Endothelial cells' activation and apoptosis induced by a subset of antibodies against human cytomegalovirus: relevance to the pathogenesis of atherosclerosis

Abstract

Background: Human cytomegalovirus (hCMV) is involved in the pathogenesis of atherosclerosis. We have previously shown in patients with atherosclerosis that antibodies directed against the hCMV-derived proteins US28 and UL122 are able to induce endothelial cell damage and apoptosis of non-stressed endothelial cells through cross-rection with normally expressed surface molecules. Our aim was to dissect the molecular basis of such interaction and to investigate mechanisms linking innate immunity to atherosclerosis.

Methodology/principal findings: We analysed the gene expression profiles in endothelial cells stimulated with antibodies affinity-purified against either the UL122 or the US28 peptides using the microarray technology. Microarray results were validated by quantitative PCR and by detection of proteins in the medium. Supernatant of endothelial cells incubated with antibodies was analysed also for the presence of Heat Shock Protein (HSP)60 and was used to assess stimulation of Toll-Like Receptor-4 (TLR4). Antibodies against UL122 and US28 induced the expression of genes encoding for adhesion molecules, chemokines, growth factors and molecules involved in the apoptotis process together with other genes known to be involved in the initiation and progression of the atherosclerotic process. HSP60 was released in the medium of cells incubated with anti-US28 antibodies and was able to engage TLR4.

Conclusions/significance: Antibodies directed against hCMV modulate the expression of genes coding for molecules involved in activation and apoptosis of endothelial cells, processes known to play a pivotal role in the pathogenesis of atherosclerosis. Moreover, endothelial cells exposed to such antibodies express HSP60 on the cell surface and release HSP60 in the medium able to activate TLR4. These data confirm that antibodies directed against hCMV-derived proteins US28 and UL122 purified from patients with coronary artery disease induce endothelial cell damage and support the hypothesis that hCMV infection may play a crucial role in mediating the atherosclerotic process.

Figure 1. Anti-hCMV antibodies induce apoptosis of endothelial cells.

Apoptosis induced in HUVECs by antibodies directed against UL122 peptide (A) and against US28 peptide (B); X axis: apoptotic index; Y axis: time expressed in hours. Data are mean of three independent experiments. Error bars = SD. Apoptosis induced by antibodies directed against the irrelevant peptide gave an apoptotic index<1 at all time points, corresponding to less than 8–12% of apoptotic cells as determined in parallel by FACS analysis.

Figure 2. FACS analysis of the expression of HSP60 at the cell surface membrane.

(A) Positive control: HUVECs stressed with glutaraldehyde for 20 minutes; (B) HUVECs treated with anti-UL122 antibodies for 9 hours; (C) HUVECs treated with anti-US 28 antibodies for 9 hours. HUVEC cells were stained with a monoclonal antibody directed against HSP60. Percentage of positive cells: (A) 44,2%; (B) 1,3%; (C): 6,7%.

Figure 3. Soluble HSP60 in the supernatant of stimulated cells.

Soluble HSP60 in the supernatant (SN) of HUVECs after stimulation with anti-US28 and anti-UL122 antibodies (Abs) at different time points. X axis: time expressed in hours. Y axis: soluble HSP60 concentration expressed in ng/ml.

Figure 4. TLR4 activation by the supernatant of treated cells.

TLR4 engagement by the SN of cells treated with the antibodies (Abs) described. Y axis: levels of TLR4 stimulation expressed as ng/mL of LPS. Data are mean of three independent experiments. Error bars = SD.

Figure 5. Gene array analysis of HUVECs stimulated with anti-hCMV antibodies.

Clustergrams of genes modulated by incubation of endothelial cells with antibodies against the US28 peptide or the UL122 peptide. Colours indicate the fold change.

Figure 6. Validation of gene array by Q-PCR.

(A) Genes selected for validation by Q-PCR in endothelial cells treated with anti-US28 peptide antibodies. CCL2, E-selectin, VCAM-1 and CXCL2 transcripts were increased by 2,46-, 5,95-, 3,76-, and 4,21-fold, respectively, compared with control endothelial cells. (B) Genes selected for validation by Q-PCR in endothelial cells treated with anti-UL122 antibodies. CCL2, E-selectin, VCAM-1 and ICAM-1 transcripts were increased 19-, 31,15-, 18,8-, and 12,7-fold compared to control endothelial cells. The level of transcript expression is reported on the vertical axis. GAPDH was selected as endogenous gene.

Figure 7. Soluble mediators released in cell culture supernatants.

(A, B) Quantification of CCL2 released in the supernatant (SN) of HUVECs stimulated with antibodies (Abs) against the irrelevant peptide and with anti-UL122 and anti-US28 affinity purified antibodies. (C, D) Quantification of soluble E-selectin in the SN of HUVECs stimulated with abs against the irrelevant peptide and with anti–UL122 and anti-US28 affinity purified antibodies. Results are expressed in pg/mL and ng/mL, respectively. Results are expressed as mean of three independent experiments.