We have identified an ENU-induced r26
mouse mutant, which is a missense mutation of the Vldlr
gene, a c.2239C>T for a p.R747X substitution. This mutation results in a truncated Vldlr protein lacking the transmembrane domain. The r26
mutant mice develop subretinal neovascularization similar to that of Vldlr−/−
mice. Therefore, the r26
mouse line is a loss-of-function mutation of Vldlr. However, unlike the Vldlr−/−
mice that lack the expression of Vldlr protein in vivo, the r26
mutant mice probably express a soluble form of Vldlr. Thus, the r26
mutant line may be a useful and complementary model for investigating the role of Vldlr signaling in the regulation of retinal angiogenesis in vivo. A recent study has reported an increased proliferation and migration of Vldlr−/−
retinal endothelial cells.14
However, the molecular and cellular mechanisms for how Vldlr controls intraretinal angiogenesis during development remain largely unknown.
Vldlr belongs to the evolutionarily conserved LDL receptor family, and it contains typical structural/functional domains like many other members of the LDL receptor family. Vldlr can be structurally divided into three regions: the extracellular region that includes ligand-binding repeats domain, the EGF-like repeat domain, the YWTD β-propeller domain, and the O-linked glycosylation domain; a single transmembrane domain region; and a short cytosolic tail region that contains the NPXY motif ().19,20
Vldlr was initially thought to be an endocytic receptor that mediates the binding and uptake of apoE-containing lipoproteins, such as VLDL and β-VLDL.21
Early studies reported that homozygous Vldlr−/−
mice had normal lipoprotein profiles without any significant phenotypes, except that the animals were somewhat smaller and leaner at a young age.9
Interestingly, recent findings reveal that retinal vasculature defects are the main phenotype observed in the Vldlr−/−
Vldlr may function in endothelial cells as an endocytic receptor to mediate the endocytosis of cell surface-bound ligands that may act as proangiogenic factors in the retina.
Figure 6. Structural domains of the Vldlr protein. In the wild-type protein, the ligand-binding repeats domain, the EGF-like repeats domain, the YWTD β-propeller domain, and the O-linked glycosylation domain are located in the extracellular space; a single (more ...)
However, Vldlr has been demonstrated to play a signaling function by its interaction with Reelin to modulate neuronal migration, neurodevelopment, and other physiological processes in the central nervous system.22
This has been well documented by the Reelin-Vldlr/ApoER2 signaling pathway. Reelin is a large secreted protein that is primarily expressed in nerve tissues. Vldlr and ApoER2 are functional Reelin receptors. Defective Reelin signaling leads to disordered laminated cortical structure.23
Indistinguishable phenotypes were uncovered among the ataxic reeler
) gene mutant mice,25,26
and mice lack both Vldlr and ApoER2,27
suggesting that Reelin, Dab1, Vldlr, and ApoER2 function in the same signaling pathway. Reelin signaling is initiated by the binding of Reelin to both Vldlr and ApoER2, and the ligand/receptor binding triggers the tyrosine phosphorylation of Dab1, thus regulating neuronal migration.28
Dab1 binds to the NPXY motif in the cytosolic domain of ApoER2 and Vldlr.29
The NPXY motif is essential for Reelin signaling, since it is the critical structural domain for Dab1 binding to both receptors.30
It has been reported recently that Vldlr-mediated Reelin signal prevents neurons from entering the marginal zone, suggesting that Vldlr provides a stop signal for migrating cortical neurons.31
However, Dab1 expression is limited to neurons, and it is unclear whether Dab1 or Dab2 is expressed in retinal endothelial cells.32
Therefore, the downstream targets of Vldlr that mediate endothelial cell migration in angiogenesis need to be explored. It is also possible that other cell types expressing Vldlr in the retina may be the key in the regulation of retinal angiogenesis.
The r26 mutation leads to a premature stop codon at amino acid residue 747 of the Vldlr protein. The truncated Vldlr-R747X protein contains only the first 746 amino acid residues and lacks the C-terminal 127 amino acids that include the single transmembrane domain. Unlike the plasma membrane localization of the full-length Vldlr protein, the r26 truncated Vldlr protein displays an intracellular distribution in transfected cells in vitro. As shown in , the r26 truncated Vldlr-R747X protein lacks the transmembrane domain. It is also possible that the truncated proteins will be secreted into the extracellular space in vivo. Regardless of the intracellular or extracellular distribution of the r26 truncated Vldlr protein in vivo, the expression level of this truncated Vldlr must be extremely low in vivo, since its protein amount in the retinal homogenates is undetectable by Western blot. Thus, the r26 mutant line is a valuable model for a loss-of-function mutation of Vldlr.
Previous reports of Vldlr−/−
mice and our present study have demonstrated that Vldlr loss-of-function mutations lead to an uncontrolled overgrowth of intraretinal vessels.10–13
Vldlr provides an essential cue to prevent retinal vascular endothelial cells from migrating into the photoreceptor cell layer. It is unknown what triggers the migration of endothelial cells into photoreceptor/subretinal space in the Vldlr mutants and what the Vldlr ligand is in retinal endothelial cells.
We hypothesize that Vldlr binds an anti-angiogenic factor to prevent the endothelial cells from migrating into the photoreceptor layer. Further investigation is needed to understand whether Vldlr has a co-receptor like ApoER2 in a Reelin-like signaling pathway in the retina and which intracellular downstream molecules, such as Dab1-like molecules, are involved in this signaling pathway. It is also necessary to determine whether Vldlr is expressed in other retinal cells besides endothelial cells to inhibit the vessel growth in photoreceptor cells or subretinal space. A better understanding of the Vldlr signaling pathway will be very helpful for developing new therapeutic methods for treating certain types of AMD, such as RAP, with abnormal intraretinal angiogenesis.