**3.1 Vascular Endothelial Growth Factor (VEGF)**

VEGF is a central mediator of angiogenesis inducing endothelial cell proliferation, sprouting and promoting vascular leakiness (Otrock et al., 2007). The VEGF family includes VEGF A, VEGF B, VEGF C, VEGF D and placenta growth factor (PlGF) each coded by a separate gene (Table 1).


Table 1. Receptor affinity and actions of VEGF family members.

The gene encoding VEGF A comprises of eight exons which by differential splicing encodes seven transcript variants that give rise to isoforms of differing amino acid length, VEGF-A206, VEGF-A189, VEGF-A183, VEGF-A165, VEGF-A148, VEGF-A145 and VEGF-A121 respectively (Bevan et al., 2008; Hoeben et al., 2004). A further variant VEGF-A110 is derived by proteolytic cleavage. The major circulating isoform VEGF-A165, is also abundant in the extracellular matrix. The VEGF polypeptides are homodimers although heteodimeric forms of VEGF-A and PlGF have also been described (DiSalvo et al., 1995). The biological functions of VEGF are mediated by binding to the tyrosine kinase receptors, VEGF receptor-1/fmslike tyrosine kinase-1 (VEGFR-1/Flt1), VEGF receptor-2/fetal liver kinase-1 (VEGFR-2/Flk-1) and VEGF receptor-3/ fms-like tyrosine kinase-4 (VEGFR-3/Flt4) (Ortega et al., 1999). The various members of the VEGF family bind to different VEGF receptors as shown in Table 1. VEGF-A (also referred to as VEGF) is expressed by mural cells including vascular smooth muscle cells and pericytes. In addition, in the kidney VEGF is expressed by both glomerular epithelial cells (podocytes) and by tubular epithelial cells (Robert et al., 2000).

Angiogenesis and the Pathogenesis of Autosomal Dominant Polycystic Kidney Disease 97

2002). The activity of Ang-4 is similar to Ang-1 as it is a Tie-2 agonist and is expressed in human kidney proximal tubule epithelial cells. Activation of Tie-2 results in a downstream activation of P13K-Akt in endothelial cells leading to a survival pathway and cell

The plasma level of Ang-2 is elevated in patients with diabetes and is associated with indices of endothelial damage and dysfunction (Lim et al., 2005). Likewise, abnormal levels of serum Ang-1 and Ang-2 in hypertension have been linked with target organ damage (Nadar et al., 2005), thus indicating a potential role for angiopoietins in exacerbation of the extrarenal complications associated with ADPKD including left ventricular hypertrophy (LVH). LVH is a major risk factor for cardiac arrhythmias, sudden death, heart failure and ischemic disease in ADPKD (Schrier, 2006). Prevention of LVH in ADPKD is consequently a key factor in patient management. The expression of Ang-1, Ang-2 and Ang-4 in different tissues including human kidney proximal tubule cells is regulated by various factors including hypoxia, VEGF, angiotensin II and estrogen (Ardelt et al., 2005; Kitayama et al.,

The polycystin proteins PC1 and PC2 have been likened to tumor suppressors associated with many types of neoplasia (Grantham, 2001). Thus, when polycystin function is impaired as in ADPKD, cells revert to a more de-differentiated state marked by high proliferative capacity (Song et al., 2009). It has been recognized for many years that angiogenesis is necessary to support tumor growth (Folkman, 1971). Moreover, many non-neoplastic diseases including macular degeneration, arthritis and endometriosis are angiogenesis dependent (Folkman, 2006). Thus a facilitative role for angiogenesis in ADPKD cyst growth is suggested. Tumor cell expression of angiogenic growth factors including VEGF is mediated by hypoxia (Pugh and Ratcliffe, 2003). Central to the hypoxia response pathway are HIF-1 and 2. HIF-1 is targeted for destruction via the ubiquitin pathway regulated by Von Hippel Lindau (VHL) protein. Inactivation of VHL results in an increase of HIF-1 and VEGF level (Na et al., 2003). In progressive renal disease human proximal tubular epithelial cells demonstrate activation of intracellular hypoxia response pathways and VEGF signaling despite attenuated expression of VEGF-A (Rudnicki et al., 2009). Growth of renal cysts results in compression of the surrounding blood vessels. Significantly, an up-regulation of hypoxia-angiogenic pathways has been reported based on a systems biology approach in ADPKD (Song et al., 2009). A further key mediator of angiogenesis is the tumor suppressor gene phosphatase and tension homolog deleted on chromosome 10 (PTEN) which is frequently deficient or inactivated in human cancers (Mirohammadsadegh et al., 2006; Ohgaki & Kleihues, 2007; Tam et al., 2007). Activation of mammalian target of rapamycin (mTOR) is a feature of ADPKD and this pathway is regulated by PTEN (Boletta, 2009; Rosner et al., 2008; Shillingford et al., 2006). Thus the literature supports similarities between tumorigenesis and ADPKD and underscores a

Abnormalities of the renal vasculature in polycystic kidneys have long been recognized based on early angiographic studies of the kidney (Cornell, 1970, Ettinger et al., 1969) Bello-

**4. Similarities between tumor growth and cyst growth in ADPKD** 

potential role for angiogenesis in ADPKD cyst growth.

**5. Evidence for angiogenesis in ADPKD kidneys** 

chemotaxis (Makinde and Agarwal, 2008).

2006, Yamakawa et al., 2004).

The VEGF receptors are expressed on vascular endothelial cells as well as on a range of nonendothelial cells including monocytes and macrophages in the case of VEGFR-1 (Koch et al., 2011). In the kidney, glomerular endothelial cells express VEGFR-1 and VEGFR-2 (Thomas et al., 2000). Expression of VEGF is upregulated in response to hypoxia through upregulation of HIF-1α transcription factors. In addition, VEGF activity is modulated by binding to heparin sulfate and through interaction with the co-receptors neuopilin 1 and neuropilin 2, although the molecular mechanisms involved at present remain unclear (Koch et al., 2011). Both animal and human studies have shown that VEGF is essential for vascular repair and maintenance of normal glomerular function in the kidney (Dumont et al., 1995; Kitamoto et al., 2001; Satchell et al., 2004; Sugimoto et al., 2003). However, over expression of VEGF is also associated with glomerular disease, indicating that maintenance of normal VEGF level is essential for renal function (Veron et al., 2010). Significantly, a link between cystogenesis and VEGF was demonstrated in an animal study showing that increased expression of VEGF in renal tubules resulted in cyst formation (Hakroush et al., 2009).

Several recent studies have supported a role for an imbalance of angiogenic growth factor levels in disease processes including tumor growth, diabetes, chronic kidney disease (CKD), and cardiovascular disease (Futrakul et al., 2008; Guo et al., 2009; Persson & Buschmann, 2011; Lim et al., 2005; Nadar et al., 2004; Nadar et al., 2005). Endothelial dysfunction is a feature of patients with ADPKD (Schrier, 2006). VEGF has been shown to play a crucial role in preservation of the microvasculature, promoting vascular proliferation and repair in experimental renal disease (Chade et al., 2006; Iliescu et al., 2009; Zhu et al., 2004). Increased plasma levels of the VEGF inhibitor, soluble VEGF receptor (sFlt1) were recently demonstrated in CKD patients supporting an imbalance of the VEGF pathway in CKD (Di Marco et al., 2009). Tubulointerstitial hypoxia and capillary rarefaction are common features of progressive renal disease. In a study of patients with progressive or stable proteinuric renal disease attenuated VEGF-A expression by proximal tubular cells, despite activation of the intracellular response signalling pathway, was shown to distinguish those patients with progressive disease (Rudnicki et al., 2009).

Patients with ADPKD are at an increased risk for development of left ventricular hypertrophy (LVH) which is a significant risk factor for sudden death (Chapman et al., 1997). Increased plasma VEGF levels have been demonstrated in patients with target organ damage, defined as stroke, previous myocardial infarction, angina, LVH, and mild renal failure (Nadar et al., 2005). Mice expressing a *vegf b* transgene develop cardiac hypertrophy, further indicating that VEGF may also play a potential role in cardiac pathology associated with ADPKD (Karpanen et al., 2008).

#### **3.2 Angiopoietins**

The members of the angiopoietin family including Ang-1, Ang-2 and Ang-4 together with their soluble Tie-2 (tyrosine kinase with immunoglobulin-like and EGF-like domains 2) receptor are endothelial cell regulators with a role in the remodeling/maturation phases of angiogenesis. In addition to expression in endothelial and vascular smooth muscle cells Ang1, Ang2 and Ang-4 are also expressed in kidney (Fiedler and Augustin, 2006; Yamakawa et al., 2004; Yuan et al., 1999). Ang-1 is a Tie-2 agonist while Ang-2 in the absence of VEGF inhibits Ang-1/Tie-2 signaling as reviewed by Fiedler et al. (Fiedler and Augustin, 2006). Conversely, under conditions of adequate VEGF, or under hypoxic conditions as may exist in and around the growing renal cysts, Ang-2 stimulates angiogenesis (Lobov et al.,

The VEGF receptors are expressed on vascular endothelial cells as well as on a range of nonendothelial cells including monocytes and macrophages in the case of VEGFR-1 (Koch et al., 2011). In the kidney, glomerular endothelial cells express VEGFR-1 and VEGFR-2 (Thomas et al., 2000). Expression of VEGF is upregulated in response to hypoxia through upregulation of HIF-1α transcription factors. In addition, VEGF activity is modulated by binding to heparin sulfate and through interaction with the co-receptors neuopilin 1 and neuropilin 2, although the molecular mechanisms involved at present remain unclear (Koch et al., 2011). Both animal and human studies have shown that VEGF is essential for vascular repair and maintenance of normal glomerular function in the kidney (Dumont et al., 1995; Kitamoto et al., 2001; Satchell et al., 2004; Sugimoto et al., 2003). However, over expression of VEGF is also associated with glomerular disease, indicating that maintenance of normal VEGF level is essential for renal function (Veron et al., 2010). Significantly, a link between cystogenesis and VEGF was demonstrated in an animal study showing that increased expression of VEGF in renal tubules resulted in cyst formation (Hakroush et al., 2009).

Several recent studies have supported a role for an imbalance of angiogenic growth factor levels in disease processes including tumor growth, diabetes, chronic kidney disease (CKD), and cardiovascular disease (Futrakul et al., 2008; Guo et al., 2009; Persson & Buschmann, 2011; Lim et al., 2005; Nadar et al., 2004; Nadar et al., 2005). Endothelial dysfunction is a feature of patients with ADPKD (Schrier, 2006). VEGF has been shown to play a crucial role in preservation of the microvasculature, promoting vascular proliferation and repair in experimental renal disease (Chade et al., 2006; Iliescu et al., 2009; Zhu et al., 2004). Increased plasma levels of the VEGF inhibitor, soluble VEGF receptor (sFlt1) were recently demonstrated in CKD patients supporting an imbalance of the VEGF pathway in CKD (Di Marco et al., 2009). Tubulointerstitial hypoxia and capillary rarefaction are common features of progressive renal disease. In a study of patients with progressive or stable proteinuric renal disease attenuated VEGF-A expression by proximal tubular cells, despite activation of the intracellular response signalling pathway, was shown to distinguish those patients with

Patients with ADPKD are at an increased risk for development of left ventricular hypertrophy (LVH) which is a significant risk factor for sudden death (Chapman et al., 1997). Increased plasma VEGF levels have been demonstrated in patients with target organ damage, defined as stroke, previous myocardial infarction, angina, LVH, and mild renal failure (Nadar et al., 2005). Mice expressing a *vegf b* transgene develop cardiac hypertrophy, further indicating that VEGF may also play a potential role in cardiac pathology associated

The members of the angiopoietin family including Ang-1, Ang-2 and Ang-4 together with their soluble Tie-2 (tyrosine kinase with immunoglobulin-like and EGF-like domains 2) receptor are endothelial cell regulators with a role in the remodeling/maturation phases of angiogenesis. In addition to expression in endothelial and vascular smooth muscle cells Ang1, Ang2 and Ang-4 are also expressed in kidney (Fiedler and Augustin, 2006; Yamakawa et al., 2004; Yuan et al., 1999). Ang-1 is a Tie-2 agonist while Ang-2 in the absence of VEGF inhibits Ang-1/Tie-2 signaling as reviewed by Fiedler et al. (Fiedler and Augustin, 2006). Conversely, under conditions of adequate VEGF, or under hypoxic conditions as may exist in and around the growing renal cysts, Ang-2 stimulates angiogenesis (Lobov et al.,

progressive disease (Rudnicki et al., 2009).

with ADPKD (Karpanen et al., 2008).

**3.2 Angiopoietins** 

2002). The activity of Ang-4 is similar to Ang-1 as it is a Tie-2 agonist and is expressed in human kidney proximal tubule epithelial cells. Activation of Tie-2 results in a downstream activation of P13K-Akt in endothelial cells leading to a survival pathway and cell chemotaxis (Makinde and Agarwal, 2008).

The plasma level of Ang-2 is elevated in patients with diabetes and is associated with indices of endothelial damage and dysfunction (Lim et al., 2005). Likewise, abnormal levels of serum Ang-1 and Ang-2 in hypertension have been linked with target organ damage (Nadar et al., 2005), thus indicating a potential role for angiopoietins in exacerbation of the extrarenal complications associated with ADPKD including left ventricular hypertrophy (LVH). LVH is a major risk factor for cardiac arrhythmias, sudden death, heart failure and ischemic disease in ADPKD (Schrier, 2006). Prevention of LVH in ADPKD is consequently a key factor in patient management. The expression of Ang-1, Ang-2 and Ang-4 in different tissues including human kidney proximal tubule cells is regulated by various factors including hypoxia, VEGF, angiotensin II and estrogen (Ardelt et al., 2005; Kitayama et al., 2006, Yamakawa et al., 2004).
