Human CMV infection of endothelial cells induces an angiogenic response through viral binding to EGF receptor and beta1 and beta3 integrins

Abstract

Human cytomegalovirus (HCMV) infection is associated with atherosclerosis, transplant vascular sclerosis, and coronary restenosis. A common theme in these vascular diseases is an increased rate of angiogenesis. Angiogenesis is a complex biological process mediated by endothelial cell (EC) proliferation, migration, and morphogenesis. Although angiogenesis is a normal process in the host, its dysregulation, after viral infection or injury to the vessel wall, is associated with plaque development in atherosclerotic patients. We now document that HCMV infection results in increased EC proliferation, motility, and capillary tube formation. The observed HCMV-induced angiogenic response depended on viral binding to and signaling through the beta(1) and beta(3) integrins and the epidermal growth factor receptor, via their ability to activate the phosphatidylinositol 3-kinase and the mitogen-activated protein kinase signaling pathways. Because a proangiogenic response drives the neovascularization observed in atherosclerotic disease, our findings identify a possible mechanism for how HCMV infection contributes to vascular disease.

Fig. 1.

HCMV infection enhanced EC proliferation. Proliferation assays were performed on mock-infected, HCMV-infected, UV-irradiated HCMV-treated, or PMA-treated HMECs. An MTS assay (A) and total cell counts (B) were performed at various times after infection. Results are plotted as the mean ± SD with significant differences of *, P < 0.05.

Fig. 2.

HCMV infection increased EC motility. HMECs were mock-infected, HCMV-infected, UV-irradiated HCMV-treated, or PMA-stimulated for 12 h. (A) Scratch assays were performed, and the average percentage scratch recovery was determined. (B) Phagokinetic motility tract assays were performed, and the average area of colloidal gold cleared per cell was determined. Results are plotted as the mean ± SD with significant differences of *, P < 0.05.

Fig. 3.

HCMV infection promoted capillary tube formation. HMECs were mock-infected, HCMV-infected, UV-irradiated HCMV-treated, or PMA-stimulated and plated onto Matrigel for 12 hpi, and then the average number of branches was determined. Results are plotted as the mean ± SD for each group with significant differences of *, P < 0.05.

Fig. 4.

HCMV infection rapidly activated ECs. HMECs were mock-infected or HCMV-infected for 0 min (mock infection) out to 120 min. Western blot analyses of pan EGFR, pEGFR, pan AKT, pAKT, pan Src, pSrc, pan ERK, pERK, and actin were performed with equal protein loading of each sample.

Fig. 5.

The PI3K and MAPK pathways are required for the HCMV-induced angiogenic response. HMECs were mock-infected or infected with HCMV after pretreatment with LY294002, U0126, DMSO, or no pretreatment. MTS (only the 96-hpi point is shown) (A), scratch motility (B), and morphogenesis (C) assays were performed. Results are plotted as the mean ± SD for each group with significant differences of *, P < 0.05.

Fig. 6.

The EGFR tyrosine kinase was required for the HCMV-induced angiogenic response, whereas the Src family of tyrosine kinases was only partially required. HMECs were mock-infected or HCMV-infected after pretreatment with AG1478, PP2, DMSO, or no pretreatment. MTS (only the 96-hpi point is shown) (A), scratch motility (B), and morphogenesis (C) assays were performed. Results are plotted as the mean ± SD for each group with significant differences of *, P < 0.05.

Fig. 7.

Viral binding to EGFR and the β₁ and β₃ integrins dictate the HCMV-induced angiogenic response. HMECs were pretreated with function-blocking antibodies to EGFR, the β₁ and β₃ integrins, or the isotype control for 1 h before mock or HCMV infection. MTS (only the 96-hpi point is shown) (A), scratch motility (B), and morphogenesis (C) assays were performed. Results are plotted as the mean ± SD for each group with significant differences of *, P < 0.05.