Pigment Epithelium-derived Factor Expression Levels Research Articles

The role of PEDF, VEGF, and Ki-67 in tissue invasion and tumor angiogenesis of medullary thyroid carcinoma

Objective: This study investigated the role of pigment epithelium-derived factor (PEDF), vascular endothelial growth factor (VEGF), nuclear factor kappa B (NF-kB), and Ki-67 proliferation index (PI) factors in Medullary thyroid carcinoma (MTC) tissue progression and their relationship with tumor angiogenesis. Immunohistochemical (IHC) analyses of PEDF expression in various human tumor samples and healthy tissues have shown that high levels of PEDF expression are associated with a positive prognosis, and low levels of PEDF suggest a poorer prognosis. With the completion of further studies showing the antitumor effects of PEDF on different types of cancer, PEDF’s potential as a therapeutic agent has become increasingly promising. To the best of our knowledge, the role of PEDF in tumor angiogenesis in MTC has not been previously investigated. Material and Methods: Thirty-seven tissue samples were retrospectively analyzed and sent/removed for pathology after total or subtotal thyroidectomy and archived between 01.01.2000 and 31.12.2015 in the Pathology Department of Hacettepe University. Relationships between categorical variables are analyzed using Pearson Chi-squared or Fisher’s Exact tests.The correlation between two numerical variables is analyzed using Spearman’s Rho correlation coefficient, and a comparison of numerical variables between two groups is made using the Mann-Whitney U test. Results: The clinicopathological features of the patients were evaluated as sporadic or hereditary, tumor node metastasis (TNM) staging, single or multifocal focus, recurrence or regional or distant metastasis, progression-free survival time (PFS), and appropriate pathology tissue samples were determined as PEDF, VEGF, NF-kB, Ki-67 staining was compared. As a result of this comparison, a moderate negative correlation was found between VEGF expression level and PFS (rho = - 0,407, p = 0,019). No statistically significant correlation was found between single or multifocal groups and Ki-67 proliferation index (PI) expression levels (p = 0.070), but this is a small p value, a variable that is likely to find significant differences in studies with larger sample groups.The relationship between PEDF, VEGF, NF- kB, Ki-67 PI factors and tumor angiogenesis as reflected by microvessel density (MVD) was also investigated. To this end, we correlated the expression levels of PEDF, VEGF, NF-kB, and Ki-67 staining of suitable pathology tissue samples of the patients with the results obtained from the staining with CD 31. As a result of this comparison, no statistically significant relationship was found. Conclusions: We demonstrated and described for the first time PEDF expression patterns in MTC tumor tissues. A direct relationship between PEDF expression level and angiogenesis, angiogenetic and clinicopathological factors does not seem to exist, at least as it pertains to the group studied. Further studies are required to fully elucidate mechanisms for tumor angiogenesis and invasion in MTC.

Ренопротекторний ефект метформіну в щурів із цукровим діабетом 2-го типу реалізується шляхом посилення експресії фактора пігментного епітелію в тканині нирок

Актуальність. Діабетична нефропатія стала основною причиною термінальної стадії ниркової недостатності. Фактор пігментного епітелію (ФПЕ) є ендогенним протизапальним фактором in vivo, що може пригнічувати експресію патогенних чинників трансформуючого фактора росту β1 и фактора росту сполучної тканини, а також продукцію білка позаклітинного матриксу в нирці за наявності цукрового діабету. Мета дослідження: вивчити вплив метформіну на експресію ФПЕ у тканині нирок щурів із цукровим діабетом 2­го типу і встановити його можливі основні захисні механізми при ураженні нирок. Матеріали та методи. Десять самців щурів лінії Sprague­Dawley були рандомізовані як нормальна контрольна група. Цукровий діабет 2­го типу індукували дієтою з високим вмістом жиру і внутрішньоперитонеальним введенням 30 мг/кг стрептозотоцину. Загалом 30 щурів із діабетом 2­го типу були розподілені на 3 групи, які отримували метформін 300 мг/(кг • д) (група МЕТ, n = 10), глібенкламід 5 мг/(кг • д) (група ГЛІ, n = 10) або фізіологічний розчин (група ФР, n = 10) шляхом введення протягом восьми тижнів. Аналізували різні біохімічні показники, дані гістопатологічного дослідження нирок і рівні експресії ФПЕ у нирковій тканині. Результати. На восьмому тижні рівні глюкози крові натще (ГН), глікованого гемоглобіну (HbA1c), тригліцеридів, екскреції альбуміну із сечею і ФПЕ, сироваткового креатиніну й азоту сечовини крові в групі МЕТ і групі ГЛІ вірогідно знизилися порівняно з групою ФР, вірогідних відмінностей у показниках глюкози крові натщесерце і глікованого гемоглобіну між групою МЕТ і групою ГЛІ не виявлено. При гістологічному дослідженні виявлено поліпшення гломерулярних патологічних змін, спричинених цукровим діабетом, після лікування МЕТ і ГЛІ порівняно з групою ФР. Крім того, зменшувалася екскреція альбуміну із сечею і ФПЕ, спостерігалася менша вираженість патологічних змін у клубочках і підвищувалася експресія білка і мРНК ФПЕ ниркової тканини в групі MЕТ порівняно з групою ГЛІ. Висновки. Метформін знижує екскрецію альбуміну із сечею в діабетичних щурів, покращує морфологію і зменшує вираженість структурних уражень подоцитів. Це може бути частково пов’язане з його роллю у відновленні експресії ФПЕ та інгібуванні екскреції ФПЕ із сечею.

Pigment epithelium-derived factor, a novel decidual natural killer cells-derived factor, protects decidual stromal cells via anti-inflammation and anti-apoptosis in early pregnancy

What is the role of pigment epithelium-derived factor (PEDF) from decidual natural killer (dNK) cells during early pregnancy? PEDF from dNK cells limits the lipopolysaccharide (LPS)-induced apoptosis and inflammation of decidual stromal cells (DSCs) to maintain DSCs homoeostasis and immune balance at the maternal-foetal interface during early pregnancy. dNK cells, which secrete PEDF, play critical roles during pregnancy via a series of key regulators. PEDF, a multifunctional endogenous glycoprotein, exhibits a wide range of biological actions upon angiogenesis, inflammation, metabolic homoeostasis, immunomodulation etc., providing potential clinical applications. Natural killer (NK) cells from decidua and peripheral blood as well as DSCs isolated from normal pregnancy (NP) during the first trimester (6-10 weeks) and the matched patients suffering recurrent miscarriage (RM) were studied. RNA-sequencing analysis of dNK cells was performed to screen for potential key genes involved in RM. The expression of PEDF in dNK cells in NP and RM was examined. A coculture system with LPS-stimulated DSCs and NK cell supernatants derived from NP or RM was established to explore the regulatory mechanisms of PEDF at the maternal-foetal interface. Peripheral blood and decidual tissues were obtained from women with NP (n = 61) and RM (n = 21). The expression levels of PEDF in NK cells and its receptor (PEDFR) on DSCs were analysed using flow cytometry, western blot and immunohistochemistry. Purified peripheral natural killer (pNK) cells were cocultured with DSCs or trophoblast cells or a combination of both cell types, and PEDF expression in pNK cells was then examined by flow cytometry. DSCs were treated with LPS, an outer-membrane component of Gram-negative bacteria, thereby mimicking an enhanced inflammatory status within decidua, and were cocultured with dNK cell supernatants from NP or RM. In the coculture system, plasmids expressing short hairpin RNA were used to silence PEDFR on DSCs and block the PEDF/PEDFR interaction. Inflammatory cytokines and apoptosis of DSCs treated as described above were assessed by flow cytometry. Western blotting was performed, and the specific signal pathway inhibitors were used to determine downstream PEDF/PEDFR signalling in early decidua. Markedly higher RNA (P < 0.001) and protein expression of PEDF (P < 0.01) was detected in normal dNK cells when compared with pNK cells. Compared with pNK cells cultured alone, PEDF expression in pNK cells was elevated after coculture with DSCs (P < 0.01) or trophoblast cells (P < 0.001). The increased pro-inflammatory cytokine, tumour necrosis factor-α and apoptosis of DSCs following LPS stimulation were suppressed by recombinant human PEDF (P < 0.001) or the supernatant of dNK cells derived from NP (P < 0.001). However, these effects were somewhat abrogated when the PEDF/PEDFR interaction was blocked with PEDFR short hairpin sRNA (P < 0.01). Furthermore, dNK cell-derived PEDF protected DSCs from LPS-induced inflammation via inhibition of nuclear factor kappa-B activation, while also protecting DSCs from LPS-induced apoptosis via promotion of extracellular signal-regulated kinase expression. Compared with NP, both significantly decreased PEDF RNA (P < 0.001) and protein expression (P < 0.001) in dNK cells, but not in pNK cells (P > 0.05), were detected in women with RM. PEDFR on DSCs was also decreased within RM compared with that within NP (P < 0.001). As a result, dNK cell-mediated anti-inflammation (P < 0.01) and anti-apoptosis (P < 0.05) for protection of LPS-treated DSCs was attenuated in patients suffering from RM. N/A. We cannot exclude the possibility that the differences in amounts of PEDF and its receptor in tissue from NP versus RM women could be caused by the miscarriage event in women with RM. Our experiments only involved human samples investigated in vitro. Experiments in animal models and human study cohorts are still needed to confirm these findings and further clarify the role of PEDF-PEDFR in NP and/or RM. To the best of our knowledge, this is the first study to demonstrate PEDF expression and function at the maternal-foetal interface in the first trimester, providing further evidence that PEDF exhibits functional diversity and has great potential for clinical application(s). The findings of selectively high expression of PEDF in normal dNK cells and the PEDF-mediated role of dNK cells during NP and RM help to further elucidate the immune mechanisms behind RM. This work was supported by the National Basic Research Programme of China (2017YFC1001403 and 2015CB943300), Nature Science Foundation from National Nature Science Foundation of China (NSFC; 31970859, 81630036, 81501334, 91542116, 31570920, 81490744 and 31171437), the Innovation-oriented Science and Technology Grant from NHC Key Laboratory of Reproduction Regulation (CX2017-2), the Programme of Shanghai Academic/Technology Research Leader (17XD1400900) and the Key Project of Shanghai Basic Research from Shanghai Municipal Science and Technology Commission (STCSM; 12JC1401600). None of the authors has any conflict of interest to declare.

Therapeutic Potential of Pigment Epithelium-derived Factor in Cancer.

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

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

ObjectivesSystemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1);...

Pigment epithelium-derived factor has a role in the progression of papillary thyroid carcinoma by affecting the HIF1α-VEGF signaling pathway.

The progression mechanism of papillary thyroid carcinoma (PTC) remains largely unknown. Accumulating evidence has suggested that various targets of pigment epithelium-derived factor (PEDF) are able to inhibit cancer progression. The aim of the present study was to examine PEDF expression in PTC patients and to investigate its relationship with aggressive clinicopathological features, as well as to explore whether PEDF affects the progression of PTC via the hypoxia-inducible factor 1α (HIF1α)-vascular endothelial growth factor (VEGF) pathway. A total of 271 patients with PTC, including 24 men and 247 women, were enrolled in the present study. Relevant patient data, including demographic features, preoperative clinical features and pathological features, were collected for analysis. The protein expression levels of PEDF in PTC tissues were detected using immunohistochemical staining, and the mRNA expression levels of PEDF, VEGF and HIF1α in 15 PTC tissues with lymph node metastasis (LNM) and 10 tissues without LNM were detected using reverse transcription-quantitative polymerase chain reaction. Immunohistochemical staining with an anti-PEDF antibody detected PEDF expression in 74.5% of the PTC tissues. PEDF expression levels were significantly correlated with LNM, extrathyroid invasion, a high TNM stage, the presence of the BRAFV600E mutation and tumor size. PEDF mRNA expression levels were significantly decreased in PTC tissues with LNM, as compared with PTC tissues without LNM, while the mRNA expression levels of HIF1α and VEGF were markedly increased in PTC tissues with LNM. Taken together, the results of the present study suggested that PEDF plays a role in the progression of PTC, and that PEDF may exert an anti-angiogenesis role by affecting the HIF1α-VEGF pathway, eventually inhibiting the metastasis of PTC.

Polymeric Nanomedicine for Combined Gene/Chemotherapy Elicits Enhanced Tumor Suppression.

Combination treatment through simultaneous delivery of DNA and anticancer drugs with nanoparticles has been demonstrated to be an elegant and efficient approach for cancer therapy. Herein, we employed a combination therapy for eliminating both the tumor cells and intratumoral neovascular network based on the nanoplatform we designed. Pigment epithelium-derived factor (PEDF) gene, a powerful antiangiogenic agent, and the clinically widely used chemotherapy agent paclitaxel (PTX) were simultaneously encapsulated in the same nanoparticle by a modified double-emulsion solvent evaporation method. The dual-drug-loaded nanoparticles (D/P-NPs) exhibited a uniform spherical morphology and released PTX and PEDF gene in a sustained manner. D/P-NPs showed an enhanced antitumor effect on C26 and A549 cells and a stronger inhibitory activity on proliferation of HUVECs. Moreover, D/P-NPs could dramatically elevate the PEDF expression levels in both C26 and A549 cells in comparison with PEDF gene loaded nanoparticles and significantly promote the cellular uptake of PTX. Additionally, microtubules were stabilized and G2/M phase arrest along with a higher subG1 cell population was induced by D/P-NPs in contrast to PTX or PTX loaded nanoparticles. Besides, D/P-NPs showed sustained release of PTX and PEDF gene in tumors as well as long-term gene expression. A significantly improved anticancer effect was also demonstrated in a C26 subcutaneous tumor model using this combinational therapy. D/P-NPs could sharply reduce the microvessel density and significantly promoted tumor cell apoptosis in vivo. More importantly, the in vivo distribution, serological and biochemical analysis, and H&E staining revealed that D/P-NPs had no obvious toxicity. Our study suggested that this novel polymeric nanomedicine had great potential for improving the therapeutic efficacy of combined gene/chemotherapy of cancer.

Expression of pigment epithelium-derived factor is associated with a good prognosis and is correlated with epithelial-mesenchymal transition-related genes in infiltrating ductal breast carcinoma.

Epithelial-mesenchymal transition (EMT) is a pivotal event in the progression of cancer towards metastasis. Given that pigment epithelium-derived factor (PEDF) inhibits angiogenesis, the present study analyzed whether PEDF expression is associated with EMT and prognosis in invasive ductal breast cancer (IDC). Immunohistochemical analysis was used to examine the expression levels of PEDF, E-cadherin, vimentin, Snail and nuclear factor-κB (NF-κB) in 119 cases of IDC. Correlations between PEDF expression and EMT-related genes, and clinicopathological features and clinical prognosis were analyzed. E-cadherin, vimentin, Snail and NF-κB expression was correlated with tumor size, lymph node metastasis and clinicopathological stage. PEDF expression was closely associated with tumor size. Spearman's rank correlation analysis revealed a positive correlation between PEDF and E-cadherin, vimentin, Snail and NF-κB expression (P<0.05). Additionally, Kaplan-Meier survival analysis demonstrated that the five-year survival rate was higher for patients with PEDF- and E-cadherin-positive tumors, but was lower for those with vimentin-, Snail- and NF-κB-positive tumors. Vimentin, E-cadherin and NF-κB levels were dependent prognostic factors of favorable outcomes in IDC, as determined by Cox multivariate analysis. PEDF expression in breast cancer was significantly associated with EMT-related genes, suggesting that it may be an EMT suppressor. However, its potential as a prognostic indicator in breast cancer warrants further investigation.

Abstract 3359: PEDF modulatory action on macrophages: A new way to curb prostate cancer development

Abstract Macrophages have been described as one of the main inflammatory components involved in prostate cancer (PCa) initiation, progression, and metastasis. PEDF (Pigment Epithelium-Derived Factor) is an anti-angiogenic factor with differentiation activities and was recently suggested as an immune-modulating factor. PEDF expression has been shown to be down-regulated in PCa compared to normal tissues. In previous studies we have demonstrated that PEDF re-expression in PCa cells curbs tumor growth in vivo and significantly prolongs the survival of tumor-bearing mice. Others have shown that PEDF expression increased the recruitment of tumor-cytotoxic macrophages into orthotopic MatLyLu rat prostate tumors suggesting a link between PEDF and inflammation in PCa. While PEDF's role in inflammation has been suggested, the precise modes of action of PEDF on macrophages still remain unknown and necessitate further investigation. Our preliminary data have showed that PEDF stimulates the migration of monocytes/macrophages using in vitro chemotaxis boyden chamber assay and towards tumor 3D spheroids. We showed that PEDF expression levels positively correlate with macrophage density in human prostate. In both cell lines and prostate specimens, we have demonstrated that PEDF directly induces the polarization of macrophages and bone marrow derived macrophages (BMDMs) towards a M1/tumor-cytotoxic pathway. As a result of their differentiation, we have found that PEDF stimulates the phagocytosis of tumor cells, which suggest another mechanism by which PCa growth is halted. We are currently investigating the molecular mechanisms by which PEDF induces migration, differentiation and phagocytic activity in macrophages. PEDF mechanisms are being investigated by expression levels of PEDF receptors (ATP5B, PNPLA2, LRP6) in macrophage cell lines and BMDMs. So far we have been identified ATP5B and PNPLA2 as the two main receptors expressed in both cell types. While all three receptors in cell lines increased, only ATP5B and PNPLA2 receptors increased in BMDMs in response to PEDF. To map the functional region involved in PEDF's inflammatory action, we are investigating the PEDF-derivative synthetic 18-mer peptide (residues 39-57). P18 has been shown to block endothelial cell chemotaxis and induces apoptosis in vitro, and to be more effective than its parental 34-mer peptide (residues 24-57) in blocking growth and angiogenesis in PCa. We were able to demonstrate that macrophages treated with P18 in comparison to PEDF, show a higher efficacy for macrophages differentiation. Using confocal microscopy we demonstrated a larger rate of phagocytosis of PCa cells by macrophages than compared to PEDF. The results of our study are of importance as they suggest that macrophages may play a key role in PEDF anti-tumor effects. A better understanding of PEDF may lead to further development of PEDF-based anticancer therapy or improvement of alternatives to chemotherapy for PCa. Citation Format: Dalia Martinez-Marin, Thomas Nelius, Olga Volpert, Stephanie Filleur. PEDF modulatory action on macrophages: A new way to curb prostate cancer development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3359. doi:10.1158/1538-7445.AM2015-3359

Hormonal regulation of pigment epithelium-derived factor (PEDF) expression in the endometrium

Pigment epithelium-derived factor (PEDF) is highly expressed in the female reproductive system and is subjected to regulation by steroid hormones in the ovary. As the uterine endometrium exhibits morphological and functional changes in response to estrogen (E2) and progesterone (P4), we aimed at characterizing the expression of PEDF in this component of the female reproductive tract and further at exploring the hormonal regulation of its expression. We found that PEDF is expressed in human and mouse endometrium. We further showed that this expression is subjected to regulation by steroid hormones, both in vivo and in vitro, as follows: E2 decreased PEDF expression and P4 increased its levels. In human endometrial samples, PEDF levels were dynamically altered along the menstrual cycle; they were low at the proliferative and early secretory phases and significantly higher at the late secretory phase. The expression levels of PEDF were inversely correlated to that of vascular endothelial growth factor (VEGF). We also showed that PEDF receptor was expressed in the endometrium and that its stimulation reduced VEGF expression. Illustrating the pattern of PEDF expression during the menstrual cycle may contribute to our understanding of the endometrial complexity.

Positive correlation between PEDF expression levels and macrophage density in the human prostate

In this study, we investigated the capacity of pigment epithelium-derived factor (PEDF) to modulate the recruitment and the differentiation of monocytes/macrophages both in vitro and in human prostate. Using Boyden chambers, we assessed PEDF effect on the migration of monocytes and chemically activated RAW 264.7 macrophages. Normal, prostatitis, and prostate cancer specimens were retrospectively selected and examined by immunohistochemistry for PEDF expression and infiltration of immune CD68 + macrophagic cells. PEDF expression and macrophage density were then correlated with each other and clinicopathological parameters. M1 and M2 differentiation markers were quantified by qRT-PCR, Western blotting, and ELISA. In chemotaxis, PEDF induced the migration of monocytes/macrophages. In immunohistochemistry, macrophages were markedly increased in prostatitis and malignant compared to normal tissues. PEDF was expressed at variable levels in the stroma and epithelium. PEDF mRNA was down-regulated in both prostate cancer and prostatitis compared to normal tissues. In correlation studies, macrophage density and PEDF expression were respectively positively and negatively associated with prostate size. Most importantly, PEDF expression positively correlated with macrophage density. Finally, PEDF stimulated the expression of iNOS, IL12, and TNFα; and inhibited IL10 and arginase 1 in mouse and human macrophages confirming a M1-type differentiation. Our data demonstrate that PEDF acts directly on monocytes/macrophages by inducing their migration and differentiation into M1-type cells. These findings suggest a possible role of macrophages in PEDF anti-tumor properties and may support further development of PEDF-based anti-cancer therapy.

Protective role of steroids on mouse primary RPE cells under hypoxic stress

AbstractPurpose Angiogenesis is a clinically critical aspect of the pathogenesis of many retinal neovascular diseases. Triamcinolone acetonide (TA) is an important anti‐angiogenic and anti‐inflammatory agent. This study has evaluated the effect of TA on the expression of pigment epithelium derived factor (PEDF), thrombospondin‐1 (TSP‐1) and vascular endothelial growth factor A (VEGF‐A) in cultured mouse RPE cells under hypoxic stress.Methods Primary cultures of mouse RPE cells were grown in culture to passage 2‐3 and were then subjected to hypoxic stress. TA (50µg/ml) was used to determine the steroid effect under normoxic (95% air/5 % carbon dioxide) and hypoxic (95% nitrogen/5% carbon dioxide) conditions. Hypoxia was continued for 48 hrs with and without TA. Cells were harvested at 0, 24 and 48 hours and both RNA and protein were extracted. Real Time PCR was used to analyze expression levels of PEDF, TSP‐1 and VEGF‐A. The expression pattern of PEDF, TSP‐1 and VEGF‐A in the presence of TA were quantifie.Results After 24 hours of hypoxia, mRNA expression levels of both PEDF and TSP‐1 were down‐regulated by 2‐fold. Hypoxic stress for 48 hours, resulted in 2.2‐fold down‐regulation of PEDF and a stronger down‐regulation of TSP‐1 of 3.4‐fold. VEGF‐A transcripts levels, on the other hand, showed an opposite response to hypoxic stress, an up‐regulation of 1.9‐fold at 24 hours and 2.7‐fold increase at 48 hours. Hypoxic RPE cells, when exposed to TA, led to the rapid recovery of PEDF and TSP‐1 transcripts at 24 hours after the initiation of hypoxia. VEGF‐A mRNA expression rebounded to control levels in TA treated hypoxic cells. Western blots showed that TSP‐1 protein levels correlated with the PCR results. TA significantly induced the expression of TSP‐1 protein in hypoxic cells, and the strongest expression was at 48 hours.Conclusion The results suggests that TA has a protective effect on PEDF and TSP‐1 levels, and on the fine balance of PEDF, TSP‐1 and VEGF‐A levels.

Therapeutic potential of bone marrow-derived mesenchymal stem cells producing pigment epithelium-derived factor in lung carcinoma

Specific and efficient gene delivery to target cells and the subsequent expression of the RNA and protein is crucial to the success of gene-based therapy for cancer. Mesenchymal stem cells (MSCs) represent novel and efficient tools for delivery of therapeutic agents to tumor cells. In this study, we evaluated the potential of bone marrow-derived mesenchymal stem cells, genetically modified to express pigment epithelium-derived factor (PEDF) for the treatment of Lewis lung carcinoma (LLC). MSCs derived from murine bone marrow were efficiently engineered to express human PEDF by adenoviral transduction, and the expression and bioactivity of the transgenic protein from engineered MSCs were confirmed in vitro. Animal experiments showed that the systemic administration of MSCs treated with PEDF dramatically reduced the growth of LLC tumors and significantly prolonged survival. Immunohistochemistry analysis of the tumors from MSC-PEDF-treated animals indicated an increase in apoptosis and a decrease in microvessel density. ELISA showed that the group of MSCs treated with PEDF had relatively higher expression levels of PEDF in tumor tissue and lower levels in serum compared with the free Ad-PEDF group. These results suggest that MSCs have potential use as effective delivery of vehicles for therapeutic genes in the treatment of LLC.

Administration of Pigment Epithelium-Derived Factor Inhibits Left Ventricular Remodeling and Improves Cardiac Function in Rats with Acute Myocardial Infarction

Oxidative stress and inflammation are involved in cardiac remodeling after acute myocardial infarction (AMI). We have found that pigment epithelium-derived factor (PEDF) inhibits vascular inflammation through its anti-oxidative properties. However, effects of PEDF on cardiac remodeling after AMI remain unknown. We investigated whether PEDF could inhibit left ventricular remodeling and improve cardiac function in rats with AMI. AMI was induced in 8-week-old Sprague-Dawley rats by ligation of the left ascending coronary artery. Rats were treated intravenously with vehicle or 10 μg PEDF/100 g b.wt. every day for up to 2 weeks after AMI. Each rat was followed until 16 weeks of age. PEDF levels in infarcted areas and serum were significantly decreased at 1 week after AMI and remained low during the observational periods. PEDF administration inhibited apoptotic cell death and oxidative stress generation around the infarcted areas at 2 and 8 weeks after AMI. Further, PEDF injection suppressed cardiac fibrosis by reducing transforming growth factor-β and type III collagen expression, improved left ventricular ejection fraction, ameliorated diastolic dysfunction, and inhibited the increase in left ventricular mass index at 8 weeks after AMI. The present study demonstrated that PEDF could inhibit tissue remodeling and improve cardiac function in AMI rats. Substitution of PEDF may be a novel therapeutic strategy for cardiac remodeling after AMI.

Inhibition of Chemical Cautery–Induced Corneal Neovascularization by Topical Pigment Epithelium–Derived Factor Eyedrops

To evaluate the effect of topically administered pigment epithelium-derived factor (PEDF) on experimentally induced corneal neovascularization (NV) in a rat model. Corneal chemical cauterization was induced in the left eye by using silver nitrate sticks in 160 anesthetized male Sprague-Dawley rats. The rats were randomly assigned to 4 groups with 40 rats each for topical administration of recombinant PEDF, chloramphenicol, chondroitin sulfate, and normal saline (as control). At different intervals (3, 10, 15, and 30 days) of the treatment, rats were euthanized and the corneas removed for immunohistochemistry and Western blot analyses to measure expression levels of PEDF, vascular endothelial growth factor (VEGF), and CD34, an endothelial maker. The right eyes were used as normal control. There were high levels of PEDF expression and low levels of VEGF and CD34 in the normal cornea. VEGF and CD34 levels were significantly induced by chemical cauterization in the groups treated with chloramphenicol, chondroitin sulfate, and normal saline, demonstrating corneal NV. The VEGF and CD34 levels reached a plateau in the cornea on the 10th day after cauterization and remained at high levels thereafter. In contrast, the PEDF treatment prevented the overexpression of VEGF and CD34 induced by the cauterization. PEDF downregulates VEGF expression and inhibits corneal NV induced by chemical cauterization. The results suggested that PEDF has therapeutic potential for corneal neovascular diseases.

The molecular impact of pigment epithelium-derived factor, PEDF, on lung cancer cells and the clinical significance

Pigment epithelium-derived factor (PEDF) is an endogenous protein factor that has been shown to act as anti-angiognesis factor. The present study aimed to determine the direct biological effects of PEDF on lung cancer cells and deduce a clinical relevance in patients with lung cancer, major cause of death worldwide in which the knowledge of PEDF remains poor. We constructed a mammalian expression system for human PEDF produced recombinant PEDF (rhPEDF) protein from 3T3 cells. The expression of PEDF was examined using SDS-PAGE and Western blot analysis. Using the rhPEDF protein, we investigated the biological function of PEDF in the lung cancer cells as well as endothelial cells. PEDF expression levels were assessed in a cohort of human lung cancer specimen (77 pairs of matched normal and tumour tissues), in association with patient clinical variables and survival, using quantitative analysis of PEDF. In vitro, we found that administration of rhPEDF on two lung cancer cell lines (A549 and SK-MES1) significantly reduced tumour cell growth (P<0.05) with no significant effect on the growth of vascular cell line (HECV). We also found that rhPEDF significantly decreased lung cancer motility and adhesion to extracellular matrix (Matrigel) when compared with the control cells (P<0.05). We showed that reduced PEDF levels in lung cancer tissues significantly correlated with lymph node metastasis and an overall poor prognosis in the lung cancer patients. PEDF suppresses the growth and motility of lung cancer cells and has a significant correlation with the clinical outcome of the patients. These results contribute to our understanding of the molecular mechanisms of PEDF and indicates a potential prognostic and therapeutic impact of PEDF in lung cancer.

Administration of pigment epithelium-derived factor (PEDF) reduces proteinuria by suppressing decreased nephrin and increased VEGF expression in the glomeruli of adriamycin-injected rats

Pigment epithelium-derived factor (PEDF) is a glycoprotein with potent neuronal differentiating activity. We, along with others, have recently found that PEDF inhibits retinal hyperpermeability by counteracting the biological effects of vascular endothelial growth factor (VEGF). However, the protective role of PEDF against nephrotic syndrome (NS), a condition of hyperpermeability in the glomerular capillaries, remains to be elucidated. In this study, we investigated whether and how PEDF reduced proteinuria in rats with adriamycin (ADR)-induced nephropathy (ADN), an experimental model of NS. ADN was induced by a single intravenous injection of doxorubicin hydrochloride (n = 12). Half the ADN rats were intravenously administrated human recombinant PEDF; the other half were given vehicle everyday for up to 14 days. Control rats (n = 6) received vehicle only. In ADN, expression levels of PEDF in isolated glomeruli were significantly decreased, which were associated with a marked proteinuria and increased urinary excretion of nephrin, an index of podocyte damage. Loss of nephrin and decreased podocyte cell number and fusion of foot processes of podocytes with nuclear factor-kappa B (NF-kappaB) activation and VEGF overexpression were also observed in the glomeruli of rats with ADN. Intravenous administration of PEDF ameliorated all of these changes in ADN rats. The present findings suggest that PEDF could reduce proteinuria by suppressing podocyte damage and decreased nephrin as well as increased VEGF expression in the glomeruli of ADN rats. Pharmacological up-regulation or substitution of PEDF may offer a promising therapeutic strategy for the treatment of nephrotic syndrome.

Abstract 225: Role Of PEDF In Regulation Of Skeletal Blood Flow And Neovascularization In Ischemic Limb Via Interaction With VEGF

[Introduction] We reported that pigment epithelium-derived factor (PEDF) plays an important role in the angiogenesis, vascular permeability and regulation of blood flow in the eye and brain via interaction with VEGF. [Objective] We examined the role of PEDF in ischemia or VEGF-related angiogenesis or regulation of blood flow in severe limb ischemia. [Methods] Unilateral hindlimb ischemia was performed in klotho-mice by binding the unilateral femoral artery. After hindlimb ischemia was surgically induced, we injected saline (control group) or VEGF (3 μg/day × 7, VEGF group) to ischemic limb from day 1 to 7. The expression level of PEDF in the ischemic tissue was examined by RT-PCR method. Furthermore, we injected recombinant mouse PEDF (4 μg/day × 7) or anti-PEDF antibody (60 μg/day × 7) in addition to VEGF (VEGF + PEDF group and VEGF + anti-PEDF group, respectively) to ischemic limb from day 1 to 7. We evaluated limb blood flow by laser doppler blood-flow (LDBF) at day 0, 3, 7, and 14, and number of CD34 positive cells in the ischemic tissue by histology as an index of ischemic angiogenesis at day 7 in all groups. [Results] Expression levels of PEDF in ischemic tissue were significantly elevated in VEGF group. In VEGF group, the ischemic/normal limb blood flow ratio by LDBF analysis and number of CD34 positive cells were significantly increased compared with control group (p &lt; 0.05). The effects of VEGF on blood flow or number of CD34 positive cells were completely abolished by administration of PEDF. Furthermore, anti-PEDF group, accelerated recover of blood flow in ischemic limb, but not number of CD34 positive cells compared in VEGF administration. [Conclusion] VEGF augmented the expression level of PEDF in the ischemic limb. VEGF-induced ischemic angiogenesis was abolished by exogenous administration of PEDF. Furthermore, anti-PEDF antibody augmented VEGF-induced increase of blood flow. These results suggest that PEDF may play an important role in regulation of skeletal blood flow and neovascularization in ischemic limb via interaction with VEGF.

Pigment Epithelium-Derived Factor Inhibits Neointimal Hyperplasia after Vascular Injury by Blocking NADPH Oxidase-Mediated Reactive Oxygen Species Generation

Pigment epithelium-derived factor (PEDF) inhibits cytokine-induced endothelial cell activation through its antioxidative properties. However, the effect of PEDF on restenosis remains to be elucidated. Because the pathophysiological feature of restenosis is characterized by increased superoxide formation and accumulation of smooth muscle cells (SMCs), PEDF may inhibit this process via suppression of reactive oxygen species generation. We investigated here whether PEDF could prevent neointimal formation after balloon injury. PEDF levels were decreased in balloon-injured arteries. Adenoviral vector encoding human PEDF (Ad-PEDF) prevented neointimal formation. Expression and superoxide generation of the membrane components of NADPH oxidase, p22(phox) and gp91(phox), in the neointima were also suppressed by Ad-PEDF. Ad-PEDF reduced G(1) cyclin (cyclin D1 and E) expression and increased p27, a cyclin-dependent kinase inhibitor. In vitro, PEDF inhibited platelet-derived growth factor-BB-induced SMC proliferation and migration by blocking reactive oxygen species generation through suppression of NADPH oxidase activity via down-regulation of p22(PHOX) and gp91(PHOX). PEDF down-regulated G(1) cyclins and up-regulated p27 levels in platelet-derived growth factor-BB-exposed SMCs as well. These results demonstrate that PEDF could inhibit neointimal formation via suppression of NADPH oxidase-mediated reactive oxygen species generation. Our present study suggests that substitution of PEDF may be a novel therapeutic strategy for restenosis after balloon angioplasty.

Focus on Molecules: Pigment epithelium-derived factor (PEDF)

Microvascular diseases are among the most clinically important diseases, and vascular abnormalities are central in the development of such diseases. Pigment epithelium-derived factor (PEDF), a potent inhibitor of angiogenesis, exerts antiangiogenic effects without affecting the structure and function of normal blood vessels. PEDF also has neurotrophic effects, which may be a potential direction for the future treatment of angiogenic diseases with lower side effects. Here, we review (i) the expression levels of PEDF in several important organs and clinically common microvascular diseases and (ii) the effects of its absence and presence on the vasculature and nerves, focusing on both angiogenic and neuroprotective aspects. These effects are both positive and negative, and have the potential to be exploited. Additionally, we summarize and compare various PEDF agents and their possible advantages and disadvantages as therapeutic agents, which, despite most still being in the experimental stage, may provide some new opportunities for future clinical treatments and interventions in PEDF-targeted microvascular diseases.