PEDF Deletion Induces Senescence and Defects in Phagocytosis in the RPE.

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 ß-galactosidase. Senescence-associated ß-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.

Pigment epithelium-derived factor engineered to increase glycosaminoglycan affinity while maintaining bioactivity.

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.

Delivery Systems of Retinoprotective Proteins in the Retina.

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.

Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex.

Mutations in myosin-VIIa (MYO7A) cause Usher syndrome type 1, characterized by combined deafness and blindness. MYO7A is proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex, but conclusive evidence is lacking. Here we report that multiple MYO7A isoforms are expressed in the mouse cochlea. In mice with a specific deletion of the canonical isoform (Myo7a-ΔC mouse), MYO7A is severely diminished in inner hair cells (IHCs), while expression in outer hair cells is affected tonotopically. IHCs of Myo7a-ΔC mice undergo normal development, but exhibit reduced resting open probability and slowed onset of MET currents, consistent with MYO7A's proposed role in tensioning the tip link. Mature IHCs of Myo7a-ΔC mice degenerate over time, giving rise to progressive hearing loss. Taken together, our study reveals an unexpected isoform diversity of MYO7A expression in the cochlea and highlights MYO7A's essential role in tensioning the hair cell MET complex.

A Functional MicroRNA Screening Method for Organ Morphogenesis.

The increasing repertoire of microRNAs expressed during organ development and their role in regulating organ morphogenesis provide a compelling need to develop methods to assess microRNA function using various in vitro and in vivo experimental models. Methods to assess microRNA function during organ morphogenesis include transfection of microRNA inhibitors (antagomirs) and activators (mimics) into mouse embryonic explanted organs using liposomes, which can potentially result in low efficiency of transfection and off-target effects. We devised a method to assess microRNA function in explanted organs by transfecting antagomirs and mimics using peptide-based nanoparticles, increasing functional microRNA targeting efficiency, and decreasing off-target effects. Our method can be applied to a variety of embryonic organs that can be explanted and provides an alternative to efficiently and functionally prioritize microRNAs during organ morphogenesis for further in vivo genetic approaches. © 2017 by John Wiley & Sons, Inc.

An integrated miRNA functional screening and target validation method for organ morphogenesis.

The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

miR-200c regulates FGFR-dependent epithelial proliferation via Vldlr during submandibular gland branching morphogenesis.

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.

MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.

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 beta1 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.

ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during submandibular gland development.

Epithelial-mesenchymal interactions during organogenesis are regulated by dynamic and reciprocal interactions between growth factors and extracellular matrix (ECM) components. Mouse embryonic submandibular gland (SMG) epithelium, isolated from its endogenous mesenchyme, undergoes branching morphogenesis when cultured ex vivo in a basement membrane extract in serum-free medium with growth factor stimulation. The resulting three-dimensional epithelial morphogenesis in the defined culture system makes this a useful model to analyze cell-cell and cell-matrix interactions, growth factor-mediated signaling and gene expression, proliferation, apoptosis, migration, lumen formation, and epithelial morphogenesis in a primary organ culture system. SMG epithelial culture is robust, reproducible, uses small amounts of reagents, and changes in gene expression are measured by real-time PCR using a limited amount of embryonic tissue. In this chapter, we describe a detailed protocol for isolating primary embryonic SMG epithelium and setting up an ECM and growth factor-dependent, serum-free assay of epithelial morphogenesis, with subsequent analysis of gene expression by real-time PCR.

Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling.

Laminin alpha 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 alpha5 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 alpha5 and alpha1 are present in epithelial clefts but as branching begins alpha5 expression increases while alpha1 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-beta1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that beta1 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 alpha5 controls SMG epithelial morphogenesis through beta1 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.

Salivary gland branching morphogenesis.

Salivary gland branching morphogenesis involves coordinated cell growth, proliferation, differentiation, migration, apoptosis, and interaction of epithelial, mesenchymal, endothelial, and neuronal cells. The ex vivo analysis of embryonic mouse submandibular glands, which branch so reproducibly and beautifully in culture, is a powerful tool to investigate the molecular mechanisms regulating epithelium-mesenchyme interactions during development. The more recent analysis of genetically modified mice provides insight into the genetic regulation of branching morphogenesis. The review begins, as did the field historically, focusing on the role of the extracellular matrix (ECM), and its components such as glycosaminoglycans, collagens, and laminins. Following sections describe the modification of the ECM by proteases and the role of cell-matrix and cell-cell receptors. The review then focuses on two major families of growth factors implicated in salivary gland development, the fibroblast growth factors (FGFs) and the epidermal growth factors (EGFs). The salivary gland phenotypes in mice with genetic modification of FGFs and their receptors highlight the central role of FGFs during salivary gland branching morphogenesis. A broader section mentions other molecules implicated from analysis of the phenotypes of genetically modified mice or organ culture experiments. The review concludes with speculation on some future areas of research.

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.

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. FGFR1b 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.