Aberrant angiogenesis (neovascularization) occurs in several diseases of the eye, including AMD and proliferative diabetic retinopathy. CNV is considered to be the defining characteristic of late-stage wet AMD, where new sprouting vessels penetrate Bruch's membrane, growing into the subretinal space and sometimes into vessels that are derived from the retinal circulation.15
The primary mediator of CNV is VEGF. VEGF binds to and activates an endothelial receptor tyrosine kinase (RTK), vascular endothelial growth factor receptor-2 (VEGFR-2/FLK-1/KDR), as well as other endothelial RTKs, including VEGFR-1 and -3.16
It is thought that VEGF induces vascular leakage and inflammation by promoting the increased production and permeability of capillary endothelial cells. VEGF is considered to play a critical role in the pathogenesis of AMD, and recent therapies targeting VEGF have shown promising results of significant improvement of central vision and reduction of CNV.11
Activation of VEGFR-2 by VEGF family ligands in endothelial cells leads to a wide variety of biological responses that emanate from several key autophosphorylation sites within the cytoplasmic domain of VEGFR-2.16
Activation of PLCγ1 in endothelial cells is considered an important mediator of angiogenic signaling of VEGFR-2.7
The initial evidence linking PLCγ1 to endothelial cell function and angiogenesis in vivo was provided by targeted deletion of PLCγ1, which resulted in early embryonic lethality in zebrafish and mice that was caused by significantly impaired vasculogenesis and erythrogenesis.9
Regulation of angiogenesis is often viewed as a balance between proangiogenic and antiangiogenic factors. Recent studies have shown that PLCγ1, a major signaling substrate of VEGFR-2, undergoes c-Cbl-mediated ubiquitination. Ubiquitination of PLCγ1 blocks its activation by VEGFR-2 and with it VEGF-induced endothelial cell proliferation and angiogenesis.10
Further analysis showed that loss of c-Cbl in mice is associated with elevated PLCγ1 activation and endothelial cell proliferation19
(Meyer RD, et al., manuscript submitted, 2010). However, to date, the biological importance of PLCγ1 in pathologic angiogenesis and the possible link between c-Cbl and PLCγ1 activation in pathologic angiogenesis has remained unknown. In the present study we used various in vitro and in vivo angiogenesis models and demonstrated that PLCγ1 activation plays a pivotal role in angiogenesis. More important, our study for the first time linked c-Cbl to the negative regulation of CNV.
The data presented in this study as well as in a manuscript submitted elsewhere (Meyer RD, et al., manuscript submitted, 2010) strongly implicate c-Cbl as a negative regulator of angiogenesis. Our observation is consistent with the conventional and well-recognized function of c-Cbl as a negative regulator of receptor tyrosine kinase signaling.20
Loss of c-Cbl is dispensable for normal embryonic development and angiogenesis,21
whereas loss of both c-Cbl and Cbl-b is lethal before embryonic day (E)10.5,22
suggesting that the proteins of the Cbl family are compensatory in their ability to regulate the activity of target proteins. Regardless of this possibility, our data show that the absence of c-Cbl led to robust and sustained angiogenesis in the eye. This finding indicates that c-Cbl specifically regulates pathologic angiogenesis and that the unique characteristics of c-Cbl may make it a good target for antiangiogenic therapy.
Although various risk factors have been linked to AMD, the etiology and pathogenesis of AMD remain largely unclear. Similarly, treatment options for AMD patients are limited, further arguing a need for new preventative and therapeutic strategies for this disease. Our present study indicates a vital role for PLCγ1 in pathologic angiogenesis of the eye. Therefore, targeting PLCγ1 may potentially offer a novel treatment for ocular neovascularization which would be more specific and longer-acting than the treatments currently available.