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Abstract —An accumulation of evidence suggests that vascular smooth muscle is composed of cell subpopulations with distinct patterns of gene expression. Much of this evidence has come from serendipitous discoveries of genes marking phenotypically distinct aortic cultures derived from 12-day-old and 3-month-old rats. To identify more systematic differences, we isolated 40 genes at random from libraries of these 2 cultures and examined message expression patterns. To determine consistency of differential expression, we measured mRNA levels in 4 sets of cultures in 6 phenotypically distinct aortic cell clones and in balloon injured rat carotid arteries to determine the relevance of these differences in vitro to in vivo biology. The following 5 consistently differentially expressed genes were identified in vitro: zonula occludens 2 (ZO-2); peroxisome proliferator-activated receptor δ (PPARδ); secreted protein, acidic and rich in cysteine (SPARC); α1(I)collagen; and A2, an uncharacterized gene. We examined these 5 clones during carotid artery injury and an inconsistently differentially expressed clone Krox-24 because, as an early response transcription factor, it could be involved in the injury response. PPARδ, A2, and Krox-24 mRNAs were upregulated during the day after injury. ZO-2 and α1(I)collagen messages were modulated for up to a month, whereas SPARC message showed no consistent change. An analysis of ZO-2 and other tight junction genes indicates that tight junctions may play a role in smooth muscle biology. These data suggest that a systematic analysis of these libraries is likely to identify a very large number of differentially expressed genes. ZO-2 is particularly intriguing both because of this tight junction gene’s pattern of prolonged over-expression after injury and because of its potential role in determining the distinctive epithelioid phenotype of smooth muscle cells identified in rat and other species.

Abstract —The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.

Abstract Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call π SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether π and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [ 35 S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 K av on Sepharose CL-2B) that was not detectable in a π SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial–type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat π SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types.