Role Of IQGAP1in Vascular Smooth Muscle Cell Adhesion And Migration Induced By Chlamydia Pneumoniae Infection

Objecti ves:Using the model of vascular smooth muscle cell (VSMC) infected with Chlamydia pneumoniae (C.pn) in vitro, to analyze the changes in the cell migration related genes expression in the rat VSMC, to observe the effects of C.pn infection on the adhesion and migration of VSMC, to investigate the roles of IQGAP1in adhesion and migration of VSMC induced by C.pn infection.Methods:1. Rat VSMCs were infected with C.pn in vitro after the culture and propagation of C.pn.2. The changes in the expression of the genes related to cell migration in the rat VSMCs infected with C.pn was detected by the Affymetrix Rat Genome2302.0microarray.3. The effects of C.pn infection on the adhesion of VSMCs were investigated by the MTT assay.4. Transwell assay was performed to observe the effects of C.pn infection on VSMC migration.5. RT-PCR was used to detect the IQGAP1mRNA expression in C.pn-infected VSMCs.6. Western blot was performed to detect the IQGAP1expression in the VSMCs infected with C.pn.7. The expression of IQGAP1in VSMCs was downregulated by means of RNA interference. RT-PCR was then used to detect the IQGAP1mRNA expression to evaluate the effects of the specific-shRNAs on silencing the expression of IQGAP1gene.8. MTT assay was used to observe the effects of RNA interference-mediated gene silencing of IQGAP1on the VSMC adhesion induced by C.pn infection.9. Transwell assay was performed to investigate the effects of RNA interference- mediated gene silencing of IQGAP1on C.pn infection-induced VSMC migration.Results:1. GeneChip Affymetrix Rat Genome2302.0Array including31000probe sets was used to analyze the expression level of over31042transcripts and variants from over28,000well-substantiated rat genes in the VSMCs with or without C.pn infection. The results of gene chip hybridization was scanned, quantified and analyzed by the software. And35genes exhibit higher differential expression level between the normal group and C.pn infection group, including12upregulated genes and23downregulated genes. The changes in the expression level of some genes involved in cell migration was observed. TLR2gene was upregulated, while TGF-β3and AKAP12genes were downregulated. Gene ontology analysis showed that30genes of the35genes are annotated to biological process, cellular component and molecular function;4genes are not annotated to these three processes. The pathway analysis indicates that7genes are involved in the cell cycle-related signaling pathways, the signaling pathways associated with the classical pathways of complement activation, the oxidative stress-related signaling pathways and the signaling pathways related to the regulation of prostaglandin synthesis.2. The ability of VSMCs to adhere to Matrigel was significantly enhanced16h after C.pn infection. The A values in the C.pn infection group were much higher than those in the normal group. The ratio of the adhesion of C.pn-infected VSMCs was162.342%±4.691%(P<0.001).3. At19h postinfection, the number of VSMCs infected with C.pn that migrated through the membranes was much larger than that of the normal (P<0.01). The index of the migration C.pn-infected VSMCs was152.653%±7.493%(P<0.01).4. The mRNA expression of IQGAP1in theVSMCs2h after C.pn infection had no significant difference with the normal. Subsequently, the IQGAP1mRNA expression increased gradually to reach a peak at12h postinfection and then decreased gradually to a level similar to the normal at48h postinfection. 5. Western blot results showed that the expression level of IQGAP1also had a tendency to increase gradually to reach the highest level at16h postinfection and then decreased gradually.6. Green fluorescence was clearly seen24h after the transfection of VSMCs with the recombinant plasmids pGPH1/GFP/Neo-IQGAP1Scrambled shRNA and pGPH1/GFP/Neo-IQGAP1shRNAs respectively under a fluorescence microscope. And no green fluorescence can be seen in the VSMCs untransfected or only transfected with the Nucleofector solution. At48h posttransfection, the intensity of green fluorescence was significantly enhanced. RT-PCR results showed that the mRNA expression of IQGAP1in the normal VSMCs had no significant difference with theVSMCs transfected with pGPHl/GFP/Neo-IQGAP1Scrambled shRNA or only with the Nucleofector solution, while the IQGAP1mRNA expression in the VSMCs with the transfection with pGPH1/GFP/Neo-IQGAP1shRNAs was reduced remarkably compared with the normal.7. The A values in the VSMCs transfected with pGPHl/GFP/Neo-IQGAP1shRNAs were much lower than those in the normal (P<0.001) and the cell adhesion ratio was68.543%±1.197%(P<0.001). The ability of VSMCs transfected with pGPH1/GFP/Neo-IQGAP1Scrambled shRNA to adhere to Matrigel had no significant difference with the normal (P>0.05). After transfection with pGPH1/GFP/Neo-IQGAP1shRNAs, VSMCs were infected with C.pn and the A values were much lower than those in the VSMCs only infected with C.pn (P<0.001). The cell adhesion ratio was104.991%±1.225%(P<0.001).8. VSMCs infected with C.pn after transfection with pGPH1/GFP/Neo-IQGAP1shRNAs were found to migrate less than the normal (P<0.01) and the migration index was69.927%±4.838%(P<0.01). The number of VSMCs transfected with pGPH1/GFP/Neo-IQGAP1Scrambled shRNA that migrated through the membranes had no significant difference with that of the normal (P>0.05). After transfection with pGPH1/GFP/Neo-IQGAPl shRNAs, VSMCs were infected with C.pn and the number of VSMCs that migrated through the membranes was much less than that of the normal (P<0.01). The migration index was109.553%±4.401%(P<0.01). Conclusions:C.pn infection may induce the migration of VSMCs possibly through upregulating the gene of toll-like receptor2and downregulating the genes of transforming growth factor beta3and a kinase (PRKA) anchor protein12, subsequently promoting the initiation and development of atherosclerosis. Moreover, C.pn infection can promote the adhesion and migration of VSMCs possibly by upregulating the IQGAP1expression, then regulating the signal pathways involved in cell migration.