n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Retinal-Neovascularization

Amyloid-beta (Aβ) is an endogenous peptide that becomes dysregulated in AMD and Alzheimer disease. Both of these disorders are marked by extracellular deposits that contain Aβ, highly branched capillary networks, and neurodegeneration. Although Aβ has been implicated in AMD and Alzheimer pathology for decades, its nonpathological function has remained unclear. We recently showed that high levels of monomeric Aβ induce blood vessel branching in embryonic zebrafish brain, and here we report that a similar mechanism may contribute to aberrant blood vessel branching in the retina of adult zebrafish.. Transgenic zebrafish expressing enhanced green fluorescence protein (EGFP) in their endothelial cells were sedated and small intraocular injections of PBS were made into one eye and either Aβ or γ-secretase inhibitor were injected into their opposite eye. A week later, the eyes were enucleated and high resolution maps of the retina vasculature were created using confocal microscopy. Comparisons were made between the treatment groups using the general linear model ANOVA.. We found that Aβ significantly affects capillary blood vessels in the retina. Small volumes of Aβ injected into the eyes of adult zebrafish induced the formation of significantly more endothelial tip cells and capillary bridges-some with loops-near the circumferential vein. These effects were dose-dependent and increased capillary bed density, though there was no effect on larger arterial vessels.. This study reveals a previously unknown role for Aβ in regulating capillary bed density, providing new insight into the normal biological function. Aβ will help in the development of therapeutic interventions for AMD and Alzheimer disease.

Topics: Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Animals, Genetically Modified; Capillaries; Dipeptides; Dose-Response Relationship, Drug; Endothelium, Vascular; Gene Expression; Green Fluorescent Proteins; Injections, Intraocular; Microscopy, Confocal; Peptide Fragments; Retinal Neovascularization; Retinal Vessels; RNA, Messenger; Zebrafish

Mutations in Norrin, the ligand of a receptor complex consisting of FZD4, LRP5 and TSPAN12, cause severe developmental blood vessel defects in the retina and progressive loss of the vascular system in the inner ear, which lead to congenital blindness and progressive hearing loss, respectively. We now examined molecular pathways involved in developmental retinal angiogenesis in a mouse model for Norrie disease. Comparison of morphometric parameters of the superficial retinal vascular plexus (SRVP), including the number of filopodia, vascular density and number of branch points together with inhibition of Notch signaling by using DAPT, suggest no direct link between Norrin and Notch signaling during formation of the SRVP. We noticed extensive vessel crossing within the SRVP, which might be a loss of Wnt- and MAP kinase-characteristic feature. In addition, endomucin was identified as a marker for central filopodia, which were aligned in a thorn-like fashion at P9 in Norrin knockout (Ndp(y/-)) mice. We also observed elevated mural cell coverage in the SRVP of Ndp(y/-) mice and explain it by an altered expression of PDGFβ and its receptor (PDGFRβ). In vivo cell proliferation assays revealed a reduced proliferation rate of isolectin B4-positive cells in the SRVP from Ndp(y/-) mice at postnatal day 6 and a decreased mitogenic activity of mutant compared with the wild-type Norrin. Our results suggest that the delayed outgrowth of the SRVP and decreased angiogenic sprouting in Ndp(y/-) mice are direct effects of the reduced proliferation of endothelial cells from the SRVP.

Topics: Animals; Calcium-Binding Proteins; Cell Proliferation; Dipeptides; Endothelial Cells; Eye Proteins; HEK293 Cells; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Confocal; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neovascularization, Physiologic; Nerve Tissue Proteins; Pseudopodia; Receptor, Notch1; Retina; Retinal Neovascularization; Retinal Vessels; Serrate-Jagged Proteins; Signal Transduction

Wet age-related macular degeneration (AMD), which accounts for most AMD-related vision loss, is characterized by choroidal neovascularization (CNV). The underlying mechanism of CNV is poorly understood, but evidence indicates pathologic recruitment of normal angiogenic signaling pathways such as the VEGF pathway. Recent evidence suggests that the VEGF pathway regulates angiogenesis in concert with Notch signaling. Here, the authors examined the role of Notch signaling in CNV in the backdrop of Notch signaling-mediated regulation of retinal angiogenesis.. Choroid sclera complexes, after laser-induced CNV, were examined for changes in CNV lesion volume and in proangiogenic and antiangiogenic gene expression after perturbation in Notch signaling. Retinal vessels and angiogenic gene expression in retinal endothelial cells were analyzed in postnatal rats after perturbations in Notch signaling. Notch signaling was activated and inhibited by intravitreal or systemic injection of Jagged1 peptide and gamma secretase inhibitor DAPT, respectively.. The authors demonstrated that activation of the canonical Notch pathway reduced the volume of CNV lesions as it attenuated the development of postnatal retinal vasculature. In contrast, inhibition of the Notch pathway exacerbated CNV lesions as it led to the development of hyperdense retinal vasculature. The authors also identified genes associated with proangiogenesis (Vegfr2, Ccr3, and Pdgfb) and antiangiogenesis (Vegfr1 and Unc5b) as targets of Notch signaling-mediated vascular homeostasis, the disruption of which might underlie CNV.. This study suggests that Notch signaling is a key regulator of CNV and thus a molecular target for therapeutic intervention in wet AMD.

Topics: Animals; Calcium-Binding Proteins; Choroidal Neovascularization; Dipeptides; Disease Models, Animal; Fluorescein Angiography; Gene Expression Regulation; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Proto-Oncogene Proteins c-sis; Rats; Rats, Sprague-Dawley; Receptors, CCR3; Receptors, Notch; Retinal Neovascularization; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Serrate-Jagged Proteins; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2; Wet Macular Degeneration