Laser microdissection-based analysis of hypoxia- and thioredoxin-related genes in human stable carotid plaques

Neovascularization in the carotid atherosclerotic plaque is a common pathogenetic feature in carotid artery stenosis. To investigate whether the neovascular region of the stable plaque differentially expresses specific genes, we analyzed the patterns of angiogenesis-related gene expression in regions of the plaque isolated by laser microdissection and examined by immunohistochemistry and real-time reverse transcription (RT)-polymerase chain reaction (PCR).

Carotid plaque samples were obtained by carotid endarterectomy in 27 clinically asymptomatic patients with high-grade internal carotid artery stenosis. Among these 27 plaque samples, 23 plaques were confirmed to be stable pathologically, and 14 stable plaques had neovascularization. The medial, shoulder, and neovascular regions of the 14 carotid plaques were determined by immunohistochemical staining. These 3 regions were microdissected, and total RNA was extracted for real-time RT-PCR analysis. The expressions of hypoxia inducible factor 1alpha, vascular endothelial growth factor-A, thioredoxin, and thioredoxin interacting protein were analyzed at mRNA level.

Real-time RT-PCR was performed on 42 laser microdissected regions of 14 plaques. The expressions of all four genes examined were significantly lower in the medial region at mRNA level. High expressions were noted in both shoulder and neovascular regions, with no significant difference between the two. Furthermore, these expression patterns were related significantly to macrophage infiltration. In conclusion, hypoxia- and thioredoxin-related genes are significantly overexpressed in human stable carotid atherosclerotic plaques and strongly correlate with macrophage infiltration rather than neovascularization. Macrophage infiltration may lead to overexpression of these genes and promote angiogenesis in stable carotid plaques.