PEDF and 34-mer peptide inhibit cardiac microvascular endothelial cell ferroptosis via Nrf2/HO-1 signalling in myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) represents a critical pathology in acute myocardial infarction (AMI), which is characterized by high mortality and morbidity. Cardiac microvascular dysfunction contributes to MIRI, potentially culminating in heart failure (HF). Pigment epithelium-derived factor (PEDF), which belongs to the non-inhibitory serpin family, exhibits several physiological effects, including anti-angiogenesis, anti-inflammatory and antioxidant properties. Our study aims to explore the impact of PEDF and its functional peptide 34-mer on both cardiac microvascular perfusion in MIRI rats and human cardiac microvascular endothelial cells (HCMECs) injury under hypoxia reoxygenation (HR). It has been shown that MIRI is accompanied by ferroptosis in HCMECs. Furthermore, we investigated the effect of PEDF and its 34-mer, particularly regarding the Nrf2/HO-1 signalling pathway. Our results demonstrated that PEDF 34-mer significantly ameliorated cardiac microvascular dysfunction following MIRI. Additionally, they exhibited a notable suppression of ferroptosis in HCMECs, and these effects were mediated through activation of Nrf2/HO-1 signalling. These findings highlight the therapeutic potential of PEDF and 34-mer in alleviating microvascular dysfunction and MIRI. By enhancing cardiac microvascular perfusion and mitigating endothelial ferroptosis, PEDF and its derivative peptide represent promising candidates for the treatment of AMI.

Roles of pigment epithelium-derived factor in exercise-induced suppression of senescence and its impact on lung pathology in mice.

Senescent cells contribute to tissue aging and underlie the pathology of chronic diseases. The benefits of eliminating senescent cells have been demonstrated in several disease models, and the efficacy of senolytic drugs is currently being tested in humans. Exercise training has been shown to reduce cellular senescence in several tissues; however, the mechanisms responsible remain unclear. We found that myocyte-derived factors significantly extended the replicative lifespan of fibroblasts, suggesting that myokines mediate the anti-senescence effects of exercise. A number of proteins within myocyte-derived factors were identified by mass spectrometry. Among these, pigment epithelium-derived factor (PEDF) exerted inhibitory effects on cellular senescence. Eight weeks of voluntary running increased Pedf levels in skeletal muscles and suppressed senescence markers in the lungs. The administration of PEDF reduced senescence markers in multiple tissues and attenuated the decline in respiratory function in the pulmonary emphysema mouse model. We also showed that blood levels of PEDF inversely correlated with the severity of COPD in patients. Collectively, these results strongly suggest that PEDF contributes to the beneficial effects of exercise, potentially suppressing cellular senescence and its associated pathologies.

Pigment epithelium-derived factor expression and role in follicular development.

RESEARCH QUESTION: What is the involvement of pigment epithelium-derived factor (PEDF), expressed in granulosa cells, in folliculogenesis? DESIGN: mRNA expression of PEDF and other key factors [Cyp19, anti-Müllerian hormone receptor (AMHR) and vascular endothelial growth factor (VEGF)] in mice follicles was examined in order to typify the expression of PEDF in growing follicles and in human primary granulosa cells (hpGC), and to follow the interplay between PEDF and the other main players in folliculogenesis: FSH and AMH. RESULTS: mRNA expression of PEDF increased through folliculogenesis, although the pattern differed from that of the other examined genes, affecting the follicular angiogenic and oxidative balance. In hpGC, prolonged exposure to FSH stimulated the up-regulation of PEDF mRNA. Furthermore, a negative correlation between AMH and PEDF was observed: AMH stimulation reduced the expression of PEDF mRNA and PEDF stimulation reduced the expression of AMHR mRNA. CONCLUSIONS: Folliculogenesis, an intricate process that requires close dialogue between the oocyte and its supporting granulosa cells, is mediated by various endocrine and paracrine factors. The current findings suggest that PEDF, expressed in granulosa cells, is a pro-folliculogenesis player that interacts with FSH and AMH in the process of follicular growth. However, the mechanism of this process is yet to be determined.

Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor in diabetic and non-diabetic retinopathy: associated factors and anatomical correlation.

BACKGROUND: This study aims to investigate the factors affecting the vitreous levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VGEF) among patients with pars plana vitrectomy (PPV). Also, this study correlates the levels of PEDF with RRD characteristics. METHODS: All patients who were scheduled for PPV for any indication were included in the study. They were divided into a case group which included patients with advanced PDR and a control group which included the remaining diagnoses. During the PPV, an undiluted vitreous sample was taken and the enzyme-linked immunosorbent assay method was utilized to measure the levels of VEGF and PEDF. RESULTS: Eighty eyes were involved. Patients diagnosed with advanced PDR and endophthalmitis exhibited higher levels of VEGF. PEDF was affected inversely by the age of the patients and PEDF levels were higher in RRD and endophthalmitis cases. In patients with RRD, the level of PEDF was higher if the tear was found inferiorly, if the macula was detached, and with a longer duration of RRD. CONCLUSIONS: This study highlights the clinical importance of those biomarkers. Anti-VEGF-based treatment is the mainstay against PDR. PEDF may show a promising predictive values regarding patients with RRD.

Synergy between PEDF and Doxorubicin in Breast Cancer Cells: Effects on Metastatic and Metabolic Pathways.

Pigment epithelium-derived factor (PEDF), a serine protease inhibitor (Serpin) family member, shows promise in inhibiting tumour growth. In our study, we explored the effects of PEDF on the efficacy of the frontline chemotherapy agent doxorubicin (Dox) in BC cells. We found that Dox+PEDF treatment significantly reduced glucose uptake in MDA-MB-231 cells compared to the control (p = 0.0005), PEDF (p = 0.0137), and Dox (p = 0.0171) alone but paradoxically increased it in MCF-7 cells. Our findings further revealed that PEDF, Dox, and Dox+PEDF substantially hindered tumour cell migration from tumour spheroids, with Dox+PEDF showing the most significant impact (p < 0.0001). We also observed notable decreases in the expression of metastatic markers (uPAR, uPA, CXCR4, MT1-MMP, TNF-α) across all treatment groups (p < 0.0001) in both cell lines. When it comes to metabolic pathways, PEDF increased phosphorylated IRS-1 (p-IRS1) levels in MDA-MB-231 and MCF-7 (p < 0.0001), while Dox decreased it, and the combination led to an increase. In MDA-MB-231 cells, treatment with PEDF, Dox, and the combination led to a notable decrease in both phosphorylated AKT (p-AKT) and total AKT levels. In MCF-7, while PEDF, Dox, and their combination led to a reduction in p-AKT, total levels of AKT increased in the presence of Dox and Dox+PEDF. Combining PEDF with Dox enhances the targeting of metastatic and metabolic pathways in breast cancer cell lines. This synergy, marked by PEDF's increasing roles in cancer control, may pave the way for more effective cancer treatments.

PEDF-derived peptide protects against Amyloid-ß toxicity in vitro and prevents retinal dysfunction in rats.

Amyloid-beta (Aß), a family of aggregation-prone and neurotoxic peptides, has been implicated in the pathophysiology of age-related macular degeneration (AMD). We have previously shown that oligomeric and fibrillar species of Aß42 exerted retinal toxicity in rats, but while the consequences of exposure to amyloid were related to intracellular effects, the mechanism of Aß42 internalization in the retina is not well characterized. In the brain, the 67 kDa laminin receptor (67LR) participates in Aß-related neuronal cell death. A short peptide derived from pigment epithelium-derived factor (PEDF), formerly designated PEDF-335, was found to mitigate experimental models of ischemic retinopathy via targeting of 67LR. In the present study, we hypothesized that 67LR mediates the uptake of pathogenic Aß42 assemblies in the retina, and that targeting of this receptor by PEDF-335 may limit the internalization of Aß, thereby ameliorating its retinotoxicity. To test this assumption ARPE-19 cells in culture were incubated with PEDF-335 before treatment with fibrillar or oligomeric structures of Aß42. Immunostaining confirmed that PEDF-335 treatment substantially prevented amyloid internalization into ARPE-19 cells and maintained their viability in the presence of toxic oligomeric and fibrillar Aß42 entities in vitro. FRET competition assay was performed and confirmed the binding of PEDF-335 to 67LR in RPE-like cells. Wild-type rats were treated with intravitreal PEDF-335 in the experimental eye 2 days prior to administration of retinotoxic Aß42 oligomers or fibrils to both eyes. Retinal function was assessed by electroretinography through 6 weeks post injection. The ERG responses in rats treated with oligomeric or fibrillar Aß42 assemblies were near-normal in eyes previously treated with intravitreal PEDF-335, whereas those measured in the control eyes treated with injection of the Aß42 assemblies alone showed pathologic attenuation of the retinal function through 6 weeks. The retinal presence of 67LR was determined ex vivo by immunostaining and western blotting. Retinal staining demonstrated the constitutional expression of 67LR mainly in the retinal nuclear layers. In the presence of Aß42, the levels of 67LR were increased, although its retinal distribution remained largely unaltered. In contrast, no apparent differences in the retinal expression level of 67LR were noted following exposure to PEDF-335 alone, and its pattern of localization in the retina remained similarly concentrated primarily in the inner and outer nuclear layers. In summary, we found that PEDF-335 confers protection against Aß42-mediated retinal toxicity, with significant effects noted in cells as well as in vivo in rats. The effects of PEDF-335 in the retina are potentially mediated via binding to 67LR and by at least partial inhibition of Aß42 internalization. These results suggest that PEDF-335 may merit further consideration in the development of targeted inhibition of amyloid-related toxicity in the retina. More broadly, our observations provide evidence on the importance of extracellular versus intracellular Aß42 in the retina and suggest concepts on the molecular mechanism of Aß retinal pathogenicity.

The Various Roles of PEDF in Cancer.

Pigment epithelium-derived factor (PEDF) is a natural immunomodulator, anti-inflammatory, anti-angiogenic, anti-tumour growth and anti-metastasis factor, which can enhance tumour response to PEDF but can also conversely have pro-cancerous effects. Inflammation is a major cause of cancer, and it has been proven that PEDF has anti-inflammatory properties. PEDF's functional activity can be investigated through measuring metastatic and metabolic biomarkers that will be discussed in this review.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Gut-liver axis calibrates intestinal stem cell fitness.

The gut and liver are recognized to mutually communicate through the biliary tract, portal vein, and systemic circulation. However, it remains unclear how this gut-liver axis regulates intestinal physiology. Through hepatectomy and transcriptomic and proteomic profiling, we identified pigment epithelium-derived factor (PEDF), a liver-derived soluble Wnt inhibitor, which restrains intestinal stem cell (ISC) hyperproliferation to maintain gut homeostasis by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, we found that microbial danger signals resulting from intestinal inflammation can be sensed by the liver, leading to the repression of PEDF production through peroxisome proliferator-activated receptor-α (PPARα). This repression liberates ISC proliferation to accelerate tissue repair in the gut. Additionally, treating mice with fenofibrate, a clinical PPARα agonist used for hypolipidemia, enhances colitis susceptibility due to PEDF activity. Therefore, we have identified a distinct role for PEDF in calibrating ISC expansion for intestinal homeostasis through reciprocal interactions between the gut and liver.

Balance of Gata3 and Ramp2 in hepatocytes regulates hepatic vascular reconstitution in postoperative liver regeneration.

BACKGROUND & AIMS: Post-hepatectomy liver failure (PHLF) leads to poor prognosis in patients undergoing hepatectomy, with hepatic vascular reconstitution playing a critical role. However, the regulators of hepatic vascular reconstitution remain unclear. In this study, we aimed to investigate the regulatory mechanisms of hepatic vascular reconstitution and identify biomarkers predicting PHLF in patients undergoing hepatectomy. METHODS: Candidate genes that were associated with hepatic vascular reconstitution were screened using adeno-associated virus vectors in Alb-Cre-CRISPR/Cas9 mice subjected to partial hepatectomy. The biological activities of candidate genes were estimated using endothelial precursor transfusion and associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) models. The level of candidates was detected in biopsies from patients undergoing ALPPS. Risk factors for PHLF were also screened using retrospective data. RESULTS: Downregulation of Gata3 and upregulation of Ramp2 in hepatocytes promoted the proliferation of liver sinusoidal endothelial cells and hepatic revascularization. Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor A (VEGFA) played opposite roles in regulating the migration of endothelial precursors from bone marrow and the formation of new sinusoids after hepatectomy. Gata3 restricted endothelial cell function in patient-derived hepatic organoids, which was abrogated by a Gata3 inhibitor. Moreover, overexpression of Gata3 led to higher mortality in ALPPS mice, which was improved by a PEDF-neutralizing antibody. The expression of Gata3/RAMP and PEDF/VEGFA tended to have a negative correlation in patients undergoing ALPPS. A nomogram incorporating multiple factors, such as serum PEDF/VEGF index, was constructed and could efficiently predict the risk of PHLF. CONCLUSIONS: The balance of Gata3 and Ramp2 in hepatocytes regulates the proliferation of liver sinusoidal endothelial cells and hepatic revascularization via changes in the expression of PEDF and VEGFA, revealing potential targets for the prevention and treatment of PHLF. IMPACT AND IMPLICATIONS: In this study, we show that the balance of Gata3 and Ramp2 in hepatocytes regulates hepatic vascular reconstitution by promoting a shift from pigment epithelium-derived factor (PEDF) to vascular endothelial growth factor A (VEGFA) expression during hepatectomy- or ALLPS (associating liver partition and portal vein ligation for staged hepatectomy)-induced liver regeneration. We also identified serum PEDF/VEGFA index as a potential predictor of post-hepatectomy liver failure in patients who underwent hepatectomy. This study improves our understanding of how hepatocytes contribute to liver regeneration and provides new targets for the prevention and treatment of post-hepatectomy liver failure.

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

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

A PEDF peptide mimetic effectively relieves dry eye in a diabetic murine model by restoring corneal nerve, barrier, and lacrimal gland function.

PURPOSE: The study investigated effectiveness of a novel PEDF peptide mimetic to alleviate dry eye-like pathologies in a Type I diabetic mouse model established using streptozotocin. METHODS: Mice were treated topically for 3-6 weeks with Ppx (a 17-mer PEDF mimetic) 2x/day or vehicle. Corneal sensitivity, tear film, epithelial and endothelial injury were measured using Cochet-Bonnet esthesiometer, phenol red cotton thread wetting, fluorescein sodium staining, and ZO1 expression, respectively. Inflammatory and parasympathetic nerve markers and activation of the MAPK/JNK pathways in the lacrimal glands were measured. RESULTS: Diabetic mice exhibited features of dry eye including reduced corneal sensation and tear secretion and increased corneal epithelium injury, nerve degeneration, and edema. Ppx reversed these pathologies and restored ZO1 expression and morphological integrity of the endothelium. Upregulation of IL-1ß and TNFα, increased activation of P-38, JNK, and ERK, and higher levels of M3ACHR in diabetic lacrimal glands were also reversed by the peptide treatment. CONCLUSION: The study demonstrates that topical application of a synthetic PEDF mimetic effectively alleviates diabetes-induced dry eye by restoring corneal sensitivity, tear secretion, and endothelial barrier and lacrimal gland function. These findings have significant implications for the potential treatment of dry eye using a cost-effective and reproducible approach with minimal invasiveness and no obvious side effects.

Knockdown of DAPK1 inhibits IL-1ß-induced inflammation and cartilage degradation in human chondrocytes by modulating the PEDF-mediated NF-κB and NLRP3 inflammasome pathway.

Osteoarthritis (OA) is a common joint disease that is characterized by inflammation and cartilage degradation. Death-associated protein kinase 1 (DAPK1) is a multi-domain serine/threonine kinase and has been reported to be involved in the progression of OA. However, its role and mechanism in OA remain unclear. Here, we found the expression of DAPK1 in OA cartilage tissues was higher than that in normal cartilage tissues. The expression of DAPK1 in chondrocytes was up-regulated by IL-1ß. Knockdown of DAPK1 promoted cell viability and anti-apoptotic protein expression, while it inhibited the apoptosis rate and pro-apoptotic protein expressions in IL-1ß-induced chondrocytes. In addition, DAPK1 inhibition reduced the levels of inflammatory cytokines and expressions of matrix metalloproteinases (MMPs), and increased the expressions of collagen II and aggrecan. The data of mechanistic investigation indicated that the expression of pigment epithelium-derived factor (PEDF) was positively regulated by DAPK1. Overexpression of PEDF attenuated the effects of DAPK1 knockdown on IL-1ß-induced cell viability, apoptosis, inflammation, and cartilage degradation. Furthermore, PEDF overexpression restored the activity of the NF-κB pathway and NLRP3 inflammasome after DAPK1 knockdown. Collectively, down-regulation of DAPK1 inhibited IL-1ß-induced inflammation and cartilage degradation via the PEDF-mediated NF-κB and NLRP3 inflammasome pathways.

PEDF Prevents Mitochondrial Function Decay and ER Stress Induced by Rotenone in Aging RPE Cells.

BACKGROUND: Neurodegenerative diseases, including age-related macular degeneration (AMD), may be linked to mitochondrial dysfunction and endoplasmic reticulum (ER) stress. We examined whether Pigment epithelium-derived factor (PEDF) could prevent changes in the structure and function of these organelles by accelerating by rotenone (ROT), a mitochondrial inhibitor, in human retinal pigment epithelium (RPE) cells of chronological age. METHODS: RPE cells from 9-20, 50-55, 60-70, and >70-year-old donors were isolated, grown as primary cultures, harvested, and treated with ROT and PEDF for electron microscope (EM), western blot analysis, and polymerase chain reaction (PCR). Reactive oxygen species (ROS) and cytoplasmic calcium [Ca2+]c and mitochondrial calcium [Ca2+]m levels were measured by flow cytometry using 2',7'-dichlorodihydrofluorescin diacetate (H2-DCF-DA), fluo-3/AM, and Rhod-2/AM, and ATP levels were measured using a luciferin/luciferase-based assay. Mitochondrial membrane potential (ΔΨm) was detected using 5,5',6,6'-tetrachloro1,1',3,3'-tetraethylbenzimid azolocarbocyanine iodide (JC-1), and susceptibility of the cells to ROT toxicity and PEDF-protective effect was determined by propidium iodide (PI) staining and lactate dehydrogenase (LDH) assay. The expression of ER stress-related genes was detected using real-time (RT)-PCR. RESULTS: We observed decay in the mitochondria of aged RPE cells, including matrix abnormalities, elongation, loss of cristae, and disruption of membrane integrity after ROT treatment. We also observed lower [Ca2+]c, higher ROS and [Ca2+]m levels, decreased ΔΨm after ROT treatment, and greater susceptibility to ROT toxicity in aged RPE cells. PEDF can protect the cristae and integrity of the mitochondrial membrane, increase ATP levels and ΔΨm, and lower ROS, [Ca2+]c, and [Ca2+]m in aged RPE cells induced by ROT. In addition, there was an increase in RDH expression in RPE cells with increasing age after PEDF treatment. Similarly, PEDF decreased the expression of ROT-induced ER stress-related genes. CONCLUSIONS: Our study provides evidence that PEDF can reduce bioenergetic deficiencies, mitochondrial decay, and ER stress in aging RPE, a condition that may trigger the onset of retinal diseases such as AMD.

Self-Renewal Inhibition in Breast Cancer Stem Cells: Moonlight Role of PEDF in Breast Cancer.

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells' self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells' in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers.

Characterization of three adults and an adolescent with Osteogenesis Imperfecta type VI and a novel founder SERPINF1 variant.

Osteogenesis imperfecta (OI) type VI is an extremely rare form of OI caused by biallelic variants in the SERPINF1 gene, which codes for the pigment-epithelium derived factor (PEDF). We report on four patients (three adults and one adolescent) with a severe deforming form of OI. All patients presented no abnormalities at birth, frequent long bone and vertebrae fractures (mainly during childhood), marked short stature, severe bone deformities, chronic mild to moderate pain, and severe limitation of mobility, with three being completely wheelchair bound. Blue sclera and dentinogenesis imperfecta were absent, although some patients presented tooth, ophthalmological, and/or cardiac features. Radiographic findings included, among others, thin diaphysis and popcorn calcifications, both of which are non-specific to this type of OI. The novel homozygous variants c.816_819del (p.Met272Ilefs*8) and c.283+2T > G in SERPINF1 were identified in three and one patient, respectively. The three patients carrying the frameshift variant were born in nearby regions suggesting a founder effect. Describing the long-term outcomes of four patients with OI type VI, this cohort adds relevant data on the clinical features and prognosis of this type of OI.

Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs.

BACKGROUND/AIMS: Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung. METHODS: For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection. RESULTS: The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters. CONCLUSION: These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

PEDF inhibits LPS-induced acute lung injury in rats and promotes lung epithelial cell survival by upregulating PPAR-γ.

BACKGROUND: The progression of acute lung injury (ALI) involves numerous pathological factors and complex mechanisms, and cause the destruction of epithelial and endothelial barriers. Pigment epithelium-derived factor (PEDF) is an angiogenesis inhibitor and a potential anti-inflammatory factor. The purpose of this study was to investigate the effect of PEDF on lipopolysaccharide (LPS)-induced ALI in rats. METHODS: In vivo, pathological and injury related factors examination were performed on rat lung to investigate the effect of PEDF on ALI. In vitro, the effect of PEDF on inflammatory injury and apoptosis of lung epithelial type II RLE-6TN cell was evaluated, and the expression of inflammatory factors and related pathway proteins and PPAR-γ (in the presence or absence of PPAR-γ inhibitors) were analyzed. RESULTS: In vivo results showed that PEDF inhibited the inflammatory factor expression (TNF-α, IL-6 and IL-1ß) and progression of ALI and reduced lung cell apoptosis in rats. In vitro results showed that PEDF could effectively inhibit LPS-stimulated inflammatory damage and apoptosis of RLE-6TN cells. PEDF inhibited the RLE-6TN cell injury by enhancing the expression of PPAR-γ. CONCLUSIONS: PEDF is an anti-inflammatory factor, which can inhibit apoptosis of lung epithelial cells by upregulating the expression of PPAR-γ and reducing LPS-induced ALI in rats.

The Impact of Pigment-Epithelium-Derived Factor on MCF-7 Cell Metabolism in the Context of Glycaemic Condition.

Studies have demonstrated that pigment-epithelium-derived factor (PEDF) is a robust inhibitor of tumour growth and development, implying that this may serve as a promising target for therapeutic intervention. However, the precise impact of PEDF on cancerous cell metabolic pathways remains uncertain despite ongoing research. In this light, this study aimed to employ a metabolomics approach for understanding the metabolic reprogramming events in breast cancer across different glycaemic loads and their response to PEDF. Gas chromatography-quadrupole mass spectrometry (GC/Q-MS) analysis revealed metabolic alterations in ER+ human cell line MCF-7 cells treated with PEDF under varying glycaemic conditions. The identification of significantly altered metabolites was accomplished through MetaboAnalyst (v.5.0) and R packages, which enabled both multivariate and univariate analyses. Out of the 48 metabolites identified, 14 were chosen based on their significant alterations in MCF-7 cells under different glycaemic conditions and PEDF treatment (p < 0.05, VIP > 0.8). Dysregulation in pathways associated with amino acid metabolism, intermediates of the TCA cycle, nucleotide metabolism, and lipid metabolism were detected, and they exhibited different responses to PEDF. Our results suggest that PEDF has a diverse influence on the metabolism of MCF-7 cells in both normo- and hyperglycaemic environments, thereby warranting studies using patient samples to correlate our findings with clinical response in the future.

ADAMTS5 promotes neovascularization via autophagic degradation of PEDF in proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) adversely affects visual function. Extracellular matrix proteins (ECM) contribute significantly to the development of PDR. A Disintegrin and Metalloproteinase with Thrombospondin motifs 5 (ADAMTS5) is a member of ECM proteins. ADAMTS5 participates in angiogenesis and inflammation in diverse diseases. However, the role of ADAMTS5 in PDR remains elusive. Multiplex beam array technology was used to analyze vitreous humor of PDR patients and normal people. ELISA and Western blot were used to detect the expression of ADAMTS5, PEDF and autophagy related factors. Immunofluorescence assay was used to mark the expression and localization of ADAMTS5 and PEDF. The neovascularization was detected by tube formation test. Our results revealed that ADAMTS5 expression was increased in the vitreous humor of PDR patients and oxygen-induced retinopathy (OIR) mice retinas. Inhibiting ADAMTS5 alleviated pathological angiogenesis and upregulated PEDF expression in the OIR mice. In addition, ADAMTS5 inhibited PEDF secretion in ARPE-19 cells in vitro studies, thereby inhibiting the migration of HMEC-1. Mechanically, ADAMTS5 promoted the autophagic degradation of PEDF. Collectively, inhibition of ADAMTS5 during OIR suppresses pathological angiogenesis. Our study provides a new approach for resolving pathological angiogenesis in PDR.