Inhibition of endothelial cell activation by the homeobox gene Gax.
BACKGROUND
cardiovascular system, strongly inhibits growth factor-stimulated phenotypic modulation of vascular smooth muscle cells in vitro and in vivo. The function of Gax in vascular endothelium is unknown, but we hypothesized that it may play a similar role there. We therefore studied Gax expression in vascular endothelial cells and its effects on proliferation and tube formation.


METHODS
Gax expression in normal endothelial cells was examined in vitro by Northern blot and reverse transcriptase polymerase chain reaction and in vivo by immunohistochemistry. A replication-deficient adenovirus was then used to express Gax in human umbilical vein endothelial cells (HUVECs). HUVEC proliferation, 3H-thymidine uptake, p21 expression, and tube formation on reconstituted basement membrane were measured at different viral multiplicities of infection.


RESULTS
Gax mRNA was detected in HUVECs by reverse transcriptase polymerase chain reaction and Northern blot analysis and in normal vascular endothelium by immunohistochemistry. Compared with controls transduced with a virus expressing beta-galactosidase, Gax strongly inhibited HUVEC proliferation and mitogen-stimulated 3H-thymidine uptake. p21 expression in HUVECs transduced with Gax was increased up to 5-fold as measured by Northern blot, and p21 promoter activity was activated by 4- to 5-fold. Tube formation on Matrigel was strongly inhibited by Gax expression.


CONCLUSIONS
Gax is expressed in vascular endothelium and strongly inhibits endothelial cell activation in response to growth factors and tube formation in vitro. These observations suggest that Gax inhibits endothelial cell transition to the angiogenic phenotype in response to proangiogenic growth factors and, as a negative regulator of angiogenesis, may represent a target for the antiangiogenic therapy of cancer.
Gorski
David H
DH
Division of Surgical Oncology, UMDNJ-Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey 08901, USA.
Leal
Alejandro J
AJ
D015152:Blotting, Northern; D002455:Cell Division; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000098:blood supply; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000737:chemistry; D018919:Neovascularization, Physiologic; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014471:Umbilical Veins
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; C081336:Meox2 protein, rat; D009124:Muscle Proteins; D049452:Green Fluorescent Proteins