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Both Protein Activation and Gene Expression Are Involved in Early Vascular Tube Formation in Vitro

Laboratory of Pathology, National Cancer Institute [A. D. G., F. C. L., V. K., E. C. K.], and Mathematical and Statistical Computing Laboratory, Center for Information Technology, NIH [V. V. P., B. L. Y., P. J. M.], Bethesda, Maryland 20892

Purpose: Gene expression and protein translation regulate and direct endothelial cell proliferation and differentiation. We initiated an unbiased global search for transcriptional changes occurring during endothelial cell vascular differentiation in vitro, focusing on genes not previously implicated in vascularization and angiogenesis.

Experimental Design: cDNA and protein from human umbilical vein endothelial cells forming vascular tubes on the basement membrane surrogate, Matrigel, were collected and subjected to a global unbiased search for alterations in expression of genes not previously linked to angiogenesis.

Results: Transcriptional inhibitors blocked vascular tube formation only when present within the first hour of incubation (P < 0.05). cDNA array analysis yielded 31 differentially regulated transcripts (of 5100 queried; false positive rate, 0.4%) from gene classes representing transcription, translational regulation, cell structure, and cell adhesion. mRNA levels of caldesmon, a cytoskeleton-associated protein not previously linked to angiogenesis, were markedly reduced during early tube formation. Caldesmon protein quantity was also markedly decreased as demonstrated by laser capture microdissection of tubule cells followed by immunoblotting. Strikingly, no significant changes in transcription of genes previously demonstrated to contribute to angiogenesis, invasion, or signal transduction contained on the array were observed. To investigate the possibility that posttranslational rather than transcriptional changes were involved in facilitating tube formation, we evaluated the activation status of two dominant signal pathways, RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT. A net 3-fold reduction in phospho-AKT and a 4-fold reduction in phospho-extracellular signal-regulated kinase-1/2 occurred in a transcription-independent fashion.

Conclusions: These data suggest that both changes in gene expression and transcription-independent activation of signal transduction pathways may be involved in vascular tube formation. A combination of transcriptional and proteomic analysis has the potential to identify novel transcription-dependent and -independent molecular targets of angiogenesis.

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