Treatment of Mid‐Pregnant Mice with KRN633, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase,Induces Abnormal Retinal Vascular Patterning in Their Newborn Pups

We previously reported that treatment of mid‐pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid‐pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633‐treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633‐treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633‐treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network     

Fibroblast growth factor 21 reverses high‐fat diet‐induced impairment of vascular function via the anti‐oxidative pathway in ApoE knockout mice

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver‐secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the β‐klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H₂O₂)‐induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H₂O₂. Administration of N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)‐deficient mice that were fed a high‐fat diet (HFD). Endothelial function, as reflected by acetylcholine‐stimulated aortic relaxation, was improved after FGF21 treatment in ApoE‐deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE‐deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD‐induced vascular injury in ApoE‐deficient mice.     

Vascular endothelial growth factor‐D modulates oxidant–antioxidant balance of human vascular endothelial cells

Vascular endothelial growth factor‐D (VEGF‐D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF‐D stimulates the expression of proteins involved in cell–matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up‐regulated expression of proteins that form the antioxidant barrier of the cell in VEGF‐D‐treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF‐D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF‐D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF‐D‐induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser‐2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.     

Anti‐angiogenic therapy: Adapting strategies to overcome resistant tumors

Healthy cells, as well as benign and malignant tumors, depend upon the body's blood supply to bring in oxygen and nutrients and carry away waste products. Using this property against tumors, anti‐angiogenic therapy targets the tumor vasculature with the aim of starving the tumor, and has demonstrated exceptional clinical efficacy against a number of tumors. This review discusses the current state of knowledge regarding anti‐angiogenic therapies presently available to patients, and garners from both preclinical and clinical literature the benefits and side effects associated with anti‐angiogenic therapies, the unfortunate mechanisms of acquired resistance to these novel therapeutics, and highlights promising next generation anti‐angiogenics that may overcome the limitations encountered with first generation therapies. J. Cell. Biochem. 111: 543–553, 2010. © 2010 Wiley‐Liss, Inc.     

Cross‐talk between the VEGF‐A and HGF signalling pathways in endothelial cells

Background information. Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre‐existing vascular bed. VEGF‐A (vascular endothelial growth factor‐A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF‐A‐driven angiogenesis, although the signalling mechanisms underlying this co‐operation are not completely understood. Results. We analysed the effects of the combination of VEGF‐A and HGF on the activation of VEGFR‐2 (VEGF receptor‐2) and c‐met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR‐2 and c‐met do not physically associate and do not transphosphorylate each other, suggesting that co‐operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF‐A₁₆₅ and HGF stimulate a similar set of MAPKs (mitogen‐activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF‐A₁₆₅ and HGF. Moreover, the combination of VEGF‐A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF‐A₁₆₅ and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF‐A₁₆₅ and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular‐like patterns in vitro in a Rho‐ or Rac‐dependent manner. Conclusions. Under angiogenic conditions, combining VEGF‐A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.     

Self‐organized developmental patterning and differentiation in cerebral organoids

Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self‐organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso‐ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso‐ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.     

血小板活化因子对大鼠黄体细胞孕酮分泌及血管内皮生长因子表达的作用

本文旨在研究血小板活化因子(platelet-activating factor,PAF)对大鼠黄体细胞孕酮分泌及血管内皮生长因子(vascularendothelial growth factor,VEGF)mRNA表达的作用.将未成年(25～28 d)Sprague-Dawley雌性大鼠颈部皮下注射50 IU孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG),48 h后注射25 IU人绒毛膜促性腺激素(human chorionicgonadotrophin.hCG)诱导卵泡发育和黄体生成,第6天(hCG注射日为第1天)收集卵巢黄体细胞,体外培养24 h后,不加或加入不同剂量(0.1 μg/mL、1 μg/mL、10 μg/mL)PAF,37℃、5%CO2培养箱内培养24 h.用放射免疫方法测定培养液中孕酮的含量,流式细胞仪和RT-PCR方法检测黄体细胞凋亡以及VEGF mRNA的表达.结果显示,PAF促进黄体细胞孕酮分泌,1 μg/mL PAF作用最强(P<0.05);PAF促进黄体细胞凋亡无明显剂量依赖性,但10 μg/mL PAF显著促进大鼠黄体细胞凋亡(P<0.05):PAF刺激黄体细胞VEGF mRNA表达,1 μg/mL PAF效果最显著(P<0.01).结果提示,PAF可通过调节黄体细胞孕酮的分泌和VEGF mRNA的表达来促进黄体形成.     

MicroRNA-9 inhibits retinal neovascularization in rats with diabetic retinopathy by targeting vascular endothelial growth factor A

Diabetic retinopathy (DR) is a leading cause of adult visual impairment and loss. This study aims to explore the effects of microRNA-9 (miR-9) on retinal neovascularization during DR by targeting the vascular endothelial growth factor A (VEGFA). DR rat models were successfully established. Retinal microvascular endothelial cells (RMECs) of DR rats were isolated and treated with miR-9 mimic, miR-9 inhibitor or small interfering RNA (siRNA)-VEGFA. The expressions of miR-9, VEGFA, and cluster of differentiation 31 (CD31) of the rats’ tissues and cells were examined. The targeting relationship between miR-9 and VEGFA was testified. The tubule formation, the cell proliferation and the periodic distribution and apoptosis were evaluated after transfection. In the retinal tissues of DR rats, miR-9 expression decreased while the expression of VEGFA and CD31 increased. Notably, miR-9 targeted and inhibited VEGFA expression. In response to the treatment of miR-9 mimic and siRNA-VEGFA, a reduction was identified in CD31 expression, tubule formation, and proliferation of RMECs and cell ratio in the S phase, but an increase was observed in apoptosis rate of RMECs. The treatment of miR-9 inhibitor reversed the manifestations. Our study demonstrated that miR-9 could inhibit retinal neovascularization of DR and tubule formation, and promote apoptosis in RMECs by targeting VEGFA.     

Changes in VEGF expression and DNA synthesis in hepatocytes from hepatectomized and tumour‐bearing mice

Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S‐phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S‐strain mice. After an appropriate period of synchronization, the C3H/S‐histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animal's flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour‐bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour‐bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.     

Inhibition of FGF‐FGFR and VEGF‐VEGFR signalling in cancer treatment

The sites of targeted therapy are limited and need to be expanded. The FGF‐FGFR signalling plays pivotal roles in the oncogenic process, and FGF/FGFR inhibitors are a promising method to treat FGFR‐altered tumours. The VEGF‐VEGFR signalling is the most crucial pathway to induce angiogenesis, and inhibiting this cascade has already got success in treating tumours. While both their efficacy and antitumour spectrum are limited, combining FGF/FGFR inhibitors with VEGF/VEGFR inhibitors are an excellent way to optimize the curative effect and expand the antitumour range because their combination can target both tumour cells and the tumour microenvironment. In addition, biomarkers need to be developed to predict the efficacy, and combination with immune checkpoint inhibitors is a promising direction in the future. The article will discuss the FGF‐FGFR signalling pathway, the VEGF‐VEGFR signalling pathway, the rationale of combining these two signalling pathways and recent small‐molecule FGFR/VEGFR inhibitors based on clinical trials.     

VEGF受体功能研究进展

血管内皮生长因子受体（VEGFR）调控心血管系统的发育。VEGFR1对于造血祖细胞的招募及单核巨噬细胞的迁移是必需的;VEGFR2、VEGFR3在调控血管及淋巴管内皮细胞的功能时发挥重要作用,而现在很多研究都聚焦于阻断VEGFR信号通路以达到阻断肿瘤血管生长的目的。     

Differential effects of cyclic stretch on bFGF‐ and VEGF‐induced sprouting angiogenesis

How mechanical factors affect angiogenesis and how they and chemical angiogenic factors work in concert remain not yet well‐understood. This study investigated the interactive effects of cyclic uniaxial stretch and two potent proangiogenic molecules [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)] on angiogenesis using a stretchable three‐dimensional (3‐D) cell culture model. Endothelial cells seeded atop a 3‐D collagen gel underwent sprouting angiogenesis while being subjected to either 10 or 20% cyclic uniaxial stretch at a frequency of either 1/12 or 1 Hz, in conjunction with an elevated concentration of bFGF or VEGF. Without the presence of additional growth factors, 10 and 20% stretch at 1 Hz induced angiogenesis and the perpendicular alignment of new sprouts, and both inductive effects were abolished by cytochalasin D (an actin polymerization inhibitor). While “10% stretch at 1 Hz,” “20% stretch at 1 Hz,” bFGF, and VEGF were strong angiogenesis stimulants individually, only the combination of “20% stretch at 1 Hz” and bFGF had an additive effect on inducing new sprouts. Interestingly, the combination of “20% stretch at a lower frequency (1/12 Hz)” and bFGF decreased sprouting angiogenesis, even though the level of perpendicular alignment of new sprouts was the same for both stretch frequencies. Taken together, these results demonstrate that both stretch frequency and magnitude, along with interactions with various growth factors, are essential in mediating formation of endothelial sprouts and vascular patterning. Furthermore, work in this area is warranted to elucidate synergistic or competitive signaling mechanisms. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:879–888, 2014     

缺氧诱导因子-1α与血管内皮生长因子及其受体2在大鼠3期压力性损伤皮肤组织中的表达及意义*

目的:分析缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)和血管内皮生长因子受体2(KDR)在不同受压时间点大鼠压力性损伤局部皮肤组织中的表达及相互关系,探讨3期压力性损伤慢性难愈的可能机制。方法:将40只SD雄性大鼠随机分为正常对照组、受压3 d、5 d、7 d、 9 d组( n=8 ),使用磁铁压迫法建立3期压力性损伤动物模型。HE染色观察皮肤组织形态;免疫组化法检测VEGF表达,Western blot 检测皮肤组织HIF-1α、VEGF、KDR蛋白表达;对数据行单因素方差分析、LSD检验。结果:①HE结果显示,与正常对照组相比,受压组大鼠表皮逐渐增厚,血管数量不断减少,胶原排列紊乱,炎症细胞浸润增加。②免疫组化结果显示:受压3 d组大鼠皮肤组织中VEGF蛋白表达量较正常对照组明显增高(P＜0.01);受压5 d、7 d和 9 d组大鼠皮肤组织中VEGF蛋白表达量均明显低于正常对照组(P＜0.05)。WB结果和免疫组化结果一致。③WB结果显示:受压3 d、5 d和7 d组大鼠皮肤组织中HIF-1α表达量均明显高于正常对照组(P＜0.01 或 P＜0.05);4组受压组大鼠皮肤组织KDR蛋白表达量均低于正常对照组(P＜0.05或P＜0.01)。结论:HIF-1α介导的VEGF和KDR蛋白表达减少引起组织血管生成减少可能是3期压力性损伤慢性难愈的重要原因之一。     

Up-regulation of vascular endothelial growth factor in response to glucose deprivation

Vascular endothelial growth factor (VEGF), also known as a vascular permeability factor (VPF), is an endothelial specific mitogen and is a potent inducer of angiogenesis. Recently it has been reported that hypoxia induces VEGF mRNA expression in various cells. Since both oxygen and glucose are required for efficient production of energy, we examined the effect of glucose deprivation on VEGF mRNA expression and VEGF protein production in U-937 (a human monocytic cell line) cells. Both the mRNA expression and secretion of VEGF increased after exposure to low glucose. Addition of L-glucose, the L-stereoisomer of D-glucose, did not prevent the up-regulation of VEGF expression. The conditioned medium from glucose-deprived cells, followed by supplementation with glucose, did not up-regulate VEGF mRNA expression in U-937 cells. The low glucose-induced VEGF mRNA expression returned to the control level after supplementation with D-glucose. Furthermore, oligomycin, a mitochondrial ATP synthase inhibitor, increased VEGF protein production. The results suggest that the up-regulation of VEGF mRNA in U-937 cells in response to glucose deprivation is not mediated by autocrine factors from the cells nor is the osmotic change of the medium mediated by the deficiency of glucose metabolism in the cells. Our results also suggest that the intracellular ATP depletion due to glucose deprivation may be one of the causes for increased VEGF mRNA expression. We speculate that local hypoglycemia may act as an essential trigger for angiogenesis through the VEGF gene expression.     

Hypoxic damage to the periventricular white matter in neonatal brain: role of vascular endothelial growth factor, nitric oxide and excitotoxicity

The present study examined factors that may be involved in the development of hypoxic periventricular white matter damage in the neonatal brain. Wistar rats (1-day old) were subjected to hypoxia and the periventricular white matter (corpus callosum) was examined for the mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS and iNOS), vascular endothelial growth factor (VEGF) and N-methyl-D-aspartate receptor subunit 1 (NMDAR1) between 3 h and 14 days after hypoxic exposure by real-time RT-PCR, western blotting and immunohistochemistry. Up-regulated mRNA and protein expression of HIF-1alpha, VEGF, NMDAR1, eNOS, nNOS and iNOS in corpus callosum was observed in response to hypoxia. NMDAR1 and iNOS expression was found in the activated microglial cells, whereas VEGF was localized to astrocytes. An enzyme immunoassay showed that the VEGF concentration in corpus callosum was significantly higher up to 7 days after hypoxic exposure. NO levels, measured by colorimetric assay, were also significantly higher in hypoxic rats up to 14 days after hypoxic exposure as compared with the controls. A large number of axons undergoing degeneration were observed between 3 h and 7 days after the hypoxic exposure at electron-microscopic level. Our findings point towards the involvement of excitotoxicity, VEGF and NO in periventricular white matter damage in response to hypoxia.     

Deregulation of XBP1 expression contributes to myocardial vascular endothelial growth factor‐A expression and angiogenesis during cardiac hypertrophy in vivo

Endoplasmic reticulum (ER) stress has been reported to be involved in many cardiovascular diseases such as atherosclerosis, diabetes, myocardial ischemia, and hypertension that ultimately result in heart failure. XBP1 is a key ER stress signal transducer and an important pro‐survival factor of the unfolded protein response (UPR) in mammalian cells. The aim of this study was to establish a role for XBP1 in the deregulation of pro‐angiogenic factor VEGF expression and potential regulatory mechanisms in hypertrophic and failing heart. Western blots showed that myocardial XBP1s protein was significantly increased in both isoproterenol (ISO)‐induced and pressure‐overload‐induced hypertrophic and failing heart compared to normal control. Furthermore, XBP1 silencing exacerbates ISO‐induced cardiac dysfunction along with a reduction of myocardial capillary density and cardiac expression of pro‐angiogenic factor VEGF‐A in vivo. Consistently, experiments in cultured cardiomyocytes H9c2 (2‐1) cells showed that UPR‐induced VEGF‐A upregulation was determined by XBP1 expression level. Importantly, VEGF‐A expression was increased in failing human heart tissue and blood samples and was correlated with the levels of XBP1. These results suggest that XBP1 regulates VEGF‐mediated cardiac angiogenesis, which contributes to the progression of adaptive hypertrophy, and might provide novel targets for prevention and treatment of heart failure.     

Hypoxia-inducible factor and vascular endothelial growth factor in the neuroretina and retinal blood vessels after retinal ischemia

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immunoreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia.