Mapping Semaphorins and Netrins in the Pathogenesis of Human Thoracic Aortic Aneurysms

Abstract

Thoracic aortic aneurysm (TAA) is a complex life-threatening disease characterized by extensive extracellular matrix (ECM) fragmentation and persistent inflammation, culminating in a weakened aorta. Although evidence suggests defective canonical signaling pathways in TAA, the full spectrum of mechanisms contributing to TAA is poorly understood, therefore limiting the scope of drug-based treatment. Here, we used a sensitive RNA sequencing approach to profile the transcriptomic atlas of human TAA. Pathway analysis revealed upregulation of key matrix-degrading enzymes and inflammation coincident with the axonal guidance pathway. We uncovered their novel association with TAA and focused on the expression of Semaphorins and Netrins. Comprehensive analysis of this pathway showed that several members were differentially expressed in TAA compared to controls. Immunohistochemistry revealed that Semaphorin4D and its receptor PlexinB1, similar to Netrin-1 proteins were highly expressed in damaged areas of TAA tissues but faintly detected in the vessel wall of non-diseased sections. It should be considered that the current study is limited by its sample size and the use of internal thoracic artery as control for TAA for the sequencing dataset. Our data determines important neuronal regulators of vascular inflammatory events and suggest Netrins and Semaphorins as potential key contributors of ECM degradation in TAA.

Keywords: Semaphorins; aneurysms; extracellular matrix; netrins; vascular remodeling.

Figure 1

Distinct up-regulation of axonal guidance pathway. (a) Graphic representation of selected up-regulated pathways in thoracic aortic aneurysm samples compared to internal thoracic artery for the genes with p < 0.05 and fold change >2. (b) Schematic of KEGG enrichment pathway for the set of genes for axon guidance signaling in thoracic aortic aneurysm (TAA). The genes indicated by a star are predicted as increased in TAA.

Figure 2

Profiling of neuronal guidance cues in TAA. (a) Transcript expression of neuronal cue genes (ligands) in thoracic aortic aneurysm compared to internal thoracic artery. Genes are clustered based on their family. (b) Transcript expression of neuronal cue genes (receptors) in thoracic aortic aneurysm compared to internal thoracic artery. Genes are clustered based on their family. (c) Profiling and analysis of mRNA copy numbers, identified by RNA-Seq, of the neuronal cue genes involved in axon guidance pathway. Genes are grouped by families of ligands and receptors. * p < 0.05, ** p < 0.01.

Figure 3

Transcript analysis and immunohistochemistry of Semaphorins and Netrins. (a,b) Elastin staining (Verhoeff-Van Gieson) and degradation score in non-diseased and TAA sections of size as indicated. Dashed boxes indicate magnified areas. Scale bars represent 1000 µm and 200 µm in low and high magnified images. Arrow indicate broken elastin fragments and stars indicate areas of complete elastin degradation. *** p < 0.001. Non-diseased (n = 4), TAA < 5.5 cm (n = 4), TAA > 5.5 cm (n = 5) (c) Quantitative PCR analysis of Semaphorin4D transcripts in non-diseased (n = 3), TAA < 5.5 cm (n = 4), TAA > 5.5 cm (n = 6). (d) Hematoxylin and Eosin (H & E) image and representative immunofluorescence staining of Semaphorin4D (red) and Dapi (blue) in non-diseased and TAA sections of human aorta. Dashed boxes indicate magnified areas of immunofluorescence staining (e) Quantification of Semaphorin4D immunofluorescence staining in non-diseased and TAA. (f) Quantitative PCR analysis of PlexinB1 transcripts in non-diseased (n = 3), TAA < 5.5 cm (n = 4), TAA > 5.5 cm (n = 6). (g) H & E image and representative fluorescence staining of PlexinB1 (red) and Dapi (blue) in non-diseased and TAA. (h) Quantification of PlexinB1 immunofluorescence staining in non-diseased and TAA. (i) Quantitative PCR analysis of Netrin-1 transcripts in non-diseased (n = 3), TAA < 5.5 cm (n = 4), TAA > 5.5 cm (n = 5). (j) H & E image and representative immunofluorescence staining of Netrin-1 (red) and Dapi (blue) in non-diseased and TAA sections of human aorta. Dashed boxes indicate magnified areas of immunofluorescence staining (k) Quantification of Netrin-1 immunofluorescence staining in non-diseased and TAA. (l) Quantitative PCR analysis of Netrin-3 transcripts in non-diseased (n = 3), TAA < 5.5 cm (n = 4), TAA > 5.5 cm (n = 5). (m) H & E image and representative immunofluorescence staining of Netrin-3 (red) and Dapi (blue) in non-diseased and TAA sections of human aorta. Dashed boxes indicate magnified areas of immunofluorescence staining. (n) Quantification of Netrin-3 immunofluorescence staining in non-diseased and TAA. * p < 0.05 ** p < 0.01 *** p <0.001. L = lumen. Scale bars represent 500 µm on H & E images. Magnified scale bars are 50 µm.