基于VEGFR2/Src/FAK通路探讨葫芦素D抗血管新生机制

目的探讨葫芦素D对VEGF诱导的血管新生的作用及其机制。方法鸡胚绒毛尿囊膜血管新生实验、大鼠胸主动脉血管内皮迁出与人血管内皮细胞划痕实验检测葫芦素D对VEGF诱导的血管新生和内皮细胞迁移的作用,Western blot技术检测葫芦素D对VEGF诱导的VEGFR2及其下游的Src、FAK磷酸化水平的影响。结果葫芦素D对VEGF诱导的鸡绒毛尿囊膜血管新生具有明显的抑制作用,并能抑制VEGF诱导的大鼠胸主动脉环模型和划痕实验中血管内皮迁移。葫芦素D能够抑制VEGF刺激的VEGFR2、Src、FAK磷酸化。结论葫芦素D具有抑制VEGF诱导的血管新生作用,其机制可能与抑制VEGFR2及其下游的细胞信号通路蛋白活化有关。     

血管内皮生长因子及其受体在抗肿瘤血管新生中的地位及应用

VEGF及其受体是肿瘤血管新生过程中最重要的调控因素 ,在抗肿瘤血管新生中具有不可替代的地位。VEGF在许多肿瘤组织及培养的肿瘤细胞株中均有高水平的表达 ,其主要的作用途径是通过旁分泌的形式作用于内皮上的特异受体flt 1和KDR ,诱导血管内皮细胞增殖和迁移 ,构建新生的肿瘤血管。以VEGF及其受体为靶向的抗肿瘤血管新生主要是针对VEGF和受体作用途径的任一环节 ,阻断VEGF对肿瘤血管的作用 ,从而达到遏制肿瘤的生长和转移的目的 ,这给临床上的肿瘤治疗开辟了新的领域。     

抗人VEGF受体Ⅱ胞外Ⅲ区单克隆抗体的生物学活性研究

目的研究抗KDR单体Ycom1D3抑制VEGF诱导脐静脉内皮细胞（HUVEC）增殖的体外生物学活性。方法采用FACS鉴定Ycom1D3与抗原结合特异性，采用免疫共沉淀测定Ycom1D3阻断VEGF165刺激KDR酪氨酸激酶受体磷酸化作用，并采用[^3H]-Thymidine掺入法、内皮细胞损伤愈合试验和内皮细胞血管腔形成实验进一步确定Ycom1D3抑制VEGF165诱导内皮细胞增殖的中和活性。结果Ycom1D3不但能与HUVEC结合，而且能阻断由VEGF165刺激HUVEC表面KDR酪氨酸激酶受体磷酸化，进而显著抑制VEGF165诱导HUVEC增殖、迁移及体外三维胶原模型上内皮细胞毛细血管样结构的形成。结论Ycom1D3可以通过封闭KDR而抑制VEGF活性，在肿瘤及其它血管新生疾病治疗中具有潜在应用前景。     

内皮祖细胞与肿瘤血管新生

血管内皮祖细胞（EPCs）是内皮细胞的前体细胞,特异性表达CD34,CD133和VEGFR-2,具有向血管内皮细胞分化的潜能。EPCs主要位于骨髓和外周血。肿瘤的生长和转移依赖于肿瘤血管新生。肿瘤细胞可合成和释放多种细胞因子,在不同因子的趋化作用下EPCs从骨髓动员至外周血循环,然后迁移和定居到肿瘤组织,经细胞因子诱导分化为成熟内皮细胞,参与肿瘤血管新生。VEGF/VEGFR-2信号途径在EPCs参与的肿瘤血管新生方面起重要作用。     

VEGF165转染大鼠血管内皮细胞对新生血管形成影响的实验研究

目的探讨血管内皮细胞生长因子（vascular endothelial growth factor，VEGF）基因与增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）共表达载体转染大鼠血管内皮细胞，植入大鼠体内，观察诱导新生血管情况，为移植组织诱导新生血管形成奠定基础。方法对质粒pIRES2-EGFP／VEGF，岱进行扩增、纯化，以脂质体法转染大鼠血管内皮细胞并植入肾被膜下。采用荧光显微镜检测增强绿色荧光蛋白在内皮细胞中的表达，用流式细胞仪检测转染效率。用RT-PCR检测VEGF mRNA的表达。免疫组化检测VEGF在内皮细胞中的表达。切取移植肾脏组织标本,HE染色观察组织形态学变化。结果荧光显微镜观察到实验组内皮细胞有特异性的EGFP表达。流式细胞仪分析转染效率为13．06％。实验组血管内皮细胞胞核和胞浆中均有VEGF表达。RT-PCR显示实验组大鼠血管内皮细胞中有人源化VEGF165基因在mRNA水平表达。移植后14d，实验组大鼠肾被膜下可见成团的新生毛细血管网形成，而对照组及空白转染组尽管血管内皮细胞仍存活，但未形成明显血窦。结论转染VEGF是促进内皮细胞早期（14d内）形成新生血管的有效途径。     

KDR反义寡核苷酸对人血管内皮细胞的作用

VEGF及其受体KDR在肿瘤血管生成中起重要作用。我们用KDR特异性反义寡核苷酸作用于人血管内皮细胞，以阻断VEGF的自分泌作用通路，观察对细胞增殖的影响、细胞超微结构的变化及检测细胞DNA含量，测定细胞分泌VEGF的能力，并检测作用后KDR mRNA和KDR蛋白的水平。结果发现未经处理的人血管内皮细胞能分泌一定量的VEGF，KDR ASODN能抑制人血管内皮细胞内KDR基因的表达，并显著抑制细胞的增殖，在一定剂量下还可诱导凋亡。结果说明KDR ASODN能显著抑制血管内皮细胞的增殖，VEGF受体KDR在人血管内皮细胞的增殖和凋亡的凋控中起重要作用。     

miR-184在碱烧伤大鼠角膜新生血管中的表达及靶基因预测分析

目的 初步探miR-184在碱烧伤诱导大鼠角膜新生血管形成过程中的作用和基因调控机制.方法 碱烧伤诱导大鼠角膜新生血管形成.基质胶上建立人脐静脉血管内皮细胞体外三维培养体系.应用qRT-PCR检测miR-184的体内外表达.生物信息学方法预测并分析miR-184靶基因.结果 初步建立起血管新生的体内外研究模型,miR-184在碱烧伤大鼠角膜新生血管角膜中的表达(0.145±0.013)较正常角膜(1.015±0.189)显著下降(P＜0.01),而在人脐静脉血管内皮细胞中相对表达量极少(0.007±0.004) (P ＜0.05).预测的靶基因与VEGF血管生成信号通路有关.结论 新生血管进程中伴随着miR-184表达量降低,提示其与血管新生密切相关,可能通过VEGF信号通路参与碱烧伤角膜新生血管形成.     

GM-CSF对诱导人血管内皮细胞血管形成的影响及VEGF的作用

目的： 研究炎性因子粒细胞-巨噬细胞集落刺激因子（GM-CSF）对诱导人血管内皮细胞血管形成的影响及血管内皮生长因子（VEGF）的作用，为探讨GM-CSF和VEGF与动脉粥样硬化斑块内血管新生及斑块稳定性之间的关系提供实验基础。方法： 在 Matrigel上诱导人脐带静脉内皮细胞（HUVECs）形成管腔结构，从而建立稳定的血管形成的体外培养体系，然后施加实验因素。分别检测rhGM-CSF的浓度效应和时间效应，及加入VEGF165的作用。然后各实验组用CD34免疫细胞化学染色，光学显微镜下计数各组管腔数。最后用统计学方法进行分析。结果： 应用Matrigel可在体外诱导培养的HUVECs形成管腔结构。rhGM-CSF作用后，培养体系的管腔数逐渐增多，呈剂量及时间依赖效应；加入VEGF165后，管腔数显著增多。结论： 应用Matrigel可在体外诱导培养的HUVECs形成管腔结构。GM-CSF可以促进体外诱导人血管内皮细胞血管形成，并在一定范围内呈剂量及时间依赖效应。VEGF可以促进体外诱导人血管内皮细胞血管形成。     

血管内皮生长因子在肿瘤血管生成中的研究

血管内皮生长因子 (VEGF)具有促进内皮细胞增殖 ,加速新生血管形成及增加血管通透性等作用。在肿瘤的生长和转移中具有重要作用 ,是判断肿瘤预后的良好指标。     

血管内皮生长因子与肺疾病

血管内皮生长因子(VEGF)是一种特异地作用于血管内皮细胞的生长因子,具有促进血管内皮细胞增殖、刺激体内新生血管生成、促进血管通透性增加及维持血管正常状态和完整性的功能,它在肺癌的发生发展、低氧性肺动脉高压及肺纤维化的形成等多种肺疾病中起重要作用.     

Treatment with anti‐NAP monoclonal antibody reduces disease severity in murine model of novel angiogenic protein‐induced or ovalbumin‐induced arthritis

Rheumatoid arthritis (RA) is a polyarticular inflammatory, angiogenic disease. Synovial angiogenesis contributes to inflammation in RA. In this study we have developed an arthritic model in rats using a novel angiogenic protein (NAP), isolated from human synovial fluid of RA patients. We produced anti‐NAP monoclonal antibodies (mAbs) and investigated the therapeutic efficacy of the same in adjuvant‐induced or NAP‐induced arthritis as a model of human RA. The treatment of arthritic rats with anti‐NAP mAbs resulted in effective amelioration of paw oedema, radiological arthritic characteristics, serum levels of vascular endothelial growth factor (VEGF) and NAP, compared to that of untreated arthritic animals. Further, profiling of angiogenic markers such as synovial microvessel density, angiogenesis, CD31, VEGF and fms‐like tyrosine kinase (Flt1) by immunohistochemistry both in arthritic and anti‐NAP mAb‐treated animals revealed the efficacy of mAb as an anti‐angiogenic functional antibody. Therefore, NAP may be an attractive target to design anti‐angiogenic and anti‐arthritic therapies to control the pathogenesis of arthritis.     

Vascular endothelial growth factor mediates angiogenic activity during the proliferative phase of wound healing.

Angiogenesis is an essential component of normal wound repair, yet the primary mediators of wound angiogenesis have not been well described. The current study characterizes the contribution of vascular endothelial cell growth factor (VEGF) to the angiogenic environment of human surgical wounds. Surgical wound fluid samples (n = 70) were collected daily for up to 7 postoperative days (POD) from 14 patients undergoing mastectomy or neck dissection. VEGF levels in surgical wound fluid were lowest on POD 0, approximating values of serum, but increased steadily through POD 7. An opposite pattern was noted for basic fibroblast growth factor-2. Fibroblast growth factor-2, which has been previously described as a wound angiogenic factor, exhibited highest levels at POD 0, declining to near serum levels by POD 3. Surgical wound fluid form all time points stimulated marked endothelial cell chemotaxis and induced a brisk neovascular response in the rat corneal micropocket angiogenesis assay. Antibody neutralization of VEGF did not affect the in vitro chemotactic or the in vivo angiogenic activity early wound samples (POD 0). In contrast, VEGF neutralization significantly attenuated both chemotactic activity (mean decrease 76 +/- 13%, P < 0.01) and angiogenic activity (5 of 5 samples affected) of later wound samples (POD 3 and 6). The results suggest a model of wound angiogenesis in which an initial angiogenic stimulus is supplied by fibroblast growth factor-2, followed by a subsequent and more prolonged angiogenic stimulus mediated by VEGF.     

Selected angiogenic cytokines in systemic lupus erythematosus patients

The objective of this study was to determine the serum concentration of angiogenic factors (vascular endothelial growth factor, VEGF; transforming growth factor beta, TGF-β1; hepatic growth factor, HGF; basic fibroblast growth factor, bFGF; tumor necrosis factor alpha, TNF-α; soluble vascular endothelial growth factor receptor 1, sVEGF-R1; soluble vascular endothelial growth factor receptor 2, sVEGF-R2), the relationships among them and to assess the relation of their levels with the applied therapy in 48 females with systemic lupus erythematosus (SLE; 37 long-term treated +11 newly diagnosed). The control group consisted of 24 healthy women. A statistically significant increase of sVEGF-R2 and significant decrease of sVEGF-R1 were observed in the subgroup of newly diagnosed SLE patients as compared to the control subjects. No significant differences were found between serum angiogenic factors in the long-term treated subgroup and the control, the long-term treated subgroup and the newly diagnosed SLE patients after a 3-month treatment, and the subgroup of newly diagnosed SLE patients before therapy and after a 3-month treatment. The significant decrease in the serum of sVEGF-R2 was revealed in the subgroup treated for a long-time as compared to the subgroup of newly diagnosed untreated SLE patients. The analysis of relationships between serum concentration of sVEGF-R1 and other cytokines levels revealed positive correlation with concentration of VEGF and TNF-α in the total group of patients. In the newly diagnosed untreated subgroup, a strong positive correlation between concentration of sVEGF-R1 and bFGF was observed. Furthermore, a moderate positive correlation between concentration of sVEGF-R1 and the level of VEGF was revealed in the long-term treated patients. The association between sVEGF-R2 and HGF was also noted in this subgroup. The obtained data suggest the necessity of further investigations to determine the importance of angiogenic factors in pathogenesis and therapy of SLE.     

Essential role for platelet-activating factor-induced NF-kappaB activation in macrophage-derived angiogenesis

Activated monocyte-macrophages have been implicated in tumor angiogenesis via their capacity to produce many potent angiogenic factors. However, the mechanisms leading to production of these angiogenic factors in macrophages remain to be elucidated. In this study, we demonstrated by use of a mouse Matrigel implantation model that mouse peritoneal macrophages induce angiogenesis. mRNA expression and protein synthesis of macrophage-derived crucial angiogenic factors such as IL-1, TNF-alpha, basic fibroblast growth factor, and vascular endothelial growth factor (VEGF) were blocked by platelet-activating factor (PAF) receptor antagonists. It was also observed that inhibitors of NF-kappaB blocked macrophage production of these angiogenic factors. Gene expression and protein synthesis of the angiogenic factors cited above were also inhibited in IkappaBalpha-mutated macrophages. VEGF is the most potent angiogenic factor in macrophage-induced angiogenesis. PAF antagonists or NF-kappaB inhibitors also inhibit the capacity of conditioned medium from LPS-stimulated human peripheral blood monocytes to induce sprouting of porcine pulmonary arterial endothelial cells. These data indicate that PAF-induced NF-kappaB activation is a common upstream pathway leading to the production of crucial macrophage-derived angiogenic factors. This will provide an important clue for a better understanding of mechanisms involved in tumor angiogenesis.     

Inhibition of angiogenesis by NSAIDs: molecular mechanisms and clinical implications

Angiogenesis, the formation of new capillary blood vessels, is a fundamental process essential for reproduction and embryonic development. It is crucial to the healing of tissue injury because it provides essential oxygen and nutrients to the healing site. Angiogenesis is also required for cancer growth and progression since tumor growth requires an increased nutrient and oxygen supply. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs worldwide for treating pain, arthritis, cardiovascular diseases, and more recently for colon cancer prevention. However, NSAIDs produce gastrointestinal ulcers and delay ulcer healing. Recently NSAIDs have been demonstrated to inhibit angiogenesis, but the underlying mechanisms are only beginning to be elucidated. The inhibition of angiogenesis by NSAIDs is a causal factor in the delay of ulcer healing, and it is becoming clear that this is also likely to be one of the mechanisms by which NSAIDs can reduce or prevent cancer growth. Based on the experimental data and the literature, the mechanisms by which NSAIDs inhibit angiogenesis appear to be multifactorial and likely include local changes in angiogenic growth factor expression, alteration in key regulators and mediators of vascular endothelial growth factor (VEGF), increased endothelial cell apoptosis, inhibition of endothelial cell migration, recruitment of inflammatory cells and platelets, and/or thromboxane A2 mediated effects. Some of these mechanisms include: inhibition of mitogen-activated protein (Erk2) kinase activity; suppression of cell cycle proteins; inhibition of early growth response (Egr-1) gene activation; interference with hypoxia inducible factor 1 and VEGF gene activation; increased production of the angiogenesis inhibitor, endostatin; inhibition of endothelial cell proliferation, migration, and spreading; and induction of endothelial apoptosis.     

Effects of modified collagen matrices on human umbilical vein endothelial cells

The most commonly used biomaterials fail to ensure sufficient angiogenesis for fast in vivo incorporation. This results in central necrosis and consequent infection. One way of obtaining a high angiogenic response is the application of vascular endothelial growth factor (VEGF). To obtain a sustained release of these cytokines, heparin was incorporated into collagen matrices using 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC) and N-hydroxysuccinmide (NHS). The functionality of the heparinized collagen matrices was then enhanced by immobilization of VEGF via its heparin-binding domain. This procedure changed in vitro degradation behavior and water-binding capacity. Accelerated endothelial cell proliferation was also achieved. A range of different heparin and EDC/NHS concentrations in combination with VEGF induced variation in endothelial cell growth and tubulogenic formation. Polymerized collagen scaffolds presented biointeractive systems with integrated angiogenic activity. This may become a useful tool in the clinical therapy of disorders connected with wound healing.     

Promoting angiogenesis via manipulation of VEGF responsiveness with notch signaling

Promoting angiogenesis via delivery of vascular endothelial growth factor (VEGF) and other angiogenic factors is both a potential therapy for cardiovascular diseases and a critical aspect for tissue regeneration. The recent demonstration that VEGF signaling is modulated by the Notch signaling pathway, however, suggests that inhibiting Notch signaling may enhance regional neovascularization, by altering the responsiveness of local endothelial cells to angiogenic stimuli. We tested this possibility with in vitro assays using human endothelial cells, as well as in a rodent hindlimb ischemia model. Treatment of cultured human endothelial cells with DAPT, a gamma secretase inhibitor, increased cell migration and sprout formation in response to VEGF stimulation with a biphasic dependence on DAPT concentration. Further, delivery of an appropriate combination of DAPT and VEGF from an injectable alginate hydrogel system into ischemic hindlimbs led to a faster recovery of blood flow than VEGF or DAPT alone; perfusion levels reached 80% of the normal level by week 4 with combined DAPT and VEGF delivery. Direct intramuscular or intraperitoneal injection of DAPT did not result in the same level of improvement, suggesting that appropriate presentation of DAPT (gel delivery) is important for its activity. DAPT delivery from the hydrogels also did not lead to any adverse side effects, in contrast to systemic introduction of DAPT. Altogether, these results suggest a new approach to promote angiogenesis by controlling Notch signaling, and may provide new options to treat patients with diseases that diminish angiogenic responsiveness.     

Induction of a potential paracrine angiogenic loop between human THP-1 macrophages and human microvascular endothelial cells during Bartonella henselae infection

Bartonella henselae is responsible for various disease syndromes that loosely correlate with the immune status of the host. In the immunocompromised individual, B. henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposi's sarcoma. We hypothesize that B. henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells. Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1beta (IL-1beta), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to live B. henselae. Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1beta induction. IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells. In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential. These data suggest that Bartonella modulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis. B. henselae induction of VEGF, IL-1beta, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.     

Modulation of angiogenic potential of collagen matrices by covalent incorporation of heparin and loading with vascular endothelial growth factor

One of the prominent shortcomings of matrices for tissue engineering is their poor ability to support angiogenesis. We report here on experiments to enhance the angiogenic properties of collagen matrices. Our aim is to achieve this goal by covalently incorporating heparin into collagen matrices and by physically immobilizing angiogenic vascular endothelial growth factor (VEGF) to the heparin. The immobilization of heparin was performed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Carboxyl groups on the heparin are activated to succinimidyl esters, which react with amino functions on the collagen to zero length cross-links. This modification leads--in addition to the incorporation of heparin--to gross changes in in vitro degradation behavior and water-binding capacity. As a first approach to testing angiogenic capabilities, endothelial cells were exposed to nonmodified and heparinized collagen matrices. This exposure leads to an increase in endothelial cell proliferation. The increase can be further enhanced by loading the (heparinized) collagen matrices with VEGF. Evaluation of the angiogenic potential of heparinized matrices was further investigated by exposing them to the chorioallantoic membrane of chicken embryos and to the subcutaneous tissue of rats. Both approaches show that heparinized matrices have substantially increased angiogenic potential. In particular, the loading of heparinized matrices with VEGF invokes a further increase in angiogenic potential. It is apparent that the physical binding of VEGF to heparin allows for a release that is beneficial to angiogenesis. By varying the heparin and EDC/NHS concentrations during the modification process and by varying the loading with VEGF, the angiogenic potential as well as the degradation behavior can be adapted to obtain matrices that fulfill specific angiogenic requirements in the field of tissue engineering.