Foxc2 transcription factor: a newly described regulator of angiogenesis

Introduction

Vascular network formation is vital for embryonic development as well as postnatal life. While endothelial progenitor cells coalesce and undergo vasculogenesis to form the primitive capillary plexus, angiogenesis, the subsequent process of vascular sprouting and remodeling, gives rise to a mature network of blood vessels, including arteries and veins (Coultas et al. 2005). While angiogenesis occurs in almost all tissues under pathological and physiological conditions, elucidation of the mechanisms for pathological angiogenesis such as tumor growth and ischemic heart disease is clearly of great importance to therapeutic development (Carmeliet 2005, Holderfield and Hughes 2008, Siekmann et al. 2008). Signaling pathways that control angiogenesis such as vascular endothelial growth factor (VEGF) and Notch have been identified, and regulation of angiogenesis is thought to be largely dependent on a balance between pro- and anti-angiogenic factors during the vascular network formation. For instance, Notch-Delta-like 4 (Dll4) ligand signaling is a newly recognized pathway in angiogenic sprouting and acts as a negative regulator of this process induced by VEGF (Kerbel 2008, Siekmann et al. 2008). Remarkably, despite extensive research in the field, the molecular mechanisms by which endothelial gene regulation is associated with angiogenic signaling pathways remain largely unknown and need further investigation (Minami and Aird 2005). Here I discuss the rapidly accumulating evidence that the transcription factor Foxc2 is a key regulator associated with multiple angiogenic pathways during vascular network formation.

Role of Foxc2 in Vascular Development

Foxc2 is a member of the Forkhead transcription factor family and has recently been implicated in vascular development and disease (Papanicolaou et al. 2008). During embryonic development, Foxc2 is required for the remodeling of the branchial arch arteries to form the aortic arch (Iida et al. 1997, Winnier et al. 1999). In addition, Foxc2 plays a critical role in the lymphatic vasculature. Of note, mutations in human FOXC2 are responsible for the autosomal dominant syndrome, lymphedema-distichiasis (Online Mendelian Inheritance in Man [OMIM] #153400), characterized by the obstruction of lymphatic drainage of the limbs and the growth of an extra set of eyelashes (distichiasis) (Fang et al. 2000). Deficiency of FOXC2/Foxc2 in human and mouse leads to abnormal lymphatic patterning, failure of lymphatic valve formation and lymphatic dysfunction (Kriederman et al. 2003, Dagenais et al. 2004, Petrova et al. 2004). Interestingly, in addition to lymphatic valve failure, mutations of human FOXC2 are also associated with venous valve failure (Mellor et al. 2007).

Although it has previously been reported that compound null mouse mutant embryos for Foxc2 and a related Foxc, Foxc1, fail to remodel the primary vascular plexus into a highly organized vascular network (Kume et al. 2001), the precise function of Foxc2 in angiogenesis has yet to be defined. Indeed, until recently, direct downstream targets of Foxc2 in this process were unknown. New studies have now provided evidence that Foxc2 is an important transcriptional regulator to control expression of multiple genes in critical aspects of angiogenesis.

Foxc2 Function in Angiogenesis

Recent studies have demonstrated that Foxc2 directly induces the transcription of two cell surface molecules in vascular endothelial cells, the chemokine receptor CXCR4 and integrin β3, by activating their promoters via Foxc-binding elements (Hayashi and Kume 2008a, Hayashi et al. 2008) (Figure 1A). These proteins are essential for endothelial cell migration, a key aspect of angiogenesis (Petit et al. 2007, Avraamides et al. 2008). Upon binding of the CXCL12 ligand, CXCR4 activates downstream components to induce cell migration, while the integrin β3 subunit forms a heterodimeric complex with the integrin αv subunit to allow interaction with extracellular matrix components such as vitronectin. The integrin β3 subunit also functionally interacts with VEGF receptor 2 (VEGF-R2) in endothelial cells. Although Foxc2 has no effect on the proliferation of endothelial cells, Foxc2 enhances endothelial cell migration in wound closure and transwell migration assays in vitro, as well as sprouting and microvessel formation in ex vivo aortic ring assay (Figure 1B) (Hayashi et al. 2008). Foxc2 can also induce the migration of Maden-Darby canine kidney (MDCK) epithelial cells by upregulating matrix metalloproteinase (MMP)-2, 9 (Mani et al. 2007). Taken together, these findings provided the first demonstration that Foxc2 is directly involved in angiogenesis.

Foxc2 and a related Fox protein, Foxc1, play a critical role in determining an arterial cell fate by acting upstream of Notch signaling during embryonic development (Seo et al. 2006, Red-Horse et al. 2007, Hong et al. 2008). Foxc1 and Foxc2 directly activate the promoter of Dll4, a novel ligand for Notch receptors that is specifically expressed in arterial endothelial cells. Moreover, Dll4 is also involved in the process of angiogenesis (Thurston et al. 2007, Holderfield and Hughes 2008, Siekmann et al. 2008). While VEGF guides sprouting capillaries through the filopodia of endothelial tip cells at the beginning of angiogenic sprouting, Dll4 expression is induced in the tip cells and precisely controls vessel branching. It remains to be determined whether the expression and activity of Foxc2 are localized in these cells during sprouting of endothelial cells. Further analysis has recently elucidated details of the molecular mechanism by which Foxc2 interacts with VEGF signaling to induce Dll4 expression (Hayashi and Kume 2008b). Significantly, two VEGF-mediated signaling pathways, the phosphoinositide 3-kinase (PI3K) and the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways, modulate the transcriptional activity of Foxc2 in regulating arterial gene expression, including Dll4 (Hayashi and Kume 2008b, Hong et al. 2008). In the compound null mutant embryos, VEGF expression is significantly upregulated compared with the wild-type (Seo et al. 2006), suggesting upregulation of a feedback response to impaired VEGF signaling.

Foxc2-dependent Paracrine Effects on Angiogenesis

Angiogenesis is modulated by various factors secreted from tissues surrounding blood vessels. For instance, while the expansion and regression of adipose tissue in adult life largely depend on blood vessel formation to meet the metabolic demand of the body, adipocytes in turn express known angiogenic factors, including VEGF, angiopoietin (Ang) and fibroblast growth factor (FGF), and control vessel growth and remodeling in a paracrine manner (Cao 2007). Xue et al. have shown that Foxc2 is a critical regulator of the close relationship between adipogenesis and angiogenesis (Xue et al. 2008). Given evidence that Ang-2, a ligand for the tyrosine kinase receptor Tie2 on endothelial cells, stimulates angiogenesis in the presence of VEGF (Tait and Jones 2004), Foxc2 indirectly controls angiogenesis as well as remodeling and maturation of the vasculature by inducing Ang-2 expression in adipocytes (Xue et al. 2008). Interestingly, both Foxc2 and Ang-2 mutant mice exhibit a similar lymphatic phenotype, the lack of lymphatic valves and improper recruitment of smooth muscle cells into lymphatic vessels (Gale et al. 2002, Petrova et al. 2004, Shimoda et al. 2007, Dellinger et al. 2008). However, the nature of functional interactions between Foxc2 and Ang-2 in the remodeling and maturation of the lymphatic vasculature is currently unclear.

Conclusion and Future Perspectives

Recent studies have now provided new insights into the molecular and cellular mechanisms underlying Foxc2-mediated transcriptional control during vascular network formation. As illustrated in Figure 2, Foxc2 is central to the regulation of critical signaling pathways in a highly coordinated process of angiogenesis, including endothelial cell migration and vascular remodeling and maturation.

Although the significance of the role of Foxc2 under adult pathological conditions such as cardiovascular disease and tumor angiogenesis remains to be elucidated, recent gene expression profiling using adipose tissue revealed that in addition to Ang-2, Foxc2 can upregulate potent angiogenic factors such as placental growth factor (PlGF), ephrinB2 and platelet-derived growth factor (PDGF) (Xue et al. 2008). These results raise the possibility that Foxc2 simultaneously controls various angiogenic signaling pathways under pathological conditions. Notably, it has recently been shown that Foxc2 expression is increased in peri-infarcted zones of the adult rat left ventricle (Philip-Couderc et al. 2008) and that Foxc2 expression is associated with human heart failure (Hannenhalli et al. 2006).

Another perspective regarding cardiovascular disease is a possible involvement of Foxc2 in the development and function of endothelial progenitor cells (EPCs). Since the identification of EPCs in human peripheral blood in 1997, substantial progress has been made in understanding EPC-mediated postnatal neovascularization (Hirschi et al. 2008, Kawamoto and Losordo 2008). The mobilization of EPCs from bone marrow and recruitment of these cells into the sites of neovascularization are critical for vascular regeneration in patients with peripheral vascular and coronary artery disease. Significantly, the recently reported signaling pathways associated with Foxc2 such as VEGF, Notch and CXCR4 are all involved in these processes (Gehling et al. 2000, Walter et al. 2005, Kwon et al. 2008). While the definition of EPCs is still a matter of debate because of the lack of unique cell surface molecules that specifically recognize EPCs (Prater et al. 2007, Hirschi et al. 2008), very little is known about transcriptional regulation in these cells. Given evidence that another Fox protein, FoxO4, plays a role in EPC apoptosis (Urbich et al. 2005), investigation of gene regulatory networks involving Foxc2 in EPCs is likely to lead to the advancement of cell-based therapies for cardiovascular regeneration. Taken together, further analysis of a possible role of Foxc2 in therapeutic angiogenesis may promote a new treatment approach for ischemic heart disease in the future.