Guoquan Gao

Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor

Pigment epithelium‐derived factor (PEDF) is a potent angiogenic inhibitor. Reduced PEDF levels are associated with diabetic retinopathy. However, the mechanism for the protective effects of PEDF against diabetic retinopathy (DR) is presently unclear. As inflammation plays a role in DR, the present study determined the effect of PEDF on inflammation. Western blot analysis and ELISA demonstrated that retinal and plasma PEDF levels were drastically decreased in rats with endotoxin‐induced uveitis (EIU), which suggests that PEDF is a negative acute‐phase protein. Intravitreal injection of PEDF significantly reduced vascular hyper‐permeability in rat models of diabetes and oxygen‐induced retinopathy, correlating with the decreased levels of retinal inflammatory factors, including VEGF, VEGF receptor‐2, MCP‐1, TNF‐α, and ICAM‐1. In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF‐α and ICAM‐1 expression under hypoxia. Moreover, down‐regulation of PEDF expression by siRNA resulted in significantly increases of VEGF and TNF‐α secretion in retinal Müller cells. These findings suggest that PEDF is a novel endogenous anti‐inflammatory factor in the eye. The decrease of ocular PEDF levels may contribute to inflammation and vascular leakage in DR.

Activation of the Wnt Pathway Plays a Pathogenic Role in Diabetic Retinopathy in Humans and Animal Models

Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of beta-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of beta-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target.

Decreased pigment epithelium-derived factor and increased vascular endothelial growth factor levels in pterygia.

Purpose. Pterygia are histologically composed of proliferating fibrovascular tissue. This study compared expression levels of an angiogenic inhibitor, pigment epithelium-derived factor (PEDF), in pterygia with those in normal corneal and conjunctival tissues. Methods. The normal human conjunctival and corneal tissues were obtained from surgery or from donor eyes without ocular diseases. Pterygia were excised by therapeutic surgery under a microscope. Pigment epithelium-derived factor and vascular endothelial growth factor (VEGF) were measured by Western blot analysis. Their cellular localizations were determined by immunohistochemistry. Results. Intensive PEDF immunostaining was detected in all the normal corneal and conjunctival samples analyzed, predominantly in the epithelium and endothelium of the cornea and in the epithelium of the limbus and conjunctiva. Under the same immunostaining conditions, pterygial samples showed negative or faint PEDF staining. In contrast, the same pterygial samples all showed intensive VEGF staining, predominantly in the epithelium and in blood vessels. Western blot analysis confirmed that the average PEDF level in pterygia was drastically lower than those in normal corneal and conjunctival tissues, respectively. In contrast, the VEGF level in pterygia was significantly higher than in the normal tissues. Conclusion. Pterygia exhibit significantly lower PEDF but higher VEGF levels than those in normal corneas and conjunctivae. The decreased PEDF level in pterygia may play a role in the formation and progression of pterygia.

Identification of a Novel Inhibitor of the Canonical Wnt Pathway

ABSTRACT Wnt signaling is known to regulate multiple processes including angiogenesis, inflammation, and fibrosis. Here, we identified a novel inhibitor of the Wnt pathway, pigment epithelium-derived factor (PEDF), a multifunctional serine proteinase inhibitor. Both overexpression of PEDF in transgenic mice and administration of PEDF protein attenuated Wnt signaling induced by retinal ischemia. Furthermore, PEDF knockdown by small interfering RNA (siRNA) and PEDF knockout in PEDF−/− mice induced activation of Wnt signaling. PEDF bound to LRP6, a Wnt coreceptor, with high affinity (Kd [dissociation constant] of 3.7 nM) and blocked the Wnt signaling induced by Wnt ligand. The physical interaction of PEDF with LRP6 was confirmed by a coprecipitation assay, which showed that PEDF bound to LRP6 at the E1E2 domain. In addition, binding of PEDF to LRP6 blocked Wnt ligand-induced LRP6-Frizzled receptor dimerization, an essential step in Wnt signaling. These results suggest that PEDF is an endogenous antagonist of LRP6, and blocking Wnt signaling may represent a novel mechanism for its protective effects against diabetic retinopathy.

Implication of dysregulation of the canonical wingless-type MMTV integration site (WNT) pathway in diabetic nephropathy

Aims/hypothesisThe wingless-type MMTV integration site (WNT) pathway mediates multiple physiological and pathological processes, such as inflammation, angiogenesis and fibrosis. The aim of this study was to investigate whether canonical WNT signalling plays a role in the pathogenesis of diabetic nephropathy.MethodsExpression of WNT ligands and frizzled receptors in the canonical WNT pathway in the kidney was compared at the mRNA level using real-time RT-PCR between Akita mice, streptozotocin-induced diabetic rats and db/db mice and their respective non-diabetic controls. Renal function was evaluated by measuring the urine albumin excretion. Human renal proximal tubular epithelial cells were treated with high-glucose medium and 4-hydroxynonenal (HNE). Levels of β-catenin, connective tissue growth factor and fibronectin were determined by western blot analysis.ResultsSome of the WNT ligands and frizzled receptors showed increased mRNA levels in the kidneys of Akita mice, streptozotocin-induced diabetic rats and db/db mice compared with their non-diabetic controls. Renal levels of β-catenin and WNT proteins were upregulated in these diabetic models. Lowering the blood glucose levels by insulin attenuated the activation of WNT signalling in the kidneys of Akita mice. In cultured human renal proximal tubular epithelial cells, both high glucose and HNE activated WNT signalling. Inhibition of WNT signalling with a monoclonal antibody blocking LDL-receptor-related protein 6 ameliorated renal inflammation and fibrosis and reduced proteinuria in Akita mice.Conclusions/interpretationThe WNT pathway is activated in the kidneys of models of both type 1 and 2 diabetes. Dysregulation of the WNT pathway in diabetes represents a new pathogenic mechanism of diabetic nephropathy and renders a new therapeutic target.

In vivo and in vitro inhibitory effect of amniotic extraction on neovascularization

Purpose: To prepare amniotic extraction (AE) and to test its antiangiogenic effect in vivo and in vitro. Methods: AE was prepared and diluted to 50, 100, and 200 μg/mL concentrations. Alkali burn-induced corneal neovascularization (NV) was produced and topically treated with different concentrations of AE or 0.1% dexamethasone for 7 days. Normal saline was used as a control. Corneal NV was visualized by heart perfusion of Chinese ink and quantified as the percentage of corneal NV area to the whole corneal area. Human umbilical vein endothelial cells (HUVECs) were primarily cultured. The effects of AE on proliferation and tube formation of HUVECs were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and in vitro angiogenesis assay. Pigment epithelium-derived factor (PEDF) in AE was detected by Western blot. Results: Relative corneal NV area in the control group was 56.6% ± 9.9%, which was significantly reduced by 50 μg/mL AE (47.6% ± 6.9%; P = 0.043) and 200 μg/mL AE (34.3% ± 7.8%; P < 0.001) and by 0.1% dexamethasone (21.1% ± 1.8%; P < 0.001). HUVEC cell proliferation was significantly decreased after treatment with AE at concentrations of 50 and 100 μg/mL compared with control (P = 0.036 and 0.001, respectively). The tube formation was significantly suppressed by 100 μg/mL AE (70.03% ± 4.35%) compared with control (100% ± 4.84%; P = 0.002). No expression of PEDF was detected in AE. Conclusion: AE inhibits NV induced by alkali burn. This effect may be elicited at least in part through the inhibiting activity of blood vessel endothelial cells and is not associated with PEDF.

High Levels of Pigment Epithelium-derived Factor in Diabetes Impair Wound Healing through Suppression of Wnt Signaling

Diabetic foot ulcer (DFU) caused by impaired wound healing is a common vascular complication of diabetes. The current study revealed that plasma levels of pigment epithelium–derived factor (PEDF) were elevated in type 2 diabetic patients with DFU and in db/db mice. To test whether elevated PEDF levels contribute to skin wound-healing delay in diabetes, endogenous PEDF was neutralized with an anti-PEDF antibody in db/db mice. Our results showed that neutralization of PEDF accelerated wound healing, increased angiogenesis in the wound skin, and improved the functions and numbers of endothelial progenitor cells (EPCs) in the diabetic mice. Further, PEDF-deficient mice showed higher baseline blood flow in the skin, higher density of cutaneous microvessels, increased skin thickness, improved numbers and functions of circulating EPCs, and accelerated wound healing compared with wild-type mice. Overexpression of PEDF suppressed the Wnt signaling pathway in the wound skin. Lithium chloride–induced Wnt signaling activation downstream of the PEDF interaction site attenuated the inhibitory effect of PEDF on EPCs and rescued the wound-healing deficiency in diabetic mice. Taken together, these results suggest that elevated circulating PEDF levels contribute to impaired wound healing in the process of angiogenesis and vasculogenesis through the inhibition of Wnt/β-catenin signaling.

PEDF inhibits growth of retinoblastoma by anti-angiogenic activity

Pigment epithelium‐derived factor (PEDF), an angiogenesis inhibitor with multiple other functions, balances angiogenesis in the eye and blocks tumor progression. Retinoblastoma, an angiogenesis‐dependent tumor, is the most common ocular cancer in children without effective treatment. It has been reported that PEDF can induce neuronal differentiation of retinoblastoma cells; however, its anti‐angiogenic potential for inhibition of retinoblastoma growth in vivo has not been elucidated. The present study was designed to investigate the effect of PEDF on growth of retinoblastoma and the possible molecular mechanism. Soluble and non‐fusion recombinant PEDF were generated in E. coli. Recombinant PEDF dose‐dependently inhibited proliferation and induced apoptosis of endothelial cells. PEDF had no effects on the proliferation and apoptosis of retinoblastoma cell line SO‐Rb50. Intraperitoneal injection of PEDF resulted in growth inhibition of heterotopic retinoblastoma xenografts at 68.78%. MVD in tumor tissues treated with PEDF was significantly decreased. These results suggested that PEDF suppressed tumor growth by blocking angiogenesis instead of a direct cytotoxic effect on tumor cells. Vascular endothelial growth factor (VEGF), a major angiogenic stimulator, was down‐regulated by PEDF in both SO‐Rb50 cells and retinoblastoma xenografts. Hypoxia‐inducible factor (HIF)‐1α, a crucial transcriptional factor for VEGF expression, was also down‐regulated by PEDF both in vitro and in vivo. PEDF reduced HIF‐1α nuclear translocation, which may be responsible for the down‐regulation of VEGF. Down‐regulation of VEGF expression in tumor cells through inhibiting HIF‐1α, thus attenuating the paracrine effect of VEGF on endothelial cell proliferation and vascular permeability in tumor tissues, may represent a mechanism for the anti‐angiogenic activity of PEDF. (Cancer Sci 2009; 100: 2419–2425)

Combination of pigment epithelium-derived factor with radiotherapy enhances the antitumor effects on nasopharyngeal carcinoma by downregulating vascular endothelial growth factor expression and angiogenesis.

Nasopharyngeal carcinoma (NPC), which has the highest incidence in South China, is mainly treated by radiotherapy. However, the survival rate remains low. Angiogenesis is closely correlated with progress of NPC. Thus, the combination of anti‐angiogenesis with radiation is an attractive strategy for NPC treatment. A heterogenic xenografted human NPC nude mice model was established to investigate the effect of pigment epithelium‐derived factor (PEDF), a potent anti‐angiogenic factor, and the combined effect of PEDF and radiotherapy on nasopharyngeal carcinoma. Pigment epithelium‐derived factor remarkably suppressed the growth of NPC by 43.52% and decreased the tumor microvessel density (MVD). Pigment epithelium‐derived factor had no effects on the proliferation and apoptosis of NPC cell lines by MTT and flow cytometry assay. However, PEDF decreased vascular endothelial growth factor (VEGF) in NPC cell lines by downregulation of hypoxia‐inducible factor 1α, a crucial transcriptional factor for VEGF expression, as demonstrated by western blotting and immunofluorescent staining assay. Interestingly, irradiation alone could also effectively downregulate VEGF and MVD of xenografted tumor, which indicates that irradiation suppresses NPC not only by killing tumor cells but also through anti‐angiogenesis. Furthermore, combined treatment of PEDF with irradiation enhanced the antitumor efficacy. The MVD and VEGF in the combined therapy were much less than in the treatment with PEDF or radiotherapy alone. Our observation demonstrated that the combination of PEDF with radiotherapy enhances the efficacy of the antitumor effect on NPC by the coordinated inhibition on angiogenesis, which implies the potential role of PEDF as an adjuvant agent for NPC treatment. (Cancer Sci 2011; 102: 1789–1798)

Kallikrein-binding protein inhibits growth of gastric carcinoma by reducing vascular endothelial growth factor production and angiogenesis.

Kallikrein-binding protein (KBP) has been identified as an endogenous angiogenic inhibitor. We previously showed that KBP inhibited rat retinal neovascularization by down-regulation of vascular endothelial growth factor (VEGF) in endothelial cells. However, its antiangiogenic potential for inhibition of gastric carcinoma and the effect on VEGF in tumor cells have not been elucidated. The present study was designed to investigate the effect of KBP on growth of gastric carcinoma and the possible molecular mechanism. Recombinant KBP dose dependently inhibited proliferation and induced apoptosis of endothelial cells, but no effect on proliferation and apoptosis of SGC-7901 gastric carcinoma cells. I.p. injection of KBP resulted in growth inhibition of both heterotopic and orthotopic gastric carcinoma xenografts at 61.4% and 52.3%, respectively. Microvessel density in tumor tissues treated with KBP was significantly decreased, suggesting that KBP suppressed tumor growth by antiangiogenesis. The expression and release of VEGF, a major angiogenic stimulator, were down-regulated by KBP in SGC-7901 cells and gastric carcinoma xenografts. RNA levels of VEGF in SGC-7901 cells were also decreased by KBP, thus suggesting the regulation at the transcriptional level. Therefore, hypoxia-inducible factor 1α (HIF-1α), a crucial transcriptional factor for VEGF expression, was examined in SGC-7901 cells treated by KBP. KBP reduced HIF-1α protein level and nuclear translocation, which may be responsible for the down-regulation of VEGF transcription. Down-regulation of VEGF expression and release in tumor cells through inhibiting HIF-1α, thus attenuating the paracrine effect of VEGF on endothelial cell proliferation and vascular permeability in tumor tissues, may represent a novel mechanism for the antiangiogenic and antitumor activity of KBP. [Mol Cancer Ther 2007;6(12):3297–306]