VEGF 家族及其在肿瘤生长中作用的研究

血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)家族是一类多功能的细胞因子,在血管生成和淋巴管生成中具有直接和间接的调控作用,可促进内皮细胞增殖、促进血管生成以及增加血管的通透性。VEGF/VEGFR轴由多重配基和受体质量叠加交错组成,并且受体与配基结合具有专一性,在不同的细胞中具有不同的细胞类型表达和功能.启动VEGF信号通路,触发了一个网状的信号过程,从而促进血管内皮细胞生长、转移和存活。进来研究发现,VEGF的一个重要作用表现为可动员内皮祖细胞从骨髓向远处转移从而形成新生血管,因而有必要设计和发展针对这一途径的抑制因子。随着研究的深入,VEGF促进肿瘤血管生成的作用和与人类癌症的发病机制的关系是确定的,因此,抑制VEGF途径被确认为是一种重要的有效的抗癌模式     

Sustained VEGF blockade results in microenvironmental sequestration of VEGF by tumors and persistent VEGF receptor-2 activation

Vascular endothelial growth factor (VEGF) blockade has been validated clinically as a treatment for human cancers, yet virtually all patients eventually develop progressive disease during therapy. In order to dissect this phenomenon, we examined the effect of sustained VEGF blockade in a model of advanced pediatric cancer. Treatment of late-stage hepatoblastoma xenografts resulted in the initial collapse of the vasculature and significant tumor regression. However, during sustained treatment, vessels recovered, concurrent with a striking increase in tumor expression of perlecan, a heparan sulfate proteoglycan. Whereas VEGF mRNA was expressed at the periphery of surviving clusters of tumor cells, both secreted VEGF and perlecan accumulated circumferential to central vessels. Vascular expression of heparanase, VEGF receptor-2 ligand binding, and receptor activation were concurrently maintained despite circulating unbound VEGF Trap. Endothelial survival signaling via Akt persisted. These findings provide a novel mechanism for vascular survival during sustained VEGF blockade and indicate a role for extracellular matrix molecules that sequester and release biologically active VEGF.     

VEGF and VEGF receptor levels in retinal and brain-derived endothelial cells

Vascular endothelial cell growth factor (VEGF) is an endothelial cell-specific angiogenic and permeability-inducing factor that has been implicated in the pathogenesis of diabetic retinopathy. The objectives of this study are to compare VEGF and VEGF receptor expression between retinal and brain-derived endothelial cells cultured in 5 or 30 mM glucose for 5 days. Our results show that expression of cell-surface VEGF receptors, assessed by flow cytometry, is higher in retinal-derived endothelial cells. RT-PCR results show that both retinal and brain-derived endothelial cells express comparable levels and types of VEGF. Exposure to 30 mM glucose for 5 days did not alter levels of VEGF or VEGF receptors. The higher level of VEGF receptor expression in retinal endothelial cells suggests that the retinal microcirculation may be more sensitive to the effects of VEGF and this may contribute to the pathogenesis of diabetic retinopathy.     

VEGF in biological control

Vascular endothelial growth factor A (VEGF-A) belongs to a family of heparin binding growth factors that include VEGF-B, VEGF-C, VEGF-D, and placental-like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well-known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non-endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non-angiogenic processes that take place in adult organs.     

Variations in transfection efficiency of VEGF165 and VEGF121-cDNA

Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF₁₂₁ and VEGF₁₆₅ isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51–0.95 pg/mL/μg protein in umbilical artery, through 2.32–2.39 pg/mL/μg protein in coronary artery, to 5.45–7.52 pg/mL/μg protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 μg VEGF₁₂₁-cDNA/in the wells)—an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.     

Engineered conformation-dependent VEGF peptide mimics are effective in inhibiting VEGF signaling pathways

Angiogenesis, or formation of new blood vessels, is crucial to cancer tumor growth. Tumor growth, progression, and metastasis are critically influenced by the production of the pro-angiogenic vascular endothelial growth factor (VEGF). Promising anti-angiogenic drugs are currently available; however, their susceptibilities to drug resistance and long term toxicity are serious impediments to their use, thus requiring the development of new therapeutic approaches for safe and effective angiogenic inhibitors. In this work, peptides were designed to mimic the VEGF-binding site to its receptor VEGFR-2. The VEGF conformational peptide mimic, VEGF-P3(CYC), included two artificial cysteine residues, which upon cyclization constrained the peptide in a loop native-like conformation to better mimic the anti-parallel structure of VEGF. The engineered cyclic VEGF mimic peptide demonstrated the highest affinity to VEGFR-2 by surface plasmon resonance assay. The VEGF peptide mimics were evaluated as inhibitors in several in vitro assays in which VEGF-dependent signaling pathways were observed. All VEGF mimics inhibited VEGFR-2 phosphorylation with VEGF-P3(CYC) showing the highest inhibitory effects when compared with unstructured peptides. Additionally, we show in several angiogenic in vitro assays that all the VEGF mimics inhibited endothelial cell proliferation, migration, and network formation with the conformational VEGF-P3 (CYC) being the best. The VEGF-P3(CYC) also caused a significant delay in tumor development in a transgenic model of VEGF(+/-)Neu2-5(+/-). These results indicate that the structure-based design is important for the development of this peptidomimetic and for its anti-angiogenic effects.     

VEGF isoforms

The Rho-family of p21 small GTPases are directly linked to the regulation of actin-based motile machinery and play a key role in the control of cell migration. Aside from the original and most well-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerous isoforms of these key proteins have been identified and shown to have specific roles in regulating various cellular motility processes. The major difficulty in addressing these isoform-specific effects is that isoforms typically contain highly similar primary amino acid sequences and thus are able to interact with the same upstream regulators and the downstream effector targets. Here, we will introduce the major members of each GTPase subfamily and discuss recent advances in the design and application of fluorescent resonance energy transfer-based probes, which are at the forefront of the technologies available to directly probe the differential, spatiotemporal activation dynamics of these proteins in live single cells. Currently, it is possible to specifically detect the activation status of RhoA vs. RhoC isoforms, as well as Cdc42 vs. TC-10 isoforms in living cells. Clearly, additional efforts are still required to produce biosensor systems capable of detecting other isoforms of Rho GTPases including RhoB, Rac2/3, RhoG, etc. Through such efforts, we will uncover the isoform-specific roles of these near-identical proteins in living cells, clearly an important area of the Rho GTPase biology that is not yet fully appreciated.     

Role of VEGF in organogenesis

The cardiovascular system, consisting of the heart, blood vessels and hematopoietic cells, is the first organ system to develop in vertebrates and is essential for providing oxygen and nutrients to the embryo and adult organs. Work done predominantly using the mouse and zebrafish as model systems has demonstrated that Vascular Endothelial Growth Factor (VEGF, also known as VEGFA) and its receptors KDR (FLK1/VEGFR2), FLT1 (VEGFR1), NRP1 and NRP2 play essential roles in many different aspects of cardiovascular development, including endothelial cell differentiation, migration and survival as well as heart formation and hematopoiesis. This review will summarize the approaches taken and conclusions reached in dissecting the role of VEGF signalling in vivo during the development of the early cardiovasculature and other organ systems. The VEGF?mediated assembly of a functional vasculature is also a prerequisite for the proper formation of other organs and for tissue homeostasis, because blood vessels deliver oxygen and nutrients and vascular endothelium provides inductive signals to other tissues. Particular emphasis will therefore be placed in this review on the cellular interactions between vascular endothelium and developing organ systems, in addition to a discussion of the role of VEGF in modulating the behavior of nonendothelial cell populations.     

VEGF in the nervous system

Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.Key words: VEGF, neuron, neurogenesis, glia, endothelial cell, blood vessel, angiogenic niche     

VEGF in the nervous system

Vascular endothelial growth factor (VEGF, VEGFA) is critical for blood vessel growth in the developing and adult nervous system of vertebrates. Several recent studies demonstrate that VEGF also promotes neurogenesis, neuronal patterning, neuroprotection and glial growth. For example, VEGF treatment of cultured neurons enhances survival and neurite growth independently of blood vessels. Moreover, evidence is emerging that VEGF guides neuronal migration in the embryonic brain and supports axonal and arterial co-patterning in the developing skin. Even though further work is needed to understand the various roles of VEGF in the nervous system and to distinguish direct neuronal effects from indirect, vessel-mediated effects, VEGF can be considered a promising tool to promote neuronal health and nerve repair.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 91-103.     

Role of VEGF in organogenesis

The cardiovascular system, consisting of the heart, blood vessels and hematopoietic cells, is the first organ system to develop in vertebrates and is essential for providing oxygen and nutrients to the embryo and adult organs. Work done predominantly using the mouse and zebrafish as model systems has demonstrated that Vascular Endothelial Growth Factor (VEGF, also known as VEGFA) and its receptors KDR (FLK1/VEGFR2), FLT1 (VEGFR1), NRP1 and NRP2 play essential roles in many different aspects of cardiovascular development, including endothelial cell differentiation, migration and survival as well as heart formation and hematopoiesis. This review will summarize the approaches taken and conclusions reached in dissecting the role of VEGF signalling in vivo during the development of the early cardiovasculature and other organ systems. The VEGF-mediated assembly of a functional vasculature is also a prerequisite for the proper formation of other organs and for tissue homeostasis, because blood vessels deliver oxygen and nutrients and vascular endothelium provides inductive signals to other tissues. Particular emphasis will therefore be placed in this review on the cellular interactions between vascular endothelium and developing organ systems, in addition to a discussion of the role of VEGF in modulating the behavior of nonendothelial cell populations.Key words: VEGF, VEGF receptors, organogenesis, mouse, angiogenesis, cardiovascular, conditional mutagenesis, Cre-loxP system     

VEGF function in vascular pathogenesis

Vascular endothelial growth factor (VEGF) has proven to be the most critical angiogenic factor identified to date; its discovery has transformed the field of angiogenesis research. After two decades of intense studies by numerous laboratories, the complex biology of VEGF is slowly being elucidated. VEGF is a collection of several different isoforms with overlapping but also unique functions. Furthermore, there are at least four different VEGF receptors, adding to the complexity of the VEGF/receptor system. VEGF exerts its effects on endothelial cells, inflammatory cells and neuronal cells, and its expression may contribute to the pathologies of diseases ranging from retinopathies to cancer. This review focuses on the biochemistry and cell biology of VEGF, with particular emphasis on the different isoforms and receptors, thereby illustrating the multifunctional nature of this growth factor. Lastly, critical considerations based on the complex biology of VEGF for developing specific and more targeted anti-VEGF therapeutic agents will be discussed.     

VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.

Note: Previously published in VEGF in Development, edited by Christiana Ruhrberg. Landes Bioscience and Springer Science+Business Media 2008; pp. 14-29.     

VEGF receptor signaling in vertebrate development

The secreted glycoprotein vascular endothelial growth factor A (VEGF or VEGFA) affects many different cell types and modifies a wide spectrum of cellular behaviors in tissue culture models, including proliferation, migration, differentiation and survival. The versatility of VEGF signaling is reflected in the complex composition of its cell surface receptors and their ability to activate a variety of different downstream signaling molecules. A major challenge for VEGF research is to determine which of the specific signaling pathways identified in vitro control development and homeostasis of tissues containing VEGF-responsive cell types in vivo.     

Assessment of VEGF and VEGF receptor concentrations in patients with benign and malignant thyroid tumors

Neoangiogenesis is a significant event in a cascade of growth and progression of solid tumors. Assessment of the tissue expression and measurement of the concentrations of angiogenic and antiangiogenic factors, contributing to this process, in body fluids, can be used not only for an early diagnosis of tumors and their staging but also as an important parameter of treatment efficiency evaluation. The aim of this study is to evaluate the concentrations of crucial angiogenic cytokine VEGF and its soluble receptors in peripheral blood of patients with benign and malignant thyroid tumors. The study comprised 35 patients with thyroid cancer and 10 patients with follicular neoplasm, both diagnosed by means of ultrasound-guided fine-needle aspiration biopsy. For these patients surgical treatment was instituted. The examined angiogenic factors were determined preoperatively and 4 weeks after the surgical procedures. The results were compared with the control group which comprised 10 healthy individuals. Analysing obtained results, we demonstrated high VEGF concentrations and low soluble VEGF receptor concentrations in patients with benign and malignant thyroid tumors. This fact confirms a vital role of VEGF in angiogenesis of thyroid tumors and a hypothetical antiangiogenic activity of its soluble receptors. Disequilibrium of the above-mentioned angiogenic factor concentrations is probably essential for the growth and progression of benign and malignant thyroid tumors.     

Greater free plasma VEGF and lower soluble VEGF receptor-1 in acute mountain sickness.

Vascular endothelial growth factor (VEGF) is a hypoxia-induced protein that produces vascular permeability, and limited evidence suggests a possible role for VEGF in the pathophysiology of acute mountain sickness (AMS) and/or high-altitude cerebral edema (HACE). Previous studies demonstrated that plasma VEGF alone does not correlate with AMS; however, soluble VEGF receptor (sFlt-1), not accounted for in previous studies, can bind VEGF in the circulation, reducing VEGF activity. In the present study, we hypothesized that free VEGF is greater and sFlt-1 less in subjects with AMS compared with well individuals at high altitude. Subjects were exposed to 4,300 m for 19-20 h (baseline 1,600 m). The incidence of AMS was determined by using a modified Lake Louise symptom score and the Environmental Symptoms Questionnaire for cerebral effects. Plasma was collected at low altitude and after 24 h at high altitude, or at time of illness, and then analyzed by ELISA for VEGF and for soluble VEGF receptor, sFlt-1. AMS subjects had lower sFlt-1 at both low and high altitude compared with well subjects and a significant rise in free plasma VEGF on ascent to altitude compared with well subjects. We conclude that increased free plasma VEGF on ascent to altitude is associated with AMS and may play a role in pathophysiology of AMS.