**4.8. HOXD1**

important for PAA formation, and the authors provide a novel model to study the molecular

The function of the *HOXB3* gene was studied after finding the function of its paralogous gene, *HOXD3*. While *HOXD3* is required for mediating the invasive and migratory behavior of endothelial cells during the early stages of neovascularization, *HOXB3* is required for the morphogenesis of new capillary tubes, suggesting that these paralogous Hox genes may perform complementary functions [53]. The authors also found that the capillary morphogenesis

The *HOXB5* gene, also known as Hox-2.1, codes for a potent transcriptional regulator present in several adult tissues. Similar to *HOXA9*, *HOXB5* has been associated with vascular alterations. In this regard, studies have shown that *HOXB5* homeobox protein regulates the expression of VEGFR2, the earliest marker of endothelial precursors, by direct binding to the *HOXB5*-binding element (HBE) in the VEGFR2 gene [66]. They also found that overexpression of HoxB5 increased the number of angioblasts during embryonic stem cell differentiation and the number of mature endothelial cells, which in turn have been associated with high expression of platelet endothelial cell adhesion molecule (PECAM) and the formation of primitive blood vessels [66]. Years later, the same research group investigated the in vivo role of HoxB5 in angiogenesis using the chick (*Gallus gallus*) chorioallantoic membrane assay. They concluded that HoxB5 exerted an activating effect on angiopoietin 2 (ANG2), which was essential for endothelial cell sprouting and vascular growth [60]. More recently, the same group investigated the role of HoxB5 overexpression during revascularization in ischemic disease using femoral artery ligation in C57BL/6 mice. They observed that HoxB5 enhanced perfusion restoration and increased capillary density in vivo via monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) upregulation and increased endothelial cell migration [67]. Furthermore, other studies have shown that HoxB5 is a transactivator of the promoter of VEGFR2, an early marker of endothelial precursors [66], which might be involved in the differentiation of mesoderm-derived precursors toward an endothelial phenotype [66, 68]. In fact, it has been described that overexpression of HoxB5 leads to differentiation of mesoderm-derived precursors toward the endothelial phenotype, which in turn lead to high expression of angiopoietin 2 (ANG2) and therefore enhance vascularization in a model of fertilized white Leghorn chicken eggs [68].

*HOXB7* has been associated with tumor progression and angiogenesis [61]. Care et al. in 2001 provided evidence that HoxB7 promotes tumor-associated angiogenesis by increasing the expression of VEGF, melanoma growth stimulatory activity/growth-related oncogene alpha, interleukin-8, and angiopoietin 2 (ANG2) in SkBr3 cells [69]. The authors concluded that HoxB7 acted as a key factor in a tumor-associated angiogenic switch [69]. In 2008, Murthi et al. identified differences in

origin of great artery defects, which are often observed in human patients.

36 Endothelial Dysfunction - Old Concepts and New Challenges

induced by *HOXB3* was mediated by ephrin A1 ligand (EFNA1) [53].

**4.5. HOXB3**

**4.6. HOXB5**

**4.7. HOXB7**

*HOXD1* is specifically expressed in mature endothelial cells compared to early-stage EPC [62, 75]. However, not only HoxD1 is expressed in these cells, but also microarray studies have revealed that several Hox genes from the cluster on chromosome 2 such as *HOXD1*, *HOXD3*, *HOXD4*, *HOXD8*, and *HOXD9* were highly expressed in blood-derived endothelial cells [62]. In particular, *HOXD1* regulates endothelial cell migration and cell adhesion on fibronectin by targeting integrin β1 (ITGβ1) in mature endothelial cells [75].

#### **4.9. HOXD3**

*HOXD3* is a member of the *HOXD* cluster on chromosome 2, and it can be induced by extracellular matrix protein, Del-1, and integrin alphavbeta5 interaction on resting endothelium. Del-1 is a protein that accumulates around angiogenic blood vessels and promotes angiogenesis in the absence of exogenous growth factors [76]. Zhong et al. showed that Del-1 initiates angiogenesis by binding to integrin alphavbeta5 on the resting endothelium, resulting in expression of HoxD3 [76]. HoxD3 was then promoting angiogenesis by inducing the expression of the pro-angiogenic molecule integrin alphavbeta3 (integrin β3) [76]. These findings provide evidence for an angiogenic switch that can be initiated in the absence of exogenous growth factors indicating that the angiogenic matrix protein Del-1 may be a useful tool for the therapy of ischemic disease [76]. A year later, Chen and Ruley demonstrated the role of HoxD3 expression in human brain vessels [52]. They showed that HoxD3 expression significantly induced cerebral angiogenesis, increased focal cerebral blood flow, and reduced vascular leakage by inducing integrin β3. These data suggest that HoxD3 plays an important role in regulating angiogenesis. Other studies reported that HoxD3 mediates the basic fibroblast growth factor (bFGF)-induced expression of integrin β3 and urokinase plasminogen activator (uPA) in HUVECs [77] and promotes angiogenesis in in vivo models [78, 79]. Furthermore, *HOXD3* has been shown to be involved in cerebral angiogenesis in mice [52].
