GroEL1, a Heat Shock protein 60 of Chlamydia pneumoniae, Impairs Neovascularization by Decreasing Endothelial Progenitor Cell Function

Yi-Wen Lin,Chun-Yao Huang,Nen-Chung Chang,Hsiao-Ya Tsai,Jui-Chi Tsai,Chi-Yuan Li,Cheng-Yen Lin,Yung-Hsiang Chen,Feng-Yen Lin,Nai-Wen Tsao,Chien-Sung Tsai,Chun-Ming Shih,Po-Hsun Huang,Francesco Bertolini

doi:10.1371/journal.pone.0084731

Abstract

The number and function of endothelial progenitor cells (EPCs) are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4) mutation) mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS) in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.

Highlights

Chlamydia pneumoniae is thought to play key roles in atherogenesis [1]
Consistent with the measurements obtained by laser Doppler imaging, anti-Von Willebrand factor (vWF) immunostaining revealed that GroEL1 administration for 8 weeks at 4 μg/kg body weight (BW) significantly decreased the number of detectable capillaries in the ischemic muscle of C57BL/B6 mice (Figure 1D) compared to untreated ischemic C57BL/B6 mice
We provide evidence that 1) heat shock protein 60 of C. pneumonia, GroEL1, impaired the recovery of capillary density, which may be mediated by TLR4 in mice; 2) GroEL1 impaired endothelial progenitor cells (EPCs) mobilization and vessel formation as well as endothelial nitric oxide synthase (eNOS) expression in ischemic tissue; 3) GroEL1 administration impaired the migration and vasculogenesis of late EPCs in vitro; 4) EPC senescence was enhanced by GroEL1, which was mediated by activation of caspases, p38 MAPK and ERK1/2; and 5) GroEL1 decreased integrin and Eselectin expression but induced inflammatory responses in EPCs

Summary

Introduction

Chlamydia pneumoniae is thought to play key roles in atherogenesis [1]. In clinical observation, C. pneumoniae accumulates in early fatty streaks in association with increasing vascular intima-media thickness [2]. Inoculation of animals with C. pneumoniae may induce low-density lipoprotein (LDL) oxidation within the neointima [5], increase the formation of atherosclerotic lesions in hypercholesterolemic conditions [6] and accelerate the formation of complex atherosclerosis [7]. The in vitro studies had confirmed these results which were obtained from clinical observation and animal studies. These evidence indicated that C. pneumoniae may increase uptake of LDL in macrophages [8,9], induce matrix metalloproteinases (MMPs) and adhesion molecules expression through activation of the lectin-like oxidized LDL receptor (LOX)-1 in human vascular endothelial cells [10,11,12], which may potentially promote the development of atherosclerosis

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Conclusion