Ocular delivery of Pigment Epithelium-Derived Factor (PEDF) as a neuroprotectant for Geographic Atrophy.

Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), that starts with atrophic lesions in the outer retina that expand to cover the macula and fovea, leading to severe vision loss over time. Pigment Epithelium-Derived Factor (PEDF) has a diverse-range of properties, including its ability to promote cell survival, reduce inflammation, inhibit angiogenesis, combat oxidative stress, regulate autophagy, and stimulate anti-apoptotic pathways, making it a promising therapeutic candidate for GA. However, the relatively short half-life of PEDF protein has precluded its potential as a clinical therapy for GA since it would require frequent injections. Therefore, we describe administration of a PEDF gene, comparing and contrasting delivery routes, viral and non-viral vectors, and consider the critical challenges for PEDF as a neuroprotectant for GA.

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

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

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

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

Kallistatin alleviates heart failure in rats by inhibiting myocardial inflammation and apoptosis via regulating sirt1.

OBJECTIVE: Heart failure (HF) is the loss of myocardial structure and function caused by various congenital or acquired heart diseases. This study explored the new target of treatment of HF by investigating the effect of Kallistatin (KS) on inflammation and apoptosis of myocardial tissue in HF rats. MATERIALS AND METHODS: We used doxorubicin to induce rat HF, and determined the success rate of modeling by detecting changes in rat heart weight and body weight, cardiac function and histology. We used two different doses (1 mg/kg, 2 mg/kg) of KS intraperitoneally injected rats and detected changes in inflammation and apoptosis of rat myocardial tissue by enzyme-linked immunosorbent assay (ELISA), Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and immunohistochemical staining. Changes in the expression of sirt1 were also detected. In addition, we cultured rat myocardial cell line, H9c2 cells, and used siRNA-sirt1 to inhibit sirt1 in H9c2 cells to clarify the mechanism of KS regulating myocardial cells. RESULTS: The body weight of HF rats treated with KS decreased while the heart weight increased. KS has also been found to reduce the concentration of brain natriuretic polypeptide (BNP) in rat serum. The results of echocardiography showed that KS effectively relieved the cardiac function of HF rats. Inflammatory factors (interleukin (IL)-1ß, IL-6, IL-8 and tumor necrosis factor (TNF)-α) and pro-apoptotic molecules (caspase3/9 and Bax) in the serum and myocardial tissue of rats treated with KS were also significantly reduced. The inhibition of sirt1 in H9c2 cells significantly reduced the anti-apoptotic effect of KS on H9c2 cells. CONCLUSIONS: KS reduces the inflammation and apoptosis of myocardial tissue in HF rats by promoting the expression of sirt1, thereby alleviating HF-induced myocardial injury.

Anti-intoxication and protective effects of a recombinant serine protease inhibitor from Lentinula edodes against acute alcohol-induced liver injury in mice.

Serine protease inhibitors (serpins) are involved in inflammation, coagulation, fibrinolysis, tumor suppression, molecular chaperone, chromatin densification, and hormone transport. However, their anti-intoxication activity has not been determined. Here, we heterologously expressed the serpin gene from Lentinula edodes in Escherichia coli and purified the recombinant serpin protein from L. edodes (rLeSPI). Then, we administered alcohol and active protein or Haiwangjinzun as a positive control to mice via gavage to evaluate the anti-intoxication activities of rLeSPI in vivo. We also investigated the protective effects of rLeSPI on acute alcohol-induced liver injury in mice by physiological and biochemical assays. The assay results for the anti-intoxication activity revealed that pretreating mice with 5 mg/kg rLeSPI for 0.5 h before gavage with Erguotou liquor (56%V EtOH, 0.15 ml/10 g) significantly prolonged the tolerance time and shortened the intoxication time relative to the results of the control group, thereby proving its anti-intoxication activities. The biochemical analysis showed that rLeSPI improved glutathione peroxidase activity, which was evidently reduced by ethanol. Additionally, rLeSPI significantly improved the activity of aldehyde dehydrogenase, which is important in alcohol metabolism, and reduced the intracellular malondialdehyde content, aspartate amino transferase, and alanine amino transferase activity. We concluded that LeSPI displayed anti-intoxication activity and exerted protective effects against acute alcohol-induced liver injury, providing new insight into the prevention of alcoholism and alcohol-related diseases.Key Points• Anti-intoxication activity of a recombinant serpin protein rLeSPI was assessed.• LeSPI displayed anti-intoxication activity in mice.• LeSPI exerted protective effects against acute alcohol-induced liver injury in mice.

Role of vaspin in porcine ovary: effect on signaling pathways and steroid synthesis via GRP78 receptor and protein kinase A†.

Vaspin, visceral-adipose-tissue-derived serine protease inhibitor, is involved in the development of obesity, insulin resistance, inflammation, and energy metabolism. Our previous study showed vaspin expression and its regulation in the ovary; however, the role of this adipokine in ovarian cells has never been studied. Here, we studied the in vitro effect of vaspin on various kinase-signaling pathways: mitogen-activated kinase (MAP3/1), serine/threonine kinase (AKT), signal transducer and activator of transcription 3 (STAT3) protein kinase AMP (PRKAA1), protein kinase A (PKA), and on expression of nuclear factor kappa B (NFKB2) as well as on steroid synthesis by porcine ovarian cells. By using western blot, we found that vaspin (1 ng/ml), in a time-dependent manner, increased phosphorylation of MAP3/1, AKT, STAT3, PRKAA1, and PKA, while it decreased the expression of NFKB2. We observed that vaspin, in a dose-dependent manner, increased the basal steroid hormone secretion (progesterone and estradiol), mRNA and protein expression of steroid enzymes using real-time PCR and western blot, respectively, and the mRNA of gonadotropins (FSHR, LHCGR) and steroids (PGR, ESR2) receptors. The stimulatory effect of vaspin on basal steroidogenesis was reversed when ovarian cells were cultured in the presence of a PKA pharmacological inhibitor (KT5720) and when GRP78 receptor was knocked down (siRNA). However, in the presence of insulin-like growth factor type 1 and gonadotropins, vaspin reduced steroidogenesis. Thus, vaspin, by activation of various signaling pathways and stimulation of basal steroid production via GRP78 receptor and PKA, could be a new regulator of porcine ovarian function.

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

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

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

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

Squamous Cell Carcinoma Biomarker Sensing on a Strontium Oxide-Modified Interdigitated Electrode Surface for the Diagnosis of Cervical Cancer.

Cervical cancer is a life-threatening complication, appearing as the uncontrolled growth of abnormal cells in the lining of the cervix. Every year, increasing numbers of cervical cancer cases are reported worldwide. Different identification strategies were proposed to detect cervical cancer at the earlier stages using various biomarkers. Squamous cell carcinoma antigen (SCC-Ag) is one of the potential biomarkers for this diagnosis. Nanomaterial-based detection systems were shown to be efficient with different clinical biomarkers. In this study, we have demonstrated strontium oxide-modified interdigitated electrode (IDE) fabrication by the sol-gel method and characterized by scanning electron microscopy and high-power microscopy. Analysis of the bare devices indicated the reproducibility with the fabrication, and further pH scouting on the device revealed that the reliability of the working pH ranges from 3 to 9. The sensing surface was tested to detect SCC-Ag against its specific antibody; the detection limit was found to be 10 pM, and the sensitivity was in the range between 1 and 10 pM as calculated by 3σ. The specificity experiment was carried out using major proteins from human serum, such as albumin and globulin. SCC-Ag was shown to be selectively detected on the strontium oxide-modified IDE surface.

Effects of Vaspin on Insulin Resistance in Rats and Underlying Mechanisms.

Insulin resistance (IR) is the main pathogenesis of metabolic syndrome and a shared pathophysiological change in conditions such as diabetes mellitus, adiposity, hypertension, and atherosclerosis. Visceral adipose tissue-derived serpin (Vaspin) is a newly discovered adipocytokine with insulin-sensitizing and anti-inflammatory effects. To examine if vaspin can improve insulin resistance in rats fed a high-fat diet via the insulin receptor substrate/phosphatidylinositol 3 kinase/protein kinase B/glucose transport (IRS/PI3K/Akt/Glut) and inhibitory κB alpha/nuclear factor-kappa B (IκBα/NF-κB) signalling pathways, thirty male Sprague-Dawley (SD) rats were randomly divided into three groups: the normal control group (NC group, n = 10), high-fat diet group (HFD group, n = 10) and vaspin intervention group (HFD + vaspin group, n = 10). Results showed that intervention with vaspin significantly decreased fasting blood glucose (FBG) and fasting insulin (FINS) concentrations in HFD - fed rats without significantly affecting body weight or triglyceride (TG) or total cholesterol (TC) levels. The areas under the intraperitoneal glucose tolerance test (IPGTT) and the insulin tolerance test (ITT) curves were significantly decreased in HFD + vaspin group compared with the HFD group, and the glucose infusion rate (GIR) showed the same trends. Western blot, real-time polymerase chain reaction (RT-PCR) and immunofluorescence staining showed that vaspin could improve insulin resistance in liver, skeletal muscle and adipose tissue by activating the IRS/PI3K/Akt/Glut signalling pathway and inhibiting the IκBα/NF-κB signalling pathway.

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

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

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

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

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

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

Kallistatin exerts anti-lymphangiogenic effects by inhibiting lymphatic endothelial cell proliferation, migration and tube formation.

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, its effects on lymphatic endothelial cells and lymphangiogenesis remain poorly understood. Lymphangiogenesis is involved in tumor metastasis via the lymphatic vasculature in various types of tumors. The aim of this study was to investigate the effects of kallistatin on lymphangiogenesis and the mechanism of action involved. Treatment with kallistatin recombinant protein or overexpression of kallistatin inhibited the proliferation, migration and tube formation of human lymphatic endothelial cells (hLECs), and induced apoptosis of hLECs. Furthermore, our results showed that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Treatment with kallistatin recombinant protein decreased the LVD in the implanted gastric xenograft tumors of nude mice. To the best of our knowledge, the present study is the first to demonstrate that kallistatin possesses anti-lymphangiogenic activity in vitro and in vivo. Moreover, kallistatin inhibited proliferation and migration of hLECs by reducing the phosphorylation of ERK and Akt, respectively. These findings suggested that kallistatin may be a promising agent that could be used to suppress cancer metastasis by inhibiting both angiogenesis and lymphangiogenesis.

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

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

Vaspin prevents elevation of blood pressure through inhibition of peripheral vascular remodelling in spontaneously hypertensive rats.

AIM: Visceral adipose tissue-derived serine protease inhibitor (vaspin) is a relatively novel adipocytokine with protective effects on metabolic diseases including obesity and type II diabetes. We have previously demonstrated that vaspin exerts anti-inflammatory and antimigratory roles through antioxidative effects in vascular smooth muscle cells. As inflammatory responses and migration of smooth muscle in peripheral vascular wall are key mechanisms for the pathogenesis of hypertension, we hypothesized that vaspin could prevent the development of hypertension in in vivo hypertensive animal model. METHODS: Vaspin (1 µg kg(-1)  day(-1) ) was administered intraperitoneally to 5-week-old male spontaneously hypertensive rats (SHR) for 4 weeks. Superior mesenteric artery was isolated and used for measurement of isometric contraction and histological analysis. RESULTS: Long-term vaspin treatment significantly prevented an elevation of systolic blood pressure (SBP) at 8 weeks of age. Vaspin had no effect on reactivity of isolated mesenteric artery from SHR. In contrast, vaspin significantly inhibited mesenteric arterial wall hypertrophy in SHR. Moreover, vaspin significantly inhibited an increase of tumour necrosis factor-α expression and a production of reactive oxygen species in isolated mesenteric artery from SHR. CONCLUSION: This study for the first time demonstrates that vaspin prevents the increase of SBP in SHR through inhibiting peripheral vascular hypertrophy possibly via antioxidative and anti-inflammatory mechanisms.

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

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

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

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

Kallistatin, a novel anti-angiogenesis agent, inhibits angiogenesis via inhibition of the NF-κB signaling pathway.

Development of a novel angiogenesis inhibitor will be essential for the improvement of therapeutics against cancer. Kallistatin had been recognized as an endogenous angiogenesis inhibitor. Here, we demonstrated kallistatin's strong anti-angiogenesis and anti-metastasis activity stimulated by breast cancer cells (MCF-7) and its mechanism of action in vitro. The anti-angiogenesis effect in vivo was evaluated by chicken chorioallantoic membrane (CAM) neovascularisation. Because of the underlying molecular mechanism of its anti-angiogenesis activity remains poorly understood. In this study, we examined whether the NF-κB signaling pathway was involved in the anti-angiogenesis and anti-metastasis activity of kallistatin. Kallistatin significantly inhibited TNF-α-induced nuclear factor-κB activation in a dose-dependent manner. Addition of kallistatin inhibited TNF-α induced IκBα degradation; phosphorylation of IκBα kinase (IKK), nuclear factor-κB-p65 protein; and nuclear translocation of p65/50. Meanwhile, we investigated the effects of kallistatin on the expression of vascular endothelial growth factor (VEGF) and other angiogenesis-related gene in human umbilical vein endothelial cells (HUVECs). We found that kallistatin decreased the expression of VEGF and some angiogenesis-related genes, which promoted angiogenesis in cancer. Taken together, we suggested that kallistatin would inhibit tumor angiogenesis via inhibition of the NF-κB signaling pathway and finally abrogate NF-κB-dependent gene expression. All the results revealed that kallistatin would have potential as a novel.

Human kallistatin administration reduces organ injury and improves survival in a mouse model of polymicrobial sepsis.

Kallistatin, a plasma protein, has been shown to exert multi-factorial functions including inhibition of inflammation, oxidative stress and apoptosis in animal models and cultured cells. Kallistatin levels are reduced in patients with sepsis and in lipopolysaccharide (LPS)-induced septic mice. Moreover, transgenic mice expressing kallistatin are more resistant to LPS-induced mortality. Here, we investigated the effects of human kallistatin on organ injury and survival in a mouse model of polymicrobial sepsis. In this study, mice were injected intravenously with recombinant kallistatin (KS3, 3 mg/kg; or KS10, 10 mg/kg body weight) and then rendered septic by caecal ligation and puncture 30 min later. Kallistatin administration resulted in a > 10-fold reduction of peritoneal bacterial counts, and significantly decreased serum tumour necrosis factor-α, interleukin-6 and high mobility group box-1 (HMGB1) levels. Kallistatin also inhibited HMGB1 and toll-like receptor-4 gene expression in the lung and kidney. Administration of kallistatin attenuated renal damage and decreased blood urea nitrogen and serum creatinine levels, but increased endothelial nitric oxide synthase and nitric oxide levels in the kidney. In cultured endothelial cells, human kallistatin via its heparin-binding site inhibited HMGB1-induced nuclear factor-κB activation and inflammatory gene expression. Moreover, kallistatin significantly reduced apoptosis and caspase-3 activity in the spleen. Furthermore, kallistatin treatment markedly improved the survival of septic mice by 23% (KS3) and 41% (KS10). These results indicate that kallistatin is a unique protecting agent in sepsis-induced organ damage and mortality by inhibiting inflammation and apoptosis, as well as enhancing bacterial clearance in a mouse model of polymicrobial sepsis.