Expression and significance of pigment epithelium-derived factor and vascular endothelial growth factor in colorectal adenoma and cancer

BACKGROUND The incidence and mortality of colorectal cancer(CRC)are among the highest in the world,and its occurrence and development are closely related to tumor neovascularization.When the balance between pigment epithelium-derived factors(PEDF)that inhibit angiogenesis and vascular endothelial growth factors(VEGF)that stimulate angiogenesis is broken,angiogenesis is out of control,resulting in tumor development.Therefore,it is very necessary to find more therapeutic targets for CRC for early intervention and later treatment.AIM To investigate the expression and significance of PEDF,VEGF,and CD31-stained microvessel density values(CD31-MVD)in normal colorectal mucosa,adenoma,and CRC.METHODS In this case-control study,we collected archived wax blocks of specimens from the Digestive Endoscopy Center and the General Surgery Department of Chengdu Second People's Hospital from April 2022 to October 2022.Fifty cases of specimen wax blocks were selected as normal intestinal mucosa confirmed by electronic colonoscopy and concurrent biopsy(normal control group),50 cases of specimen wax blocks were selected as colorectal adenoma confirmed by electronic colonoscopy and pathological biopsy(adenoma group),and 50 cases of specimen wax blocks were selected as CRC confirmed by postoperative pathological biopsy after inpatient operation of general surgery(CRC group).An immunohistochemical staining experiment was carried out to detect PEDF and VEGF expression in three groups of specimens,analyze their differences,study the relationship between the two and clinicopathological factors in CRC group,record CD31-MVD in the three groups,and analyze the correlation of PEDF,VEGF,and CD31-MVD in the colorectal adenoma group and the CRC group.The F test or adjusted F test is used to analyze measurement data statistically.Kruskal-Wallis rank sum test was used between groups for ranked data.The chi-square test,adjusted chi-square test,or Fisher's exact test were used to compare the rates between groups.All differences between groups were compared using the Bonferroni method for multiple comparisons.Spearman correlation analysis was used to test the correlation of the data.The test level(α)was 0.05,and a two-sided P<0.05 was considered statistically significant.RESULTS The positive expression rate and expression intensity of PEDF were gradually decreased in the normal control group,adenoma group,and CRC group(100%vs 78%vs 50%,χ^(2)=34.430,P<0.001;++~++vs+~++vs-~+,H=94.059,P<0.001),while VEGF increased gradually(0%vs 68%vs 96%,χ^(2)=98.35,P<0.001;-vs-~+vs++~+++,H=107.734,P<0.001).In the CRC group,the positive expression rate of PEDF decreased with the increase of differen-tiation degree,invasion depth,lymph node metastasis,distant metastasis,and TNM stage(χ^(2)=20.513,4.160,5.128,6.349,5.128,P<0.05);the high expression rate of VEGF was the opposite(χ^(2)=10.317,13.134,17.643,21.844,17.643,P<0.05).In the colorectal adenoma group,the expression intensity of PEDF correlated negatively with CD31-MVD(r=-0.601,P<0.001),whereas VEGF was not significantly different(r=0.258,P=0.07).In the CRC group,the expression intensity of PEDF correlated negatively with the expression intensity of CD31-MVD and VEGF(r=-0.297,P<0.05;r=-0.548,P<0.05),while VEGF expression intensity was positively related to CD31-MVD(r=0.421,P=0.002).CONCLUSION It is possible that PEDF can be used as a new treatment and prevention target for CRC by upregulating the expression of PEDF while inhibiting the expression of VEGF.

Expression of Pigment Epithelium-derived Factor in Bladder Tumour Is Correlated with Interleukin-8 yet Not with Interleukin-1α

Pigment epithelium-derived factor （PEDF） is an antiangiogenic factor which is effective in tumour inhibition in a variety of tumours and has not yet been studied in bladder tumour before. In this study the expression of PEDF, interleukin-1α （IL-1α） and -8 （IL-8） in bladder tumours was investigated. Immunohistochemistry was performed on 64 bladder tumour and 23 normal uroepithelium samples. Expression change of the factors was compared with clinicopathological parameters. Correlations between PEDF, IL-1α and IL-8 were analyzed. None of the factors was in relation to gender, tumour occurrence, and size or onset pattern. PEDF （P=0.014） and IL-1α （P=0.049） expression was down-regulated with grade progression. PEDF expression was lower in normal uroepithelium than in papillary urothelial neoplasm of low malignant potential （PUNLMP） （P=0.000） and carcinoma （P=0.009） whilst IL-1α （P=0.000 and P=0.000 respectively） and IL-8 （P=0.000 and P=0.023 respectively） expression was higher in the same grouping. PEDF expression had a negative correlation with IL-8 in PUNLMP （P=0.049, r=-0.578） as well as in tumour grouping （P=0.033, r=-0.276）. Deranged expressional change of PEDF, IL-1α and IL-8 could be in relation to loss of differentiation from normal uroepithelium to papillary lesion and eventually to carcinoma.

Pigment epithelium-derived factor protects retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction

AIM： To investigate the potential of pigment epitheliumderived factor（PEDF） to protect the immortalized rat retinal ganglion cells-5（RGC-5） exposed to Co Cl2-induced chemical hypoxia. METHODS： After being differentiated with staurosporine（SS）, RGC-5 cells were cultured in four conditions： control group cells cultured in Dulbecco ＇s modified eagle medium（DMEM） supplemented with 10% fetal bovine serum, 100 μmol/m L streptomycin and penicillin（named as normal conditions）; hypoxia group cells cultured in DMEM containing 300 μmol/m L Co Cl2; cells in the group protected by PEDF were first pretreated with 100 ng/m L PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/m L PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species（ROS） was measured by dichloro-dihydro-fluorescein diacetate（DCFH-DA） probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores（m PTPs） and membrane potential（Δψm） were tested as cellular adenosine triphosphate（ATP） level and glutathione（GSH）. Also, the expression and distribution of Cyt C and apoptosis inducing factor（AIF） were observed.RESULTS： SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 μmol/m L Co Cl2 triggered death of 30% of the total cells in cultures within 24 h. At the same time, pretreatment with 100 ng/m L PEDF significantly suppressed the cell death induced by hypoxia（P〈0.05）. The apoptosis induced by treatment of Co Cl2 was that induced cell death accompanied with increasing intracellar ROS and decreasing GSH and ATP level. PEDF pretreatment suppressed these effects（P〈0.05）. Additionally, PEDF treatment inhibited the opening of m PTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway.CONCLUSION： Pretreatment of RGC-5 cells with 100 ng/m L PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of m PTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level＇s reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.

Effect of local injection of pigment epithelium-derived factor on angiogenesis, inflammatory response and apoptosis in the retina of diabetic rats

Objective: To study the effect of local injection of pigment epithelium-derived factor (PEDF) on angiogenesis, inflammatory response and apoptosis in the retina of diabetic rats. Methods:Adult male SD rats were selected as experimental animals and randomly divided into control group, DM group and PEDF group, DM group and PEDF group were made into diabetes models through intraperitoneal injection of streptozotocin, and PEDF group were given local injection of PEDF. The contents of angiogenesis molecules and inflammatory response molecules as well as the expression of apoptosis genes in retinal tissue were measured after intervention. Results: mTOR, VEGF, VEGFR, Ang-2, Tie-2, NF-κB, COX2, iNOS, SDF-1 and CXCR4 contents as well as ASK1, JNK, p38MAPK and Caspase-3 mRNA expression in retinal tissue of DM group were significantly higher than those of control group whereas Survivin and Bcl-2 mRNA expression were significantly lower than those of control group;mTOR, VEGF, VEGFR, Ang-2, Tie-2, NF-κB, COX2, iNOS, SDF-1 and CXCR4 contents as well as ASK1, JNK, p38MAPK and Caspase-3 mRNA expression in retinal tissue of PEDF group were significantly lower than those of DM group whereas Survivin and Bcl-2 mRNA expression were significantly higher than those of control group. Conclusion: Local injection of PEDF has inhibitory effect on the angiogenesis, inflammatory response and apoptosis in the retina of diabetic rats.

Pigment epithelium derived factor(PEDF)prevents methyl methacrylate monomer-induced cytotoxicity in H9c2 cells

Acrylic bone cements are currently the most frequently and extensively used materials in orthopedic implant treatment. However, adverse effects have been described of acrylic bone cement on the cardiovascular system. In the present study, we examined the cytotoxicity of bone cement ingredient methyl methacrylate（MMA） to cardiomyocytes and the potential detoxifying effect of pigment epithelium-derived factor（PEDF） in H9c2 cells.We found that high concentration of MM A（〉 120 mmol/L） led to necrotic cell death in H9c2 cells. However, MMA at low concentrations（30-90 mmol/L） caused apoptosis. Pretreatment of PEDF prevented MMA-induced cytotoxicity. In addition, PEDF enhanced total superoxide dismutase activities, and decreased MMA-induced production of malonaldehyde. Furthermore, MMA-induced downregulation of Akt activity was suppressed by PEDF.PEDF also increased the levels of peroxisome proliferator activated receptor gamma（PPARγ）and lysophosphatidic acids（LPA） through PEDF receptor. These results indicated that PEDF inhibited MMA-induced cytotoxicity through attenuating oxidative stress, activating the phosphatidylinositol 3-kinase（PI3K）/Akt pathway and/or PEDF receptorLPA-PPARy pathways in H9c2 cells. PEDF may be explored as a candidate therapeutic agent for alleviating bone cement implantation syndrome during orthopedic surgery.

Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization （CNV）, and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor （PEDF） could inhibit CNV.Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure （intravitreal injection）; infusing liposomes with the naked plasmid DNA of PEDF into the vitreous （lipofectamine ＋ PEDF）; infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately （retrobular injection）; infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately （vein injection）. The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively. Conclusions Ultrasound-microbubble technique could increase PEDF gene transfer into rats＇ retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.

Anti-inflammatory effects of a synthetic peptide derived from pigment epithelium-derived factor on H2O2-induced corneal injury in vitro

Background The common pathological characteristics of corneal injury include inflammatory factors activation, vascular endothelial cells or inflammatory cells infiltration into lesions, corneal edema, corneal neovascularization （CNV）, and scar formation. PEDF-34 is the functional fragment of pigment epithelium-derived factor （PEDF） that has anti-angiogenic and anti-inflammatory properties and contains an N-terminal 34-amino acid peptide. This study was to investigate the anti- inflammatory effects of PEDF-34 on H202-induced corneal injury in vitro. Methods After cultured in H202 （0.1 mmol/L） for 2 hours, human corneal fibroblasts （HCFs） and human umbilical vein endothelial cells （HUVECs） were treated with PEDF-34-nanoparticles （NPs） at different concentrations （0.1, 0.5, 1.0, 2.0 μg/ml） or 2.0 μg/ml controI-NPs for 24 hours. The viable cells were quantified using the MTT assay. Western blotting or ELISA analysis was performed for measuring the human vascular endothelial growth factor （VEGF） and intercellular adhesion molecule-1 （ICAM-1） expression of both HCFs and HUVECs. VEGF and nuclear factor KB （NF-KB） mRNA levels of HCFs were semi-quantified by RT-PCR. Results The survival rates of HCFs or HUVECs stimulated by H202 did not decrease significantly （P 〉0.05） compared to those in the normal conditions. As compared to controI-NP group, PEDF-34-NPs had dose-dependent inhibitive effect on HUVECs with the MTT assay, but not HCFs. Western blotting analysis showed that the VEGF and ICAM-1 levels in the HCFs and HUVECs stimulated by H202 were significantly higher than those in the normal conditions, which were decreased dramatically in those treated with PEDF-34-NPs. RT-PCR analysis revealed that the VEGF mRNA and NF-KB mRNA levels increased in H202-stimulated HCFs, while both of them decreased in PEDF-34-NP groups dose dependently. Conclusions PEDF-34-NPs may play an important role in regulating the NF-kB pathway, inhibiting inflammatory activity. PEDF-34-NPs may be a potential new drug for treating corneal injury in the future.

Role of pigment epithelium-derived factor on proliferation and migration of choroidal capillary endothelium induced by vascular endothelial growth factor in vitro

Background Pigment epithelium-derived factor （PEDF） is expressed in several normal organs and identified as an inhibitor of neovascularization. In the present study, we investigated the effect of PEDF in an in vitro model of ocular choroidal neovascularization. Methods Microdissection was used to isolate the human choroidal endothelial cells （CECs）, followed by the use of superparamagnetic beads （Dynabeads） coated with the CD31 antibody, which selectively binds to the endothelial cell surface. The mitogenic and motogenic effects of vascular endothelial growth factor （VEGF） on cultured choroidal capillary endothelial cells were examined in the presence or absence of PEDF （1, 10, 100, and 1000 ng/ml） using cell counts and migration assays. Results Cells bound to the beads were isolated using a magnetic particle concentrator and they were successfully cultured and characterized to be endothelial cells that possessed greater than 95% immunoreactivity to von Willebrand factor. PEDF suppressed the proliferation and migration of VEGF-induced choroidal capillary endothelial cells. However, the concentration of PEDF which we used has little effect on normal CECs. Conclusions PEDF played an important role on the growth and migration of VEGF-stimulated choroidal endothelial cell These findings suggest that PEDF may be an effective approach to the treatment of choroidal neovascular disorders.

Predictive Value of Serum Vascular Endothelial Growth Factor and Pigment Epithelium-derived Factor in Ovarian Hyperstimulation Syndrome

Objective To explore whether the serum concentrations of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （PEDF） could serve as the predictors of ovarian hyperstimulation syndrome （OHSS） in patients undergoing in vitro fertilization-embryo transfer （IVF-ET）. Methods Enzyme-linked immunoadsordent assay （ELISA） was employed to measure the serum concentrations of VEGF and PEDF on the day of hCG administration, oocyte retrieval and embryo transfer, respectively. Based on OHSS classification of the criteria of Golan, 85 patients were divided into three groups. Patients in group A （n=10） showed symptoms of severe OHSS and patients in group B （n=13） suffered from moderate OHSS. The control group （group C, n=62） contained patients without symptoms of OHSS as well as patients with mild OHSS.Results In groups A, B and C, serum concentrations of PEDF on the day of hCG administration （h-PEDF）（166.54 ± 102.81 pg/ml, 159.45 ±136. 77 pg/ml, 172.05±170.95 pg/ml, P=0.48）, oocyte retrieval （o-PEDF）（176.91 ± 103.37 pg/ml, 122.52± 92.54 pg/ml, 179.82±177.47 pg/ml, P=0.27） and embryo transfer （e-PEDF）（169.02± 240.08 pg/ml, 136.80 ±139.21pg/ml, 157.38 ±222.54 pg/ml, P=0.95）, h-VEGF （175.55 ± 103.54 pg/ml, 218.84 ±179.70pg/ml, 153.39±145.06 pg/ml, P=0.36） and o- VEGF （171.93 ± 128.55 pg/ml, 220.36±149.82 pg/ml, 138. 74 ±% 139.30 pg/ml, P=0. 15） showed no significant differences. There was a statistical difference in serum concentration of e-VEGF between group A （197.04±156.63 pg/ml） and group C （110.69±49.55 pg/ml）（P=0.008）. The serum level of estradiol showed a positive correlation with the count of large follicles （r=0. 744）. The ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF were calculated and showed a clear difference among groups A, B and C （4.04±3.39, 2.10±2.14, 1.05± 4.80, P〈0.001; 4.54 5.69, 2.29 ±1.67, 0.94 ±0.59, P〈0.001; 5.43±6.16, 1.81±1.36, 2.42±2.60, P=0.04）. Conclusion While neither serum concentrations of VEGF nor PEDF can be used as an OHSS predictor, the ratios of h-VEGF/h-PEDF, o-VEGF/o-PEDF and e-VEGF/e-PEDF may have great predictive value.

色素上皮细胞衍生因子对甲状腺乳头状癌细胞作用的研究

目的探讨重组色素上皮细胞衍生因子(rPEDF)对甲状腺乳头状癌细胞(TPC-1)增殖、侵袭和表达有关的血管活性因子的影响。方法采用四甲基偶氮唑蓝(MTT)法检测癌细胞增殖率,观察不同浓度rPEDF对TPC-1细胞增殖活性的影响;细胞侵袭实验检测rPEDF抑制甲状腺乳头状癌细胞侵袭性;酶联免疫吸附试验(ELISA)检测血管内皮细胞生长因子(VEGF)与血小板反应素-1(TSP-1)的变化。结果甲状腺乳头状癌细胞穿透数为(91.4±20.3)%明显多于rPEDF处理过的细胞(43.6±13.8)%的2.1倍,加入促血管生成因子(VEGF)后,细胞穿透数减少至72.4%[(72.4±16.5)%,P<0.05],表明rPEDF能有效抑制甲状腺乳头状癌细胞的体外侵袭性,至少部分是在VEGF调节的作用下。TPC-1PEDF组(将rPEDF加入甲状腺乳头状癌细胞TPC-1中)中VEGF表达量为(0.38±0.04)IU/mL,与未加rPEDF蛋白的甲状腺乳头状癌细胞作对照[TPC-1con组,(2.11±0.02)IU/mL]比较,平均减少5.6倍(P<0.01),同时TSP-1的表达量TPC-1PEBF组为(1298±116)IU/mL,与TPC-1con组的(251±21)IU/mL比较,平均表达上调5.2倍(P<0.01)。结论 rPEDF能抑制甲状腺乳头状癌细胞增殖、侵袭并改变其表达。

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells

AIM: To investigate the regulation of vascular endothelial growth factors(VEGF) and pigment epithelium-derived factor(PEDF) expression by autophagy in retinal pigment epithelium(RPE) cells on exposure to hypoxia. METHODS: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O_2/5% CO_2/94% N_2 for 24 h; the hypoxia + 3-methyladenine(3-MA) group was pretreated with 10 mmol/L 3-MA for 1 h and then in the hypoxic incubator for 24 h; and the hypoxia + chloroquine(CQ) group was pretreated with 50 μmol/L CQ for 1 h and then in the hypoxic incubator for 24 h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope(TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. RESULTS: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3 B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3 B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. CONCLUSION: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.

Inhibition of choroidal neovascularization by lentivirusmediated PEDF gene transfer in rats

AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.

Protective effects of piperine on the retina of mice with streptozotocin-induced diabetes by suppressing HIF-1/VEGFA pathway and promoting PEDF expression

AIM:To evaluate the protective mechanisms of piperine in the retina of mice with streptozotocin-induced diabetes.METHODS:In experiments in vitro,stimulation by chemical hypoxia was established in ARPE-19 cells.Then,the expression of hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA),and pigment epithelium-derived factor(PEDF)was assessed at the m RNA and protein levels.In experiments in vivo,diabetes mellitus was established by intraperitoneally injecting 150 mg/kg streptozotocin once.After 3 wk of the onset of diabetes,15 mg/kg piperine was intraperitoneally injected once daily for 1 or 3 wk.Then,the retinal morphology and m RNA and protein expression were assessed.RESULTS:In hypoxia,1-100μmol/L piperine significantly decreased the expression of VEGFA m RNA and increased the expression of PEDF m RNA without affecting HIF-1αm RNA.Meanwhile,100μmol/L piperine substantially decreased the protein level of VEGFA and increased the protein level of PEDF.The HIF-1αprotein level was also hampered by piperine.In the diabetic retina of mice,the morphological damage was alleviated by piperine.Likewise,the retinal vascular leakage was substantially decreased by piperine.Further,the protein levels of HIF-1αand VEGFA were significantly reduced by piperine.Moreover,the level of the antiangiogenic factor of PEDF dramatically increased by piperine.CONCLUSION:Piperine may exert protective effects on the retina of mice with diabetes via regulating the pro-antiangiogenic homeostasis composed of HIF-1/VEGFA and PEDF.

Contemporary Approaches for Identifying Rare Bone Disease Causing Genes

Recent improvements in the speed and accuracy of DNA sequencing, together with increasingly sophisti- cated mathematical approaches for annotating gene networks, have revolutionized the field of human genetics and made these once time consuming approaches assessable to most investigators. In the field of bone research, a particularly active area of gene discovery has occurred in patients with rare bone disorders such as osteogenesis imperfecta （OI） that are caused by mutations in single genes. In this perspective, we highlight some of these technological advances and describe how they have been used to identify the genetic determinants underlying two previously unexplained cases of OI. The widespread availability of advanced methods for DNA sequencing and bioinformatics analysis can be expected to greatly facilitate identification of novel gene networks that normally function to control bone formation and maintenance.