Epigenetic control of tumor angiogenesis

The term “epigenetic” is used to refer to heritable alterations in chromatin that are not due to changes in DNA sequence. Different growth factors and vascular genes mediate the angiogenic process, which is regulated by epigenetic states of genes. The aim of this article is to analyze the role of epigenetic mechanisms in the control and regulation of tumor angiogenetic processes. The reversibility of epigenetic events in contrast to genetic aberrations makes them potentially suitable for therapeutic intervention. In this context, DNA methyltransferase (DNMT) and HDAC inhibitors indirectly—via the tumor cells—exhibit angiostatic effects in vivo, and inhibition of miRNAs can contribute to the development of novel anti‐angiogenesis therapies.     

Genetic analysis of blood vessel formation role of endothelial versus smooth muscle cells

Formation of new blood vessels is vital during embryogenesis, essential for reproduction and wound healing during adulthood, and required to rescue tissue during ischemia. Neovascularization may, however, also contribute to the pathogenesis of several disorders, including tumorigenesis, diabetic vasculopathy, and chronic inflammation. Initially, blood vessels form as endothelium-lined channels by in situ differentiation of endothelial cells. Subsequently, they sprout and remodel into a highly organized and interconnected vascular network. During further maturation of the blood vessels, a sheet of primitive vascular smooth muscle cells surrounds the endothelium-lined channels, which controls endothelial cell function and provides structural support. Recent molecular analyses have identified candidate molecules that affect these processes. Their in vivo role has been further established by targeted gene manipulation in transgenic mice. This review highlights recent developments in the genetic analysis of blood vessel formation, as deduced from analysis of gene-inactivated mice. (Trends Cardiovasc Med 1997;7:271–281). © 1997, Elsevier Science Inc.     

A model system for the study of human retinal angiogenesis: activation of monocytes and endothelial cells and the association with the expression of the monocarboxylate transporter type 1 (MCT-1)

Aims/hypothesis. The growth of retinal vessels is associated with a number of disease conditions, including diabetic retinopathy and proliferative vitreo-retinopathy. In this study we describe a model of human retinal angiogenesis and show how this may be used to explain the mechanisms that are associated with the growth of new retinal vessels. Methods. A 4 mm diameter disc of retinal tissue was placed within a fibrin matrix and the appearance was monitored daily by light microscopy. Immunohistochemical techniques were used for the detection of, glial fibrillary acidic protein, CD68, the Ki-67 antigen, vascular endothelial growth factor, monocarboxylate transporter type 1 and von Willebrand's factor. Results. Vessels were evident extending from the periphery of the explant and the activation of endothelial cells was shown by immuno-peroxidase staining of paraffin embedded sections of the explants for the expression of the Ki-67 antigen, a marker of cell proliferation. The expression of glial fibrillary acidic protein and von Willebrand's factor increased with duration in culture and the presence of activated macrophages or microglia or both was shown by positive immunoreactivity for CD68 and Ki-67 and were identified by day 3. The presence of endogenous vascular endothelial growth factor and the activation of monocarboxylate transporter type 1 by vascular endothelial growth factor, showed the involvement of specific growth factors. Conclusion/interpretation. The explant model provides evidence for the involvement of macrophages and glial fibrillary acidic protein activation in human retinal angiogenesis and for the expression of monocarboxylate transporter type 1, which is likely to be important in the use of lactate in the hypoxic retina. [Diabetologia (1999) 42: 870–877] Received: 9 December 1998 and in final revised form: 26 January 1999     

Insulin-like growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor

Summary The release of growth factors from ischaemic retina has been hypothesized as the central stimulus for retinal neovascularization in proliferative diabetic retinopathy. Two of the growth factors implicated are insulin-like growth factor-I and basic fibroblast growth factor. We examined the effect of insulin-like growth factor-I on in vivo neovascularization using the established angiogenic model of the rabbit cornea (n=30), and also compared the effects of insulin-like growth factor-I and basic fibroblast growth factor using two new in vivo systems. Either supraphysiologic concentrations of each growth factor (600 g) were injected intravitreally into pigmented rabbits (n=21) or porous polyfluorotetraethylene chambers filled with an emulsion containing collagen and growth factor (500 ng) were placed on the retina surface (n=8). Our results demonstrate that when insulin-like growth factor-I was implanted together with a slow release carrier into the pocket of the normally avascular cornea, insulin-like growth factor-I (10 g/pellet) induced angiogenesis in all rabbits. This degree of angiogenesis was comparable to that previously shown for basic fibroblast growth factor. For the intravitreal studies, the fibrotic component was greater in the basic fibroblast growth factor injected eyes, whereas the vascular component was accentuated in the eyes injected with insulin-like growth factor-I. Light and electron microscopy demonstrated areas of vascular proliferation in both groups. Porous polyfluorotetraethylene chamber studies with insulin-like growth factor-I and basic fibroblast growth factor demonstrated vascular proliferation in the vicinity of the chamber similar to the intravitreal injected eyes, but to a lesser degree than the injected eyes. Our experiments overall support the angiogenic potential of both insulin-like growth factor-I and basic fibroblast growth factor and support distinct but complimentary roles for each growth factor in the pathogenesis of proliferative diabetic retinopathy.     

胰岛素对糖尿病视网膜血管新生的影响和调控机制

胰岛素作为促进合成代谢、调节血糖最主要的激素,担任着严格控制糖尿病患者血糖、延缓甚至避免糖尿病慢性并发症发生、发展的重要角色;同时,其亦可通过增强血管内皮生长因子基因的转录和表达,促进视网膜内皮细胞的增殖和血管新生,该功能可能是胰岛素导致糖尿病视网膜病变恶化的原因.因此,胰岛素可能是糖尿病治疗的一把双刃剑.     

Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives

Retinal neovascularization and macular edema are central features of diabetic retinopathy, the major cause of blindness in the developed world. Current treatments are limited in their efficacy and are associated with significant adverse effects. Characterization of the molecular and cellular processes involved in vascular growth and permeability has led to the recognition that the angiogenic growth factor and vascular permeability factor vascular endothelial growth factor (VEGF) plays a pivotal role in the retinal microvascular complications of diabetes. Therefore, VEGF represents an exciting target for therapeutic intervention in diabetic retinopathy. This review highlights the current understanding of the mechanisms that regulate VEGF gene expression and mediate its biological effects and how these processes may become altered during diabetes. The cellular and molecular alterations that characterize experimental models of diabetes are considered in relation to the influence of high glucose-mediated oxidative stress on VEGF expression and on the mechanisms of VEGF's actions under hyperglycemic induction. Finally, potential therapeutic strategies for preventing VEGF overexpression or blocking its pathological effects in the diabetic retina are considered.     

原发性肝癌血管形成的免疫组化和形态学研究

目的研究原发性肝癌血管形成及血管起源。方法用病理及CD34、UEA-1、Collagen Ⅳ免疫组化的方法对21例肝癌及癌旁组织中血管形成进行原位检测和观察。结果阳性反应可见血管星棕色或棕黄色条带。癌旁组织血窦内皮细胞CD34及UEA-1染色阴性,炎症组织内的血管染色阳性。部分肿瘤血管与癌旁组织血管不相连;部分肿瘤血管与癌旁组织血管相连。Collagen Ⅳ在肝癌组织中有较强的表达,癌旁组织中表达很弱,但在门脉区炎症组织内表达可较强。结论CD34、UEA-1、Collagen Ⅳ染色与肿瘤血管形成有关,部分肝癌血管可能起源于毛细血管化的癌旁肝血窦。     

Release of the angiogenesis inhibitor angiostatin in patients with proliferative diabetic retinopathy: association with retinal photocoagulation

Aims/hypothesis. Proliferative diabetic retinopathy is a major debilitating disease causing most cases of blindness in humans in the Western world. Photocoagulation is the established therapy of proliferative diabetic retinopathy, although the molecular mechanisms of its effects are still not known. Recently angiostatin has been characterized as a potent inhibitor of neovascularization. Apart from a possible down-regulation of angiogenic cytokines, release of angiostatin could initiate the anti-angiogenic effects of retinal photocoagulation.¶Methods. We investigated the regulation of angiostatin and the angiogenic cytokines vascular endothelial growth factor and basic fibroblast growth factor in vivo by comparing vitreal concentrations of 18 control patients and 34 patients with proliferative diabetic retinopathy with and without previous photocoagulation. Concentrations of basic fibroblast growth factor and angiostatin were additionally measured in serum, while vascular endothelial growth factor is known to be regulated locally in the eye. Cytokines were measured by immunological methods.¶Results. Angiostatin could be detected in 2 out of 18 control patients and in 25 out of 34 diabetic patients (p < 0.00 001). Most importantly, production of angiostatin in human vitreous correlated significantly with previous retinal photocoagulation (p < 0.0001) in patients with proliferative diabetic retinopathy. Only two patients (one control and one diabetic) had detectable serum concentrations of angiostatin. Additionally patients with proliferative diabetic retinopathy and with previous photocoagulation had significantly lower concentrations of vascular endothelial growth factor (0.9 ± 0.1 ng/ml; p < 0.0001) than diabetic patients without previous photocoagulation (4.0 ± 0.8 ng/ml). The investigation of vitreal and serum basic fibroblast growth factor concentrations yielded no significant differences between the groups.¶Conclusion/interpretation. Angiostatin is not a regularly expressed angiogenesis inhibitor in human vitreous. The alterations we observed suggest that local release of angiostatin and down-regulation of vascular endothelial growth factor mediate the therapeutic effects of retinal photocoagulation in proliferative diabetic retinopathy. [Diabetologia (2000) 43: 1404–1407]     

Enhanced angiogenesis following allogeneic blood transfusion1

Blood transfusions are associated with recurrence of solid cancers. Angiogenesis is essential for cancer growth. Our aim was to determine for the first time in a prospective cohort study the effect of prestorage allogeneic leucodepleted SAGM (saline, adenine, glucose, mannitol) red cell transfusion on angiogenic factor levels and in vitro angiogenesis. Forty pretransfusion adult hospital inpatients were selected consecutively. Serum vascular endothelial growth factor (VEGF) and endostatin were measured in each patient before and after prestorage allogeneic leucodepleted SAGM red cell transfusion. All samples were exposed to an in vitro endothelial cell proliferation assay and 10 sample groups were also exposed to an in vitro whole angiogenesis assay. The median number of units transfused was 2 (minimum–maximum, 2–4). Twenty‐nine (73%) patients had a rise in VEGF, with an overall increase of 118 pg/ml (quartiles ?5, 306; P < 0.01). Twenty‐eight (70%) patients had a decrease in endostatin, with an overall reduction of 1.2 ng/ml (quartiles 4.0, 0.0; P = 0.017). There was an overall 33% increase in endothelial cell proliferation (P < 0.01) and a 9.4% increase in in vitro whole assay angiogenesis (P < 0.01). Prestorage allogeneic leucodepleted SAGM red cell transfusions are associated with a favourable angiogenic factor imbalance and an elevation in in vitro angiogenesis.     

Manipulating angiogenesis in medicine

Blood vessels nourish organs with vital nutrients and oxygen and, thus, new vessels form when the embryo needs to grow or wounds are to heal. However, forming new blood vessels is a complex and delicate process, which, unfortunately, is often derailed. Thus, when insufficient vessels form, the tissue becomes ischaemic and stops to function adequately. Conversely, when vessels grow excessively, malignant and inflamed tissues grow faster. It is now becoming increasingly evident that abnormal vessel growth contributes to the pathogenesis of numerous malignant, ischaemic, inflammatory, infectious and immune disorders. With an in-depth molecular understanding, we should be better armamented to combat such angiogenic disorders in the future. That such therapeutic strategies might change the face of medicine is witnessed by initial evidence of success in the clinic.     

麝黄消瘤方对小鼠肝癌血管生成的抑制作用

目的:观察麝黄消瘤方(SHXLF)对肝癌H22模型小鼠癌细胞侵袭和转移的影响,探讨其作用机制。方法:以小鼠H22肝癌淋巴道转移模型为对象,观察其淋巴结指数、脾指数、胸腺指数,光镜下观察肝癌H22细胞局部侵袭和淋巴结转移程度。免疫组化SP法观察局部瘤组织血管内皮细胞生长因子(VEGF)表达水平和微血管密度(MVD)。结果:麝黄消瘤方可使局部瘤组织的侵袭和腘窝淋巴结转移程度及淋巴结指数有降低趋势;大中剂量组可使胸腺指数明显升高(P<0.05)。中药各治疗组可明显降低肝癌组织VEGF表达水平和MVD(P<0.05)。结论:SHXLF可抑制H22肝癌小鼠癌组织在局部及淋巴结的侵袭转移程度。其作用机制可能是通过调整免疫功能,杀伤癌细胞,抑制肿瘤血管生成(降低VEGF和MVD)而实现的。     

Role of the microenvironment in promoting angiogenesis in acute myeloid leukemia

Angiogenesis is a crucial event in the survival and progression of solid tumors. To determine whether angiogenesis in acute myeloid leukemia (AML) is an intrinsic property of leukemic cells, the vascularity of bone marrow biopsies was determined. Bone marrow vascularity in newly diagnosed or post-chemotherapy AML patients was increased 4-fold (P < 0.01) and 8.7-fold (P < 0.01), respectively, relative to controls. Vascular endothelial growth factor (VEGF) expression by AML blast cells was assessed by immunohistochemistry, and bone marrow cell supernatants were assayed for secretion of VEGF, fibroblast growth factor-2 (FGF-2), and endostatin by enzyme-linked immunosorbent assay. Diffuse cytoplasmic and strong extracellular VEGF immunoreactivity was seen in bone marrow aspirates from AML patients, but not controls. In contrast, there was no difference in the levels of VEGF, FGF-2, and endostatin secreted by mononuclear cells cultured from bone marrows of AML patients compared to normal controls following two days of culture in vitro. Total angiogenic potential of bone marrow cell supernatants was assessed by endothelial sprouting in vitro and by a chick chorioallantoic membrane assay. No differences were found between 2-day conditioned medium from normal and AML bone marrow mononuclear cells in either assay. Our data show a discrepancy between bone marrow vascularity and VEGF expression in vivo and VEGF expression and angiogenesis from 2-day conditioned medium ex vivo. This suggests that angiogenesis in AML likely represents a response to microenvironmental factors in vivo, rather than being an intrinsic property of leukemic cells.     

An in vitro model of angiogenesis: Basic features

This report describes a model of angiogenesis which develops in admixtures (co-cultures) of human umbilical vein endothelial cells (HUVEC) and human diploid fibroblasts of dermal origin from adult patients. The system does not require the addition of further growth factors other than those normally present in endothelial growth medium (EGM), nor matrix proteins, and cell growth and proliferation are allowed to occur in a standard low (2%) concentration of fetal calf serum. Angiogenesis was specifically stimulated in response to vascular endothelial growth factor (VEGF), resulting in an increased development of structures resembling a microvasculature bed. Alternatively, angiogenesis was inhibited by addition of an excess of neutralising anti-VEGF antibodies, and the anti-angiogenic drugs such as suramin. We briefly show that stimulatory and inhibitory activities can be easily and quickly quantified by image analysis. Tubule formation was confirmed by confocal and electron microscopy, and the development and disposition of these structures within the co-cultures has been analysed immunochemically to show expression of specific endothelial cell determinants, such as PECAM-1. On this and a number of other criteria, the findings validate this in vitro process as a model of in vivo angiogenesis that can be quantified to assay stimulatory and inhibitory agents, signals and drugs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple angiogenesis stimulators in a single malignancy: implications for anti-angiogenic tumour therapy

Anti-angiogenesis is likely to develop into a novel therapeutic approach for patients with solid malignancies. Most current clinical trials evaluate anti-angiogenic drugs aimed primarily against single angiogenesis stimulators. Here, we show that a single solid malignancy, i.e., a human embryonal rhabdomyosarcoma, produces in vivo at least three biologically active angiogenesis stimulators (vascular endothelial growth factor, basic fibroblast growth factor and interleukin-8). This suggests that tumour angiogenesis results from the activity of multiple, rather than a single angiogenesis stimulator(s). We, furthermore, show that a combination of anti-angiogenic drugs is more effective in inhibiting tumour-induced endothelial cell growth than a single agent. Our results imply that clinical anti-angiogenic strategies for the treatment of solid malignancies may be most effective when multiple rather than single anti-angiogenic drugs are used. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vascular endothelial growth factor (VEGF) in children, adolescents and young adults with Type 1 diabetes mellitus: relation to glycaemic control and microvascular complications.

AIMS: To evaluate serum levels of vascular endothelial growth factor (VEGF) in a large group of children, adolescents and young adults with Type 1 diabetes mellitus to investigate whether increased VEGF concentrations are associated with long-term glycaemic control and microvascular complications. METHODS: The study involved 196 patients with Type 1 diabetes mellitus (age range 2-24 years, onset of diabetes before the age of 12 years, duration of disease longer than 2 years), without clinical and laboratory signs of microvascular complications; they were divided into three groups (group 1 - n = 37, age < 6 years; group 2 - n = 71, age 6-12 years; group 3 - n = 88, age > 12 years). Fifty-three adolescents and young adults (age 16.1-29.7) with different grades of diabetic retinopathy and microalbuminuria were also selected (group 4). A total of 223 healthy controls were matched for age and sex with each group of patients with diabetes mellitus. RESULTS: VEGF serum levels were significantly increased in pre-school and pre-pubertal children with diabetes as well as in pubertal patients compared to controls. VEGF concentrations were markedly increased in adolescents and young adults with microvascular complications compared with both healthy controls and diabetic patients without retinopathy or nephropathy. Multivariate analysis showed that elevation of VEGF in serum was an independent correlate of complications. One-year mean HbA1c values were significantly correlated with VEGF concentrations (r = 0.372; P < 0.01). Children with HbA1c levels greater than 10% had significantly higher VEGF concentrations when compared with matched patients whose HbA1c levels were lower than 10%. In poorly controlled diabetic children (HbA1c > 10%), long-term (2 years) improvement of glycaemic control (aiming at HbA1c < 7%) resulted in a significant reduction of VEGF levels. CONCLUSIONS: VEGF serum concentrations are increased in prepubertal and pubertal children with diabetes. Glycaemic control influences VEGF serum levels. Severity of microvascular complications is associated with marked increase of VEGF concentrations in the serum of these patients.     

Evaluation of a combination digital retinal camera with spectral-domain optical coherence tomography (SD-OCT) that might be used for the screening of diabetic retinopathy with telemedicine: A pilot study

Aims

Pilot study to determine whether an instrument combining a non-mydriatic retinal camera and spectral domain optical coherence tomography (SD-OCT) is effective for screening patients with diabetic retinopathy (DR).