Case report on two diabetic donor eyes with no retinopathy: Clinicopathological and molecular studies

We were intrigued to analyze donor eyes of two individuals without retinopathy even after 40 years of type 2 diabetes mellitus. Targeted molecular factors associated with angiogenesis and the key antioxidant enzymes in retinal tissue were analyzed. Accordingly PEDF, Adiponectin and Paraoxonase 2 showed augmented mRNA expression in both the retina with no significant change in VEGF expression. Vitreous showed increased PEDF protein in donor 1 and Adiponectin in donor 2 with no change in VEGF protein. This study highlights the profile of specific molecular factors that contribute to the non-development of diabetic retinopathy changes in these individuals.

Effects of PEDF on proliferation, apoptosis and invasion of squamous lung carcinoma cells in high-glucose environment / 西安交通大学学报(医学版)

Objective: To study the effects of pigment epithelial-derived factor (PEDF) on the proliferation, apoptosis and invasion of squamous lung carcinoma cells in high-glucose environment so as to explore the significance of PEDF in the development, prognosis and treatment of lung cancer associated with diabetes. Methods: SK-MES-1 lung squamous carcinoma cells were cultured and divided into negative control group; high-glucose group; and PEDF+high glucose groups 1, 2 and 3. The cell morphological changes were observed under the inverted microscope. Then proliferation inhibition rates of SK-MES-1 cells in all the groups were observed by MTT assay. The cell cycle and cell apoptosis rates were detected by flow cytometry. The number of penetration cells was determined by cell invasion experiment. Expression of VEGF in culture supernatant in each group was detected by ELISA. Results: ① Compared with that in the negative control group, the proliferation inhibition rate and apoptosis rate in high-glucose group were low, the percentage of cells blocked in G0/G1 phase was decreased, the number of penetration cells was increased and the concentration of VEGF was increased (P<0.05). ② With the increase of PEDF intervention concentration, the proliferation inhibition rate and apoptosis rate in each group increased, the percentage of G0/G1 phase increased, the number of penetration cells decreased, and the concentration of VEGF decreased (P<0.05). Conclusion: ① The development of squamous cell carcinoma of the lung is promoted in high glucose. ② PEDF can inhibit the proliferation of lung squamous cell carcinoma cells in high-glucose environment, promote early apoptosis and reduce the invasiveness in the concentration-dependent manner. PEDF is predicted to be a target therapeutic drug for lung cancer complicated with diabetes mellitus.

Protective effects of PEDF gene modified human umbilical cord mesenchymal stem cells in rat with retinal ischemia-reperfusion injury / 眼科新进展

Objective To observe the effects of pigment epithelial-derived factor gene-modified human umbilical cord mesenchymal stem cells (PEDF-MSCs) on the expression of pigment epithelial-derived factor (PEDF) and vascular endothehal growth factor (VEGF) in a rat model of retinal ischemia-reperfusion injury (RIRI) and its protection on retinal ganglion cells.Methods Lentivirus (LV) labeled with green fluorescent protein (GFP) was used as a vector to transfect the PEDF gene into human umbilical cord mesenchymal stem cells (hUCMSCs) at a multiplicity of infection (MOI) of 1,10,20,and 50 in vitro,and then the expression of PEDF gene and protein in cells transfected with the best MOI value was detected by RT-PCR and ELISA.The healthy male SD rats were randomly divided into normal group (N group) and experimental group.The RIRI model was made by high intraocular pressure in the experimental group,and the RIRI rats with PBS treatment were allocated as the PBS group,with hUC-MSCs treatment as M group and LV-PEDF-MSCs treatment as P group,and the N group was left untreated.All rats were sacrificed on day 5,and the number of retinal ganglion cells were counted by Nissl staining,the thickness of the retina was calculated,as well as the expression of PEDF and VEGF mRNA in rat retina was detected by RT-PCR.Results The transfection efficiency was as high as 75.8％ under fluorescence microscope.The results of RT-PCR showed that the relative expression of PEDF mRNA in PEDF-MSCs supernatant (4.34 ± 0.29) was significantly higher than that in hUCMSCs (1.08 ± 0.15),and the difference was statistically significant (P ＜ 0.05);moreover,the results of ELISA showed that PEDF protein expression in PEDF-MSCs supernatant [(83.09 ± 7.58)μg · L-1] was significantly higher than that in hUCMSCs [(12.30 ±1.24) μg · L-1],and the difference was statistically significant (P ＜ 0.05).Nissl staining results showed that the number of ganglion cells in group PBS decreased after model establishment.After 5 days of treatment,the number of ganglion cells in P group and M group was higher than that in PBS group,and the difference was statistically significant (both P ＜ 0.05);and P group was higher than M group,with the significant difference (P ＜ 0.05).And this was true of the thickness of the retina.RT-PCR showed that the expression of PEDF mRNA in P group was significantly up-regulated,but VEGF mRNA expression was significantly down-regulated,and the differences were statistically significant when compared with PBS and M group (both P ＜ 0.05).Conclusion Intravitreal injection of PEDF-MSCs can up-regulate the expression of PEDF but down-regulate the expression of VEGF in the retina of RIRI rats,which can protect the retinal ganglion cells against RIRI.

PEDF Inhibits Non?small Cell Lung Cancer Proliferation by Down?regulating VEGF Expression / 中国医科大学学报

Objective To analyze the mechanism of pigment epithelium?derived factor(PEDF)regulation in lung cancer cell proliferation and migration by vascular endothelial growth factor(VEGF). Methods VEGF expression was observed after overexpression or silencing of PEDF. Proliferation and migration were analyzed by MTT and transwell assays. Real?time PCR and Western blotting were performed to investigate the mechanism underlying PEDF regulation of the VEGF/SRC/FAK pathways. Results PEDF could inhibit the proliferation and migration of A549 cells by VEGF. Conclusion PEDF can be considered as a potential therapeutic target for lung cancer.

Effect and mechanism of PEDF on proliferationof human umbilical veinendothelial cellsand lung cancer SK-MES-1 cells / 安徽医科大学学报

Objective To investigate the effect and potential mechanism of pigment epithelium derived factor(PEDF) acting upon SK-MES-1 cell and human umbilical vein endothelial cells(HUVECs).Methods CCK-8 was used to detect the effect of varying concentrations of PEDF upon HUVECs and SK-MES-1 cell, measuring the degree of cell proliferation and inhibition effect across varying times.The flow cytometry tests were carried out to invest gate the apoptosis of these two kinds of cells when exposed to varying concentration of PEDF.qRT-PCR were carried out to assess the vascular endothelial growth factor(VEGF) gene expression level in these two kinds of cells after treatment of PEDF.Results CCK-8 results revealed that PEDF had a concentration-dependent and time-dependent cell proliferation inhibition effect on SK-MES-1 cell and HUVECs(P<0.05);Flow cytometry showed that the apoptosis of the cells in the treatment group were higher than that of control group(P<0.05), and the apoptosis rate of high concentration group was higher than that of the low concentration group(P<0.05);qRT-PCR results showed that PEDF was able to inhibit expression of mRNA of VEGF in both HUVECs and SK-MES-1 cell compared with control samples(P<0.05).Conclusion The antitumor properties of PEDF is mainly related to the inhibition of tumor angiogenesis and direct effects on tumor cells, the effect of PEDF on HUVECs and SK-MES-1 cell maybe related to the effects of PEDF on downregulating expression of VEGF.

Research progress of pigment epithelium-derived factor ,insulin resistance and type 2 diabetes / 中国糖尿病杂志

Pigment epithelium-derived factor (PEDF ) was originally found fromretinal pigment epithelium. It is confirmed that it plays an anti-angiogenic and apoptosis role in diabetic microangiopathy. Recently ,PEDF has been found to have a close relationship with insulin resistance ,type 2 diabetes and diabetic macroangiopathy. But the mechanism is unclear. More and more researches focused on its role in oxidative stress. PEDF may become a new target for diabetes and diabetic complication treatment and also a predictive factor for the disease.

Inhibition of Tumor Angiogenesis by Targeted Induction of Endothelial Cell Apoptosis / 中山大学学报(医学科学版)

Angiogenesis is one of the important pathological characteristics in the development of tumor growth. Hence ,an?ti-angiogenes is has become a hot topic in the field of cancer research. The current strategy for anti-angiogenesis therapy is to restore the angiogenic balance which is broken in the tumor via either block of proangiogenic factorsor application of angiogenic inhibitors. Endogenous angiogenic inhibitors show more promising prospects compared with proangiogenic factor antagonists. However ,the un?derlying mechanisms for the angiogenic inhibitors remain to be thoroughly elucidated. There are two kinds of endogenous angiogenic inhibitors,one is the hydrolyzed fragments of precursor protein,such as plasminogen Kringle 5(K5),angiostatin/kringle 1~4,end?ostatin,etc;the other is cell secreted proteins,such as pigment epithelium-derived factor(PEDF),kallikrein-binding protein (KBP/kallistatin),antithrombin,etc. Here we summarized the research progresses on the biological functions,underlying mecha?nisms of tumor angiogenesis and application prospects of K5,PEDF,and KBP,so as to provide insights into the antiangiogenic ther?apies of tumor in the future.

Administration of Pigment Epithelium-Derived Factor Inhibits Airway Inflammation and Remodeling in Chronic OVA-Induced Mice via VEGF Suppression

PURPOSE: Pigment epithelium-derived factor (PEDF) is a recently discovered antiangiogenesis protein. PEDF possesses powerful anti-inflammatory, antioxidative, antiangiogenic, and antifibrosis properties. It has been reported that PEDF can regulate vascular endothelial growth factor (VEGF) expression. This study aimed to evaluate whether recombinant PEDF protein could attenuate allergic airway inflammation and airway remodeling via the negative regulation of VEGF using a murine model of chronic ovalbumin (OVA)-induced asthma and BEAS-2B human bronchial epithelial cells. METHODS: In an in vivo experiment, mice sensitized with OVA were chronically airway challenged with aerosolized 1% OVA solution for 8 weeks. Treated mice were given injections of recombinant PEDF protein (50 or 100 microg/kg body weight) via the tail vein. In an in vitro experiment, we investigated the effects of recombinant PEDF protein on VEGF release levels in BEAS-2B cells stimulated with IL-1beta. RESULTS: Recombinant PEDF protein significantly inhibited eosinophilic airway inflammation, airway hyperresponsiveness, and airway remodeling, including goblet cell hyperplasia, subepithelial collagen deposition, and airway smooth muscle hypertrophy. In addition, recombinant PEDF protein suppressed the enhanced expression of VEGF protein in lung tissue and bronchoalveolar lavage fluid (BALF) in OVA-challenged chronically allergic mice. In the in vitro experiment, VEGF expression was increased after IL-1beta stimulation. Pretreatment with 50 and 100 ng/mL of recombinant PEDF protein significantly attenuated the increase in VEGF release levels in a concentration-dependent manner in BEAS-2B cells stimulated by IL-1beta. CONCLUSIONS: These results suggest that recombinant PEDF protein may abolish the development of characteristic features of chronic allergic asthma via VEGF suppression, providing a potential treatment option for chronic airway inflammation diseases such as asthma.

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogen-esis in retinal pigment epithelium (RPE). RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

The mechanism of PDEF inhibit invasion and metastasis of prostate cancer cells with down-reg uLated HIF-1α / 实用医学杂志

Objective To explore the mechanism that PDEF inhibition the invasion and metastasis of prostate cancer cells by down-reg uLated HIF-1α. Methods PEDF and PBS as the experimental group and control group were add to the c uLture medium of human prostate cancer cell line (PC3), the wound healing and transwell experiment were carried out to observed the ability of invasion and metastasis. And HIF-1α expression was detect by RT-QPCR. ResuLts The res uLts of wound healing and transwell showed that PEDF inhibit the ability of invasion and metastasis of prostate cancer. PEDF down-reg uLated the expression of HIF-1α. Conclusions PEDF inhibit the invasion and metastasis of prostate cancer by down-reg uLated the expression of HIF-1α.It will be the new target of tumor intervention and for the treatment of prostate cancer and other malignant tumors to provide adequate scientific basis and efficient drug candidates.

The Expression of Extracellular Matrix mRNA in the Chondrocytes of the Femoral Head Affected by Avascular Necrosis

PURPOSE: The aim of this study was to evaluate the expression of type II collagen, aggrecan, VEGF-A and PEDF mRNAs in the human chondrocytes derived from the articular cartilage of the femoral heads with avacular necrosis (AVN). MATERIALS AND METHODS: We cultured human chondrocytes that were primarily derived from the articular cartilage of femoral heads with AVN. We evaluated the mRNA expression of type II collagen, aggrecan, VEGF-A and PEDF. RESULTS: The chondrocytes of the AVN group showed decreased expressions of type II collagen mRNA and aggrecan mRNA (p0.05). CONCLUSION: The cartilage matrix's formation ability was found to be decreased in the chondrocytes of the femoral heads affected by AVN.

Inhibition of retinal angiogenesis by PEDF / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effect of PEDF on retinal neovascularization in mice. METHODS: 40 7-day-old C57BL/6J mice was exposed to 750mL/L oxygen for 5 days and then to normal situation to produce the murine model of oxygen-induced retinopathy(OIR). One eye of the mouse was regarded as experimental one and the other served as control. Eyes in experimental group received intravitreal injection of PEDF and eyes in control group received intravitreal injection of PBS at postnatal day 12. All mice were executed at postnatal day 17. The changes of retinal vessels of mice were observed by ADPase histochemical technique. The inhibitory effect of PEDF on retinal neovascularization was evaluated by counting the endotheliocyte nuclei of new vessels which extended from retina to vitreous in the tissue slice of HE staining. RESULTS: Neovascularization was reduced, retinal blood vessels distributed regularly and non-perfusion areas were not found in eyes of experimental group compared with control group. The number of endotheliocyte nuclei of new vessels extending from retina to vitreous was significantly less in the eyes of experimental group (10.18±1.74) than that in control group (38.89±2.98) (P<0.01). Retinal toxicity and inflammatory reactions were not found in tissue slice.CONCLUSION: PEDF inhibits retinal angiogenesis in OIR and the feasibility should be determined for use of PEDF in ocular angiogenesis treatment.

Detection of pigment epithelium derived factor gene mutation in a human malignant melanoma cell line A375 / 中华皮肤科杂志

Objective To detect the mutations of pigment epithelium derived factor (PEDF) gene in a human malignant melanoma cell line A375. Methods A375 cells and control melanocytes obtained from circumcised prepuce were cultured, genomic DNA was extracted from these cells. All eight exons of PEDF gene were scanned by single strand conformation polymorphism analysis ofpolymerase chain reaction products (PCR-SSCP) in both A375 cells and control melanocytes. DNA sequencing was performed for the PCR products separated into electrophoretic bands with altered mobility. Results Altered mobility was observed with SSCP analysis in amplicons of exon 3, 4, 5, 6 and 7, with the most obvious alteration occurred in exon 5 and 6. DNA sequencing revealed mutations in both exon 5 and 6. The common type of mutations was single base-deletion in exon 5 and single base-substitution in exon 6. Conclusion Mutations of PEDF gene may contribute to the development of human malignant melanoma.

Effect of Alcoholon the Expression of VEGF-A,PEDF,and VEGFR-2 of Osteoblast in Avascular Necrosis of Femoral Head / 대한정형외과학회잡지

PURPOSE: The aim of this study was to determine the effect of alcohol on the expression of VEGF-A, PEDF, and VEGFR-2 in human osteoblasts. MATERIALS AND METHODS: Human osteoblasts primarily derived from the intertrochanteric region of the femur with osteonecrosis and fracture (control) were cultured with alcohol (0, 20, 100, 150 mM). The level of cell proliferation and the expression levels of VEGF-A mRNA, PEDF mRNA, and VEGFR-2 mRNA was evaluated according to the alcohol concentrations and the culture periods. RESULTS: Osteoblasts with the added alcohol showed an early increase in cell population, and a subsequent decrease or steady level thereafter compared with those without alcohol (p<0.05). The osteoblasts in the osteonecrosis group showed an increase in VEGF-A mRNA and PEDF mRNA expression at high alcohol concentrations (100, 150 mM), resulting in an decreased VEGF-A/PEDF ratio, while those in the control group showed an increase in VEGF-A mRNA expression and a decrease in PEDF mRNA expression, resulting in an increase in the VEGF-A/PEDF ratio (p<0.05). CONCLUSION: Alcohol stops the proliferation of osteoblasts and can cause an imbalance between VEGF-A and PEDF, thereby inhibiting the neovascularization of osteonecrosis.

Expression of Angiogeneis-related Factors in Retinal Pigment Epithelial Cells under Hypoxia

PURPOSE: To elucidate the mechanism of neoangiogenesis of human retinal pigment epithelium (HRPE) under hypoxia. METHODS: HRPE cells were cultured for 2 and 24 hours in a hypoxic chamber. Expression and production of the angiogenic factor, vascular endothelial growth factor (VEGF) and the anti-angiogenic factor, pigment epithelium-derived factor (PEDF) were analyzed by RT-PCR and Western blot, respectively. Neoangiogenesis was induced by adding culture supernatant harvested from cells exposed to hypoxic conditions. Neoangeogenesis was measured with a tube formation assay that uses ECV 304 cells and with a migration assay that uses human dermal microvascular endothelial cells. RESULTS: Competitive RT-PCR showed that the expression of the PEDF gene in HRPE cells under hypoxic state decreased compared to normoxic state (p<0.01) but the expression of the VEGF gene increased (p<0.01) when exposed to hypoxic conditions. These results corresponded to those of the Western blot analysis which revealed a significant increase of VEGF production (p<0.01) and a decrease of PEDF production (p<0.01). Moreover, the tube formation and migration assays demonstrated that angiogenesis was increased by exposure to hypoxic stress. Taken together, HRPE cells under hypoxic stress produce more VEGF and less PEDF, which lead to neoangiogenesis. CONCLUSIONS: These results suggest that the subretinal neovascularization that occurs under hypoxic stress might be caused by an imbalance of angiogenesis-related factors in HRPE cells.