijanu sE&ndash;selektina u ispitanika II stepena gojaznosti (41,5 (36,58&ndash;49,48) vs. 32,1 (26,1&ndash;43,64) ng/mL, P=0,00). Nije uočena statistički značajna razlika u medijani serumske koncentracije sE&ndash;selektina između ispitanika I i II stepena gojaznosti (34,34 (22,49&ndash;36,62) vs. 32,1 (26,1&ndash;43,64) ng/mL, P=0,12). Gojazni ispitanici su imali statistički značajno vi&scaron;e vrednosti medijane serumske koncentracije sICAM&ndash;1 u poređenju sa medijanom serumske koncetracije sICAM&ndash;1 učesnika u kontrolnoj grupi (266,8 (245,8&ndash;326,73) vs.183,32 (167,9&ndash;208,57), P=0,00). U ispitivanoj grupi gojaznih uočena je statistički značajna razlika u medijani koncentracije sICAM&ndash;1 između ispitanika u I, II i III stepena gojaznosti (200,6 (190,26 - 264,4) vs. 278,5 (219,54 - 343,24) vs. 329,6 (259,2 - 350,34) ng/mL, P=0,00). Učestalost IMK Z&ndash;skor &gt; 1 je bila statistički značajno eća u gojaznih ispitanika u odnosu na kontrolnu grupu (36/60 vs. 7/30, P=0,00). Ispitanici sa IMK Z&ndash;skor &gt; 1 su imali statistički značajno vi&scaron;u medijanu koncentracije sICAM&ndash;1 u odnosu na ispitanike kod kojih je IMK Z&ndash;skor &le; 1 (295,4 (238,46&ndash;340,38) vs. 244,2 (227,35&ndash;260,38), P=0.00). Regresionom analizom (R2=0,71, korigovani R2=0,59) je utvrđeno da su parametri hsCRP (&beta;=0,45, P=0,00), HOMA&ndash;IR (&beta;=0,44, P=0,035) i ISI (&beta;=&ndash;0,36, P=0,028) nezavisno i statistički značajno povezani sa serumskom koncentracijom sE&ndash;selektina. Regresionom analizom (R2=0,65, korigovani R2=0,56) je utvrđeno da parametri ITM (&beta;=0,55, P=0,00), trigliceridi (&beta;=0,30, P=0,00), HDL holesterol (&beta;=&ndash;0,31, P=0,00), odnos TG/HDL&ndash;holesterol (&beta;=0,33, P=0,01), hsCRP (&beta;=0,31, P=0,00) i fibrinogen (&beta;=0,34, P=0,00) su nezavisno i statistički značajno povezani sa serumskom koncentracijom sICAM&ndash;1. U faktorskoj analizi je izdvojeno pet faktora &ldquo;gojaznost&rdquo;, &ldquo;insulinska rezistencija&rdquo;, &ldquo;aterogeni faktor&rdquo;, &ldquo;endotelna disfunkcija i vaskularna inflamacija&rdquo; i &ldquo;metabolički faktor&rdquo; koji obja&scaron;njavaju 69.72% ukupne varijanse ispitivanog uzorka. U multivarijabilnom modelu sa svim faktorima zajedno kojim je obja&scaron;njeno ukupno 75% varijanse, jedino je faktor gojaznost imao statički značajan i nezavistan uticaj na vrednost IMK Z&ndash;skor &gt; 1 (OR=2,74 (CI 1,18&ndash;6,33), P=0,019). U faktoru gojaznost su se izdvojili parametri: FAT trunk (%), FAT (%), OS (cm), ITM (kg/m2), LDL &ndash; holesterol (mmol/L), SP (mmHg), HOMA1&ndash;%B, fibrinogen (g/L), ApoB/apoA-I i hsCRP (mg/L). Univarijantom logističkom regresijom je uočeno da porast u koncentraciji LDL&ndash;H (OR=5,33 (CI 1,9&ndash;14,2), P=0,02) i koncentraciji hsCRP&ndash;a (OR=2,53 (CI 1,3&ndash;3,98),P=0,017) povećava rizik za postojanje vrednosti IMK Z&ndash;skor &gt; 1. ZAKLJUČAK: Cirkuli&scaron;uće serumske koncentracije biomarkera disfunkcije vaskularnog endotela, sE&ndash;selektina i sICAM&ndash;1, su značajno vi&scaron;e kod ispitanika sa hiperalimentacionim tipom gojaznosti u odnosu na njihove koncentracije u normalno uhranjenih ispitanika. U gojaznih ispitanika, koncentracija sE&ndash;selektina je povezana sa vrednostima indeksa insulinske rezistencije i biomarkera inflamacije, dok je koncentracija sICAM&ndash;1 značajno povezana sa udelom masne mase u ukupnoj telesnoj masi, vrednostima biomarkera inflamacije i proaterogenih lipidskih parametara. Ispitanici kod kojih postoji uvećanje abdominalnih masnih depoa i ukupnog udela masnog tkiva u telesnoj masi, vrednosti SKP, koncentracije LDL &ndash; holesterola, vrednosti lipoproteinskog indeksa ApoAI/apoB, bazalne insulinemije i biomarkera inflamacije, imaju trostruko povećan rizik od supkliničkog stadijuma ateroskleroze. U gojaznih osoba prilikom procene rizika od aterosklerotskih KVB, potrebno je utvrditi fenotipske osobine vaskularnog endotela i eventualno postojanje supkliničkog stadijuma ateroskleroze, da bi se definisale adekvatne preventivne mere i sagledale potencijalne terapijske mogućnosti.</p> / <p>INTRODUCTION: Obesity is a chronic, multifactorial and complex disease associated with an increased risk of atherosclerotic cardiovascular diseases (CVD). Vascular endothelial dysfunction is an early event in the pathophysiological continuum of atherosclerotic process. The prolonged exposure of vascular endothelium to classical and obesity associated risk factors (insulin resistance, dyslipidemia, proinflammatory state) could further promote deterioration of endothelial function and progression of atherosclerosis to subclinical or clinical form of disease. OBJECTIVE: The aim of the study was to compare the concentration of soluble forms of adhesion molecules, intracellular adhesion molecule-1 (sICAM-1) and E-selectin (sE-selectin), between obese subjects and normal weight healthy subjects, as well as to determine the possible existence of differences in concentration of sICAM-1 and sE-selectin among subjects with subclinical stage of atherosclerosis (assessed by measuring the thickness of the intima media complex of the carotid artery (IMT)), and subjects who have a normal value of IMT. Also, the aim was to determine the association between the parameters of body composition (total body fat mass and fat mass intra-abdominal depots), circulating concentrations of sICAM-1 and sE-selectin, and value of IMT in obese subjects. MATERIALS AND METHODS: The study included 60 obese nondiabetic subjects, without preexisting CVD and other associated comorbidity, and 30 healthy normal weight age and sex matched participants. All subjects underwent anthropometric measurements, analysis of the components of body composition (bioelectrical impedance analysis, Tanita Body Composition Analyzer BC - 418 MA III), laboratory analysis of blood samples (automated analyzer systems) with determining the parameters of glucose metabolism (basal and 2 h during the oral glucose tolerance test), lipids and lipoproteins, inflammation and homocysteine. Serum concentrations of sICAM-1 and sE-selectin were determined by ELISA (R &amp; D Systems, Inc., Minneapolis, USA). The values of IMK were determined by carotid duplex ultrasound (Aloka &ndash; ProSound ALPHA 10). IMK Z-score was calculated using the measured and the normal expected values of IMT for each patient. Subclinical stage of atherosclerosis was defined as the value of IMT Z-score greater than 1 (corresponding to the 95th sex-age-specific percentile of IMT measurements). RESULTS: Obese subjects had significantly higher median sE-selectin serum concentrations compared to median serum concentrations of sE-selectin in the normal weight subjects (36.2 (33.21-43.7) vs 25.14 (23.1-29.48) ng/mL, P=0.00). Morbid obesity subjects had significantly higher sE-selectin median serum concentration compared to the median sE-selectin concentration in moderate obese subjects (41.5 (36.58-49.48) vs 34.34 (22.49-36.62) ng/mL, P=0.00), and compared to the median sE-selectin concentration in severely obese subjects (41.5 (36.58-49.48) vs. 32.1 (26.1-4364) ng / mL, P=0.00). Obese subjects had significantly higher median sICAM-1 serum concentration compared to median sICAM-1 serum concentration in the control group (266.8 (245.8-326.73) vs. 183.32 (167.9-208.57), P=0.00). In the obese group, we observed a statistically significant difference in median sICAM-1 serum concentrations between moderate, severely and morbid obese subjects (200.6 (190.26-264.4) vs. 278.5 (219.54-343.24) vs. 329.6 (259.2-350.34) ng/mL, P=0.00). The frequency of IMT Z-score&gt; 1 was significantly higher in the obese group compared to control group (36/60 vs. 7/30, P=0.00). Subjects with IMT Z-score&gt; 1 had significantly higher median concentrations of sICAM-1 compared to those in which the IMK Z-score &le; 1 (295.4 (238.46-340.38) vs. 244.2 ( 227.35-260.38), P=0.00). In regression analysis (R2=0.71, adjusted R2=0.59), hsCRP (&beta;=0.45, P=0.00), HOMA-IR (&beta;=0.44, P=0.035) and ISI (&beta;=-0.36, P=0.028) were independently and significantly associated with serum sE-selectin concentration. In regression analysis (R2=0.65, adjusted R2=0.56), BMI (&beta;=0.55, P=0.00), triglycerides (&beta;=0.30, P=0.00), HDL cholesterol (&beta;=-0.31, P=0.00), the ratio of TG/HDL-cholesterol ratio (&beta;=0.33, P=0.01), hsCRP (&beta;=0.31, P=0.00 ) and fibrinogen (&beta;=0.34, P=0.00) were independently and significantly associated with serum sICAM-1 concentration. In the Factor analysis, five factors &quot;obesity&quot;, &quot;insulin resistance&quot;, &quot;atherogenic factor,&quot; &quot;endothelial dysfunction and vascular inflammation&quot; and &quot;metabolic factor&quot; explained 69.72% of the total variance of the test sample. In a multivariate model with all the factors together (75% of the total variance), &quot;obesity&quot; factor was significantly and independently associated with IMT Z-score&gt; 1 (OR=2.74 (CI 1.18-6.33), P=0.019). The &quot;obesity&quot; factor consisted of parameters: trunk fat (%), fat (%), waist (cm), BMI (kg/m2), LDL &ndash; cholesterol (mmol/L), systolic blood presure (mmHg), HOMA1-% B, fibrinogen (g/L), Apo B/apoA-I and hsCRP (mg/L). Logistic regression analysis showed that independent predictors of IMT Z-score&gt; 1 were LDL-cholesterol (OR=5.33(CI 1.9-14.2), P=0.02) and hsCRP (OR=2.53 (CI 1.3-3.98), P=0.017). CONCLUSION: Circulating serum concentrations of endothelial dysfunction biomarkers, sE-selectin and sICAM-1, were significantly higher in obese subjects compared to concentration in the normal weight subjects. In obese subjects, the concentration of sE-selectin was associated with insulin resistance and biomarkers of inflammation, whereas sICAM-1 concentration was associated with fat mass, inflammation biomarkers and the proatherogenic lipid parametars. In individuals with increased abdominal fat depots and total proportion of fat mass in the body weight, values of SBP, LDL-C, ApoB/apoA-I, basal insulin levels and biomarkers of inflammation, there is threefold increased risk of subclinical stages of atherosclerosis. In order to define an adequate preventive measures and possible therapeutic options for atherosclerotic CVD in obese subjects, it is necessary to assess the phenotypic characteristics of vascular endothelium and possible presence of subclinical stage of atherosclerosis.</p>

No description available.

In der vorliegenden Arbeit sind Untersuchungen vorgestellt, die sich mit Wirkungen und Interaktionen von zwei ubiquitär im menschlichen Körper vorkommenden Gasen befassen, i.e. Sauerstoff und Stickstoffmonoxid. Im Falle beider Substanzen ermöglicht die geringe Größe der Moleküle eine freie Diffusion über Membranen hinweg, eine Eigenschaft, die für die Funktion der Signaltransduktion geradezu prädestiniert. Aus den vorgelegten Untersuchungen lassen sich die folgenden Folgerungen ableiten: * Stickstoffmonoxid wirkt in-vitro selektiv bakteriostatisch auf Bakterien, die üblicherweise Früh- und Neugeborene besiedeln. Dabei hängt die Selektivität von den jeweiligen bakteriellen Verteidigungsmechanismen ab, die bakteriostatische Wirkung liegt in einem Konzentrationsbereich, der außerhalb desjenigen liegt, der derzeit klinisch angewendet wird. * Hyperoxie führt im Ganztiermodell der unreifen Ratte zu einer zerebralen Hochregulation von iNOS und damit zur Synthese von NO. Soweit dies anhand der Synthese von Peroxynitrit als definitivem Schädigungsmechanismus beurteilbar ist, wird trotz entsprechender iNOS-Expression wenig bis gar kein Peroxynitrit gebildet. Da das Zusammentreffen von NO und Sauerstoff sonst regelhaft zur Entstehung von Peroxynitrit führt, müssen im Gehirn der unreifen Ratte ausreichende antioxidative Schutzmechanismen präsent sein, die diese Reaktion verhindern. * Im in-vitro-Modell der Gasäquilibrierung von Nabelschnur-PMN zeigte sich unter Hyperoxie das ausgeprägteste Aktivierungsmuster aller verglichenen Sauerstoffkonzentrationen. Dies stand im Gegensatz zur Exposition adulter Zellen, hier fand sich eine größere Hyperoxietoleranz bei gleichzeitig stärkster Aktivierung unter Hypoxiebedingungen. Welche Bedeutung diesen Ergebnissen im klinischen Umgang mit Neugeborenen zukommt muß derzeit noch offen bleiben. Allerdings häufen sich Hinweise aus experimentellen Studien, die darauf hindeuten, daß ein restriktiver Umgang mit hohen Sauerstoffkonzentrationen auch im klinischen Umfeld gerechtfertigt sein könnte. / The present investigations deal with the effects and interactions of gases, which are ubiquitous in the human body i.e. oxygen and nitric oxide. Both substances are small enough to freely diffuse across biological membranes. This ability predestines both molecules for the function of signal transduction. The results of our investigations lead to conclusions as follows: * Nitric oxide has selective bacteriostatic effects in-vitro on some bacterial strains typically isolated from preterm and term newborn infants. Selectivity depends on the presence of bacterial defense mechanisms. The bacteriostatic effect takes place at concentrations above those currently used in clinical practice. * Hyperoxia leads to upregulation of iNOS and subsequent NO production in an animal model of the immature rat. Despite this upregulation of iNOS synthesis there is no increased production of peroxynitrite which is known to cause cellular and DNA damage. Since the combination of NO and high concentrations of oxygen lead to peroxynitrite formation on a regular basis, effective antioxidant mechanisms appear to prevent peroxynitrite formation in the brain of the immature rat. * The most pronounced activation of cord blood polymorphonuclear cells (PMN) during conditions of hyperoxia, normoxia, and hypoxia was found for exposure towards high oxygen concentrations in an in-vitro model of gas equilibration. As opposed to that, hypoxia was the most potent trigger for adult PMN. It remains to be determined which clinical implications must be derived from these results. However, increasing experimental evidence indicates that exposure towards high oxygen concentrations should be restricted also in clinical practice and not only in preterm infants, but also in term newborns.

1 Cardiovascular risks in chronic airway disease in childhood The aim of this thesis was to evaluate cardiovascular risk by using a combined diagnostic approach by measuring RHI and specific biochemical markers in patients with chronic respiratory disease, where we could assume a possible risk of CVD. A total of 119 probands were examined, including 22 patients with cystic fibrosis (CF) and 52 asthma patients. We evaluated RHI using a new plethysmographic method that has a number of advantages over the ultrasonographic methods used in other studies, including non-invasiveness, high sensitivity, low biological variability and objectivity due to automatic processing. Of the biochemical parameters, we measured 4 biomarkers in relation to endothelial dysfunction (ED): hsCRP, ADMA, E-selectin, and VCAM-1. We compared RHI and biomarkers in CF and asthma patients with healthy controls and sought mutual correlations. We did not prove a statistically significant difference in RHI between the test groups with CF children but we confirmed the decreasing trend of RHI since adolescence and significantly lower RHI values in CF adults, confirming the progressive development of atherogenesis and worsening of ED with age. Biochemical parameters showed significantly higher levels of hsCRP, sVCAM-1 and E-selectin in CF...

Erkrankungen des Herz-Kreislaufsystems sind die häufigste Todesursache in Deutschland. Eine gestörte Funktion des Gefäßendothels spielt bei der Entstehung von Herz-Kreislauferkrankungen eine Schlüsselrolle. Das Risiko einer kardiovaskulären Erkrankung ist bei Diabetikern stark erhöht. Der Transkriptionsfaktor COUP-TFII spielt eine essentielle Rolle im Glukosemetabolismus. Gleichzeitig ist er für die Differenzierung von Endothelzellen von großer Bedeutung. Für die Differenzierung und Aufrechterhaltung des arteriellen und venösen Phänotyps von Endothelzellen sind dabei maßgeblich der NOTCH-Signalweg und insbesondere die Transkriptionsfaktoren HEY2 (arteriell) und COUP-TFII (venös) verantwortlich. Gesteigerte Glukosespiegel könnten somit Auswirkungen auf die Differenzierung von Endothelzellen haben und damit einen neuen Mechanismus für das erhöhte Risiko von Gefäßerkrankungen bei Diabetikern darstellen. Im Rahmen der Arbeit konnte die exklusive Expression von COUP-TFII im Zellkern von humanen venösen Endothelzellen nachgewiesen werden. Humane arterielle Endothelzellen zeigten keine Expression von COUP-TFII. Außerdem konnte im Rahmen der Arbeit erstmals die spezifische Expression von COUP-TFII in humanen Endothelzellen der Koronararterie nachgewiesen werden. Die Untersuchung der COUP-TFII Promotoraktivität konnte das Expressionsmuster von COUP-TFII bestätigen. Der Promotor zeigte sowohl in den venösen Endothelzellen der humanen Nabelschnur als auch in den humanen Endothelzellen der Koronararterie Aktivität. Die kurzzeitige Stimulation von venösen Endothelzellen mit Glukose führte zu einem starken Anstieg der COUP-TFII Expression. Eine Translokation von COUP-TFII aus dem Zellkern in das Zytoplasma konnte nicht nachgewiesen werden. Die Langzeitstimulation führte interessanterweise zu einer Verminderung der COUP-TFII Expression und zu einer Erhöhung der Expression von E-Selektin. In beiden Fällen zeigte sich keine Beeinträchtigung der Expression durch Insulin. Die durchgeführten Untersuchungen schließen eine Beteiligung des AKT-Signalweges an der Regulation aus. Es zeigte sich jedoch, dass humane venöse Endothelzellen als Insulin-sensitives Gewebe mit funktionsfähigem AKT-Signalweg einzustufen sind. Stimulationsversuche mit L-Glukose zeigten keine Regulation der COUP-TFII Expression. Eine osmotische Wirksamkeit der hohen Glukosekonzentration auf die Expression von COUP-TFII konnte somit ausgeschlossen werden. Die Deletionsanalyse des COUP-TFII Promotors konnte einen Glukose-sensitiven Bereich innerhalb des COUP-TFII Promotors identifizieren. Weiterhin konnte die Repression der Aktivität des COUP-TFII Promotors durch Hypoxie nachgewiesen werden. Eine der wichtigen Aufgaben von Endothelzellen ist die von der endothelialen NO-Synthase (eNOS) katalysierte Bildung von Stickstoffmonoxid (NO). NO hemmt die Expression des Adhäsionsmoleküls E-Selektin. Eine verringerte NO-Produktion hat die Ausbildung einer endothelialen Dysfunktion zur Folge. In dieser Arbeit konnte erstmals eine Erhöhung der eNOS Expression nach Verminderung der Expression von COUP-TFII in humanen venösen Endothelzellen gezeigt werden. Diese könnte die Ursache für die Verminderung der E-Selektin Expression nach Herabregulation von COUP-TFII sein. Durch die Anwendung einer Plattenkegel-Viskometer-Apparatur konnte gezeigt werden, dass die NO-Abgabe entscheidend von den Strömungsbedingungen und Scherkräften abhängig ist. Die Stimulation arterieller Endothelzellen mit laminarer oder oszillatorischer Schubspannung führte zu einer Erhöhung der NO-Abgabe. Turbulente Schubspannung zeigte dagegen keinen Einfluss auf die NO-Abgabe. Durch Überexpression von COUP-TFII in Kombination mit laminarer Schubspannung wurde die NO-Abgabe weiter gesteigert. Die gezeigte direkte Regulation der HEY2 und COUP-TFII Promotoraktivität durch geänderte Strömungsbedingungen spielt in diesem Prozess möglicherweise eine bedeutende Rolle. Die beschriebene Regulation von COUP-TFII durch Glukose in Endothelzellen könnte eine neue Erklärung für die gesteigerte Rate an Gefäßerkrankungen von Typ2-Diabetikern darstellen. Bei der Regulation der endothelialen NO-Synthase und E-Selektin durch COUP-TFII handelt es sich möglicherweise um einen neuen, anti-adhäsiven Feedback-Mechanismus, der zur Verringerung der Leukozyten-Adhäsion an Endothelzellen und damit zur Gefäßprotektion beitragen könnte. Die differentielle Expression der arteriellen Markergene HEY2 und CD44 konnte in humanen venösen und arteriellen Endothelzellen gezeigt werden. Die Untersuchung der Expression von FOXC1 legt nahe, dass es sich bei diesem Transkriptionsfaktor ebenfalls um ein in Endothelzellen arteriovenös differentiell exprimiertes Gen handelt. Die differentielle Exprimierung von HEY2 in Endothelzellen konnte auf transkriptioneller Ebene zusätzlich durch ein HEY2 Promotor Funktionsassay gezeigt werden. Die Überexpression der NOTCH1 intrazellulären Domäne führte zur Induktion der endogenen Expression der NOTCH-Zielgene HEY1 und HEY2 in HEK 293T Zellen. In dem Zelltyp durchgeführte Reportergenassays zeigten ebenfalls eine deutliche Aktivierung des HEY2 Promotors durch die Überexpression der NOTCH1 intrazellulären Domäne. Durch eine Deletionsanalyse konnte der Bereich, der für die Aktivierung verantwortlichen DNA-Sequenz-Motive stark eingegrenzt werden. Weiterhin konnte die Induktion des HEY2 Promotors durch VEGF und seine Repression durch einen γ-Sekretase Inhibitor nachgewiesen werden. Die Überexpression der NOTCH1 intrazellulären Domäne führte zur Verringerung der mRNA- und Protein-Expression von COUP-TFII in HEK 293T Zellen. Dieses Ergebnis konnte zusätzlich durch ein COUP-TFII Promotor Aktivitätsassay nach Überexpression des NOTCH-Zielgens HEY2 gezeigt werden. Die Deletionsanalyse des COUP-TFII Promotors lässt eine direkte Inhibition von COUP-TFII durch HEY2 vermuten. Die Überexpression von COUP-TFII führte zu einer starken Induktion der COUP-TFII mRNA- und Protein-Expression, jedoch weder in HEK 293T Zellen noch in Endothelzellen zu einer Änderung der HEY2 Promotoraktivität. Die Überexpression von FOXC1 und FOXC2 bewirkte eine Inhibition der HEY2 Promotoraktivität in HEK 293T Zellen. Die in der Arbeit gezeigte hohe Expression von FOXC1 in venösen Endothelzellen könnte somit in Kombination mit COUP-TFII für die komplette Repression der Aktivität des HEY2 Promotors in venösen Endothelzellen verantwortlich sein. Die durchgeführte Deletionsanalyse des HEY2 Promotors legt eine direkte Bindung von FOXC1 und FOXC2 an den HEY2 Promotor nahe. Die erzielten Ergebnisse dieser Arbeit sprechen im Kontext mit der Literatur für eine zelltypspezifische Regulierung/Aktivierung des NOTCH-Signalweges und lassen folgendes Modell zur Differenzierung des venösen oder arteriellen endothelialen Phänotyps vermuten: Die Determinierung des Phänotyps wird entschieden durch das Gleichgewicht der Expression der Interaktionspartner des NOTCH-Signalweges. Der VEGF Co-Rezeptor NRP1 und der VEGFR2 sind die entscheidenden Aktivatoren des NOTCH-Signalweges. Die Balance der Bindung des Repressors COUP-TFII an den NRP1 und VEGFR2 Promotor sowie des Aktivatorkomplexes NICD/RBP-JК an den NRP1 Promotor sind damit entscheidend für die Aktivität des NOTCH-Signalweges. NRP1 bindet VEGF und steigert gleichzeitig dessen Bindung an den VEGFR2. Dies führt zur Induktion von DLL4. Die Bindung von DLL4 an die NOTCH1/4 Rezeptoren führt zur Abspaltung der NOTCH intrazellulären Domäne (NICD) des Rezeptors. Die NICD wandert in den Zellkern und aktiviert dort zusammen im Komplex mit dem Transkriptionsfaktor RBP-JК die Gene HEY1, HEY2 und NRP1. Die Transkriptionsfaktoren HEY1 und HEY2 reprimieren über einen Feedback-Mechanismus direkt die Aktivität des COUP-TFII Promotors.