Identification of Macrophage Genotype and Key Biological Pathways in Circulating Angiogenic Cell Transcriptome

Abstract

Background: Circulating angiogenic cells (CAC) have been identified as important regulators of vascular biology. However, there is still considerable debate about the genotype and function of CAC.

Methods and results: Data from publicly available gene expression data sets were used to analyse the transcriptome of in vitro cultured CAC (CAC_iv). Genes and pathways of interest were further evaluated using qPCR comparing CAC_iv versus CD14⁺ monocytic cells. The CAC_iv transcriptome strongly related to tissue macrophages, and more specifically to regulatory M2c macrophages. The cytokine expression profile of CAC_iv was predominantly immune modulatory and resembled the cytokine expression of tumor-associated macrophages (TAM). Pathway analysis revealed previously unrecognized biological processes in CAC_iv, such as riboflavin metabolism and liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR)/retinoid X receptor (RXR) pathways. Analysis of endothelial-specific genes did not show evidence for endothelial transdifferentiation.

Conclusions: CAC_iv are genotypically similar to regulatory M2c macrophages and lack signs of endothelial differentiation.

Figure 1

CAC_iv gene signature. 70 genes were significantly upregulated versus 107 genes were significantly downregulated in CAC_iv versus CD14⁺ monocytic cells. The upregulated (red) and downregulated (blue) genes express a close lineage relationship with macrophages and monocytes, respectively. We highlighted those genes of the CAC_iv signature that are specific for either macrophages or monocytes. ^∗: very specific. Mϕ: macrophage; Mo: monocyte.

Figure 2

Expression of CAC_iv signature related genes in myeloid cells. To demonstrate the lineage relationships between CAC_iv and other cell types, we highlighted those genes of the CAC_iv signature that were found to be significantly upregulated and also specific for a particular cell type. ^∗: very specific for macrophages. Mϕ: macrophage; Mo: monocyte; EC: endothelial cell; DC: dendritic cell; Ocl: osteoclast.

Figure 3

Gene expression analysis. Gene expression of (a) M2c macrophage-related, (b) endothelial, (c) cytokine/cytokine receptor and (d) cholesterol transporter pathway genes in CAC_iv (green) vs. CD14⁺ monocytes (red) vs. human umbilical vein endothelial cells (HUVEC) (grey). Only the relevant statistical significance between CACiv and CD14⁺ monocytes is depicted. NRQ: normalized relative quantity. ^∗∗ p < 0.01; ^∗∗∗ p < 0.001.

Figure 4

CAC_iv differentiate towards a predominanty M2 macrophage genotype. Boxplots comparing the relative expression of the genes in (a) the monocyte-specific gene signature (MONO), (b) the macrophage-specific gene signature (MACRO), (c) the LPS- and IFN-γ-stimulated M1 macrophage-specific gene signature (MACRO M1), and (d) the IL4-stimulated M2 macrophage-specific gene signature (MACRO M2), between CAC_iv and CD14⁺ monocytic cells (MONO). Positive values signify a stronger degree of similarity of the genetic expression of CAC_iv or CD14⁺ monocytic cells to the specified cell type.

Figure 5

Analysis of EC-associated genes in the CAC_iv profile. (a) Boxplot comparing the relative expression of an endothelial gene set between CAC_iv and CD14⁺ monocytic cells. There is no significant difference in similarity of the genetic expression of CAC_iv or CD14⁺ monocytic cells to an EC-specific gene signature. (b) UHC analysis highlighting the relative expression of EC-associated genes in CAC_iv (grey) and monocytes (black). RNASE1 is indicated as most differentially expressed in CAC_iv compared to CD14⁺ monocytic cells. We observed a lack of consistency in EC-associated gene expression between different CAC_iv culture samples. Red: upregulation; green: downregulation.

Figure 6

CAC_iv -related cytokine and chemokine/chemokine receptor expression profile. Gene expression plot depicting (a) cytokines or (b) chemokines and chemokine receptors that were differentially overexpressed in CAC_iv (grey) vs. monocytes (black). The influence (y-axis) represents the number of standard deviations (SD) the gene expression of each gene exceeds the null hypothesis that there would be no difference between both groups (z-score). Genes with an influence of ≥1.96 show a statistically significant differential gene expression (p < 0.05). ^∗ p < 0.05.

Figure 7

Summary of IPA analysis. The main upregulated canonical pathways are shown. Riboflavin metabolism and LXR/RXR and FXR/RXR activation remain statistically significant even after correction for multiple comparisons (Threshold indicates false discovery rate corrected p-value of 0.05). Bottom axis depicts the ratio of the number of genes upregulated in de CAC_iv signature divided by the total number of genes involved in a given pathway (squares).