Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo.

Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.

A link between smooth muscle cell death and extracellular matrix degradation during vascular atrophy.

OBJECTIVE: High blood flow induces neointimal atrophy in polytetrafluoroethylene (PTFE) aortoiliac grafts and a tight external PTFE wrap of the iliac artery induces medial atrophy. In both nonhuman primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins at ≤4 days. We hypothesized that matrix loss would be linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy. METHODS: DNA microarray analysis (Sentrix Human Ref 8; Illumina, San Diego, Calif; ∼23,000 genes) was performed on arterial tissue from the wrap model (n = 9) and graft neointima from the graft model (n = 5) 1 day after wrapping or the switch to high flow, respectively. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied after Fas ligand-induced cell death in cultured smooth muscle cells and organ cultured arteries. RESULTS: Microarray analysis showed 15 genes were regulated in the same direction in both atrophy models: 9 upregulated and 6 downregulated. Seven of nine upregulated genes were confirmed by qRT-PCR in both models. Upregulated genes included the ECM-degrading enzymes ADAMTS4, tissue plasminogen activator (PLAT), and hyaluronidase 2; possible growth regulatory factors, including chromosome 8 open reading frame 4 and leucine-rich repeat family containing 8; a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1); a dead cell removal factor (ficolin 3); and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five downregulated genes were confirmed but only in one or the other model. Of the seven upregulated genes, ADAMTS4, PLAT, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 were also upregulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with Z-VAD inhibited FasL-mediated cell death, but not gene induction. CONCLUSION: Seven gene products were upregulated in two distinctly different in vivo nonhuman primate vascular atrophy models. Induction of cell death by FasL in vitro induced six of these genes, including the ECM-degrading factors ADAMTS4, hyaluronidase 2, and PLAT, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

A Marfan syndrome gene expression phenotype in cultured skin fibroblasts.

BACKGROUND: Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. RESULTS: We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. CONCLUSION: Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 x 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.

Synectin-dependent gene expression in endothelial cells.

Synectin (GIPC1), a receptor scaffold protein, has been isolated by our laboratory as a syndecan-4 cytoplasmic domain binding partner that regulates important aspects of cell motility (Gao Y, Li M, Chen W, Simons M. J Cell Physiol 184: 373-379, 2000; Tkachenko E, Elfenbein A, Tirziu D, Simons M. Circ Res 98: 1398-1404, 2006). Moreover, synectin plays a major role in arterial morphogenesis and in growth factor signaling in arterial endothelial cells by regulating Rac1 activity (Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh J, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, Simons M. Dev Cell 10: 783-795, 2006). The present study was carried out to characterize changes in synectin-dependent gene expression induced by homozygous disruption of the gene in endothelial cells. Using a combination of suppression subtraction hybridization and high throughput microarray technology, we have identified aberrant biological processes of transcriptional regulation in synectin(-/-) primary endothelial cells including abnormal basal regulation of genes associated with development, cell organization and biogenesis, intracellular tracking, and cell adhesion. Analysis of gene expression following FGF2 treatment demonstrated significant abnormalities in transcription, cytoskeletal organization and biogenesis, and protein modification and transport in synectin(-/-) compared with synectin(+/+) endothelial cells. These results confirm synectin involvement in FGF2-dependent signal transduction and provide insights into synectin-dependent gene expression in the endothelium.

Bone morphogenetic protein 4: potential regulator of shear stress-induced graft neointimal atrophy.

OBJECTIVE: Placement in baboons of a distal femoral arteriovenous fistula increases shear stress through aortoiliac polytetrafluoroethylene (PTFE) grafts and induces regression of a preformed neointima. Atrophy of the neointima might be controlled by shear stress-induced genes, including the bone morphogenetic proteins (BMPs). We have investigated the expression and function of BMPs 2, 4, and 5 in the graft neointima and in cultured baboon smooth muscle cells (SMCs). METHODS: Baboons received bilateral aortoiliac PTFE grafts and 8 weeks later, a unilateral femoral arteriovenous fistula. RESULTS: Quantitative polymerase chain reaction showed that high shear stress increased BMP2, 4, and 5 messenger RNA (mRNA) in graft intima between 1 and 7 days, while noggin (a BMP inhibitor) mRNA was decreased. BMP4 most potently (60% inhibition) inhibited platelet-derived growth factor-stimulated SMC proliferation compared with BMP2 and BMP5 (31% and 26%, respectively). BMP4 also increased SMC death by 190% +/- 10%. Noggin reversed the antiproliferative and proapoptotic effects of BMP4. Finally, Western blotting confirmed BMP4 protein upregulation by high shear stress at 4 days. BMP4 expression demonstrated by in situ hybridization was confined to endothelial cells. CONCLUSIONS: Increased BMPs (particularly BMP4) coupled with decreased noggin may promote high shear stress-mediated graft neointimal atrophy by inhibiting SMC proliferation and increasing SMC death.

Atherosclerotic plaque smooth muscle cells have a distinct phenotype.

OBJECTIVE: The present study addresses the question, "Are plaque smooth muscles cells (SMCs) genetically distinct from medial SMCs as reflected by the ability to maintain a distinctive expression phenotype in vitro?" METHODS AND RESULTS: Multiple cell strains were developed from carotid endarcterectomy specimens, and quadruplicate array hybridizations were completed for each sample. A new normalization protocol was developed and used to analyze the data. Permutation analysis suggests that most of the significant differences in expression could not have occurred by chance. A broad pattern of significant expression differences, consisting of almost 5% of the genes probed, was detected. Quantitative polymerase chain reaction (QPCR) confirmation was found in 70% of a subset of genes selected for validation. CONCLUSIONS: The SMC cultures were nearly indistinguishable by morphological features, population doubling time, and sensitivity to cell death induced by Fas cross-linking. Surprisingly, array expression analysis identified differences so extensive that we conclude that plaque and medial SMCs are distinctly different SMC cell types.