Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells

Abstract

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function.

Fig. 1

Cytotoxicity assay after 72 h of EPC treatment with mCRP and pCRP. a Viable cells were measured by the CytoTox-Glo™ cytotoxicity assay. The bars represent the relative luminesce unit (RLU) calculated for the viable cells, i.e. dead cells deducted from total cells, mean and SD of n = 3. b. Histograms of one representative experiment showing the binding of the apoptosis marker Annexin V to EPCs using a FITC-labelled Annexin V antibody detected by flow cytometry

Fig. 2

The effect of mCRP (1 μg/ml) or pCRP (5 μg/ml) on phenotype and function of EPCs. a Binding of ulex lectin and uptake of AcLDL after 72 h of culture of EPCs in the presence or absence of mCRP or pCRP visualised with FITC-labelled ulex lectin and Dil-labelled AcLDL, respectively. The photomicrographs show a typical optical field from one representative experiment. The bar graph shows the percentage of double-positive cells, mean and SD of n = 3. b The number of colony forming units (CFU-Hill) visualised by Giemsa staining. The bars represent the total number of colonies per well in a 24-well plate, mean and SD of n = 3. c Endothelial tube formation assay after 72 h of culture of EPCs in the presence or absence of mCRP or pCRP. The treated EPCs were co-cultured with HUVECs for additional 4, 16 and 24 h, respectively. The photomicrographs show the results of a representative experiment. The length of the tubuli was assessed after 16 h. The box plot represents the mean (dotted line), median (solid line) and SD of three independent experiments

Fig. 3

a Principal component analysis (PCA) of the source of variation in the sample gene expression. This was an exploratory multivariate statistical technique that was used to simplify the complex microarray changes that occur in three individuals (patient 10, 18 and 20) and in response to different valency CRP. This is done by reducing the dimensionality of the data matrix by finding r new variables (that sum the expression of multiple genes into single axes); shown here are the average signal of each sample along the three-dimensional virtual space of the first three principal components. The plot clearly shows that the variation in gene expression is dominated by the CRP treatment and not the patient source. b Hierarchical clustering of the gene expression response to CRP valency; 320 differentially expressed genes are plotted according to their degree of respective co-expression. Columns represent samples, while rows represent genes. The treatment of each sample is listed at the bottom. The degree of correlation between genes (left) or samples (top) is plotted in a tree view fashion

Fig. 4

Ingenuity pathways analysis. Network of up-regulated genes in response to 1 μg/ml mCRP. Networks of gene/gene product interaction were generated using IPA (Ingenuity^® Systems, http://www.ingenuity.com). Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one published reference. Solid edges represent a direct relationship and dashed edges represent an indirect relationship. The red node colour represents up-regulation in response to mCRP and the colour intensity indicates the degree of up-regulation. The shape of each node represents the functional class of the gene product, as shown in the legend

Fig. 5

Ingenuity pathways analysis. Network of up-regulated genes in response to 5 μg/ml pCRP. Networks of gene/gene product interaction were generated using IPA (Ingenuity^® Systems, http://www.ingenuity.com). Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one published reference. Solid edges represent a direct relationship and dashed edges represent an indirect relationship. The red node colour represents up-regulation in response to pCRP. The shape of each node represents the functional class of the gene product, as shown in the legend of Fig. 4

Fig. 6

Validation of the expression of selected highly upregulated genes in mCRP (1 μg/ml) and pCRP (5 μg/ml)-treated EPCs by quantitative real-time PCR. Validation of gene expression by quantitative real-time PCR. The mean in gene expression from EPCs derived from three individual cord blood donors was obtained using differences in cycle threshold between the gene and 18 s (ΔCt). The fold change in difference (ΔΔCt) in gene expression in the treated samples compared to the PBS control samples was determined (2ΔΔCt) and expressed in the diagram as mean and SEM of n = 3. Grey bars represent pCRP and black bars represent mCRP-treated EPCs

Fig. 7

The effect of mCRP (1 mg/ml), pCRP (5 mg/ml) or IFNa2A (1 ng/ml) on phenotype and function of EPCs. a Binding of ulex lectin and uptake of acetylated LDL after 72 h of culture of EPCs in the presence or absence of pCRP, mCRP and IFNa2A. The bar graph shows the percentage of cells double positive for binding of ulex lectin and uptake of acetylated LDL as compared to the total number of cells in three random optical fields (20× magnification). Mean and SD of n = 3. The statistical significance was determined by one-way ANOVA with Tukey’s post hoc test, ***p<0.001. b The bar graph shows the number of colony forming units (CFU-Hills) visualised by Giemsa staining, formed by EPCs that had been cultured in the presence of pCRP, mCRP or IFNalpha2 for 72 h (mean and SD of three individual donors, n = 3). The statistical significance was determined by one-way ANOVA with Tukey’s post hoc test, **p < 0.01. c The boxplot shows the lengths of the tubuli formed in a co-culture of HUVECs and EPCs (n = 3) in an endothelial tube formation assay. The EPCs had been cultured in the presence of pCRP, mCRP or IFNa2A for 72 h prior to the assay. The photomicrographs were taken when a clear tubuli network was observed after 16 h of incubation. The statistical significance was determined by ANOVA on ranks with Dunn’s post hoc test, *p < 0.05