Harun Ülger

The relationship between circulating endothelial microparticles and arterial stiffness and atherosclerosis in children with chronic kidney disease

BACKGROUND Endothelial dysfunction is an important factor in the pathogenesis of atherosclerosis, and endothelial microparticles (EMPs) are considered as markers of endothelial dysfunction. In this study, we aimed to examine the relationship between EMPs and arterial stiffness and atherosclerosis in children with chronic kidney disease (CKD). METHODS This cross-sectional study included 37 dialysis patients (12 haemodialysis, 25 peritoneal dialysis), 33 pre-dialysis patients and 18 healthy controls. Both in vivo and in vitro (HUVECs) evaluations were used for the study. Circulating EMPs were measured by flow cytometry. The carotid artery intima-media thickness (cIMT) and pulse wave velocity (PWV) were measured by using high-resolution ultrasound. Study groups were compared for circulating EMP, cIMT and PWV. The relationship between EMPs and arterial stiffness and atherosclerosis was evaluated. RESULTS The levels of PWV, cIMT, CD144 + EMP and CD146 + EMP in the dialysis group were significantly higher than those in the pre-dialysis and control groups (P < 0.05). Additionally, the levels of cIMT, CD144 + EMP and CD146 + EMP in the pre-dialysis group were significantly higher than those in the control group (P < 0.05). In all CKD patients, the CD144 + EMP was significantly positively associated with blood pressures, age, known duration of disease, CRP and PTH, and was significantly negatively associated with haemoglobin, GFR and albumin. The CD146 + EMP was significantly positively associated with blood pressures, age and CRP. In a multiple linear regression analysis, in the CKD group, cIMT was independently related to mean blood pressure and dialysis duration. PWV was independently related to the CD144 + EMP and mean blood pressure. CONCLUSION Our results suggest that endothelial damage starts in the early stage of CKD, that the endothelial dysfunction becomes overt with the increase of cardiovascular risk factors and that EMPs may be a reliable marker of the subclinical atherosclerosis and arterial stiffness.

Protection by free oxygen radical scavenging enzymes against salicylate-induced embryonic malformations in vitro

Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals. In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated. Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr. ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml. Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 micromol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud. When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations. Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL. There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione. The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid. An enhanced production of these metabolites in embryonic tissues may be directly related to the increased risk of congenital malformations.

Labelling of Rat Endothelial Cells with Antibodies to vWF, RECA-1, PECAM-1, ICAM-1, OX-43 and ZO-1

Labelling with endothelium specific monoclonal antibodies, von Willebrand Factor (vWF), rat endothelial cell antigen‐1 (RECA‐1), platelet‐endothelial cell adhesion molecule‐1 (PECAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), OX‐43 and zonula occludentes‐1 (ZO‐1), was investigated in cryostat sections of vessels from rats of different ages using a confocal microscope. The results showed that labelling of the vWF was positive in endothelial cells from adult, fetal and different ages of embryonic rat. Labelling with RECA‐1 was weakly positive in adult rat aorta and lung endothelial cells but not in embryonic yolk sac endothelial cells. Labelling using PECAM‐1, ICAM‐1 and OX‐43 was negative in both adult and embryonic endothelial cells. ZO‐1 showed positive but very weak reactivity in embryonic yolk sac endothelial cells. The expression of vWF on vessels from adult and 19.5‐day fetal tissues was strongly positive. However, the expression of vWF in embryonic endothelial cells was dependent on the gestational age. While the 11.5‐day yolk sac vessels stained weakly, staining gradually increased in 13.5‐, 15.5‐ and 17.5‐day‐old yolk sac vessels. The results suggest that vWF is a reliable endothelial cell marker in rat vascular endothelial cells, including both fetal and embryonic stages.

The growth promoting effects of bFGF, PD-ECGF and VEGF on cultured postimplantation rat embryos deprived of serum fractions.

Serum components in which embryos are cultured in vitro are very important for normal embryonic development. In this study, rat serum was fractionated using Macrosep filters to study the effect of a single growth factor. The fractionated serum, both that containing only material greater than 30 kDa molecular weight (> 30 kDa) and that from which material between 30 kDa and 50 kDa had been removed (< 30 kDa + > 50 kDa), caused significant embryonic growth retardation. Addition of different concentrations of basic fibroblast growth factor (bFGF, 18 kDa), vascular endothelial growth factor (VEGF, 45 kDa) and platelet‐derived endothelial growth factor (PD‐ECGF, 45 kDa), to fractionated serum (bFGF to > 30 kDa serum and VEGF or PD‐ECGF to < 30 kDa + > 50 kDa serum) partially restored embryonic growth and development according to a morphological scoring system and protein assay. This restoration was clear by all criteria, as well as in yolk sac vascularisation and heart development. The growth promoting effects of all 3 factors were significant but did not reach the level seen in embryos grown in whole rat serum. The effect of these growth factors was also investigated on anembryonic yolk sac development using a concentration for which maximum whole embryonic growth was seen (128 ng/ml bFGF, 1.6 ng/ml VEGF and 4 ng/ml PD‐ECGF), and significant anembryonic yolk sac development was found. These findings suggest that the angiogenic factors may have a growth promoting effect on total embryonic development and vascularisation.

The Effect of Vascular Endothelial Growth Factor on in vitro Embryonic Heart Development in Rats

In vitro effects of vascular endothelial growth factor (VEGF) on heart development and total embryonic growth were investigated in 84 rat embryos (obtained from nine pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), in <30 kDa + >50 kDa serum fractions [retenate (R)], and in R + VEGF. After 24‐h culture, the embryos from each group were harvested and divided into two groups. One group was analysed morphologically and biochemically to obtain embryo protein content, the second group was serially sectioned and examined by light microscopy. Morphological score, embryo protein content, somite number and crown‐rump length of embryos indicated that embryos cultured in R had significant embryonic retardation, whereas the addition of VEGF to R increased embryonic growth and development. The morphological scores for WRS, R and R + VEGF were 57.7 ± 0.87, 46.6 ± 1.90 and 52.1 ± 0.97, somite numbers were 26.5 ± 0.47, 20.1 ± 0.63 and 24.4 ± 0.46, crown‐rump lengths were 3 ± 0.07, 2.4 ± 0.06 and 2.7 ± 0.06 mm, and embryo protein contents were 160.5 ± 7.41, 98.2 ± 4.81 and 141.1 ± 10.96 μg per embryo, respectively. The results of histological examination of heart development were similar. The hearts of embryos grown in R were unseptated and tubular. The atrioventricular endocardial cushions were incompletely developed. The addition of VEGF to R improved heart development. There were no gross morphological differences in the cardiac development between embryos grown in WRS and R + VEGF. In both groups, development of the muscular interventricular septum had begun. Development of the atrioventricular cushions was also similar in both groups and had caused narrowing of the atrioventricular canals, but the atrial septation was not observed.

Influence of gilaburu (Viburnum opulus) juice on 1,2-dimethylhydrazine (DMH)-induced colon cancer.

In this study, the effects of gilaburu (Viburnum opulus) juice on colon tumorogenesis were investigated. Eight weeks old Balb-C male mice received subcutaneous injections of 1,2-dimethylhydrazine (DMH) (20 mg/kg body weight) once a week for 12 weeks. Both the sham control (group 1) and the DMH control (group 2) groups received drinking water alone, whereas the mice of groups 3 and 4 received gilaburu juice for 30 weeks (started with first DMH injection) and for 18 weeks (started after last DMH injection), respectively. Eighteen weeks after the last DMH injection, all mice were killed and the histogenesis of colon tumors was investigated from the paraffin-embedded sections of colon, which were stained with hematoxylin–eosin. The sites and incidences of tumoral lesions (low-grade dysplasia, high-grade dysplasia, intramucosal carcinoma and invasive carcinoma) were analyzed and compared with control. The results showed that the body weights of the mice were similar in all the groups. No tumoral lesions were found in group 1. Colon tumors developed in all DMH-treated mice (groups 2, 3 and 4). In these groups, the greatest numbers of tumor lesions were detected in the distal colon, followed by the mid-colon and only a few in the proximal colon. There was a reduction in the mean total number of tumor lesion in groups 3 (8.5) and 4 (8.3), when compared to group 2 (11.3). The incidence of invasive carcinoma in group 3 was significantly lower than group 2 (p < 0.05). On the basis of these results, we conclude that gilaburu juice may be useful for the prevention of colon cancer at the initiation stage.

Erythropoietin improves brain development in short-term hypoxia in rat embryo cultures

BACKGROUND Hypoxic ischemic encephalopathy continues to be a significant cause of death and disability worldwide. Erythropoietin (EPO) has the potential to lessen neurologic sequelae due to hypoxia-ischemia. METHODS The in vitro effects of EPO on total embryonic development and brain VEGF receptor (VEGFR) expressions were investigated in 50 rat embryos at 9.5 days of gestation that were cultured in whole rat serum (WRS). According to the study protocol, the embryos were divided into two groups. The first group is comprised hypoxia, 100 and 50 U/ml EPO after hypoxia groups. Group 2 comprised control (WRS) and WRS+EPO. After 48-h culture, the embryos from each group were harvested to be analyzed according to a morphological scoring system and also genetically to measure brain VEGFR expression. RESULTS The mean morphological scores for the embryos grown in control, WRS+EPO, hypoxia, and in the presence of 100 and 50 U/ml EPO in hypoxic medium were 55.30±7.22, 52.10±5.27, 23.0±4.60, 36.20±5.07, and 19.70±5.07, respectively. Expressions of VEGFR-1, -2, -3 were significantly elevated in the 100U/ml EPO and WRS+EPO groups compared to the hypoxia group (p<0.05). CONCLUSIONS These results support the conclusion that (1) VEGFR-1, -2, -3 may increase with EPO treatment in hypoxic conditions, (2) VEGF and EPO may be part of a self-regulated physiological protection mechanism to prevent neuronal injury including in utero neural tube defects.

Teratogenicity of edoferon kappa A, a molecule derived from salicylate, in cultured rat embryos: differences from salicylate and interaction with free oxygen radical scavenging enzymes.

The effect of edoferon kappa A (E‐KA), a non‐specific immunomodulatory and anti‐neoplastic chemical substance derived from the methyl form of salicylate (acetyl salicylic acid; ASA), on mammalian embryos was studied and compared to the effects of ASA. Rat embryos were cultured in vitro from 9.5 days of gestation for 48 h. E‐KA (0.1–12.8 mg/ml) and ASA (0.1–0.6 mg/ml) were added to the whole rat serum. To investigate the interaction of these molecules with antioxidant agents, the lowest effective concentrations of E‐KA (0.6 mg/ml) and ASA (0.3 mg/ml) for all parameters were added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 μmol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. E‐KA and ASA decreased growth and development in a concentration‐responsive manner. There was also a concentration‐related increase in overall dysmorphology (haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud). There were no statistically significant differences between the control and embryos grown in the presence of 0.1–0.4 mg/ml E‐KA, although the effects of ASA started at a concentration of 0.2 mg/ml. Embryos showed significant growth retardation in all scoring criteria and severe malformations when 0.5–3.2 mg/ml E‐KA and 0.3–0.6 mg/ml ASA were added. When SOD was added, there was a significant decrease in the incidence of malformations and growth and developmental parameters were increased but this decrease never reached the control level. We concluded that E‐KA has direct toxic effects on the developing embryo but at much higher concentrations than ASA, and the teratogenic effects of these molecules might be related to free oxygen radicals.

The Age-related Development of Maxillary Sinus in Children

Introduction:Paranasal sinuses are complex structures and show individual variation. Providing normative values for paranasal sinus size and their changes related to age could be helpful in evaluating the presence of some diseases related to sinonasal region. The purpose of the current study was to investigate the development of maxillary sinuses and evaluate the volume changes according to age and sex by using stereological and ellipsoidal formula methods after that to compare these approaches with each other in children. Materials and Methods:This retrospective volumetric computed tomography (CT) study was carried out on 361 individuals (180 females, 181 males) between 0 and 18 years old (10 females, 10 males in each group, only 14 age group includes 11 males) with no signs of sinus pathology volumetric estimations determined on CT images using point-counting approach of stereological methods and ellipsoid formula by using morphometric data. Results:Maxillary sinus volume measurements that were obtained using 2 methods were increased with age in both sexes until 16 years old. There was a significant correlation determined between 2 methods (ICC 0.894–1.000 for right and 0.862–0.999 for left maxillary sinus measurements). According to the sex, the right and left mean maxillary sinuses volumes were determined at 8.30 ± 5.19 and 8.57 ± 5.53 cm3 in male and at 7.60 ± 4.57 and 7.99 ± 4.73 cm3 in female by using ellipsoid formula respectively. By the stereological method these values were 8.28 ± 5.26, 8.44 ± 5.35 cm3 and 7.64 ± 4.55, 7.85 ± 4.73 cm3 respectively. There was no statistically significant difference between the volume of maxillary sinuses with sex and side using both methods. Conclusions:This study presents the basic data for studies relative to the development of the maxillary sinus in children according to 2 methods. The current study demonstrated that the point-counting method and ellipsoid formula are both effective in determining volume estimation of maxillary sinuses and are well suited for CT studies.

Effect of Anti-Basic Fibroblast Growth Factor (Anti-bFGF) on In Vitro Embryonic Development in Rat

In this study, we aimed at the in vitro effects of anti‐fibroblast growth factor‐2 (anti‐FGF‐2 or anti‐bFGF) on embryo culture in rats. In vitro effects of anti‐bFGF on total embryonic development were investigated in 40 rat embryos (which were divided into four groups) (obtained from five pregnant females) at 9.5 days of gestation that were cultured in whole rat serum (WRS), and in WRS+ 2.5, 5, and 10 μg/ml anti‐bFGF. After 48 h of culturing, the embryos from each group were harvested to be analysed morphologically according to a morphological scoring system and biochemically to obtain the embryo protein content. The morphological score, embryo protein content, somite number and crown‐rump length of embryos indicated that embryos cultured in WRS+ anti‐bFGF had significant embryonic retardation. Mean morphological scores for the embryos grown in WRS, in the presence of 2.5, 5 and 10 μg anti‐FGF‐2 were 61.4 ± 1.64, 46.3 ± 8.42, 27 ± 2.58 and13.6 ± 0.96 respectively. These results suggest that bFGF is very important for normal embryonic development and rat anti‐bFGF neutralizes bFGF effect.