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Molecular responses of choroidal endothelial cells to elastin derived peptides through the elastin-binding protein (GLB1).

ABSTRACT: Neovascular AMD involves the activation of choroidal endothelial cells to increase their inflammatory and angiogenic behaviors. Elastin derived peptides (EDPs) can elicit some of these phenotypic changes in endothelial cells. This investigation was performed to follow up on those findings by determining a receptor for these peptides in the human eye as well as evaluating the effects of elevated EDPs on choroidal cells in vitro and in vivo.The expression of elastin receptor genes including GLB1 was analyzed using reverse transcription PCR. Migration of choroidal endothelial cells was quantified in the presence of inhibitors to different EDP binding proteins. C57BL6 mice were injected with EDPs and studied by electroretinography, transmission electron microscopy, and microarray analysis.An alternatively spliced form of beta-galactosidase (GLB1) is present on human choroidal endothelial cells and acts as a receptor for EDPs. Elevated levels of circulating EDPs do not affect retinal function in the mouse, but do increase the expression and deposition of collagen IV in the RPE/choroid complex.EDPs may play a role in neovascular AMD by binding to and inducing neovascular phenotypes in choroidal endothelial cells through their receptor, GLB1. These peptides also cause an increased mRNA expression and deposition of collagen IV in the RPE/choroid, which may alter diffusion properties between the retina and choriocapillaris.

SUBMITTER: Skeie JM

PROVIDER: S-EPMC3288713 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Molecular responses of choroidal endothelial cells to elastin derived peptides through the elastin-binding protein (GLB1).

Skeie Jessica M JM Hernandez Jasmine J Hinek Aleksander A Mullins Robert F RF

Matrix biology : journal of the International Society for Matrix Biology 20111202 2

<h4>Purpose</h4>Neovascular AMD involves the activation of choroidal endothelial cells to increase their inflammatory and angiogenic behaviors. Elastin derived peptides (EDPs) can elicit some of these phenotypic changes in endothelial cells. This investigation was performed to follow up on those findings by determining a receptor for these peptides in the human eye as well as evaluating the effects of elevated EDPs on choroidal cells in vitro and in vivo.<h4>Methods</h4>The expression of elastin ...[more]

PMID: 22178079

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Project description:Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 ?g). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 ?M, PEDF 336 (100 ?M) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

Project description:BackgroundEndothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.MethodsWe transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.ResultsWe report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.ConclusionECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

Dataset Information

Molecular responses of choroidal endothelial cells to elastin derived peptides through the elastin-binding protein (GLB1).

Publications

Molecular responses of choroidal endothelial cells to elastin derived peptides through the elastin-binding protein (GLB1).

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