Oncology
Inhibition of endothelial cell activation by the homeobox gene Gax

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Abstract

Background. Angiogenesis is critical to tumor growth. Gax, a homeobox transcription factor whose expression in the adult is restricted mainly to the 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.

Materials and 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 β-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.

Introduction

Vascular remodeling plays a critical role in the biology of tumors, whose growth without a blood supply is limited to less than 1 mm in diameter by diffusion of oxygen and nutrients through the interstitial fluids [1]. To overcome this limitation, tumors secrete proangiogenic factors, such as vascular endothelial growth factor (VEGF) [2] and basic fibroblast growth factor (bFGF) [3], to stimulate the ingrowth of new blood vessels 1, 4. To form new tumor vasculature, endothelial cells undergo profound phenotypic changes, many of which are similar to the phenotypic changes tumor cells undergo when invading the surrounding stroma 1, 5, 6. They degrade their basement membrane and invade the surrounding tissue, migrate towards the proangiogenic stimulus secreted by the tumor, and then form tubular structures and finally neovasculature 1, 7. Although the receptors and signaling pathways activated by proangiogenic factors and cytokines have been extensively studied in endothelial cells 8, 9, much less is known about the molecular biology of the downstream transcription factors that regulate the tissue-specific gene expression controlling endothelial cell growth and differentiation and are activated by these signaling pathways. These transcription factors represent a common mechanism that can be influenced by the interaction of multiple signaling pathways and therefore might represent targets for the antiangiogenic therapy of cancer.

To understand the transcriptional control of tumor-induced angiogenesis and thereby potentially identify new ways to target it therapeutically, we decided to study the role of homeobox transcription factors in regulating the phenotypic changes that occur in endothelial cells when stimulated with proangiogenic factors. Because of their ubiquitous role as regulators of cell proliferation, migration, and differentiation, as well as body plan formation and organogenesis during embryogenesis in vertebrates and invertebrates 10, 11 and as oncogenes and tumor suppressors in various human cancers 12, 13, of all the various classes of transcription factors, we considered homeobox genes as especially likely to be important in regulating endothelial cell phenotype during angiogenesis.

Among homeobox genes, Gax (Growth Arrest-specific homeoboX) has several characteristics that suggest it as a candidate for a role as an inhibitor of the endothelial cell phenotypic changes that occur as a result of stimulation by proangiogenic factors. Originally isolated from vascular smooth muscle [14], in the adult Gax expression is largely restricted to the cardiovascular system 14, 15. In vascular smooth muscle cells, Gax expression is downregulated by mitogens 14, 16 and upregulated by growth arrest signals 14, 17. Consistent with this observation, Gax expression induces G1 cell cycle arrest [18] and inhibits vascular smooth muscle cell migration, downregulating the expression of integrins, αVβ3 and αvβ5 [19], both of which are associated with the synthetic state in vascular smooth muscle cells and the angiogenic phenotype in endothelial cells 19, 20. In vivo, Gax expression in arteries inhibits proliferative restenosis of the arterial lumen after injury [21]. Because Gax expression is largely confined to the cardiovascular system and mesoderm-derived structures 15, 22, we considered it likely that Gax is also expressed in endothelial cells because endothelial cells are also derived from mesoderm. Because of its activities in vascular smooth muscle cells, we further hypothesized that Gax may be involved in inhibiting the phenotypic changes that occur in endothelial cells in response to stimulation with proangiogenic factors. In this report, we show that Gax is also expressed in vascular endothelial cells and inhibits endothelial cell cycle activation and tube formation in response to proangiogenic factors, suggesting that it has a role as a negative regulator of angiogenesis.

Section snippets

Cells and cell culture

Human umbilical vein endothelial cells were obtained from Cambrex Biosciences (Walkersville, MD) and cultured as previously described [23] according to manufacturer’s instructions in EGM-2 medium (Cambrex Biosciences, Walkersville, MD). For experiments, recombinant VEGF165 (R & D Systems, Minneapolis, MN) was substituted in the media at the concentrations indicated for the proprietary VEGF solution.

Plasmid and adenoviral constructs

The Gax cDNA was maintained in pBluescript SK+ vectors and excised as needed for use as probes

Gax is expressed in human vascular endothelium

Because we hypothesized that Gax is expressed in endothelial cells as well as vascular smooth muscle cells, we first examined Gax expression in cultured human vascular endothelial cells and detected Gax expression in HUVECs by Northern blot (Fig. 1A) and by RT-PCR using human Gax-specific primers (Fig. 1B). Next, to verify that Gax protein is expressed in the endothelium of normal human blood vessels, we subjected a section of human kidney from a nephrectomy specimen to immunohistochemistry

Discussion

The primary target of proangiogenic factors secreted by tumor cells, and many antiangiogenic factors, is the vascular endothelial cell 1, 30. During angiogenesis, whether physiologic or tumor-induced, endothelial cells undergo distinct changes in phenotype and gene expression, including activation of proteolytic enzymes to degrade basement membrane, sprouting, proliferation, tube formation, and production of extracellular matrix 1, 4, 31. Endothelial proliferation accompanies cell invasion and

Acknowledgements

The authors would like to thank Dr. Kenneth Walsh (Boston University) for supplying the adenoviral constructs expressing Gax and β-galactosidase, as well as the p21 promoter-Luciferase constructs and the p21 cDNA. Thanks are also due to Dr. Daniel Medina (UMDNJ-Robert Wood Johnson Medical School and The Cancer Institute of New Jersey) for supplying the adenoviral construct expressing GFP. The work described in this article was supported by grants from the Foundation of UMDNJ, the New Jersey

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