HMGB-1 directly mediates retinal endothelial cell death.

<p>(<b>A</b>) Expression of HMGB-1 receptors (TLR4 and RAGE) in HMGB-1-treated (5 and 10 µg/ml) and control (PBS-treated) retinal endothelial cells. (<b>B</b>) Treatment with HMGB-1 (10 µg/ml) increases cell death in primary human retinal endothelial cell cultures. All experiments were repeated at least three times (*P<0.05, **P<0.01). (<b>C</b>) Endothelial cell death was determined using annexin V and annexinV/PI to identify apoptotic and necrotic cells, respectively.</p

Glia mediates the effect of HMGB-1.

<p>(<b>A</b>) Primary pericytes co-cultured with glia were treated with HMGB-1 (5 µg/mL, 24 hours). Pericyte survival was assessed using an apoptosis assay kit. All experiments were repeated at least three times (**P<0.01). (<b>B</b>) Representative confocal images.</p

List of PCR primers.

<p>List of PCR primers.</p

HMGB-1 does not directly mediate pericyte death.

<p>(<b>A</b>) Treatment with HMGB-1 (10 µg/ml) does not increase cell death in pure primary human pericyte cultures. All experiments were repeated at least three times. (<b>B</b>) Pericyte death was determined using annexin V as a marker of apoptotic cells and annexinV/propidium iodide (PI) to identify necrotic cells.</p

HMGB-1 can affect retinal endothelial cell activity.

<p>(<b>A</b>) Expression of <i>CCL2</i> and <i>CCL5</i> chemokines, <i>IL-1β</i> and <i>IL-6</i> cytokines, as well as cell adhesion molecule <i>ICAM-1</i> in HMGB-1-treated (5 and 10 µg/ml) and control (PBS-treated) retinal endothelial cells. Gene expression was assessed using quantitative RT-PCR in cells exposed to HMGB-1 or PBS after 24 hours (*P<0.05, **P<0.01, n = 6). (<b>B</b>) Comparative analysis of chemokine and cytokine expression in HMGB-1 treated retinal endothelial cells.</p

There was no association found between the level of HMGB-1 and neovascularization in the retina.

<p>Neovascularization was induced by subretinally injecting Matrigel either with PBS (<b>A</b>) or mixed with HMGB-1 (<b>B</b>). Neovascularization was allowed to develop for 10 days. Blood vessels were labeled with DiI and visualized by fluorescence microscopy (white arrows indicate neovascularization in the Matrigel injected area. Ch, choroids; M, Matrigel, R retina. Scale bar: 200 µm). (<b>C</b>) Quantitative analysis shows no difference in the levels of neovascularization between HMGB1-treated eyes compared to control eyes. (<b>D</b>) <i>VEGF-A</i> expression was not changed in glial cells (astrocytea and microglia) treated with HMGB-1 and PBS (control). (<b>E</b>) Examination by western blot of vitreous humor from both hyperoxia-injured and room air control animals (obtained from postnatal days 15 and 17 pups) did not show a statistically significant difference between them. Results of analysis of western blots (top) are expressed as a percentage of corresponding value in the control eyes ± SEM (n = 6).</p

Significant positive correlations between vitreous fluid levels of vascular endothelial growth factor (VEGF) and levels of matrix metalloproteinase-1 (MMP-1) (A) and MMP-9 (B) and between vitreous fluid levels of MMP-1 and MMP-9 (C) in vitreous samples from 16 proliferative diabetic retinopathy and 24 nondiabetic control patients analyzed with mosaic multiplex matrix metalloproteinase (MMPs) enzyme-linked immunosorbent assay.

<p>Significant positive correlations between vitreous fluid levels of vascular endothelial growth factor (VEGF) and levels of matrix metalloproteinase-1 (MMP-1) (A) and MMP-9 (B) and between vitreous fluid levels of MMP-1 and MMP-9 (C) in vitreous samples from 16 proliferative diabetic retinopathy and 24 nondiabetic control patients analyzed with mosaic multiplex matrix metalloproteinase (MMPs) enzyme-linked immunosorbent assay.</p

Interactions among Vascular-Tone Modulators Contribute to High Altitude Pulmonary Edema and Augmented Vasoreactivity in Highlanders

<div><h3>Background</h3><p>The interactions among various biomarkers remained unexplored under the stressful environment of high-altitude. Present study evaluated interactions among biomarkers to study susceptibility for high altitude pulmonary edema (HAPE) in HAPE-patients (HAPE-p) and adaptation in highland natives (HLs); both in comparison to HAPE-free sojourners (HAPE-f).</p> <h3>Methodology/Principal Findings</h3><p>All the subjects were recruited at 3500 m. We measured clinical parameters, biochemical levels in plasma and gene expression using RNA from blood; analyzed various correlations between and among the clinical parameters, especially arterial oxygen saturation (SaO₂) and mean arterial pressure (MAP) and biochemical parameters like, asymmetric dimethylarginine (ADMA), serotonin (5-HT), 8-iso-prostaglandin F2α (8-isoPGF2α), endothelin-1 (ET-1), plasma renin activity (PRA), plasma aldosterone concentration (PAC), superoxide dismutase (SOD) and nitric oxide (NO) in HAPE-p, HAPE-f and HLs. ADMA, 5-HT, 8-isoPGF2α, ET-1 levels, and PAC were significantly higher (p<0.0001, each), whereas SOD activity and NO level were significantly lower in HAPE-p than HAPE-f (p≤0.001). Furthermore, ADMA, 5-HT, 8-isoPGF2α, NO levels and PAC were significantly higher (p<0.0001), whereas ET-1 level significantly (p<0.0001) and SOD activity non-significantly (p>0.05) lower in HLs than HAPE-f. The expression of respective genes differed in the three groups. In the correlations, SaO₂ inversely correlated with ADMA, 5-HT and 8-isoPGF2α and positively with SOD in HAPE-p (p≤0.009). MAP correlated positively with 5-HT and 8-isoPGF2α in HAPE-p and HLs (p≤0.004). A strong positive correlation was observed between ADMA and 5-HT, 5-HT and 8-isoPGF2α (p≤0.001), whereas inverse correlation of SOD with ET-1 in HAPE-p and HLs (p≤0.004), with 5-HT and 8-isoPGF2α in HAPE-p (p = 0.01) and with 5-HT in HLs (p = 0.05).</p> <h3>Conclusions/Significance</h3><p>The interactions among these markers confer enhanced vascular activity in HLs and HAPE in sojourners.</p> </div

Proliferative diabetic retinopathy epiretinal membranes.

<p>Negative control slide that was treated identically with an irrelevant antibody showing no labeling (A). Immunohistochemical staining matrix metalloproteinase-9 (MMP-9) showing immunoreactivity in endothelial cells (arrows), intravascular leukocytes (B) and stromal cells (C). Immunohistochemical staining for CD45 showing immunoreactivity in leukocytes expressing the leukocyte common antigen CD45 (D) Double immunohistochemistry for CD45 (brown) and MMP-9 (red) showing cells co-expressing CD45 and MMP-9 (arrows). No counterstain was applied (E) (original magnification X40).</p

Clinical characteristics of the HAPE-p, HAPE-f and HLs.

<p>Data are presented as mean ± standard deviation and are compared by Student's <i>t</i>-test. n, number of subjects; SBP, systolic blood pressure; DBP, diastolic blood pressure; MAP, mean arterial pressure; RR, respiratory rate; PR, pulse rate; SaO₂, arterial oxygen saturation; PASP, pulmonary artery systolic pressure.</p