VEGF and SDF-1 are not only angiogenesis stimulators, but also mobilizers of BM stem cells

presented as %; microalbuminuria was defined as >3 mg/dl in spot urine albumin specific-dipstick; statistically significant differences between the groups were marked with “”. doi:10.1371/journal.pone.0126190.t001 differences between the subjects in exposition to the pollution that could affect serum levels of measured cytokines. Fasting blood samples were drawn from all the participants in sitting position from forearm vein and subsequently were used to measure standard clinical parameters and serum concentration of endostatin, VEGF, IL-8, angiogenin, and bFGF. The study was conducted in accordance with the Declaration of Helsinki principles and was approved by the Ethics Committee of the Medical University of Gdask, Poland. Written informed consent was received from all the participants. Sample preparation Serum was obtained from blood samples after centrifugation at 500xg for 15 minutes. Then serum was apportioned into 0.5 ml aliquots, and stored at -80C until analysis. 3 / 17 Angiogenesis in Hypertension TCh = total cholesterol, LDL = low-density lipoprotein, HDL = high-density lipoprotein, TG = triglicerides doi:10.1371/journal.pone.0126190.t002 Analysis of concentration of selected mediators affecting angiogenesis Angiogenin and endostatin concentration were measured with ELISA Quantikine tests according to the manufacturer instructions. The tests’ sensitivities were 6 pg/ml and 0.063 ng/ml, respectively. VEGF, IL-8, and bFGF were analyzed with flow cytometer using Cytometric Bead Array Human Soluble Protein Flex Sets. The test sensitivities for measured proteins were as follow: 4.5 pg/ml, 1.2 pg/ml, and 3.4 pg/ml. Both methods, CBA and ELISA interpolate concentrations of the measured cytokines from the standard curves, quantifying their levels in analyzed samples and provide comparable sensitivity and specificity. The main advantage of CBA technique in (S)-(-)-Blebbistatin web comparison with traditional ELISA is low sample volume required and low time to results ratio, as multiple cytokines can be measured in 1 tube simultaneously. Nevertheless, endostatin and angiogenin were measured with conventional ELISA, because they are present in human serum in significantly higher concentrations than VEGF, IL-8 and bFGF. Measurement of serum endostatin and angiogenin concentrations requires multiple dilutions of the samples that makes VEGF, IL-8 and bFGF undetectable, as they are present in serum in about 1000 fold lower levels than endostatin and angiogenin. 1 Statistical analysis The results were calculated with Statistica 10.0 software. As indicated by the distribution of the variables, data were analyzed with Mann-Whitney U test and Spearman’s rank correlation for nonparametric data. In addition, multiple regression analyses were performed to verify if age, body mass index and serum lipids affect levels of studied cytokines. Values of p<0.05 were considered statistically significant. Results Hypertension is associated with increased serum concentration of endostatin, VEGF and IL-8 Analysis showed that patients with hypertension were characterized by higher serum levels of endostatin, VEGF and IL-8, than healthy individuals. The same differences were found when the data were reanalyzed in PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 order to express the amount of the cytokine of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 interest in relation to the amount of total serum protein for each evaluated sample. The data are presented as medians, 2575% percentiles and minimum–maximum. Statistical significance is marked with “”. Endostatin concentration