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      Differential Expression of the α1 Type VIII Collagen Gene by Smooth Muscle Cells from Atherosclerotic Plaques of Apolipoprotein-E-Deficient Mice

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          We have investigated the morphology, growth and gene expression of smooth muscle cell (SMC) cultures derived from advanced atherosclerotic plaques and from non-plaque-containing aorta of individual apolipoprotein-E-deficient mice. The initial outgrowth of cells was faster from plaques (P) than from non-plaque segments (NP), but the cells in P cultures divided more slowly than NP cells in subcultures. By the 6th passage, the general growth pattern, morphology, ploidy and response to mitogenic factors of the cells were no longer consistently different in P and NP cultures. However, by the use of differential display, several transcripts were identified that were differentially expressed in three independent pairs of P and NP cultures. One of the transcripts, from a type VIII collagen gene, was elevated in all the P cultures compared to their NP counterparts even at the 40th passage. The α1 type VIII collagen transcripts were also readily detectable by RT-PCR in freshly dissected plaques, but not in the normal parts of aortas from the apolipoprotein-E-deficient mice. In situ hybridization showed that the transcripts were limited to the fibrous cap of plaques. Thus, SMCs from atherosclerotic plaques produce type VIII collagen and this differential expression continues when the cells are maintained in tissue culture.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.

            Author and article information

            J Vasc Res
            Journal of Vascular Research
            S. Karger AG
            June 2000
            24 May 2000
            : 37
            : 3
            : 158-169
            aDepartment of Pathology and Laboratory Medicine, and bDepartment of Biochemistry, University of North Carolina, Chapel Hill, N.C., USA
            25727 J Vasc Res 2000;37:158–169
            © 2000 S. Karger AG, Basel

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            Page count
            Figures: 7, References: 42, Pages: 12
            Research Paper


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