Chlamydia pneumoniae infection promotes vascular endothelial cell angiogenesis through an IQGAP1-related signaling pathway.

Chlamydia pneumoniae (C. pneumoniae) infection plays a potential role in angiogenesis. However, it is still an enigma how C. pneumoniae is involved in this process. Therefore, we investigated the effect of C. pneumoniae infection on angiogenesis, and then explored the roles of IQGAP1-related signaling in C. pneumoniae infection-induced angiogenesis. C. pneumoniae infection significantly enhanced angiogenesis as assessed by the tube formation assay possibly by inducing vascular endothelial cell (VEC) migration in the wound healing and Transwell migration assays. Subsequently, immunoprecipitation, Western blot and tube formation assay results showed that the phosphorylation of both IQGAP1 and N-WASP was required for the angiogenesis induced by C. pneumoniae infection. Our co-immunoprecipitation study revealed that IQGAP1 physically associated with N-WASP after C. pneumoniae infection of VECs. Actin polymerization assay further showed that in C. pneumoniae-infected VECs, both IQGAP1 and N-WASP were recruited to filamentous actin, and shared some common compartments localized at the leading edge of lamellipodia, which was impaired after the depletion of IQGAP1 by using the small interference RNA. Moreover, the knockdown of IQGAP1 also significantly decreased N-WASP phosphorylation at Tyr256 induced by C. pneumoniae infection. We conclude that C. pneumoniae infection promotes VEC migration and angiogenesis presumably through the IQGAP1-related signaling pathway.

Chlamydia pneumoniae infection promotes vascular smooth muscle cell migration through a Toll-like receptor 2-related signaling pathway.

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima is proposed to be a key event in the development of atherosclerosis. Recently, we reported that Chlamydia pneumoniae infection is involved in VSMC migration. However, the exact mechanisms for C. pneumoniae infection-induced VSMC migration are not yet well elucidated. In this study, we examined the role of the Toll-like receptor 2 (TLR2) activation-related signaling pathway in VSMC migration induced by C. pneumoniae infection. An Affymetrix-based gene expression array was conducted to identify the changes of gene expression in rat primary VSMCs (rVSMCs) infected with C. pneumoniae. Both the microarray analysis and quantitative real-time reverse transcription (RT)-PCR revealed that TLR2 mRNA expression was strongly upregulated 12 h after C. pneumoniae infection. RT-PCR and Western blot analysis further showed that the expression levels of TLR2 mRNA and protein significantly increased at the different time points after infection. Immunocytochemical analysis suggested a TLR2 recruitment to the vicinity of C. pneumoniae inclusions. Cell migration assays showed that the TLR2-neutralizing antibody could significantly inhibit C. pneumoniae infection-induced rVSMC migration. In addition, C. pneumoniae infection stimulated Akt phosphorylation at Ser 473, which was obviously suppressed by the PI3K inhibitor LY294002, thereby inhibiting rVSMC migration caused by C. pneumoniae infection. Furthermore, both the infection-induced Akt phosphorylation and rVSMC migration were suppressed by the TLR2-neutralizing antibody. Taken together, these data suggest that C. pneumoniae infection can promote VSMC migration possibly through the TLR2-related signaling pathway.

[Promoting effect of Chlamydia pneumoniae infection on human laryngeal carcinoma HEp-2 cell adhesion and migration].

OBJECTIVE: To explore the effect of Chlamydia pneumoniae (C.pn) infection on human laryngeal carcinoma cell line HEp-2 cell adhesion and migration, to further clarify the role and mechanism of C.pn infection in tumor metastasis. METHODS: HEp-2 cells were infected with C.pn after the culture and propagation of C.pn. The cytopathic effect was observed by microscopy. Morphological characteristics of C.pn inclusions in HEp-2 cells were examined by fluorescence microscopy and acridine orange staining. The ultrastructural changes of C.pn inclusions in the HEp-2 cells were examined by transmission electron microscopy (TEM). Cell adhesion assay was performed to investigate the effect of C.pn infection on the adhesion of HEp-2 cells to collagen I. Wound-healing assay and transwell assay were performed to explore the effect of C.pn infection on HEp-2 cell migration. RESULTS: At 72 h post-infection, C.pn infected-HEp-2 cells were swollen and partially desquamated. Numerous vacuoles (inclusions) were observed and C.pn inclusions occupied almost the whole cytoplasm of the HEp-2 cells. Grape-like C.pn inclusions were observed in the HEp-2 cells stained with acridine orange under a fluorescence microscope at 72 h after infection. Under TEM, there were more mature pear-shaped elementary bodies, but less larger and round reticulate bodies in the HEp-2 cells infected with C.pn for 72 h. In the cell adhesion assay, the A value in C.pn infection group was 0.669 ± 0.011, significantly higher than that in the control group (0.558 ± 0.005) at 2 h after infection (P < 0.001). The cell adhesion ratio in the C.pn infection group was 119.89%. The migration distance of C.pn infected-HEp-2 cells in the wound-healing assay was significantly longer than that of control cells at 24 h after infection (P < 0.05). HEp-2 cells infected with C.pn for 12 h migrated more than the control cells in the transwell assay (23.40 ± 2.41 vs 10.40 ± 1.67) (P < 0.001). CONCLUSIONS: C.pn infection can significantly promote HEp-2 cell adhesion to collagen I and migration of HEp-2 cells, indicating that C.pn infection may play an important role in promoting the metastasis of laryngeal cancer.

Differential expression of ubiquitin-conjugating enzyme E2r in the developing ovary and testis of penaeid shrimp Marsupenaeus japonicus.

In order to identify genes involved in oogenesis and spermatogenesis in penaeid shrimp Marsupenaeus japonicus, a modified annealing control primer (ACP) system was adapted to identify genes differentially expressed in ovary and testis at different developmental stages. By using 20 pairs of ACP primers, 8 differentially expressed genes were obtained. One of these genes is ubiquitin-conjugating enzyme E2r (UBE2r). Bioinformatics analyses show that this gene encodes a protein of 241 amino acids with a predicted molecular mass of 27.4 kDa. Real time PCR analyses demonstrated that the expression level changed significantly in the developing testis and ovary. In the stage 2 of testis, it reached its highest expression level, the lowest expression level present in the stage 1 of ovary. The significantly different expression levels in developing testis and ovary suggest that UBE2r has an important role in oogenesis and spermatogenesis. This article is the first report of UBE2r in crustaceans and also is the first report showing that UBE2r is differentially expressed at different stages of the developing ovary and testis in an animal.

Molecular cloning of proliferating cell nuclear antigen and its differential expression analysis in the developing ovary and testis of penaeid shrimp Marsupenaeus japonicus.

To understand the molecular mechanisms of gonadal development and maturation in penaeid shrimp Marsupenaeus japonicus, eight differentially expressed genes were obtained using a modified annealing control primer system. One of these genes is a proliferating cell nuclear antigen (PCNA). Bioinformatics analyses showed that full-length cDNA of M. japonicus PCNA (mjPCNA) consists of 75 bp of 5' untranslated region, 783 bp of coding region, and 65 bp of 3' untranslated region (excluding the polyA tail), encoding a protein of 260 amino acids with a predicted molecular mass of 28.85 kDa and an isoelectric point of 4.59. Real-time polymerase chain reaction analyses demonstrated that the gene expression level changed significantly in the developing testis and ovary. In stage 1 of ovary and testis, mjPCNA showed its lowest level during development and reached its highest expression level in stage 2 of ovary and testis. In stages 4 and 5 of ovary and the stage 3 of testis, mjPCNA held a steady expression level. Data suggest that PCNA plays an important role in the testis and ovary development, especially in the process of mitosis and meiosis.