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Microarray-based gene expression of rabbit basilar artery smooth muscle cells exposed to hemodynamic insult

ABSTRACT: High-flow causes the remodeling of arteries, in which smooth muscle cells play an important role. To know the profile of smooth muscle gene expression under high-flow conditions in vivo, flow of rabbit basilar artery was increased by ligation of both common carotid arteries. Microarrays were performed to profile the gene expression of smooth muscle cells isolated from rabbit basilar artery. Expression profiles indicate 43603 differentially expressed genes in smooth muscle cells exposed to high-flow insult compared with the sham control, of which 1470 genes were upregulated and 780 genes downregulated using 2 fold-changes and P<0.05 as a cut-off. Bilateral common carotid arteries of female New Zealand White rabbits were ligated to increase vascular flow.The control group was performed the same procedure to expose the CCAs without ligation. Rabbits were euthanized at day 5 after ligation or exposure of bilateral CCAs in both groups (n=3 for each group). The rabbits used and all procedures in this study were approved by the local Institutional Animal Care and Use Committee. Smooth muscle cells were isolated. After euthanization of rabbits, the whole basilar arteries were removed. The arteries were cleaned in PBS buffer,cannulated and perfused at a constant flow with a cocktail which contains PBS and 0.4 mg/ml elastase (Sigma) and 1 mg/ml collagenase (type 1A, Sigma). After an incubation time of 45 min, the tissue left was removed and stored in PBS. SMCs were released from the artery by trituration. Then Total RNA was extracted and gene chip tests were performed.

ORGANISM(S): Oryctolagus cuniculus

SUBMITTER: Zhaolong Zhang

PROVIDER: E-GEOD-61212 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:IRF9 is ubiquitously expressed and mediates the effects of IFNs, previous study showed that IRF9 played an important role in immunity and cell fate decision. Our recent study revealed that IRF9 involved in cardiac hypertrophy, hepatic steatosis and insulin resistance. However, the function of IRF9 in VSMC and neointima formation was largely unknown. We found that IRF9 expression was significantly increased in the VSMCs of mouse carotid artery. More importantly, we generated SMC-specific IRF9 overexpression transgenic mice (IRF9 TG) and found that IRF9 TG significantly increased VSMC proliferation, migration and neointima formation compared with NTG mice in response to injury. To evaluate the underlying mechanism by which IRF9 promotes VSMC proliferation and migration after vascular injury, IRF9 TG and NTG mice were subjected to wire-injury and the carotid arteries were collected at 14 days post-injury. We combined 3-5 vessels for one sample, and 3 samples for each phenotype. Subsequently, a total of 400ng RNA was used following Affymetrix instruction and 10 ug of cRNA were hybridized for 16 hr at 45°. GeneChips were scanned using the Scanner 7G and the data was analyzed with Expression Console using Affymetrix default analysis settings and global scaling as normalization method. RMA analysis was employed to evaluate the gene expression. We used microarrays to detect the global gene expression in the carotid arteries of smooth muscle cell specific IRF9 transgenic mice(IRF9 TG) compared with non transgenic control mice (NTG) at 14 days post-injury and identified distinct classes of altered genes. non-transgenic controls mice (NTG) and smooth muscle specific IRF9 transgenic mice (IRF9 TG) were subjected to wire-injury and the carotid ateries were collected at 14 days post-injury. We combine 3-5 vessels in one tube and for a single Affymetrix microarray. Total RNA was extracted and a total of 400ng RNA was used following Affymetrix instruction. 3 biological samples for each genotype.

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Microarray-based gene expression of rabbit basilar artery smooth muscle cells exposed to hemodynamic insult

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