SPP1/osteopontin: a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?

Abstract

Degenerative ascending aortic aneurysm (AscAA) is a silent and potentially fatal disease characterized by excessive vascular inflammation and fibrosis. We aimed to characterize the cellular and molecular signature for the fibrotic type of endothelial mesenchymal transition (EndMT) that has previously been described in degenerative AscAA. Patients undergoing elective open-heart surgery for AscAA and/or aortic valve repair were recruited. Gene expression in the intima-media of the ascending aorta was measured in 22 patients with non-dilated and 24 with dilated aortas, and candidate genes were identified. Protein expression was assessed using immunohistochemistry. Interacting distal gene enhancer regions were identified using targeted chromosome conformation capture (HiCap) in untreated and LPS-treated THP1 cells, and the associated transcription factors were analyzed. Differential expression analysis identified SPP1 (osteopontin) as a key gene in the signature of fibrotic EndMT in patients with degenerative AscAA. The aortic intima-media expression of SPP1 correlated with the expression of inflammatory markers, the level of macrophage infiltration, and the aortic diameter. HiCap analysis, followed by transcription factor binding analysis, identified ETS1 as a potential regulator of SPP1 expression under inflammatory conditions. In conclusion, the present findings suggest that SPP1 may be involved in the development of the degenerative type of AscAA. KEY MESSAGES: In the original manuscript titled "SPP1/osteopontin, a driver of fibrosis and inflammation in degenerative ascending aortic aneurysm?" by David Freiholtz, Otto Bergman, Saliendra Pradhananga, Karin Lång, Flore-Anne Poujade, Carl Granath, Christian Olsson, Anders Franco-Cereceda, Pelin Sahlén, Per Eriksson, and Hanna M Björck, we present novel findings on regulatory factors on osteopontin (SPP1) expression in immune cells involved in degenerative ascending aortic aneurysms (AscAA). The central findings convey: SPP1 is a potential driver of the fibrotic endothelial-to-mesenchymal transition in AscAA. SPP1/osteopontin expression in AscAA is predominately by immune cells. ETS1 is a regulatory transcription factor of SPP1 expression in AscAA immune cells.

Keywords: Aneurysm; Ascending aorta; Fibrosis; Inflammation; Osteopontin.

Fig. 1

a EMT genes with differing expression in non-dilated and dilated ascending aortas from participants with TAV (i.e., TAV-specific dilatation genes). An alpha cut-off value of 0.5 was used. The log2 fold difference is shown on the x-axis, and the − log10 p-values for participants with dilated TAV (n = 24) and non-dilated TAV (n = 22) on the y-axis. b Correlation between the aortic intima-media mRNA expression of SPP1 and the ascending aortic diameters (mm), P = 0.0001, Pearson r = 0.52, (TAV n = 46, BAV n = 65). TAV, tricuspid aortic valve; BAV, bicuspid aortic valve

Fig. 2

a Osteopontin and CD68 protein expression in participants with non-dilated (n = 19) or dilated (n = 17) ascending aortas with tricuspid aortic valves (magnification × 20, scale bar 20 μm). b Correlations of the gene expression of SPP1 with inflammatory and immune cell markers (IL1B, CD168, CD4, and CD163) and smooth muscle cell markers (ACTA2, CALD1, CNN1, and MYH11) in non-dilated (n = 22) and dilated (n = 24) ascending aortas

Fig. 3

Single-cell genomic analysis of SPP1 expression in Plaqview 2.0 software [31], performed using publicly available data regarding thoracic aortic aneurysm tissue (Li et al. [21]). EC, endothelial cell; FB, fibroblast; NK, natural killer cell; SMCs, smooth muscle cells; Mø, macrophage

Fig. 4

Results of the HiCap analysis of SPP1 promoter interactions in THP1 cells stimulated or not with LPS. The results are mapped to their chromosomal positions. Gray bars indicate distal elements interacting with the SPP1 promoter. The samples were analyzed in duplicate, and the distal elements identified in both samples with a significance of p < 0.001 were considered to show significant interactions (red bars)

Fig. 5

a Correlations of SPP1 expression with that of the top five putative transcription factors that were differentially expressed in non-dilated and dilated ascending aortas. Red boxes indicate negative correlations; green boxes indicate positive correlations, and gray boxes indicate non-significant relationships. Pearson r-values are shown; n = 22. b Correlation between the aortic intima-media mRNA expression of ETS1 and the ascending aortic dimensions (mm). p = 0.0001, Pearson r = 0.52, n = 46

Fig. 6

TF binding analysis using the CUT&RUN-method for the interaction between SPP1 and ETS1 in THP1 cells, treated with LPS (2 h) or not (0 h) (a) and in dilated ascending aortas (b). ETS1 binding is demonstrated using a 2% agarose gel. c ETS1 and SPP1 mRNA expression in differentiated THP1 cells transfected with ETS1 and control siRNA, respectively, prior to 2 h LPS stimulation