Characterization of differential gene expression in quiescent and invasive human arterial smooth muscle cells.
Proliferation, migration and invasion of smooth muscle cells (SMCs) are essential pathogenic processes in the development of a broad spectrum of cardiovascular disorders, like arteriosclerosis, restenosis after percutaneous transluminal angioplasty and stent implantation as well as transplant vessel disease. As an in vitro model mimicking these processes, the Boyden chamber was employed to characterize the diverging migratory and invasive potentials of proliferating and nonproliferating human arterial SMCs (haSMCs). Using this model, differential gene expression of both phenotypes was analyzed by a cDNA array system (Clontech human cardiovascular array). With these arrays, 558 cardiovascular-associated genes could be compared. Further, gene expression was exactly quantified by real-time RT-PCR. Protein expression was analyzed by ELISA and Western blotting. In total, 47 genes were differentially expressed more than 1.5 times. Most of the differentially regulated genes in this study were associated with the extracellular matrix (ECM) and cell motility. In detail, the respective groups were matrix-organizing proteins, ECM proteins, cell adhesion proteins, extracellular communication and cytoskeleton motility proteins. Genes known to be differentially regulated during haSMC migration and invasion, like TIMP 2, TIMP 3, and MMP 3, were confirmed by the array data. Reduced expression of several cytoskeletal proteins, like vimentin, fibronectin, cytokeratins and beta1 integrin, was shown in the invasive phenotype. Further, angio-associated protein, alpha E-catenin and atrial brain natriuretic peptide receptor were downregulated whereas TFPI 2 was strongly upregulated in invasive haSMCs. In conclusion, several relevant potential candidate genes for the quiescent and the invasive SMC phenotype were identified and genes already known to be differentially regulated by previous analysis were confirmed.
Medical Clinic I, University Hospital RWTH Aachen, Aachen, Germany. ruediger.blindt@post.rwth-aachen.de
Rüdiger
R
Blindt
Felix
F
Vogt
Daniel
D
Lamby
Ute
U
Zeiffer
Nicole
N
Krott
Kristina
K
Hilger-Eversheim
Peter
P
Hanrath
Jürgen
J
vom Dahl
Anja-Katrin
AK
Bosserhoff
D001161:Arteriosclerosis / Q000473:pathology; D002455:Cell Division; D002465:Cell Movement; D002478:Cells, Cultured; D023903:Coronary Restenosis / Q000473:pathology; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics; D007633:Keratins / Q000235:genetics; D008323:Mammary Arteries; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics; D014746:Vimentin / Q000235:genetics
D005353:Fibronectins; D019012:Integrin beta1; D019717:Tissue Inhibitor of Metalloproteinase-3; D014746:Vimentin; D019716:Tissue Inhibitor of Metalloproteinase-2; D007633:Keratins; D019278:Matrix Metalloproteinase 3