Fatih Yesildal

Sodium nitrite provides angiogenic and proliferative effects in vivo and in vitro.

Background Angiogenesis is the formation of new blood vessels from pre-existing vasculature. Many factors and substances may stimulate angiogenesis and exhibit proliferative effect. In this study, we aimed to investigate the angiogenic and proliferative effects of sodium nitrite. Material/Methods The angiogenic activity of sodium nitrite was examined in vivo in the chick chorioallantoic membrane (CAM) model and in vitro in tube formation assay of human umbilical vein endothelial cells (HUVECs). The proliferative activity of sodium nitrite was also determined through MTT assay on HUVECs. Results In CAM assay, sodium nitrite had an angiogenic effect especially at high concentrations compared with the control group and this was statistically significant. There was a proliferative effect on HUVECs in the presence of sodium nitrite for 24 and 48 h, and this was statistically significant (p<0.05). Comparing the tube length/area ratio values, there was statistically significant increase in the sodium nitrite group compared to the control group (p<0.05). Conclusions The results provide evidence that sodium nitrite induces angiogenesis in vitro and in vivo.

Comparison of four automated serum vitamin B12 assays.

Abstract Background: Diagnosis of vitamin B12 deficiency is generally based on the measurement of serum vitamin B12 levels. However, in selected cases functional indices of vitamin B12, such as methylmalonic acid (MMA) and homocysteine (HCY), are needed. Here we compare the performance of four automated total vitamin B12 assays and also investigate how these assays relate to functional indices of vitamin B12 status. Methods: Total vitamin B12, MMA and HCY were measured in 69 serum samples from routine vitamin B12 assay requests. Serum vitamin B12 analysis was performed using four different immunoassay autoanalyzers: DxI 800 Unicel (Beckman Coulter, USA), ADVIA Centaur XP (Siemens Diagnostics, Tarrytown, NY, USA), Roche Cobas E601 (Roche Diagnostics, Germany), Architect i2000sr (Abbott Laboratories, Abbott Park, IL, USA). Serum MMA levels were determined by liquid chromatography-mass spectrometry (LC-MS) and serum homocysteine levels were determined by high pressure liquid chromatography (HPLC) methods. Results: Four immunoassay methods were comparable and correlated with each other. Correlation coefficients (r) ranged from 0.898 to 0.987, p<0.001. Highest correlation was observed between Roche Cobas – Architect i2000sr and poorest correlation was observed between DxI 800 Unicel – ADVIA Centaur comparison. DxI 800 Unicel assay demonstrated high mean bias [–122 pg/mL (–616–125 pg/mL)] and a concordance correlation coefficient (CCC) of 0.9161, lower than the others. MMA and HCY were correlated with the vitamin B12 results. The correlation coefficients with their 95% CI indicated that there was no statistically significant difference between the four methods according to their relationship with MMA and HCY. Conclusions: Total B12 assays correlate very well with each other. However, results of DxI 800 Unicel were lower compared to the other three autoanalyzers. All total vitamin B12 methods show similar relationships with HCY and MMA. Standardization of serum vitamin B12 assays is still not completed and further standardization studies are needed. Laboratory professionals and clinicians should be aware of this disagreement between assay methods and they should use these tests as ancillary tests.

Effects of triethylene glycol dimethacrylate (TEGDMA) on the odontoclastic differentiation ability of human dental pulp cells

Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

A novel model of retinopathy of prematurity in normobaric hyperoxic conditions.

AIM To examine changes in retinal vasculature after treatment with different oxygen concentrations from common retinopathy of prematurity (ROP) models and to determine a novel and practical ROP model. METHODS A sample of 14 newborn Sprague-Dawley rats was used. The study group (n=7) was exposed to 95% oxygen for 4h per day followed by normoxic laboratory conditions for 20h. This cycle was repeated for 14d. The control group (n=7) was subjected to normobaric normoxic conditions. On postnatal day 14 (P14), the two groups were placed in room air for 7d. On P21, the two groups were examined using indirect ophthalmoscopy. All eyes were enucleated for immunofluorescence (IF) staining of the vasculature of retinas and analysis of vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1α), placental growth factor (PLGF) in vitreous humor, and then the rats were sacrificed by decapitation. All procedures were repeated using another litter of 14 pups. RESULTS In the study group and under normobaric hyperoxic conditions, retinal neovascularization and peripheral avascular retina were determined in 85% of the rats through indirect ophthalmoscopic examination. Also IF staining of retina of the study group showed retarded peripheral vascular growth. The difference between the two groups for VEGF, HIF-1α and PLGF concentrations of vitreous humor was statistically significant (P=0.003, 0.007, 0.027 respectively). CONCLUSION Fluctuating oxygen concentrations are primarily responsible for retinal neovascularization. Our new ROP model is practical and applicable for all retinal neovascularization studies, considering the laboratory procedures.

A practical ID-LC-MS/MS method for the most commonly analyzed steroid hormones in clinical laboratories

Abstract Background Analysis of steroid hormones rapidly and reliably remains a challenge in clinical laboratories as this plays an important role in evaluation of many endocrine disorders. The aim of this study was to create a steroid profiling panel by using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method which was composed of the most commonly analyzed steroid hormones in clinical laboratories. Materials and methods Protein precipitation was performed for sample preparation. Ultra performance liquid chromatography (UPLC) system and an analytical column with C18 selectivity was chosen for chromatographic seperation. Atmospheric pressure chemical ionization (APCI) ion source was preferred for ionization, and tandem MS with triple quadrupole was used. MS scan was performed using the selected reaction monitoring mode in positive polarity. During the method validation process, test performance was evaluated for each steroid hormone, and 40 serum samples were used for method comparison with immunoassays available in our core laboratory. Results An isotope dilution (ID)-LC-MS/MS method was developed, in which 13 steroids can be analyzed in the same run. Test performance was quite good for the 11 steroids (cortisol, DHEA, DHEAS, total testosterone, progesterone, androstenedione, 11-deoxycortisol, cortisone, corticosterone and dihydrotestosterone) while estradiol and aldosterone performance was suboptimal considering the precision and trueness. Conclusion This ID-LC-MS/MS method would be useful in clinical laboratories, especially for the immunoassays having insufficient test performance and when checking for interferences in available immunoassays.

Evaluation of the effects of different treatment modalities on angiogenesis in heart failure patients with reduced/mid-range ejection fraction via VEGF and sVEGFR-1

Objectives: To investigate the clinical significance of VEGF, sVEGFR-1 in heart failure reduced ejection fraction (HFrEF) and heart failure mid-range ejection fraction (HFmrEF) patients. Methods: A total of 104 people consisting of HFrEF and HFmrEF patients (n=54) and healthy (n=50) subjects were included in this comparative cross-sectional study. The study took place in Gulhane Training and Research Hospital, Ankara, Turkey, between 2011 and 2013. Serum VEGF, sVEGFR-1, plasma pro-BNP analysis and transthoracic echocardiography were performed. Results: The average sVEGFR-1 level of the HFrEF and HFmrEF patients was significantly higher than the control group (0.185±0.122; 0.141±0.120; p=0.013). The average sVEGFR-1 level of the HFrEF and HFmrEF patients using beta-blocker was significantly higher than the HFrEF and HFmrEF patients not using it (p=0.015). There was a significant and positive correlation between sVEGFR-1 and N-terminal pro-brain natriuretic peptide (pro-BNP) levels in the group with HF (r=0.211, p=0.044). Conclusion: It increases awareness about the role of sVEGFR-1 in HFrEF anf HFmrEF patients and the need for further studies. Beta-blocker may have a negative effect on angiogenesis in HFrEF and HFmrEF via increasing sVEGFR-1 levels. Additionally, Pro-BNP may contribute to inhibiting angiogenesis by increasing sVEGFR-1 levels and sVEGFR-1 may be an important biomarker in HFrEF and HFmrEF.

Abstract B153: sVEGFR-1 functionalized nanoparticles for diagnosis of cancer using magnetic resonance imaging.

Cancer is considered to be an important public health problem throughout the world. Early diagnosis of cancer and recurrent tumors are extremely important for reducing mortality rates and also for following up the response to treatment. The increased need of oxygen leads to new vascular structures in and around the tumor. The most important components of this angiogenic process are vascular endothelial growth factor (VEGF) and its receptors which are overexpressed in tumor cells. The aim of this study is to develop soluble VEGF receptor-1 (sVEGFR-1) functionalized iron oxide nanoparticles for diagnosis of early stages of tumors or metastases by MRI. A single dose of N-nitroso-N-methylurea (NMU) at a concentration of 50 mg/kg was injected to inbred rats intraperitoneally on the 21st day of their birth to create breast cancer. In this study, in vivo characteristics of functionalized nanoparticles were studied by comparing rats with tumors and the healthy controls. Approximately 70 days later, tumor formed rats were administered with nanoparticles functionalized with sVEGFR-1, intravenously and scanned with MRI. MR images were taken before the treatment with nanoparticles (0 minute), and at 30th and 60th minutes after treatment. The evaluation of MRI data revealed that the images of metastases were hypointense at 30th minute with respect to the 0 minute and 60th minute images (p Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B153. Citation Format: Taner Ozgurtas, Fatih Yesildal, Fevzi Nuri Aydin, Melis Sardan, Gozde Uzunalli, Murat Kocaoglu, Ayse Begum Tekinay, Mustafa Ozgur Guler, Rukan Genc, Salih Deveci. sVEGFR-1 functionalized nanoparticles for diagnosis of cancer using magnetic resonance imaging. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B153.