Both protein activation and gene expression are involved in early vascular tube formation in vitro.
OBJECTIVE
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.


METHODS
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.
Grove
Andrew D
AD
Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892.
Prabhu
Vinay V
VV
Young
B Lynn
BL
Lee
Frank C
FC
Kulpa
Vyta
V
Munson
Peter J
PJ
Kohn
Elise C
EC
D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004789:Enzyme Activation; D004790:Enzyme Induction; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D009024:Morphogenesis / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000235:genetics / Q000502:physiology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D009924:Organ Culture Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D014471:Umbilical Veins / Q000166:cytology
D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019384:Nucleic Acid Synthesis Inhibitors; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D020543:Proteome; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; C494918:AKT1 protein, human