br Introduction Retinal neovascularization is the

Introduction Retinal neovascularization is the most common cause of moderate to severe vision loss in all age groups; i.e., retinopathy of prematurity (ROP) in children, diabetic retinopathy in adults and age-related macular degeneration in the elderly (Andreoli and Miller, 2007; Chen and Smith, 2007; Ferrara and Kerbel, 2005). These various retinal neovascular pathologies, in turn, may cause vitreous hemorrhage, retinal detachment and/or neovascular glaucoma affecting vision (Aiello et al., 1994). A large body of evidence shows that vascular endothelial growth factor A (VEGFA) is one of the major factors linked to the pathogenesis of proliferative retinopathy (Alon et al., 1995). The VEGF family consists of 7 structurally related molecules (VEGF-A through F and placental growth factor) that bind to 3 tyrosine kinase receptors (VEGFR1, 2 and 3) with different affinities (Karkkainen and Petrova, 2000). VEGFA has been the focus of many current anti-angiogenesis regimens as it was the most characterized angiogenic and permeability factor implicated in various pathologies including retinal neovascularization (Kuiper et al., 2008). However, the anti-VEGFA therapies have been observed to have some deleterious effects such as an increase in the expression of connective tissue growth factor (CTGF), which promotes a switch from angiogenesis to fibrosis (Kuiper et al., 2008). In addition, 82% of the tractional retinal detachments (TRDs) were reported linked to anti-VEGFA injections (Arevalo et al., 2008). Furthermore, besides VEGFA, other molecules such as VEGFC and VEGFD have been reported to play a role in angiogenesis and lymphangiogenesis via activation of VEGFR2 and VEGFR3 (Achen et al., 1998; Cao et al., 1998). Although, the initial studies have reported that the expression of VEGFR3 is restricted to lymphatic endothelium (Jeltsch et al., 1997; Kaipainen et al., 1995), it is now recognized that the VEGFC–VEGFR3 signaling also modulates the branching morphogenesis of blood vessels (Tammela et al., 2008). Despite these advances in our understanding of the role of VEGFC in angiogenesis and lymphangiogenesis, its involvement in pathological retinal angiogenesis is not explored. The cyclic adenosine monophosphate (cAMP)-response element-binding protein (CREB) is a transcription factor that belongs to the basic leucine-zipper family and binds to a specific regulatory element known as cAMP-response element (CREs) in the promoters of its target genes and enhances their expression (Shaywitz and Greenberg, 1999). CREB mediates hypoxia-induced expression of a number of genes, including VEGFA and its receptors (Kang et al., 2014; Leonard et al., 2008; Morishita et al., 1995; Wu et al., 2007). Many studies have also demonstrated that VEGFA, FGF2 and hepatocyte growth factor by phosphorylating increase CREB transcriptional transactivation activity in endothelial Cy3.5 maleimide Supplier (Abramovitch et al., 2004; Hoot et al., 2010; Kottakis et al., 2011; Zhao et al., 2011). Furthermore, it has been reported that overexpression of constitutively active CREB aggravates tumor angiogenesis (Abramovitch et al., 2004). Despite these clues linking CREB to the modulation of angiogenesis, its developmental or pathological angiogenesis is not known. In the present study, we used a mouse model of retinopathy to understand the role of CREB in pathological retinal neovascularization. The mouse model of retinopathy which involves an initial phase of hyperoxia-induced vessel loss, followed by a phase of hypoxia-induced pathological neovascularization (Smith et al., 1994), allowed us to decipher the role of VEGFC and CREB axis in pathological retinal neovascularization. Our results for the first time demonstrate that CREB by modulating DLL4–NOTCH1 signaling plays a crucial role in pathological retinal angiogenesis.
Materials and Methods
Results
Discussion Hypoxia and hypoxia-inducible factors are important cues in the modulation of retinal neovascularization (Arjamaa and Nikinmaa, 2006; Gariano and Gardner, 2005). It should be pointed out that hypoxia by itself provokes a constellation of signaling events that promote angiogenesis (Konisti et al., 2012; Semenza, 2003). Although a large body of evidence shows that VEGFA plays a major role in pathological retinal neovascularization (Carmeliet et al., 1996; Ferrara et al., 1996), the anti-VEGFA therapies have failed in the reduction of the overall severity of the disease and a number of patients suffering from age-related macular degeneration or cancer do not respond to these regimens (Jubb and Harris, 2010; Lux et al., 2007). In this regard, to understand the role of other molecules in the modulation of pathological retinal angiogenesis, we studied the capacity of VEGFA-related molecules. We found that hypoxia induces the expression of VEGFC as potently as VEGFA in the retina. Interestingly, VEGFC expression was also found in angiogenic ECs during retinal development (Tammela et al., 2008). VEGFC induces the proliferation, migration, sprouting and tube formation of HRMVECs, the findings suggesting its role in retinal angiogenesis. As CREB mediates hypoxia-induced expression of a number of growth factors (Kang et al., 2014; Leonard et al., 2008; Morishita et al., 1995; Wu et al., 2007) and its activity potentiates tumor angiogenesis (Abramovitch et al., 2004), we explored its role in VEGFC-induced HRMVEC proliferation, migration, sprouting and tube formation. Our results indicate that VEGFC stimulates CREB activation in HRMVECs. In addition, blockade of CREB activation by its dominant negative mutant blunted VEGFC-induced HRMVEC proliferation, migration, sprouting and tube formation. These findings infer that CREB mediates VEGFC-induced angiogenic events in HRMVECs. Although the involvement of CREB in tumor angiogenesis has been demonstrated (Abramovitch et al., 2004; Chen et al., 2005), its role in hypoxia-induced retinal neovascularization is not known. Therefore, as CREB mediates VEGFC-induced HRMVEC sprouting and tube formation, we next investigated the functional significance of VEGFC–CREB axis in retinal neovascularization. The observations that suppression of VEGFC levels attenuates hypoxia-induced retinal EC proliferation, tip cell formation and neovascularization suggest a role for VEGFC in pathological retinal neovascularizaion. VEGFC has also been reported to play a role in developmental and tumor angiogenesis (De Palma et al., 2003; Ober et al., 2004). Second, our findings show that hypoxia induces CREB activation in retina in VEGFC-dependent manner and suppression of its levels inhibits hypoxia-induced EC proliferation, tip cell formation and neovascularization.