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The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for β-galactosidase. Senescence-associated β-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE. [ABSTRACT FROM AUTHOR]

Pigment epithelium-derived factor (PEDF) is a secreted protein that is essential in tissue homeostasis and is involved in multiple functions in the eye, such as antiangiogenesis and neuroprotection. However, short retention in the retinal microenvironment can limit its therapeutic effects. In this study, we modified the amino acid sequence of PEDF to increase its affinity for heparin and hyaluronic acid (HA), which are negatively charged extracellular matrix (ECM) molecules. HA is the major component of the vitreous humor. We selectively converted neutral or anionic residues into cationic residues to obtain engineered PEDF (ePEDF). Using in vitro binding assays, we demonstrate that ePEDF had higher affinity for heparin and HA than wild-type PEDF (wtPEDF). ePEDF exhibited antiangiogenic and retinal survival bioactivities. It inhibited endothelial cell proliferation and tube formation in vitro. In an ex vivo model mimicking retinal degeneration, ePEDF protected photoreceptors from cell death. The findings suggest that protein engineering is an approach to develop active PEDF with higher ECM affinity to potentially improve its retention in the retina microenvironment and in turn make it a more efficient therapeutic drug for retinal diseases. • Short retention in the vitreous humor prevents the translation of pigment epithelium-derived factor (PEDF) protein therapy. • PEDF engineering increases cationic property and enhances binding to heparin and HA, which are abundant in the vitreous humor. • In vitro and ex vivo models evaluate antiangiogenic and neuroprotective properties of engineered PEDF. [ABSTRACT FROM AUTHOR]

Summary: Proteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here, we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Specifically, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA treatment, increasing MT-MMP and proproliferative gene expression via β1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis. [Copyright &y& Elsevier]

L7644 --> 505.29 --> The gene Serpinf1 encodes for Pigment Epithelium‐Derived Factor (PEDF), which protects photoreceptors from cell death. The Serpinf1 null mouse exhibits normal retinal function; however, PEDF deficiency increases retinal degeneration susceptibility in rd10 mouse. CRX is a transcription factor that regulates expression of several photoreceptor specific genes, including opsins and phosphodiesterase. This study aims to explore whether regulation of CRX activation is one mechanism by which PEDF exerts photoreceptor survival. Serpinf1‐/‐, Serpinf1+/‐ and Serpinf1+/+ mice at 3 months of age were used. We prepared cultures of mouse retinal explants. They were treated with recombinant human PEDF. Zaprinast (a PDE inhibitor) was added to induce photoreceptor death. We determined cell death using PSVue‐550, a visible fluorescent probe for detection of phosphatidylserine on the surface of cells. Subcellular distribution of CRX, PDE6A and pan‐acetylation was detected by immunofluorescence. The transcriptional levels of Crx, Pde6a, Opn1mw and Opn1sw were assessed using qPCR. PEDF treatment induced the expression of Crx and its regulated genes Pde6a, Opn1mw and Opn1sw. PEDF also enhanced the CRX immunoreactivity in the nuclei of photoreceptors. In contrast, the nuclear CRX immunoreactivity was suppressed in photoreceptors from retinas of Serpinf1‐/‐ mice deficient of PEDF. PDE6A immunoreactivity also decreased in Serpinf1‐/‐ photoreceptors compared to those from Serpinf1+/+ mice. Histone acetylation was detected in discrete photoreceptors in Serpinf1+/+ but was undetectable in the Serpinf1‐/‐ retinas. Zaprinast‐induced photoreceptor death was more pronounced in Serpinf1‐/‐ retinal explants than in wild type controls. PEDF pre‐treatment prior to the zaprinast treatment improved photoreceptor survival and enhanced CRX immunoreactivity in both Serpinf1‐/‐ and Serpinf1+/+ retinal explants. Deletion in PEDF in the Serpinf1 null mice accelerated retinal degeneration induced by zaprinast, which was prevented by exogenous addition of PEDF in retinal explants. The findings imply that PEDF activated the CRX‐associated transcription factor network. They provide a novel insight linking extracellular PEDF to nuclear CRX during photoreceptor survival. [ABSTRACT FROM AUTHOR]

R4747 --> 501.2 --> Retinitis pigmentosa (RP) is one of the most common inherited diseases of the retina (retinopathies) that is estimated to affect 1 in 4,000 people in the United States and worldwide. This collection of genetic diseases leads to progressive photoreceptor cell degeneration and ultimately vision loss, with more than 60 genes implicated in molecular pathways towards photoreceptor cell death. Currently, there are no treatments available and, due to its vast diverse genetic background, a putative gene‐based therapy is challenging. Consequently, mutation‐independent strategies to protect photoreceptors against continued damage and progressive vision loss are appealing approaches. One of them is the use of survival, neurotrophic and neuroprotective factors that target common signaling pathways of cell death in the different types of RP. The aim of this study is to optimize methods to evaluate the efficacy of compounds to protect photoreceptor cells against death. We used PSVue® to optimize the cell death read‐out in cultured retinal explants and in retinas of a mouse model for a human inherited autosomal recessive RP, the rd10 mouse. Retinal degeneration in the rd10 mice is caused by a spontaneous mutation in the gene for the β‐subunit of phosphodiesterase. PSVue® is a robust probe for detecting phosphatidyl serine on the surface of cells undergoing death. We used retinal tissue explanted from wild type mouse eyes, and cultured it ex vivo. Induction of photoreceptor cell death was achieved with Zaprinast, an inhibitor of phosphodiesterase, to mimic the rd10 photoreceptor cell death. Low probe concentrations and short incubation times resulted in a reproducible detection of photoreceptor death in the retina by confocal super resolution microscopy. The PSVue® detection method yielded lower fluorescence background than the TUNEL detection method. Pigment epithelium‐derived factor (PEDF), a photoreceptor survival protein, was used as control to prevent photoreceptor cell death induced by Zaprinast. PEDF supplementation prior to Zaprinast treatment decreased cell death detection, as expected. We used the mouse model rd10 to evaluate photoreceptor death in vivo. PSVue® eye‐drops were administered to the left eye of rd10 mice of P17‐P25 postnatal ages, and fluorescence was detected by fundoscopy using MICRON III a day later. HBBS eye‐drops were administered to the right eye as a control reference. The fluorescence of the probe detected in the back of mouse eyes increased between ages P17 and P23 relative to that of HBBS‐treated eyes. The results revealed a peak of photoreceptor cell degeneration that occurred at P25 days of age in living rd10 mouse models. We have optimized methods to determine photoreceptor cell death in the ex vivo and in vivo experimental systems described here using a fluorescent probe. The methodologies are simple, fast, and complementary, and together they will prove to be suitable for screening factors and drugs against RP. [ABSTRACT FROM AUTHOR]

Abstract: Laminin α chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin α5 in mouse submandibular glands (SMGs). Lama5 −/− SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs α5 and α1 are present in epithelial clefts but as branching begins α5 expression increases while α1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-β1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that β1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3 −/−:Itga6 −/− SMGs have a similar phenotype to Lama5 −/−. Our findings suggest that laminin α5 controls SMG epithelial morphogenesis through β1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling. [Copyright &y& Elsevier]

Retinoprotective proteins play important roles for retinal tissue integrity. They can directly affect the function and the survival of photoreceptors, and/or indirectly target the retinal pigment epithelium (RPE) and endothelial cells that support these tissues. Retinoprotective proteins are used in basic, translational and in clinical studies to prevent and treat human retinal degenerative disorders. In this review, we provide an overview of proteins that protect the retina and focus on pigment epithelium-derived factor (PEDF), and its effects on photoreceptors, RPE cells, and endothelial cells. We also discuss delivery systems such as pharmacologic and genetic administration of proteins to achieve photoreceptor survival and retinal tissue integrity. [ABSTRACT FROM AUTHOR]

The regulation of epithelial proliferation during organ morphogenesis is crucial for normal development, as dysregulation is associated with tumor formation. Non-coding microRNAs (miRNAs), such as miR-200c, are post-transcriptional regulators of genes involved in cancer. However, the role of miR-200c during normal development is unknown. We screened miRNAs expressed in the mouse developing submandibular gland (SMG) and found that miR-200c accumulates in the epithelial end buds. Using both loss- and gain-of-function, we demonstrated that miR-200c reduces epithelial proliferation during SMG morphogenesis. To identify the mechanism, we predicted miR-200c target genes and confirmed their expression during SMG development. We discovered that miR-200c targets the very low density lipoprotein receptor (Vldlr) and its ligand reelin, which unexpectedly regulate FGFR-dependent epithelial proliferation. Thus, we demonstrate that miR-200c influences FGFR-mediated epithelial proliferation during branching morphogenesis via a Vldlr-dependent mechanism. miR-200c and Vldlr may be novel targets for controlling epithelial morphogenesis during glandular repair or regeneration. [ABSTRACT FROM AUTHOR]

Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Here, we report that ex vivo branching of intact submandibular glands decreases when either FGFR2 expression is downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial budding, suggesting that multiple signaling pathways exist. We immunolocalized FGF receptors and analyzed changes in FGFR, FGF and MMP gene expression to identify the mechanisms of FGF-mediated morphogenesis. FGFRlb and FGFR2b are present throughout the epithelium, although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, our data suggests that FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis. [ABSTRACT FROM AUTHOR]