CIITA downregulates collagen type I promoters and activates MHC II promoter in human SMCs. A, COL1A1 promoter construct (−311/+114) (0.5 μg) was cotransfected with GFP construct (0.1 μg) and/or pcDNA3 empty vector or CIITA type I, III, and IV expression vectors (0.5 μg) into human aortic SMCs. B, COL1A2 promoter construct (pGL3-Col-Luc, −357/+55) (0.5 μg) was cotransfected with GFP construct (0.1 μg) and/or pcDNA3 empty vector or CIITA type I, III, and IV expression vectors as in A. C, pGL3-HLA-DRA promoter construct (pGL-300-DRα) was cotransfected with GFP construct and either pcDNA3 empty vector or CIITA type I, III, and IV expression vectors as in A. Luciferase activity, corrected for transfection efficiency with GFP, was normalized by protein concentration and is shown as the mean±SD (n=3). The data were evaluated for significance by ANOVA followed by Sheffe's post hoc analysis using SPSS software (*P<0.01).

CIITA isoforms and MHC II genes are not affected by simvastatin treatment of human SMCs. Aortic human SMCs were pretreated with 1 μmol/L simvastatin or vehicle for 18 hours, followed by 100 U/mL IFN-γ for 24 hours. RNA and proteins were extracted, as described in the expanded Materials and Methods section (supplemental data). TaqMan probes and specific primers were used to amplify cDNA for MHC II (HLA-Drα) (A), CIITA type III and IV (B), RFX5 (C), and COL1A2 (D). Each graph shows results from a representative experiment performed in duplicate repeated 4 times and values represent mean±SD. The data from at least 4 experiments were evaluated for significance by ANOVA followed by Sheffe's post hoc analysis using SPSS software (*P<0.001, **P<0.01, **P<0.05). IFN-γ significantly increased MHC II, RFX5, and CIITA (*P<0.001). Simvastatin decreased COL1A2 (**P<0.01) and RFX5 (***P<0.05). Protein levels were detected by Western blotting and antibodies for RFX5 (C) and CIITA (B).

IFN-γ stimulated SMCs express great levels of HLA-Drα. RNA was extracted from human aortic SMCs that were treated with or without 100 U/mL IFN-γ at various times (2, 4, 6, 24, and 48 hours). TaqMan probe and specific primers were used to amplify cDNA for HLA-Dra (A and B) by real-time PCR. Each experiment was repeated at least 3 times and values represented as mean±SD. The data were evaluated for significance by ANOVA followed by Sheffe's post hoc analysis using SPSS software (*P<0.01). C, Cytoplasmic proteins were extracted from human aortic SMCs that were induced with IFN-γ (100 U/mL) at different times (2, 4, 6, and 24 hours). Proteins (20 mg) were separated in 10% SDS gel and electroblotted to a nitrocellulose membrane. The membrane was incubated with HLA-Dra antibody (FL-254; Santa Cruz Biotechnology) that detected a correct size band of 32 kDa after 24 hours of IFN-γ treatment. The chemiluminescence was measured with a Kodak Image station.

Type I Collagen α1(I) and α2(I) mRNA is repressed at 48 hours. RNA was extracted from human aortic SMCs that were treated with or without 100 U/mL of IFN-γ for 0, 24, and 48 hours. TaqMan probe and specific primers were used to amplify cDNA for COL1A 1 (A) and COL1A 2 (B) by real-time PCR. Each experiment was repeated at least 3 times and values represented mean±SD (n=5). The data were evaluated for significance by ANOVA followed by Sheffe's post hoc analysis using SPSS software (*=P<0.01). C, Cytoplasmic proteins were extracted from human aortic SMCs that were induced with IFN-γ (100 U/mL) at different times (0, 2, 24, and 48 hours). Proteins (20 μg) were separated in 4% Novex Tris–Glycin gel (Invitrogen) and SDS denaturing buffer and electroblotted to a nitrocellulose membrane. The membrane was incubated with anti-collagen type I (Rockland no. 103) at 1:5000 dilution. The antibody has been shown to be specific for α1(I) and not α2(I).50 The chemiluminescence was measured with a Kodak image station.

Detection of CIITA type I, III, and IV isoforms by real-time quantitative PCR in human aortic SMCs. A, Schematic representation of primer design. The first and second exons of CIITA types are depicted. The first exon encodes for the 5′ untranslated region (UTR) and the 5′ end of each isoform. Transcription of each individual isoform is directed by 3 different promoters (PI, PIII, PIV). Position of the forward specific primer is shown in the first exon (I F, III F, IV F). For CIITA type III and IV, the TaqMan probes were designed complementary to the first and second exon borders. Reverse primer was common (III) and complementary to the second exon. For CIITA type I, the TaqMan probe and the reverse primer were within the first exon. The size of the amplicons predicted from the cDNA sequence is shown. B, Aortic smooth muscle cell cDNA was amplified with CIITA isoform specific primers by real-time PCR and SYBR green detection. The expression of CIITA type I, III, and IV was measured by SYBR green cycle threshold (Ct) and the products of PCR were also resolved by 1.5% agarose gel electrophoresis. C, RNA from dendritic cells, isolated from human peripheral blood, was extracted and converted to cDNA. Dendritic cell cDNA was amplified with CIITA-specific primers, as in B, and served as a positive control for the expression of all 3 CIITA isoforms. The products of PCR were detected by 1.5% agarose gel. D, Aortic SMCs were treated for 24 hours with or without recombinant IFN-γ (100 U/mL). RNA from 3 wells of cultured cells was extracted, converted to cDNA, and subjected to real-time PCR with TaqMan probes/primers specific for CIITA isoforms. Specific CIITA plasmids were used to construct standard curves and for conversion of Ct values to copy numbers. Results are presented as absolute values and numbers of molecules that are expressed after IFN-γ stimulation. Results are normalized to 18 S ribosomal RNA and error bars represent mean of quadruplicates±SD. The data (n=4) were evaluated for significance by ANOVA followed by Sheffe's post hoc analysis using SPSS software. CIITA type III and CIITA type IV expression was significantly higher than CIITA type I expression. This difference was statistically significant (*P<0.01).