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Transcriptional profiling of laser capture microdissected rat arterial elements: fenoldopam-induced vascular toxicity
Deidre A Dalmas1, Marshall S Scicchitano, Yifeng Chen
1Department of Safety Assessment, 709 Swedeland Road, Mail Stop UE0376, King of Prussia, Pennsylvania 19406, USA. deidre.a.dalmas@gsk.com
Abstract:
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.
Insights
Laser capture microdissection (LCM) precisely isolates vascular cells for gene expression analysis. This method reveals molecular changes in endothelial cells and smooth muscle cells following vasotoxic drug exposure.
Area of Science:
- Vascular Biology
- Toxicology
- Molecular Biology
Background:
- Understanding molecular mechanisms of vascular injury is crucial.
- Transcriptional profiling of specific vascular elements aids in studying vascular toxicity.
- Laser capture microdissection (LCM) is a technique for isolating cells from tissue.
Purpose of the Study:
- To assess the feasibility of using LCM to evaluate differential gene expression in specific vascular cells.
- To investigate molecular changes in mesenteric arteries (MA) after exposure to a vasotoxic drug, Fenoldopam.
- To analyze gene expression in endothelial cells (EC) and vascular smooth muscle cells (VSMC) at different time points post-treatment.
Main Methods:
- Laser capture microdissection (LCM) was used to isolate EC and VSMC from rat mesenteric arteries.
- RNA was extracted, linearly amplified (LA), and hybridized to Affymetrix GeneChips for transcriptional profiling.
- Differential gene expression analysis was performed on samples from control rats and rats treated with Fenoldopam.
Main Results:
- LCM successfully enriched specific vascular elements, confirmed by unique gene-expression profiles.
- Fenoldopam treatment induced differential expression of hundreds of genes in both EC and VSMC at 1 and 4 hours post-treatment.
- Analysis identified affected gene functions and pathways, including novel genes linked to vascular function.
Conclusions:
- Tissue-element enrichment using LCM combined with linear amplification (LA) and GeneChip analysis provides a refined method for studying injury-mediated transcriptome changes.
- This approach allows for detailed assessment of molecular responses in distinct vascular cell types.
- The study identified specific genes and pathways affected by vasotoxic injury in EC and VSMC.
