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Transcriptional Profiling of Laser Capture Microdissected Rat Arterial Elements: Fenoldopam-induced Vascular Toxicity as a Model System

¹ Department of Safety Assessment
² Discovery Technology Group, GlaxoSmithKline, King of Prussia, Pennsylvania, USA

Correspondence: Address correspondence to: Deidre A. Dalmas, Department of Safety Assessment, 709 Swedeland Road, Mail Stop UE0376, King of Prussia, Pennsylvania 19406; e-mail:deidre.a.dalmas{at}gsk.com.

Transcriptional profiling of specific elements of vasculature from animal models of vascular toxicity is an approach to gain insight into molecular mechanisms of vascular injury. Feasibility of using laser capture microdissection (LCM) to evaluate differential gene expression in selected elements of mesenteric arteries (MA) from untreated rats and rats given a single vasotoxic dose of 100 mg/kg Fenoldopam and euthanized 1 or 4 hours postdose was assessed. Regions of MA (endothelial cells [EC] and vascular smooth muscle cells [VSMC]) were selectively microdissected from optimal-cutting-temperature (O.C.T.)-embedded-frozen tissue sections. RNA was isolated, linearly amplified (LA), and hybridized to Affymetrix GeneChips®. Enrichment for specific vascular elements was evident by unique gene-expression profiles. Statistical analysis indicated that Fenoldopam treatment resulted in differential expression of 333 versus 458 genes in EC and 371 versus 618 genes in VSMC at the 1-hour or 4-hour time point, respectively. Analysis of regulated EC and VSMC genes common to both time points identified several gene functions or pathways affected by treatment. Several genes were identified in EC and/or VSMC that have not been previously linked to vascular structure or function. These data indicate that tissue–element-enrichment by LCM in conjunction with LA and GeneChip analysis offers a refined approach for assessment of injury-mediated transcriptome changes in distinct elements of the vasculature.

Key Words: Fenoldopam • vascular injury • laser capture microdissection • endothelial cells • vascular smooth muscle cells • transcriptional profiling

Abbreviations: aRNA, amplified RNA • bp, basepairs • DA1, dopamine 1 receptor • DAB, 3,3-dimethylaminobenzidine • DIG, Digoxiginin • EC, endothelial cells • ECM, extracellular matrix • FFPE, formalin-fixed paraffin-embedded • GPCR, G-protein coupled receptor • H&E, hematoxylin & eosin • HO-1, heme oxygenase 1 • HRP, horse radish peroxidase • IHC, immunohistochemistry • IL-6, interleukin-6 • ISH, nonisotopic in situ hybridization • IVT, in vitro transcription • LA, linear amplification • LCM, laser capture microdissection • MA, mesenteric arteries • MAS, microarray analysis suite • msec, millisecond • mM, millimeter • MT, metallothionine • mW, milliwatts • NOS, nitric-oxide synthase • NOV, nephroblastoma overexpressed gene • O.C.T., optimal cutting temperature • PDE, phoshodiesterase • RP, riboprobes • RT, reverse transcriptase • RT-PCR, reverse transcriptase polymerase chain reaction • TaqMan, quantitative real-time RT-PCR • VCAM, vascular cell adhesion molecule-1 • VEGF, vascular endothelial growth factor • VSMC, vascular smooth muscle cells • 18S, 18S ribosomal RNA

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