Volume 7, Issue 5, Pages 1059-1069 (May 2001) Transcriptome Analysis Reveals a Role of Interferon-γ in Human Neointima Formation  Dietlind Zohlnhöfer, Thomas Richter, Franz-Josef Neumann, Thomas Nührenberg, Rainer Wessely, Richard Brandl, Alexander Murr, Christoph A. Klein, Patrick A. Baeuerle  Molecular Cell  Volume 7, Issue 5, Pages 1059-1069 (May 2001) DOI: 10.1016/S1097-2765(01)00239-8

Figure 1 cDNA Array Analysis of Gene Expression Four Clontech Atlas microarrays containing a total of 2435 human cDNAs were hybridized with cDNA labeled with dig-dUPT prepared from RNA from in-stent neointima (n = 10) and from control media (n = 11) as described in Experimental Procedures. Spots indicate the mean of the relative expression of the two examined groups. (A) The expression of all examined genes in this study is shown. (B) Shown is the expression of the 223 differentially expressed genes that were more than 2.5-fold induced or reduced in neointima and showed a descriptive p value <0.05 in the Wilcoxon test Molecular Cell 2001 7, 1059-1069DOI: (10.1016/S1097-2765(01)00239-8)

Figure 2 Cluster Image of the 223 Genes whose mRNAs Were Differentially Expressed between Neointima and Control This subset of genes was clustered into four groups on the basis of their expression in different cell types. The expression pattern of each gene in this set is displayed here as a horizontal strip. Each column represents the average mRNA expression level of the examined group. For each gene, the average of the mRNA level of neointima (n = 10), control (n = 11), proliferating SMCs (n = 3), and blood samples (n = 10) normalized to the mRNA expression level of the housekeeping genes is represented by a gray value according to the signal intensity scale at the bottom. (A) Group I contained genes only expressed in neointima specimens. (B) Group II contained genes expressed simultaneously in neointima and proliferating SMCs. (C) Group III consisted of genes whose mRNA was expressed in neointima as well as in blood. (D) Group IV contained genes whose mRNA was overexpressed in control specimens Molecular Cell 2001 7, 1059-1069DOI: (10.1016/S1097-2765(01)00239-8)

Figure 3 IFN-γ-Associated Cluster of 37 Genes that Were Regulated in Neointima Versus Control Each column represents a single specimen, and each row represents a single gene. Brackets indicate independent hybridization experiments. Gray values correspond to signal intensity as shown at the bottom of the figure Molecular Cell 2001 7, 1059-1069DOI: (10.1016/S1097-2765(01)00239-8)

Figure 4 Upregulation of IRF-1 Protein in Coronary In-Stent and in Carotid Restenosis Specimen Immunostaining with antibodies against the transcription factor IRF-1 demonstrates a nuclear staining pattern of most smooth muscle cells in coronary specimens ([C], arrows; brown reaction product) and a nuclear and perinuclear cytoplasmatic staining in carotid specimens ([E], arrows; brown reaction product), whereas there is no immunostaining in healthy control media (F). Neointima is mainly composed of α-actin-positive smooth muscle cells ([C]; red reaction product). There were few CD3-positive T cells ([D], arrows; brown reaction product). The experiment shown is representative of four independent experiments for coronary and six independent experiments for carotid neointima specimens. The bars represent 100 μm Molecular Cell 2001 7, 1059-1069DOI: (10.1016/S1097-2765(01)00239-8)

Figure 5 Effect of IFN-γ on the Survival of Cultured Proliferating SMCs Flow cytometry analysis of spontaneous (A and C) and H2O2-induced apoptosis (B and D). Cells were double stained by annexin V and PI at 6 hr after treatment with 100 μmol/L H2O2. A representative analysis of five independent experiments is shown Molecular Cell 2001 7, 1059-1069DOI: (10.1016/S1097-2765(01)00239-8)

Figure 6 The Effect of an IFN-γ Receptor Null Mutation on the Development of Neointima in a Mouse Model of Restenosis (A–D) Representative microphotographs of cross-sectioned mouse carotid arteries from wild-type (wt) and IFN-γR−/− knockout mice are shown for the untreated artery (control) and the contralateral ligated artery (ligated) at 4 weeks after ligation. The elastica-van-Giesson staining procedure was used. The bars represent a length of 100 μm. (E) Data from 16 wild-type and 11 IFN-γR−/− mice are shown as mean ± SEM (bars) and analyzed by the t test for unpaired samples. The scale gives the thickness of media and neointima in μm. Open columns, untreated and ligated carotid arteries from wild-type mice; filled columns, untreated and ligated carotid arteries from IFN-γR−/− mice. The shaded area indicates the thickness of neointima Molecular Cell 2001 7, 1059-1069DOI: (10.1016/S1097-2765(01)00239-8)