Blood was sampled for examination from patients who had relapsing UC or CD. Among 21 UC patients, 16 were on their 1^st relapse (mean time from diagnosis [mtd] was 1 yr), and 5 on 2^nd relapse (mtd = 3 yr), whereas all the 18 CD patients were on their 1^st relapse (mtd = 4 yr). Control sera were obtained from 16 healthy volunteers. They received no systemic or locally applied corticosteroids, and their medication was stable for the last four weeks before sampling. All the patients were on 5ASA oral therapy. Patients diagnosed as having UC had left side colitis (Table 1). Crohn’s patients had colonic localization and a non-stricturating, non-penetrating form of the disease (Table 1) according to the Vienna classification (1998), and an elevated C-reactive protein level (CRP>10 mg/L), without abscess or sign of infection (all of the patients had negative abdominal ultrasound). In cases which raised the suspicion of abdominal or pelvic abscess, CT or MRI (to exclude the presence of perianal / perirectal abscesses) was performed. Five milliliters of blood were taken from patients of UC, CD and control subjects respectively, placed into collecting tubes, and centrifuged at 2 000 r/min for 10 min. The sera were removed and mixed, and kept frozen at -80°C until use.

Freshly isolated umbilical cords were obtained from the Department of Obstetrics and Gynecology (University of Debrecen, Medical and Health Science Center). Umbilical cords were immersed into a sterile flask containing culture medium (components are described below) and carried to the cell culture room. Both ends of the cords were bound and the ends of the umbilical vein were cannulated. The vein vessel was washed through the cannulas by sterile PBS, followed by Hanks’ Balanced Salt Solution (HBSS), and M199 media (all purchased from Sigma, Budapest, Hungary). Then, the vein vessel was filled with 1 g/L collagenase in M199, and placed in a CO₂ incubator for 12 min at 37 °C. Endothelial cells were isolated in a sterile centrifuge tube from the vessel by washing twice with 20 mL M199 medium. Cell suspension was centrifuged at 1000 r/min for 15 min, and the supernatant was discarded and the cells were resuspended in the culture medium.

The cells (2 ×10⁹ cells/L) were inoculated into a plastic culture dish. The culture medium contained the following components: 500 mL/L normal human serum, 20 g/L HEPES, 10 g/L L-glutamine, 10 g/L amphotericin, 10 g/L penicillin, 450 mL/L sterile filtered M199. One day after inoculation, dishes were washed with 37 °C HBSS, and put into the culture media again. After 4-5 d of culturing, during which time cells grew extensively over the entire space of the dish, cells were washed with HBSS, then digested with 1 mL tripsin-EDTA (tripsin : EDTA = 1 : 25). The cells were suspended in 1 mL culture medium and collected into a sterile flask, then mixed well. The cells were inoculated into the culture dish and the cell propagation and collection procedure were repeated again as described above. Then, cells were passed onto culturing plates at about 2 ×10⁹ cell/L concentration. Cell monolayers that reached 20%-30% or 70%-80% confluency were used for the experiments. The cells were kept in 50% normal, 50% UC or 50% CD sera dissolved in culturing medium for 2, 4, 8, 12, 20, 24, 36, and 48 h, then processed for visualization of eNOS or iNOS.

After removal of the culture media, cells were washed with ice-cold PBS, then harvested by a cell scraper into 50 µL RIPA lysis buffer (0.01 mol/L NaH₂PO₄, 10 g/L Nonident P-40, 10 g/L Na-deoxycholate, 1 g/L Na-dodecyl sulfate (SDS), 0.15 mol/L NaCl, 2 mmol/L EDTA), containing 100 kU/L protease inhibitor mix (Sigma-Aldrich, Budapest, Hungary). Cell lysis was accelerated by continuous shaking. Five microliters of this mixture were added to 45 µL RIPA buffer for protein measurement. All protein solutions were frozen and kept at -80 °C until use.

The protein concentration of the lysates was determined by the BCA method using BSA as standard in an ELISA reader at 540 nm. Data were analyzed by GraphPad Prism software using linear regression to determine the protein concentration (g/L). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the conditions of Laemmli (1970) on 100 g/L acrylamide gels in a Bio-Rad MiniProtean apparatus[3]. Samples for immunoblot analysis were diluted in the RIPA buffer, dissolved in an equal volume of 2 ×SDS sample buffer (100 mmol/L Tris pH 6.8, 2 g/L bromophenol blue, 200 mL/L glycerol, 200 mL/L β-mercaptoethanol) and boiled for 5 min. From each solution, aliquots (30 µg protein) were applied to SDS-PAGE and electrophoresed at 120 V for 1 h. Separated proteins were blotted onto a nitrocellulose membrane (Pharmacia, Vienna, Austria) at 100 V for 90 min during the continuous mixing and cooling of the transfer buffer (192 mmol/L glycine, 25 mmol/L Tris-base, 200 mL/L methanol). Membranes were rinsed twice in 0.1 mol/L PBS (pH 7.6) containing 1 g/L Tween 20 (PBS-Tween), then blocked for 1 h with 50 mL/L non-fat dried milk in PBS-Tween. After 3 × 5 min of washing with PBS-Tween, the membranes were incubated overnight at 4 °C with the anti-eNOS (diluted into 1∶5000, Cat. No.: 482726, Merck-Calbiochem, Darmstadt, Germany) or anti-iNOS (1∶2000, Cat. No.: AB5384, Chemicon, Temecula, CA) polyclonal antibodies raised in rabbits. After washing with PBS-Tween for 3 ×5 min, membranes were incubated with horseradish peroxidase conjugate of anti-rabbit IgG (diluted into 1∶5000 in PBS-Tween, Vector, Bulingame, CA) for 30 min. After washing the membranes subsequently with PBS-Tween for 2 ×5 min, and once with PBS for 5 min. The blots were developed with the enhanced chemiluminescent (ECL) reagent kit (Pierce, Rockford, IL) and the immunoreactions were visualized on X-ray films.

Densitometry of the blots was performed and the scans were analyzed by the Volume Analyse feature of the Molecular Analyst Software (Bio-Rad, Hercules, CA). The densities for the NOS bands were normalized in each blot to the NOS-IR band obtained from the sample of 2 h treatment with normal serum and expressed as percentage of this control. Means of the percentages obtained from three independent experiments were presented in figures as mean ± SEM. The significance of the data between groups (normal, UC, CD) was tested by analysis of variance (ANOVA). Differences were considered significant for values of P < 0.01.

Cells were cultured on coverglasses coated with sterile filtered 100 g/L gelatine in PBS. Cultures showing 20%-30% confluence were used for detection of proliferation, cell distribution and apoptosis/necrosis characteristics, whereas those with 70%-80% confluency were used for visualization of NOS isoforms. After treatments, cells were fixed in an ascendant ethanol gradient (500 ml/L, 700 mL/L, 960 mL/L), then rehydrated with PBS. For specific detection of eNOS and iNOS proteins, non-specific binding sites of primary antibodies were blocked by incubation of the fixed cells for 1 h with 50 mL/L normal goat serum in PBS also containing 1 g/L bovine serum albumin (BSA), 1 g/L Triton-X 100 and 0.1 g/L Na-azide (referred to as solution-A below). NOS primary antibodies used for immunocytochemistry were the same as those used for immunoblotting. Both eNOS and iNOS antibodies were applied at 1∶200 dilution in solution-A overnight at 4 °C. After extensive washing with PBS (3×5 min), cells were incubated with FITC conjugated anti-rabbit IgG diluted to 1∶40 in solution-A for 30 min at room temperature. Cells were washed for 3 ×5 min with PBS and cover glasses were mounted on microscope slides (with one drop of PBS-glycerol (1∶1) solution on them).

For evaluation of the proliferation activity, HUVECs were incubated in a medium containing 500 mL/L normal, 500 mL/L UC or 500 mL/L CD sera, or in a medium containing 500 mL/L normal sera plus 10^-4 mol/L nitro-L-arginine methyl-ester (L-NAME, Sigma-Aldrich), an NOS inhibitor. Cells were exposed to a monoclonal antibody (at 1∶100 dilution) specific for the proliferation marker Ki-67 antigen (clone No. MIB-1, Dako, Denmark) for 2 h at room temperature. In this case, FITC-conjugated anti-mouse IgG (1∶40 in solution-A, Dako) was used as a secondary antibody. For testing of the effect of normal and IBD sera on the viability of HUVEC, apoptotic/necrotic cells were assessed by the annexin-V-biotin kit (Cat. No. 1828690, Boehringer-Mannheim, Germany). Briefly, after removal of the incubation media and washing with PBS, cells were labeled with the annexin-V-biotin/propidium iodide solution (1∶50 annexin-V-biotin stock solution diluted in a 0.01 mol/L HEPES buffer, pH 7.4, containing 0.14 mol/L NaCl, 0.005 mol/L CaCl₂, and 1 mg/L propidium iodide) for 15 min. After subsequent washing with HEPES, cells were fixed with methanol-ethanol (1∶1) solution for 1 min, and labeled with 5 mg/L avidin-fluorescein for 15 min. After consecutive washing with HEPES and PBS, cells were put on microscope slides as described above. Preparations used for the assay of proliferation activity and assessment of apoptosis/necrosis were stained prior to covering with 10 g/L toluidin blue in PBS. The latter labeled the cell nuclei and helped to determine total cell numbers for calculation of apoptotic/necrotic indices. The integrity of the cytoskeleton was examined by visualization of actin filaments by incubation with 0.2 mg/L phalloidin-FITC (Sigma-Aldrich) for 1 h.

Examination was carried out and digital images were captured with an Olympus AX-70 fluorescence microscope. In each case, the brightness-contrast of the images was set in the same manner by the Jasc software Paintshop Pro 7.0. Images were taken from Ki-67 immunoreactive (IR), annexin-labeled, and propidium iodide reactive cell nuclei, respectively. The same sites of the preparations were also captured in bright visual illumination to count the toluidin blue stained nuclei (total cell number). The images were quantitatively analyzed by the computer image analysis software Olympus analysis 2.11 (SiSoft), developed and validated by Olympus (Tokyo, Japan). At least 10³ cells were counted for each data point and the ratios of proliferative, apoptotic and necrotic cells were given as the percentage of total cell numbers.

Statistical analyses were performed to compare the data obtained from the different treatments groups of IBD and normal sera using the ANOVA test. P < 0.05 was

considered as significant.

Western blots from HUVEC lysates detected eNOS immunoreactive bands at approximately 160 kU, corresponding to the molecular mass of human eNOS (Figure 1A). HUVEC was cultured in a medium containing 500 mL/L normal human serum. The amount of eNOS did not change significantly during the experimental period (0-48 h). However, in the presence of 500 mL/L UC serum, eNOS level increased after 8 h, and peaked at 12 h (relative optical density value was 188% ± 23% and 229 %± 26%, P < 0.01, respectively). At 12 h, eNOS level appeared to have doubled compared to the normal level at 2 h (100% ± 15%). Over the 12 h period in the presence of 500 mL/L UC serum, eNOS level began to decline (36 h, 153% ±23%, P < 0.01), and reached control level at 48 h (100%± 8%). In the presence of 500 ml/L CD serum, the level of eNOS was close to normal until 8 h, and decreased after 12 h (41% ± 12%, P < 0.01), reaching the lowest level at 20-24 h (18% ± 16% and 23% ± 15%, P < 0.01, respectively), and then slowly increased rising close to normal at 48 h (65% ± 8%, Figure 1A, B).

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Figure 1 eNOS content in HUVEC treated with IBD sera (Western blot. Mean ± SE, n = 3; bP<0.01 vs normal).

Localization of eNOS in HUVEC by immunofluorescence revealed that the enzyme mostly accumulated in the nuclear area of the cytoplasm and was also detected at the periphery of the cells (Figure 2). Normal human serum had no effect on the distribution and intensity of eNOS immunoreactivity in HUVEC. Prolonged treatment (12 h) with UC serum extended eNOS immunoreactive area and simultaneously enhanced slightly the intensity of eNOS immunofluorescence in HUVEC. In contrast, in the

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Figure 2 eNOS in HUVEC (immunoreaction was visualized by FITC. bar: 10 µm).

presence of CD serum (after more than 4 h), eNOS immunoreactivity seemed to contract toward the centre of the cells and its immunofluorescence intensity decreased (Figure 2). At the end of the experimental period (48 h treatment), no visible difference could be detected in eNOS immunofluorescence of HUVEC in the presence of normal, UC or CD sera.

Western blots in HUVEC lysates detected faint iNOS immunoreactive bands at around 130 kU, corresponding to the molecular mass of human iNOS (Figure 3A). HUVEC was cultured in the presence of 500 mL/L normal human serum, the relatively low level of iNOS expression did not change significantly during the 48 h incubation period. In the presence of 500 mL/L UC serum, iNOS level increased at 4 h (relative optical density value was 225% ± 20%, P < 0.01), peaked at 8 h (300% ± 21%, P < 0.01), and then declined to about normal level at 48 h (117% ± 15%). In the presence of 500 mL/L CD serum the iNOS level in HUVEC increased after 2 h (201% ±19%, P < 0.01) and peaked at 8 h (275% ± 27%, P < 0.01). From 12 h iNOS level declined and was not distinguishable from the control during the 24-48 h period (at 48 h: 92% ± 18%, Figure 3A, B).

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Figure 3 iNOS content in HUVEC (Western blot. Mean ± SE, n=3; bP<0.01 vs normal).

In the presence of 500 mL/L normal serum, HUVEC exhibited faint iNOS immunofluorescence (Figure 4). Expression of iNOS in HUVEC by immunofluorescence could obviously be detected in the presence of IBD sera during the 4-12 h period. Figure 4 shows that iNOS was dispersed in the cytoplasm, accumulated in certain areas showing a high intensity of fluorescent signal. Both UC and CD sera significantly increased iNOS immunofluorescence at the 4-12 h period. Before and after this time interval, iNOS could not be detected unequivocally in HUVEC by immunofluorescence (Figure 4).

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Figure 4 iNOS in HUVEC (immunoreaction was visualized by FITC. bar: 10 µm).

Ten to fifteen percent of HUVEC was observed to be in the state of proliferation in the cultures with 20%-30% confluency at the beginning of the experiments as revealed by Ki-67 immunostaining (Figure 5). During the 2 to 48 h period no difference was detected in the ratio of Ki-67-IR/toluidin blue stained cell nuclei in cultures in the presence of 500 mL/L normal (at 48 h: 19% ± 10%) or 500 mL/L UC sera (at 48 h: 10% ± 3%). In the presence of 500 mL/L CD serum a significant increase in the number of proliferating cells was apparent at 20 h (41% ± 12%, P < 0.01). About 50%-65% of HUVEC nuclei exhibited Ki-67 immunofluorescence in the presence of CD serum for 24-48 h (Figure 5). The presence of 10^-4 mol/L L-NAME in the medium containing 500 ml/L normal serum, increased the proliferation activity significantly from 24 h (42% ± 5%, P < 0.01) to 48 h (48% ± 7%, P < 0.01).

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Figure 5 Proliferation activity of HUVEC (Ki-67 immunoreactivity was visualized by FITC. bar: 50 µm. mean ± SE, n = 3; bP<0.01 vs normal).

By the annexin-V-biotin method, early apoptotic cells revealed fluorescent signals only on the cell membrane (Figure 6A), whereas necrotic cells showed propidium iodide binding to DNA (nuclear fluorescent signal, Figure 6B). Both signals could be detected in late apoptotic cells (Figure 6C). During the experimental period (2-48 h), the number of apoptotic cells was low (1%-3%) in the presence of 500 mL/L normal serum, but a continuous increase of apoptotic cell numbers was observed in the presence of 500 mL/L CD serum (2%-5%, with a maximum level at 36 h), which proved not to be significant (Figure 7A). In the presence of UC serum the apoptotic index did not differ from that of the control. Incubation of HUVEC with normal or CD sera slightly increased the number of necrotic cells during the examination period (2-48 h), but no significant changes were observed between the necrotic indices (Figure 7B). Actin filaments visualized by phalloidin-FITC seemed to be intact in cells independent of the media used and the time of incubation (Figure 6D).

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Figure 6 HUVEC viability. A: Early apoptotic cells; B: necrotic cells; C: Late apoptotic cell; D: Phalloidin conjugated with TRITC labeled actin filaments. bars: 10 µm.

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Figure 7 HUVEC 48 h incubation with CD sera (mean ± SE, n = 3).

HUVEC cultures of 20%-30% confluency were used also to investigate the effect of IBD sera on the distribution and shape of the cells. Cells visualized by eNOS immunofluorescence were partly found to be accumulated in islands exhibiting polygonal shape and attached to each other. In addition, single cells exhibiting long processes were also present. In the presence of 500 ml/L normal or 500 ml/L UC sera cells did not change in distribution and shape over the 48 h experimental period. However, in the presence of 500 mL/L CD serum, after 20 h cells were distributed in a two dimensional reticular network, which changed the shape of the culture reminiscent of the appearance of early vascular vessels in tissues (Figure 8).

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Figure 8 HUVEC visualized by eNOS immunofluorescence in culture. bar: 50 µm.