Design of vascular endothelium-specific drug-targeting strategies for the treatment of cancer

Tumor endothelial cells are actively involved in the neovascularization processes that accompany tumor growth. Their easy accessibility for systemically applied therapeutics makes them interesting targets for therapeutic intervention. Especially for drug targeting-based therapeutics that often consist of macromolecular moieties, the tumor endothelium is considered a much better target than the tumor cells located behind the vascular wall barrier. In this review, the general principles underlying the development and choices in the development of vascular drug-targeting strategies are discussed. An overview of target epitopes identified in the past two decades is followed by a summary of those strategies that directly or indirectly induced tumor blood flow blockade in vivo. The demonstrated therapeutic success in pre-clinical animal models in debulking large tumor masses and inhibiting tumor outgrowth warrant further development of these therapeutic approaches. Yet, more effort should be put in studies in which the efficacy of different effector activities aimed at the same target, of one effector activity aimed at different targets, and of multiple target strategies are be compared. Combining these data with proper inventories on the molecular basis of tumor endothelial heterogeneity in general will make possible the development of tumor vascular drug-targeting strategies towards clinical application.     

肿瘤血管靶向药物的研究进展

肿瘤血管在实体瘤的发生发展中具有重要的作用,靶向肿瘤血管的新药研发已成为一个热点领域.抗肿瘤血管的治疗策略分为肿瘤新生血管生成抑制剂（tumor angiogenesis inhibitor,TAI）和肿瘤血管靶向药物（vascular targeting agents,VTAs）两方面的研究.肿瘤新生血管生成抑制剂旨在抑制肿瘤新生血管生成的过程,而肿瘤血管靶向药物则是通过快速而有选择性地破坏肿瘤血管功能,使肿瘤血供受阻,导致肿瘤坏死.VTA类药物分为两类：一是小分子抑制剂（small molecule agents）,利用肿瘤血管和正常组织血管存在的差别选择性地破坏肿瘤血管;另一种是生物制剂（biological agents）,借助能够特异结合肿瘤血管的配体将毒素、凝血诱导剂、凋亡诱导分子等运送到肿瘤血管,引起血管阻塞使肿瘤坏死.     

A transgenic Tie2-GFP athymic mouse model; a tool for vascular biology in xenograft tumors

We report the generation of a transgenic Tie2-GFP athymic nude mouse, carrying green fluorescent blood vessels throughout the body. This transgenic mouse is a tool for studies in vascular biology, and is especially of interest for imaging of tumor angiogenesis and the study of anti-angiogenesis strategies in (human) xenografts. Intravital microscopy identified the presence of blood conducting structures that are not lined by endothelial cells. Dedifferentiation of aggressive tumor cells can lead to acquisition of endothelial characteristics. This process of tumor cell plasticity, also referred to as vasculogenic mimicry, has been suggested to contribute to the circulatory system in a tumor. In plastic EW7 Ewing sarcoma tumors in these Tie2-GFP mice, we observed blood flow in both regular blood vessels and non-fluorescent tumor cell-lined channels, visualizing in vivo hemodynamics in vasculogenic channels. These results demonstrate that the transgenic Tie2-GFP athymic mouse model is a valuable tool for vascular biology research.     

Vascular disrupting agents

A clear definition for vascular targeting agents (VTAs) and vascular disrupting agents (VDAs) has separated the two as distinct methods of cancer treatment. VDAs differ from VTAs (antiangiogenesis drugs) in their mechanism of action. VTAs attempt to keep new blood vessels from forming and do not act on blood vessels that already feed existing tumors. In contrast, VDAs cause the vascular structure inside a solid tumor to collapse, depriving the tumor of blood and oxygen it needs to survive. Therefore, VDAs are an attractive way to approach the cancer problem by combating developed tumors. The following review discusses six small molecule VDAs, namely DMXAA, ZD6126, TZT1027, CA4P, AVE8062, and Oxi4503, their synthesis, biological mechanism of action, and current clinical status.     

Green tea catechin inhibits ephrin-A1-mediated cell migration and angiogenesis of human umbilical vein endothelial cells

Angiogenesis, the formation of new blood vessels from preexisting capillaries, is essential for tumor progression and metastasis. During tumor neovascularization, vascular endothelial growth factor and ephrin (Eph) families emerge as critical mediators of angiogenesis. The green tea catechin epigallocatechin gallate (EGCG), a tyrosine kinase inhibitor, has been demonstrated in previous studies to be an effective antiangiogenesis agent. However, the inhibitory effect of green tea catechins on ephrin-A1-mediated tumor angiogenesis has not been demonstrated yet. Thus, in this study, we investigated the molecular mechanism of ephrin-A1-mediated cell migration and angiogenesis, as well as the inhibitory effects of EGCG. Here we show that ephrin-A1 mediates endothelial cell migration and regulates vascular remodeling in tumor neovascularization in vitro. We also demonstrated that ephrin-A1-mediated cell migration required the activation of extracellular-regulated kinase (ERK-1/2) but not of phosphatidylinositol-3-kinase. The green tea catechin EGCG inhibited ephrin-A1-mediated endothelial cell migration, as well as tumor angiogenesis, in a dose-dependent manner. Furthermore, EGCG inhibited the ephrin-A1-mediated phosphorylation of EphA2 and ERK-1/2. Taken together, these data indicated that activation of ERK-1/2 plays an essential role in ephrin-A1-mediated cell migration. EGCG inhibited ephrin-A1-mediated endothelial migration and angiogenesis. It suggests a novel antiangiogenesis application of EGCG in cancer chemoprevention.     

Vascular remodeling marks tumors that recur during chronic suppression of angiogenesis

The potential for avoiding acquired resistance to therapy has been proposed as one compelling theoretical advantage of antiangiogenic therapy based on the normal genetic status of the target vasculature. However, previous work has demonstrated that tumors may resume growth after initial inhibition if antiangiogenic blockade is continued for an extended period. The mechanisms of this recurrent growth are unclear. In these studies, we characterized molecular changes in vasculature during apparent resumption of xenograft growth after initial inhibition by vascular endothelial growth factor blockade, "metronome" topotecan chemotherapy, and combined agents in a xenograft murine model of human Wilms' tumor. Tumors that grew during antiangiogenic blockade developed as viable clusters surrounding strikingly remodeled vessels. These vessels displayed significant increases in diameter and active proliferation of vascular mural cells and expressed platelet-derived growth factor-B, a factor that functions to enhance vascular integrity via stromal cell recruitment. In addition, remodeled vessels were marked by expression of ephrinB2, required for proper assembly of stromal cells into vasculature. Thus, enhanced vascular stability appears to characterize tumor vessel response to chronic antiangiogenesis, features that potentially support increased perfusion and recurrent tumor growth.     

Effects of bradykinin on the hemodynamics of tumor and granulating normal tissue microvasculature.

Bradykinin (BK) is an important endogenous mediator of microvascular flow modulation. Since the structure of the microcirculation is very different in tumor tissues than in normal tissues, bradykinin may elicit different responses in tumors. This study was designed to test the hypothesis that local administration of bradykinin increases blood flow preferentially in normal tissue relative to adjacent tumor tissue, resulting in a "vascular steal" phenomenon. Microvessel diameters (D), velocities (Vc), length densities, shear rates, and intermittent flow frequencies were measured every 10 min before, during, and after 40 min exposure to BK in rats with dorsal flap window chambers 9 days after tumor implantation. Separate studies were made of normal vessels outside the tumor margin, the hypervascular tumor periphery, and the tumor center. Bradykinin was administered with a suffusion medium flowing over the tissue at 1-2 ml/min with a BK concentration of 1.6 x 10(7) M. Administration of BK created five distinct changes in normal and tumor vessel function that varied over time, but coincidentally reached a maximum effect after 20 min exposure to BK. In normal vessels, increased Vc and D led to increased flow, which reached a peak 20 min after onset of suffusion with BK. In contrast, in centrally located tumor vessels, decreased D and Vc were observed in most vessels during the initial 10-20 min of suffusion. In addition, there was a significant increase in intermittent flow frequency in tumor central vessels, which peaked after 20 min of suffusion with BK. These five separate observations that coincided at 20 min of suffusion are consistent with a "vascular steal" phenomenon. The increase in normal microvessel D and Vc at 20 min suggests that BK causes vasodilation in arterioles. The coincident decrease in tumor microvessel D and Vc suggests that tumor feeding vessels are less able to respond to BK by vasodilating. The concomitant increase in intermittent flow frequency in tumor vessels suggests that a reduction in pressure drop occurred after 20 min exposure to BK, which is also consistent with "vascular steal." Since BK is also known to increase vascular permeability, it is possible that increases in interstitial fluid pressure brought on by exposure to BK contributed to the observed reduction in tumor blood flow. In normal vessels, reduced D and Vc, relative to peak values, were noted after 40 min suffusion with BK. Adherence of leukocytes to the vessel walls was prominent and microthrombi were also observed during this period. No evidence of such adhesion was seen in tumor vessels, although microthrombi were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tumor blood flow differs between mouse strains: consequences for vasoresponse to photodynamic therapy

Fluctuations in tumor blood flow are common and attributed to factors such as vasomotion or local vascular structure, yet, because vessel structure and physiology are host-derived, animal strain of tumor propagation may further determine blood flow characteristics. In the present report, baseline and stress-altered tumor hemodynamics as a function of murine strain were studied using radiation-induced fibrosacomas (RIF) grown in C3H or nude mice. Fluctuations in tumor blood flow during one hour of baseline monitoring or during vascular stress induced by photodynamic therapy (PDT) were measured by diffuse correlation spectroscopy. Baseline monitoring revealed fluctuating tumor blood flow highly correlated with heart rate and with similar median periods (i.e., ～9 and 14 min in C3H and nudes, respectively). However, tumor blood flow in C3H animals was more sensitive to physiologic or stress-induced perturbations. Specifically, PDT-induced vascular insults produced greater decreases in blood flow in the tumors of C3H versus nude mice; similarly, during baseline monitoring, fluctuations in blood flow were more regular and more prevalent within the tumors of C3H mice versus nude mice; finally, the vasoconstrictor L-NNA reduced tumor blood flow in C3H mice but did not affect tumor blood flow in nudes. Underlying differences in vascular structure, such as smaller tumor blood vessels in C3H versus nude animals, may contribute to strain-dependent variation in vascular function. These data thus identify clear effects of mouse strain on tumor hemodynamics with consequences to PDT and potentially other vascular-mediated therapies.     

Blood flow dynamics after photodynamic therapy with verteporfin in the RIF-1 tumor

In the present study, the effects of photodynamic therapy (PDT) with verteporfin on tumor blood flow and tumor regrowth were compared as verteporfin distributed in different compartments within the RIF-1 tumor. Tissue distribution of verteporfin was examined by fluorescence microscopy, and blood flow measurements were taken with a laser Doppler system. It was found that, at 15 min after drug administration, when verteporfin was mainly confined within the vasculature, PDT induced a complete arrest of blood flow by 6 h after treatment. PDT treatment at a longer drug-light interval (3 h), which allowed the drug to diffuse to the tumor interstitium, caused significantly less flow decrease, only to 50% of the initial flow in 6 h. A histological study and Hoechst 33342 staining of functional tumor vasculature confirmed the primary vascular damage and the decrease in tumor perfusion. The regrowth rate of tumors treated with 15-min interval PDT was 64% of that of the control group. However, when tumors were treated with 3-h interval PDT, the regrowth rate was not significantly different from that of the control, indicating that only the 15-min interval PDT caused serious damage to the tumor vascular bed. These results support the hypothesis that temporal pharmacokinetic changes in the distribution of the photosensitizer between the tumor parenchyma and blood vessels can significantly alter the tumor target of PDT.     

神经节苷脂GD3与肿瘤的血管生成作用(英文)

 血管生成作用 (angiogenesis)是实体瘤 (solidtumor)生长和扩散的必要条件 .实体瘤的微血管密度与肿瘤的恶性程度成正相关 ,而且也与病人的预后密切相关 .因此 ,对抗血管生成作用是一种很有吸引力的肿瘤疗法 .神经节苷脂GD3在多种类型的肿瘤中超常表达 .一般认为 ,神经节苷脂GD3有增强肿瘤本身及邻近组织中的血管生成作用 ,从而促进肿瘤的演进和转移 .最近的研究工作为这一假设提供了有力的实验证据 .应用GD3合酶的反意DNA转染肿瘤细胞从而抑制细胞中的GD3合酶的表达 ,极大地降低了细胞的内源GD3含量 .进一步的研究证明 ,抑制肿瘤细胞的GD3合成明显地降低了该肿瘤细胞的血管内皮生长因子 (VEGF)的水平 ,并使血管生成作用降至最小限度 .这些实验说明GD3在肿瘤的血管生成中具有重要的作用 .此外 ,GD3作为肿瘤的一种相关抗原 ,它与血管生成因子的协同效应将在未来的联合基因疗法中起到重要的作用     

基质衍生的血管生成抑制剂研究进展

血管的生成与肿瘤密切相关,抑制肿瘤血管生成可以调节肿瘤的生长。体内存在着内源性的促血管生成因子和抑制因子的平衡,当促血管形成因子增强就会产生新生血管供肿瘤生长,而当抑制因子增强则会抑制肿瘤的生长。本文即对细胞外基质衍生的内源性血管生成抑制因子TSP、内皮他丁、Arresten;Canstatin、Endorepellin、Fibulin、Tumstatin等的特性、应用和作用机制等作一总结。     

肿瘤新生血管及分子靶向治疗新策略

肿瘤血管靶向治疗是基于肿瘤新生血管与正常血管的不同,药物专一识别并阻断肿瘤新生血管,使肿瘤细胞“饿死”,而不影响正常细胞。从1971年Folkman提出“饿死肿瘤”的假说到2004年第一个血管靶向药物上市,记载着30多年领域发展的传奇经历。当今,肿瘤血管已成为生物医学和临床研究的热点,新的发现层出不穷。该文重点介绍肿瘤血管新靶点、新机制、新药物与未来发展。     

Vascular injury and modulation of MAPKs: a targeted approach to therapy of restenosis

Cardiovascular interventions that restore blood circulation to ischemic areas are accompanied by significant tissue damage, which triggers a vascular remodeling response that may result in restenosis of blood conduits. Early endothelial dysfunction and/or impairment is the early event of a cascade that leads, through an inflammatory response and dedifferentiation of medial smooth muscle cells with abundant deposition of extracellular matrix, to intimal hyperplasia. Here we present the molecular and cellular mechanisms of intimal hyperplasia secondary to vascular injury and discuss the potential role of therapeutic modulation of the intracellular signaling pathways that differentially effect vascular endothelial and smooth muscle cells. The role of mitogen-activated protein kinases (MAPKs) and the outcome of their modulation in these processes are highlighted here as they provide a promising therapeutic target for prevention of restenosis.     

Vascular targeted therapies in oncology

Neovascularization is intimately involved in tumor survival, progression, and spread, factors known to contribute significantly to treatment failures. Thus, strategies targeting the tumor blood vessel support network may offer not only unique therapeutic opportunities in their own right, but also novel means of enhancing the efficacies of conventional anticancer treatments. This article reviews one such therapeutic approach directed at the tumor blood vessel support network. Vascular disrupting therapies seek the destruction of the established neovasculature of actively growing tumors. The goal of these therapies is to cause a rapid and catastrophic shutdown in the vascular function of the tumor in order to arrest the blood flow and produce tumor cell death as a result of oxygen and nutrient deprivation and the build up of waste products. The authors’ work is supported by the National Cancer Institute (Public Health Service grants CA084408 and CA089655) and the Danish Cancer Society.     

Regulation of tumor angiogenesis by the microtubule-binding protein CLIP-170

Angiogenesis, the expansion of preexisting blood vessels, is a complex process required for tumor growth and metastasis. Although current antiangiogenic strategies have shown promising results in several cancer types, identifi-cation of additional antiangiogenic targets is required to improve the therapeutic response. Herein, we show that the microtubule-binding protein CLIP-170 (cytoplasmic linker protein of 170 kDa) is highly expressed in breast tumor samples and correlates positively with blood vessel density. Depletion of CLIP-170 significantly impaired vascular endothelial tube formation and sprouting in vitro and inhibited breast tumor growth in mice by decreasing tumor vascularization. Our data further show that CLIP-170 is important for the migration but not the proliferation of vascular endothelial cells. In addition, CLIP-170 promotes the polarization of endothelial cells in response to the angiogenic stimulus. These findings thus demonstrate a critical role for CLIP-170 in tumor angiogenesis and suggest its potential as a novel antiangiogenic target     

Frequency modulation of mesenteric and renal vascular resistance

The hypothesis was tested that low-frequency vasomotions in individual vascular beds are integrated by the cardiovascular system, such that new fluctuations at additional frequencies occur in arterial blood pressure. In anesthetized rats (n = 8), the sympathetic splanchnic and renal nerves were simultaneously stimulated at combinations of frequencies ranging from 0.075 to 0.8 Hz. Blood pressure was recorded together with mesenteric and renal blood flow velocities. Dual nerve stimulation at low frequencies (<0.6 Hz) caused corresponding oscillations in vascular resistance and blood pressure, whereas higher stimulation frequencies increased the mean levels. Blood pressure oscillations were only detected at the individual stimulation frequencies and their harmonics. The strongest periodic responses in vascular resistance were found at 0.40 +/- 0.02 Hz in the mesenteric and at 0.32 +/- 0.03 Hz (P < 0.05) in the renal vascular bed. Thus frequency modulation of low-frequency vasomotions in individual vascular beds does not cause significant blood pressure oscillations at additional frequencies. Furthermore, our data suggest that sympathetic modulation of mesenteric vascular resistance can initiate blood pressure oscillations at slightly higher frequencies than sympathetic modulation of renal vascular resistance.     

Chemical proteomic and bioinformatic strategies for the identification and quantification of vascular antigens in cancer

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels (“vascular targeting”) is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation.     

Differential Mechanisms Associated with Vascular Disrupting Action of Electrochemotherapy: Intravital Microscopy on the Level of Single Normal and Tumor Blood Vessels

Electropermeabilization/electroporation (EP) provides a tool for the introduction of molecules into cells and tissues. In electrochemotherapy (ECT), cytotoxic drugs are introduced into cells in tumors, and nucleic acids are introduced into cells in gene electrotransfer. The normal and tumor tissue blood flow modifying effects of EP and the vascular disrupting effect of ECT in tumors have already been determined. However, differential effects between normal vs. tumor vessels, to ensure safety in the clinical application of ECT, have not been determined yet. Therefore, the aim of our study was to determine the effects of EP and ECT with bleomycin on the HT-29 human colon carcinoma tumor model and its surrounding blood vessels. The response of blood vessels to EP and ECT was monitored in real time, directly at the single blood vessel level, by in vivo optical imaging in a dorsal window chamber in SCID mice with 70 kDa fluorescently labeled dextrans. The response of tumor blood vessels to EP and ECT started to differ within the first hour. Both therapies induced a vascular lock, decreased functional vascular density (FVD) and increased the diameter of functional blood vessels within the tumor. The effects were more pronounced for ECT, which destroyed the tumor blood vessels within 24 h. Although the vasculature surrounding the tumor was affected by EP and ECT, it remained functional. The study confirms the current model of tumor blood flow modifying effects of EP and provides conclusive evidence that ECT is a vascular disrupting therapy with a specific effect on the tumor blood vessels.     

The enigmatic role of angiopoietin-1 in tumor angiogenesis

A tumor vasculature is highly unstable and immature, characterized by a high proliferation rate of endothelial cells, hyper-permeability, and chaotic blood flow. The dysfunctional vasculature gives rise to continual plasma leakage and hypoxia in the tumor, resulting in constant on-sets of inflammation and angiogenesis. Tumors are thus likened to wounds that will not heal. The lack of functional mural cells, including pericytes and vascular smooth muscle cells, in tumor vascular structure contributes significantly to the abnormality of tumor vessels. Angiopoietin-1 (Ang 1) is aphysiological angiogenesis promoter during embryonic development. The function of Angl is essential to endothelial cell survival, vascular branching, and pericyte recruitment. However, an increasing amount of experimental data suggest that Angl-stimulated association of mural cells with endothelial cells lead to stabilization of newly formed blood vessels. This in turn may limit the otherwise continuous angiogenesis in the tumor, and consequently give riseto inhibition of tumor growth. We discuss the enigmatic role of Angl in tumor angiogenesis in this review.