The present study involving fresh plasma was approved by normolipidemic volunteers and the Wuhan Blood Centre (Wuhan, China; authorization: 2010–8) and conformed to the guidelines outlined in the Declaration of Helsinki.

HUVECs and epithelial HEp-2 cells were obtained from Tongji Medical College (Wuhan, China). C. pneumoniae strain AR-39 was purchased from the American Type Culture Collection (Rockville, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A cell lipid peroxide assay kit was purchased from Genmed Scientifics, Inc. (Shanghai, China). A quantitative polymerase chain reaction (qPCR) kit was purchased from Takara Bio Inc. (Otsu, Shiga, Japan). Fluorescein isothiocyanate (FITC)-conjugated specific anti-chlamydial monoclonal antibodies were obtained from Dako Ltd. (Copenhagen, Denmark), and mouse polyclonal anti-SR-A1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rat monoclonal anti-CD36 antibodies were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Rabbit polyclonal anti-ACAT1 antibodies were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Rabbit monoclonal anti-ABCG1 antibodies were obtained from Epitomics (Burlingame, CA, USA). Mouse monoclonal anti-ABCA1 antibodies were obtained from Abcam PLC (Cambridge, UK). Mouse polyclonal anti-β-actin antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from AntGene Biotech Co., Ltd. (Wuhan, China).

C. pneumoniae strain AR-39 was propagated in HEp-2 cells in the presence of 2 μg/ml cycloheximide using centrifugation-driven infection as described previously (18). Infected HEp-2 cells were harvested subsequent to incubation for 72 h at 37°C and 5% CO₂, and disrupted by freezing, thawing and ultrasonication. Following centrifugation at 500 × g at 4°C for 30 min to remove the cell debris, the supernatant containing elementary bodies was collected and suspended in sucrose-phosphate-glutamic acid buffer and stored at −70°C in aliquots until use. Chlamydial inclusion forming units (IFU) were assessed by counting the chlamydial inclusions formed in the HEp-2 cells using FITC-conjugated anti-chlamydial antibodies.

HUVECs were seeded onto six-well plates at a density of 1×10⁶ cells per well in DMEM containing 10% FBS at 37°C in 5% CO₂. In the presence of 50 mg/l LDL, HUVECs were cultured alone or with C. pneumoniae (1×10⁶ IFU) for 48 h. In certain experiments, HUVECs were incubated in six-well plates with 50 mg/l ox-LDL for 48 h.

Human LDL (density, 1.006–1.063 g/ml) was isolated from the blood of healthy donors using density-gradient ultracentrifugation as described previously (19). The isolated LDL was dialyzed in phosphate-buffered saline for 24 h and was condensed using polyoxyl 20,000. The concentration of LDL was measured using the Bradford assay, and the LDL underwent sterile filtration (0.22 mm) prior to storage in the dark at 4°C for no longer than 2 weeks before use. LDL was analyzed using 4–20% SDS-PAGE gradient gel and Sudan black B staining to reveal a single protein. The preparations of ox-LDL from human plasma were performed as described previously (20). Briefly, LDL was diluted to a concentration of 200 mg/dl in medium M199 and incubated with 5 μmol/l CuSO₄, CuCl₂ or copper acetate at 37°C for 24 h, as indicated. The reactions were terminated using the addition of 100 μmol/l EDTA. The oxidation state of LDL was measured using the PeroXOquant Quantitative Peroxide Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Peroxidation products contained in the LDL and ox-LDL were <5 and 200–300 μmol/l H₂O₂, respectively. Contamination of LDL and the modified LDL preparations by the lipopolysaccharide (LPS) endotoxin was assessed using a Limulus Amebocyte Lysate kit (BioWhittaker^®; Lonza, Walkersville, MD, USA). All LDL preparations had an LPS content of <50 pg/mg protein.

The determination of LDL oxidation was performed as described previously (21). The determination of TBARS involves the measurement of malondialdehyde (MDA) derived from the hydroperoxidation of unsaturated fatty acids containing three or more double bonds (22). In brief, 2.5 ml 10% trichloroacetic acid was added to a tube with 0.5 ml HUVEC supernatant or cell lysates with or without C. pneumoniae infection, in order to precipitate the protein. Following centrifugation, 100 μl supernatant was mixed with 50 μl 0.67% TBA. Subsequent to vortexing and boiling for 15 min, samples were cooled under running water. Absorbance was measured at 532 nm in a Perkin Elmer Lambda 2 spectrophotometer (Perkin Elmer, Überlingen, Germany) against a blank control with distilled water. The concentration of oxidation products was determined as MDA (nmol/ml) according to a standard curve of MDA reacting with TBA using the following formula: MDA (nmol/ml) = absorbance × 53.4188. Experiments were repeated three times.

Cells were harvested and disrupted using ultrasonication. Intracellular TC and free cholesterol (FC) were detected using zymochemistry as described previously (23). TC and FC content was determined using a standard curve (312.5, 625, 1,250 and 2,500 μmol/l standard cholesterol) and expressed as μmol/g protein. CE content was calculated by subtracting the content of FC from that of TC. Experiments were repeated three times.

Total RNA was isolated using TRIzol^® reagent (Takara Bio, Inc.). cDNA was synthesized using a PrimeScript™ RT Reagent kit (Takara Bio, Inc.) according to the manufacturer’s instructions, and qPCR was performed using a Taq polymerase (Takara Bio, Inc.). The oligonucleotide primers for SR-A1, CD36, ACAT1, ABCA1, ABCG1 and β-actin were as follows: SR-A1, 5′-GCAGTTCTCATC CCTCTCAT-3′ (forward) and 5′-GGTATTCTCTTGGAT TTTGCC-3′ (reverse); CD36, 5′-TGCCTCTCCTAG TTGAAAAC-3′ (forward) and 5′-GCAACAAACGATCAC CACAC-3′ (reverse); ACAT1, 5′-TCCCAGGAATCCCAC TGTAA-3′ (forward) and 5′-ACGAAGAGCACGGGA TAGAA-3′ (reverse); ABCA1, 5′-TATGAGGGCCAGATC ACCTC-3′ (forward) and 5′-GCTGGCTTGTTTTGCTT TTC-3′ (reverse); ABCG1, 5′-CAGGAAGATTAGACACTG TGG-3′ (forward) and 5′-GAAAGGGGAATGGAGAGAAG-3′ (reverse); β-actin, 5′-GTCCACCTTCCAGCAGATGT-3′ (forward) and 5′-CACCTTCACCGTTCCAGTTT-3′ (reverse).

Total protein was extracted from the cells and the protein content was measured in triplicate using the Bicinchoninic Acid Protein Assay kit (Pierce Biotechnology, Inc.). Absorption was measured at 562 nm. Protein samples (60 μg) were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Invitrogen Life Technologies, Grand Island, NY, USA). Membranes were incubated at 4°C overnight with the following antibodies: Mouse polyclonal anti-SR-A1 (dilution, 1:200), rat monoclonal anti-CD36 (dilution, 1:500), rabbit polyclonal anti-ACAT1 (dilution, 1:200), mouse monoclonal anti-ABCA1 (dilution, 1:1,000), rabbit monoclonal anti-ABCG1 (dilution, 1:2,000) and mouse polyclonal anti-β-actin (dilution, 1:1,000). The membranes were then incubated with HRP-conjugated secondary anti-rabbit, -rat and -mouse antibodies, each at a dilution of 1:2,000, at room temperature for 2 h. Proteins were detected using the enhanced chemiluminescence method. For quantification, SR-A1, CD36, ACAT1, ABCA1 and ABCG1 protein levels were normalized to those of β-actin.

Quantitative data are expressed as the mean ± standard derivation. Direct comparisons between two groups were performed using the Student’s t-test. Data from more than two groups were compared using analysis of variance with repeated measures, followed by the Student-Newman-Keuls multiple comparison test. A value of P<0.05 was considered to indicate a statistically significant difference.

Lipid peroxidation was quantified using the measurement of TBARS. As shown in Fig. 1A, lipid peroxidation products were increased in the supernatant of C. pneumoniae-infected HUVECs (1×10⁶ IFU). MDA production was observed to increase from 4.61±0.90 nmol/ml in the control HUVEC supernatant to 7.16±0.40 nmol/ml in the infected HUVEC supernatant (P<0.05). In contrast to the supernatant, no significant difference in LDL oxidation was observed in the infected HUVEC lysates (Fig. 1B; P>0.05).

Cholesterol is accumulated either as CE in cytoplasmic vesicles or as FC in the membrane (24). To determine whether intracellular cholesterol metabolism was affected by C. pneumoniae in HUVECs, the levels of intracellular TC and CE were assessed using lipid mass quantification. LDL-treated HUVECs were used as a negative control, ox-LDL-treated HUVECs were used as a positive control and LDL-treated HUVECs infected with C. pneumoniae were used as the infection group. As shown in Table I, C. pneumoniae infection was found to increase the levels of TC and CE compared with the negative control (P<0.05). In addition, levels of intracellular TC and CE were increased in the ox-LDL-treated HUVECs compared with those in the negative control group (P<0.05). C. pneumoniae infection was not observed to significantly increase TC and CE levels compared with the positive controls (P>0.05).

The mRNA and protein expression of lipoprotein receptors was assessed using qPCR and western blot analyses, respectively. SR-A1 and CD36 have been demonstrated to participate in lipoprotein uptake (25). As shown in Fig. 2A and B, SR-A1 and CD36 mRNA expression was upregulated by up to 1.8- and 2.0-fold in the C. pneumoniae-infected HUVECs compared with that in the negative controls (P<0.05). In addition, ox-LDL was found to upregulate the mRNA expression of SR-A1 and CD36 by up to 2.0- and 1.5-fold, respectively (P<0.05). However, no significant difference was observed in the mRNA expression of SR-A1 and CD36 between the C. pneumoniae-infected HUVECs and the positive controls (P>0.05). Furthermore, C. pneumoniae infection was found to increase the protein expression of SR-A1 and CD36 by 3.53- and 2.40-fold, respectively, relative to the negative control group (P<0.05). SR-A1 and CD36 protein expression was also increased by up to 4.12- and 2.11-fold, respectively, in the positive control group relative to that in the negative controls (P<0.05) (Fig. 2C and D). No significant difference was observed in the protein expression of SR-A1 and CD36 between the C. pneumoniae-infected HUVECs and the positive controls (P>0.05).

The conversion of cholesterol to CE is catalyzed by the enzyme ACAT. In order to determine the role of ACAT1 in the C. pneumoniae-induced perturbation of intracellular cholesterol homeostasis in HUVECs, the expression of ACAT1 was assessed using qPCR and western blot analyses. As shown in Fig. 3A and B, C. pneumoniae infection (1×10⁶ IFU) was found to significantly increase the mRNA and protein expression of ACAT1 by up to 1.5- and 1.9-fold, respectively, compared with the LDL-treated HUVECs (P<0.05). In addition, ox-LDL upregulated ACAT1 mRNA expression by up to 1.58-fold and ACAT1 protein expression by up to 2.03-fold relative to the negative control. No significant difference was observed in ACAT1 mRNA and protein expression between the C. pneumoniae-infected HUVECs and the positive controls (P>0.05). These data show that ACAT1 may be involved in the C. pneumoniae-induced increases in CE levels in HUVECs.

ABCA1 and ABCG1 are critical cell surface proteins that mediate endothelial cholesterol efflux (15). In order to investigate the expression and regulation of ABCA1 and ABCG1 in C. pneumoniae-infected HUVECs, the mRNA and protein levels of ABCA1 and ABCG1 were detected. As shown in Fig. 4A and B, the mRNA expression of both ABCA1 and ABCG1 was reduced by 40% in the C. pneumoniae-infected HUVECs compared with that in the LDL-treated HUVECs (P<0.05). Furthermore, in the ox-LDL-treated HUVECs, the mRNA expression of ABCA1 and ABCG1 was reduced by 50% (P<0.05). In addition, ABCA1 and ABCG1 protein expression was reduced by 62 and 48% in the C. pneumoniae-infected HUVECs (P<0.05; Fig. 4C and D). However, no significant difference was observed between the C. pneumoniae-infected group and the positive control group (P>0.05).