High glucose conditions had no effect on apoptosis of retinal AC but enhanced their DNA synthesis and GFAP expression.

<p>(A) The rate of apoptosis was determined by TdT-dUTP Terminal Nick-End Labeling (TUNEL) staining. Positive cells were counted using a fluorescence microscope and calculated as percentage of total cell number per field. (B) Level of cleaved caspase-3 was determined by FACS analysis. Shaded areas show staining in the absence of primary antibody. (C) The percentage of retinal AC undergoing active DNA synthesis was determined using FACScan flow cytometery. Data are presented as Mean ± SEM. *<i>p</i><0.05; <i>n</i> = 3 (NG vs. HG and NG vs. NG+L-Glu). EdU is 5-ethynyl-2′-deoxyuridine. (D) Level of GFAP in retinal AC was determined by FACS analysis. High glucose conditions increased the mean fluorescence intensity of GFAP by 1.5-fold in retinal AC compared with normal glucose and osmolarity control conditions (P<0.05, n = 3). Shaded areas show staining in the absence of primary antibody.</p

Attenuation of retinal AC migration and network formation in Matrigel under high glucose conditions.

<p>(A) High glucose conditions inhibited the organization and network formation of retinal AC in Matrigel. (B) The quantitative assessment of the data. Data are the mean number of branch points from 5 high-power fields (×100) ± SEM. *<i>p</i><0.05; n = 5 (NG vs. HG and NG vs. NG+L-Glu). (C) Transwell migration of retinal AC under different glucose conditions. High glucose conditions significantly inhibited migration of retinal AC compared with normal glucose and osmolarity control. Data are presented as mean ± SEM. n = 3,*<i>p</i><0.05 (NG vs. HG). (D) Conditioned medium was collected from retinal AC incubated under normal glucose, high glucose, or osmolarity control for five days. The effects of these conditioned medium on the migration of retinal EC was determined in a transwell migration assay. Please note a significant decrease in migration of retinal EC incubated with conditioned medium from retinal AC cultured under high glucose conditions. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG)</p

The impact of high glucose conditions on various down-stream signaling pathways.

<p>The status of various signaling pathways in retinal AC under various glucose conditions was determined as described in Methods. Levels of active Src (A), Akt (B), ERK (C), p38 (D), JNK1 (E), Fyn (F) and p65 (G) in retinal AC under different glucose conditions were determined by Western blot analysis from equal amounts of cell lysates. The total level for each protein is shown in the lower part of each panel. The quantitative assessment of the data for each blot is shown below the blot. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu). Levels of active Fyn (F) in retinal AC under different glucose conditions was determined by Western blot analysis of immunoprecipitates from equal amounts of retinal AC lysates.</p

High glucose conditions enhanced the adhesion of retinal AC to extracellular matrix (ECM) proteins.

<p>Adhesion of retinal AC to fibronectin (A) and vitronectin (B) was determined by measuring the number of adherent cells in each well coated with different concentration of ECM proteins. Number of adherent cells was quantified by measuring the cellular phosphatase activity as described in Methods. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG). No significant adhesion to collagen I or laminin was observed (not shown). The impact of different glucose conditions on formation of actin stress fibers and focal adhesions in retinal AC. (C) Examination of actin stress fibers and focal adhesions in retinal AC. Retinal AC were stained with anti-vinculin (red), phalloidin (green), and DAPI (×630). Scale bar  = 20 µm. (D) Coherency of stress fibers was assessed using image J software. Data are presented as Mean ± SEM. (E) The number of focal adhesions was quantified per cell. Cells (5 to 10) in each condition were analyzed. Data are presented as Mean ± SEM. n ≥5; <i>p</i>>0.05 (NG vs. HG).</p

The effect of different glucose conditions on the expression of antioxidant enzymes.

<p>The expression of HO-1 (A) and Prdx2 (B) was determined by Western blot analysis. The quantification of data is also shown. The β-actin was used as loading control. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu).</p

The effects of various glucose conditions on PDGF-mediated migration of retinal AC.

<p>Migration of retinal AC incubated under different glucose conditions for five days was determined using transwell assay. Transwell assay was performed with basal medium or medium containing PDGF-AA (A) or PDGF-BB (B) in lower compartment. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (Basal vs. PDGF-AA or PDGF-BB). Please note PDFG-AA only enhanced the migration of AC under high glucose conditions. However, PDGF-BB enhanced AC migration under various glucose conditions.</p

Effects of NAC on the migration of retinal AC under different glucose conditions and capillary morphogenesis of retinal EC.

<p>(A) Incubation of retinal AC cultured under high glucose with NAC restored basal migration. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG). (B) Retinal EC were resuspended in conditioned medium collected from retinal AC incubated under different glucose conditions with or without NAC and plated on Matrigel to analyze capillary morphogenesis. (C) The quantitative assessment of capillary morphogenesis of retinal EC. Data are the mean number of branch points from 8 high-power fields (×100) ± SEM. *<i>p</i><0.05; n = 8 (NG vs. HG and HG vs. HG+NAC). NAC had no effect on capillary morphogenesis of retinal EC in the absence of AC conditioned medium (not shown).</p

Circulating retinol-binding protein 4 levels in gestational diabetes mellitus: a meta-analysis of observational studies

<div><p></p><p>Retinol-binding protein 4 (RBP4) is a novel adipocyte-derived cytokine playing an important role in the regulation of energy metabolism and insulin sensitivity. Although the association between RBP4 and metabolic dysfunction is well established, studies on the relationship between circulating RBP4 levels and the risk of gestational diabetes mellitus (GDM) have yielded inconclusive results. We performed a meta-analysis to investigate whether women with GDM had higher circulating RBP4 levels than the normglycemic pregnant women. PubMed, Web of Science and EMBASE were searched up to 1 August 2014. A total of 14 studies comprised of 884 women with GDM and 1251 normglycemic pregnant women were included. The overall results suggested that maternal circulating RBP4 levels were significantly higher in GDM than their normal controls (SMD: 0.49 μg/ml, 95% CI: 0.23–0.75 μg/ml, <i>p</i> < 0.001, random effect model). However, stratified results indicated that this significant difference only existed in the second/third trimester and was limited to Asian populations. Furthermore, subgroup analysis according to matched maternal age and BMI still demonstrated that GDM had higher circulating RBP4 levels than the normal controls. Our findings suggested that Asian women with GDM had increased circulating RBP4 levels in their second/third trimester.</p></div

The effect of different glucose conditions on the expression and secretion of thrombospondins 1 and 2.

<p>(A) Levels of TSP1 and TSP2 in cell lysates and conditioned medium collected from retinal AC were determined by Western blot analysis. The β-actin was used as loading control for cell lysates. Quantification for band intensity is also shown. (B): TSP1 in cell lysates. (C): TSP1 in conditioned medium. (D): TSP2 in cell lysates. (E): TSP2 in conditioned medium. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu). Please note a significant decrease in the amount of TSP1 and TSP2 secreted by retinal AC under high glucose and osmolarity control conditions. No differences were observed in the levels of fibronectin, osteopontin, SPARC, periostin, and tenascin-C (not shown).</p

Increased expression of inflammatory cytokines in retinal AC under different glucose conditions.

<p>The mRNA levels for inflammatory cytokines in retinal AC under different glucose conditions were investigated by quantitative real time PCR. TNF-α (A), MCP-1 (B) and IL-1β (C) levels are shown. n = 5; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu). (D) The iNOS protein level was determined by Western and the quantification of the data is also shown. The β-actin was used as loading control. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu).</p