S. Tsai1,2, D. Ramnarain1, M. Kanchwala4, C. Xing4, J. Herz3 1University Of Texas Southwestern Medical Center,Vascular Surgery,Dallas, TX, USA 2Dallas Veterans Affairs Medical Center,Vascular Surgery,Dallas, TX, USA 3University Of Texas Southwestern Medical Center,Molecular Genetics,Dallas, TX, USA 4University Of Texas Southwestern Medical Center,Clinical Sciences,Dallas, TX, USA
Introduction: LDL receptor related protein 1 (LRP1) is an integral regulator of vascular wall structural stability. A mouse model generated by targeted deletion of LRP1 in smooth muscle cells (smLRP1-/-) in the context of hyperlipidemia is characterized by aggressive aortic atherosclerosis and aneurysmal degeneration. Even under normolipidemic conditions, smLRP1-/- mice have evidence of structural abnormalities in the aortic wall. The objective of this study was to understand the enhanced gene expression patterns and signaling pathways driving the changes in aortic wall structure using whole transcriptome sequencing.
Methods: The aortas of 6 smLRP1-/- mice and their wild type littermate controls were harvested into RNAlater. The adventitia and peri-adventitial tissues were stripped using micro-dissection techniques. RNA was extracted and pooled RNA from each genotype was submitted for whole transcriptome analysis, which was performed in duplicate. Whole transcriptome data was mapped with TopHat and differential expression analysis was carried out using EdgeR. Genes with false detection rate (FDR) <0.05 (999 genes) were used for pathway/network analysis using Ingenuity Pathway Analysis (IPA).
Results: LRP1 deficiency in aortic smooth muscle cells (SMCs) was confirmed by immunohistochemistry of descending thoracic aortic cross sections from smLRP1-/- and wild type mice. Elastin staining revealed diffuse elastin breaks in the smLRP1-/- aortas, accompanied by thickened medial layers of the aortic wall (Figure). Among the genes that were significantly upregulated in the smLRP1-/- aorta were 15 collagen genes, including the fibrillar collagens Col1a1 (14.92 fold change, FDR<0.01), Col1a2 (6.39 fold change, FDR<0.01), and Col3a1 (5.82 fold change, FDR<0.01), as well as collagens associated with the basement membrane and regulation of cell migration, including Col4a1, Col4a2, Col4a4, and Col15a1. Elastin gene expression was also upregulated (4.01 fold change, FDR<0.01), in addition to expression of MMP14 (34.19 fold change, FDR=0.01), an activator for MMP2, consistent with observed elastin degradation and partial compensatory upregulation of elastin. Pathway analysis using IPA software demonstrated upregulation of genes involved in cell migration and cell proliferation pathways, converging on erk1/2, which is known to undergo enhanced phosphorylation/activation in the absence of LRP1.
Conclusion: Our findings demonstrate that the major aortic wall structural changes associated with LRP1 deficiency in SMCs are driven by enhanced expression of pro-fibrotic genes, in addition to genes involved in cell migration and cell proliferation, as well as a shift towards elastin degradation.
