ECM-related gene expression profile in vascular smooth muscle cells from human saphenous vein and internal thoracic artery.

UNLABELLED: Currently, Saphenous vein (SV) and internal thoracic artery (ITA) are still the most common graft materials in Coronary Artery Bypass Grafting (CABG) whereas SV graft have a lower long-term patency than ITA. Vascular smooth muscle cells (VSMCs) phenotype conversion, proliferation and migration may play a key role in mechanism of vein graft restenosis. To explore differential gene expression profile in VSMCs from SV and ITA will help to further elucidate the mechanism of VSMCs in vein graft restenosis after CABG and to provide new thread of gene therapy. METHODS: VSMCs from paired SV and ITA were cultured for experiments of Affymetrix microarrays and verification using FQ RT-PCR, while the database for annotation, visualization and integrated discovery bioinformatics resources (DAVID 2.0) was utilized for bioinformatics analysis of differential gene expression profile between SV VSMCs and ITA VSMCs. RNA of tunica media from SV and ITA segments were extracted for FQ RT-PCR to display differential expression of PLAT RESULTS: 54,613 probe sets were examined by gene microarray experiments. In SV VSMCs, 1,075 genes were up-regulated and 406 of them were higher than two-fold; 1,399 genes were down-regulated and 424 of them were lower than two-fold as compare with ITA VSMCs.14 ECM-related genes differentially expressed were verificated and listed as following: COL4A4, COL11A1, FN1, TNC, THBS, FBLN, MMP3, MMP9, TIMP3, WNT5A, SGCD were higher whereas COL14A1, ELN, PLAT lower in SV VSMCs than ITA VSMCs. In addition, PLAT was lower in tunica media from SV segments than ITA. CONCLUSION: VSMCs from SV and ITA have distinct phenotypes characteristics. Both promoting and inhibiting migration ECM-related genes were higher in VSMCs from SV as compared with ITA, suggesting that VSMCs from SV have more potential migrating capability whereas less PLAT both in SV VSMCs and vascular tissue,implying that SV may prone to be restenosis after CABG.

Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells.

Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.

[p16INK4a expression mediated by recombinant adenovirus can induce senescence of A549 cells].

OBJECTIVE: To construct E1-deletion and replication-defective human type 5 recombinant adenovirus vector and to study the effect of p16INK4a on proliferation and aging of A549 cells. METHODS: p16INK4a cDNA was cloned into pAdCMV to construct recombinant pAdCMV p16INK4a, which was co-transfected into 293 cell together with pJM17. The recombinant p16INK4a adenovirus (Ad-p16INK4a) was generated by homologous recombination and identified with duplex PCR. Lung cancer cell A549, which has a homozygous deletion of p16INK4a gene, was infected with the prepared Ad-p16INK4a virus. X-gal staining and TRAP-ELISA were used for detecting senescence-associated beta-galactosidase and telomerase activities in A549 cells. RESULTS: Immunohistochemical staining and Western blot indicated that p16INK4a gene was transferred into A549 cell with more than 95% efficiency by recombinant adenovirus and p16INK4a protein was expressed at a high level- p16INK4a could markedly inhibit growth of A549 cells, induced expression of senescence-associated beta-galactosidase and suppressed telomerase activity in A549 cells. CONCLUSION: Recombinant adenovirus vector could efficiently mediate transfer and expression of foreign genes in human cell and could be used for gene immunization and gene therapy; p16INK4a could inhibit A549 cell growth and induce its replicative senescence.