Short peptides derived from pigment epithelium-derived factor attenuate retinal ischemia reperfusion injury through inhibition of apoptosis and inflammatory response in rats.

Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.

Evaluation of pigment epithelium-derived factor concentration in equine amniotic membrane homogenate and its in-vitro vascular endothelial growth factor inhibition effect in tears of dogs with vascularized ulcerative keratitis.

Background: Corneal neovascularization can result from many pathological processes affecting the ocular surface leading to disturbances and opacifications that reduce corneal clarity and may impact vision. In veterinary medicine, the use of topical corticosteroid is contraindicated in the presence of ulcerative keratitis, and there is sparse research regarding safe medical alternatives to inhibit corneal neovascularization in dogs to improve visual outcome. Aim: To investigate the pigment epithelium-derived factor (PEDF) concentration in equine amniotic membrane homogenate (EAMH) and its in-vitro vascular endothelial growth factor (VEGF) inhibition in tears of dogs with vascularized ulcerative keratitis. Methods: Homogenates from 10 equine amniotic membranes (AM) were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of equine PEDF and VEGF. Forty tear samples were collected from both eyes of dogs diagnosed with vascularized ulcerative keratitis, and 50 samples from healthy dogs. Samples from affected eyes were allocated to G1 - affected undiluted tears; G2 - affected tears diluted with phosphate-buffer solution; G3 - affected tears treated with low-concentrated EAMH; and G4 - affected tears treated with high-concentrated EAMH. Tears from the unaffected contralateral eyes were composed in G5, while G6 was composed by tears from healthy dogs (control). The presence and levels of VEGF were evaluated in all groups by Western blot and ELISA. Results: The PEDF:VEGF ratio in EAMH was 110:1. An increase in VEGF levels was observed in tears from eyes with vascularized corneal ulcers (G1) as well as in contralateral tears (G5), compared to normal dogs (G6). High-concentrated EAMH provided a greater decrease in VEGF levels in-vitro compared to low-concentrated EAMH. Conclusion: EAMHs exhibited high concentrations of PEDF in comparison to VEGF and were able to partially decrease VEGF levels in tears of dogs with vascularized ulcers, in-vitro. Our results suggest that VEGF concentration is elevated in tears of dogs with active vascularized ulcerative keratitis in both affected and contralateral eyes compared to that of healthy dogs.

A Role for Low-Density Lipoprotein Receptor-Related Protein 6 in Blood Vessel Regression in Wound Healing.

Objective: The healing of skin wounds is typified by a pattern of robust angiogenesis followed by vascular regression. Pigment epithelium-derived factor (PEDF), a recognized endogenous antiangiogenic protein, regulates vascular regression in resolving wounds through an unknown receptor. Among the multiple receptors for PEDF that have been identified, low-density lipoprotein receptor-related protein 6 (Lrp6) has been described as a regulator of angiogenesis in multiple systems. The purpose of the current study was to determine if the Lrp6 receptor plays a role in vessel regression in wounds. Approach: Excisional skin wounds were prepared on C57BL/6 mice. RT-PCR and immunoblots were performed to measure Lrp6 expression over a time course of wound healing. Immunohistochemistry was performed to localize Lrp6 in both recombinant PEDF (rPEDF)-treated and control wounds. To examine whether Lrp6 is critical to the regulation of capillary regression in vivo, wounds were treated with Lrp6 siRNA to minimize its presence in wounds. Immunohistochemistry for CD31 was performed to quantify blood vessel density. Results: PCR and immunoblots revealed significant increases in Lrp6 expression during the vascular regression phase of wound healing. Lrp6 was found to colocalize with CD31+ endothelial cells in wounds. The addition of rPEDF to wounds caused an increase in Lrp6-CD31+ endothelial cell colocalization. Inhibition of Lrp6 by siRNA impeded the vascular regression phase of healing. Innovation: This study is the first to demonstrate an association between Lrp6 and vessel regression in wound healing. Conclusion: Lrp6 is expressed in wounds in a temporal and spatial manner that suggests it may be a receptor for PEDF during vascular regression. PEDF increases Lrp6 expression in the wound vasculature, and inhibition of Lrp6 blocked vascular regression in wounds. The results suggest that Lrp6 is important to vascular regression in wounds, possibly through direct interaction with PEDF.

Retinol binding protein 3 is increased in the retina of patients with diabetes resistant to diabetic retinopathy.

The Joslin Medalist Study characterized people affected with type 1 diabetes for 50 years or longer. More than 35% of these individuals exhibit no to mild diabetic retinopathy (DR), independent of glycemic control, suggesting the presence of endogenous protective factors against DR in a subpopulation of patients. Proteomic analysis of retina and vitreous identified retinol binding protein 3 (RBP3), a retinol transport protein secreted mainly by the photoreceptors, as elevated in Medalist patients protected from advanced DR. Mass spectrometry and protein expression analysis identified an inverse association between vitreous RBP3 concentration and DR severity. Intravitreal injection and photoreceptor-specific overexpression of RBP3 in rodents inhibited the detrimental effects of vascular endothelial growth factor (VEGF). Mechanistically, our results showed that recombinant RBP3 exerted the therapeutic effects by binding and inhibiting VEGF receptor tyrosine phosphorylation. In addition, by binding to glucose transporter 1 (GLUT1) and decreasing glucose uptake, RBP3 blocked the detrimental effects of hyperglycemia in inducing inflammatory cytokines in retinal endothelial and Müller cells. Elevated expression of photoreceptor-secreted RBP3 may have a role in protection against the progression of DR due to hyperglycemia by inhibiting glucose uptake via GLUT1 and decreasing the expression of inflammatory cytokines and VEGF.

Type I pig collagen enhances the efficacy of PEDF 34-mer peptide in a mouse model of laser-induced choroidal neovascularization.

BACKGROUND: Pigment epithelium-derived factor (PEDF)-derived 34-mer peptide (PEDF34, Asp44-Asn77) has anti-angiogenic activity but has limitations in clinical application because of an inverted bell-shaped dose-effect relationship and a short half-life. In this study, we attempted to mitigate these problems by mixing PEDF34 with type I collagen. METHODS: The anti-angiogenic activity of the PEDF34/atelocollagen mixture was evaluated by HUVEC tube formation assay and in a laser-induced choroidal neovascular (CNV) mouse model. PEDF34 and/or collagen were administrated using intravitreal injections or eye drops. CNV lesion size was quantified using FITC-dextran-perfused retinal whole mounts. Western blot analysis and inhibitor assays were used to define the action mechanisms of PEDF34 and the mixture. RESULTS: Collagen broadened the effective dose range of PEDF34 in the tube formation assay by > 250 times (from 0.2 to 50 nM). In the CNV model, five intravitreal injections of PEDF34 were required for therapeutic effect, whereas the mixture had a significant therapeutic effect following a single injection. Eye drops of the mixture showed significantly stronger CNV-suppressive effects than drops of PEDF34 alone. The anti-angiogenic activity of PEDF34 might be mediated by inhibition of ERK and JNK activation by VEGF, and collagen potentiated these effects. CONCLUSIONS: Collagen can serve as a carrier and reservoir of PEDF34. PEDF peptide/collagen mixture is easy to prepare than conventional methods for maintaining the therapeutic effect of PEDF peptide.

Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine Experimental Autoimmune Uveoretinitis.

In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclera-choroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU.

Alginate Bead-Encapsulated PEDF Induces Ectopic Bone Formation In Vivo in the Absence of Co-Administered Mesenchymal Stem Cells.

BACKGROUND: Bone defects can be severely debilitating and reduce quality of life. Osteoregeneration can alleviate some of the complications in bony defects. For therapeutic use in future, a single factor that can cause potent bone regeneration is highly preferred as it will be more costeffective, any off-target effects will be more easily monitored and potentially managed, and for ease of administration which would lead to better patient compliance and satisfaction. OBJECTIVE: We demonstrate that pigment epithelium-derived factor (PEDF), one such factor that is known to be potent against angiogenesis, promotes osteoblastogenesis in mesenchymal stem cells in vitro, but does not need co-encapsulation of cells in alginate bead scaffolds for osteogeneration in vivo. RESULTS: Osteogenic differentiation by PEDF in vitro was confirmed with immunoblotting and immunocytochemical staining for bone markers (alkaline phosphatase, osteocalcin, osteopontin, collagen I), calcified mineral deposition, and assay for alkaline phosphatase activity. PEDF-mediated bone formation in a muscle pocket in vivo model was confirmed by microcomputed tomography (microCT), histology (haematoxylin and eosin, Alcian blue staining), immunostaining for bone markers and for collagen I-processing proteins (heat shock protein 47 and membrane type I matrix metalloproteinase). CONCLUSION: PEDF therefore presents itself as a promising biological for osteogeneration.

Glycoprotein Nonmelanoma Clone B Regulates the Crosstalk between Macrophages and Mesenchymal Stem Cells toward Wound Repair.

The process of wound repair requires the coordinated participation of multiple types of cells, which are sequentially recruited during the healing process. In response to tissue injury, both macrophages and mesenchymal stem cells (MSCs) are recruited to the site of injury, where they participate in the repair process. Despite considerable understanding of the role of each cell type in the process of wound repair, the nature of the dynamic interplay between these two cell types and how this interaction influences the process of wound repair are not well understood. Here, using an in vivo model of cutaneous wound healing in mice, we provide evidence that GPNMB is functionally important in promoting the recruitment of MSCs to the site of skin injury, which in turn modulates inflammatory responses by directing the M2 polarization of macrophages in acute wound healing. Furthermore, we show that GPNMB activity is impaired in a diabetic wound environment, which is associated with impaired MSC recruitment that is reversed by the topical administration of recombinant GPNMB protein to the wounds of diabetic mice. Our study provides important insight into the crosstalk between macrophages and endogenous MSCs toward wound repair.

Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.

The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf, and the axon growth promoter semaphorin 7a (sema7a), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y (npy), small proline-rich protein 1a (sprr1a), and vasoactive intestinal peptide (vip) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea-TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea.

Core-shell fibrous scaffold as a vehicle for sustained release of retinal pigmented epithelium-derived factor (PEDF) for photoreceptor differentiation of conjunctiva mesenchymal stem cells.

Coaxial electrospinning technique was introduced as a flexible and promising technique for the fabrication of core-shell fibrous scaffold from poly ethylene glycol/poly caprolactone (PEG/PCL), where retinal pigmented epithelium-derived factor (PEDF) was encapsulated in the core, for photoreceptor differentiation of conjunctiva mesenchymal stem cells (CJMSCs) seed on scaffolds. The morphology and structure of fibers were characterized using SEM and TEM and photoreceptor differentiation was examined by quantitative real time PCR (qPCR). Release study showed that, a sustained release of PEDF from PEG/PCL scaffold was observed over 14 days. qPCR analysis demonstrated that rhodopsin (as a main photoreceptor gene) was significantly expressed in CJMSCs cultured on scaffold loaded with PEDF. According to the result, the core-shell scaffold loaded with PEDF (PEG + PEDF)/PCL) has superior control over factor release profile and has a potential for guiding photoreceptor differentiation of mesenchymal stem cells and promoting retinal regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3514-3519, 2017.

Evaluation of tolerance to lentiviral LV-RPE65 gene therapy vector after subretinal delivery in non-human primates.

Several approaches have been developed for gene therapy in RPE65-related Leber congenital amaurosis. To date, strategies that have reached the clinical stages rely on adeno-associated viral vectors and two of them documented limited long-term effect. We have developed a lentiviral-based strategy of RPE65 gene transfer that efficiently restored protein expression and cone function in RPE65-deficient mice. In this study, we evaluated the ocular and systemic tolerances of this lentiviral-based therapy (LV-RPE65) on healthy nonhuman primates (NHPs), without adjuvant systemic anti-inflammatory prophylaxis. For the first time, we describe the early kinetics of retinal detachment at 2, 4, and 7 days after subretinal injection using multimodal imaging in 5 NHPs. We revealed prolonged reattachment times in LV-RPE65-injected eyes compared to vehicle-injected eyes. Low- (n = 2) and high-dose (n = 2) LV-RPE65-injected eyes presented a reduction of the outer nuclear and photoreceptor outer segment layer thickness in the macula, that was more pronounced than in vehicle-injected eyes (n = 4). All LV-RPE65-injected eyes showed an initial perivascular reaction that resolved spontaneously within 14 days. Despite foveal structural changes, full-field electroretinography indicated that the overall retinal function was preserved over time and immunohistochemistry identified no difference in glial, microglial, or leucocyte ocular activation between low-dose, high-dose, and vehicle-injected eyes. Moreover, LV-RPE65-injected animals did not show signs of vector shedding or extraocular targeting, confirming the safe ocular restriction of the vector. Our results evidence a limited ocular tolerance to LV-RPE65 after subretinal injection without adjuvant anti-inflammatory prophylaxis, with complications linked to this route of administration necessitating to block this transient inflammatory event.

PEDF plus DHA modulate inflammation and stimulate nerve regeneration after HSV-1 infection.

Herpes simplex virus type-1 (HSV-1) infection leads to impaired corneal sensation and, in severe cases, to corneal ulceration, melting and perforation. Here, we explore the potential therapeutic action of pigment epithelial-derived factor (PEDF) plus docosahexaenoic acid (DHA) on corneal inflammation and nerve regeneration following HSV-1 infection. Rabbits inoculated with 100,000 PFU/eye of HSV-1 strain 17Syn+ were treated with PEDF + DHA or vehicle. PEDF + DHA treatment resulted in a biphasic immune response with stronger infiltration of CD4+T cells, neutrophils and macrophages at 7-days post-treatment (p.t.) that was significantly decreased by 14 days, compared to the vehicle-treated group. Screening of 14 immune-related genes by q-PCR showed that treatment induced higher expression of IFN-γ and CCL20 and inhibition of IL-18 by 7 days in the cornea. PEDF + DHA-treated animals developed less dendritic corneal lesions, opacity and neovascularization. Corneal nerve density increased at 12-weeks p.t. with functional recovery of corneal sensation. Treatment with PEDF + DHA that was postponed by 3 weeks also showed increased nerve density when compared to vehicle. Our data demonstrate that PEDF + DHA promotes resolution of the inflammatory response to the virus and, most importantly, induces regeneration of damaged corneal nerves vital for maintaining ocular surface homeostasis.

Recovery of Corneal Sensitivity and Increase in Nerve Density and Wound Healing in Diabetic Mice After PEDF Plus DHA Treatment.

Diabetic keratopathy decreases corneal sensation and tear secretion and delays wound healing after injury. In the current study, we tested the effect of treatment with pigment epithelium-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) on corneal nerve regeneration in a mouse model of diabetes with or without corneal injury. The study was performed in streptozotocin-induced diabetic mice (C57BL/6). Ten weeks after streptozotocin injection, diabetic mice showed significant decreases of corneal sensitivity, tear production, and epithelial subbasal nerve density when compared with age-matched normal mice. After diabetic mice were wounded in the right eye and treated in both eyes with PEDF+DHA for 2 weeks, there was a significant increase in corneal epithelial nerve regeneration and substance P-positive nerve density in both wounded and unwounded eyes compared with vehicle-treated corneas. There also was elevated corneal sensitivity and tear production in the treated corneas compared with vehicle. In addition, PEDF+DHA accelerated corneal wound healing, selectively recruited type 2 macrophages, and prevented neutrophil infiltration in diabetic wounded corneas. These results suggest that topical treatment with PEDF+DHA promotes corneal nerve regeneration and wound healing in diabetic mice and could potentially be exploited as a therapeutic option for the treatment of diabetic keratopathy.

Pigment epithelium-derived factor attenuates myocardial fibrosis via inhibiting Endothelial-to-Mesenchymal Transition in rats with acute myocardial infarction.

Endothelial mesenchymal transition (EndMT) plays a critical role in the pathogenesis and progression of interstitial and perivascular fibrosis after acute myocardial infarction (AMI). Pigment epithelium-derived factor (PEDF) is shown to be a new therapeutic target owing to its protective role in cardiovascular disease. In this study, we tested the hypothesis that PEDF is an endogenous inhibitor of EndMT and represented a novel mechanism for its protective effects against overactive cardiac fibrosis after AMI. Masson's trichrome (MTC) staining and picrosirius red staining revealed decreased interstitial and perivascular fibrosis in rats overexpressing PEDF. The protective effect of PEDF against EndMT was confirmed by co-labeling of cells with the myofibroblast and endothelial cell markers. In the endothelial cells of microvessels in the ischemic myocardium, the inhibitory effect of PEDF against nuclear translocation of ß-catenin was observed through confocal microscopic imaging. The correlation between antifibrotic effect of PEDF and inactivation of ß-catenin was confirmed by co-transfecting cells with lentivirus carrying PEDF or PEDF RNAi and plasmids harboring ß-catenin siRNA(r) or constitutive activation of mutant ß-catenin. Taken together, these results establish a novel finding that PEDF could inhibit EndMT related cardiac fibrosis after AMI by a mechanism dependent on disruption of ß-catenin activation and translocation.

Span poly-L-arginine nanoparticles are efficient non-viral vectors for PRPF31 gene delivery: An approach of gene therapy to treat retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common cause of inherited blindness in adults. Mutations in the PRPF31 gene produce autosomal dominant RP (adRP). To date there are no effective treatments for this disease. The purpose of this study was to design an efficient non-viral vector for human PRPF31 gene delivery as an approach to treat this form of adRP. Span based nanoparticles were developed to mediate gene transfer in the subretinal space of a mouse model of adRP carrying a point mutation (A216P) in the Prpf31 gene. Funduscopic examination, electroretinogram, optomotor test and optical coherence tomography were conducted to further in vivo evaluate the safety and efficacy of the nanosystems developed. Span-polyarginine (SP-PA) nanoparticles were able to efficiently transfect the GFP and PRPF31 plasmid in mice retinas. Statistically significant improvement in visual acuity and retinal thickness were found in Prpf31A216P/+ mice treated with the SP-PA-PRPF31 nanomedicine.

Stimulation of bone regeneration with pigment epithelium-derived factor microparticles: evidence in silico, in vitro and in vivo.

The occurrence of bone defects can be due to a variety of factors not limited to bone fractures and tumours. Most diseased bone is removed and the patient fitted with prosthetics, prior to use of certain factors such as bone morphogenetic proteins (BMPs) to aid healing. Recently, the protein pigment epithelium-derived factor (PEDF) and the polysaccharide chitosan have been found to have promising effects on the regeneration of bone, with the major advantage of these agents being their safety to date. A study was performed to determine whether the combination of both chitosan and PEDF would enhance greater bone regeneration effects. Post-formulation, in silico tests (particle sizing and surface charge determination) were followed by several cell-based assays (microparticle cellular uptake, cytotoxicity, mitochondrial abundance, bone mineral formation, colony formation in matrigel, and colony formation in collagen I matrix), and finally in vivo testing where microparticles were injected periosteally in the hindlimb. Collectively, these findings support the idea that PEDF microencapsulated within chitosan promotes bone regeneration, and has potential for bone trauma management. Future studies will examine the ability of this promising bone regeneration microparticle to heal bone in disease states such as fracture and tumour-mediated osteolysis.

Modulating of ocular inflammation with macrophage migration inhibitory factor is associated with notch signalling in experimental autoimmune uveitis.

The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno-associated virus (AAV8) (Y733F)-chicken ß-actin (CBA)-MIF or AAV8 (Y733F)-CBA-enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP-injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta-like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF-injected EAU mice. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.

Antiangiogenic and Neurogenic Activities of Sleeping Beauty-Mediated PEDF-Transfected RPE Cells In Vitro and In Vivo.

Pigment epithelium-derived factor (PEDF) is a potent multifunctional protein that inhibits angiogenesis and has neurogenic and neuroprotective properties. Since the wet form of age-related macular degeneration is characterized by choroidal neovascularization (CNV), PEDF would be an ideal candidate to inhibit CNV and support retinal pigment epithelial (RPE) cells. However, its short half-life has precluded its clinical use. To deliver PEDF to the subretinal space, we transfected RPE cells with the PEDF gene using the Sleeping Beauty transposon system. Transfected cells expressed and secreted biologically active recombinant PEDF (rPEDF). In cultures of human umbilical vein endothelial cells, rPEDF reduced VEGF-induced cumulative sprouting by ≥47%, decreased migration by 77%, and increased rate of apoptosis at least 3.4 times. rPEDF induced neurite outgrowth in neuroblastoma cells and protected ganglion and photoreceptor cells in organotypic retinal cultures. In a rat model of CNV, subretinal transplantation of PEDF-transfected cells led to a reduction of the CNV area by 48% 14 days after transplantation and decreased clinical significant lesions by 55% and 40% after 7 and 14 days, respectively. We showed that transplantation of pigment epithelial cells overexpressing PEDF can restore a permissive subretinal environment for RPE and photoreceptor maintenance, while inhibiting choroidal blood vessel growth.

Pigment Epithelium-Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia.

Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor-positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-α and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies.

Norrin protected blood-brain barrier via frizzled-4/ß-catenin pathway after subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: Norrin and its receptor Frizzled-4 have important roles in the blood-brain barrier development. This study is to investigate a potential role and mechanism of Norrin/Frizzled-4 on protecting blood-brain barrier integrity after subarachnoid hemorrhage (SAH). METHODS: One hundred and seventy-eight male Sprague-Dawley rats were used. SAH model was induced by endovascular perforation. Frizzled-4 small interfering RNA was injected intracerebroventricularly 48 hours before SAH. Norrin was administrated intracerebroventricularly 3 hours after SAH. SAH grade, neurological scores, brain water content, Evans blue extravasation, western blots, and immunofluorescence were used to study the mechanisms of Norrin and its receptor regulation protein TSPAN12, as well as neurological outcome. RESULTS: Endogenous Norrin and TSPAN12 expression were increased after SAH, and Norrin was colocalized with astrocytes marker glial fibrillary acidic protein in cortex. Exogenous Norrin treatment significantly alleviated neurobehavioral dysfunction, reduced brain water content and Evans blue extravasation, promoted ß-catenin nuclear translocation, and increased Occludin, VE-Cadherin, and ZO-1 expressions. These effects were abolished by Frizzled-4 small interfering RNA pretreated before SAH. CONCLUSIONS: Norrin protected blood-brain barrier integrity and improved neurological outcome after SAH, and the action of Norrin appeared mediated by Frizzled-4 receptor activation, which promoted ß-catenin nuclear translocation, which then enhanced Occludin, VE-Cadherin, and ZO-1 expression. Norrin might have potential to protect blood-brain barrier after SAH.