Academic literature on the topic 'Endotel'

ABSTRACT Endothelial dysfunction play a major role in the pathogenesis of vascular diseases, especially atherosclerosis. Endothelial dysfunction causes endothelial cells are activated and resulting homeostasis changes in endothelial cell. Rats were induced by LPS 0.5 mg/kg and treated with ALA at doses of 30 mg/kg, 60 mg/kg and 120 mg/kg BW for 2 weeks which given 1 hour before the LPS administration. This study show that LPS causes morphological changes in rat aorta based on histochemical staining show that endothelial cell are irregular, not homogeneous and increase permeability but in group that received show that ALA can improve the morphological of endothelial cell by reduces ROS, regenerates of exogenous and endogenous antioxidants. Administration of ALA and LPS reduce the rats body weight but not significant compared to the control group. Therefore can be concluded that administration of ALA can prevent endothelial dysfunction. Keywords: Endothel dysfunction, alpha lipoic acid, lipopolysaccharide ABSTRAK Disfungsi endotel berperan penting pada pathogenesis penyakit vascular, khususnya aterosklerosis. Disfungsi endotel menyebabkan sel endotel teraktivasi dan terjadi perubahan homeostatis pada sel endotel. Tikus diinduksi dengan LPS 0.5 mg/Kg BB dan selanjutnya di terapi dengan ALA pada dosis 30mg/Kg BB, 60mg/Kg BB, dan 120mg/Kg BB selama 2 minggu 1 jam sebelum pemerian LPS. Penelitian ini menujukan bahwa LPS dapat menyebabkan perubahan morfologi pada sel nedotel berdasakan pada hasil pewarnaan jaringan yang menunjukan sel endotel yang tersusun tidak beraturan, tidak homogeny dan terjadi peningkatan permeabilitas tapi pada kelompok perlakuan yang mendapatkan terapi ALA menunjukan ALA dapat memperbaiki morfologi sel endotel dengan mengurangi ROS, meningkatkan antioksidan eksogen dan endogen. Pemberian ALA dan LPS tidak menyebabkan perubahan berat badan tikus yang signifikan di bandingkan dengan kelompok control. Sehingga dapat disimpulkan pemberian ALA dapat mencegah disfungsi endotel. Kata Kunci: disfungsi endotel, histokimia, alpha lipoic acid, lipopolisakarida

Sel endotel melapisi lumen pembuluh darah sehingga menyebabkan paparan langsung aliran darah dan timbul gaya hemodinamik shear stress. Vascular Endothelial (VE) Cadherin merupakan salah satu struktur penghubung antar sel yang berperan mencegah terlepasnya sel endotel dari membran dasar. Paparan glukosa tinggi menyebabkan stress oksidatif sehingga sel endotel mengalami apoptosis dan nekrosis dan terlepas. Penelitian ini bertujuan mempelajari efek paparan glukosa tinggi dan fluid shear stress terhadap morfologi, struktur VE-Cadherin dan densitas sel endotel pada kultur sel endotel HUVECs (Human Vein Endothelial Cells Culture).Metode Penelitian eksperimental laboratorium dengan metode HUVECs yang dipapar d-glukosa 22 mM selama 7 hari. Shear stress dibangkitkan dengan alat cone and plate 10 dyne/cm2 selama 5, 8, 12 dan 15 menit. Pulasan VE-Cadherin dengan imunohistokimia. Data dianalisis dengan metode statistik. Signifikan pada p<0,05.Hasil Shear stress selama 15 menit menyebabkan perubahan bentuk sel endotel menjadi lebih panjang dan inti sel lebih pipih. Paparan glukosa tinggi dan fluid shear stress menyebabkan penurunan skor VE-Cadherin dan densitas sel endotel secara signifikan Penurunan skor VE-Cadherin berpengaruh langsung terhadap penurunan densitas sel endotel.Kesimpulan. Paparan glukosa tinggi dan fluid shear stress menyebabkan kerusakan struktur VE-Cadherin sehingga terjadi peningkatan apoptosis dan nekrosis sel endotel.

Lapisan endotelium terdiri dari satu lapisan sel endotel yang melapisi dinding vaskular yang menghadap ke lumen. Sel endotel menghasilkan mediator vasoaktif Endothelium Derived Relaxing Factor (EDRF) yaitu nitrogen monoksida (NO). Disfungsi sel endotel ditunjukkan dengan rendahnya kadar NO dan dapat menyebabkan hipertensi. Pada penelitian ini diteliti disfungsi sel endotel pada mencit putih jantan. Disfungsi sel endotel diinduksi dengan pemberian NaCl 3 %. Pada penelitian ini digunakan dosis ekstrak terpurifikasi daun Toona sureni (5, 10, 20) mg/kg BB yang diberikan secara oral selama 21 hari bersamaan dengan NaCl. Para meter yang diamati adalah kadar NO dalam serum. Hasil penelitian menunjukan bahwa pemberian ekstrak terpurifikasi daun T sureni terbukti dapat memproteksi disfungsi sel endotel diinduksi NaCl 3 %, ditandai dengan meningkatkan kadar nitrogen monoksida mencit putih jantan dibandingkan kadar NO mencit kontrol positif (hanya diberi NaCl 3 %) dan berbeda nyata p < 0,05.

Atherosklerosis merupakan suatu penyakit degeneratif. Proses Atherosklerosis terjadi melalui beberapa tahap. Kejadian awal yang berperan pada proses atherosklerosis adalah kerusakan atau disfungsi endotel karena kadar lemak LDL. LDL Lipoprotein) adalah kolesterol “jahat” yang potensial menyumbat pembuluh darah. Akan tetapi, hal tersebut dapat dicegah dengan pemberian antioksidan. Oleh karena itu, perlu adanya altermatif pengobatan yang bersifat preventif yang bahannya berasal dari buah-buahan. Quersetin adalah salah satu zat aktif kelas flavonoid yang secara biologis amat kuat. Antioksidan ini dapat mencegah kerusakan sel sel endotel atau jaringan pembuluh darah. Pada saat bersamaan, antioksidan akan meningkatkan koesterol “baik” (High Density Lipoprotein / HDL) yang bermanfaat untuk mencegah penyakit jantung dan pembuluh darah. Penelitian ini bertujuan mengetahui peranan quercetin sebagai antioksidan dalam mencegah penurunan jumlah sel endotel aorta tikus yang diberi diet aterogenik. Jenis penelitian ini menggunakan experimental laboratorik pada hewan coba tikus dengan menggunakan desain penelitian Control Group Post Test Design. Variabel penelitian ini adalah jumlah sel endotel aorta. Hasil uji statistik One Way Anova menunjukkan bahwa terdapat perubahan jumlah sel endotel aorta tiap taraf perlakuan yang signifikan. Kesimpulan dari penelitian ini adalah pemberian quercetin dengan dosis 56 mg paling potensial dalam pencegahan penurunan jumlah sel endotel aorta tikus. Kata Kunci :

Latar Belakang : Preeklampsia adalah penyulit kehamilan yang memiliki angka mortalitas dan morbiditas yang tinggi. Preeklampsia ditandai dengan adanya hipertensi gestasional onset baru dan proteinuria. Salah satu teori mengenai mekanisme terjadinya preeklampsia adalah adanya disfungsi endotel. Disfungsi endotel menyebabkan menurunnya kadar nitrit oksida. Tujuan : mengetahui lebih lanjut tentang konsumsi cokelat hitam untuk mencegah preeklampsia Metode : Menggunakan studi literatur dari jurnal baik nasional maupun internasional dengan cara meringkas topik pembahasan dan membandingkan hasil yang disajikan dalam artikel. Hasil : Cokelat hitam memiliki kadar flavonoid yang tinggi. Flavonoid dalam cokelat hitam dapat membantu untuk meningkatkan kadar nitrit oksida sehingga mencegah terjadinya disfungsi endotel. Kesimpulan : Konsumsicokelat hitam dapat mencegah terjadinya preeklampsia.

<p>Cardiovascular risk factors have been known to play a role in vascular endothelial damage. Endothelial damage can cause a condition called endothelial dysfunction. Endothelial dysfunction is the initial process of various cardiovascular problems such as coronary heart disease, stroke, peripheral artery disease, and others. Early stage endothelial damage can actually be detected, but often patients present at the stage of cardiovascular problems. Various examinations of endothelial function start from invasive, non invasive, until biomarkers marking the occurrence of dysfunction can be done. So that we can prevent or reduce the occurrence of further damage</p>

Latar belakang. Trombomodulin(TM) adalah reseptor sel endotel untuk trombin. Pada penyakit malaria tropikadan malaria tertiana, TM disekresi sesudah kerusakan sel-sel endotel yang terinfeksi P.falciparum atau P.vivax.Tujuan. Mengetahui apakah TM dapat mendeteksi kerusakan sel endotel pada malaria tropika atau malariatertiana dan apakah TM berhubungan dengan berat ringan malaria tropika?Metode. Penelitian menggunakan metode analitik potong lintang, di lima RS di provinsi Sulawesi Utara,Juni-September 2006 pada kasus malaria tropika/malaria tertiana umur 2 tahun-13 tahun. PemeriksaanTM secara ELISA menggunakan kit Fujirebio TM (FU/ml). Analisis data dengan uji t independen dankoefisien korelasi Spearmen Rank.Hasil. Tiga puluh pasien malaria tropika (TM 0,060-0,180FU/ml), 2 pasien malaria tertiana (TM 0,068-0,075FU/ml). Uji t terdapat perbedaan bermakna TM pada malaria tropika dan malaria tertiana (p=0,044).Malaria berat 11 penderita (TM 0,086-0,162 FU/ml). Uji t terdapat perbedaan sangat bermakna TMpada malaria tropika dengan komplikasi dan tanpa komplikasi (p=0,009). Uji Spearmen Rank TMberhubungan positif bermakna dengan derajat parasitemia (rs=0,686, p=0,001).Kesimpulan. Trombomodulin dapat digunakan untuk mendeteksi kerusakan sel endotel, TM pada malariatropika lebih tinggi dari malaria tertiana dan tm berbeda bermakna pada malaria tropika dengan komplikasidan tanpa komplikasi serta berhubungan bermakna dengan derajat parasitemia.

Pendahuluan. Terdapat peningkatan mortalitas akibat penyakit kardiovaskular (PKV) sebesar 50% pada pasien artritis reumatoid (AR). Trigliserida pascaprandial (TGPP) saat ini dikaitkan dengan risiko penyakit jantung iskemik, infark miokard, stroke iskemik, kematian, serta peningkatan kadar molekul adhesi. Kadar molekul adhesi yang meningkat merupakan tanda terjadinya aktivasi endotel, proses awal pada terbentuknya lesi aterosklerosis. Belum ada penelitian tentang peran TGPP dalam risiko kardiovaskular pada pasien AR. Penelitian ini ingin mengetahui hubungan antara TGPP dengan penanda biologis aktivasi endotel.Metode. Penelitian ini adalah studi potong lintang, yang menggunakan analisis korelasi dengan analisis multivariat. Sampel diambil dengan metode consecutive sampling. Pada subjek penelitian dilakukan pemeriksaan profil lipid dan penanda aktivasi endotel. Sebagai penanda biologis aktivasi endotel digunakan sICAM-1 dan sE-selectin. Dilakukan uji korelasi antara TGPP dengan sE-selectin dan sICAM-1.Hasil. Tidak terdapat korelasi antara TGPP dengan kadar sE-selectin dan sICAM-1 pada analisis multivariat. HDL mempengaruhi kadar sICAM-1 (R2=0,087). Sementara itu kadar sE-selectin dipengaruhi oleh DAS-28 (R2=0,174), indeks massa tubuh (R2=0,125), dan gula darah pascaprandial (R2=0,138).Simpulan. Tidak ditemukan kaitan antara TGPP dengan kadar sE-selectin dan sICAM-1 pada pasien AR.

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.

Sulfur content in the logitstic fuels such as diesel, jet fuel and coal poses great challenge, as it leads to severe deactivation and poisioning of the reforming catalysts. The utilization of logistic fuel in high efficiency fuel cells as hydrogen-rich reformates, therefore, necessitates that the sulfur (mostly present as organosulfur species) be either eliminated totally, or its level be reduced below such levels as not to mitigate the long-term sustained performance of the reforming catalysts. Besides, SOFC anodes are also quickly poisoned by sulfur present even in trace amounts (1–2 ppm). Thus, desulfurization becomes an important and integral component in harnessing clean power from these abundunt fuel resources. Recently, we have developed a series of novel sulfur sorbents with agile scavengers dispersed thoroughly and uniformly on lightweight, highly periodic nanoporous biomimetic or zeolitic inert matrices. These formulations have been tested in a range of temperatures for durations ranging from 12 to 100 hours in sulfur-bearing gas streams. In this presentation, their synthesis, characterization (before and after sulfidation) by XRD, SEM/EDS, RBSE imaging, on-line real-time quantification in gas chromatography in conjunction with systematic chemical analyses to gauge their efficacy in sulfur removal will be discussed.