Laboratory Investigation
Early Changes of Gene Expression Profiles in the Rat Model of Arterial Injury

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Abstract

Purpose

Restenosis caused by intimal hyperplasia (IH) remains a significant drawback for vascular interventions. It is crucial to understand the molecular mechanisms that control activation of smooth muscle cells (SMCs) after the injury in order to develop strategies to prevent IH. The purpose of the present study was to investigate the early alterations in arterial-wall gene expression after balloon injury in the rat carotid artery with focus on the induction of an inflammatory response.

Materials and Methods

Twenty-four male Sprague–Dawley rats were subjected to injury of the left common carotid artery by using a 2-F Fogarty catheter. The arteries were harvested 5, 10, and 20 hours after injury. Uninjured arteries from an additional eight rats were used as controls. RNA was isolated and used for genome-wide microarray expression analysis, followed by validation of selected genes with quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed on the cross-sectioned vessels.

Results

Analysis of gene expression by microarrays showed that the most differentially expressed genes were primarily associated with inflammation, cell proliferation, migration, and adhesion. As confirmed by qRT-PCR, microarray data showed a significant (P < .005) upregulation of cytokines and chemokines (IL-6, CCL2, CXCL1, AIMP1, and CD44) just 5 hours after injury. Immunohistochemistry demonstrated that CCL2 and the adhesion receptor CD44 were expressed by SMCs in the early response to injury and in the absence of leukocyte infiltration.

Conclusions

Arterial injury is followed by an early induction of inflammatory genes in the vessel wall that appears to be confined to SMCs.

Section snippets

Study Design

As shown on the flowchart depicting the study design (Fig 1), a total of 32 rats were used in the study. The animals were euthanized with isoflurane 5, 10, or 20 hours after vascular injury, and the injured left common carotid arteries (CCAs) were harvested (n = 8 animals in each group). Arteries were rinsed with phosphate-buffered saline solution to remove blood. Uninjured left CCAs from an additional eight animals were used as controls. Total RNA was isolated from whole arteries (n = 4 per

Time-Dependent Changes in Gene Expression Profiles

A total of 2,480 genes were differentially expressed at one or more of the designated sampling times (P < .005; more than twofold change in gene expression). In the group of genes known to be associated with an inflammatory response, 74% were upregulated at one or more time intervals (Fig 2). Four temporal clusters of gene expression were revealed (Fig 3a): genes that reached maximum expression at 5 hours after injury and then decreased (cluster I), genes upregulated at 5 hours with sustained

Discussion

Restenosis and vein graft disease remains a limitation of long-term patency after vascular reconstructions. These interventions trigger vessel wall repair processes with activation of the SMCs and inflammatory responses, which lead to the formation of IH and lesions that jeopardize patency of the reconstructed vessel segment. To develop strategies to prevent IH, it is crucial to understand the molecular pathways that control SMC function in vessel wall healing. Although previous studies have

Acknowledgments

The authors thank Mariette Lengquist for excellent help with double immunostaining.

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    The work was supported by grants from the Ministry of Education and Science of the Russian Federation (project P1308), RFBR (project 14-04-01833), Swedish Heart–Lung Foundation, Swedish Research Council, and Cardiovascular Program funded by the Stockholm Council and Karolinska Institutet. None of the authors have identified a conflict of interest.

    An Appendix, Figures E1 and E2, and Tables E1–E3 are available online at www.jvir.org. A list of gene and protein abbreviations used is provided in the Appendix.

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