The unique role of bone marrow adipose tissue in ovariectomy-induced bone loss in mice.

BACKGROUND: Accumulation of bone marrow adipose tissue (BMAT) is always seen in osteoporosis induced by estrogen deficiency. Herein, we aimed to investigate the mechanisms and consequences of this phenomenon by establishing a mouse model of osteoporosis caused by ovariectomy (OVX)-mimicked estrogen deficiency. METHODS: Micro-CT, osmium tetroxide staining, and histological analyses were performed to examine the changes in bone microstructure, BMAT and white adipose tissue (WAT) in OVX mice compared to sham mice. The osteogenesis and adipogenesis of primary bone marrow stromal cells (BMSCs) isolated from sham and OVX mice were compared in vitro. The molecular phenotypes of BMAT and WAT were determined and compared by quantitative PCR (qPCR). Bone marrow adipocyte-conditioned medium (BMA CM) was prepared from sham or OVX mice for coculture assays, and BMSCs or bone marrow monocytes/macrophages (BMMs) were isolated and subjected to osteoblast and osteoclast differentiation, respectively. Cell staining and qPCR were used to assess the effects of BMAT on bone metabolism. RESULTS: OVX-induced estrogen deficiency induced reductions in both cortical and trabecular bone mass along with an expansion of BMAT volume. At the cellular level, loss of estrogen inhibited BMSC osteogenesis and promoted BMSC adipogenesis, whereas addition of estradiol exerted the opposite effects. In response to estrogen deficiency, despite the common proinflammatory molecular phenotype observed in both fat depots, BMAT, unlike WAT, unexpectedly exhibited an increase in adipocyte differentiation and lipolytic activity as well as the maintenance of insulin sensitivity. Importantly, BMAT, but not WAT, presented increased mRNA levels of both BMP receptor inhibitors (Grem1, Chrdl1) and Rankl following OVX. In addition, treatment with BMA CM, especially from OVX mice, suppressed the osteoblast differentiation of BMSCs while favoring the osteoclast differentiation of BMMs. CONCLUSION: Our study illustrates that OVX-induced estrogen deficiency results in bone loss and BMAT expansion by triggering imbalance between the osteogenesis and adipogenesis of BMSCs. Furthermore, expanded BMAT, unlike typical WAT, may negatively regulate bone homeostasis through paracrine inhibition of osteoblast-mediated bone formation and promotion of osteoclast-mediated bone resorption.

Dual Pigment Epithelium-derived Factor and Hepatocyte Growth Factor Overexpression: A New Therapy for Pulmonary Hypertension.

Pulmonary hypertension (PH) is a disease characterized by advanced pulmonary vasculature remodeling that is thought to be curable only through lung transplantation. The application of angiogenic hepatocyte growth factor (HGF) is reported to be protective in PH through its anti-vascular remodeling effect, but excessive HGF-mediated immature neovascularization is not conducive to the restoration of pulmonary perfusion because of apparent vascular leakage. As a canonical antiangiogenic molecule, pigment epithelium-derived factor (PEDF) inhibits angiogenesis and reduces vascular permeability in a variety of diseases. However, the effect of PEDF on HGF-based PH treatment remains to be determined. In this study, monocrotaline-induced PH rats and endothelial cells isolated from rat and human PH lung tissues were used. We assessed PH progression, right cardiac function, and pulmonary perfusion in HGF- and/or PEDF-treated rats with PH. Additionally, the receptor and mechanism responsible for the role of PEDF in HGF-based PH therapy were investigated. In this study, we found that HGF and PEDF jointly prevent PH development and improve right cardiac function in rats with PH. Moreover, PEDF delivery increases the pulmonary perfusion in PH lungs and inhibits immature angiogenesis and vascular endothelial (VE)-cadherin junction disintegration induced by HGF without affecting the therapeutic inhibition of pulmonary vascular remodeling by HGF. Mechanistically, PEDF targets VE growth factor receptor 2 and suppresses its phosphorylation at Y951 and Y1175 but not Y1214. Finally, VE growth factor receptor 2/VE protein tyrosine phosphatase/VE-cadherin complex formation and Akt and Erk1/2 inactivation were observed in rat and human PH lung endothelial cells. Collectively, our data indicate that PEDF additively enhances the efficacy of HGF against PH, which may provide new insights into treatment strategies for clinical PH.

Roles of pigment epithelium-derived factor in cardiomyocytes: implications for use as a cardioprotective therapeutic.

OBJECTIVES: Cardiovascular diseases are the leading cause of death worldwide, with patients having limited options for treatment. Pigment epithelium-derived factor (PEDF) is an endogenous multifunctional protein with several mechanisms of action. Recently, PEDF has emerged as a potential cardioprotective agent in response to myocardial infarction. However, PEDF is also associated with pro-apoptotic effects, complicating its role in cardioprotection. This review summarises and compares knowledge of PEDF's activity in cardiomyocytes with other cell types and draws links between them. Following this, the review offers a novel perspective of PEDF's therapeutic potential and recommends future directions to understand the clinical potential of PEDF better. KEY FINDINGS: PEDF's mechanisms as a pro-apoptotic and pro-survival protein are not well understood, despite PEDF's implication in several physiological and pathological activities. However, recent evidence suggests that PEDF may have significant cardioprotective properties mediated by key regulators dependent on cell type and context. CONCLUSIONS: While PEDF's cardioprotective activity shares some key regulators with its apoptotic activity, cellular context and molecular features likely allow manipulation of PEDF's cellular activity, highlighting the importance of further investigation into its activities and its potential to be applied as a therapeutic to mitigate damage from a range of cardiac pathologies.

Combination of pigment epithelium derived factor with anti-vascular endothelial growth factor therapy protects the neuroretina from ischemic damage.

Ocular ischemia is a vision-threatening disease, and is a medical condition associated with many ocular diseases. Anti-VEGF therapy has limitations related to its side effects and suppression of physiological revascularization. Pigment epithelium derived factor (PEDF) has anti-angiogenesis and neurotrophic neuroprotective functions and is a promising agent in the treatment of ischemia-induced retinal neurodegeneration. The purpose of this study is to investigate the effect of PEDF and anti-VEGF and the combined therapy on the ischemic rat eye model ex vivo. In this study, the PEDF protein, anti-VEGF drug (Avastin) or the combination of PEDF and Avastin were intravitreally injected immediately after eye enucleation. Then the eyes were incubated in Dulbecco's modified eagle medium (DMEM) at 4 â„ƒ for 14 h. After that the eyes were fixed immediately by formalin. VEGF, PEDF and glial fibrillary acidic protein (GFAP) were detected by immunohistochemical (IHC) staining. The IHC staining intensity was evaluated for each eye. Compared to the groups treated by vehicle, PEDF, and anti-VEGF alone, the value of staining intensity of VEGF and GFAP was significantly reduced in the retina and choroidal vessels of the PEDF/Anti-VEGF treatment group. The intravitreally injected PEDF protein can locate in the retina and the choroidal vessels. Compared to the vehicle-treatment group, both the PEDF-treatment and the PEDF/Anti-VEGF treatment groups showed significantly decreased number of TUNEL-positive nuclei, and the PEDF/Anti-VEGF treatment group had the least TUNEL-positive nuclei. Combination of PEDF and an anti-VEGF drug (Avastin) is a possible therapeutic strategy against ischemic retinal and choroidal diseases.

Pigment epithelium-derived factor, an anti-VEGF factor, delays ovarian cancer progression by alleviating polarization of tumor-associated macrophages.

Ovarian cancer (OC) is one of the most dangerous gynecological malignancies with no effective treatment so far. Pigment epithelium-derived factor (PEDF) has been reported to have ideal anti-tumor effects, but its relationship with the regulation of tumor-associated macrophage polarization is currently unclear. In this study, the mRNA expression of PEDF and macrophage markers were determined in OC tissues from clinic patients and five OC (A2780, SKOV3, CAOV3, OVCAR3, and OVCA433) cell lines through quantitative reverse transcription PCR. Afterwards, tumor growth, cell proliferation and apoptosis, and macrophage polarization in OC tumor-bearing mice with PEDF overexpression were recorded and investigated. Finally, the polarization of macrophages was explored in the presence of lentiviral PEDF overexpression, adipose triglyceride lipase (ATGL) and laminin receptor (LR) knockdown, and mitogen-activated protein kinase (MAPK) pathway inhibition. Our results suggest that PEDF mRNA level is significantly decreased in OC tissues and cells and has a significant negative correlation with OC progression and the level of tumor-related macrophage markers. Furthermore, OC tumors overexpressing PEDF show suppressed growth viability and increased apoptosis rate. The fluorescence activated cell sorting (FACS) analysis reveals that PEDF can promote macrophage polarization in OC tumors towards M1 subtype. Mechanistically, we found that ATGL and extracellular-regulated kinase 1/2 (ERK1/2) signaling are involved in the regulation of macrophage polarization in OC tumors by PEDF. Taken together, these data indicate that the role of PEDF in regulating the polarization of tumor-associated macrophages may make it a potential therapeutic strategy for the treatment of OC in the future.

A short peptide derived from pigment epithelial-derived factor exhibits an angioinhibitory effect.

BACKGROUND: Pigment epithelial-derived factor (PEDF), a 50 kDa secreted glycoprotein, exhibits distinct effects on a range of cell types. PEDF has been shown to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis and widely accepted as a promising agent for treatment eye diseases related to neovascularization. A pool of short peptide fragments derived from PEDF reportedly manifests angioinhibitory activity. This study aims to determine the minimal PEDF fragment which can exert the anti-VEGF effect. METHODS: A series of shorter synthetic peptides, derived from the 34-mer (PEDF amino acid positions Asp44-Asn77), were synthesized. An MTT assay was used to evaluate the ability of the 34-mer-derived peptides to inhibit VEGF-induced proliferation of multiple myeloma RPMI8226 cells. Cell apoptosis was monitored by annexin V-FITC staining. Western blot analysis was used to detect phosphorylated kinases, including c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and the expression of apoptosis-associated proteins, including p53, bax and caspase-3. VEGF-mediated angiogenesis of human umbilical vein endothelial cells (HUVECs), rat aortic ring and mouse cornea were used to detect the angioinhibitory activity of the PEDF-derived peptides. RESULTS: The MTT assay showed that the anti-VEGF effect of a 7-mer (Asp64-Ser70) was 1.5-fold greater than the 34-mer. In addition, massive apoptosis (37%) was induced by 7-mer treatment. The 7-mer induced JNK phosphorylation in RPMI8226 cells. Cell apoptosis and apoptosis-associated proteins induced by the 7-mer were blocked by pharmacological inhibition of JNK, but not p38 MAPK. Moreover, the 7-mer prevented VEGF-mediated angiogenesis of endothelial cells (ECs), including tube formation, aortic EC spreading and corneal neovascularization in mice. CONCLUSIONS: This is the first study to show that the PEDF 7-mer peptide manifests anti-VEGF activity, further establishing its potential as an anti-angiogenic agent.

Secreted PEDF modulates fibroblast collagen synthesis through M1 macrophage polarization under expanded condition.

Tissue expansion is widely used to obtain new skin tissue for repairing defects in the clinical practice of plastic surgery. One major complication can be dermal thinning during expansion, which usually leads to skin rupture. Collagen synthesis can determine dermal thickness and can be influenced by macrophage polarization during expansion. The aim of the study was to test whether pigment epithelium-derived factor (PEDF) could be a modulator of collagen synthesis in fibroblasts by regulating macrophage polarization during skin expansion. Our results showed that PEDF mRNA expression was increased in expanded human and mouse epidermis. PEDF protein levels were elevated in the subcutaneous exudates of a rat skin expansion model. Increased PEDF mRNA expression was accompanied by dermal thinning during a three-week expansion protocol. Subcutaneous injection of PEDF in vivo further resulted in dermal thinning and cell number increase of M1 macrophage in the expanded skin. PEDF also promoted macrophage polarization in vitro to the M1 subtype under hypoxic conditions. PEDF did not influence collagen gene expression in fibroblasts directly, but attenuated collagen synthesis in a macrophage-mediated manner. Additionally, blockage of PEDF receptors on macrophages with inhibitors rescued collagen synthesis in fibroblasts. Our research demonstrated PEDF elevation in expanded skin leads to dermal thinning through M1 macrophage-mediated collagen synthesis inhibition in fibroblasts. Our results could form a basis for the development of novel strategies to improve skin integrity in expanded skin by using PEDF.

Pathogenic Activation and Therapeutic Blockage of FcαR-Expressing Polymorphonuclear Leukocytes in IgA Pemphigus.

Pathomechanisms in IgA pemphigus are assumed to rely on Fc-dependent cellular activation by antigen-specific IgA autoantibodies; however, models for the disease and more detailed pathophysiologic data are lacking. In this study, we aimed to establish in vitro models of disease for IgA pemphigus, allowing us to study the effects of the interaction of anti-keratinocyte IgA with cell surface FcαRs. Employing multiple in vitro assays, such as a skin cryosection assay and a human skin organ culture model, in this study, we present mechanistic data for the pathogenesis of IgA pemphigus, mediated by anti-desmoglein 3 IgA autoantibodies. Our results reveal that this disease is dependent on FcαR-mediated activation of leukocytes in the epidermis. Importantly, this cell-dependent pathology can be dose-dependently abrogated by peptide-mediated inhibition of FcαR:IgA-Fc interaction, as confirmed in an additional model for IgA-dependent disease, that is, IgA vasculitis. These data suggest that IgA pemphigus can be modeled in vitro and that IgA pemphigus and IgA vasculitis are FcαR-dependent disease entities that can be specifically targeted in these experimental systems.

PEDF Gene Deletion Disrupts Corneal Innervation and Ocular Surface Function.

Purpose: The cornea is richly innervated by the trigeminal ganglion (TG) and its function supported by secretions from the adjacent lacrimal (LG) and meibomian glands (MG). In this study we examined how pigment epithelium-derived factor (PEDF) gene deletion affects the cornea structure and function. Methods: We used PEDF hemizygous and homozygous knockout mice to study effects of PEDF deficiency on corneal innervation assessed by beta tubulin staining, mRNA expression of trophic factors, and PEDF receptors by adjacent supporting glands, corneal sensitivity measured using a Cochet-Bonnet esthesiometer, and tear production using phenol red cotton thread wetting. Results: Loss of PEDF was accompanied by reduced corneal innervation and sensitivity, increased corneal surface injury and tear production, thinning of the corneal stroma and loss of stromal cells. PEDF mRNA was expressed in the cornea and its supporting tissues, the TG, LG, and MG. Deletion of one or both PEDF alleles resulted in decreased expression of essential trophic support in the TG, LG, and MG including nerve growth factor, brain-derived neurotrophic growth factor, and GDNF with significantly increased levels of NT-3 in the LG and decreased EGF expression in the cornea. Decreased transcription of the putative PEDF receptors, adipose triglyceride lipase, lipoprotein receptor-related protein 6, laminin receptor, PLXDC1, and PLXDC2 was also evident in the TG, LG and MG with the first three showing increased levels in corneas of the Pedf+/- and Pedf-/- mice compared to wildtype controls. Constitutive inactivation of ERK1/2 and Akt was pronounced in the TG and cornea, although their protein levels were dramatically increased in Pedf-/- mice. Conclusions: This study highlights an essential role for PEDF in corneal structure and function and confirms the reported rescue of exogenous PEDF treatment in corneal pathologies. The pleiotropic effects of PEDF deletion on multiple trophic factors, receptors and signaling molecules are strong indications that PEDF is a key coordinator of molecular mechanisms that maintain corneal function and could be exploited in therapeutic options for several ocular surface diseases.

Pigment epithelium-derived factor (PEDF) and derived peptides promote survival and differentiation of photoreceptors and induce neurite-outgrowth in amacrine neurons.

Pigment epithelium-derived factor (PEDF) is a cytoprotective protein for the retina. We hypothesize that this protein acts on neuronal survival and differentiation of photoreceptor cells in culture. The purpose of the present study was to evaluate the neurotrophic effects of PEDF and its fragments in an in vitro model of cultured primary retinal neurons that die spontaneously in the absence of trophic factors. We used Wistar albino rats. Cell death was assayed by immunofluorescence and flow cytometry through TUNEL assay, propidium iodide, mitotracker, and annexin V. Immunofluorescence of cells for visualizing rhodopsin, CRX, and antisyntaxin under confocal microscopy was performed. Neurite outgrowth was also quantified. Results show that PEDF protected photoreceptor precursors from apoptosis, preserved mitochondrial function and promoted polarization of opsin enhancing their developmental process, as well as induced neurite outgrowth in amacrine neurons. These effects were abolished by an inhibitor of the PEDF receptor or receptor-derived peptides that block ligand/receptor interactions. While all the activities were specifically conferred by short peptide fragments (17 amino acid residues) derived from the PEDF neurotrophic domain, no effects were triggered by peptides from the PEDF antiangiogenic region. The observed effects on retinal neurons imply a specific activation of the PEDF receptor by a small neurotrophic region of PEDF. Our findings support the neurotrophic PEDF peptides as neuronal guardians for the retina, highlighting their potential as promoters of retinal differentiation, and inhibitors of retinal cell death and its blinding consequences. Cover Image for this issue: https://doi.org/10.1111/jnc.15089.

PEDF inhibits lymphatic metastasis of nasopharyngeal carcinoma as a new lymphangiogenesis inhibitor.

Nasopharyngeal carcinoma (NPC) is one of the most malignant tumors in southern China and Asia, and lymph node metastasis is an important cause for treatment failure. Lymphangiogenesis is a crucial step in lymphatic metastasis of NPC, while little is known about lymphangiogenesis in NPC. Similar to angiogenesis, lymphangitic neovascularization is a process of balance between pro-lymphangiogenesis and anti-lymphangiogenesis factors, but there are few studies on endogenous lymphangiogenesis inhibitors. Pigment epithelium-derived factor (PEDF) is a well-known effective endogenous angiogenesis inhibitor. However, the relationship between PEDF and lymphangiogenesis remains unknown. Our present study reveals that PEDF is lowly expressed in human NPC tissues with poor prognosis and is negatively correlated with lymphatic vessel density (LVD). Consistently, PEDF inhibits lymphangiogenesis and lymphatic metastasis of NPC in vivo experiments. Mechanistically, PEDF inhibits the proliferation, migration, and tube formation of lymphatic endothelial cells and promotes cell apoptosis. On the other hand, PEDF reduces the expression and secretion of vascular endothelial growth factor C (VEGF-C) of NPC cells through the nuclear factor-κB (NF-κB) signaling pathway. Our findings indicate that PEDF plays a vital role in lymphatic metastasis by targeting both lymphatic endothelial cells and NPC cells, and PEDF may represent a novel therapeutic target for NPC.

Pigment Epithelium-Derived Factor Enhances the Suppressive Phenotype of Regulatory T Cells in a Murine Model of Dry Eye Disease.

Pigment epithelium-derived factor (PEDF) is a widely expressed 50-kDa glycoprotein belonging to the serine protease inhibitor family, with well-established anti-inflammatory functions. Recently, we demonstrated the immunoregulatory role played by PEDF in dry eye disease (DED) by suppressing the maturation of antigen-presenting cells at the ocular surface following exposure to the desiccating stress. In this study, we evaluated the effect of PEDF on the immunosuppressive characteristics of regulatory T cells (Tregs), which are functionally impaired in DED. In the presence of PEDF, the in vitro cultures prevented proinflammatory cytokine (associated with type 17 helper T cells)-induced loss of frequency and suppressive phenotype of Tregs derived from normal mice. Similarly, PEDF maintained the in vitro frequency and enhanced the suppressive phenotype of Tregs derived from DED mice. On systemically treating DED mice with PEDF, moderately higher frequencies and significantly enhanced suppressive function of Tregs were observed in the draining lymphoid tissues, leading to the efficacious amelioration of the disease. Our results demonstrate that PEDF promotes the suppressive capability of Tregs and attenuates their type 17 helper T-cell-mediated dysfunction in DED, thereby playing a role in the suppression of DED.

PEDF Attenuates Ocular Surface Damage in Diabetic Mice Model Through Its Antioxidant Properties.

PURPOSE: To investigate the antioxidative effect and mechanism of pigment epithelium-derived factor (PEDF) on the ocular surface damage in diabetic mice. METHODS: C57BL/6 mice were injected intraperitoneally with streptozocin to generate diabetic models and then 50 nM PEDF or artificial tears were used to treat the diabetic mice. Treatment was given three times a day for eight weeks. Corneal epithelial damage, corneal sensitivity, and tear volume were quantified by fluorescein staining, esthesiometer, and phenol red cotton thread, respectively. Animals were sacrificed at 16 weeks after diabetes and the whole globe specimens were subjected to histochemical staining. Reactive oxygen species (ROS) generation was detected by 2',7-dichlorodihydrofluorescein probe. The levels of receptor for advanced glycation end products (RAGE) and superoxide dismutase 1 (SOD1) were examined by quantitative real-time PCR and western blotting. RESULTS: Topical application of PEDF improved corneal epithelial damage, increased corneal sensitivity, and tear volume in diabetic mice. ROS levels in the cornea were significantly higher in the diabetic mice than in the normal mice. Moreover, PEDF attenuated the accumulation of ROS, decreased the expression of RAGE, and elevated SOD1 expression in the cornea. CONCLUSIONS: Topical application of PEDF can alleviate diabetes-related ocular surface damage and increase tear volume, along with the improvement of oxidative stress status.

Ethacrynic acid, a loop diuretic, suppresses epithelial-mesenchymal transition of A549 lung cancer cells via blocking of NDP-induced WNT signaling.

Lung cancer is one of the leading causes of death in cancer patients. Epithelial-mesenchymal transition (EMT) plays an important role in lung cancer progression. Therefore, for lung cancer treatment, it is crucial to find substances that inhibit EMT. Ethacrynic acid (ECA) is a diuretic that inhibits cellular ion flux and exerts anticancer effects. However, the effects of ECA on EMT in lung cancer remain unclear. We examined the effects of ECA on sphingosylphosphorylcholine (SPC) or TGF-ß1-induced EMT process in A549 and H1299 cells via reverse transcription polymerase chain reaction and Western blotting. We found that ECA inhibited SPC-induced EMT and SPC-induced WNT signalling in EMT. We observed that SPC induces the expression of NDP [Norrie disease protein] and WNT-2, whereas ECA suppressed their expression. SPC-induced WNT activation, EMT, migration, and invasion were suppressed by NDP small-interfering RNA (siNDP), but NDP overexpression (pNDP) enhanced these events in A549 and H1299 cells. Accordingly, NDP expression may influence lung cancer prognosis. In summary, our results revealed that ECA inhibited SPC or TGF-ß1-induced EMT in A549 and H1299 lung cancer cells by downregulating NDP expression and inhibiting WNT activation. Therefore, ECA might be a new drug candidate for lung cancer treatment.

Pigment Epithelium-Derived Factor (PEDF) Fragments Prevent Mouse Cone Photoreceptor Cell Loss Induced by Focal Phototoxicity In Vivo.

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor.

Tofacitinib inhibits the development of experimental autoimmune uveitis and reduces the proportions of Th1 but not of Th17 cells.

Purpose: Tofacitinib is a pan-Janus kinase (JAK) inhibitor that suppresses cytokine signaling and in turn, the cells that participate in inflammatory immunopathogenic processes. We examined the capacity of tofacitinib to inhibit the induction of experimental autoimmune uveitis (EAU) and related immune responses. Methods: EAU was induced in B10.A mice with immunization with bovine interphotoreceptor retinoid-binding protein (IRBP), emulsified in complete Freund's adjuvant (CFA), and a simultaneous injection of pertussis toxin. Tofacitinib, 25 mg/kg, was administered daily, and the vehicle was used for control. EAU development was assessed by histological analysis of the mouse eyes, and related immune responses were assessed by (i) the levels of interferon (IFN)-γ and interleukin (IL)-17, secreted by spleen cells cultured with IRBP; (ii) flow cytometric analysis of intracellular expression by spleen, or eye-infiltrating CD4 or CD8 cells of IFN-γ, IL-17, and their transcription factors, T-bet and RORγt. In addition, the inflammation-related cell markers CD44 and CD62L and Ki67, a proliferation marker, were tested. The proportions of T-regulatory cells expressing FoxP3 were determined by flow cytometric intracellular staining, while levels of antibody to IRBP were measured with enzyme-linked immunosorbent assay (ELISA). Results: Treatment with tofacitinib significantly suppressed the development of EAU and reduced the levels of secreted IFN-γ, but not of IL-17. Further, treatment with tofacitinib reduced in the spleen and eye-infiltrating cells the intracellular expression of IFN-γ and its transcription factor T-bet. In contrast, treatment with tofacitinib had essentially no effect on the intracellular expression of IL-17 and its transcription factor, RORγt. The selective effect of tofacitinib treatment was particularly evident in the CD8 population. Treatment with tofacitinib also increased the population of CD44, but reduced the populations of cells producing CD62L and Ki67. Treatment with tofacitinib had no effect on the proportion of FoxP3 producing regulatory cells and on the antibody production to IRBP. Conclusions: Treatment with tofacitinib inhibited the development of EAU, reduced the production of IFN-γ, but had essentially no effect on the production of IL-17.

Pigment epithelium-derived factor attenuates angiogenesis and collagen deposition in hypertrophic scars.

Scar forming wounds are often characterized by higher levels of vascularity than non-scarring wounds and normal skin, and inhibition of angiogenesis has been shown to inhibit scar formation in some model systems. The rabbit ear hypertrophic scar (HS) model has been widely used to study the mechanisms that underlie the development of HS as well as the effectiveness of various treatments. Although the rabbit ear HS model is well characterized in terms of scar formation, the rate and level of angiogenesis has not been investigated in this model, and the cause-effect relationship between angiogenesis and rabbit HSs has not been examined. In the current study, full-thickness excisional wounds were created on the ventral side of New Zealand White rabbit ears to induce HS formation, and the dynamic pattern of angiogenesis and the expression of angiogenic regulatory factors were examined over time. Blood vessel density was found to peak at 2.7% on day 14 post-wounding, decreasing to 1.7% by day 28. mRNA levels of the proangiogenic factor VEGF-A peaked at day 14, while the expression of the antiangiogenic factors pigment epithelium-derived factor (PEDF) and thrombospondin 1 (TSP1) peaked at day 28 post-wounding. To examine whether inhibition of angiogenesis influences HS formation in this model, wounds were treated with exogenous soluble antiangiogenic agents including recombinant PEDF (rPEDF) and a functional PEDF peptide (PEDF-335). rPEDF and PEDF-335 were administered intradermally from day 4 post-wounding every 3 days until day 19. Intradermal injection of rPEDF or PEDF-335 both led to decreased angiogenesis and decreased collagen deposition at the wound site. The results support the utility of antiangiogenic therapies, including rPEDF/PEDF-335, as a potential new strategy for the prevention or treatment of HSs.

Norrin restores blood-retinal barrier properties after vascular endothelial growth factor-induced permeability.

Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through ß-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/ß (GSK-3α/ß) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a ß-catenin-dependent manner.

Norrin Protects Retinal Ganglion Cells from Excitotoxic Damage via the Induction of Leukemia Inhibitory Factor.

PURPOSE: To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is a secreted protein that protects RGC from N-methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2. METHODS: Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment. RESULTS: After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice. CONCLUSIONS: Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.

Effects of a combinatorial treatment with gene and cell therapy on retinal ganglion cell survival and axonal outgrowth after optic nerve injury.

After an injury, axons in the central nervous system do not regenerate over large distances and permanently lose their connections to the brain. Two promising approaches to correct this condition are cell and gene therapies. In the present work, we evaluated the neuroprotective and neuroregenerative potential of pigment epithelium-derived factor (PEDF) gene therapy alone and combined with human mesenchymal stem cell (hMSC) therapy after optic nerve injury by analysis of retinal ganglion cell survival and axonal outgrowth. Overexpression of PEDF by intravitreal delivery of AAV2 vector significantly increased Tuj1-positive cells survival and modulated FGF-2, IL-1ß, Iba-1, and GFAP immunostaining in the ganglion cell layer (GCL) at 4 weeks after optic nerve crush, although it could not promote axonal outgrowth. The combination of AAV2.PEDF and hMSC therapy showed a higher number of Tuj1-positive cells and a pronounced axonal outgrowth than unimodal therapy after optic nerve crush. In summary, our results highlight a synergistic effect of combined gene and cell therapy relevant for future therapeutic interventions regarding optic nerve injury.