Identification of a by-product of nitric oxide synthase activity in human acute brain injury with in vivo proton magnetic resonance spectroscopy

BACKGROUND AND PURPOSE: Laboratory studies have been used to identify nitric oxide as a notable mediator in neuronal death after acute brain injury. To our knowledge, this has not previously been confirmed with in vivo study in humans. Our purpose was to seek in vivo evidence for the induction of nitric oxide synthase (NOS) in human acute brain injury by using proton MR spectroscopy. METHODS: In vitro proton MR spectra were obtained in neural extracts from 30 human cadavers, and in vivo spectra were obtained in 20 patients with acute brain injury and in a similar number of control subjects. RESULTS: We identified a unique peak at 3.15 ppm by using in vivo proton MR spectroscopy in eight of 20 patients with acute brain injury but not in 20 healthy volunteers (P &lt; .002). On the basis of in vitro data, we have tentatively assigned this peak to citrulline, a NOS by-product. CONCLUSION: To our knowledge, our findings suggest, for the first time, that excitotoxicity may occur in human acute brain injury. Confirmation with the acquisition of spectra in very early acute cerebral injury would provide a rationale for the use of neuroprotective agents in these conditions, as well as a new noninvasive method for quantification. <br /

Integrated mutation, copy number and expression profiling in resectable non-small cell lung cancer

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to identify critical genes involved in non-small cell lung cancer (NSCLC) pathogenesis that may lead to a more complete understanding of this disease and identify novel molecular targets for use in the development of more effective therapies.</p> <p>Methods</p> <p>Both transcriptional and genomic profiling were performed on 69 resected NSCLC specimens and results correlated with mutational analyses and clinical data to identify genetic alterations associated with groups of interest.</p> <p>Results</p> <p>Combined analyses identified specific patterns of genetic alteration associated with adenocarcinoma vs. squamous differentiation; <it>KRAS </it>mutation; <it>TP53 </it>mutation, metastatic potential and disease recurrence and survival. Amplification of 3q was associated with mutations in <it>TP53 </it>in adenocarcinoma. A prognostic signature for disease recurrence, reflecting <it>KRAS </it>pathway activation, was validated in an independent test set.</p> <p>Conclusions</p> <p>These results may provide the first steps in identifying new predictive biomarkers and targets for novel therapies, thus improving outcomes for patients with this deadly disease.</p

Vascular Endothelial Growth Factor Receptor-3 Directly Interacts with Phosphatidylinositol 3-Kinase to Regulate Lymphangiogenesis

Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCc1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis

Direct interaction between PI3K p85 and VEGFR-3 in metastatic small cell lung carcinoma.

<p>VEGFR-3/PI3K complexes (<i>red</i>) detected by <i>in situ</i> PLA in lung tumor area and lymphatic vessels (<i>green,</i> podoplanin). Lymphatic vessel VEGFR-3/PI3K signals (<i>red</i>) and their representative co-staining with podoplanin (<i>green</i>) in metastatic (<i>A, top and middle panel,</i> respectively) and non-metastatic (<i>B, top and middle panel,</i> respectively) in small cell lung cancer tissue. VEGFR-3/PI3K signals were detected in cancer cells surrounding the lymphatic vessels in the metastatic samples (<i>A</i>, <i>top and bottom panel</i>) as well as the lymphatic vessels; whereas the low signal in non-metastatic samples was detected mostly away from the lymphatic vessels (<i>B</i>, <i>bottom panel</i>), and not in tumor cells inside or surrounding the non-metastatic lymphatic vessels (<i>B</i>, <i>top and middle panels</i>). Bar, 25 µm. DAPI (<i>blue</i>); <i>C.</i> Quantification of PLA signals (at least 5 different regions in each sample) in the lymphatic vessels (LV) (<i>C</i>, <i>left</i>) in lymph node negative (LN-; n = 7; two samples were excluded because there were no lymphatic vessels present in the sections examined) and lymph node positive (LN+; n = 10) samples using Olink Imaging Software. Plotted are mean values for individual patients. Line represents median value. *<i>P</i>&lt;0.05.</p

Blocking PI3K inhibits VEGF-C-induced LEC tube formation and migration.

<p>Effect of anti-VEGFR-3 antibody (hF4-3C5, 20 µg/ml), selective PI3Kγ inhibitor (AS252424, 5 µM), Raf/MEK inhibitor (PD98059, 5 µM) and PLCγ1 inhibitor (U-73122, 1 µM) on VEGF-C-induced (200 ng/ml) LEC tube formation (<i>A</i>) and migration (<i>B</i>, <i>left panel</i>). Effect of PLCγ1 siRNA or non-targeting control (NTC) on LEC VEGF-C-induced migration (<i>B</i>, <i>right panel</i>). Control serum-free (with DMSO as appropriate) LEC is indicated by ‘0′. *<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01, ***<i>P</i>&lt;0.001, compared to VEGF-C treated cells/NTC. n = 3. Data shown as mean±s.e.m.</p

VEGF-C induces PI3Kγ-dependent P70S6K (A), eNOS (B), and PLCγ1 (C) phosphorylation via VEGFR-3 in LECs.

<p>Western blotting analysis of phosphorylated P70S6K (<i>A, top left</i>) and eNOS (S1177) (<i>B, top left</i>) in prostatic LECs following 15 minute stimulation with ligand: VEGF-C (100 ng/ml), VEGF-A (100 ng/ml), VEGF-C156S (250 ng/ml), VEGF-D (250 ng/ml) and VEGF-E (100 ng/ml). Time- and concentration-dependent phosphorylation of P70S6K (<i>A, top right</i>), eNOS (S1177) (<i>B, top right</i>), and PLCγ1 (Tyr783) (<i>C, top left</i>) in LECs in response to VEGF-C. The effect of inhibition of VEGFR-3 (hF4-3C5), VEGFR-2 (IMC-1121b) or VEGFR-1 (IMC-18F1) on phosphorylation of P70S6K (<i>A, bottom left</i>), eNOS (S1177) (<i>B, bottom left</i>); and VEGFR-3 (hF4-3C5) on PLCγ1 (Tyr783) (<i>C, bottom right</i>) on LEC response to VEGF-C (100 ng/ml). The effect of AS252424, LY294002, PD98059, and U-73122 on phosphorylation of P70S6K (<i>A, top right</i>), eNOS (S1177) (<i>B, bottom right</i>), and PLCγ1 (Tyr783) (<i>C, bottom right</i>) on LECs in response to VEGF-C. The effect of VEGF-C on phosphorylation of PLCγ2 (Tyr759, Tyr1217) in LECs (<i>C, bottom left</i>). Control serum-free vehicle treated LEC lysate is indicated by ‘0′ in all blots. n = 3. Densitometry analysis is shown under each blot in italics; where integrated intensity of phosphorylated molecules was firstly compared to that of the total target protein for each sample, and then expressed as fold change in integrated density compared to either serum-free (A, <i>top left and right panels</i>; B, <i>top left and right panels</i>; C, <i>top and bottom left panels</i>) or VEGF-C treated control samples (A, <i>bottom left and right panels</i>; B, <i>bottom left and right panels</i>; C, <i>bottom and bottom left panels</i>).</p

Direct interaction between PI3K p85 and VEGFR-3 in metastatic melanoma (A), breast (B) and colon (C) cancers.

<p>PI3K p85/VEGFR-3 complexes (<i>red</i>) detected by <i>in situ</i> PLA in tumor cells and lymphatic vessels (<i>green,</i> podoplanin). Bar, 50 µm. Nuclei stained with DAPI (<i>blue</i>).</p

Differential expression of VEGF ligands and receptors in prostate cancer

BACKGROUND\ud Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer.\ud \ud METHODS\ud The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer.\ud \ud RESULTS\ud Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue.\ud \ud CONCLUSIONS\ud These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer

VEGF-C induces LEC tube formation.

<p><i>A</i>, Prostatic LEC tube length at 4.5 hours post VEGF-C treatment was quantified using ImageJ. VEGF-C significantly increased the number of tubes formed compared to vehicle control. Data expressed as mean±s.e.m., n = 3, ***<i>P</i>&lt;0.001 using One-way ANOVA, Bonferroni post-analysis. <i>B</i>, Western blotting analysis of VEGFR-2 and VEGFR-3 expression in lung, neonatal dermis and prostate LECs. β-tubulin was used as a loading control.</p