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Brain insulin signal anomalies are implicated in Alzheimer's disease (AD) pathology. In this background, metformin, an insulin sensitizer's neuroprotective effectiveness, has been established in the prior findings. In the present investigation, combining an epigenetic modulator, romidepsin, and metformin will improve the gene expressions of neurotrophic factors and reduce AD-associated biochemical and cellular changes by loading them mainly into a nanocarrier surface-modified framework for improved therapeutic effectiveness and bioavailability. In the present investigation, the mediated intra-cerebroventricular streptozocin (3 mg/kg) AD of the model was loaded with metformin and romidepsin into a poloxamer stabilized polymer nanocarrier system. Free combination drug therapy (Romidepsin 25 mg/kg and metformin 5 mg/kg) reduced biochemical and cellular variations over three weeks, respectively, compared to either free treatment (Romidepsin 50 mg/kg and metformin 10 mg/kg). The nanoformulations (Romidepsin 25 mg/kg and Metformin 5 mg/kg), as shown by enhanced significantly reduce stress and high neurotrophic factors, has also exerted superior neurological effectiveness than the free combination of drugs. Eventually, through the Poloxamer stable polymeric nanocarrier framework, the synergistic neuroprotective efficacy of metformin and romidepsin has improved., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acute kidney injury (AKI) is a common clinical condition associated with diverse etiologies and abrupt loss of renal function. In patients with sepsis, rhabdomyolysis, cancer, and cardiovascular disorders, the underlying disease or associated therapeutic interventions can cause hypoxia, cytotoxicity, and inflammatory insults to renal tubular epithelial cells (RTECs), resulting in the onset of AKI. To uncover stress-responsive disease-modifying genes, here we have carried out renal transcriptome profiling in three distinct murine models of AKI. We find that Vgf nerve growth factor inducible gene up-regulation is a common transcriptional stress response in RTECs to ischemia-, cisplatin-, and rhabdomyolysis-associated renal injury. The Vgf gene encodes a secretory peptide precursor protein that has critical neuroendocrine functions; however, its role in the kidneys remains unknown. Our functional studies show that RTEC-specific Vgf gene ablation exacerbates ischemia-, cisplatin-, and rhabdomyolysis-associated AKI in vivo and cisplatin-induced RTEC cell death in vitro Importantly, aggravation of cisplatin-induced renal injury caused by Vgf gene ablation is partly reversed by TLQP-21, a Vgf-derived peptide. Finally, in vitro and in vivo mechanistic studies showed that injury-induced Vgf up-regulation in RTECs is driven by the transcriptional regulator Sox9. These findings reveal a crucial downstream target of the Sox9-directed transcriptional program and identify Vgf as a stress-responsive protective gene in kidney tubular epithelial cells., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Kim et al.)

Aims: Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secretory neurotrophic factor protein that promotes repair after neuronal injury. The microglia cell surface receptor (triggering receptor expressed on myeloid cells-2; TREM2) regulates the production of pro- and antiinflammatory mediators after stroke. Here, we study MANF and TREM2 expression after middle cerebral artery occlusion (MCAo) and explore if docosahexaenoic acid (DHA) treatment exerts a potentiating effect., Methods: We used 2 hours of the MCAo model in rats and intravenously administered DHA or vehicle at 3 hours after the onset of MCAo. Neurobehavioral assessment was performed on days 1, 3, 7, and 14; MANF and TREM2 expression was measured by immunohistochemistry and Western blotting., Results: MANF was upregulated in neurons and astrocytes on days 1, 7, and 14, and TREM2 was expressed on macrophages in the ischemic penumbra and dentate gyrus (DG) on days 7 and 14. DHA improved neurobehavioral recovery, attenuated infarct size on days 7 and 14, increased MANF and decreased TREM2 expression in ischemic core, penumbra, DG, and enhanced neurogenesis on Day 14., Conclusion: MANF and TREM2 protein abundance is robustly increased after MCAo, and DHA treatment potentiated MANF abundance, decreased TREM2 expression, improved neurobehavioral recovery, reduced infarction, and provided enhanced neuroprotection., (© 2020 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

The flower of Hibiscus syriacus Linn is a well-known traditional Chinese medicine (TCM) and health food in China, which has been used to treat dysentery, vaginal discharge, and hemorrhoids. In this study, five polyphenols (compounds 1-5) and five fatty acids (compounds 6-10) were isolated from the ethanol extract of the flower of H. syriacus. The isolated compounds were characterized by spectroscopic techniques. Polyphenols, an important type of natural product, have variety of biological activities. Here, we employed LPS or H 2 O 2 -treated SH-SY5Y cell models to test the neuroprotective effect of compounds 1-10. Results found compounds 1-5 (concentration range was around 20 μM on LPS model, concentration range was around 13 μM on H 2 O 2 model), not compounds 6-10, exhibited neuroprotective effect in LPS or H 2 O 2 -treated SH-SY5Y cell. PCR analysis showed that compounds 1-5 can effectively improve the mRNA expression of synapse-related gene and neurotrophic factors (Syp, NGF and BDNF) in LPS-treated SH-SY5Y cell. In addition, compounds 1-5 decreased the levels of ROS and MDA and increased the activities of SOD, GSH-Px and CAT in LPS-treated SH-SY5Y cell. Furthermore, compounds 1-5 inhibited neuroinflammation (TNF-α, IL-1β and IL-6) in LPS-treated SH-SY5Y cell. In conclusion, the polyphenols in the flower of H. syriacus could be a promising candidate for preventive effect of neuroinflammation., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, and its prognosis is still poor. Mesencephalic astrocyte-derived neurotrophic factor (MANF) plays a key role in endoplasmic reticulum stress. ER stress plays a key role in HCC carcinogenesis. To confirm the clinical and prognostic value of MANF in HCC, we investigated the expression level of MANF in HCC as recorded in databases, and the results were verified by experiment. Survival analysis was probed by the Kaplan-Meier method. Cox regression models were used to ascertain the prognostic value of MANF in HCC tissue microarray. The diagnostic value of MANF in HCC was evaluated by receiver operating characteristic curve analysis. Potential correlation between MANF and selected genes was also analyzed. Results showed that MANF was overexpressed in HCC. Patients with high MANF expression levels had a worse prognosis and higher risk of tumor recurrence. Furthermore, the expression level of MANF had good diagnostic power. Correlation analysis revealed potential regulatory networks of MANF in HCC, laying a foundation for further study of the role of MANF in tumorigenesis. In conclusion, MANF was overexpressed in HCC and related to the occurrence and development of HCC. It is a potential diagnostic and prognostic indicator of HCC., Competing Interests: The authors report no conflicts of interest in this work., (Copyright © 2020 Jingyi He et al.)

Introduction: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells., Objective: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation., Materials and Methods: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors., Results: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample., Conclusions: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.

Peripheral nerve regeneration following injury is often slow and impaired, which results in weakened and denervated muscle with subsequent atrophy. Human Wharton's jelly mesenchymal stem cells (hWJ-MSC) have potential regenerative properties which, however, remain unknown in mouse nerve recovery. This study investigated the effect of the topical application of hWJ-MSC onto repairing transected sciatic nerves in a mouse model. Human adipocyte-derived stem cells (hADSC) were used as a positive control. The sciatic nerve of BALB/c mice was transected at a fixed point and repaired under the microscope using 10-0 sutures. hWJ-MSC and hADSC were applied to the site of repair and mice were followed up for 1 year. The hWJ-MSC group had significantly better functional recovery of five-toe spread and gait angles compared with the negative control and hADSC groups. hWJ-MSC improved sciatic nerve regeneration in a dose-dependent fashion. The hWJ-MSC group had a better quality of regenerated nerve with an increased number of myelinated axons throughout. hWJ-MSC appear to be safe in mice after 1 year of follow-up. hWJ-MSC also expressed higher levels of neurotrophic factor-3, brain-derived neurotrophic factor, and glial-derived neurotrophic factor than hADSC. hWJ-MSC may promote better nerve recovery than hADSC because of this upregulation of neurotrophic factors.

Nerve growth factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro-NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates that pro-NGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF is highly similar, and zebrafish NGF protein has been reported in mature and two-precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF-positive cells. NGF mRNA was visualized by in situ hybridization on whole-mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize pro-NGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF-positive cells, anti-NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double-immunolabeling against NGF/proliferative cell nuclear antigen (PCNA; proliferation marker) and NGF/microtube-associated protein2 (MAP2; neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish, and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF-positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store pro-NGF., (© 2019 Anatomical Society.)

Neural stem/progenitor cells hold valuable potential for the treatment of neurodegenerative disorders. The modulation of intrinsic growth factor expression, such as neurotrophins and their receptors, is a necessary step in achieving neural stem cells (NSCs) therapy. The statins have recently been reported to provide both anti‑inflammatory and neuroprotective effects. In the developing and mature nervous systems, neurotrophic factors are known to impact neuronal growth and survival. In this study, we investigated for a positive effect of lovastatin on the expression of neurotrophins in the neonatal rat hippocampus‑derived NSCs. NSCs were isolated and cultured up to passage three. To confirm cellular identity, immunocytochemical evaluation and flow cytometry analysis were performed using specific antibodies. To determine the optimum concentration of lovastatin, the MTT assay was used. Neurotrophin expression was evaluated using quantitative real‑time reverse transcription‑polymerase chain reaction (RT‑qPCR). Flow cytometry results demonstrated that NSCs were positive for nestin, a marker for neural progenitor cells. An increase in cellular viability was observed with a 24 h exposure of lovastatin. Moreover, results showed an increase in mRNA expression for all neurotrophins compared to the control group. Taken together, the results of this study add to the growing body of literature on the neuroprotective effects of statins in neurological disorders. Lovastatin is a promising therapeutic agent for the treatment of neurodegenerative disorders.

To evaluate the effectiveness of epigallocatechin gallate (EGCG) in inhibiting corneal neovascularization in rat alkaline burn model. Corneal neovascularization model was induced by sodium hydroxide alkaline burn injury in SD rats. Rats were randomly divided into two groups and were given intraperitoneal injection with EGCG or PBS per day for up to 14 days respectively. Corneal inflammation and neovascularization area were assessed on days 3, 7, and 14 after cauterization with digital photographs. Vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) mRNA levels were measured by reverse transcription-polymerase chain reaction (qRT-PCR). The nuclear transfactor-Κb (NF-κB) subunit P65 protein was assayed by immunohistochemistry. The differences of corneal inflammation scores between two groups were significant. The area of CNV between two groups had no significant difference on day 3 but have significant difference on days 7 and 14.The PDEF mRNA expression in EGCG group was significantly higher and the expression of VEGF mRNA was lower than those in PBS group. The results of immunohistochemistry showed from day 7, expression of NF-κB P65protein was suppressed considerably in EGCG group. This study demonstrates that EGCG inhibits corneal neovascularization in a rat model induced by alkali burn.

Background: Spinocerebellar ataxia 17 (SCA17) belongs to the family of neurodegenerative diseases caused by polyglutamine (polyQ) expansion. In SCA17, polyQ expansion occurs in the TATA box binding protein (TBP) and leads to the misfolding of TBP and the preferential degeneration in the cerebellar Purkinje neurons. Currently there is no effective treatment for SCA17. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a recently identified neurotrophic factor, and increasing MANF expression ameliorated SCA17 neuropathology in TBP-105Q knock-in (KI) mouse model, indicating that MANF could be a therapeutic target for treating SCA17., Methods: In this study, we screened a collection of 2000 FDA-approved chemicals using a stable cell line expressing luciferase reporter, which is driven by MANF promoter. We identified several potential candidates that can induce the expression of MANF. Of these inducers, piperine is an agent that potently induces the luciferase expression or MANF expression., Results: Addition of piperine in both cellular and mouse models of SCA17 alleviated toxicity caused by mutant TBP. Although mutant TBP is primarily localized in the nuclei, the polyQ expansion in TBP is able to induce ER stress, suggesting that nuclear misfolded proteins can also elicit ER stress as cytoplasmic misfolded proteins do. Moreover, piperine plays its protective role by reducing toxicity caused by the ER stress., Conclusion: Our study established piperine as a MANF-based therapeutic agent for ER stress-related neuropathology in SCA17.

Purpose: To evaluate the angiogenic properties of corneal derived mesenchymal stromal cells (Co-MSC)., Methods: Co-MSCs were extracted from human cadaver, and wild-type (C57BL/6J) and SERPINF1-/- mice corneas. The MSC secretome was collected in a serum-free medium. Human umbilical vein endothelial cell (HUVEC) tube formation and fibrin gel bead assay (FIBA) sprout formation were used to assess the angiogenic properties of Co-MSC secretome. Complete corneal epithelial debridement was used to induce corneal neovascularization in wild-type mice. Co-MSCs embedded in fibrin gel was applied over the debrided cornea to evaluate the angiogenic effects of Co-MSCs in vivo. Immunoprecipitation was used to remove soluble fms-like tyrosine kinase-1 (sFLT-1) and pigment epithelium-derived factor (PEDF, SERPINF1 gene) from the Co-MSC secretome., Results: Co-MSC secretome significantly inhibited HUVECs tube and sprout formation. Co-MSCs from different donors consistently contained high levels of antiangiogenic factors including sFLT-1 and PEDF; and low levels of the angiogenic factor VEGF-A. In vivo, application of Co-MSCs to mouse corneas after injury prevented the development of corneal neovascularization. Removing PEDF or sFLT-1 from the secretome significantly diminished the antiangiogenic effects of Co-MSCs. Co-MSCs isolated from SERPINF1-/- mice had significantly reduced antiangiogenic effects compared to SERPINF1+/+ (wild-type) Co-MSCs., Conclusions: These results illustrate the direct antiangiogenic properties of Co-MSCs, the importance of sFLT-1 and PEDF, and their potential clinical application for preventing pathologic corneal neovascularization.

Understanding how nerves spontaneously innervate tissues or regenerate small injuries is critical to enhance material-based interventions to regenerate large scale, traumatic injuries. During embryogenesis, neural and vascular tissues form interconnected, complex networks as a result of signaling between these tissue types. Here, we report that human endothelial cells (HUVECs) secrete brain-derived neurotrophic factor (BDNF), which significantly stimulated axonal growth from chicken or rat dorsal root ganglia (DRGs). HUVEC-conditioned medium was sufficient to enhance axonal growth, demonstrating that direct cell-cell contact was not required. When BDNF was neutralized, there was a significant reduction in axonal growth when incubated in HUVEC-conditioned medium and in direct co-culture with HUVECs. These data show that HUVECs secrete neurotrophic factors that significantly enhance axonal growth, and can inform future in vivo studies to direct or pattern the angiogenic response in regenerating tissues to encourage re-innervation.

Parkinson's disease (PD) is a progressive and age-associated neurodegenerative disorder. Patients at different stages of the disease course have distinguished features, mainly in the number of dopaminergic neurons. Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered neurotrophic factor, being deemed as a hopeful candidate for PD treatment. Here, we evaluated the efficacy of CDNF in protecting dopaminergic function using the 6-OHDA-induced PD rat model suffering from different levels of neuronal loss and the recombinant adeno-associated virus 8 (AAV8) as a carrier for the CDNF gene. The results showed that AAV8-CDNF administration significantly improved the motor function and increased the tyrosine hydroxylase (TH) levels in PD rats with mild lesions (2 weeks post lesion), but it had limited therapeutic effects in rats with severe lesions (5 weeks post lesion). To better improve the recovery of motor function in severely lesioned PD rats, we employed a strategy using the CDNF gene along with the aromatic amino acid decarboxylase (AADC) gene. This combination therapeutic strategy indeed showed an enhanced benefit in restoring the motor function of severely lesioned PD rats by providing the neuroprotective effect of CDNF and dopamine enhancing effect of AADC as expected. This study may provide a basis for future clinical application of CDNF in PD patients at different stages and offer a new alternative strategy of joint use of CDNF and AADC for advanced PD patients in clinical trials.

SOX6 plays important roles in cell proliferation, differentiation, and cell fate determination. It has been confirmed that SOX6 is a tumor suppressor and downregulated in various cancers, including esophageal squamous cell carcinoma, hepatocellular carcinoma, and chronic myeloid leukemia. Netrin-1 is highly expressed in various human cancers and acts as an anti-apoptotic and proangiogenic factor to drive tumorigenesis. The role of SOX6 and netrin-1 in regulating the growth of ovarian tumor cells still remains unclear. Real-time polymerase chain reaction and western blot were used to determine the SOX6 messenger RNA and protein levels, respectively, in ovarian cancer cells and tumor tissues. Stable transfection of SOX6 was conducted to overexpress SOX6 in PA-1 and SW626 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Invasion of ovarian cancer cells and migration of human umbilical vein endothelial cells were confirmed by Transwell assays. To overexpress netrin-1, ovarian cancer cells with SOX6 restoration was transduced with netrin-1 lentiviral particles. PA-1 xenografts in a nude mice model were used to conduct in vivo evaluation of the role of SOX6 and its relationship with netrin-1 in tumor growth and angiogenesis. In this study, we found significantly reduced SOX6 levels in PA-1, SW626, SK-OV-3, and CaoV-3 ovarian cancer cell lines and human tumor tissues in comparison with normal human ovarian epithelial cells or matched non-tumor tissues. SOX6 overexpression by stable transfection dramatically inhibited proliferation and invasion of PA-1 and SW626 cells. Also, conditioned medium from PA-1 and SW626 cells with SOX6 restoration exhibited reduced ability to induce human umbilical vein endothelial cells migration and tube formation compared with conditioned medium from the cells with transfection control. Furthermore, an inverse relationship between SOX6 and netrin-1 expression was observed in PA-1 and SW626 cells. Overexpression of netrin-1 in ovarian cancer cells with forced SOX6 expression remarkably abrogated the inhibitory effect of SOX6 on proliferation, invasion of the cells, and tumor xenograft growth and vascularity in vivo. Human umbilical vein endothelial cell migration and tube formation were enhanced in the conditioned medium from the ovarian cancer cells transduced with netrin-1 lentivirus particles. Our observations revealed that SOX6 is a tumor suppressor in ovarian cancer cells, and SOX6 exerts an inhibitory effect on the proliferation, invasion, and tumor cell-induced angiogenesis of ovarian cancer cells, whereas nerin-1 plays an opposite role and its expression is inversely correlated with SOX6. Moreover, our findings suggest a new role of SOX6 and netrin-1 for understanding the progression of ovarian cancer and have the potential for the development of new diagnosis and treatment strategies for ovarian cancer.

Purpose: Netrin-4, a secreted protein, is found in the basement membrane of blood vessels and acts as a key regulator of angiogenesis. Here we investigated the role of Netrin-4 in the mouse-model of suture-induced corneal hem- and lymphangiogenesis., Methods: Corneal hem- and lymphangiogenesis were induced in Netrin-4-deficient (Ntn4-/-) and wild-type (WT) mice by placing three 11-0 nylon sutures intrastromally. Fourteen days after suturing, the vascularized area was analyzed via corneal flat mount immunohistochemistry. Messenger RNA levels for VEGF-A, VEGF-C, Lyve-1, Netrin-4, Unc5H2, "deleted in colon cancer" receptor, and Neogenin in treated and nontreated mouse corneas, cultured human corneal keratocytes (HCK) and epithelial cells (HCEC+HCET) were analyzed by quantitative PCR., Results: In wild-type mice, Netrin-4 mRNA expression in the cornea decreased in growing corneal neovascularization after suturing. Correspondingly, Ntn4-/- mice showed an increased vascularized area compared to that in WT mice. Expression of VEGF-A mRNA was higher in Ntn4-/- versus WT mice. There was no Netrin-4 expression in lymphatic vessels and the area of lymphatic vascularization did not differ between Ntn4-/- and WT mice, nor did expression of VEGF-C and Lyve-1 mRNA. Human corneal epithelial cells showed mainly Netrin-4 mRNA expression, which increased after stimulation, while HCK demonstrated Unc5H2 mRNA expression. Expression of VEGF-A, Netrin-4, Unc5H2, and Neogenin mRNA in HCEC and HCK did not differ significantly between the serum-free condition and VEGF-A or Netrin-4 stimulation., Conclusions: Absence of Netrin-4 increased corneal hemangiogenesis but not lymphangiogenesis in the mouse-model of suture-induced neovascularization. Netrin-4 acted as an antiangiogenic factor in the cornea, with which the healthy cornea is enriched via its expression by corneal epithelial cells.

Background: Inflammation is a major cause of cartilage destruction and leads to the imbalance of metabolic activities in the arthritic joint. Pigment epithelium-derived factor (PEDF) has been reported to have both pro- and anti-inflammatory activities in various cell types and to be upregulated in the arthritic joint, but its role in joint destruction is unclear. Our aim was to investigate the role of PEDF in cartilage degeneration under inflammatory conditions., Methods: PEDF was ectopically expressed in primary human articular chondrocytes, and catabolic gene expression and protein secretion in response to the pro-inflammatory cytokine interleukin 1 beta (IL-1β) were evaluated. Metatarsal bones from PEDF-deficient and wild type mice were cultured in the presence or absence of IL-1β. Cartilage matrix integrity and matrix metalloproteinases MMP-1, MMP-3, and MMP-13 were evaluated. PEDF-deficient and wild type mice were evaluated in the monosodium iodoacetate (MIA) inflammatory joint destruction animal model to determine the role of PEDF in inflammatory arthritis in vivo. Student's t-tests and Mann-Whitney tests were employed where appropriate, for parametric and non-parametric data, respectively., Results: We showed that PEDF protein levels were higher in human osteoarthritis samples compared to normal samples. We demonstrated that ectopic PEDF expression in primary human articular chondrocytes exacerbated catabolic gene expression in the presence of IL-1β. In whole bone organ cultures, IL-1β induced MMP-1, MMP-3 and MMP-13 protein production, and caused significant cartilage matrix loss. Interestingly, Toluidine Blue staining showed that PEDF-deficient bones from 29 week old animals, but not 10 week old animals, had reduced matrix loss in response to IL-1β compared to their wild type counterparts. In addition, PEDF-deficiency in 29 week old animals preserved matrix integrity and protected against cell loss in the MIA joint destruction model in vivo., Conclusion: We conclude that PEDF exacerbates cartilage degeneration in an age-dependent manner under an inflammatory setting. This is the first study identifying a specific role for PEDF in joint inflammation and highlights the multi-faceted activities of PEDF.

Midkine is a multifunctional cytokine and growth factor displaying proinflammatory and pro-tumorigenic activity. Its association with bowel diseases has not been fully elucidated. Our purpose was to delineate midkine expression pattern by RT-qPCR in inflamed/cancerous bowel (n=208) and whole blood (n=150) in colorectal cancer (CRC), Crohn's disease (CD), and ulcerative colitis (UC) and to evaluate midkine dynamics in early postoperative period following colorectal surgery. The expression of midkine was significantly up-regulated in stage III CRC and independently associated with lymph node metastasis. The expression of midkine in whole blood was up-regulated solely in N1 CRC. Midkine expression in cancer-free tissue (CRC) was also elevated and dependent on CRC advancement. In IBD, inflammation increased the bowel expression of midkine solely in UC, in a manner proportional to the disease clinical activity. Large and small bowel differed with respect to the expression of midkine in quiescent tissue (higher in small bowel) and to its correlation pattern with chemokines (in a large bowel) and angiogenic factors and cell cycle regulators (in a small bowel). Circulating midkine and its expression in whole blood dropped directly following colorectal surgery; however, the concentration of midkine in serum was restored on postoperative day three. Midkine is involved in bowel inflammation in UC and lymph node metastasis in CRC, rendering midkine an attractive target for their treatment. Owing to midkine elevation in early postoperative period and its overexpression in tumor-adjacent tissue, targeting midkine might be considered also as a prevention of CRC recurrence following curative tumor resection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Netrin 1 was initially identified as an axon guidance factor, and recent studies indicate that it inhibits chemokine-directed monocyte migration. Despite its importance as a neuroimmune guidance cue, the role of netrin 1 in osteoclasts is largely unknown. Here we detected high netrin 1 levels in the synovial fluid of rheumatoid arthritis patients. Netrin 1 is potently expressed in osteoblasts and synovial fibroblasts, and IL-17 robustly enhances netrin 1 expression in these cells. The binding of netrin 1 to its receptor UNC5b on osteoclasts resulted in activation of SHP1, which inhibited VAV3 phosphorylation and RAC1 activation. This significantly impaired the actin polymerization and fusion, but not the differentiation of osteoclast. Strikingly, netrin 1 treatment prevented bone erosion in an autoimmune arthritis model and age-related bone destruction. Therefore, the netrin 1-UNC5b axis is a novel therapeutic target for bone-destructive diseases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurotrophins are secreted proteins that are synthesized as pre-pro-neurotrophins on the rough endoplasmic reticulum, which are subsequently processed and then secreted as mature proteins. During synthesis, neurotrophins are sorted in the trans-Golgi apparatus into 2 pathways of secretion; the constitutive and the regulated pathways. Neurotrophins in the constitutive pathway are secreted cautiously without any trigger, while in the regulated pathway of secretion an external stimulus elevates the calcium concentration intracellularly leading to neurotrophin release. The regulation of sorting and secretion of neurotrophins is critical for several processes in the body, such as synaptic plasticity, neurodegenerative disorders, demyelination disease, and inflammation. The purpose of this review is to summarize the current mechanisms of neurotrophin sorting and secretion.

In a number of human cancers, NTN1 upregulation inhibits apoptosis induced by its so-called dependence receptors DCC and UNC5H, thus promoting tumor progression. In other cancers however, the selective inhibition of this dependence receptor death pathway relies on the silencing of pro-apoptotic effector proteins. We show here that a substantial fraction of human breast tumors exhibits simultaneous DNA methylation-dependent loss of expression of NTN1 and of DAPK1, a serine threonine kinase known to transduce the netrin-1 dependence receptor pro-apoptotic pathway. The inhibition of DNA methylation by drugs such as decitabine restores the expression of both NTN1 and DAPK1 in netrin-1-low cancer cells. Furthermore, a combination of decitabine with NTN1 silencing strategies or with an anti-netrin-1 neutralizing antibody potentiates tumor cell death and efficiently blocks tumor growth in different animal models. Thus, combining DNA methylation inhibitors with netrin-1 neutralizing agents may be a valuable strategy for combating cancer., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Otosclerosis is a relatively common heterogenous condition, characterized by abnormal bone remodelling in the otic capsule leading to fixation of the stapedial footplate and an associated conductive hearing loss. Although familial linkage and candidate gene association studies have been performed in recent years, little progress has been made in identifying disease-causing genes. Here, we used whole-exome sequencing in four families exhibiting dominantly inherited otosclerosis to identify 23 candidate variants (reduced to 9 after segregation analysis) for further investigation in a secondary cohort of 84 familial cases. Multiple mutations were found in the SERPINF1 (Serpin Peptidase Inhibitor, Clade F) gene which encodes PEDF (pigment epithelium-derived factor), a potent inhibitor of angiogenesis and known regulator of bone density. Six rare heterozygous SERPINF1 variants were found in seven patients in our familial otosclerosis cohort; three are missense mutations predicted to be deleterious to protein function. The other three variants are all located in the 5'-untranslated region (UTR) of an alternative spliced transcript SERPINF1-012 RNA-seq analysis demonstrated that this is the major SERPINF1 transcript in human stapes bone. Analysis of stapes from two patients with the 5'-UTR mutations showed that they had reduced expression of SERPINF1-012 All three 5'-UTR mutations are predicted to occur within transcription factor binding sites and reporter gene assays confirmed that they affect gene expression levels. Furthermore, RT-qPCR analysis of stapes bone cDNA showed that SERPINF1-012 expression is reduced in otosclerosis patients with and without SERPINF1 mutations, suggesting that it may be a common pathogenic pathway in the disease., (© The Author 2016. Published by Oxford University Press.)

Loss of pigment epithelium-derived factor (PEDF, SERPINF1) in cancer cells is associated with poor prognosis and metastasis, but the contribution of stromal PEDF to cancer evolution is poorly understood. Therefore, we investigated the role of fibroblast-derived PEDF in melanoma progression. We demonstrate that normal dermal fibroblasts expressing high PEDF levels attenuated melanoma growth and angiogenesis in vivo, whereas PEDF-depleted fibroblasts exerted tumor-promoting effects. Accordingly, mice with global PEDF knockout were more susceptible to melanoma metastasis. We also demonstrate that normal fibroblasts in close contact with PEDF-null melanoma cells lost PEDF expression and tumor-suppressive properties. Further mechanistic investigations underlying the crosstalk between tumor and stromal cells revealed that melanoma cells produced PDGF-BB and TGFβ, which blocked PEDF production in fibroblasts. Notably, cancer-associated fibroblasts (CAF) isolated from patient-derived tumors expressed markedly low levels of PEDF. Treatment of patient CAF and TGFβ-treated normal fibroblasts with exogenous PEDF decreased the expression of CAF markers and restored PEDF expression. Finally, expression profiling of PEDF-depleted fibroblasts revealed induction of IL8, SERPINB2, hyaluronan synthase-2, and other genes associated with tumor promotion and metastasis. Collectively, our results demonstrate that PEDF maintains tumor-suppressive functions in fibroblasts to prevent CAF conversion and illustrate the mechanisms by which melanoma cells silence stromal PEDF to promote malignancy. Cancer Res; 76(8); 2265-76. ©2016 AACR., (©2016 American Association for Cancer Research.)

RNA binding proteins required for the maintenance of myelin and axoglial junctions are unknown. Herein, we report that deletion of the Quaking (QKI) RNA binding proteins in oligodendrocytes (OLs) using Olig2-Cre results in mice displaying rapid tremors at postnatal day 10, followed by death at postnatal week 3. Extensive CNS hypomyelination was observed as a result of OL differentiation defects during development. The QKI proteins were also required for adult myelin maintenance, because their ablation using PLP-CreERT resulted in hindlimb paralysis with immobility at ∼30 d after 4-hydroxytamoxifen injection. Moreover, deterioration of axoglial junctions of the spinal cord was observed and is consistent with a loss of Neurofascin 155 (Nfasc155) isoform that we confirmed as an alternative splice target of the QKI proteins. Our findings define roles for the QKI RNA binding proteins in myelin development and maintenance, as well as in the generation of Nfasc155 to maintain healthy axoglial junctions., Significance Statement: Neurofascin 155 is responsible for axoglial junction formation and maintenance. Using a genetic mouse model to delete Quaking (QKI) RNA-binding proteins in oligodendrocytes, we identify QKI as the long-sought regulator of Neurofascin alternative splicing, further establishing the role of QKI in oligodendrocyte development and myelination. We establish a new role for QKI in myelin and axoglial junction maintenance using an inducible genetic mouse model that deletes QKI in mature oligodendrocytes. Loss of QKI in adult oligodendrocytes leads to phenotypes reminiscent of the experimental autoimmune encephalomyelitis mouse model with complete hindlimb paralysis and death by 30 d after induction of QKI deletion., (Copyright © 2016 the authors 0270-6474/16/364106-15$15.00/0.)

Gap junctions are present in both vertebrates and invertebrates from nematodes to mammals. Although the importance of gap junctions has been documented in many biological processes, the molecular mechanisms underlying gap junction dynamics remain unclear. Here, using the C. elegans PLM neurons as a model, we show that UNC-44/ankyrin acts upstream of UNC-33/CRMP in regulation of a potential kinesin VAB-8 to control gap junction dynamics, and loss-of-function in the UNC-44/UNC-33/VAB-8 pathway suppresses the turnover of gap junction channels. Therefore, we first show a signal pathway including ankyrin, CRMP, and kinesin in regulating gap junctions.

The therapeutic effects of conventional treatments for advanced colorectal cancer with colorectal peritoneal carcinomatosis (CRPC) and malignant ascites are not very encouraging. Vascular endothelial growth factor-A/vascular permeability factors (VEGF-A/VPF) play key roles in the formation of malignant ascites. In previous work, we demonstrated that pigment epithelium-derived factor (PEDF) antagonized VEGF-A and could repress tumor growth and suppress metastasis in several cancer types. Thus, PEDF may be a therapeutic candidate for treating malignant ascites. Mesenchymal stem cells (MSCs) are promising tools for delivering therapeutic agents in cancer treatment. In the study, MSCs derived from bone marrow were efficiently engineered to secrete human PEDF by adenoviral transduction. Then, intraperitoneal Ad-PEDF-transduced MSCs were analyzed with respect to CRPC and malignant ascites in a CT26 CRPC model. MSCs engineered to secrete PEDF through adenoviral transduction significantly inhibited tumor metastasis and malignant ascites formation in CT26 CRPC mice. Antitumor mechanisms of MSCs-PEDF (MSCs transduced with Ad-PEDF: MOI 500) were associated with inhibiting tumor angiogenesis, inducing apoptosis, and restoring the VEGF-A/sFLT-1 ratio in ascites. Moreover, MSC-mediated Ad-PEDF delivery reduced production of adenovirus-neutralizing antibodies, prolonged PEDF expression, and induced MSCs-PEDF migration toward tumor cells. As a conclusion, MSCs engineered to secrete PEDF by adenoviral transduction may be a therapeutic approach for suppressing tumor metastasis and inhibiting malignant ascites production in CRPC.

Brain-derived neurotrophic factor (BDNF) regulates neural cell survival mainly by activating TrkB receptors. Several lines of evidence support a key role for BDNF-TrkB signaling in survival of adult retinal ganglion cells in animal models of optic nerve injury (ONI), but the neuroprotective effect of exogenous BDNF is transient. Glial cells have recently attracted considerable attention as mediators of neural cell survival, and TrkB expression in retinal glia suggests its role in neuroprotection. To elucidate this point directly, we examined the effect of ONI on TrkB(flox/flox):glial fibrillary acidic protein (GFAP)-Cre+ (TrkB(GFAP)) knockout (KO) mice, in which TrkB is deleted in retinal glial cells. ONI markedly increased mRNA expression levels of basic fibroblast growth factor (bFGF) in wild-type (WT) mice but not in TrkB(GFAP) KO mice. Immunohistochemical analysis at 7 days after ONI (d7) revealed bFGF up-regulation mainly occurred in Müller glia. ONI-induced retinal ganglion cell loss in WT mice was consistently mild compared with TrkB(GFAP) KO mice at d7. On the other hand, ONI severely decreased TrkB expression in both WT and TrkB(GFAP) KO mice after d7, and the severity of retinal degeneration was comparable with TrkB(GFAP) KO mice at d14. Our data provide direct evidence that glial TrkB signaling plays an important role in the early stage of neural protection after traumatic injury., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate-based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG+GFs), compared to SCI animals without biomaterial treatment.

Neurotrophins are important for neuronal health and function. Here, statins, inhibitors of HMG-CoA reductase and cholesterol lowering drugs, were found to stimulate expression of neurotrophins in brain cells independent of the mevalonate pathway. Time-resolved fluorescence resonance energy transfer (FRET) analyses, computer-derived simulation, site-directed mutagenesis, thermal shift assay, and de novo binding followed by electrospray ionization tandem mass spectrometry (ESI-MS) demonstrates that statins serve as ligands of PPARα and that Leu331 and Tyr 334 residues of PPARα are important for statin binding. Upon binding, statins upregulate neurotrophins via PPARα-mediated transcriptional activation of cAMP-response element binding protein (CREB). Accordingly, simvastatin increases CREB and brain-derived neurotrophic factor (BDNF) in the hippocampus of Ppara null mice receiving full-length lentiviral PPARα, but not L331M/Y334D statin-binding domain-mutated lentiviral PPARα. This study identifies statins as ligands of PPARα, describes neurotrophic function of statins via the PPARα-CREB pathway, and analyzes the importance of PPARα in the therapeutic success of simvastatin in an animal model of Alzheimer's disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: There are conflicting data regarding whether netrin-1 retards or accelerates atherosclerosis progression, as it can lead either to monocyte repulsion from or retention within plaques depending on its cellular source. We investigated the effect of aspirin, which is widely used in cardiovascular prophylaxis, on the synthesis of different isoforms of netrin-1 by endothelial cells under pro-inflammatory conditions, and defined the net effect of aspirin-dependent systemic modulation of netrin-1 on atherosclerosis progression., Experimental Approach: Netrin-1 synthesis was studied in vitro using human endothelial cells stimulated with TNF-α, with or without aspirin treatment. In vivo experiments were conducted in ApoE(-/-) mice fed with a high-fat diet (HFD), receiving either aspirin or clopidogrel., Key Results: TNF-α-induced NF-κB activation up-regulated the nuclear isoform of netrin-1, while simultaneously reducing secreted netrin-1. Down-regulation of the secreted isoform compromised the chemorepellent action of the endothelium against monocyte chemotaxis. Aspirin counteracted TNF-α-mediated effects on netrin-1 synthesis by endothelial cells through COX-dependent inhibition of NF-κB and concomitant histone hyperacetylation. Administration of aspirin to ApoE(-/-) mice on HFD increased blood and arterial wall levels of netrin-1 independently of its effects on platelets, accompanied by reduced plaque size and content of monocytes/macrophages, compared with untreated or clopidogrel-treated mice. In vivo blockade of netrin-1 enhanced monocyte plaque infiltration in aspirin-treated ApoE(-/-) mice., Conclusions and Implications: Aspirin counteracts down-regulation of secreted netrin-1 induced by pro-inflammatory stimuli in endothelial cells. The aspirin-dependent increase of netrin-1 in ApoE(-/-) mice exerts anti-atherogenic effects by preventing arterial accumulation of monocytes., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Purpose: To investigate the potential of a pigment epithelial-derived factor (PEDF) peptide 44-mer to promote limbal regeneration in a rabbit partial limbal deficiency model., Methods: Limbal excision (180°) was created surgically, and topical application of 44-mer-containing ointment once a day for 2 weeks was started immediately after injury. Limbal barrier function was inspected at 2 and 6 months after treatment. Corneal neovascularization was observed under slit-lamp microscope. The presence of goblet cells on the corneal surface was examined using impression cytology. The resulting repair tissue was assessed by immunohistochemical staining with antibodies for putative limbal stem cell (LSC) markers ΔNp63α and ABCG2. Cells harvested from the regenerated tissue were analyzed for colony-forming capacity and expression of LSC markers by immunostaining assay and quantitative real-time PCR (qPCR)., Results: Eyes treated with the 44-mer blocked vascularization and goblet cell migration onto the corneal surface. By means of immunohistochemical staining and cell isolation in the repair tissue, we showed that LSCs were widely distributed at the regenerated tissue after 44-mer treatment. The repaired limbus contributed robustly to corneal wound healing as effectively as undamaged limbus., Conclusions: We demonstrated that 44-mer regenerates a functional limbus-like structure on limbal excision wounds. Our finding suggests that the PEDF peptide derivative may be an innovative strategy for tissue engineering and repair therapy in partial LSC deficiency diseases.

Background: Capillary pericytes (cPCs), the mural cells of microvessels, play an important role in the formation and maintenance of microvessels; however, little is known about the mechanisms of how cPCs regulate angiogenesis. To identify factors that modulate cPC function, genes whose levels were altered in cPCs during neovessel formation were identified through a microarray screen., Methods and Results: Ninjurin1 (nerve injury-induced protein, Ninj1) was selected as a candidate factor for angiogenesis regulation. Ninj1 was expressed in capillary cells including endothelial cells (cECs) and was expressed at a higher level in cPCs. Hypoxia induced the gene expression of Ninj1 in addition of vascular endothelial growth factor (VEGF) in cPCs. When cPCs were co-incubated with a thoracic aorta in a three-dimensional Matrigel system, the length of the EC-tubes sprouting from the aorta was increased. Small interfering RNA-mediated downregulation of Ninj1 in cPCs enhanced these cPCs-mediated angiogenic effects, whereas overexpression of Ninj1 attenuated their effects. The production of angiogenic growth factors, such as VEGF and angiopoietin 1, by cPCs was enhanced by the downregulation of Ninj1, and reduced by the overexpression of Ninj1., Conclusions: Ninj1 is a novel regulator for the angiogenic effect of PCs. Specifically, Ninj1 negatively regulates the formation of neovessels, that is, the EC-tube, by reducing the trophic effects of cPCs.

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

Background: The anti-inflammatory effect of the cerebral dopamine neurotrophic factor (CDNF) was shown recently in primary glial cell cultures, yet such effect remains unknown both in vivo and in 6-hydroxydopamine (6-OHDA) models of Parkinson's disease (PD). We addressed this issue by performing an intranigral transfection of the human CDNF (hCDNF) gene in the critical period of inflammation after a single intrastriatal 6-OHDA injection in the rat., Methods: At day 15 after lesion, the plasmids p3xNBRE-hCDNF or p3xNBRE-EGFP, coding for enhanced green florescent protein (EGFP), were transfected into the rat substantia nigra (SN) using neurotensin (NTS)-polyplex. At day 15 post-transfection, we measured nitrite and lipoperoxide levels in the SN. We used ELISA to quantify the levels of TNF-α, IL-1β, IL-6, endogenous rat CDNF (rCDNF) and hCDNF. We also used qRT-PCR to measure rCDNF and hCDNF transcripts, and immunofluorescence assays to evaluate iNOS, CDNF and glial cells (microglia, astrocytes and Neuron/Glial type 2 (NG2) cells). Intact SNs were additional controls., Results: In the SN, 6-OHDA triggered nitrosative stress, increased inflammatory cytokines levels, and activated the multipotent progenitor NG2 cells, which convert into astrocytes to produce rCDNF. In comparison with the hemiparkinsonian rats that were transfected with the EGFP gene or without transfection, 6-OHDA treatment and p3xNBRE-hCDNF transfection increased the conversion of NG2 cells into astrocytes resulting in 4-fold increase in the rCDNF protein levels. The overexpressed CDNF reduced nitrosative stress, glial markers and IL-6 levels in the SN, but not TNF-α and IL-1β levels., Conclusion: Our results show the anti-inflammatory effect of CDNF in a 6-OHDA rat of Parkinson's disease. Our results also suggest the possible participation of TNF-α, IL-1β and IL-6 in rCDNF production by astrocytes, supporting their anti-inflammatory role.

Introduction: Giant cell arteritis (GCA) is characterized by intimal hyperplasia leading to ischaemic manifestations that involve large vessels. Neurotrophins (NTs) and their receptors (NTRs) are protein factors for growth, differentiation and survival of neurons. They are also involved in the migration of vascular smooth muscle cells (VSMCs). Our aim was to investigate whether NTs and NTRs are involved in vascular remodelling of GCA., Methods: We included consecutive patients who underwent a temporal artery biopsy for suspected GCA. We developed an enzymatic digestion method to obtain VSMCs from smooth muscle cells in GCA patients and controls. Neurotrophin protein and gene expression and functional assays were studied from these VSMCs. Neurotrophin expression was also analysed by immunohistochemistry in GCA patients and controls., Results: Whereas temporal arteries of both GCA patients (n = 22) and controls (n = 21) expressed nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB) and sortilin, immunostaining was more intense in GCA patients, especially in the media and intima, while neurotrophin-3 (NT-3) and P75 receptor (P75NTR) were only detected in TA from GCA patients. Expression of TrkB, a BDNF receptor, was higher in GCA patients with ischaemic complications. Serum NGF was significantly higher in GCA patients (n = 28) vs. controls (n = 48), whereas no significant difference was found for BDNF and NT-3. NGF and BDNF enhanced GCA-derived temporal artery VSMC proliferation and BDNF facilitated migration of temporal artery VSMCs in patients with GCA compared to controls., Conclusions: Our results suggest that NTs and NTRs are involved in vascular remodelling of GCA. In GCA-derived temporal artery VSMC, NGF promoted proliferation and BDNF enhanced migration by binding to TrkB and p75NTR receptors. Further experiments are needed on a larger number of VSMC samples to confirm these results.

Purpose: We aimed to elucidate the effects of two epigenetic inhibitors, 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A (TSA), on several key secretory mediators of diabetic retinopathy (DR) in human retinal endothelial cells (HRECs) and human retinal pigment epithelial (HRPE) cells treated with high glucose or interleukin-1β (IL-1β)., Methods: HRECs and HRPE cells were incubated in 30 mM D-glucose or 10 ng/ml IL-1β with or without the presence of various concentrations of 5-aza-dC or TSA. The production of pigment epithelium derived factor (PEDF), vascular endothelial cell growth factor (VEGF), intercellular cell adhesion molecule-1 (ICAM-1), IL-1β, and matrix metalloproteinase 2 (MMP2) was evaluated at the mRNA and protein levels using real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively., Results: In the 30 mM D-glucose and the 10 ng/ml IL-1β condition, the expression of VEGF, ICAM-1, IL-1β, and MMP2 was induced in the HRECs and the HRPE cells. PEDF was downregulated in the HRPE cells but upregulated in the HRECs. However, the PEDF-to-VEGF ratio, which is thought to be critical in DR, was downregulated in both cell types. 5-aza-dC dose-dependently alleviated VEGF, ICAM-1, IL-1β, and MMP2 and reversed PEDF or the PEDF/VEGF ratio in both cell types. TSA had similar effects as 5-aza-dC on the target mediators. However, ICAM-1 production was aggravated in the HRECs while remaining unchanged in the HRPE cells after TSA was administered., Conclusions: Our results demonstrated that 5-aza-dC and TSA enhance the protective PEDF and the PEDF/VEGF ratio and ameliorate the adverse effects of diabetic stimuli in vitro, suggesting that these two drugs may be of potential therapeutic value in DR.

Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, has been shown to have neuroprotective, neurotrophic and anti-inflammatory properties in both animal and cellular models of neurodegenerative disorders. In a previous study of ours, we demonstrated that TSA inhibited the proliferation and increased the differentiation of neuronal precursor cells (NPCs). However, the effects of TSA on Schwann cells (SCs) have not yet been fully elucidated. Thus, in the present study, using SCs derived from adult rat sciatic nerves, we investigated the effects of TSA on the survival, proliferation, migration and myelination of SCs. We found that TSA significantly induced SC death when used at high concentrations. We also observed that TSA promoted the proliferation of SCs in a time-dependent manner. In addition, TSA inhibited the migration of SCs. Moreover, RT-PCR revealed that TSA increased the mRNA expression of several neurotrophic factors and inhibited the expression of genes associated with myelination, including myelin basic protein (MBP) and myelin protein zero (MPZ). Taken together, our results suggest that TSA plays an important role in regulating the growth and biological function of SCs. These data may contribute to our understanding of TSA-based treatment of neurodegenerative diseases.

Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5'-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization.

There is currently no cure for advanced castration-refractory prostate cancer (CRPC) despite the recent approval of several new therapeutic agents. We report here the anti-tumor effect of the angio-inhibitory pigment epithelium-derived factor (PEDF) in the metastatic LNCaP-derivative CRPC CL1 model and explore PEDF anti-neoplasic efficacy in combination with low-dose chemotherapy. Androgen-sensitive LNCaP and CRPC PC3 cell lines were examined as comparison. Using a retroviral expression system, we showed that PEDF limited the proliferation of all prostatic cell lines tested; an effect attributed to interleukin 8 (IL8)-CXCR1/IL8RA inhibition. PEDF also reduced the number and size of 3D tumor spheroids in vitro, but only induced cell differentiation in CRPC spheroids. Similarly, PEDF inhibited the migration of CRPC cells suggesting both anti-proliferative and anti-migratory functions. In vivo, PEDF decreased by 85% and 65% the growth of subcutaneous (s.c.) PC3 and CL1 tumors, respectively. In the CL1 orthotopic model, tumor intake with lethal metastases was found in all animals; nevertheless, PEDF prolonged the median survival of tumor-bearing mice (95% confidence interval: 53 ± 0.001 to 57 ± 1 days). Accordingly, PEDF delayed the emergence of skeletal-related event in intra-tibial xenografts. Next, we evaluated low-dose docetaxel (DTX; 5, 1, 0.5 mg/kg) or cyclophosphamide (CTX; 10-20 mg/kg) on established s.c. PC3 tumors that conditionally express PEDF anti-tumoral epitope/NT3. Although NT3-DTX-5 mg/kg combination was inefficient, NT3-DTX-1 mg/kg and -0.5 mg/kg inhibited by 95% and 87.8%, respectively, tumor growth compared with control and induced tumor stasis. Both NT3-CTX combinations were advantageous. Inversely, PEDF-DTX-5 mg/kg and PEDF-CTX-10 mg/kg delayed the most CL1 tumor growth (15, 11 and 5 days for PEDF-DTX-5 mg/kg, PEDF-CTX-10 mg/kg and single treatments, respectively) with elevated apoptosis and serum thrombospondin-1 as possible mechanism and marker, respectively. As well, both PEDF-CTX-10 mg/kg and PEDF-DTX-5 mg/kg prolonged significantly the survival of tumor-bearing mice compared with single treatments. Metastases were reduced in PEDF-DTX-5 mg/kg compared with other treatments, suggesting that PEDF-DTX delayed metastases formation. Our results advocate that PEDF/low-dose chemotherapy may represent a new therapeutic alternative for CRPC.

Unlabelled: Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis among solid tumors and despite increased knowledge of the molecular mechanisms contributing to progression and metastasis, minimal progress has been done in establishing new targeted therapies for this deadly disease. The expression of the multifunctional growth/differentiation factor midkine (MK) promotes a variety of cellular functions leading to increased angiogenesis, proliferation, migration, and survival. Moreover, MK is intensively discussed as a potential new-therapy target and as biomarker for cancer progression and chemotherapeutic resistance in multiple cancers. Therefore, the present study investigated the molecular role of MK in pancreatic cancer. It was found that MK is elevated in PDAC and differentially expressed in other histologic subtypes of pancreatic cancer, whereas normal pancreatic cells did not express MK, thus making it an attractive candidate for targeted therapies. As a secreted growth/differentiation factor, MK was investigated as a biomarker in clinical serum specimens using ELISA. In addition, knockdown studies of MK revealed a link to proliferation and migration status in vitro. Finally, upstream signaling pathways were analyzed, with TNF-α and EGF being the main inductors of MK expression in PDAC., Implications: This study presents novel MK functions and new upstream signaling effectors that induce its expression to promote PDAC and therefore defines an attractive new therapeutic target in pancreatic cancer., (©2014 AACR.)

This study aims to investigate the expression of neurotrophin-4/5 (NT-4/5) in the pallium and hippocampus in juvenile rats with intraventricular injection of Streptococcus pneumoniae. We used 40 SPF SD rats (3 weeks old, regardless of gender) in this study. We drew 50 μL cerebrospinal fluid, and then, we injected 50 μL normal saline and S. pneumoniae suspension (10(8) CFU/mL) in the brain pool (normal control group and infection group, respectively). After 24 h, the cerebrospinal fluid was collected for bacterial culture and white blood cell count. The immunohistochemical staining was conducted at the day 1, 2, and 5, and the expression of NT-4/5 in rat brain tissue was observed. Compared with the normal control group, NT-4/5 expression in the pallium and hippocampus of rats in the 24-h infection group was significantly increased (both P < 0.05). NT-4/5 expression in the pallium and hippocampus in the 5-day infection group was significantly lower than that in the 24-h infection group (P < 0.05), and they were significantly higher than the normal control group (P < 0.05). After intraventricular injection of S. pneumoniae, the expression of NT-4/5 in the pallium and hippocampus in juvenile rats was increased, especially during early disease course.

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with important roles in regulation of inflammation and angiogenesis. It is produced by various cell types, including endothelial cells (EC). However, the cell autonomous impact of PEDF on EC function needs further investigation. Lung EC prepared from PEDF-deficient (PEDF-/-) mice were more migratory and failed to undergo capillary morphogenesis in Matrigel compared with wild type (PEDF+/+) EC. Although no significant differences were observed in the rates of apoptosis in PEDF-/- EC compared with PEDF+/+ cells under basal or stress conditions, PEDF-/- EC proliferated at a slower rate. PEDF-/- EC also expressed increased levels of proinflammatory markers, including vascular endothelial growth factor, inducible nitric oxide synthase, vascular cell adhesion molecule-1, as well as altered cellular junctional organization, and nuclear localization of β-catenin. The PEDF-/- EC were also more adhesive, expressed decreased levels of thrombospondin-2, tenascin-C, and osteopontin, and increased fibronectin. Furthermore, we showed lungs from PEDF-/- mice exhibited increased expression of macrophage marker F4/80, along with increased thickness of the vascular walls, consistent with a proinflammatory phenotype. Together, our data suggest that the PEDF expression makes significant contribution to modulation of the inflammatory and angiogenic phenotype of the lung endothelium.

Background: Netrin-1 and its receptor UNC5B play important roles in angiogenesis, embryonic development, cancer and inflammation. However, their expression patttern and biological roles in bladder cancer have not been well characterized. The present study aims to investigating the clinical significance of PKC α, netrin-1 and UNC5B in bladder cancer as well as their association with malignant biological behavior of cancer cells., Methods: Netrin-1 and UNC5B expression was examined in 120 bladder cancer specimens using immunohistochemistry and in 40 fresh cancer tissues by western blot. Immunofluorescence was performed in cancer cell lines. PKC α agonist PMA and PKC siRNA was employed in bladder cancer cells. CCK-8, wound healing assays and flow cytometry analysis were used to examine cell proliferation, migration and cell cycle, respectively., Results: Netrin-1 expression was positively correlated with histological grade, T stage, metastasis and poor prognosis in bladder cancer tissues. Immunofluorescence showed elevated netrin-1 and decreased UNC5B expression in bladder cancer cells compared with normal bladder cell line. Furthermore, cell proliferation, migration and cell cycle progression were promoted with PMA treatment while inhibited by calphostin C. In addition, PMA treatment could induce while calphostin C reduce netrin-1 expression in bladder cancer cells., Conclusions: The present study identified netrin-1/UNC5B, which could be regulated by PKC signaling, was important mediators of bladder cancer progression.

Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function.

Purpose: Retinal pigmented epithelium derived from human induced pluripotent stem (iPS) cells (iPS-RPE) may be a source of cells for transplantation. For this reason, it is essential to determine the functional competence of iPS-RPE. One key role of the RPE is uptake and processing of retinoids via the visual cycle. The purpose of this study is to investigate the expression of visual cycle proteins and the functional ability of the visual cycle in iPS-RPE., Methods: iPS-RPE was derived from human iPS cells. Immunocytochemistry, RT-PCR, and Western blot analysis were used to detect expression of RPE genes lecithin-retinol acyl transferase (LRAT), RPE65, cellular retinaldehyde-binding protein (CRALBP), and pigment epithelium-derived factor (PEDF). All-trans retinol was delivered to cultured cells or whole cell homogenate to assess the ability of the iPS-RPE to process retinoids., Results: Cultured iPS-RPE expresses visual cycle genes LRAT, CRALBP, and RPE65. After incubation with all-trans retinol, iPS-RPE synthesized up to 2942 ± 551 pmol/mg protein all-trans retinyl esters. Inhibition of LRAT with N-ethylmaleimide (NEM) prevented retinyl ester synthesis. Significantly, after incubation with all-trans retinol, iPS-RPE released 188 ± 88 pmol/mg protein 11-cis retinaldehyde into the culture media., Conclusions: iPS-RPE develops classic RPE characteristics and maintains expression of visual cycle proteins. The results of this study confirm that iPS-RPE possesses the machinery to process retinoids for support of visual pigment regeneration. Inhibition of all-trans retinyl ester accumulation by NEM confirms LRAT is active in iPS-RPE. Finally, the detection of 11-cis retinaldehyde in the culture medium demonstrates the cells' ability to process retinoids through the visual cycle. This study demonstrates expression of key visual cycle machinery and complete visual cycle activity in iPS-RPE.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are of great interest as a cellular therapeutic agent for regenerative and immunomodulatory purposes. The aim of this study was to investigate whether ADSCs transplantation could promote nerve repair in rats of cerebral ischemia-reperfusion (I/R) injury. We isolated and cultured human ADSCs, and then measured cell surface antigens by flow cytometry and immunofluorescence. Healthy SD rats were randomly divided into sham group, MCAO group, MCAO+vehicle group and MCAO+ADSCs group. Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion (MCAO). Then the human ADSCs were transplanted into the brain of rats 24 h after MCAO. The mRNA level of BDNF (brain derived neurotrophic factor, BDNF), NGF (nerve growth factor, NGF) and bFGF (basic fibroblasts growth factor, bFGF) were detected by real-time PCR at different time points (d7, d14, d21 and d28 after MCAO). Meanwhile, the neurological deficit scores were estimated. The neurological deficit of rats in MCAO+ADSCs group attenuated at d7 in contrast to the MCAO+vehicle group (P<0.05). Subsequently, they were dramatically ameliorated with the time especially at d28. At d7, d14, d21 and d28 after ADSCs transplantation, BDNF, NGF and bFGF mRNA in MCAO+ADSCs group were strikingly higher than those in MCAO+vehicle group, and these two groups both reached the peak at d14. The western blotting results showed that BDNF and Bcl-2 expressed higher in MCAO+ADSCs group than MCAO+vehicle group. Therefore, our current results suggest that ADSCs promote nerve repair after injury through elevating the expression of neurotrophic factors and inhibiting the apoptosis of neural cells.

Neurotrophins are established molecular mediators of neuronal plasticity in the adult brain. We analyzed the impact of aging on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) protein isoforms, their receptors, and on the expression patterns of multiple 5' exon-specific BDNF transcripts in the rat cortex and hippocampus throughout the life span of the rat (6, 12, 18, and 24 months of age). ProNGF was increased during aging in both structures. Mature NGF gradually decreased in the cortex, and, in 24-month-old animals, it was 30% lower than that in adult 6-month-old rats. The BDNF expression did not change during aging, while proBDNF accumulated in the hippocampus of aged rats. Hippocampal total BDNF mRNA was lower in 12-month-old animals, mostly as a result of a decrease of BDNF transcripts 1 and 2. In contrast to the region-specific regulation of specific exon-containing BDNF mRNAs in adult animals, the same BDNF RNA isoforms (containing exons III, IV, or VI) were present in both brain structures of aged animals. Deficits in neurotrophin signaling were supported by the observed decrease in Trk receptor expression which was accompanied by lower levels of the two main downstream effector kinases, pAkt and protein kinase C. The proteolytic processing of p75NTR observed in 12-month-old rats points to an additional regulatory mechanism in early aging. The changes described herein could contribute to reduced brain plasticity underlying the age-dependent decline in cognitive function.

Netrin-1 is a secreted protein that directs long-range axon guidance during early stages of neural circuit formation and continues to be expressed in the mammalian forebrain during the postnatal period of peak synapse formation. Here we demonstrate a synaptogenic function of netrin-1 in rat and mouse cortical neurons and investigate the underlying mechanism. We report that netrin-1 and its receptor DCC are widely expressed by neurons in the developing mammalian cortex during synapse formation and are enriched at synapses in vivo. We detect DCC protein distributed along the axons and dendrites of cultured cortical neurons and provide evidence that newly translated netrin-1 is selectively transported to dendrites. Using gain and loss of function manipulations, we demonstrate that netrin-1 increases the number and strength of excitatory synapses made between developing cortical neurons. We show that netrin-1 increases the complexity of axon and dendrite arbors, thereby increasing the probability of contact. At sites of contact, netrin-1 promotes adhesion, while locally enriching and reorganizing the underlying actin cytoskeleton through Src family kinase signaling and m-Tor-dependent protein translation to locally cluster presynaptic and postsynaptic proteins. Finally, we demonstrate using whole-cell patch-clamp electrophysiology that netrin-1 increases the frequency and amplitude of mEPSCs recorded from cortical pyramidal neurons. These findings identify netrin-1 as a synapse-enriched protein that promotes synaptogenesis between mammalian cortical neurons.

In Parkinson's disease, cell death of dopamine neurons in the substantia nigra progresses and neuroprotective therapy is required to halt neuronal loss. In cellular and animal models, selegiline [(-)deprenyl] and rasagiline, inhibitors of type B monoamine oxidase (MAO)-B, protect neuronal cells from programmed cell death. In this paper, the authors review their recent results on the molecular mechanisms by which MAO inhibitors prevent the cell death through the induction of antiapoptotic, prosurvival genes. MAO-A mediates the induction of antiapoptotic bcl-2 and mao-a itself by rasagiline, whereas a different mechanism is associated with selegiline. Rasagiline and selegiline preferentially increase GDNF and BDNF in nonhuman primates and Parkinsonian patients, respectively. Enhanced neurotrophic factors might be applicable to monitor the neurorescuing activity of neuroprotection.