Thrombus organization and healing in the swine experimental aneurysm model. Part I. A histological and molecular analysis

Daniel Lee M.D., Ichiro Yuki M.D., Yuichi Murayama M.D., Alexander Chiang B.S., Ichiro Nishimura D.M.SC., D.M.D., Harry V. Vinters M.D., Chiachien J. Wang Ph.D., Yih-Lin Nien M.D., Akira Ishii M.D., Benjamin M. WU D.D.S., Ph.D and Fernando Viñuela M.D.
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Object

The authors describe the process of thrombus organization in the swine surgical aneurysm model.

Methods

Lateral carotid artery aneurysms with immediately induced thrombosis were created in 31 swine for a time-course study. Aneurysms were evaluated at 1, 3, 7, 14, 30, and 90 days after they were created. Histological analyses included quantitative immunohistochemical studies and evaluation of collagen deposition. Complementary DNA microarray analysis was performed for gene expression profiling. The lists of up- and downregulated genes were cross-matched with lists of genes known to be associated with cytokines or the extracellular matrix. The expression of selected genes was quantified using real-time polymerase chain reaction. Functional clustering was performed with the Expression Analysis Systematic Explorer (EASE) bioinformatics package.

Results

Histological analysis demonstrated leukocyte and macrophage infiltration in the thrombus at Day 3, myofibroblast infiltration at Days 7 to 14, and progressive collagen deposition and contraction thereafter. Tissue organization occurred in a centripetal fashion. A previously undescribed reticular network of connective tissue was observed at the periphery of the aneurysm at Day 3. Macrophages appeared critical to this thrombus organization. A total of 1109 genes were significantly changed from reference time zero during the time course: CXCL14, which produces a monocyte-specific chemokine, was upregulated over 100-fold throughout the time course; IGF1 was upregulated fourfold at Day 7, whereas IGFBP2 was downregulated approximately 50% at Days 7 and 14. Osteopontin (SPP1) upregulation increased from 30-fold at Day 30 to 45-fold at Day 14. The EASE analysis yielded eight functional classes of gene expression.

Conclusions

This investigation provides a detailed histological and molecular analysis of thrombus organization in the swine aneurysm model. The companion study will describe the effect of embolic bioabsorbable polymers on this process.

Abbreviations used in this paper:α-SMA = α–smooth muscle actin; CCA = common carotid artery; EASE = Expression Analysis Systematic Explorer; ECM = extracellular matrix; IGF = insulin-like growth factor; IGFBP = IGF binding protein; PCR = polymerase chain reaction; TAMRA = 6-carboxytetramethylrhodamine; TGF = transforming growth factor; UCLA = University of California, Los Angeles; VEGF = vascular endothelial growth factor.

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Contributor Notes

Address reprint requests to: Daniel Lee, M.D., Division of Interventional Neuroradiology, UCLA Medical Center and David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, Los Angeles, California 90095-1721. email: daniell@mednet.ucla.edu.
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