Dissertations / Theses on the topic 'Endotel'

UVOD: Gojaznost je hronično, multifaktorijalno i kompleksno oboljenje povezano sa povećanim rizikom od aterosklerotskih kardiovaskularnih bolesti (KVB). Disfunkcija vaskularnog endotela predstavlja rani događaj u patofiziološkom 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 – intracelularnog adhezivnog molekula –1 (sICAM–1) i E selektina (sE–selektin), između ispitanika sa hiperalimentacionim tipom gojaznosti i normalno uhranjenih zdravih ispitanika, kao i utvrditi eventualno postojanje razlika u koncentraciji sICAM–1 i sE–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), cirkulišućih koncentracija biomarkera disfunkcije vaskularnog endotela (sICAM–1 i sE–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 – 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–1 i sE–selektina je vršeno ELISA tehnikom (R&D Systems, Inc. Minneapolis, USA). Vrednosti IMK–a su određivane pomoću karotidnog dupleks ultrazvuka (Aloka SSD–650 US system, Tokyo), a na osnovu izmerenih (IMK) i normalno očekivanih vrednosti IMK za svakog ispitanika je izračunavan IMK Z–skor. Supklinički stadijum ateroskleroze je definisan kao vrednost IMK Z–skora veća od 1 (što 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 više vrednosti medijane serumske koncentracije sE–selektina u poređenju sa medijanom serumske koncetracije sE–selektina učesnika u kontrolnoj grupi (36,2 (33,21–43.7) vs. 25,14 (23,1–29,48) ng/mL, P=0,00). Gojazni ispitanici III stepena gojaznosti su imali statistički značajno višu medijanu serumske koncenracije sE–selektina u odnosu na medijanu sE–selektina u ispitanika I stepena gojaznosti (41,5 (36,58–49,48) vs. 34,34 (22,49–36,62) ng/mL, P=0,00), odnosno medijanu sE–selektina u ispitanika II stepena gojaznosti (41,5 (36,58–49,48) vs. 32,1 (26,1–43,64) ng/mL, P=0,00). Nije uočena statistički značajna razlika u medijani serumske koncentracije sE–selektina između ispitanika I i II stepena gojaznosti (34,34 (22,49–36,62) vs. 32,1 (26,1–43,64) ng/mL, P=0,12). Gojazni ispitanici su imali statistički značajno više vrednosti medijane serumske koncentracije sICAM–1 u poređenju sa medijanom serumske koncetracije sICAM–1 učesnika u kontrolnoj grupi (266,8 (245,8–326,73) vs.183,32 (167,9–208,57), P=0,00). U ispitivanoj grupi gojaznih uočena je statistički značajna razlika u medijani koncentracije sICAM–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–skor > 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–skor > 1 su imali statistički značajno višu medijanu koncentracije sICAM–1 u odnosu na ispitanike kod kojih je IMK Z–skor ≤ 1 (295,4 (238,46–340,38) vs. 244,2 (227,35–260,38), P=0.00). Regresionom analizom (R2=0,71, korigovani R2=0,59) je utvrđeno da su parametri hsCRP (β=0,45, P=0,00), HOMA–IR (β=0,44, P=0,035) i ISI (β=–0,36, P=0,028) nezavisno i statistički značajno povezani sa serumskom koncentracijom sE–selektina. Regresionom analizom (R2=0,65, korigovani R2=0,56) je utvrđeno da parametri ITM (β=0,55, P=0,00), trigliceridi (β=0,30, P=0,00), HDL holesterol (β=–0,31, P=0,00), odnos TG/HDL–holesterol (β=0,33, P=0,01), hsCRP (β=0,31, P=0,00) i fibrinogen (β=0,34, P=0,00) su nezavisno i statistički značajno povezani sa serumskom koncentracijom sICAM–1. U faktorskoj analizi je izdvojeno pet faktora “gojaznost”, “insulinska rezistencija”, “aterogeni faktor”, “endotelna disfunkcija i vaskularna inflamacija” i “metabolički faktor” koji objašnjavaju 69.72% ukupne varijanse ispitivanog uzorka. U multivarijabilnom modelu sa svim faktorima zajedno kojim je objašnjeno ukupno 75% varijanse, jedino je faktor gojaznost imao statički značajan i nezavistan uticaj na vrednost IMK Z–skor > 1 (OR=2,74 (CI 1,18–6,33), P=0,019). U faktoru gojaznost su se izdvojili parametri: FAT trunk (%), FAT (%), OS (cm), ITM (kg/m2), LDL – holesterol (mmol/L), SP (mmHg), HOMA1–%B, fibrinogen (g/L), ApoB/apoA-I i hsCRP (mg/L). Univarijantom logističkom regresijom je uočeno da porast u koncentraciji LDL–H (OR=5,33 (CI 1,9–14,2), P=0,02) i koncentraciji hsCRP–a (OR=2,53 (CI 1,3–3,98),P=0,017) povećava rizik za postojanje vrednosti IMK Z–skor > 1. ZAKLJUČAK: Cirkulišuće serumske koncentracije biomarkera disfunkcije vaskularnog endotela, sE–selektina i sICAM–1, su značajno više kod ispitanika sa hiperalimentacionim tipom gojaznosti u odnosu na njihove koncentracije u normalno uhranjenih ispitanika. U gojaznih ispitanika, koncentracija sE–selektina je povezana sa vrednostima indeksa insulinske rezistencije i biomarkera inflamacije, dok je koncentracija sICAM–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 – 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.
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 & D Systems, Inc., Minneapolis, USA). The values of IMK were determined by carotid duplex ultrasound (Aloka – 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> 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> 1 had significantly higher median concentrations of sICAM-1 compared to those in which the IMK Z-score ≤ 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 (β=0.45, P=0.00), HOMA-IR (β=0.44, P=0.035) and ISI (β=-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 (β=0.55, P=0.00), triglycerides (β=0.30, P=0.00), HDL cholesterol (β=-0.31, P=0.00), the ratio of TG/HDL-cholesterol ratio (β=0.33, P=0.01), hsCRP (β=0.31, P=0.00 ) and fibrinogen (β=0.34, P=0.00) were independently and significantly associated with serum sICAM-1 concentration. In the Factor analysis, five factors "obesity", "insulin resistance", "atherogenic factor," "endothelial dysfunction and vascular inflammation" and "metabolic factor" 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), "obesity" factor was significantly and independently associated with IMT Z-score> 1 (OR=2.74 (CI 1.18-6.33), P=0.019). The "obesity" factor consisted of parameters: trunk fat (%), fat (%), waist (cm), BMI (kg/m2), LDL – 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> 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.

Uvod: Sepsa je jedan od vodećih uzroka smrtnosti u jedinicama intenzivnog lečenja i van njih uprkos implementaciji novih dijagnostičkih i terapijskih protokola širom sveta. Multiorganska disfunkcija (MODS), koja predstavlja najtežu formu nepovoljnog toka sepse, je u osnovi svojih patofizioloških dešavanja obeležena promenama, koje se dešavaju na nivou kapilara, pre svega u endotelu. Poremećaji koagulacije koji se javljaju kao posledica ovih promena u endotelu su prepoznati kao jedan od dijagnostičkih kriterijuma prema najnovijim smernicama za dijagnostiku i lečenje sepse, međutim njihov značaj u predviđanju toka i ishoda ovog oboljenja još uvek nije precizno definisan. Cilj istraživanja: Odrediti koncentraciju markera endotelne aktivacije, aktivacije koagulacije, aktivnost prirodnih inhibitora koagulacije i funkcionalnost fibrinolize kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za postavljanje dijagnoze sepse i predikciju pojave komplikacija. Ispitati mogućnost upotrebe markera endotelne disfunkcije i pokazatelja poremećaja mehanizma hemostaze za procenu ishoda kod obolelih od sepse. Materijal i metode: Istraživanje je sprovedeno analitičkom metodom u formi studije preseka, a obuhvatilo je pacijente lečene na Odeljenju anestezije i reanimacije Urgentnog centra Kliničkog centra Vojvodine i na Klinici za infektivne bolesti Kliničkog centra Vojvodine, u Novom Sadu. Istraživanje je sprovođeno tokom 2012. i 2013. godine u trajanju od dve godine. U studiju je bilo uključeno 180 ispitanika od kojih je 150 imalo postavljenu dijagnozu sepse,a 30 ispitanika su činili kontrolnu grupu su klinički i biohemijski zdravih ispitanika, dobrovoljni davaoci krvi. Ispitanici su kategorisani u četiri grupe u odnosu na kliničko stanje i laboratorijske nalaze unutar prvih 24 časa od prijema: bolesnici sa sepsom, teškom sepsom, septičkim šokom i multiorganskom disfunkcijom na prijemu. Nakon kategorizacije ispitanika, izračunati su APACHE II i SOFA numerički pokazatelji procene težine bolesti ispitanika. U roku 24 časa od trenutka postavljanja dijagnoze sepse, iz uzoraka krvi ispitanika, izvršene su predviđene laboratorijske analize u cilju praćenja endotelne aktivacije, aktivacije koagulacije i inhibicije antikoagulantnih mehanizama. U toku 48 časova od prijema, bolesnici koji nisu imali MODS na prijemu su intenzivno praćeni u cilju evidentiranja razvoja multiorganske disfunkcije, dok su bolesnici koji su imali MODS praćeni radi evidentiranja perzistiranja ili eventualne rezolucije MODS-a. Zdravstveno stanje bolesnika je praćeno tokom 28 dana od trenutka uključivanja u studiju i nakon tog perioda je evidentiran ishod lečenja u smislu preživljavanja ili smrtnog ishoda. Statistička analiza je izvršena pomoću statističkog paketa IBM SPSS 20 Statistics. Podaci su predstavljeni tabelarno i grafički, a statistička značajnost određivana je na nivou p< 0,05. Rezultati: Vrednosti bioloških markera endotelne aktivacije i aktivacije koagulacije su statistički značajno povišene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji, dok su vrednosti prirodnih inhibitora koagulacije statistički značajno snižene kod obolelih od sepse u odnosu na njihove vrednosti u zdravoj populaciji. Vrednosti APTT-a, PT-a, D-dimera, fibrinogena, prirodnih inhibitora koagulacije i markera endotelne aktivacije (endokan i vWF antigena i aktivnosti) imaju značajan i veoma visok dijagnostički potencijal. Vrednosti biomarkera endotelne disfunkcije i pokazatelja poremećaja hemostaznog mehanizma su značajni prediktori komplikacija kod bolesnika sa sepsom. APTT, PT, D-dimer, broj trombocita, vrednosti priorodnih inhibitora koagulacije, trombomodulina, endokana i ETP-a su jednako validni u inicijalnoj proceni toka kliničke slike sepse kao i prediktivni APACHE II i SOFA skorovi. Koncentracija trombomodulina, D-dimera, ETP-a i PC su dobri prediktori nastanka MODS-a u prvih 48 časova u toku sepse. Endokan, PT, APTT, koncentracija fibrinogena, prirodnih inhibitora koagulacije i vrednosti ETP-a su značajni u predikciji mortaliteta kod bolesnika sa sepsom. Zaključci: Ukoliko bi pokazatelji aktivacije endotela i mehanizma hemostaze bili inkorporirani u određeni sistem skorovanja u cilju procene težine bolesti u smislu ishoda kod bolesnika sa sepsom, to bi moglo doneti doprinos boljoj klasifikaciji bolesnika, te primeni pravovremene i adekvatne terapije u cilju postizanja pozitivnog ishoda kod bolesnika sa sepsom. Prilikom interpretacije pokazatelja inflamacije i koagulacije neophodno je steći uvid u celokupnu sliku pro-i antikoagulantnih dešavanja koja se odvijaju tokom sepse, odnosno adekvatno proceniti pravac toka disbalansa mehanizma hemostaze da bi se eventualnim terapijskim merama mogao postići pozitivan učinak.
Introduction: Sepsis is one of the main causes of death in intensive care units and other hospital wards in spite of implementation of new sepsis treatment guidelines in everyday hospital practice worldwide. Changes that occur in the microvasculature, affecting primarily endothelial cell, are the basis of the pathophysiology of multiorgan dysfunction (MODS) in sepsis. Coagulation abnormalities which occur as a consequence of endothelial changes are recognized as diagnostic criteria for sepsis, but significance of these changes in the outcome prognosis and prediction of the course of sepsis is still not accurately defined. Aims: Evaluation of hemostasis related parameters and endothelial activation biomarkers values in patients with sepsis and healthy volunteers. Determination whether the levels of hemostasis-related parameters and biomarkers of endothelial activation have diagnostic significance and are they associated with MODS development and persistence in the first 48 hours of hospitalization and 28-day mortality in patients with sepsis. Material and methods: This is cross-sectional study conducted in 2012 and 2013 in the Department of Anesthesia and Reanimation at the Emergency Center of the Clinical Center of Vojvodina and in the Clinic of Infectious Disease at the Clinical Center of Vojvodina. 150 patients who fulfilled criteria for diagnosis of sepsis were included in the study. Patients were divided into 4 groups: sepsis, severe sepsis, septic shock and MODS. 30 healthy volunteers, blood donors were the control group. After the categorization of patients, during the first 24 hours of hospitalization, predictive APACHE II and SOFA scores were calculated. Hemostasis related parameters and endothelial activation biomarkers concentrations were determined within the first 24 hours of the onset of the disease. To assess the development of complication of the disease, patients were monitored for 48 hours for MODS development and persistence or resolution and for 28 days from the onset of sepsis for outcome assessment. Data were analyzed using SPSS 20.0 software and are presented in tables and graphs, statistical significance was set at p< 0,05. Results: Biomarkers of endothelial and coagulation activation are significantly higher in patients with sepsis in comparison to their values in healthy volunteers, while concentrations of natural anticoagulants are significantly lower in patients with sepsis than in healthy volunteers. APTT, PT, D-dimer, fibrinogen, natural anticoagulants and biomarkers od endothelial activation (endocan and vWF antigen and activity) have diagnostic significance in patients with sepsis. Hemostasis related parameters and endothelial activation biomarkers are good prognostic factors for complication development in patients with sepsis. APTT, PT, D-dimer, platelet count, natural anticoagulants, thrombomodulin, endocan and ETP are equally valuable in early prediction of sepsis development as APACHE II and SOFA scores. Thrombomodulin, D-dimer, ETP and PC are good predictors of MODS development during the first 48 hours from sepsis onset. Endocan, PT, APTT, fibrinogen concentration, values of natural anticoagulants and ETP values are significant in 28-day mortality prediction in patients with sepsis. Conclusion: A combination of markers of endothelial dysfunction with widely used ICU scores and organ failure assessment could contribute to an early recognition of complication development and consequent death in patients with sepsis. It is necessary to obtain the full insight in pro-and anticoagulant dynamic evaluation while interpreting coagulation and inflammation processes in sepsis development, in order to accurately lead early resuscitation therapy.

Die Aktivierung von Endothelzellen durch Bakterien und ihre Produkte trägt wesentlich zur Ausbildung klinischer Symptome in bakteriellen Infektionen bei. Die Freisetzung von Chemo- und Zytokinen führt im Konzert mit der Expression von Adhäsionsmolekülen durch das Endothel zur Rekrutierung und Aktivierung von Granulozyten. Zur Regulation der Entzündungsreaktion tragen parakrine und systemische Effekte, ausgelöst durch die Liberation von vasoaktiven Substanzen und Zytokinen durch Endothelzellen, bei. Der Zusammenbruch der endothelialen Barrierefunktion, gekennzeichnet durch den Verlust der Permselektivität der endothelialen Grenzschicht, verursacht Ödembildung. In dieser Arbeit wurde die molekulare Interaktion von Bakterien und ihren Produkten mit Endothelzellen untersucht. Effekte auf die Rekrutierung von Granulozyten und die endotheliale Barrierefunktion wurden charakterisiert. Dabei konnten aktivierte Signalwege identifiziert werden. Darauf basierend folgte die Entwicklung erster therapeutischer Ansätze. Zusammengefasst erbrachten diese experimentellen Untersuchungen neue Erkenntnisse zum Verständnis der Bakterien-Endothel Interaktion.
Activation of endothelial cells by bacteria and their products contributed significantly to clinical signs of bacterial infections. Liberation of chemo- and cytokines in concert with expression of adhesion molecules by the endothelium resulted in recruitment of granulocytes. Paracrine and systemic effects of vasoactive agents and cytokines secreted by endothelial cells contributed the regulation of inflammation. Loss of endothelial barrier function induced edema formation. This postdoctoral lecture qualification addressed the molecular interaction of bacteria and their products with endothelial cells. The recruitment of granulocytes, the regulation of endothelial barrier function and activated signalling pathways in endothelial cells were analyzed. Based on these experiments new therapeutic strategies have been tested. In summary, extended these experimental investigations the understanding of bacterial-endothelial interaction.

UVOD: Procesi koji obuhvataju endotelnu disfunkciju, oksidativni stres, hroničnu inflamaciju, hiperaktivnost i aktivaciju trombocita te narušavanje ravnoteže procesa koagulacije i fibrinolize od najranijih faza razvoja dijabetes melitusa tip 2 (T2DM) promovišu 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 što može biti uslovljeno rezistencijom na primenjenu antitrombocitnu terapiju. Praćenje efekata antitrombocitne terapije i blagovremeno identifikovanje rezistentnih bolesnika ima za cilj optimizaciju primenjene antitrombocitne terapije što 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 bolešću 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 bolešću arterijskih krvnih sudova u odnosu na efikasnost ove terapije u nedijabetičnoj populaciji bolesnika sa bolešću 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, nepušača 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–selektina i sP-selektina je vršeno ELISA tehnikom (R&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 što 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 viši u bolesnika sa T2DM u odnosu na bolesnike bez dijabetesa (45,1±18,1vs.31,8±10,5ng/ml; p<0,001) i kontrolnu grupu zdravih ispitanika (45,1±18,1vs.27,2±11,2ng/ml; p<0,001). Plazmatski nivo vWF Ag, bio je statistički značajno viši u bolesnika sa T2DM u odnosu na grupu ispitanika bez dijabetesa (172±75,2vs. 146±40,6%; p=0,045), kao i u odnosu na kontrolnu grupu zdravih (172±75,2vs.130±33,8%; p=0,007). Nivo sPselektina bio je statistički značajno viši kod bolesnika s T2DM u odnosu na ispitanike u grupi dijabetesa (95,2±31,8vs.84,0±21,8 ng/ml; p=0,042) i kontrolnoj grupi (95,2±31,8vs.76,7±16,2ng/ml; p=0,004). Uočeno je da je %rP statistički bio značajno viši u grupi dijabetičara u odnosu na grupu ispitanika bez dijabetesa (3,47±1,30vs.2,30±1,30%; p<0,001) i kontrolnu grupu zdravih (3,47±1,30vs.2,29±1,23%; p<0,001). Bolesnici sa T2DM imali su statistički značajno više vrednosti ADP testa (70,3±22,0vs.56,9±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±12,4vs. 48,6±12,6 %; p<0,001). U grupi ispitanika sa T2DM vrednost TRAP testa statistički značajno pozitivno koreliše 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<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 koreliše 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 – selektina (r=0,560;p <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 ±19,91 vs. 94,54±16,67 vs. 112,00±10,22 vs. 128,92±15,69U;p<0,001), dok se %SATadp od prvog do četvrtog kvartila značajno smanjivao (40,44±13,33 vs. 31,20±11,82 vs. 33,16±7,03 vs. 21,53±10,16%). ZAKLJUČAK: Koncentracije cirkulišućih biomarkera endotelne aktivacije, sE – selektina i vWF Ag-a, solubilnog biomarkera trombocitne aktivacije, sP – selektina, kao i procenat retikulisanih trombocita, %rP, markera trombocitnog prometa, značajno su povišene kod bolesnika sa bolešću 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 viši 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 što je uslovilo i trend učestalijeg ponavljanja ishemijskih ataka u odnosu na bolesnike bez dijabetesa. Međusobna povezanost ispitivanih biomarkera endotelne i trombocitne aktivacije (sE – selektina, vWF Aga, sP – 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 lošem odgovoru na antitrombocitnu terapiju klopidogrelom.
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&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±18,1vs.31,8±10,5ng/ml;p<0,001) and healthy control group (45,1±18,1vs.27,2±11,2ng/ml; p<0,001). vWF Ag was significantly higher in diabetic patients than in non-diabetics (172±75,2vs. 146±40,6%; p=0,045) and healthy controls (172±75,2vs.130±33,8%; p=0,007). sP-selectin was also significantly higher in patients with T2DM than in non-diabetics (95,2±31,8vs.84,0±21,8 ng/ml; p=0,042) and healthy controls (95,2±31,8vs.76,7±16,2ng/ml; p=0,004). %rP was significantly higher in group of patients with T2DM than in nondiabetic patients (3,47±1,30vs.2,30±1,30%; p<0,001) and healthy control group (3,47±1,30vs.2,29±1,23%; p<0,001). T2DM patients had statistically higher values of ADP test (70,3±22,0vs.56,9±19,7U; p=0,002) compared to patients without diabetes, and significantly lower %DPAadp (31,6±12,4vs. 48,6±12,6 %; p<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<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 – selectin (r=0,560;p <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 ±19,91 vs. 94,54±16,67 vs. 112,00±10,22 vs. 128,92±15,69U;p<0,001) while %DPAadp decreased (40,44±13,33 vs. 31,20±11,82 vs. 33,16±7,03 vs. 21,53±10,16%). CONCLUSION: Concentration of circulating biomarkers of endothelial activation, sE-selectin and vWF Ag, soluble marker of platelet activation, sP – 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 – selectin, vWF Ag, sP – 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.

Die durch Lipopolysaccharid (LPS) induzierte frühe Immunantwort ist ein wesentlicher Mechanismus der Infektabwehr durch die angeborene Immunität. Bei starker LPS-Exposition kann es andererseits zur Ausbildung eines septischen Syndroms kommen. Der endothelialen Sekretion von Interleukin-8 (IL-8/CXCL8), das als Chemokin die Migration neutrophiler Granulozyten vermittelt, kommt dabei herausragende Bedeutung zu. Zielsetzung dieser Arbeit war es, die Relevanz der Rho-Proteine RhoA, Rac1 und Cdc42 für die LPS-induzierte intrazelluläre Signaltransduktion mittels Überexpression inaktiver Mutanten dieser Proteine zu untersuchen. Diese Untersuchung wurde erschwert durch die schlechte Transfizierbarkeit der Endothelzelllinie HPMEC-ST1.6R, die nahezu alle Charakteristika primärer Endothelzellen aufweist. Deshalb wurde eine Methode etabliert, die durch Kotransfektion des Grünen Fluoreszenzproteins (GFP) die flusszytometrische Selektion der transfizierten Zellen anhand ihrer GFP-bedingten Fluoreszenz und die Messung der Expression von CXCL8 allein in dieser Population ermöglicht. Damit wurde nachgewiesen, dass die inaktiven Mutanten RhoAN19, Rac1N17 und Cdc42N17 jeweils die LPS-induzierte Expression von CXCL8 vermindern. Die größte Reduktion der CXCL8-Expression um 38 % der Positivkontrolle zeigte sich nach Transfektion der Mutante Rac1N17. Die Zelllinie CHO-3E10 exprimiert einen artifiziellen Reporter unter der Kontrolle eines Fragments aus der Verstärkerregion des Gens für das Endotheliale Leukozyten-Adhäsionsmolekül ELAM-1 (CD62E). Die Transfektion jeder einzelnen der inaktiven Varianten der drei GTP-bindenden Proteine in Zellen der Linie CHO-3E10 reduzierte die Expression des Reporterproteins nach Stimulation mit LPS signifikant. Die stärkste Reduktion der Reporterexpression um 51 % der Positivkontrolle ergab sich unter Rac1N17. Zusammengefasst zeigt die Studie, dass die Überexpression der inaktiven Mutanten RhoAN19, Rac1N17 und Cdc42N17 zu einer Abnahme der endothelialen Expression von CXCL8 führt. Darüberhinaus ergab sich im Vergleich zu den Mutanten RhoAN19 und Cdc42N17 die stärkste Reduktion der CXCL8-Expression in Endothelzellen nach Transfektion der Mutante Rac1N17.
The early immune response induced by Lipopolysaccaride (LPS) is a crucial mechanism in fighting off infections by the innate immunity. On the other side high amounts of LPS can lead to the development of a sepsis. In this process the endothelial secretion of interleukin-8 (IL-8/CXCL8), which causes the migration of neutrophilic granulocytes to the site of infection is highly important. The aim of this study was to analyze the relevance of each of the three Rho-proteins RhoA, Rac1 and Cdc42 for the intracellular signal transduction resulting in CXCL8-expression by means of overexpressing inactive mutants of these proteins. Cells of the human microvascular endothelial cell line HPMEC-ST1.6R show most characteristics of primary endothelial cells and are extremely difficult to transfect. Therefore a method was established, which allowed sorting of successfully transfected cells by cotransfecting a gene encoding for green fluorescence protein (GFP). This method permitted measuring intracellular expression of CXCL8 in the population successfully transfected with plasmids encoding for RhoAN19, Rac1N17 or Cdc42N17 mutants. This experiments demonstrated that the inactive mutants RhoAN19 Rac1N17 or Cdc42N17 each decreased the LPS-induced expression of CXCL8. Quantitative comparision showed the greatest reduction of 38 % in CXCL8-expression due to transfection of the Rac1N17 mutant. The LPS-inducible reporter cell line CHO-3E10 used in this study expresses the human CD25-antigene as an artificial reporter protein under the control of a fragment from the enhancer region of the gene for the human endothelial leukocytic adhesionmolecule ELAM-1 (CD62E). Transfecting each of the inactive mutants RhoAN19, Rac1N17 or Cdc42N17 in CHO-3E10 cells significantly reduced the LPS-induced expression of the reporter protein. The greatest reduction in reporter expression of 51 % resulted from transfection with the Rac1N17 mutant. In conclusion, this study demonstrates that overexpression of nonfunctional GTP-binding proteins RhoAN19, Rac1N17 or Cdc42N17 leads to a decrease in endothelial CXCL8-expression. Moreover, CXCL8-expression in endothelial cells transfected with the Rac1N17 mutant was most efficiently reduced when compared to the other mutants.

Ischemia/reperfusion (IR) injury is a pathological condition caused by an initial interruption of blood, its biomechanical stimulus, and O2 supply followed by the restoration of perfusion and the accompanying oxygen, nutrient supply and shear stress. Clinically, ischemia/reperfusion injury is almost unavoidable in liver resection surgery, transplantation, and in blood transfusion after hemorrhagic shock. This pathology, associated with microvascular dysfunction and the “no reflow phenomenon” causes a wide range of derangements in the liver, which may finally lead to hepatic failure in case of severe injury. Sinusoidal dysfunction is, in part, due to the reduction in vasodilator molecules, such as nitric oxide. The decrease in NO bioavailability is due to both, a reduction in its production by eNOS in endothelial cells and an increase of its - scavenging by ROS like O2 . Thus, therapies focused on increasing eNOS expression or reducing ROS production may be useful in preventing microcirculatory derangements associated with IR injury. Despite the importance of the liver sinusoid in health and disease scarce reports investigated the pathophysiological consequences of sinusoidal cell deregulation on hepatic IR injury. This has conduced to the erroneous idea that hepatocytes should be the main target for protection and therefore most of the therapies have focused on this cell type. Although IR injury has a clear negative impact in clinical practice, nowadays no pharmacological treatment is available, possibly due to the lack of microcirculatory protection of the tested drugs. Thus, we hypothesize that therapies focused in preserving the sinusoidal endothelial phenotype during IR will conduce not only to a better microcirculation but also to a global improvement of the whole liver. With this background, the global aim of the present PhD thesis was to characterize the liver microcirculation in the setting of ischemia-reperfusion injury and evaluate possible drugs that, through an improvement in the hepatic sinusoid, could maintain a correct hepatic phenotype during IR. The results derived from this doctoral thesis demonstrate the deleterious effects of ischemia/reperfusion injury on hepatic microcirculation both in cold and warm ischemia situations. In particular, the in vivo effects of warm IR include acute microcirculatory injury associated with an increase in intrahepatic vascular resistance, portal hypertension and reduced hepatic perfusion. To prevent this damage affecting all cell types in the liver sinusoid two therapeutic strategies have been evaluated: a recombinant form of the antioxidant human manganese superoxide dismutase (rMnSOD) in the context of cold storage for transplantation and the vasoprotective drug simvastatin in a model of partial warm ischemia. The papers included in this doctoral thesis demonstrate how both drugs are effective in improving liver endothelial function. Those effects are due, in part, to the maintenance of KLF2 expression and the vasoprotective pathways derived of this transcription factor; the maintenance of nitric oxide bioavailability and the reduction in superoxide levels. The endothelial and microcirculatory hepatic protection achieved by those drugs further conduces to a prevention of liver inflammation mediated by adhesion molecules, and thus to a reduction in hepatic parenchymal injury and a global decrease in cell death. In conclusion, all the findings here described support the idea that preservation of the hepatic sinusoidal phenotype and function in essential to prevent ischemia/reperfusion injury and therefore to maintain liver function.
En la present tesi doctoral es demostren els efectes deleteris del dany per isquèmia-reperfusió sobre la microcirculació hepàtica tant en condicions de preservació en fred com d’isquèmia calenta. En particular, els efectes in vivo de l’isquèmia-reperfusió en calent inclouen dany microcirculatori agut associat amb un increment de la resistència vascular intrahepàtica, hipertensió portal i reducció de la perfusió hepàtica. Per prevenir aquests danys que afecten als diferents tipus cel·lulars del sinusoid hepàtic s’han avaluat dues estratègies terapèutiques: l’antioxidant rMnSOD en el context de la preservació en fred per transplantament i el vasoprotector simvastatina en un model d’isquèmia calenta. Els articles inclosos en la present tesi doctoral demostren com ambdós fàrmacs són efectius millorant la funció endotelial hepàtica. Aquests efectes es deuen, en part, al manteniment de les vies vasoprotectores derivades del factor de transcripció KLF2 , al manteniment de la biodisponibilitat d’òxid nítric i a la reducció dels nivells del radical lliure superòxid. Aquesta protecció de l’endoteli i la microcirculació hepàtica s’associa amb una prevenció de l’inflamació mitjançada per mol·lècules d’adhesió i finalment condueix a una reducció del dany hepàtic i una menor mort cel·lular.

La patologia associada a l’hemostàsia primària ha estat clàssicament relacionada amb diàtesi hemorràgica (malaltia de von Willebrand, Síndrome de Bernard- Soulier, malaltia de Glanzmann, trombopènia, etc) on s’ha valorat el paper de les plaquetes i del factor von Willebrand en una defectuosa agregació i/o adhesió plaquetària. També ha estat àmpliament estudiat el paper de les plaquetes en la trombosi arterial. Ja que el paper de les plaquetes és fonamental en l’inici i la formació del coàgul és possible que tinguin un paper important en la patogènia de la trombosi venosa. Treballs experimentals en ratolins demostren que les plaquetes i el FvW juguen un paper molt important en l’inici i el desenvolupament de la trombosi venosa [7]. En aquesta tesi es vol investigar sobre el paper d’aquests paràmetres en patologia trombòtica venosa humana. Recentment s’ha introduït el concepte d’hiperagregabilitat plaquetària on trobem individus amb plaquetes que agreguen a dosis molt petites d’inductor (ADP o/i Epinefrina). La agregabilitat a dosis baixes d’epinefrina (EPI) i/o d’ADP en la població normal dona lloc a dues poblacions diferenciades: individus amb hiperreactivitat plaquetària (agregació >60%) i individus amb hiporreactivitat plaquetària (agregació <40%) [18,19]. La reactivitat plaquetària (hipo i hiperreactivitat plaquetària) es pot mesurar a) mitjançant l’agregació plaquetària activada per agonistes en plasma ric en plaquetes (PRP) com s’ha indicat anteriorment o b) mitjançant test que usen sang total. Aquests test en sang total poden ser o bé agregacions en sang total (usem l’analitzador Multiplate®) o bé mitjançant test basats en l’adhesió plaquetària (usem l’analitzador PFA-100®). El PFA-100® és un analitzador que mesura el funcionalisme plaquetari. Es mesura en segons (temps d’oclusió) i aquest temps d’oclusió és inversament proporcional a la capacitat funcional de les plaquetes. El nostre grup ha descrit que temps d’oclusió curts de l’analitzador PFA-100® s’associen a risc trombòtic venós [20] suggerint que els temps d’oclusió mesurats en el PFA-100® podrien ser un marcador d’adhesió plaquetària. La hiperreactivitat plaquetària s’ha trobat associada a trombosis arterial i venosa i està relacionada amb els nivells de Serotonina, del seu transportador (SERT) i de VAMP 8 [19,21]. Aquesta hiperreactivitat sembla ser un fenomen global, on individus amb un increment d’agregació plaquetar en resposta a l’ EPI tenen probablement també una resposta elevada a altres agonistes plaquetaris. S’ha descrit que una coestimulació de serotonina i una concentració submàxima d’ EPI augmenta l’agregació en una proporció més alta que quan l’indueixes només amb EPI mantenint-se en la població la resposta bimodal [19]. El nombre d’hiperreactius quan s’utilitzen els dos inductors augmenta de manera significativa. Quan uses serotonina com a agonista sol, els individus tenen una resposta modesta, recolzant el fenomen que les plaquetes son reactives a la serotonina nomes si són activades per altres agonistes com exemple EPI. El mateix succeeix quan afegeixes serotonina a l’ADP, s’augmenta també l’hiperrectivitat plaquetària i s’ha observat que es produeix un efecte general pro coagulant [4]. Altres estudis defineixen també la hiperreactivitat plaquetària com un fenomen global [22]. Descriuen que individus sans amb hiperreactivitat plaquetària a l’ EPI tenen una funció plaquetària augmentada tant a nivell d’adhesió (s’incrementa l’aglutinació en resposta a baixes dosis de ristocetina) com d’activació (s’incrementa l’expressió de la P-selectina després de l’alliberament granular) i d’agregació (s’incrementa l’agregació en resposta a múltiples estímuls diferents). En individus hiperreactius s’ha observat nivells més elevats de SERT i un augment en l’afinitat d’unió de serotonina-SERT [19]. També en individus amb hiperreactivitat plaquetària els nivells de VAMP 8 estan augmentats [21]. Els polimorfismes rs1010 de VAMP8 i el 5-HTTLPR de SLC6A4 del transportador de la serotonina estan relacionats amb els nivells d’aquestes proteïnes, amb la hiperreactivitat plaquetària i amb el risc d’infart de miocardi [23-25]. A més a més diversos estudis han demostrat que la disminució d’ADAMTS13 i l’augment del factor von Willebrand són factors de risc de trombosi arterial [26- 30], però hi ha poca evidència d’aquesta relació i la trombosi venosa mostrant altrament resultats controvertits [31]. Es valora també la relació entre el grup sanguini ABO i els nivells d’aquestes proteïnes. Per altra banda, ja que el nostre grup ha descrit que temps d’oclusió curts de l’analitzador PFA-100® s’associen a risc trombòtic venós [20] es valora també la relació entre el grup sanguini ABO i aquest fenotip. Per tots aquests motius esmentats anteriorment creiem que es plausible plantejar la hipòtesi de la implicació de l’hemostàsia primària en l'etiopatogènia de la trombosi venosa.
The role of platelet in bleeding has been studied widely but little is known about their role in venous thrombosis (VT). Recently, platelet hyperreactivity has been described as associated with thrombotic risk. Platelet hyperreactivity is measured by aggregation in platelet rich plasma (platelet hyper-aggregability) or through test with whole blood as PFA-100® system. Recently, our group found that platelet function (measured by PFA-100® system) was associated with VT. The objective of my research is to evaluate the role of platelets in VT and the role of plasmatic biomolecules in primary hemostasis as human vesicle associated membrane protein 8 (VAMP8), serotonin, serotonin transporter (SERT), a disintegrin and metalloproteinase with thrombospodin-1-like domains (ADAMTS13) and von Willebrand factor (vWf). The main conclusions in the first article were that platelet hyper-aggregability is not independently associated with VT risk. The correlation between platelet hyper-aggregability and platelet function was modest (only 12% of PFA-100® values was estimated to be due to platelet aggregation). Although, no association was found between platelet hyper-aggregability and VT, however our group found an association between platelet hyperreactivity (measured by PFA-100® system) and VT. This finding motivated us to examine platelet hyperreactivity following treatment with some biomolecules related to this phenotype and platelet aggregation as VAMP8, SERT and serotonin. In the second article we reported that VAMP8 and SERT levels were independently associated with VT in women. We did not find a correlation between these biomolecules and PFA-100® values. Endothelium plays an important role in primary hemostasis. Endothelial cell and platelets synthesize vWf that is necessary for platelet adhesion. ADAMTS13 is the regulator of vWf. Both are associated with arterial thrombosis but little is known about their relation with VT. The third article reports that low levels of ADAMTS13 were associated with high VT risk in women and that vWf levels were blood group dependant while ADAMTS13 were not. Finally, the fourth article reports that a genome-wide association study (GWAS) identified susceptibility loci for PFA-100® phenotypes. Our results suggest that ABO locus is the main determinant of PFA-100® phenotypes.

Die In-vitro-Kultivierung von cornealem Endothel, einer funktionalen, beim Menschen nicht regenerierbaren Schicht in der Hornhaut des Auges, eröffnet weitreichende Möglichkeiten zum Zell- und Gewebeersatz. Dieser Artikel beschreibt aktuelle und künftige Optionen für zellbasierte Therapieansätze sowie die Bedeutung unbegrenzt proliferationsfähiger (immortalisierter) Zellpopulationen als Modellsystem für die Entwicklung neuartiger Methoden. In diesem Zusammenhang werden schaltbare Zellkulturträger als Möglichkeit zur schonenden Gewinnung transplantierbarer „cell sheets“ vorgestellt. Darüber hinaus wird die serumfreie Kultivierung als wichtige Voraussetzung für eine Anwendung am Menschen diskutiert
The in vitro cultivation of corneal endothelium – a functional, non-regenerable layer of the human cornea – is a promising approach for cell and tissue replacement. This paper introduces options for cell-based therapies and points out the importance of immortalised cell populations as a model system to develop tissue engineering strategies. In particular, the use of stimuli-responsive cell culture carriers for the gentle harvesting of “cell sheets” is described. Furthermore, serum-free cultivation is discussed as a prerequisite for future applications

In response to nutritional variation, white adipose tissue (WAT) undergoes a physiological remodelling that involves qualitative and quantitative changes in resident cells and is coordinated with angiogenesis. In a condition of chronic over nutrition WAT expansion is associated to insufficient vascularisation which in turn leads to local hypoxia, inflammation and adipocytes death (hallmark of obesity). Currently, enhanced WAT angiogenesis is believed to be a promising intervention to ameliorate obesity associated metabolic dysfunctions. However, we still lack understanding of the cell intrinsic function of endothelial cells in WAT remodelling. Here we take advantage of our mouse model of PTEN (a dual lipid/protein phosphatase that counterbalance the activity of PI3K) deletion in ECs to promote vessel growth, in a cell autonomous manner. To this end, we crossed Ptenflox/flox mice with PdgfbiCreERT2 transgenic mice that express a tamoxifen-inducible Cre recombinase in ECs; 4-hydroxytamoxifen was administered in vivo at postnatal day 1 (P1) and P2 to activate Cre expression. Increased ECs proliferation, induced by PTEN loss, promotes vascular hyperplasia exclusively in WAT and leads to a progressive loss of WAT mass. PTEN null ECs undergo a metabolic switch towards an oxidative metabolism; in vivo inhibition of - oxidation is sufficient to revert both vascular hyperplasia and loss of WAT mass. Enhanced adipose vascularisation prevents from high fat diet induced WAT hypertrophy, limits body weight gain and improves glucose tolerance. Taken together our results suggest that, under obesogenic stimuli, more functional ECs prevent unhealthy WAT expansion and consequently the onset of obesity related comorbidity.

El endotelio disfuncional presenta, entre otras caracteristicas, alteración en los mecanismos de vasodilatación, complicaciones trombóticas, disminución de la resistencia al estrés oxidativo, aumento de la expresión de moléculas de adhesión y de la secreción de moléculas proinflamatorias. El factor de transcripción endotelial KLF2 juega un importante papel en la regulación del fenotipo protector endotelial y su expresión depende de las fuerza hemodinámicas generadas por el flujo sanguíneo y de la administración exógena de estatinas. La hipertensión portal y el daño hepático por I/R son dos condiciones patológicas asociadas a disfunción endotelial. Los trastornos estructurales característicos de la cirrosis hepática, la mayor causa prevalente de hipertensión portal en nuestro entorno, se acompañan de variaciones en las fuerzas hemodinámicas que pueden modificar la expresión de KLF2 y su programa transcripcional vasoprotector. Asímismo, durante la isquemia asociada a la preservación de injertos hepáticos para transplante, la interrupción de las fuerzas hemodinámicas generadas por el flujo sanguíneo podría resultar en la reducción de los programas endoteliales vasoprotectores, que se debería en parte a la pérdida de expresión de KLF2. Los trabajos de investigación de la presente tesis doctoral amplian el conocimiento de los mecanismos moleculares responsables de la disfunción endotelial hepática, demostrando: 1. Que KLF2 está muy expresado en los hígados cirróticos y que su expresión se induce en las fases tempranas de la progresión de la enfermedad, representando un mecanismo compensador para mejorar los desórdenes vasculares característicos de los hígados cirróticos. 2. Que los hígados preservados en condiciones de transplante muestran un descenso tiempo-dependiente de KLF2, acompañado de daño hepático y aumentada resistencia vascular. Además, demostran que la modulación farmacologica de la expresión de KLF2 puede ser beneficiosa tanto en el tratamiento de la hipertensión portal como en la preservación de los injertos hepáticos para transplante.

La enfermedad cardiovascular (CV) es la principal causa de muerte de los pacientes con enfermedad renal crónica terminal (ERCT). Es especialmente llamativa la diferencia que existe, con respecto la población general, en el grupo de pacientes más jóvenes, siendo ésta de hasta 50 veces superior. A pesar de las mejoras técnicas implementadas en diálisis o de las estrategias farmacológicas disponibles, no se ha conseguido reducir la mortalidad de origen CV en este grupo de pacientes. Esta elevada prevalencia de enfermedad CV (en forma de alteraciones microvasculares o macrovasculares -aterosclerosis o arteriosclerosis-) tiene como nexo común la disfunción endotelial (DE), se encuentra agravada en la enfermedad renal crónica (ERC) y se manifiesta presentando un fenotipo proinflamatorio, prooxidante y protrombótico. Esta Tesis Doctoral (TD) plantea la hipótesis de que es posible mejorar la DE presente en la uremia mediante nuevos enfoques de modulación farmacológica a partir de la identificación de nuevas potenciales dianas terapéuticas. Los objetivos son: explorar los efectos de la modulación de distintos sistemas antioxidantes-antiinflamatorios, y la posibilidad de intervención sobre los cambios epigenéticos asociados a la exposición crónica de las células endoteliales (CE) al medio urémico. La metodología se centra en el estudio de los cambios y capacidad de modulación de la DE. Para ello, se ha utilizado un modelo in vitro de cultivo de CE procedentes de venas de cordón umbilical humano (HUVECs), incubadas con suero de pacientes con ERCT en programa de diálisis (diálisis peritoneal o hemodiálisis) y donantes sanos tras la exposición a los distintos compuestos estudiados. Las técnicas utilizadas en el primer trabajo incluyen: inmunofluorescencia para medir la expresión de ICAM-1, técnicas de fluorescencia para la determinación de la generación de radicales libres de oxígeno (ROS), técnicas de ELISA y Western blot para el estudio de la expresión del factor de transcripción NFкB y de la íva p38MAPK y fl uorimetría para la medición de glutatión (GSH). Las técnicas utilizadas en el segundo trabajo incluyen: análisis proteómico para detectar proteínas expresadas de manera diferencial; Western blot e inmunofluorescencia para la cuantificación de la histona desacetilasa (HDAC) tipo 1 (HDAC1) y tipo 2 (HDAC2), y de la vía PI3-kinasa/AKT; inmunofluorescencia para medir la expresión de ICAM-1, Toll Like Receptor 4 (TLR4), factor von Willebrand (FvW) y técnicas de fluorescencia para la determinación de la generación de (ROS). Los resultados del primer trabajo demuestran que la modulación farmacológica de los sistemas antioxidantes, mediante la potenciación de la vía de la glutatión peroxidasa (GPX) inducen una mayor respuesta antioxidante y antiinflamatoria que las estrategias que potencian la vía de la superóxido dismutasa (SOD) o los distintos flavonoides, que obtienen unos resultados parciales. Los resultados del segundo demuestran que el medio urémico induce cambios en la expresión de proteínas a nivel de las CE. Algunos de estos cambios son revertidos por el fármaco defibrotide (DF), con reconocidas propiedades protectoras del endotelio en otros contextos. De las proteínas sobreexpresadas en las CE expuestas al medio urémico y que son normalizadas por el DF destacamos, por su relevancia biológica, HDAC1 y HDAC2. El DF, regulando la sobreexpresión de HDAC1 y HDAC2, logra una mejoría del fenotipo proinflamatorio, protrombótico y prooxidante, y una disminución de la actividad de la inmunidad innata, de las CE expuestas al medio urémico. Así pues, esta TD permite concluir que es posible mejorar la DE presente en la ERC in vitro mediante un abordaje distinto al disponible hasta la fecha, con estrategias que se centren en contrarrestar el entorno prooxidante, así como algunos de los cambios epigenéticos observados en estas CE, abriendo las expectativas de posibles nuevas dianas terapéuticas.
Cardiovascular disease (CV) is the leading cause of death of patients with terminal chronic kidney disease (ERCT). This high prevalence of CV disease (in the form of microvascular or macrovascular alterations - atherosclerosis or arteriosclerosis) has as a common link endothelial dysfunction (ED), is aggravated in chronic kidney disease (CKD) and manifests itself by presenting a proinflammatory, prooxidant and prothrombotic phenotype. This Doctoral Thesis (TD) hypothesizes that it is possible to improve the ED present in uremia through new pharmacological modulation approaches based on the identification of new potential therapeutic targets. The objectives are: to explore the effects of the modulation of different antioxidant-anti-inflammatory systems, and the possibility of intervention on epigenetic changes associated with chronic exposure of endothelial cells (EC) to the uremic environment. he results of the first work show that the pharmacological modulation of antioxidant systems, through the potentiation of the glutathione peroxidase (GPX) pathway, induces a greater antioxidant and anti-inflammatory response than the strategies that enhance the superoxide dismutase (SOD) pathway or the different flavonoids, which obtain partial results. The results of the second show that the uremic medium induces changes in protein expression at the EC level. Some of these changes are reversed by the drug defibrotide (DF), with recognized endothelial protective properties in other contexts. Of the overexpressed proteins in the EC exposed to the uremic environment and which are normalized by the DF, we highlight, due to their biological relevance, HDAC1 and HDAC2. The DF, regulating the overexpression of HDAC1 and HDAC2, achieves an improvement of the proinflammatory, prothrombotic and prooxidant phenotype, and a decrease in the activity of innate immunity, of the EC exposed to the uremic environment. Thus, this TD allows us to conclude that it is possible to improve the ED present in CKD in vitro through a different approach to that available to date, with strategies that focus on counteracting the prooxidant environment, as well as some of the epigenetic changes observed in these CE, opening the expectations of possible new therapeutic targets.

Blood vessels distribute nutrients and oxygen to every single cell in the body. Endothelial cells define the vessel wall, and thus they are ideally located to crucially modulate nutrient availability and act as metabolic gatekeepers of the organism. In recent years, mitochondrial dynamics has emerged as a bioenergetic adaptation process to cellular metabolic demands. Mitofusins are GTPase-like proteins implicated in external mitochondrial membrane fusion. Our hypothesis is that mitochondrial fusion in endothelial cells is implicated in energy balance and metabolic control. In order to address this hypothesis, we generated mice lacking either Mitofusin 1 (Mfn1) or Mitofusin 2 (Mfn2) into adulthood by breeding a tamoxifen-inducible endothelial Cre line (PdgfbiCreERT2) with Mfn1 or Mfn2 floxed animals (hereafter called Mfn1ΔEC and Mfn2ΔEC respectively). Mfn2iΔEC mice showed a progressive reduction (25%) in body weight when compared to control counterparts. Intestinal nutrient absorption, food intake and locomotor activity were unaltered in knockout mice. However, enhanced energy expenditure and a shift towards lipid oxidation was observed, while the thermogenesis capacity was not different between groups. Consistent with this phenotype, Mfn2iΔEC mice exhibited lower fat mass and improved glucose tolerance and insulin sensitivity in the face of unaltered insulin release. Collectively, these results indicate that loss of Mfn2 in endothelial cells causes a lean phenotype as the consequence of enhanced lipid metabolism. However, endothelial Mfn1 deletion did not alter systemic metabolism. Upon high-fat diet administration, Mfn2iΔEC mice showed complete resistance to its obesogenic effects. In concordance with lower body weight due to reduced adiposity, mutant mice exhibited improved glucose homeostasis. Moreover, induction of endothelial Mfn2 ablation in established obesity reduced body weight to standard diet control levels and improved metabolic alterations. Interestingly, Mfn1iΔEC mice do not show any metabolic alteration when fed high-fat diet. Aged Mfn2iΔEC mice preserved young-like health-span parameters. Indeed, mutant mice exhibited improved age-associated physiological parameters such as kidney function or anaemia. Diverse motor and cognitive parameters were also preserved in old Mfn2i∆EC mice. Collectively, our results indicate that Mfn2 in endothelial cells is implicated in systemic energy homeostasis control as well as in ageing progression in mice.

Das corneale Endothel bildet die innere, einschichtige Zelllage der Cornea und ist für die Aufrechterhaltung der cornealen Transparenz zuständig. Krankheiten oder Verletzungen des cornealen Endothels können zu schweren Beeinträchtigungen des Sehvermögens führen und eine corneale Transplantation erforderlich machen. Der während und nach der Operation auftretende endotheliale Zellverlust erschwert das Überleben des Transplantates. Darum besteht ein Hauptziel des cornealen Tissue Engineerings in der Bereitstellung von transplantierbaren humanen cornealen Endothelzellsheets (HCEC-Sheets) mit einer adäquaten Zelldichte. Thermo-responsive Zellkulturträger fanden für die schonende, enzymfreie Gewinnung von Zellsheets für verschiedene Gewebetypen bereits Verwendung. HCEC stellen in diesem Kontext einen besonderen Fall dar, da sie eine starke Adhäsion zu ihrem Kultursubstrat ausbilden, was deren schonende, thermisch induzierte Ablösung als funktionelles Zellsheet erschwert. Im Rahmen dieser Arbeit wurde ein neuartiger thermo-responsiver Zellkulturträger entwickelt. Dieser basiert auf dem durch Elektronenbestrahlung immobilisierten und vernetzten thermo-responsiven Polymer Poly(vinylmethylether) (PVME) sowie dem alternierenden Co-Polymer Poly(vinylmethylehter-alt-maleinsäureanhydrid) (PVMEMA) als biofunktionalisierbare Komponente. Die Kombination dieser Polymere führte zur Etablierung eines thermo-responsiven Zellkulturträgers, dessen physikochemische und biomolekulare Eigenschaften in weiten Grenzen einstellbar und dadurch an die spezifischen Anforderungen von HCEC anpassbar waren. Das PVME-PVMEMA-Blend ermöglichte die Bildung konfluenter HCEC-Monolayer mit den morphologischen Grundlagen für ein funktionelles corneales Endothelgewebe. Durch Inkorporation von Poly(N-isopropylacrylamid) (PNiPAAm) als weitere thermo-responsive Polymerkomponente konnte das Ablösungsverhalten funktioneller HCEC-Sheets weiter verbessert werden. In einem weiteren Schritt erfolgte der Transfer abgelöster HCEC-Sheets auf ein planares, biofunktionalisiertes Kultursubstrat sowie auf endothelfreie porcine Corneae. Die HCEC-Sheets wurden auch nach dem Transfer umfassend biologisch analysiert. Diese Arbeit legt einen Grundstein für die Bereitstellung klinisch anwendbarer Alternativen für das Tissue Engineering von cornealem Gewebe.

INTRODUCTION. It has been demonstrated that Glucagon-like peptide-1 (GPL-1) has a protective effect on endothelial cells. GLP-1 improves endothelial function in diabetes, however the mechanisms underlying the GLP-1 protective effects have not yet been fully elucidated. Additionally, it has been proposed that GLP-1 could restore high glucose - endoplasmic reticulum (ER) stress induction. Recent evidences claim a resistance of GLP-1 action that has been shown in pancreatic 13-cells of diabetic patients. A proposed mechanism to explain this resistance to the GLP-1 action in diabetes is the activation of PKCI3, induced by hyperglycaemia, which is able to reduce the expression of GLP-1 receptor. AIM. The aim of this thesis project was to decipher if GLP-1 acute treatment is able to counteract chronic high glucose-induced damage in Human umbilical Vein Endothelial cells (HUVECs). METHODS. In this study HUVECs were cultured for 21 days under normal glucose (5mmol/L, NG) or high glucose (25mmol/L glucose, HG) concentrations. GLP-1 and Ruboxistaurin were added alone or in combination, 1 hour before cell harvesting. Analysis of GLP-1 receptor protein levels as well as of gene expression of different ER stress-related genes, proliferation markers, antioxidant cell response-related genes and PKA subunits was performed. ROS production was also measured in HUVECs exposed to mentioned treatments. RESULTS. GLP-1 receptor expression was reduced in HUVECs exposed to chronic high glucose concentrations and it was partially restored after treatment with the chemical PKCI3 specific inhibitor, Ruboxistaurin. GLP-1, added as an acute treatment in endothelial cells, had the capacity to induce the expression of detoxifying enzymes Nrf2 targets, to increase transcript levels of scavenger genes, to attenuate the high glucose-induced PKA subunits expression, ER stress and also the apoptotic phenotype of HUVECs only when high glucose-induced PKCI3 overexpression was reduced by Ruboxistaurin. In the same direction, ROS production induced by high glucose was reduced by GLP-1 in the presence of PKCI3 inhibitor. CONCLUSIONS. This study suggests that PKCI3 increase, induced by high glucose, could have a role in endothelial GLP-1 resistance, reducing GLP-1 receptor levels and disrupting GLP-1 canonical pathway.
INTRODUCCIÓN. Se ha demostrado que el Glucagon-like peptide-1 (GLP-1) tiene un efecto protector sobre las células endoteliales. GLP-1 mejora la función endotelial en la diabetes, sin embargo los mecanismos subyacentes a los efectos protectores de GLP-1 aún no han sido completamente aclarada. Además, se ha propuesto que el GLP-1 podría restaurar la función del retículo endoplasmático (ER), cuyo estés es inducido en condiciones de alta glucosa. Evidencias recientes afirman que existe una resistencia a las propiedades beneficiosas del GLP-1. Esto se ha demostrado en las células beta del páncreas de pacientes diabéticos. Un mecanismo propuesto para explicar esta resistencia a la acción de GLP-1 en la diabetes es la activación de PKCβ, inducida por la hiperglucemia, que se ha visto está involucrada en la reducción de la expresión del receptor del GLP-1 en el endotelio glomerular de modelos animales de diabetes. OBJETIVO. El objetivo de este proyecto de tesis fue descifrar si el tratamiento agudo con GLP-1 puede contrarrestar el daño inducido por condiciones de alta glucosa crónica en las células endoteliales humanas de la vena umbilical (HUVECs) y también corroborar los efectos de dicha molécula en caso de que se inhiba la activación de PKCI3 inducida por las altas concentraciones de glucosa. MÉTODOS. En este estudio las células HUVEC se cultivaron durante 21 días bajo las dos condiciones de glucosa normal (5 mmol/L, NG) o alta glucosa (25 mmol/L, HG). Se añadieron GLP-1 y Ruboxistaurin, el inhibidor específico de PKCI3, solos o en combinación, 1 hora antes de la recolección de células. Se realizó un análisis de los niveles de proteína del receptor del GLP-1, así como de la expresión génica de diferentes relacionados con el estrés del ER, la proliferación, el proceso de apoptosis, y también los genes relacionados con la respuesta antioxidante. La producción de ROS fue además medida en las HUVECs expuestas a los diferentes tratamientos mencionados. RESULTADOS. La expresión del receptor del GLP-1 fue reducida en las HUVECs expuestas a concentraciones de alta glucosa crónica y fue parcialmente restaurada después del tratamiento con el inhibidor específico de PKCI3, Ruboxistaurin. GLP-1, añadido como un tratamiento agudo en las células endoteliales, tuvo la capacidad de inducir la expresión de enzimas desintoxicantes que son dianas de Nrf2, el regulador más importante de la respuesta antioxidante en las células. Además, el GLP-1 aumentó los niveles de transcriptos de los marcadores de estrés de ER inducido por la alta glucosa y los marcadores de proliferación en las HUVECs sólo cuando la sobreexpresión PKCβ inducida por la alta glucosa se redujo en presecia de su inhibidor. En la misma dirección, la producción de ROS inducida por la alta glucosa disminuyó cuando las HUVECs se trataron con GLP-1 en presencia del inhibidor de PKCI3. CONCLUSIONES. Este estudio sugiere que el aumento de PKCβ, inducido por la alta glucosa, podría tener un papel en la resistencia a las acciones protectoras del GLP-1 a nivel endotelial, reduciendo los niveles del receptor del GLP-1 e interrumpiendo su vía canónica.

El procés de disseminació metastàsic en el càncer colorectal és un procés complex multifactorial. La present tesi ha pretès aprofundir en dos aspectes: els mecanismes de defensa immunològic a través de la caracterització dels fenòmens de interacció leucocitària, i el procés de mobilització tumoral a través de la detecció i pronòstic de les cèl·lules neoplàsiques circulants.En la present tesi s'ha confirmat que existeix una deficient interacció dels leucòcits circulants amb l'endoteli dels vasos tumorals respecte a l'endoteli no tumoral, tant de rodament en condicions basals com d'adhesió rere l'administració d'un estímul inflamatori. S'ha demostrat que la molècula implicada en els fenòmens d'adhesió leucocitària es ICAM-1, ja que el seu immunobloqueig reverteix els fenòmens d'adhesió induïts per LPS. S'ha demostrat a través de la tècnica del doble marcatge amb anticossos que la deficient interacció leucòcit-endoteli a nivell tumoral no pot atribuir-se a una menor expressió de les diferents molècules d'adhesió implicades en aquest fenomen. Al intentar avaluar els mecanismes responsables de la deficient interacció leucòcit endoteli es va descartar la participació de l'antigen carcinoembrionari, els productes derivats de la ciclooxigenasa i la sintasa induïble de l'òxid nítric. No obstant es va demostrar que la inhibició no selectiva de la sintasa de l'òxid nítric augmentava de manera dosi-depenent el rodament leucocitari i l'adhesió post-LPS a nivell del teixit tumoral. Efecte similar es va observar al procedir al immunobloqueig del factor transformant B1. Per tant, dels resultats obtinguts, es pot ressaltar que tant l'òxid nítric com el factor de creixement transformant B1 participen, al menys de manera parcial, en els fenòmens d'escapament dels sistemes de immunovigilància a nivell tumoral.La segona part de la tesi doctoral ha confirmat en primer lloc que es possible detectar la existència de cèl·lules neoplàsiques circulants en sang perifèrica de malalts amb càncer colorectal a través de la detecció de RNA missatger de l'antigen carcinoembrionari. Aquesta tècnica s'ha confirmat sensible (detecta una cèl·lula neoplàsica per cada 107 leucòcits) i específica (negativa en els controls sans). Emprat aquesta tècnica s'ha intentat avaluar un dels aspectes més controvertits de la cirurgia laparoscòpica en el càncer colorectal, la disseminació tumoral. Mitjançant estudis de detecció de cèl·lules neoplàsiques circulants en sang perifèrica, portal i en el líquid peritoneal, s'ha descartat el potencial efecte deleteri atribuït a la cirurgia laparoscòpica. No es varen observar diferències significatives entre el grup de cirurgia convencional respecte al grup de cirurgia laparoscòpica, en el número de pacients amb determinacions positives en els tres territoris avaluats rere la resecció quirúrgica . Finalment, es va intentar establir la significació pronòstica de la detecció de cèl·lules neoplàsiques circulants en pacients amb càncer colorectal mitjançant el seu seguiment a llarg termini. Tant en el grup global de pacients amb càncer colorectal com en aquells en els que s'havia realitzat una resecció curativa , la determinació preoperatòria de cèl·lules circulants no s'associava a un pitjor pronòstic a llarg termini (supervivència global i supervivència lliure de malaltia). Tenint en compte que estudis previs havien demostrat que la cirurgia afavoria la mobilització de cèl·lules neoplàsiques en el càncer colorectal, es va valorar la significació pronòstica de la detecció de cèl.lules neoplàsiques a les 24 hores de la cirurgia. Novament, la detecció postoperatòria no s'associava a un pitjor pronòstic a llarg termini.

El procés de disseminació metastàsic en el càncer colorectal és un procés complex multifactorial. La present tesi ha pretès aprofundir en dos aspectes: els mecanismes de defensa immunològic a través de la caracterització dels fenòmens de interacció leucocitària, i el procés de mobilització tumoral a través de la detecció i pronòstic de les cèl·lules neoplàsiques circulants.
En la present tesi s'ha confirmat que existeix una deficient interacció dels leucòcits circulants amb l'endoteli dels vasos tumorals respecte a l'endoteli no tumoral, tant de rodament en condicions basals com d'adhesió rere l'administració d'un estímul inflamatori. S'ha demostrat que la molècula implicada en els fenòmens d'adhesió leucocitària es ICAM-1, ja que el seu immunobloqueig reverteix els fenòmens d'adhesió induïts per LPS. S'ha demostrat a través de la tècnica del doble marcatge amb anticossos que la deficient interacció leucòcit-endoteli a nivell tumoral no pot atribuir-se a una menor expressió de les diferents molècules d'adhesió implicades en aquest fenomen. Al intentar avaluar els mecanismes responsables de la deficient interacció leucòcit endoteli es va descartar la participació de l'antigen carcinoembrionari, els productes derivats de la ciclooxigenasa i la sintasa induïble de l'òxid nítric. No obstant es va demostrar que la inhibició no selectiva de la sintasa de l'òxid nítric augmentava de manera dosi-depenent el rodament leucocitari i l'adhesió post-LPS a nivell del teixit tumoral. Efecte similar es va observar al procedir al immunobloqueig del factor transformant B1. Per tant, dels resultats obtinguts, es pot ressaltar que tant l'òxid nítric com el factor de creixement transformant B1 participen, al menys de manera parcial, en els fenòmens d'escapament dels sistemes de immunovigilància a nivell tumoral.
La segona part de la tesi doctoral ha confirmat en primer lloc que es possible detectar la existència de cèl·lules neoplàsiques circulants en sang perifèrica de malalts amb càncer colorectal a través de la detecció de RNA missatger de l'antigen carcinoembrionari. Aquesta tècnica s'ha confirmat sensible (detecta una cèl·lula neoplàsica per cada 107 leucòcits) i específica (negativa en els controls sans). Emprat aquesta tècnica s'ha intentat avaluar un dels aspectes més controvertits de la cirurgia laparoscòpica en el càncer colorectal, la disseminació tumoral. Mitjançant estudis de detecció de cèl·lules neoplàsiques circulants en sang perifèrica, portal i en el líquid peritoneal, s'ha descartat el potencial efecte deleteri atribuït a la cirurgia laparoscòpica. No es varen observar diferències significatives entre el grup de cirurgia convencional respecte al grup de cirurgia laparoscòpica, en el número de pacients amb determinacions positives en els tres territoris avaluats rere la resecció quirúrgica . Finalment, es va intentar establir la significació pronòstica de la detecció de cèl·lules neoplàsiques circulants en pacients amb càncer colorectal mitjançant el seu seguiment a llarg termini. Tant en el grup global de pacients amb càncer colorectal com en aquells en els que s'havia realitzat una resecció curativa , la determinació preoperatòria de cèl·lules circulants no s'associava a un pitjor pronòstic a llarg termini (supervivència global i supervivència lliure de malaltia). Tenint en compte que estudis previs havien demostrat que la cirurgia afavoria la mobilització de cèl·lules neoplàsiques en el càncer colorectal, es va valorar la significació pronòstica de la detecció de cèl.lules neoplàsiques a les 24 hores de la cirurgia. Novament, la detecció postoperatòria no s'associava a un pitjor pronòstic a llarg termini.

Orientador : Gilberto de Nucci
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Neste trabalho investigamos os efeitos de D-, L-nitro arginina metil éster (LNAME), D-, L-arginina, L-arginina meti! éster (L-AME) e L-arginina etil éster (L-AEE) em edema de pata em rato; descrevemos também os efeitos de Larginina, L-NAME e NG-monometil L-arginina (L-NMMA) em mastócitos isolados do peritôneo de rato. L-NAME (0.15 J.µmol/pata) reduziu significativamente o edema de pata induzido por BK e 5-HT. L-arginina, D-arginina e D-NAME, nestas mesmas doses, não interferiram na. resposta edematogênica induzida por BK e 5-HT. Em contraste, L- e O-arginina, L- e D-NAME, L-AME e L-AEE, na dose de 15 µmol/pata, aumentaram significativamente o edema de pata induzido por BK e 5-HT. O pré-tratamento dos animais com uma mistura de mepiramina (antagonista H1) e metisergida (antagonista de 5-HT) aboliu o aumento produzido pelo lNAME, L-arginina e L-AME no edema de pata induzido por BK. Resultado semelhante foi observado em animais que tiveram seus estoques de histamina e serotonina cronicamente depletados. A indometacina (inibidor da ciclooxigenase) e WEB 2086 (antagonista de PAF) não tiveram efeito no aumento produzido por L-NAME no edema de pata induzido por BK e 5-HT. L-NAME e L-arginina interferiram na detecção de histamina por fluorimetria. L-arginina, L-NMMA e L-NAME, em concentração de 300 µM, não afetaram significativamente a liberação de histamina in vitro induzida por composto 48/80, quando medida através de radio-imunoensaio. L-arginina, L-NMMA e L-NAME induziram liberação de histamina em 40% dos experimentos apenas e esta liberação foi significativamente menor ( < 10%) quando comparada com à liberação de histamina induzida por composto 48/80 ( > 40%). Estes resultados indicam que a inibição observada com doses baixas de L-NAME é devido a inibição da síntese de NO. O aumento observado com doses altas de todos os análogos de arginina estudados é devido à degranulação de mastócitos in vivo. Contudo, este fenômeno provavelmente se deve à carga catiônica que estas substâncias apresentam, independente da via L-arginina-NO.
Abstract: The effects caused by L-arginine (L-ARG), D-arginine (D-ARG), Nw-nitro-L-arginine methyl ester (L-NAME), nitro-D-arginine methyl ester (D-NAME), L-arginine methyl ester (L-AME) and L-arginine ethyl ester (L-AEE) on the rat hind paw oedema induced by bradykinin (BK) and 5-hydroxytryptamine (5-HT) were investigated. We have also examined the effects caused by L-ARG, L-NMMA and L-NAME on In vitro rat mast cell activation, as measured by histamine release. The rat paw volume was measured with a hydroplethysmometer at 15, 30, 60 and 120 min after the subplantar injection of the inflammatory agent. Histamine release from peritoneal rat mast cells was measured either fluorometrically or by radioimmunoassay. L-NAME (0.15 µmol/paw) significantly reduced both BKand 5-HT-induced oedema. At this dose L-ARG, D-ARG and D-NAME had no effect on the oedematogenic responses induced by these agents. In contrast, L-NAME, L-ARG, D-ARG, D-NAME, L-AME and L-AEE, at the dose of 15 µmol/paw, significantly potentiated both BK- and 5-HT-induced oedema. Pre-treatment of the animais with both mepyramine and methysergide abolished the potentiation caused by L-NAME, L-AME and L-ARG on paw oedema induced by BK. Similar results were observed in rats chronically treated with the histamine releasing agent compound 48/80. Indomethacin and WEB 2086 had no effect on the potentiation induced by L-NAME in both BK and 5-HT-induced oedema. L-NAME and L-ARG dose dependently (1.5-150 µM) decreased histamine detection by fluorescence. L-NAME, L-NNMA and L-ARG, at the concentration of 300 µM, did not significantly affect compound 48/80-induced histamine release from rat peritoneal mast cells as detected by radioimmunoassay. L-NAME, L-NMMA and L-ARG induced histamine release in only 40% of the experiments. Histamine release induced by L-NAME, L-NMMA and L-ARG was significantly smaller (<10%) than that induced by compound 48/80 (>40%). Our results indicate that the inhibition of rat paw oedema observed with low doses of L-NAME is due to NO synthesis inhibition. The potentlation 01 BK-and 5-HT -induced paw oedema observed with higher doses of ali arginine analogues is caused by in vivo mast cell degranulation. However, this latter phenomen is probably due to the cationic charge of these substances rather than to theinvo/vement of NO.
Mestrado
Mestre em Farmacologia

Die Manifestation der Artherosklerose an den Herzkranzarterien ist die koronare Herzerkrankung (KHK), ein pathologischer Prozess dessen Auswirkungen in Industrieländern die Krankheits- und Todesursachenstatistiken anführen1. Der Myokardinfarkt mit thrombotischem Verschluss einer Koronararterie ist dabei eine der häufigsten Komplikationen. Die zeitnahe Revaskularisation ist die wichtigste therapeutische Maßnahme zur Reduzierung des postischämischen Myokardschadens, wobei auch durch die Reperfusion selbst eine Schädigung der Myozyten und des Endothels stattfindet. Mit Wiedereröffnung der verschlossenen Herzkranzgefäße setzt eine gesteigerte inflammatorische Reaktion ein, begleitet von erhöhter Leukozytenmigration in myokardiales Gewebe. Der Rezeptor der advanced glycation end products (RAGE) wurde in verschiedenen Vorarbeiten im Kontext der myokardialen Ischämie und Reperfusion beschrieben. In dieser Arbeit wurden die postischämische Interaktion von RAGE und Leukozyten und deren funktionelle Relevanz in einem Mausmodell analysiert. Tiere, die defizient für das endotheliale Adhäsionsmolekül ICAM-1 oder für RAGE sind, sowie doppeldefiziente Tiere wurden einer 20-minütigen Okklusion der LAD unterzogen. Nach einer darauf folgenden 15-minütigen Reperfusion wurde die Leukozytenrekrutierung mittels Rhodamin-G6 Infusion fluoreszenzmikroskopisch untersucht. Außerdem wurde in WT und in ICAM-1/RAGE-defizienten Tieren die linksventrikuläre Funktion nach 45 Minuten myokardialer Ischämie und 24 Stunden Reperfusion mittels invasiver intrakardialer Druckmessung untersucht. In RAGE- oder ICAM-1-defizienten Tieren kam es zu einer gleichwertigen Verminderung der Leukozytenrekrutierung (Abb. 8) im Vergleich zu Wildtyp-Tieren. In ICAM-1/RAGE-/- Tieren kam es zu einer additiven Reduktion. Ebenso zeigten RAGE/ICAM-1-/-Tiere eine verbesserte postischämische LV-Funktion im Vergleich zu WT-Kontrollen. Um zwischen endothelial und leukozytär exprimiertem RAGE zu differenzieren, wurden Knochenmark-Chimären mit WT und ICAM-1/RAGE-/- Tieren generiert. Hierzu wurden Wildtyp-Mäusen nach Bestrahlung Knochenmark von ICAM-1/RAGE-defizienzenten Tieren transplantiert, um Mäuse mit leukozytärer ICAM-1/RAGE-Defizienz zu generieren. Umgekehrt wurde ICAM-1/RAGE-defizienten Tieren Knochenmark von Wildtyp-Tieren transplantiert. Dadurch erhielten wir Mäuse mit ausschließlich auf dem Endothel fehlender ICAM-1/RAGE-Expression. In diesen Knochenmarkschimären konnten wir zeigen, dass das Fehlen des endothelialen RAGE zu einer deutlichen Reduktion der Leukozytenadhäsion führt, das Fehlen von leukozytärem RAGE hingegen kaum zu einer Änderung der Leukozytenadhäsion. Somit konnten wir zeigen, dass nicht das leukozytäre sondern endotheliales RAGE einen redundanten Effekt für die Leukozytenrekrutierung darstellt. Wir konnten nachweisen, dass diese ICAM-1/RAGE-Defizienz zu einer signifikanten Verbesserung der linksventrikulären Funktion führt. Ein möglicher Mechanismus ist die Interaktion zwischen leukozytärem Mac-1 und endothelialem RAGE. Die reduzierte inflammatorische Reaktion ist verantwortlich für die verbesserte linksventrikuläre Funktion und könnte als neuartiger therapeutischer Ansatz von Nutzen sein.

El endotelio corneal es una monocapa de células poligonales (mayormente hexagonales) situada en la parte más interna de la córnea, en contacto directo con el humor acuoso. Su función es el control del paso de agua y nutrientes desde la cámara anterior del globo ocular hacia las capas más anteriores de la córnea, preservando el estado de deshidratación parcial necesario para el mantenimiento de la transparencia corneal. Es una capa sin capacidad de regeneración en la mayoría de especies y, por lo tanto, cualquier alteración que sitúe su densidad celular en valores por debajo de las 500-800 células/mm2 se traducirá en edema corneal y concomitantemente, en aumento del grosor corneal. Las células del endotelio corneal pueden visualizarse in vivo mediante biomicroscopía, microscopía especular y microscopía confocal, y se ha descrito su apariencia normal en diferentes especies animales y en el ser humano. Además, se ha observado que diferentes factores fisiológicos tales como la edad o la raza, entre otros, pueden hacer variar la densidad celular, así como el tamaño y la forma de las células endoteliales. El conocimiento de la apariencia fisiológica del endotelio corneal en cada especie animal, y como se ve afectada por diferentes factores fisiológicos, supone la base para detectar y comprender las diferentes alteraciones patológicas que pueden afectar a esta capa. Por otro lado, hay numerosos procesos primarios y secundarios del endotelio, como, por ejemplo, la Distrofia de Fuchs, que pueden producir ceguera en el humano y en la especie canina, de las que aún se desconocen sus causas iniciales. Es por ello necesario encontrar modelos animales que permitan el estudio de dichas patologías de manera natural, pudiendo los resultados aplicarse a diversas especies. Los objetivos de la presente tesis doctoral son múltiples. En la especie ovina, describir la apariencia normal del endotelio corneal, así como determinar si existen variaciones en dicha capa o en el grosor corneal en función de la región estudiada de la córnea, la edad o la raza. En la especie caprina, describir su endotelio y el grosor corneal, así como revisar las variaciones en función del cuadrante corneal y la edad. En el cárabo común, un ave rapaz, describir por primera vez su endotelio corneal y valorar cómo la edad afecta a las células endoteliales. Y, finalmente, en la especie canina, determinar si el índice cefálico influye en los parámetros endoteliales y/o el grosor corneal.
The corneal endothelium is a monolayer of polygonal cells (mostly hexagonal in shape) located on the inner surface of the cornea, in contact with the aqueous humor. This layer governs fluid and solute transport from the anterior chamber to the other layers of the cornea, preserving the slightly dehydrated state that is required for corneal transparency. The corneal endothelium does not regenerate in most species and therefore, if endothelial cell density drops below 500-800 cells/mm2 due to any disturbance, corneal edema will appear and concomitantly, corneal thickness will increase. The corneal endothelium cells can be visualized in vivo by means of biomicroscopy, specular microscopy and confocal microscopy and its normal appearance has been described in different animal species and in humans. Moreover, several physiological factors such as aging or race, among others, can lead to changes in cell density, size and shape of endothelial cells. The knowledge of the normal appearance of the corneal endothelium in each animal species, and how it is affected by different physiological factors, is the basis for detecting and understanding the pathologies that may alter this layer. On the other hand, many primary and secondary alterations of the corneal endothelium, such as Fuchs’ Dystrophy, can lead to blindness in humans and dogs although its initial cause is not known. For this reason, it is essential to search for animal models that allow the study of these pathologies. The purposes of this thesis are numerous. In sheep, the aim is to describe the normal appearance of the corneal endothelium as well as determine if the quadrant of the cornea, the breed or the age of the animal influence on corneal endothelial parameters and/or corneal thickness. In goats, the objective is to study the caprine endothelium and corneal thickness, and to report the variations observed in the endothelium due to aging or different corneal regions. In the tawny owl, a bird of prey, the purpose of the study is to describe its corneal endothelium and determine if aging has any effect on endothelial parameters. Finally, in the dog, the aim is to evaluate if skull index has any impact on corneal endothelium and/or corneal thickness.