Tenomodulin Inhibits Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy

We aimed to determine the anti-angiogenic effect of tenomodulin (TeM) on retinal neovascularization in an oxygen-induced retinopathy (OIR) mouse model. OIR was induced in C57BL/6 mice by exposing seven-day-old mice to 75% oxygen for five days followed by room air for five days. Control mice were exposed to room air from birth until postnatal day 17. Mice received intravitreal injections of 1 μg of TeM in one eye and PBS in the contralateral eye at P7 before being exposed to 75% oxygen. Eyes were collected at postnatal day 17. Retinal blood vessel patterns were visualized by fluorescein angiography. We quantified the number of neovascular nuclei that were present beyond the inner limiting membrane (ILM) using histological methods with a masked approach. Furthermore, double immunohistochemical staining of TeM was performed on retinas to identify nuclei protruding into the vitreous cavity. Western blot was used to detect exogenous TeM protein. The central nonperfusion area (NPA, mm²) of TeM-injected eyes was significantly different from that of OIR and PBS-injected eyes, and the number of nuclei in new blood vessels breaking through the ILM in each retinal cross-section significantly differed from that of OIR eyes and PBS-injected control eyes. Cellular nuclei of new blood vessels protruding into the vitreous cavity were also observed in TeM-injected retinas by immunohistochemistry. Western blotting revealed a 16-kDa immunoreactive protein, indicating incorporation of an exogenous TeM fragment into the retina. Our data shows that TeM can effectively inhibit pathological angiogenesis in mouse eyes; indicating its potential role in prevention and treatment of ocular neovascularization.     

TNFSF15 Inhibits Blood Retinal Barrier Breakdown Induced by Diabetes

Tumor necrosis factor superfamily 15 (TNFSF15) is an endogenous neovascularization inhibitor and an important negative regulator of vascular homeostasis. This study aimed to explore the potential role of TNFSF15 in diabetic retinopathy. Vitreous TNFSF15 and VEGF levels in proliferative diabetic retinopathy (PDR) patients were detected by ELISA. Retinal expression of TNFSF15 and the content of tight junction proteins (TJPs) in rats were detected by immunohistochemistry and Western blot, respectively. The blood retinal barrier (BRB) permeability was evaluated using Evans Blue (EB) dye. The TNFSF15/VEGF ratio was decreased in the vitreous fluid of patients with PDR relative to the controls, even though the expression levels of TNFSF15 were higher. TNFSF15 was dramatically decreased one month later after diabetes induction (p < 0.001), and then increased three months later and thereafter. TNFSF15 treatment significantly protected the BRB in the diabetic animals. Diabetes decreased TJPs levels in the retina, and these changes were inhibited by TNFSF15 treatment. Moreover, TNFSF15 decreased activation of VEGF both in mRNA and protein levels caused by diabetes. These results indicate that TNFSF15 is an important inhibitor in the progression of DR and suggest that the regulation of TNFSF15 shows promise for the development of diabetic retinopathy treatment strategies.     

Complement Factor B Mediates Ocular Angiogenesis through Regulating the VEGF Signaling Pathway

Complement factor B (CFB), a 95-kDa protein, is a crucial catalytic element of the alternative pathway (AP) of complement. After binding of CFB to C3b, activation of the AP depends on the proteolytic cleavage of CFB by factor D to generate the C3 convertase (C3bBb). The C3 convertase contains the catalytic subunit of CFB (Bb), the enzymatic site for the cleavage of a new molecule of C3 into C3b. In addition to its role in activating the AP, CFB has been implicated in pathological ocular neovascularization, a common feature of several blinding eye diseases, however, with somewhat conflicting results. The focus of this study was to investigate the direct impact of CFB on ocular neovascularization in a tightly controlled environment. Using mouse models of laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR), our study demonstrated an increase in CFB expression during pathological angiogenesis. Results from several in vitro and ex vivo functionality assays indicated a promoting effect of CFB in angiogenesis. Mechanistically, CFB exerts this pro-angiogenic effect by mediating the vascular endothelial growth factor (VEGF) signaling pathway. In summary, we demonstrate compelling evidence for the role of CFB in driving ocular angiogenesis in a VEGF-dependent manner. This work provides a framework for a more in-depth exploration of CFB-mediated effects in ocular angiogenesis in the future.     

Optimal Efficacy and Safety of Humanized Anti-Scg3 Antibody to Alleviate Oxygen-Induced Retinopathy

The retinopathy of prematurity (ROP), a neovascular retinal disorder presenting in premature infants, is the leading causes of blindness in children. Currently, there is no approved drug therapy for ROP in the U.S., highlighting the urgent unmet clinical need for a novel therapeutic to treat the disease. Secretogranin III (Scg3) was recently identified as a disease-selective angiogenic factor, and Scg3-neutralizing monoclonal antibodies were reported to alleviate pathological retinal neovascularization in mouse models. In this study, we characterized the efficacy and safety of a full-length humanized anti-Scg3 antibody (hAb) to ameliorate retinal pathology in oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, by implementing histological and functional analyses. Our results demonstrate that the anti-Scg3 hAb outperforms the vascular endothelial growth factor inhibitor aflibercept in terms of efficacy and safety to treat OIR mice. Our findings support the development of anti-Scg3 hAb for clinical application.     

Rice Bran and Vitamin B6 Suppress Pathological Neovascularization in a Murine Model of Age-Related Macular Degeneration as Novel HIF Inhibitors

Pathological neovascularization in the eye is a leading cause of blindness in all age groups from retinopathy of prematurity (ROP) in children to age-related macular degeneration (AMD) in the elderly. Inhibiting neovascularization via antivascular endothelial growth factor (VEGF) drugs has been used for the effective treatment. However, anti-VEGF therapies may cause development of chorioretinal atrophy as they affect a physiological amount of VEGF essential for retinal homeostasis. Furthermore, anti-VEGF therapies are still ineffective in some cases, especially in patients with AMD. Hypoxia-inducible factor (HIF) is a strong regulator of VEGF induction under hypoxic and other stress conditions. Our previous reports have indicated that HIF is associated with pathological retinal neovascularization in murine models of ROP and AMD, and HIF inhibition suppresses neovascularization by reducing an abnormal increase in VEGF expression. Along with this, we attempted to find novel effective HIF inhibitors from natural foods of our daily lives. Food ingredients were screened for prospective HIF inhibitors in ocular cell lines of 661W and ARPE-19, and a murine AMD model was utilized for examining suppressive effects of the ingredients on retinal neovascularization. As a result, rice bran and its component, vitamin B6 showed inhibitory effects on HIF activation and suppressed VEGF mRNA induction under a CoCl₂-induced pseudo-hypoxic condition. Dietary supplement of these significantly suppressed retinal neovascularization in the AMD model. These data suggest that rice bran could have promising therapeutic values in the management of pathological ocular neovascularization.     

Matrix Metalloproteinases MMP-2 and MMP-9, Their Inhibitors TIMP-1 and TIMP-2, Vascular Endothelial Growth Factor and sVEGFR-2 as Predictive Markers of Ischemic Retinopathy in Patients with Systemic Sclerosis—Case Series Report

Systemic sclerosis (SSc) is an autoimmune connective tissue disorder associated with multiple organ involvement. The aim of the study was to present two SSc patients who were diagnosed with ischemic retinopathy in both eyes. As a background to our case study, we decided to investigate the imbalance of angiogenesis factors in 25 SSc patients in relation to 25 healthy controls. Assays of matrix metalloproteinases-2 and -9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1) and -2 (TIMP-2), vascular endothelial growth factor (VEGF), and soluble VEGF receptor-2 (sVEGFR-2) in blood serum and tears were performed. A significantly increased levels of MMP-9 in serum and tears, (p = 0.0375 and p < 0.001, respectively) as well as VEGF/sVEGFR-2 ratio in tears (p < 0.001) were found in the whole SSc patients group compared with controls, while reduced levels of these parameters in patients with ischemic sclerodermic retinopathy were noted. We also observed decreased level MMP-2 in tears and increased levels of TIMP-2 in blood serum and tears of SSc patients with retinal ischemic changes. MMP-9, MMP-2, TIMP-2, and VEGF/sVEGFR-2 may play a crucial role in ischemic retinal degeneration or retinal reorganization in SSc.     

VEGF and FGF2 Improve Revascularization,Survival, and Oocyte Quality of Cryopreserved,Subcutaneously-Transplanted Mouse Ovarian Tissues

This study was conducted to investigate the effect of the vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) on revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue. Autologous subcutaneous transplantation of vitrified-thawed mouse ovarian tissues treated with (experimental group) or without (control group) VEGF and FGF2 was performed. After transplantation to the inguinal region for two or three weeks, graft survival, angiogenesis, follicle development, and oocyte quality were examined after gonadotropin administration. VEGF coupled with FGF2 (VEGF/FGF2) promoted revascularization and significantly increased the survival rate of subcutaneously-transplanted cryopreserved ovarian tissues compared with untreated controls. The two growth factors did not show long-term effects on the ovarian grafts. In contrast to the untreated ovarian grafts, active folliculogenesis was revealed as the number of follicles at various stages and of mature oocytes in antral follicles after gonadotropin administration were remarkably higher in the VEGF/FGF2-treated groups. Although the fertilization rate was similar between the VEGF/FGF2 and control groups, the oocyte quality was much better in the VEGF/FGF2-treated grafts as demonstrated by the higher ratio of blastocyst development. Introducing angiogenic factors, such as VEGF and FGF2, may be a promising strategy to improve revascularization, survival, and oocyte quality of cryopreserved, subcutaneously-transplanted mouse ovarian tissue.     

改良法氧诱导的早产儿视网膜病变动物模型的建立

目的改良法建立氧诱导的早产儿视网膜病变动物模型,并检测视网膜组织中血管内皮生长因子受体2(Vascular endothelial growth factor receptor 2,VEGFR2)基因mRNA的表达。方法将54只7日龄Wistar幼鼠随机分为空气对照组和高氧实验组,高氧实验组饲养环境维持氧气流量为0.5～0.75 L/min,氧浓度为(80±2)%,空气对照组一直处于空气中饲养。每组分别取12、14、17日龄幼鼠,经心腔灌注后,剥离视网膜,行ADP酶染色,观察幼鼠视网膜血管形态;每组分别取17日龄幼鼠,取眼杯,经HE染色计数突破视网膜内界膜的内皮细胞核数目;每组分别取12、14、17日龄幼鼠视网膜组织,经RT-PCR检测VEGFR2基因mRNA的表达。结果 ADP酶染色镜下观察可见,视网膜血管在高氧环境中收缩闭塞,在相对缺氧环境中迂曲扩张并生成大量病理性新生血管;空白对照组和高氧实验组突破视网膜内界膜的内皮细胞核数分别为2.650±0.875和31.450±0.686,两组间差异有统计学意义(P<0.05);高氧实验组VEGFR2基因mRNA的表达先显著下降(12日龄),后显著增加(14日、17日龄),对照组VEGFR2基因mRNA一直维持在较低水平,两组间差异有统计学意义(P<0.05)。结论已成功建立了氧诱导的早产儿视网膜病变动物模型,为进一步研究下调或阻断VEGFR2的药物对ROP等新生血管疾病的疗效奠定了基础。     

Concurrent Physiological and Pathological Angiogenesis in Retinopathy of Prematurity and Emerging Therapies

Retinopathy of prematurity (ROP) is an ocular vascular disease affecting premature infants, characterized by pathological retinal neovascularization (RNV), dilated and tortuous retinal blood vessels, and retinal or vitreous hemorrhages that may lead to retinal detachment, vision impairment and blindness. Compared with other neovascular diseases, ROP is unique because of ongoing and concurrent physiological and pathological angiogenesis in the developing retina. While the disease is currently treated by laser or cryotherapy, anti-vascular endothelial growth factor (VEGF) agents have been extensively investigated but are not approved in the U.S. because of safety concerns that they negatively interfere with physiological angiogenesis of the developing retina. An ideal therapeutic strategy would selectively inhibit pathological but not physiological angiogenesis. Our group recently described a novel strategy that selectively and safely alleviates pathological RNV in animal models of ROP by targeting secretogranin III (Scg3), a disease-restricted angiogenic factor. The preclinical profile of anti-Scg3 therapy presents a high potential for next-generation disease-targeted anti-angiogenic therapy for the ROP indication. This review focuses on retinal vessel development in neonates, the pathogenesis of ROP and its underlying molecular mechanisms, including different animal models, and provides a summary of current and emerging therapies.     

Vascular Endothelial Growth Factor,a Key Modulator of the Anti-Tumor Immune Response

During tumor growth, angiogenesis is required to ensure oxygen and nutrient transport to the tumor. Vascular endothelial growth factor (VEGF) is the major inducer of angiogenesis and appears to be a key modulator of the anti-tumor immune response. Indeed, VEGF modulates innate and adaptive immune responses through direct interactions and indirectly by modulating protein expressions on endothelial cells or vascular permeability. The inhibition of the VEGF signaling pathway is clinically approved for the treatment of several cancers. Therapies targeting VEGF can modulate the tumor vasculature and the immune response. In this review, we discuss the roles of VEGF in the anti-tumor immune response. In addition, we summarize therapeutic strategies based on its inhibition, and their clinical approval.     

AS1411 Nucleolin-Specific Binding Aptamers Reduce Pathological Angiogenesis through Inhibition of Nucleolin Phosphorylation

Proliferative retinopathies produces an irreversible type of blindness affecting working age and pediatric population of industrialized countries. Despite the good results of anti-VEGF therapy, intraocular and systemic complications are often associated after its intravitreal use, hence novel therapeutic approaches are needed. The aim of the present study is to test the effect of the AS1411, an antiangiogenic nucleolin-binding aptamer, using in vivo, ex vivo and in vitro models of angiogenesis and propose a mechanistic insight. Our results showed that AS1411 significantly inhibited retinal neovascularization in the oxygen induced retinopathy (OIR) in vivo model, as well as inhibited branch formation in the rat aortic ex vivo assay, and, significantly reduced proliferation, cell migration and tube formation in the HUVEC in vitro model. Importantly, phosphorylated NCL protein was significantly abolished in HUVEC in the presence of AS1411 without affecting NFκB phosphorylation and -21 and 221-angiomiRs, suggesting that the antiangiogenic properties of this molecule are partially mediated by a down regulation in NCL phosphorylation. In sum, this new research further supports the NCL role in the molecular etiology of pathological angiogenesis and identifies AS1411 as a novel anti-angiogenic treatment.     

Therapeutic Efficacy of a Novel Acetylated Tetrapeptide in Animal Models of Age-Related Macular Degeneration

It has been shown previously that a novel tetrapeptide, Arg-Leu-Tyr-Glu (RLYE), derived from human plasminogen inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis, suppresses choroidal neovascularization in mice by an inhibition of VEGF receptor-2 (VEGFR-2) specific signaling pathway. In this study, we report that a modified tetrapeptide (Ac-RLYE) showed improved anti-choroidal neovascularization (CNV) efficacy in a number of animal models of neovascular age-related macular degeneration (AMD) which include rat, rabbit, and minipig. The preventive and therapeutic in vivo efficacy of Ac-RLYE via following intravitreal administration was determined to be either similar or superior to that of ranibizumab and aflibercept. Assessment of the intraocular pharmacokinetic and toxicokinetic properties of Ac-RLYE in rabbits demonstrated that it rapidly reached the retina with minimal systemic exposure after a single intravitreal dose, and it did not accumulate in plasma during repetitive dosing (bi-weekly for 14 weeks). Our results suggested that Ac-RLYE has a great potential for an alternative therapeutics for neovascular (wet) AMD. Since the amino acids in human VEGFR-2 targeted by Ac-RLYE are conserved among the animals employed in this study, the therapeutic efficacies of Ac-RLYE evaluated in those animals are predicted to be observed in human patients suffering from retinal degenerative diseases.     

The Plasminogen–Activator Plasmin System in Physiological and Pathophysiological Angiogenesis

Angiogenesis is a process associated with the migration and proliferation of endothelial cells (EC) to form new blood vessels. It is involved in various physiological and pathophysiological conditions and is controlled by a wide range of proangiogenic and antiangiogenic molecules. The plasminogen activator–plasmin system plays a major role in the extracellular matrix remodeling process necessary for angiogenesis. Urokinase/tissue-type plasminogen activators (uPA/tPA) convert plasminogen into the active enzyme plasmin, which in turn activates matrix metalloproteinases and degrades the extracellular matrix releasing growth factors and proangiogenic molecules such as the vascular endothelial growth factor (VEGF-A). The plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of uPA and tPA, thereby an inhibitor of pericellular proteolysis and intravascular fibrinolysis, respectively. Paradoxically, PAI-1, which is expressed by EC during angiogenesis, is elevated in several cancers and is found to promote angiogenesis by regulating plasmin-mediated proteolysis and by promoting cellular migration through vitronectin. The urokinase-type plasminogen activator receptor (uPAR) also induces EC cellular migration during angiogenesis via interacting with signaling partners. Understanding the molecular functions of the plasminogen activator plasmin system and targeting angiogenesis via blocking serine proteases or their interactions with other molecules is one of the major therapeutic strategies scientists have been attracted to in controlling tumor growth and other pathological conditions characterized by neovascularization.     

Pemafibrate Prevents Retinal Dysfunction in a Mouse Model of Unilateral Common Carotid Artery Occlusion

Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.     

Fibroblast Growth Factor Type 1 Ameliorates High-Glucose-Induced Oxidative Stress and Neuroinflammation in Retinal Pigment Epithelial Cells and a Streptozotocin-Induced Diabetic Rat Model

Diabetic retinopathy (DR) is a common complication of diabetes that causes severe visual impairment globally. The pathogenesis of DR is related to oxidative stress and chronic inflammation. The fibroblast growth factor type 1 (FGF-1) mitogen plays crucial roles in cell function, development, and metabolism. FGF-1 is involved in blood sugar regulation and exerts beneficial antioxidative and anti-inflammatory effects on various organ systems. This study investigated the antioxidative and anti-inflammatory neuroprotective effects of FGF-1 on high-glucose-induced retinal damage. The results revealed that FGF-1 treatment significantly reversed the harmful effects of oxidative stress and inflammatory mediators in retinal tissue in a streptozotocin-induced diabetic rat model. These protective effects were also observed in the in vitro model of retinal ARPE-19 cells exposed to a high-glucose condition. We demonstrated that FGF-1 attenuated p38 mitogen-activated protein kinase and nuclear factor-κB pathway activation under the high-glucose condition. Our results indicated that FGF-1 could effectively prevent retinal injury in diabetes. The findings of this study could be used to develop novel treatments for DR that aim to reduce the cascade of oxidative stress and inflammatory signals in neuroretinal tissue.     

可溶性血管内皮生长因子受体2片段的表达及其对血管内皮细胞增殖的影响

目的探讨可溶性血管内皮生长因子受体2(sKDR)抑制血管内皮细胞增殖及在血管生成中的作用。方法提取脐静脉内皮细胞(HUVEC)总RNA,扩增KDR基因膜外1~4结构域,构建原核表达载体pQE40-KDR,转化E.coli M15,经IPTG诱导表达,镍离子柱亲和层析纯化后复性,用Western blot检测sKDR蛋白的表达,MTT比色法和鸡胚尿囊膜(CAM)试验分别检测其对HUVEC增殖的影响及其对血管生成的作用。结果经RT-PCR扩增得到了1150 bp左右的sKDR片段,并在pQE40原核表达系统中表达了sKDR蛋白,以包涵体形式存在。纯化后蛋白电泳呈现相对分子质量50000左右的单一条带,纯化蛋白占总蛋白的98%,蛋白含量为80μg/ml。Western blot证实其为重组sKDR蛋白。MTT检测结果显示,sKDR可抑制血管内皮生长因子(VEGF)刺激的HUVEC增殖,并阻滞VEGF诱导的CAM血管增生。结论已成功构建sKDR原核表达载体,并在大肠杆菌M15中获得表达,纯化的sKDR片段具有与VEGF结合的生物学功能,有望成为基因治疗肿瘤血管形成的理想靶点。     

Resveratrol,an Inhibitor Binding to VEGF,Restores the Pathology of Abnormal Angiogenesis in Retinopathy of Prematurity (ROP) in Mice: Application by Intravitreal and Topical Instillation

Retinopathy of prematurity (ROP) is a severe eye disease leading to blindness. Abnormal vessel formation is the pathological hallmark of neovascular ROP. In forming vessels, vascular endothelial growth factor (VEGF) is an important stimulator. The current anti-ROP therapy has focused on bevacizumab, a monoclonal antibody against VEGF, and pazopanib, a tyrosine kinase inhibitor on the VEGF receptor (VEGFR). Several lines of evidence have proposed that natural compounds may be more effective and safer for anti-VEGF function. Resveratrol, a common natural compound, binds to VEGF and blocks its interaction with VEGFR, thereafter suppressing angiogenesis. Here, we evaluate the efficacy of intravitreal injection, or topical instillation (eye drops), of resveratrol into the eyes of mice suffering from oxygen-induced retinopathy, i.e., developing ROP. The treatment of resveratrol significantly relieved the degree of vascular distortion, permeability and hyperplasia; the efficacy could be revealed by both methods of resveratrol application. In parallel, the treatments of resveratrol inhibited the retinal expressions of VEGF, VEGFR and CD31. Moreover, the applied resveratrol significantly relieved the damage caused by oxygen radicals through upregulating the level of superoxide dismutase (SOD) and downregulating the level of malondialdehyde (MDA) in the retina. Taken together, the potential therapeutic benefit of resveratrol in pro-angiogenic diseases, including retinopathy, can be considered.     

The FGFR Family Inhibitor AZD4547 Exerts an Antitumor Effect in Ovarian Cancer Cells

The dysregulation of fibroblast growth factor (FGF) signaling has been implicated in tumorigenesis, tumor progression, angiogenesis, and chemoresistance. The small-molecule AZD4547 is a potent inhibitor of FGF receptors. This study was performed to investigate the antitumor effects and determine the mechanistic details of AZD4547 in ovarian cancer cells. AZD4547 markedly inhibited the proliferation and increased the apoptosis of ovarian cancer cells. AZD4547 also suppressed the migration and invasion of ovarian cancer cells under nontoxic conditions. Furthermore, it attenuated the formation of spheroids and the self-renewal capacities of ovarian cancer stem cells and exerted an antiangiogenic effect. It also suppressed in vivo tumor growth in mice. Collectively, this study demonstrated the antitumor effect of AZD4547 in ovarian cancer cells and suggests that it is a promising agent for ovarian cancer therapy.