Application of mechanical stimuli has been shown to alter gene expression in bladder smooth muscle cells (SMC). To date, only a limited number of "stretch-responsive" genes in this cell type have been reported. We employed oligonucleotide arrays to identify stretch-sensitive genes in primary culture human bladder SMC subjected to repetitive mechanical stimulation for 4 h. Differential gene expression between stretched and nonstretched cells was assessed using Significance Analysis of Microarrays (SAM). Expression of 20 out of 11,731 expressed genes ( approximately 0.17%) was altered >2-fold following stretch, with 19 genes induced and one gene (FGF-9) repressed. Using real-time RT-PCR, we tested independently the responsiveness of 15 genes to stretch and to platelet-derived growth factor-BB (PDGF-BB), another hypertrophic stimulus for bladder SMC. In response to both stimuli, expression of 13 genes increased, 1 gene (FGF-9) decreased, and 1 gene was unchanged. Six transcripts (HB-EGF, BMP-2, COX-2, LIF, PAR-2, and FGF-9) were evaluated using an ex vivo rat model of bladder distension. HB-EGF, BMP-2, COX-2, LIF, and PAR-2 increased with bladder stretch ex vivo, whereas FGF-9 decreased, consistent with expression changes observed in vitro. In silico analysis of microarray data using the FIRED algorithm identified c-jun, AP-1, ATF-2, and neurofibromin-1 (NF-1) as potential transcriptional mediators of stretch signals. Furthermore, the promoters of 9 of 13 stretch-responsive genes contained AP-1 binding sites. These observations identify stretch as a highly selective regulator of gene expression in bladder SMC. Moreover, they suggest that mechanical and growth factor signals converge on common transcriptional regulators that include members of the AP-1 family.

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Cancer therapies based on the inhibition of angiogenesis by endostatin have recently been developed. We demonstrate that a mutated form of human endostatin (P125A) can inhibit the angiogenic switch in the C3(1)/Tag mammary cancer model. P125A has a stronger growth-inhibitory effect on endothelial cell proliferation than wild-type endostatin. We characterize the angiogenic switch, which occurs during the transition from preinvasive lesions to invasive carcinoma in this model, and which is accompanied by a significant increase in total protein levels of vascular endothelial growth factor (VEGF) and an invasion of blood vessels. Expression of the VEGF 188 mRNA isoform, however, is suppressed in invasive carcinomas. The VEGF receptors fetal liver kinase-1 (Flk-1) and Fms-like tyrosine kinase-1 (Flt-1) become highly expressed in epithelial tumor and endothelial cells in the mammary carcinomas, suggesting a potential autocrine effect for VEGF on tumor cell growth. Angiopoietin-2 mRNA levels are also increased during tumor progression. CD-31 Key words: angiogenesis; endostatin; vascular endothelial growth factor; mammary cancerThe growth and metastatic spread of solid tumors is critically dependent on the formation of new blood vessels through angiogenesis. 1,2 During this process, the effects of proangiogenic factors dominate over the antiangiogenic factors, resulting in the angiogenic switch. 2 A large variety of growth factors modulate tumor angiogenesis, including those that alter the extracellular matrix as well as those that promote endothelial cell proliferation and migration. 3 Among the proangiogenic factors described, vascular endothelial growth factor (VEGF) has been shown to be frequently upregulated in a variety of tumors 4 and has been well characterized. 4,5 The 2 high-affinity VEGF receptors fetal liver kinase-1 (Flk-1) and Fms-like tyrosine kinase-1 (Flt-1), members of the tyrosine kinase receptor family, transduce the activity of this growth factor. 5 The activation of intracellular signaling pathways by these receptors involves their autophosphorylation and, as a consequence, the stimulation of endothelial cell proliferation (especially in the case of the Flk-1 receptor), migration, tubule formation, and angiogenesis in vivo. [5][6][7] Although the expression of VEGF receptors was initially localized to endothelial cells, recent studies have shown that epithelial and tumor cells from various tissues also express VEGF receptors and respond to stimulation by VEGF. 8 -10 Another family of vascular growth factors, angiopoietins, has recently been characterized. 11 Angiopoietin-1 mediates cell-cell interactions between endothelial cells and other neighboring cells, such as smooth muscle cells. 12,13 In contrast to VEGF, angiopoietin-1 does not directly stimulate endothelial cell growth. Angiopoietin-2 is typically expressed at sites of vascular remodeling in the adult 14 and in tumors, where it stimulates endothelial cell growth and angiogenesis. 11,14 Moreover, angiopoietin-2 acts in a complementary a...