**4.3. HOXA13**

both in vitro and in vivo studies using mice [46, 47]. Similar results were demonstrated by Hansen et al. who confirmed that *HOXA3* is a potent inducer of angiogenesis in vivo and also promotes direct keratinocyte migration [48]. These results suggest that *HOXA3* potentiates two key processes involved in efficient wound repair: angiogenesis and reepithelialization [46, 48]. Gene transfer studies of *HOXA3* suggest that this gene also functions as a potent inducer of wound repair in genetically modified diabetic animals. A single application of protein HoxA3 resulted in complete healing of wounds after 42 days, while wounds treated with the control plasmid without *HOXA3* (β gal) required 77 days for complete tissue repair. In addition, it was demonstrated that secreted protein HoxA3 or HoxA5, coming from respective genes and derived from composite skin constructs, exhibits decreased expression of CCL-2 and CxCL-12 inflammatory mediators, which play a key role in the attraction of monocytes, macrophages, and other wound immune cells [48]. Thus, reduced recruitment of leukocytes mediated by *HOXA3* may contribute to the prolonged integrity and viability of the composite skin constructs expressing *HOXA3*, by reducing inflammation during wound healing process. Taken together, the combined actions of HoxA3 on endothelial cells and keratinocytes lead to increased angiogenesis, normal epidermal differentiation, reduced expression of inflammatory mediators, and reduced graft contraction. These effects suggest that HoxA3 may have therapeutic benefits in wound repair by improving the integrity of composite skin grafts.

**Figure 2.** HOX genes modulate the expression of crucial target genes to promote the differentiation of mature endothelial cells. Hox genes promote the differentiation of endothelial progenitor cells, which exhibit an immature

vascular endothelial growth factor receptor 2 (VEGFR2 or KDR), CD31, von Willebrand factor (vWF), and the lectin-type oxidized LDL receptor 1 (LOX-1). To promote this phenotype, some Hox genes upregulate crucial genes such as fetal liver kinase 1 (Flk1), angiopoietin 2 (ANG2), ephrin type-B receptor 4 (EphB4), and FI3K receptor, whereas other Hox genes downregulate other factors such as hypoxia-induced factor type 1α (HIF1α), cyclooxygenase-2 (cox-2), ephrin

), into mature endothelial cells that express endothelial nitric oxide synthase (eNOS),

The *HOXA9* gene code for two different proteins, HA-9A and HA-9B isoform A (HA-9A) and HoxA9 protein isoform B (HA-9B) [49] that share a common homeodomain [15]. The expression of HA-9A has been observed exclusively during fetal development, whereas the HA-9B has been found not only in fetal but also in adult organism and specifically in endothelial cells [49, 50].

In 2004, Bruhl et al. showed that *HOXA9* was able to regulate angiogenesis [51]. These authors using human umbilical vein endothelial cells (HUVECs) with sense/antisense oligonucleotides or siRNA for this gene observed that *HOXA9* expression was essential for endothelial cell migration and tube formation. Also, they evaluated the regulation of ephrin type-B (Eph) receptor B4

**4.2. HOXA9**

phenotype (CD70+

CD34+

type-a receptor 1 (EphwA1), and VEGFR2.

Oct-4+

34 Endothelial Dysfunction - Old Concepts and New Challenges

The central function of the placenta is to allow the formation of a vascular labyrinth, a juxtaposed series of finely branched blood vessels and trophoblast that regulate the exchange of nutrients and residues while maintaining the separation of maternal and fetal blood supplies. The study by Shaut et al. showed a morphological alteration in the labyrinth endothelial cells, branching of the vessels, and in the integrity of the vessels when *HOXA13* was dysfunctional [58, 59]. These findings suggest that *HOXA13* regulates a number of genes in the vascular endothelium required for vessel adhesion and branching, providing a functional explanation of the mean gestational lethality exhibited by *HOXA13* mutant mice. The same authors identified that EphA6 and EphA7 were direct transcriptional targets of *HOXA13* in the genital tubercle vascular endothelia [59]. Altogether, these findings provide a new genetic pathway to consider when placental pathologies or placental evolutionary ontogeny are characterized. Evidence for this coordination is observed in the labyrinth endothelium, where the genes required for cell adhesion and vascular branching are affected concomitantly by the loss of *HOXA13* function, including Neuropilin-1, Enpp2, Lyve1, Caveolin-1, Foxf1, and Tie2, resulting in reduced levels of provascular factors required for the vascular development of the labyrinth [58].

Besides HoxA genes, the HoxB and HoxD loci have also been involved in endothelial and angiogenesis regulation processes [60]. HUVECs, for example, express several genes from these loci [7], and it has been shown that some of these genes inhibit in vitro proliferation of HUVECs, whereas others have been associated with increased capillary morphogenesis and vasculogenesis [61].

#### **4.4. HOXB1**

Previous studies have revealed an overlap between HoxA1 and HoxB1 functions during the specification of the rhombomeres, a transiently divided segment of the developing neural tube, from which neural crest cells emerge. It has been demonstrated that both HoxA1 and HoxB1 functions are required for the heart development [62, 63]. HoxB1−/− embryos were previously described as embryos with normal pharyngeal arch arteries and cardiac neural crestderived tissue remodeling [64]. However, more recently, Roux et al. observed one HoxB1 mutant embryo with an aortic arch artery defect, which is characteristic of a developmental failure of the left pharyngeal arch arteries (PAA) [65]. These data suggest that *HOXB1* is important for PAA formation, and the authors provide a novel model to study the molecular origin of great artery defects, which are often observed in human patients.

the expression of HoxB7 between micro- and macrovascular endothelial cells [70]. They observed higher expression of HoxB7 in macrovascular HUVECs and placenta compared to microvascular endothelial cells such as human placental endothelial cell (HPEC) line, human microvascular endothelial cells (HMVEC), and freshly isolated placental microvascular endothelial cells (PLEC). Storti et al. found that HoxB7 was expressed in 10 out of 22 multiple myeloma patients analyzed at the diagnosis related to high bone marrow angiogenesis [61]. They also found that HoxB7 was overexpressed in about 40% of myeloma cell lines compared with normal plasma cells [61]. Furthermore, they observed that HoxB7 overexpression in multiple myeloma cells significantly modified their transcriptional and angiogenic profile by upregulating VEGF, fibroblast growth factor 2 (FGF2), metalloproteinase-2 (MMP-2), platelet-derived growth factor A (PDGFA), and WNT5a, while HoxB7 also downregulates thrombospondin-2, an inhibitor of angiogenesis [61]. Finally, the homeobox gene HoxB7 is overexpressed across a range of cancers and promotes tumorigenesis by inducing cell proliferation, survival, invasion, and tumor angiogenesis in pancreatic adenocarcinoma [71], cervical cancer [72], glioblastoma tumors [73], and breast cancer [74].

Hox Genes in Adult Tissues and Their Role in Endothelial Cell Differentiation and Angiogenesis

http://dx.doi.org/10.5772/intechopen.76745

37

*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

*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

targeting integrin β1 (ITGβ1) in mature endothelial cells [75].

**4.8. HOXD1**

**4.9. HOXD3**

angiogenesis in mice [52].

#### **4.5. HOXB3**

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

#### **4.6. HOXB5**

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].

#### **4.7. HOXB7**

*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 the expression of HoxB7 between micro- and macrovascular endothelial cells [70]. They observed higher expression of HoxB7 in macrovascular HUVECs and placenta compared to microvascular endothelial cells such as human placental endothelial cell (HPEC) line, human microvascular endothelial cells (HMVEC), and freshly isolated placental microvascular endothelial cells (PLEC). Storti et al. found that HoxB7 was expressed in 10 out of 22 multiple myeloma patients analyzed at the diagnosis related to high bone marrow angiogenesis [61]. They also found that HoxB7 was overexpressed in about 40% of myeloma cell lines compared with normal plasma cells [61]. Furthermore, they observed that HoxB7 overexpression in multiple myeloma cells significantly modified their transcriptional and angiogenic profile by upregulating VEGF, fibroblast growth factor 2 (FGF2), metalloproteinase-2 (MMP-2), platelet-derived growth factor A (PDGFA), and WNT5a, while HoxB7 also downregulates thrombospondin-2, an inhibitor of angiogenesis [61]. Finally, the homeobox gene HoxB7 is overexpressed across a range of cancers and promotes tumorigenesis by inducing cell proliferation, survival, invasion, and tumor angiogenesis in pancreatic adenocarcinoma [71], cervical cancer [72], glioblastoma tumors [73], and breast cancer [74].
