**2. Angiogenic promoting factors**

vascular hypertrophy and structural remodeling, which are characteristic of PH (Budhiraja et

Both processes, angiogenesis and remodeling of pulmonary arterioles, appear to share the participation of similar molecules. In fact, some of them, known to increase pulmonary blood vessels tone during hypoxic exposure, are also involved in generating pulmonary blood vessels. In this framework, three main research cellular targets can be identified: the endothe‐ lium, the smooth muscle cells of the wall of pulmonary arterioles and, adventitial connective

PH has been related with malfunction of potassium channels and over-regulation of calcium channels on the cell membrane of vascular muscle cells as well as endothelial dysfunction. The latter, as it pertains with decreasing NO and prostacyclin (vasodilators) delivering and increment of vasocontrictors, such as endothelin-1 (ET-1) Inagami *et al.,*

In adult subjects, angiogenesis could be taken as a compensatory process during hypoxic exposure. This process would be in addition to the well-known mechanisms of augmenting blood perfusion in less vascularized areas in the lung, hyperventilation and erythropoiesis. Those areas might be a potentially available area for *de novo* vascular formation. Human beings living in highlands, have developed a greater lung capacity through evolution, as an adaptive mechanism to hypobaric hypoxia (Frisancho, 1970). Also, high landers show increased lung capillary density than lowlanders, which was interpreted as an effect of hypoxia on angio‐

Ooi et al (2000) and Howell et al (2002, 2003) determined that hypoxic hypoxia (10% oxygen) provoked augmentation in volume and total length in pulmonary blood vessels in rats, together with an increment in endothelial superficial area and cells numbers. In another work, chronically hypoxic rats, which exhibited a greater degree of vascular development through angiogenesis, had higher cardiac mass index values than the correspondent ones in animals subjected to hypoxia and hypercapnia or normoxia (Howell et al., 2004). In mice subjected to hypoxia, Pascaud et al (2003) gave evidence that inhibition of hypoxia-induced angiogenesis,

Angiogenesis is regulated by angiogenic and anti-angiogenic molecules (Pascaud et al., 2003; Maharaj and D`Amore, 2007). Genetic and epigenetic factors permit blood vessels formation (Pauling and Vu, 2004). Taraseviciene-stewart et al (2001) found that plexiform lesions in idiopathic pulmonary hypertension (iPAH) were associated with disordered angiogenesis due to exaggerated mitosis of endothelial cells. Tuder and Voelkel (2002) reported endothelial cell proliferation in cases of primary PH, and, since these cells exhibited markers of angiogenesis, the authors named this process a "disordered or misguided angiogenesis". In this context, endothelium progenitor cells are now claimed to be therapeutic targets in PH due to their possible involvement in PH pathobiology. Nitric oxide synthases (eNOs), vascular endothelial growth factor (VEGF), angiopoietins and their receptor tie-2, which are associated with vascular remodeling, were all expressed in a monocrotaline induced model of PH (Cho et al.,

al., 2004; Humbert et al., 2004; reviewed by Hernández and Sandino de, 2011).

tissue cells.

104 Pulmonary Hypertension

1995; Voelkel and Tuder, 2000)

genesis (Bisschop et al, 2010).

enhanced the degree of PH.

The hypoxia inducible factor (HIF) system is oxygen sensitive. It includes HIF-1 and HIF-2. They bind and activate many of the same genes, but differ in their participation during hypobaric hypoxia. For instance, HIF-2 alone is responsible for the liberation of erythropoietin through stimulation of connective tissue cells in the kidney (Paliege et al., 2010) and other hematological reactions to hypoxia (reviewed by Tissot van Patot and Gassmann, 2011). HIF-1 is a key molecule in regulating the expression of several angiogenic molecules. Among the hypoxia-inducible genes which have important HIF-1 binding sites and are believed to participate in the pathogenesis of PH and angiogenesis as well, are those encoding VEGF (Liu et al., 1995), the VEGF receptor 1 (Gerber et al, 1997), and ET-1 (Hu et al., 1998). Expression of VEGF and its receptor Fetal Liver Kinase 1 receptor (FLK-1) was enhanced in hypoxic pulmo‐ nary hypertensive rats (Christou et al., 1998). Rats maintained under hypoxic hypoxia (exposure to 10% oxygen) showed augmented volume and total length of pulmonary blood vessels (Howell et al. 2002, 2003; Ooi et al., 2000). In another study, rats exposed to hypobaric hypoxia, exhibited reduced Angiopoietin-1/Tie2 and VEGF expression, together with a diminished number of arterial blood vessels in the lung (Yamamoto et al., 2008).

Sands et al (2012) gave insight on the interaction of members of the VEGF family in hypoxic PH. They found that VEGFB and the placental growth factor (PGF) inhibit or potentiate the actions of VEGFA, according to their relative concentrations, which change in the lungs of rats subjected to hypoxic hypoxia. The same authors stated that the abovementioned effects *in vivo* depend on specific concentrations of VEGF and PGF within the alveolar wall during adaptation to hypoxia. In this context, human subjects developing acute mountain sickness had higher levels of plasma VEGF on ascend to altitude as compared to their own values obtained under normoxic conditions. This variation did not occur in healthy subjects (Tissot van Patot et al., 2005).

VEGF exerts its angiogenic effect through four different pathways, which include NO participation (proposed by Gramatikoff, 1999). In a previous study, adrenomedullin mRNA expression was greater in the lungs of chickens with pulmonary hypertension compared with those without pulmonary hypertension (Gómez et al., 2008) and Vadivel et al (2010) demon‐ strated that adrenomedullin promotes angiogenesis in the lung. Hence, two vasodilators of pulmonary blood vessels, NO and adrenomedullin, may also have angiogenic properties.

Fahra et al (2011) found that CD34(+) and CD133(+) progenitor cell numbers are higher in the bone marrow, blood, and pulmonary arteries of pulmonary hypertensive subjects as compared to healthy controls. The blood levels of (hypoxia inducible myeloid-activating factors such as erythropoietin, the stem cell factor (SCF), and hepatocyte growth factor (HGF) were higher than normal levels in diseased individuals, and related to PH severity. Similarly, Davie et al (2004) found that progenitor cells from bone marrow origin contribute to neo-vascularization in pulmonary arteries of hypoxic calves, based on quantitative morphometric analyses of lung tissue from normoxic and hypoxic individuals. Their results showed adventitial growth in *vasa vasorum* of pulmonary arteries. This change was attributed to the transformation of cells from the bone marrow, which would be mobilized into the circulation, and differentiate into endothelial and smooth muscle cells. This model was supposed to entail an increase in the expression of c-kit(+), VEGF, fibronectin, and thrombin, as responses to hypoxia.

(Dhanabal et al., 1999). Endostatin interferes with the assembly of the actin cytoskeleton of endothelial cells and inhibits their proliferation and participation in angiogenesis (Abdollahi et al., 2004; Skovseth et al., 2005). Endostatin expression on the surface of the smooth muscle cells of pulmonary arterioles in the mouse, is provoked by hypoxic exposure (Paddenberg et al., 2006). NO, in culture, induces endostatin production. Hence, endothelial apoptosis is also induced (Deininger et al., 2003). In human endothelial cell cultures, addition of endostatin reduces angiogenic activity by reducing HIF-1α, VEGF, VEGFR2, HGH and EGFR gene expression. Furthermore, expression of some anti-angiogenic genes, such HIF1An (a HIF antagonist), Kininogen, TSP-1 and a Vasostatin precursor is increased (Abdollahi et al., 2004). However, it appears that the mode of action of endostatin, in this context, needs to be fully

Angiogenesis and Pulmonary Hypertension http://dx.doi.org/10.5772/56054 107

Adiponectin (ADPN) reinforces the vasodilatory action of NO in pulmonary blood vessels (Summer et al, 2009), and as stated, if NO contributes to the control of angiogenesis, ADPN will be a potential inhibitor of vascularization. Also, Le Cras et al (2003) found that alpha transforming growth factor (TGF-α) overexpression induced a diminution in pulmonary vascular development, which was accompanied by severe PH and vascular remodeling.

Oxidative stress is higher in the lungs of pulmonary hypertensive chickens (Iqbal y col., 2001a, b), which has been associated, in another species, with decreasing numbers of blood vessels (Murfee and Schmid-Schonbein, 2008).Oxidative stress, occurring in chronically hypoxic mice,

**4. Highlights on vascular development under hypoxia or normoxia in the**

Angiogenesis in the mature pulmonary circulation could be a structural adaptation that may have important beneficial consequences for gas exchange (Howell et al., 2003). In contrast, Yamamoto et al (2008) reported a chronic hypoxia-induced pulmonary blood vessels loss, an

Although there is no definitive evidence of less vascularized areas in the avian lung, it is feasible that during hypoxia, vasculogenesis and angiogenesis occur to augment the gas exchange area. Hypoxia exposure for 3 to 4 weeks, does not affect pulmonary growth in current commercial chicken strains (Vásquez and Hernández, 2011; Areiza et al., 2011). These findings appear to be controversial, since alveolar and vascular neo-formation should both increase in the pulmonary response to hypoxia (reviewed by Bhattacharya, 2008). If this is not the case, less vascularized areas within the lung could gain new blood vessels, which might not significantly affect the whole organ´s weight. It should be noticed that arteriogenesis might

The final goal of angiogenesis in the lung, should be to construct a complete functional arteriovenous tree. Therefore, vascular density would be represented by different types of blood vessels, such as arterioles or pre-capillaries. In this framework, using the chicken as a model,

can enhance endostatin production (Deininger et al., 2003) (Paddenberg et al., 2006).

elucidated (Deininger et al., 2003; Skovseth et al., 2005).

event sometimes called "rarefaction" or "pruning".

also play a role in neo-vascularization (Deindi and Schaper, 2008)

**chicken model**

PH is linked to myeloid abnormalities, some of which may be related to increased production of HIF-inducible factors by a diseased pulmonary vasculature, but findings in non-affected subjects, suggest that myeloid abnormalities may be intrinsic to the disease process. Proangiogenic cell progenitors, and endothelial cells that have pathologic expression of hypoxiainducible factor 1 alpha (HIF-1 alpha), were shown to be quantitatively higher in PH bone marrow, blood, and pulmonary arteries than in healthy controls. Also, the HIF-inducible myeloid-activating factors erythropoietin, stem cell factor (SCF), and hepatocyte growth factor (HGF) showed higher than normal levels in blood of pulmonary hypertensive subjects, and related to disease severity (Samar et al., 2011). In addition, endothelial progenitor cells with angiogenic capacity were found in the pulmonary circulation (Yoder, 2011). Epidermal growth factor promotes both angiogenesis and vascular remodeling (Janakidevi et al., 1995; Toby et al., 2010).

Bone morphogenetic protein (BMP) is compromised in endothelial repair within the pulmo‐ nary microvessels, and its expression is reduced in hypoxic PH, due to an increment in endothelial cell-derived Gremlin 1 and Gremlin 2, which antagonize BMP 2 type receptor expression (Cahill et al., 2012).
