Intratumoral Heterogeneity, Chemoresistance and Lymph Node Landing Zone Prognosis in Testicular Tumors Based on Histopathological Characteristics.

BACKGROUND: Existing data on the histopathological correlation of testicular tumors with lymph node prognosis have been poorly explored. We aimed to investigate the relationship of the histopathological properties of testicular tumors with lymph nodes and their involvement with chemoresistance and heterogeneity of testicular tumors. METHODS: Patients with non-seminomatous germ cell tumor (NSGCT) were selected for histopathological correlation of testicular tumor with lymph nodes and its relationship with chemoresistance and heterogeneity. Histopathological and radiological parameters associated with the risk of chemoresistance and tumor progression were measured pre- and post-chemotherapy. Binomial logistic regression and Kaplan-Meier analysis were implemented to determine the predictors of progression and adverse overall patient survival. All categorical variables were analyzed using the Chi-square test, while Pearson's R coefficient determined the correlation. RESULTS: Male patients who were diagnosed with NSGCT from March 2017 to December 2018 at Guwahati Medical College, Guwahati, India, were included in this study. Lymph node groups were predominantly incriminated with the EYST or EYS groups and minimally linked with the pure E and YCS groups. Furthermore, the highest number of lymph node stations was associated with pre-chemotherapy. In salvage chemotherapy in the form of VIP, we found exciting outcomes, as approximately 41% of cases responded positively, especially in the EYS group. CONCLUSION: Our study classifies NSGCT according to the most favorable histopathological grouping and explores the tumoral response in different intrinsic and extrinsic variables. Our analysis can serve as a triumphant histopathological nomogram for a sublime management protocol to deal with the onerous histological pairing in NSGCT.

CRISPR screen identifies GATAD1 as a synthetic lethal target with CDK4/6 inhibitors in estrogen receptor-positive breast cancer.

Estrogen receptor-positive (ER+) breast cancer represents approximately two-thirds of all breast cancers and has a sustained risk of late disease recurrence. Combining cyclin-dependent kinase 4/6 (CDK4/6) inhibitors with anti-estrogen therapies significantly improves ER+ advanced breast cancer clinical outcomes. Despite promising clinical outcomes, intrinsic or acquired resistance to CDK4/6 inhibitors has limited their success. We used CRISPR to screen MCF-7 cells to explore the targets whose inhibition is synthetic lethal with CDK4/6 inhibitors in ER+ breast cancer cells. We found that GATA zinc finger domain containing 1 (GATAD1) is a new synthetic lethal target with CDK4/6 inhibitors in ER+ breast cancer cells. Mechanistically, GATAD1 promotes cell proliferation by transcriptionally inhibiting p21 in ER+ breast cancer cells. GATAD1 depletion decreased the phosphorylation of CDK2/4 and RB transcriptional corepressor 1 (RB1), inducing cell cycle arrest. P21 overexpression abolished the enhanced proliferation induced by GATAD1 overexpression. Our results identify GATAD1 as a therapeutic target in ER+ breast cancer, which is beneficial to provide a novel treatment strategy.

A Multicenter, Phase II Trial of Schedule Modification for Nab-Paclitaxel in Combination with Ramucirumab for Patients with Previously Treated Advanced Gastric or Gastroesophageal Junction Cancer: The B-RAX Trial (JACCRO GC-09).

BACKGROUND: This study investigated whether schedule modification of bi-weekly nanoparticle albumin-bound paclitaxel (nab-PTX) plus ramucirumab (RAM) is efficacious against gastric cancer (GC) or gastroesophageal junction cancer (GJC). PATIENTS AND METHODS: Patients with unresectable GC or GJC who were previously treated with fluoropyrimidine-containing regimens received nab-PTX (100 mg/m2) on days 1, 8, and 15 and RAM (8 mg/kg) on days 1 and 15 of a 28-day cycle. Based on the incidence of severe adverse events (AEs) during the first cycle, patients were modified to bi-weekly therapy from the second cycle. The primary endpoint was progression-free survival (PFS) in the bi-weekly therapy population. Based on the hypothesis that bi-weekly nab-PTX plus RAM would improve PFS from 4.5 to 7.0 months, 40 patients were required for power of 0.8 with a one-sided α of 0.05. RESULTS: Of the 81 patients enrolled, 47 patients (58%) were assigned to bi-weekly therapy. Patient characteristics were Eastern Cooperative Oncology Group performance status of 1 (19%) and diffuse type (45%). Median PFS was 4.7 months (95% confidence interval [CI] 3.7-5.6 months) and overall response rate was 25% (95% CI 11-39%). Severe AEs of grade 3 or worse were mainly neutropenia (83%) and hypertension (23%). EQ-5D scores were maintained during the treatment. In patients who continued standard-schedule therapy, median PFS was 2.7 months (95% CI 1.8-4.0 months). CONCLUSIONS: The primary endpoint for PFS was statistically not met, but modification of nab-PTX plus RAM to a bi-weekly schedule might be a feasible treatment option as second-line treatment for advanced GC/GJC patients, especially elderly patients, with severe AEs during the first cycle.

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

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

Changes in Plasma VEGF and PEDF Levels in Patients with Central Serous Chorioretinopathy.

Background and Objectives: Retinal pigment epitheliopathy and hyperpermeability of choroidal vessels were postulated to be involved in the pathogenesis of central serous chorioretinopathy (CSC). Imbalanced levels of vascular endothelial growth factor (VEGF) and pigment-epithelium-derived factor (PEDF) were previously implicated in the development of chorioretinal diseases characterized by increased vascular permeability. We aimed to compare the plasma levels of proangiogenic VEGF and antiangiogenic PEDF for 26 patients with acute CSC, 26 patients with chronic CSC, and 19 controls. Materials and Methods: VEGF and PEDF levels were measured using a multiplex immunoassay or enzyme-linked immunosorbent assay. Correlations with disease duration were assessed. Results: VEGF levels differed between groups (p = 0.001). They were lower in patients with acute CSC (p = 0.042) and chronic CSC (p = 0.018) than in controls. PEDF levels were similar in all groups. The VEGF-to-PEDF ratio was lower in CSC patients than in controls (p = 0.04). A negative correlation with disease duration was noted only for PEDF levels in the group with chronic CSC (rho = -0.46, p = 0.017). Discussion: Our study confirmed that patients with CSC have imbalanced levels of VEGF and PEDF. This finding may have important implications for the pathogenesis of CSC. VEGF-independent arteriogenesis rather than angiogenesis may underlie vascular abnormalities in these patients.

PEDF relieves kidney injury in type 2 diabetic nephropathy mice by reducing macrophage infiltration.

INTRODUCTION: Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-angiogenic, antioxidant and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its exact role in diabetic kidneys remains unclear. P78-PEDF is an active peptide sequence consisting of 44 amino acids with biological activity similar to that of PEDF. The present study aimed to investigate whether PEDF can alleviate renal damage in type 2 diabetic nephropathy mice by inhibiting macrophage infiltration. MATERIAL AND METHODS: The db/db mice were randomly divided into a diabetes PEDF intervention group (DM-P78-PEDF), a diabetes empty carrier intervention group (DM-Vehicle), and a diabetes mellitus group (DM). Subsequently, they were injected subcutaneously P78-PEDF (0.3 µg/g/d) and PBS for 6 weeks. The ratio of kidney weight to body weight was observed in the mice. An automatic biochemical analyser was used to determine fasting blood glucose (GLU), blood urea nitrogen (UREA), serum creatinine (CREA), and haemoglobin (Hb) content. Histological and ultrastructural pathological changes in the kidneys were examined through H&E and PAS staining. Kidney tissue levels of interleukin-1ß (IL-1ß), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) were determined by ELISA. Expression of the macrophage infiltration and typing as well as that of PEDF, NF-kB, and TLR4 was evaluated in the kidneys. RESULTS: PEDF was located in glomeruli, and the expression of PEDF protein and mRNA in the kidney of diabetic mice declined significantly. Compared with diabetic mice treated with vehicle, continuous infusion of P78-PEDF could reduce blood urea nitrogen, serum creatinine (CREA), renal macrophage recruitment, inflammatory cytokines, and histological changes and restore the expression of TLR4/NF-κB signalling pathway-related factors in diabetic mice. CONCLUSION: These findings highlight the importance of P78-PEDF peptide as a potential treatment in the occurrence and development of diabetic renal injury.

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients >60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

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

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

Comparison of Topical Pigment Epithelium-Derived Factor (PEDF) with Topical Bevacizumab for Accelerating the Regression of Corneal Neovascularization in an Experimental Model of Rabbit Corneal Angiogenesis.

Purpose: To evaluate the anti-angiogenic effect of topical administration of Pigment epithelium-derived factor (PEDF) on the reduction of corneal neovascularization (NV) in comparison to topical Bevacizumab.Methods: 18 eyes of 18 New Zealand rabbits were enrolled. Corneal NV was induced by a 7-0 silk suture. After suture removal, rabbits were randomly divided into three groups. In every group, one eye randomly treated with topical bevacizumab or topical PEDF or saline for 14 days. The area and length of neovascularization were measured by Image J. Histological studies were done in three groups.Results: After 14 days, the mean decrease of corneal NV length was 1.84 ± 0.17 mm (P < .001) in PEDF group and 1.6 ± 0.07 mm (P < .001) in bevacizumab group which was significantly more than the saline group (P = .001 and P < .001, respectively). There was no significant difference between PEDF and bevacizumab group in the reduction of corneal NV length (P = .85). The mean decrease of corneal NV area was 4.94 ± 0.55 mm2 (P < .001) in PEDF group and 4.23 ± 0.29 mm2 in the bevacizumab group (P < .001). PEDF and bevacizumab significantly decreased corneal NV area in comparison to the saline group (p = .017, p = .001, respectively). The mean decrease of corneal NV area did not show a significant difference between PEDF and bevacizumab groups (P = .72).Conclusion: Topical PEDF might be an effective and safe treatment option as bevacizumab in a short-term use, indicating that it is as good as the standard. However, long-term effect is required to be investigated.

Pigment Epithelium-Derived Factor as a Possible Treatment Agent for Choroidal Neovascularization.

Choroidal neovascularization (CNV) is a sight-threatening disease and is characterized by the formation of pathological neovascularization in the choroid which extends into the subretinal space. Exudative age-related macular degeneration (AMD) is the formation of CNV in the macular area which leads to irreversible blindness. Continuous leakage and hemorrhage of the CNV lesion may eventually result in scarring or later fibrosis, which could result in photoreceptor cell atrophy. The current strategy for treating CNV is the use of antivascular endothelial growth factor (VEGF) agents. Many studies have demonstrated the efficacy of intravitreal anti-VEGF therapy. Other studies have also reported the side effects of single anti-VEGF treatment. And long-term inhibition of a single system may result in collateral damage to other visual elements. Pigment epithelium-derived factor (PEDF) is a 50 kDa protein that was first isolated from the conditioned medium of human RPE cells. PEDF has both antiangiogenesis and neuroprotective functions for photoreceptor cells. It may be a potential ocular antiangiogenic agent. This review outlines the distribution of PEDF in the eye, the mechanism of antiangiogenesis, the protective effect on the retina, and the relationship between PEDF and VEGF.

Initial results from a first-in-human gene therapy trial on X-linked retinitis pigmentosa caused by mutations in RPGR.

Retinal gene therapy has shown great promise in treating retinitis pigmentosa (RP), a primary photoreceptor degeneration that leads to severe sight loss in young people. In the present study, we report the first-in-human phase 1/2, dose-escalation clinical trial for X-linked RP caused by mutations in the RP GTPase regulator (RPGR) gene in 18 patients over up to 6 months of follow-up (https://clinicaltrials.gov/: NCT03116113). The primary outcome of the study was safety, and secondary outcomes included visual acuity, microperimetry and central retinal thickness. Apart from steroid-responsive subretinal inflammation in patients at the higher doses, there were no notable safety concerns after subretinal delivery of an adeno-associated viral vector encoding codon-optimized human RPGR (AAV8-coRPGR), meeting the pre-specified primary endpoint. Visual field improvements beginning at 1 month and maintained to the last point of follow-up were observed in six patients.

Pigment Epithelium-Derived Factor Improves Paracellular Blood-Brain Barrier Integrity in the Normal and Ischemic Mouse Brain.

Pigment epithelium-derived factor (PEDF) is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. In the brain, blood-brain barrier (BBB) function is essential for homeostasis. Its impairment plays a crucial role in the pathophysiology of many neurological diseases, including ischemic stroke. We investigated (a) whether PEDF counteracted vascular endothelial growth factor (VEGF)-induced BBB disruption in the mouse brain, (b) the time course and route of BBB permeability and the dynamics of PEDF expression after cerebral ischemia, and (c) whether intraventricular infusion of PEDF ameliorated brain ischemia by reducing BBB impairment. C57Bl6/N mice received intraparenchymal injections of CSF, VEGF, or a combination of VEGF and PEDF. PEDF increased paracellular but not transcellular BBB integrity as indicated by an increase in the tight junction protein claudin-5. In another group of mice undergoing 60-min middle cerebral artery occlusion (MCAO), transcellular BBB permeability (fibrinogen staining in the absence of a loss of claudin-5) increased as early as 6 h after reperfusion. PEDF immunofluorescence increased at 24 h, which paralleled with a decreased paracellular BBB permeability (claudin-5). PEDF after MCAO originated from the blood stream and endogenous pericytes. In the third experiment, the intraventricular infusion of PEDF decreased edema and cell death after MCAO, potentially mediated by the improvement of the paracellular route of BBB permeability (claudin-5) in the absence of an amelioration of Evans Blue extravasation. Together, our data suggest that PEDF improves BBB function after cerebral ischemia by affecting the paracellular but not the transcellular route. However, further quantitative data of the different routes of BBB permeability will be required to validate our findings.

Novel anti-angiogenic PEDF-derived small peptides mitigate choroidal neovascularization.

Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 µg). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 µM, PEDF 336 (100 µM) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

Triple-threat activity of PEDF in bone tumors: Tumor inhibition, tissue preservation and cardioprotection against doxorubicin.

Pigment epithelium-derived factor (PEDF) is known for its osteogenic properties, but its effects against primary and secondary bone tumors have not comprehensively been demonstrated. We show the ubiquitous expression of PEDF in murine embryonic tissue. Continuous administration of PEDF in pregnant mice for five days did not adversely affect foetal health, despite PEDF's known potent antiangiogenic properties. In the case of the devastating childhood bone cancer osteosarcoma, PEDF has direct anticancer activity per se, and protects against the toxicity of doxorubicin in the heart, small intestine and testes. PEDF demonstrated anti-proliferative and pro-apoptotic effects against human prostate and breast cancer cells, tumors which are known to metastasize to bone as the preferred secondary site. Caspase-2 was activated in both tumor cell types by PEDF. In models of prostate and breast cancer in bone, PEDF significantly reduced tumor volumes. When combined with zoledronic acid, continuously-administered PEDF significantly reduced breast tumor volume at the bone, and was able to preserve the quality of bone better than the combination therapy. These multiple positive findings make PEDF an ideal endogenous and safe biological for possible future clinical testing.

Therapeutic Potential of Pigment Epithelium-derived Factor in Cancer.

Pigment epithelium-derived factor (PEDF) is one of the serine protease inhibitors with multifunctional properties, which is produced by various types of organs and tissues. There is an accumulating body of evidence that PEDF plays an important role in the maintenance of tissue homeostasis. Indeed, PEDF not only works as an endogenous inhibitor of angiogenesis, but also suppresses oxidative stress, inflammatory and thrombotic reactions in cell culture systems, animal models, and humans. Furthermore, we, along with others, have found that PEDF inhibits proliferation of, and induces apoptotic cell death in, numerous kinds of tumors. In addition, circulating as well as tumor expression levels of PEDF have been inversely associated with tumor growth and metastasis. These observations suggest that supplementation of PEDF proteins and/or enhancement of endogenous PEDF expression could be a novel therapeutic strategy for the treatment of cancer. Therefore, in this paper, we review the effects of PEDF on diverse types of cancer, and discuss its therapeutic perspectives.

Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2.

Retinal ganglion cells (RGCs) undergo rapid cell death by apoptosis after injury but can be rescued by suppression of caspase-2 (CASP2) using an siRNA to CASP2 (siCASP2). Pigment epithelium-derived factor (PEDF), has neuroprotective and anti-angiogenic functions and protects RGC from death. The purpose of this study was to investigate if suppression of CASP2 is a possible mechanism of neuroprotection by PEDF in RGC. Adult rat retinal cells were treated in vitro with sub-optimal and optimal concentrations of siCASP2 and PEDF and levels of CASP2 mRNA and RGC survival were then quantified. Optic nerve crush (ONC) injury followed by intravitreal injections of siCASP2 or PEDF and eye drops of PEDF-34 were also used to determine CASP2 mRNA and protein reduction. Results showed that PEDF and PEDF-34 significantly suppressed CASP2 mRNA in culture, by 1.85- and 3.04-fold, respectively, and increased RGC survival by 63.2 ± 3.8% and 81.9 ± 6.6%, respectively compared to cells grown in Neurobasal-A alone. RGC survival was significantly reduced in glial proliferation inhibited and purified RGC cultures suggesting that some of the effects of PEDF were glia-mediated. In addition, intravitreal injection of PEDF and eye drops of PEDF-34 after ONC also suppressed CASP2 mRNA levels by 1.82- and 3.89-fold and cleaved caspase-2 (C-CASP2) protein levels by 4.98- and 8.93-fold compared to ONC + PBS vehicle groups, respectively, without affecting other executioner caspases. Treatment of retinal cultures with PEDF and PEDF-34 promoted the secretion of neurotrophic factors (NTF) into the culture media, of which brain-derived neurotrophic factor (BDNF) caused the greatest reduction in CASP2 mRNA and C-CASP2 protein. The neuroprotective effects of PEDF were blocked by a polyclonal antibody and PEDF suppressed key elements in the apoptotic pathway. In conclusion, this study shows that some of the RGC neuroprotective effects of PEDF is regulated through suppression of CASP2 and downstream apoptotic signalling molecules.

Photoreceptor degeneration in microphthalmia (Mitf) mice: partial rescue by pigment epithelium-derived factor.

Dysfunction and loss of the retinal pigment epithelium (RPE) are hallmarks of retinal degeneration, but the underlying pathogenetic processes are only partially understood. Using mice with a null mutation in the transcription factor gene Mitf, in which RPE deficiencies are associated with retinal degeneration, we evaluated the role of trophic factors secreted by the RPE in retinal homeostasis. In such mice, the thickness of the outer nuclear layer (ONL) is as in wild type up to postnatal day 10, but then is progressively reduced, associated with a marked increase in the number of apoptotic cells and a decline in staining for rhodopsin. We show that retinal degeneration and decrease in rhodopsin staining can be prevented partially in three different ways: first, by recombining mutant-derived postnatal retina with postnatal wild-type RPE in tissue explant cultures; second, by adding to cultured mutant retina the trophic factor pigment epithelium-derived factor (PEDF; also known as SERPINF1), which is normally produced in RPE under the control of Mitf; and third, by treating the eyes of Mitf mutant mice in vivo with drops containing a bioactive PEDF 17-mer peptide. This latter treatment also led to marked increases in a number of rod and cone genes. The results indicate that RPE-derived trophic factors, in particular PEDF, are instrumental in retinal homeostasis, and suggest that PEDF or its bioactive fragments may have therapeutic potential in RPE deficiency-associated retinal degeneration.

Pigment epithelium-derived factor: a key mediator in bone homeostasis and potential for bone regenerative therapy.

OBJECTIVES: Pigment epithelium-derived factor (PEDF), a multifunctional endogenous glycoprotein, has a very wide range of biological actions, notably in bone homeostasis. The question has been raised regarding the place of PEDF in the treatment of bone disorders and osteosarcoma, and its potential for tumour growth suppression. METHODS: The PubMed database was used to compile this review. KEY FINDINGS: Pigment epithelium-derived factor's actions in osteoid tissues include promoting mesenchymal stem cell commitment to osteoblasts, increasing matrix mineralisation, and promoting osteoblast proliferation. It shows potential to improve therapeutic outcomes in treatment of multiple cancer types and regrowth of bone after trauma or resection in animal studies. PEDF may possibly have a reduced adverse effect profile compared with current osteo-regenerative treatments; however, there is currently very limited evidence regarding the safety or efficacy in human models. SUMMARY: Pigment epithelium-derived factor is very active within the body, particularly in osseous tissue, and its physiological actions give it potential for treatment of both bone disorders and multiple tumour types. Further research is needed to ascertain the adverse effects and safety profile of PEDF as a therapeutic agent.

PEDF Reduces the Severity of Herpetic Simplex Keratitis in Mice.

Purpose: The purpose of this study was to explore the effects of pigment epithelium derived factor (PEDF) and PEDF-derived peptides Mer44 and Mer34 on the severity of herpetic simplex keratitis (HSK) in mice. Methods: Adult C57BL/6 mice were infected ocularly with the herpes simplex virus type 1 (HSV-1, McKrae strain) and injected subconjunctivally with PEDF, Mer44, or Mer34. Corneal nerve degeneration, neovascularization, sensitivity, neutrophils, macrophages and CD4+ T-cell infiltration, virus contents, and expressions of VEGF, PEDF, and proinflammatory factors were evaluated during acute period. The direct inhibitory effect of PEDF on HSV-1 replication was further evaluated in cultured monkey Vero cells. Results: Following HSV-1 infection, corneal PEDF expression decreased at 3 and 7 days postinfection (dpi) but increased at 15 dpi, and returned to the similar level of normal mice at 45 dpi, which was accompanied with the progress of corneal nerve degeneration and neovascularization. Exogenous PEDF application attenuated corneal nerve degeneration and neovascularization and improved the impaired corneal sensitivity. Moreover, PEDF attenuated the neutrophils, but not macrophage or CD4+ T-cell infiltration, with the reduced expressions of IL-1ß, IL-6, TNF-α, and VEGF. In addition, PEDF inhibited the replication of HSV-1 both in vitro and in mice. Mer44 attenuated corneal nerve degeneration more significantly than Mer34, whereas Mer34 inhibited corneal neovascularization. Conclusions: PEDF and its derived peptides reduce the severity of herpetic simplex keratitis in mice, representing the potential therapeutic approach to control HSK lesions.

Neuralized1a regulates asymmetric division in mouse Lewis lung carcinoma cells.

Asymmetric division (ASD), the unique characteristic of normal stem cells, is regarded as a stemness marker when applied to the study of cancer stem cells (CSCs). However, the role of ASD in the self-renewal of CSCs and its regulation remain largely unknown. Here, we first established a mouse Lewis lung carcinoma CSC cell line that could undergo asymmetric division (LLC-ASD cells) derived from the parental mouse Lewis lung carcinoma cancer cells (LLC-Parental cells). In vitro assessment of stemness by RT-qPCR and western blot analysis of stem cell markers, clonogenic assay (p < 0.001), single cell spheroid formation assay (p < 0.05) and 96-well-plate single-cell cloning assay (p < 0.01) indicated that the LLC-ASD cells exhibited stronger stemness features in comparison to the LLC-Parental cells. In vivo, tumorigenicity of LLC-ASD cells, transplanted subcutaneously to the nude mice, was increased compared to that of LLC-parental cells (p < 0.05). Further, Neuralized1a, a regulator of ASD in normal stem cells, was highly expressed in the LLC-ASD cells. Silencing Neuralized1a expression in LLC-ASD cells by siRNA weakened the stemness features measured by the in vitro assays listed above (p < 0.05). The tumorigenic ability was also decreased in the nude mice upon Neuralized1a silencing (p < 0.05). Collectively, the present study suggests that Neuralized1a regulates the stemness of LLC-ASD cells which could be the new marker and therapeutic target of CSCs.