PEDF peptide plus hyaluronic acid stimulates cartilage regeneration in osteoarthritis via STAT3-mediated chondrogenesis.

Aims: Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA). Methods: Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers. Results: The 29-mer promoted expansion and chondrogenic differentiation of BM-MSCs cultured in different defined media. MIA injection caused chondrocyte death throughout the AC, with cartilage degeneration thereafter. The 29-mer/HA treatment induced extensive chondrocyte regeneration in the damaged AC and suppressed MIA-induced synovitis, accompanied by the recovery of cartilage matrix. Pharmacological inhibitors of PEDF receptor (PEDFR) and signal transducer and activator of transcription 3 (STAT3) signalling substantially blocked the chondrogenic promoting activity of 29-mer on the cultured BM-MSCs and injured AC. Conclusion: The 29-mer/HA formulation effectively induces chondrocyte regeneration and formation of cartilage matrix in the damaged AC.

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.

Potential Benefits of Integrin αvß3 Antagonists in a Mouse Model of Experimental Dry Eye.

PURPOSE: The purpose of this study was to extensively evaluate the efficacy of integrin αvß3 antagonists for the treatment of experimental dry eye (EDE). METHODS: Vitronectin, an αvß3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvß3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction. RESULTS: The inhibitory effects of αvß3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvß3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE. CONCLUSIONS: Topical applications of αvß3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvß3 may be a novel promising strategy to treat patients with dry eye disease.

The Therapeutic Effects of a PEDF-Derived Short Peptide on Murine Experimental Dry Eye Involves Suppression of MMP-9 and Inflammation.

Purpose: To evaluate the efficacy of a pigment epithelium-derived factor (PEDF)-derived short peptide 29-mer, on the treatment and prevention of experimental dry eye (EDE). Methods: C57BL/6 mice were housed in a low humidity controlled environment chamber for 14 days to induce EDE. The 29-mer was administered topically to their eyes, for treatment or dosing, from the point of housing in the controlled environment chamber. The efficacy of the 29-mer on EDE was evaluated in terms of corneal epithelial integrity, tear secretion, and the density of conjunctival goblet cells. PEDF and inflammatory factors, including tumor necrosis factor-α, IL-1ß, IL-6, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase-9, and macrophage infiltration, were examined by real-time polymerase chain reaction, Western blotting, and immunostaining. The involvement of the PEDF receptor/PNPLA2 on the 29-mer effects was evaluated by a specific inhibitor, atglistatin. Rabbit corneal epithelial cells were exposed to hyperosmotic medium to induce inflammatory responses. Results: The levels of PEDF protein increased in the corneal epithelium of EDE, compared with the nonstressed mice. The 29-mer showed a therapeutic effect on EDE and prevented the development of EDE, accompanied by amelioration of the inflammatory factors. The 29-mer effects of inflammatory relief were dramatically reversed by atglistatin. The 29-mer also suppressed the expression of matrix metalloproteinase-9 and proinflammatory cytokines in rabbit corneal epithelial cells induced by hyperosmolarity. Conclusions: Through this animal study, we provide a proof of concept of the anti-inflammatory domain of PEDF having potential to treat dry eye disease. Translational Relevance: This study shows the 29-mer has novel potential as an ophthalmic drop treatment for dry eye disease.

Interleukin-20 is involved in dry eye disease and is a potential therapeutic target.

BACKGROUND: Dry eye disease (DED) is a common disease in ophthalmology, affecting millions of people worldwide. Recent studies have shown that inflammation is the core mechanism of DED. IL-20 is a proinflammatory cytokine involved in various inflammatory diseases. Therefore, we aimed to explore the role of this cytokine in the pathogenesis of DED and evaluate the therapeutic potential of the anti-IL-20 monoclonal antibody (mAb) 7E for DED treatment. METHODS: Clinical tear samples from patients with DED and non-DED controls were collected and their IL-20 protein levels were determined. We established three DED animal models to explore the role of IL-20 and the efficacy of IL-20 antibody in DED. Benzalkonium chloride (BAC)-induced over-evaporative DED, extra-orbital lacrimal gland excision (LGE)-induced aqueous tear-deficient DED, and desiccating stress (DS)-induced combined over-evaporative and aqueous tear-deficient DED animal models were established to investigate the role of IL-20. The anti-IL-20 antibody 7E was established to neutralize IL-20 activity. The effects of IL-20 or 7E on human corneal epithelial cells and macrophages under hyperosmotic stress were analyzed. 7E was topically applied to eyes to evaluate the therapeutic effects in the DED animal models. RESULTS: IL-20 was significantly upregulated in the tears of patients with DED and in the tears and corneas of DED animal models. Under hyperosmotic stress, IL-20 expression was induced via NFAT5 activation in corneal epithelial cells. 7E suppressed hyperosmotic stress-induced activation of macrophages. IL-20 induced cell death in corneal epithelial cells and 7E protected cells from hyperosmotic stress-induced cell death. Blocking IL-20 signaling with 7E protected mice from BAC-induced, LGE-induced, and DS-induced DED by reducing DED symptoms and inhibiting inflammatory responses, macrophage infiltration, apoptosis, and Th17 populations in the conjunctiva and draining lymph nodes. CONCLUSIONS: Our results demonstrated the functions of IL-20 in DED and presented a potential therapeutic option for this condition.

Breast carcinoma-amplified sequence 2 regulates adult neurogenesis via ß-catenin.

BACKGROUND: Breast carcinoma-amplified sequence 2 (BCAS2) regulates ß-catenin gene splicing. The conditional knockout of BCAS2 expression in the forebrain (BCAS2 cKO) of mice confers impaired learning and memory along with decreased ß-catenin expression. Because ß-catenin reportedly regulates adult neurogenesis, we wondered whether BCAS2 could regulate adult neurogenesis via ß-catenin. METHODS: BCAS2-regulating neurogenesis was investigated by characterizing BCAS2 cKO mice. Also, lentivirus-shBCAS2 was intracranially injected into the hippocampus of wild-type mice to knock down BCAS2 expression. We evaluated the rescue effects of BCAS2 cKO by intracranial injection of adeno-associated virus encoding BCAS2 (AAV-DJ8-BCAS2) and AAV-ß-catenin gene therapy. RESULTS: To show that BCAS2-regulating adult neurogenesis via ß-catenin, first, BCAS2 cKO mice showed low SRY-box 2-positive (Sox2+) neural stem cell proliferation and doublecortin-positive (DCX+) immature neurons. Second, stereotaxic intracranial injection of lentivirus-shBCAS2 knocked down BCAS2 in the hippocampus of wild-type mice, and we confirmed the BCAS2 regulation of adult neurogenesis via ß-catenin. Third, AAV-DJ8-BCAS2 gene therapy in BCAS2 cKO mice reversed the low proliferation of Sox2+ neural stem cells and the decreased number of DCX+ immature neurons with increased ß-catenin expression. Moreover, AAV-ß-catenin gene therapy restored neuron stem cell proliferation and immature neuron differentiation, which further supports BCAS2-regulating adult neurogenesis via ß-catenin. In addition, cells targeted by AAV-DJ8 injection into the hippocampus included Sox2 and DCX immature neurons, interneurons, and astrocytes. BCAS2 may regulate adult neurogenesis by targeting Sox2+ and DCX+ immature neurons for autocrine effects and interneurons or astrocytes for paracrine effects. CONCLUSIONS: BCAS2 can regulate adult neurogenesis in mice via ß-catenin.

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.

Integrin αv and Vitronectin Prime Macrophage-Related Inflammation and Contribute the Development of Dry Eye Disease.

Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of dry eye disease (DED) remains unclear. In a murine model of DED, we found increased αv integrin expression in ocular surface macrophages. The αv integrins inhibitor c(RGDfK) ameliorated the corneal damage caused by DED, suggesting a pathogenic role for αv integrin. Because tear hyperosmolarity induces ocular inflammation in DED, a hyperosmolar culture of murine bone marrow-derived macrophages (BMDMs) is used to reproduce inflammation in vitro. However, the expression of proinflammatory cytokine mRNA was minimal, even though αv integrin was induced. In searching for components that are involved in αv integrin-mediated inflammation but that are missing from the culture model, we showed that the levels of vitronectin (VTN), a binding ligand of αv integrins, were increased in the tear fluid and conjunctival stroma of DED animals. The addition of VTN prominently enhanced hyperosmolarity-induced inflammation in BMDMs. Mechanistically, we showed that VTN/αv integrins mediated NF-κB activation to induce inflammatory gene expression in the BMDMs. Our findings indicate that interaction the of VTN with αv integrins is a crucial step in the inflammatory process in DED and suggests a novel therapeutic target.

Autoantibody of NRIP, a novel AChR-interacting protein, plays a detrimental role in myasthenia gravis.

BACKGROUND: Nuclear receptor interaction protein (NRIP) co-localizes with acetylcholine receptor (AChR) at the neuromuscular junction (NMJ), and NRIP deficiency causes aberrant NMJ architecture. However, the normal physiological and pathophysiological roles of NRIP in NMJ are still unclear. METHODS: We investigated the co-localization and interaction of NRIP with AChR-associated proteins using immunofluorescence and immunoprecipitation assay, respectively. The binding affinity of AChR-associated proteins was analysed in muscle-restricted NRIP knockout mice and NRIP knockout muscle cells (C2C12). We further collected the sera from 43 patients with myasthenia gravis (MG), an NMJ disorder. The existence and features of anti-NRIP autoantibody in sera were studied using Western blot and epitope mapping. RESULTS: NRIP co-localized with AChR, rapsyn and α-actinin 2 (ACTN2) in gastrocnemius muscles of mice; and α-bungarotoxin (BTX) pull-down assay revealed NRIP with rapsyn and ACTN2 in complexes from muscle tissues and cells. NRIP directly binds with α subunit of AChR (AChRα) in vitro and in vivo to affect the binding affinity of AChR with rapsyn and rapsyn with ACTN2. In 43 patients with MG (age, 58.4 ± 14.5 years; female, 55.8%), we detected six of them (14.0%) having anti-NRIP autoantibody. The presence of anti-NRIP autoantibody correlated with a more severe type of MG when AChR autoantibody existed (P = 0.011). The higher the titre of anti-NRIP autoantibody, the more severe MG severity (P = 0.032). The main immunogenic region is likely on the IQ motif of NRIP. We also showed the IgG subclass of anti-NRIP autoantibody mainly to be IgG1. CONCLUSIONS: NRIP is a novel AChRα binding protein and involves structural NMJ formation, which acts as a scaffold to stabilize AChR-rapsyn-ACTN2 complexes. Anti-NRIP autoantibody is a novel autoantibody in MG and plays a detrimental role in MG with the coexistence of anti-AChR autoantibody.

Pigment Epithelium-Derived Factor Peptide Promotes Corneal Nerve Regeneration: An In Vivo and In Vitro Study.

Purpose: To investigate the potential of a pigment epithelium-derived factor (PEDF) peptide 44-mer to promote nerve regeneration in a rabbit corneal nerve injury model to demonstrate its neurotrophic ability in cultivated mouse trigeminal neuron cells. Methods: Subconjunctival or intrastromal injection of 44-mer on the cornea was performed in a rabbit model of corneal nerve injury created by corneal epithelial debridement. Immunocytochemical analysis (44-mer, anti-tubulin III, SMI312, CD11b, and α-SMA) and in vivo confocal microscopy were performed. Corneal sensation was estimated using a Cochet-Bonnet corneal esthesiometer. Primary cultivated mouse trigeminal neurons were used to examine the in vitro neurotrophic ability of 44-mer. The cellular morphology and the immunocytochemical staining with anti-tubulin III and SMI312 in different concentrations of 44-mer were compared, and a quantitative assessment of neurite outgrowth was performed. Results: Immunohistochemical staining showed the retention of 44-mer in the corneal stroma for at least 7 days after a single dose of corneal intrastromal injection and promoted corneal nerve regeneration revealed by in vivo confocal microscopy. Corneal esthesiometer demonstrated gradual recovery of the corneal sensation in 44-mer-treated eyes with a lower corneal touch threshold than wounded vehicles and closer to baseline at 3 weeks after corneal injury (P < 0.001). In vitro studies showed a dose-dependent neurotrophic effect of 44-mer in cultivated trigeminal neuron cells. Conclusions: The 44-mer showed in vivo and in vitro corneal neurotrophic abilities. Our results suggest that intrastromal injection of 44-mer into the corneal stroma may have a potential role in treating diseases related to corneal nerve damage.

The Use of Autologous Serum to Reverse Severe Contact Lens-induced Limbal Stem Cell Deficiency.

PURPOSE: To describe the efficacy of autologous serum (AS) eye drops to reverse severe contact lens (CL)-induced limbal stem cell (LSC) deficiency (LSCD). METHODS: This is a prospective, uncontrolled, interventional case series that enrolled 20 eyes of 14 consecutive patients diagnosed with severe CL-induced LSCD at presentation, based on clinical examination, at a tertiary referral center for the period December 2016 to December 2018. All eyes underwent AS treatment for at least 2 weeks with a follow-up for at least 2 months. Demographic data and treatment outcomes were collected and analyzed. RESULTS: The mean patient age at presentation was 30.5 years (range, 19-49 years). The mean duration of soft contact lens wear was 15.6 years (SD, 7.58 years; range, 5-31 years). All study eyes had pain and blurred vision at presentation. All eyes had recurrent or persistent corneal epithelial defect, stromal scarring and opacity, and superficial vascularization and peripheral pannus at presentation. Aggressive treatment with AS succeeded in all eyes. Signs and symptoms of LSCD stabilized in all eyes within 2 weeks and resolved in 6 eyes (30.0%) in 2 weeks, 9 eyes (45.0%) in 4 weeks, and 5 eyes (25.0%) in 8 weeks. The mean follow-up time was 9.45 ± 1.79 weeks (range, 8-24 weeks). CONCLUSIONS: Early identification and aggressive treatment of the ocular surface disease with AS can medically reverse severe CL-induced LSCD and prevent the need for surgical intervention.

Deficiency of nuclear receptor interaction protein leads to cardiomyopathy by disrupting sarcomere structure and mitochondrial respiration.

BACKGROUND: Cardiomyopathy is a common and lethal complication in patients with limb-girdle muscular dystrophy (LGMD), one of the most prevalent forms of muscular dystrophy. The pathogenesis underlying LGMD-related cardiomyopathy remains unclear. NRIP (gene name DCAF6), a Ca2+-dependent calmodulin binding protein, was reduced in dystrophic muscles from LGMD patients. Mice lacking NRIP exhibit a myopathic phenotype resembling that in LGMD patients, making NRIP deficiency a potential culprit leading to cardiomyopathy. This study aimed to determine if NRIP deficiency leads to cardiomyopathy and to explore the underlying molecular mechanisms. METHODS AND RESULTS: NRIP expression was reduced in both human and mouse failing hearts. Muscle-specific NRIP knockout (MCK-Cre::Dcaf6flox/flox) mouse heart and isolated cardiomyocytes exhibited markedly reduced contractility. Transmission electron microscopy revealed abnormal sarcomere structures and mitochondrial morphology in MCK-Cre::Dcaf6flox/flox hearts. Protein co-immunoprecipitation and confocal imaging revealed that NRIP interacts with α-actinin 2 (ACTN2) at the Z-disc. We found that NRIP facilitated ACTN2-mediated F-actin bundling, and that NRIP deficiency resulted in reduced binding between Z-disc proteins ACTN2 and Cap-Z. In addition, NRIP-deficiency led to increased mitochondrial ROS and impaired mitochondrial respiration/ATP production owing to elevated cellular NADH/NAD+ ratios. Treatment with mitochondria-directed antioxidant mitoTEMPO or NAD+ precursor nicotinic acid restored mitochondrial function and cardiac contractility in MCK-Cre::Dcaf6flox/flox mice. CONCLUSIONS: NRIP is essential to maintain sarcomere structure and mitochondrial/contractile function in cardiomyocytes. Our results revealed a novel role for NRIP deficiency in the pathogenesis of LGMD and heart failure. Targeting NRIP, therefore, could be a powerful new approach to treat myocardial dysfunction in LGMD and heart failure patients.

The Psoriasis Therapeutic Potential of a Novel Short Laminin Peptide C16.

Psoriasis is a chronic inflammatory skin disease characterized by excessive growth of keratinocytes and hyperkeratosis in the epidermis. An abnormality of the non-lesional epidermis at an early stage of psoriasis is involved in triggering inflammatory cell infiltration into the dermis. Integrin α5ß1 acts as a receptor for fibronectin and has been found to be overexpressed in non-lesional psoriatic epidermis. To investigate whether α5ß1 integrin has a potential as a drug target for psoriasis treatment, the α5ß1 integrin-binding peptide, C16, was used to obstruct the HaCat keratinocyte cellular responses induced by fibronectin (Fn) in culture and psoriasis-like skin inflammation induced in mice by imiquimod (IMQ). The C16 exhibited antagonistic activity against α5ß1 integrin in HaCat cells, with evidence of suppression of the Fn-mediated proliferative, cytoskeletal, and inflammatory responses. Topical treatment with C16 greatly reduced the IMQ-induced epidermal hyperplasia, infiltration of neutrophils/macrophages, and expression of pro-inflammatory mediators in mouse skin. The C16SP (C16-derived short peptide; DITYVRLKF) also exhibited antagonistic activity, suppressing α5ß1 integrin activity in culture, and reducing IMQ-induced skin inflammation. Taken together, this study provides the first evidence that α5ß1 integrin may be a potential drug target for psoriasis. The synthetic C16 peptide may serve as an agent for psoriasis therapy.

Pigment epithelium-derived factor peptide promotes limbal stem cell proliferation through hedgehog pathway.

Expansion of limbal epithelial stem cells (LSCs) is crucial for the success of limbal transplantation. Previous studies showed that pigment epithelium-derived peptide (PEDF) short peptide 44-mer could effectively expand LSCs and maintain them in a stem-cell state, but the mechanism remained unclear. In the current study, we found that pharmacological inhibition of Sonic Hedgehog (SHh) activity reduced the LSC holoclone number and suppressed LSC proliferation in response to 44-mer. In mice subjected to focal limbal injury, 44-mer facilitated the restoration of the LSC population in damaged limbus, and such effect was impeded by the SHh or ATGL (a PEDF receptor) inhibitor. Furthermore, we showed that 44-mer increased nuclear translocation of Gli1 and Gli3 in LSCs. Knockdown of Gli1 or Gli3 suppressed the ability of 44-mer to induce cyclin D1 expression and LSC proliferation. In addition, ATGL inhibitor suppressed the 44-mer-induced phosphorylation of STAT3 at Tyr705 in LSC. Both inhibitors for ATGL and STAT3 attenuated 44-mer-induced SHh activation and LSC proliferation. In conclusion, our data demonstrate that SHh-Gli pathway driven by ATGL/STAT3 signalling accounts for the 44-mer-mediated LSC proliferation.

Pigment epithelium-derived factor peptide reverses mouse age-related meibomian gland atrophy.

Age-related meibomian gland (MG) atrophy, characterized by decreased meibocyte proliferation, is one of the causes of meibomian gland dysfunction (MGD), which leads to dry eye disease. Currently, there is no available treatment effectively preventing or reversing the decreased cell proliferation and acinar tissue atrophy. In this study, we investigated the therapeutic effects of a pigment epithelium-derived factor (PEDF) peptide in treating this condition. We found abundant expression of PEDF in the nucleus of acinar basal cells, but not in mature meibocytes, and that the expression levels were significantly decreased in the aged mice. We next treated the aged mice (15-month old) with atrophic MGs using a synthetic PEDF-derived peptide 29-mer (residues 93-121). We found that 29-mer effectively stimulated acinar basal cell proliferation and the following mature meibocyte proliferation in the atrophied MGs. In addition, the treatment increased ΔNp63 and Lrig1 expressions in acinar basal cells. Finally, the aged mice receiving the treatment showed MG growth and improved tear film break-up time. In conclusion, the 29-mer treatment is effective in promoting MG acinar basal cell proliferation and enlarging the acinar size of MG, as well as improving MG function in aged mice, suggesting a therapeutic potential of the PEDF-derived short peptide in ameliorating age-related MGD.

PEDF-derived peptide promotes tendon regeneration through its mitogenic effect on tendon stem/progenitor cells.

BACKGROUND: Tendon stem/progenitor cells (TSPC) exhibit a low proliferative response to heal tendon injury, leading to limited regeneration outcomes. Exogenous growth factors that activate TSPC proliferation have emerged as a promising approach for treatment. Here, we evaluated the pigment epithelial-derived factor (PEDF)-derived short peptide (PSP; 29-mer) for treating acute tendon injury and to determine the timing and anatomical features of CD146- and necleostemin-positive TSPC in the tendon healing process. METHODS: Tendon cells were isolated from rabbit Achilles tendons, stimulated by the 29-mer and analyzed for colony-forming capacity. The expression of the TSPC markers CD146, Oct4, and nestin, induced by the 29-mer, was examined by immunostaining and western blotting. Tendo-Achilles injury was induced in rats by full-thickness insertion of an 18-G needle and immediately treated topically with an alginate gel, loaded with 29-mer. The distribution of TSPC in the injured tendon and their proliferation were monitored using immunohistochemistry with antibodies to CD146 and nucleostemin and by BrdU labeling. RESULTS: TSPC markers were enriched among the primary tendon cells when stimulated by the 29-mer. The 29-mer also induced the clonogenicity of CD146+ TSPC, implying TSPC stemness was retained during TSPC expansion in culture. Correspondingly, the expanded TSPC differentiated readily into tenocyte-like cells after removal of the 29-mer from culture. 29-mer/alginate gel treatment caused extensive expansion of CD146+ TSPC in their niche on postoperative day 2, followed by infiltration of CD146+/BrdU- TSPC into the injured tendon on day 7. The nucleostemin+ TSPC were located predominantly in the healing region of the injured tendon in the later phase (day 7) and exhibited proliferative capacity. By 3 weeks, 29-mer-treated tendons showed more organized collagen fiber regeneration and higher tensile strength than control tendons. In culture, the mitogenic effect of the 29-mer was found to be mediated by the phosphorylation of ERK2 and STAT3 in nucleostemin+ TSPC. CONCLUSIONS: The anatomical analysis of TSPC populations in the wound healing process supports the hypothesis that substantial expansion of resident TSPC by exogenous growth factor is beneficial for tendon healing. The study suggests that synthetic 29-mer peptide may be an innovative therapy for acute tendon rupture.

Choroidal metastasis from esophageal squamous cell carcinoma.

We presented a rare case of a sole choroidal metastatic tumor from esophageal squamous cell carcinoma (ESCC) without other organ metastasis in Taiwan. A 43-year-old male with ESCC was referred for a 1-month history of decreased vision in his left eye. A 5.7 mm thick, yellow choroidal tumor occupied posterior pole, featured with pinpoint hyperfluorescence on angiography and subretinal fluid on optical coherence tomography. Positron emission tomography showed a singular hypermetabolic focus in the left eye. The tumor regressed with complete response and the vision preserved after radiation with total 57.60 gray applied by tomotherapy. The gastrointestinal system is the third most common metastatic origin in Taiwan while esophageal cancer metastasizing to choroid is rare. The discrepancy between the high prevalence of primary ESCC and the rareness of choroidal metastasis from ESCC is undetermined.

Muscle-restricted nuclear receptor interaction protein knockout causes motor neuron degeneration through down-regulation of myogenin at the neuromuscular junction.

BACKGROUND: Nuclear receptor interaction protein (NRIP) is a calcium/calmodulin (CaM) binding protein. Nuclear receptor interaction protein interacts with CaM to activate calcineurin and CaMKII signalling. The conventional NRIP knockout mice (global knockout) showed muscular abnormality with reduction of muscle oxidative functions and motor function defects. METHODS: To investigate the role of NRIP on neuromuscular system, we generated muscle-restricted NRIP knockout mice [conditional knockout (cKO)]. The muscle functions (including oxidative muscle markers and muscle strength) and lumbar motor neuron functions [motor neuron number, axon denervation, neuromuscular junction (NMJ)] were tested. The laser-captured microdissection at NMJ of skeletal muscles and adenovirus gene therapy for rescued effects were performed. RESULTS: The cKO mice showed muscular abnormality with reduction of muscle oxidative functions and impaired motor performances as global knockout mice. To our surprise, cKO mice also displayed motor neuron degeneration with abnormal architecture of NMJ. Specifically, the cKO mice revealed reduced motor neuron number with small neuronal size in lumbar spinal cord as well as denervating change, small motor endplates, and decreased myonuclei number at NMJ in skeletal muscles. To explore the mechanisms, we screened various muscle-derived factors and found that myogenin is a potential candidate that myogenin expression was lower in skeletal muscles of cKO mice than wild-type mice. Because NRIP and myogenin were colocalized around acetylcholine receptors at NMJ, we extracted RNA from synaptic and extrasynaptic regions of muscles using laser capture microdissection and showed that myogenin expression was especially lower at synaptic region in cKO than wild-type mice. Notably, overexpression of myogenin using intramuscular adenovirus encoding myogenin treatment rescued abnormal NMJ architecture and preserved motor neuron death in cKO mice. CONCLUSIONS: In summary, we demonstrated that deprivation of NRIP decreases myogenin expression at NMJ, possibly leading to abnormal NMJ formation, denervation of acetylcholine receptor, and subsequent loss of spinal motor neuron. Overexpression of myogenin in cKO mice can partially rescue abnormal NMJ architecture and motor neuron death. Therefore, muscular NRIP is a novel trophic factor supporting spinal motor neuron via stabilization of NMJ by myogenin expression.

Pigment epithelium-derived factor short peptides facilitate full-thickness cutaneous wound healing by promoting epithelial basal cell and hair follicle stem cell proliferation.

A previous study by our group showed that a 44-amino-acid fragment of pigment epithelium-derived factor (PEDF) facilitated corneal epithelial wound healing. In the present study this fragment was shortened to obtain peptides of 18, 20 and 29 amino acids in length, and their promoting effects on the healing of full-thickness skin wounds were assessed. Peptides were delivered periodically by topical application to punch wounds of mice. The wound healing speed was evaluated by measuring the reduction of wound areas at 4 and 7 days after injury. Histological analysis with Masson's trichrome staining was used to confirm epithelialization and dermal collagen deposition. Proliferation of epithelial basal cells was documented by 5-bromo-2'-deoxyuridine incorporation. Hair follicle stem cells were identified by immunostaining for leucine-rich repeat-containing G protein-coupled receptor 6. The results indicated that the 20- and 29-amino-acid short peptides significantly reduced the time required for wound healing compared to the vehicle. Histological analysis confirmed faster epithelial cell coverage of open wounds. Treatment with the PEDF peptide fragments also contributed to granulation, tissue formation by increasing the fibroblast population and enhancing collagen deposition in the dermis. Wounds treated with PEDF peptide fragments contained more basal cells proliferated in the epithelium. Moreover, hair follicle stem cells were also stimulated to proliferate by peptide exposure. In conclusion, the present study reported the identification of two short peptides that can enhance the healing of full-thickness skin wounds following topical application. The underlying mechanisms may involve activation of basal cell proliferation and mobilization of hair follicle stem cells.

Pigment epithelium-derived factor (PEDF) peptide promotes the expansion of hepatic stem/progenitor cells via ERK and STAT3-dependent signaling.

Hepatic stem/progenitor cells (HPC) have been considered as a potential cell source of an alternative to liver transplantation. Production of large numbers of autologous HPC from small pieces of live tissue is crucial for the application of HPC-based liver therapy. In this study, we demonstrated that a synthetic 44-amino acid peptide (44-mer) derived from pigment epithelium-derived factor (PEDF) can facilitate the production of a large number of actively dividing HPC from normal adult rat livers in a 35-day culture period. The phenotypic properties of HPC were characterized by morphological observation, colony formation and high expression of classical HPC markers including epithelial cell adhesion molecule (EpCAM), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and tumor-associated calcium signal transducer (TROP2). The 44-mer stimulated HPC proliferation in vitro and in mouse livers injured by a single intraperitoneal injection of carbon tetrachloride. In addition, the 44-mer induced the phosphorylation of ERK1/2 and STAT3 in HPC. Blocking the activity of ERK or STAT3 with pharmacological inhibitors attenuated the effects of the 44-mer on the induction of HPC proliferation. The long-term expanded HPC still possessed a bipotent ability to differentiate towards bile duct cells and mature hepatocytes. These results imply that the PEDF peptide may be a simple and effective agent to improve HPC-based liver therapy.