Dataset Information

Recombinant Osteopontin Stabilizes Smooth Muscle Cell Phenotype via Integrin Receptor/Integrin-Linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage in Rats.

ABSTRACT: Recombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH).One hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked kinase small interfering RNA was intracerebroventricularly injected 48 hours before SAH. The integrin receptor antagonist fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), focal adhesion kinase inhibitor Fib-14, and Rac-1 inhibitor NSC23766 were administered 1 hour before SAH induction. rOPN was administered via the intracerebroventricular and nasal route after SAH. SAH grade, neurological scores, brain water content, brain swelling, hematoxylin and eosin staining, India ink angiography, Western blots, and immunofluorescence were used to study the mechanisms of rOPN on the vascular smooth muscle phenotypic transformation.The marker proteins of vascular smooth muscle phenotypic transformation ?-smooth muscle actin decreased and embryonic smooth muscle myosin heavy chain (SMemb) increased significantly at 24 and 72 hours in the cerebral arteries after SAH. rOPN prevented the changes of ?-smooth muscle actin and SMemb and significantly alleviated neurobehavioral dysfunction, increased the cross-sectional area and the lumen diameter of the cerebral arteries, reduced the brain water content and brain swelling, and improved the wall thickness of cerebral arteries. These effects of rOPN were abolished by GRGDSP, integrin-linked kinase small interfering RNA, and NSC23766. Intranasal application of rOPN at 3 hours after SAH also reduced neurological deficits.rOPN prevented the vascular smooth muscle phenotypic transformation and improved the neurological outcome, which was possibly mediated by the integrin receptor/integrin-linked kinase/Rac-1 pathway.

SUBMITTER: Wu J

PROVIDER: S-EPMC4846549 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Recombinant Osteopontin Stabilizes Smooth Muscle Cell Phenotype via Integrin Receptor/Integrin-Linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage in Rats.

Wu Jiang J Zhang Yang Y Yang Peng P Enkhjargal Budbazar B Manaenko Anatol A Tang Jiping J Pearce William J WJ Hartman Richard R Obenaus Andre A Chen Gang G Zhang John H JH

Stroke 20160322 5

<h4>Background and purpose</h4>Recombinant osteopontin (rOPN) has been reported to be neuroprotective in stroke animal models. The purpose of this study is to investigate a potential role and mechanism of nasal administration of rOPN on preserving the vascular smooth muscle phenotype in early brain injury after subarachnoid hemorrhage (SAH).<h4>Methods</h4>One hundred and ninety-two male adult Sprague-Dawley rats were used. The SAH model was induced by endovascular perforation. Integrin-linked k ...[more]

PMID: 27006454

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Project description:The diagnosis of cerebral vasospasm after Subarachnoid-Hemorrhage is currently very difficult, additional tools such as blood biomarkers are necessary. We tested the ability of gene expression profiles of blood cells to predict vasospasm. 32 patients suffering subarachnoid-hemorrhage were included in this prospective monocentre study. They were grouped according to have a complicated cerebral vasospasm (Vasospasm) or not (Control) and Paired according to age (+/- 10 years), sex, Fisher grade (+/- 1), location, smoking (at least 3 first parameters). Gene expression profiles of blood cells were determined using 25,000~gene microarray. Blood sample: 2.5 mL harvested in PAXgene® Blood RNA tubes (PreAnalytix) RNA extraction: PAXgene® Blood RNA kit (Qiagen). We used a Universel Reference RNA (Stratagene). RNA amplification and labelling: kit Amino Allyl MessageAmp II (Ambion). We hybridized 4 microarrays per patient using pangenomic microarrays from the "Réseau National des Génopôles" (Illkirch, France). 2 slides were hybridized with reference RNA labelled Cy3 and patient RNA labelled Cy5, and 2 slides were hybridized with reference RNA labelled Cy5 and patient RNA labelled Cy3. Hybridation : Agilent protocol with few modifications : 750 ng of each labelled RNA were hubriddized at 60°C during 17 hours in an Aglient hybridization oven. After washings, Slides were scanned with a GenePix 4000B scanner (Molecular Devices). Image intensity data were extracted with GenePix Pro 6.0 analysis software. Quantification of Cy3 and Cy5 and selection of good spots were performed using the MAIA software (Novikov E and Barillot E. Software package for automatic microarray image analysis (MAIA). The ACUITY software was then used to normalize log ratios Cy3/Cy5 with Lowess non linear normalization, to filter out genes not present in at least 3 slides out of 4, to evaluate the reproducibility of the 4 microarrays of each patient (hierarchical clustering, Self Organizing Maps). Statistical analyses to insure reproducibility was performed using Excel (correlation coefficients, ANOVA). Only slides that passed all reprocubility tests were validated.

Dataset Information

Recombinant Osteopontin Stabilizes Smooth Muscle Cell Phenotype via Integrin Receptor/Integrin-Linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage in Rats.

Publications

Recombinant Osteopontin Stabilizes Smooth Muscle Cell Phenotype via Integrin Receptor/Integrin-Linked Kinase/Rac-1 Pathway After Subarachnoid Hemorrhage in Rats.

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