Endogenous angiogenesis inhibitors and their therapeutic implications

A number of endogenous inhibitors targeting the tumor vasculature have recently been identified using in vitro and in vivo antiangiogenesis models. While many of these angiogenesis inhibitors display a broad spectrum of biological actions on several systems in the body, several inhibitors including angiostatin, endostatin, and serpin antithrombin seem to act specifically on the proliferating endothelial cell compartment of the newly formed blood vessels. The discovery of these specific endothelial inhibitors not only increases our understanding of the functions of these molecules in the regulation of physiological and pathological angiogenesis, but may also provide an important therapeutic strategy for the treatment of cancer and other angiogenesis dependent diseases, including diabetic retinopathy and chronic inflammations. Systemic administration of these angiogenesis inhibitors in animals significantly suppresses the growth of a variety of tumors and their metastases. However, their production as functional recombinant proteins has been proven to be difficult. In addition, high dosages of these inhibitors are required to suppress tumor growth in animal studies. Other disadvantages of the antiangiogenic protein therapy include repeated injections, prolonged treatment, transmission of toxins and infectious particles, and high cost for manufacturing large amounts of protein molecules. Thus, alternative strategies need to be developed in order to improve the clinical settings of antiangiogenic therapy. Developments of these strategies are ongoing and they include identification of more potent inhibitors, antiangiogenic gene therapy, improvement of protein/compound half-lives in the circulation, increase of their concentrations at the disease location, and combinatorial therapies with approaches including chemotherapy, radiotherapy, and immunotherapy. Despite the above-mentioned disadvantages, a few inhibitors have entered into the early stages of clinical trials and they may bring new hopes for the treatment of cancer and other angiogenesis dependent diseases.     

Endostatin: Current concepts about its biological role and mechanisms of action

Endogenous inhibitors of angiogenesis are proved to be a major factor preventing the emergence of clinically manifested stages of human cancer. The protein endostatin, a 20-kD proteolytic fragment of type XVIII collagen, is one of the most active natural inhibitors of angiogenesis. Endostatin specifically inhibits the in vitro and in vivo proliferation of endothelial cells, inducing their apoptosis through inhibition of cyclin D1. On the surface of endothelial cells, endostatin binds with the integrin alpha(5)beta(1) that activates the Src-kinase pathway. The binding of endostatin with integrins also down-regulates the activity of RhoA GTPase and inhibits signaling pathways mediated by small kinases of the Ras and Raf families. All these events promote disassembly of the actin cytoskeleton, disorders in cell-matrix interactions, and decrease in endotheliocyte mobility, i.e., promote the suppression of angiogenesis. Endostatin displays a high antitumor activity in vivo: it inhibits the progression of more than 60 types of tumors. This review summarizes results of numerous studies concerning the biological activity and action mechanism of endostatin.     

内皮抑素与肿瘤治疗

内皮抑素(endostatin)是近年来发现的,天然产生的新血管生成抑制因子。它在体内由胶原ⅩⅧ经酶解产生,可以抑制新血管生成,在肿瘤动物模型中显示出显著的抑瘤活动。通过抑制肿瘤相关的新血管的形成来治疗肿瘤,是目前出现的新疗法,内皮抑素是这种疗法中很有前景的侯选药物之一。在美国已经开始了用内皮抑素治疗肿瘤的Ⅰ期临床试验,关于它的临床前基础实验也在广泛开展。本文综述了内皮抑素在肿瘤治疗中的应用基础研究 。     

Autophagy and angiogenesis inhibition

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.     

Endostatin gene therapy enhances the efficacy of paclitaxel to suppress breast cancers and metastases in mice

Chemotherapy combined with antiangiogenic therapy is more effective than chemotherapy alone. The aim of this study was to investigate whether endostatin, a potent anti-angiogenic agent, could enhance the efficacy of paclitaxel to combat breast cancer. An expression plasmid encoding mouse endostatin (End-pcDNA3.1) was constructed, which produced intense expression of endostatin and inhibited angiogenesis in the chorioallantoic membrane assay. 4T1 breast tumors were established in BALB/c mice by subcutaneous injection of 1 × 10⁵ 4T1 cells. The End-pcDNA3.1 plasmid diluted in the transfection reagent FuGENE^TM was injected into the tumors (around 100 mm²), and paclitaxel was injected i.p. into the mice. Endostatin gene therapy synergized with paclitaxel in suppressing the growth of 4T1 tumors and their metastasis to the lung and liver. Both endostatin and paclitaxel inhibited tumor angiogenesis and induced cell apoptosis. Despite the finding that endostatin was superior to paclitaxel at inhibiting tumor angiogenesis, paclitaxel was nevertheless more effective at inducing tumor apoptosis. The combination of paclitaxel and endostatin was more effective in suppressing tumor growth, metastases, angiogenesis, and inducing apoptosis than the respective monotherapies. The combinational therapy with endostatin and paclitaxel warrants future investigation as a therapeutic strategy to combat breast cancer.     

A pharmacologically based multiscale mathematical model of angiogenesis and its use in investigating the efficacy of a new cancer treatment strategy

Tumor angiogenesis is the process by which new blood vessels are formed and enhance the oxygenation and growth of tumors. As angiogenesis is recognized as being a critical event in cancer development, considerable efforts have been made to identify inhibitors of this process. Cytostatic treatments that target the molecular events of the angiogenesis process have been developed, and have met with some success. However, it is usually difficult to preclinically assess the effectiveness of targeted therapies, and apparently promising compounds sometimes fail in clinical trials.We have developed a multiscale mathematical model of angiogenesis and tumor growth. At the molecular level, the model focuses on molecular competition between pro- and anti-angiogenic substances modeled on the basis of pharmacological laws. At the tissue scale, the model uses partial differential equations to describe the spatio-temporal changes in cancer cells during three stages of the cell cycle, as well as those of the endothelial cells that constitute the blood vessel walls.This model is used to qualitatively assess how efficient endostatin gene therapy is. Endostatin is an anti-angiogenic endogenous substance. The gene therapy entails overexpressing endostatin in the tumor and in the surrounding tissue. Simulations show that there is a critical treatment dose below which increasing the duration of treatment leads to a loss of efficacy.This theoretical model may be useful to evaluate the efficacy of therapies targeting angiogenesis, and could therefore contribute to designing prospective clinical trials.     

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution.

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C-terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 A resolution. The structure reveals a compact fold distantly related to the C-type lectin carbohydrate recognition domain and the hyaluronan-binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.     

重组人内皮抑素的结构改造及抗肿瘤活性变化

内皮抑素是一种内源性的血管生成和肿瘤生长抑制剂 .运用定点突变技术对人内皮抑素基因工程菌中的内皮抑素基因进行改造 ,将内皮抑素中的GRIRGAD改为RGDRGD序列 ,提高了内皮抑素的抗肿瘤活性 .裸鼠体内抑瘤试验发现 ,野生与诱变内皮抑素的抑瘤率分别为 4 0 6 6 %和5 1 0 5 % ,诱变内皮抑素抑瘤率比野生内皮抑素提高了近 11个百分点 .病理切片HE染色诱变组肿瘤的血管生成比野生组明显减少 ,坏死灶增多 .免疫组化试验中 ,诱变组在微血管密度MVD(P <0 0 5 )、血管内皮生长因子VEGF(P <0 0 5 )、增殖细胞核抗原PCNA(P >0 0 5 )三个指标上均比野生组减小 .体外细胞实验中 ,MTT结果表明 ,野生组内皮抑素半数抑制浓度IC50 =185 μg ml,诱变组内皮抑素IC50 =2 7μg ml,诱变组抑瘤活性是野生组的 6倍 .细胞迁移抑制实验表明 ,野生组与诱变组内皮抑素均对肿瘤细胞迁移有抑制作用 ,野生组迁移抑制率为 6 8 95 % ;诱变组迁移抑制率为89 94 % .上述结果说明 ,内皮抑素除抑制血管生成外 ,对肿瘤细胞的生长和迁移也有一定的抑制作用 .诱变内皮抑素通过RGDRGD序列与整合素结合而提高了抗肿瘤活性 .     

Amyloid endostatin induces endothelial cell detachment by stimulation of the plasminogen activation system

Endostatin is a fragment of collagen XVIII that acts as an inhibitor of tumor angiogenesis and tumor growth. Anti-tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study, we have investigated the cellular effects of insoluble endostatin. We previously found that insoluble endostatin shows all the hallmarks of amyloid aggregates and potently stimulates tissue plasminogen activator-mediated formation of the serine protease plasmin. We here show that amyloid endostatin induces plasminogen activation by endothelial cells, resulting in vitronectin degradation and plasmin-dependent endothelial cell detachment. Endostatin-mediated stimulation of plasminogen activation, vitronectin degradation, and endothelial cell detachment is inhibited by carboxypeptidase B, indicating an essential role for carboxyl-terminal lysines. Our results suggest that amyloid endostatin may inhibit angiogenesis and tumor growth by stimulating the fibrinolytic system.     

Autophagy and Angiogenesis Inhibition

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.

Addendum to:

Kringle 5 of Human Plasminogen, an Angiogenesis Inhibitor, Induces Both Autophagy and Apoptotic Death in Endothelial Cells

T.M.B. Nguyen, I.V. Subramanian, A. Kelekar and S. Ramakrishnan

Blood 2007; 109:4793-802     

癌抑素(canstatin)在肿瘤治疗中的研究现状

癌抑素(canstatin,Cans)是继血管生成抑素(angiostatin)、内皮抑素(endostatin)和肿瘤抑素(tumstatin)之后,新近发现的一种内源性肿瘤血管生成抑制因子,因其具有高效的抗肿瘤血管形成活性而成为肿瘤治疗中的研究热点。本文就Cans的结构、功能、作用机制及在肿瘤治疗中的研究进展作一综述。     

Glioma angiogenesis

Brain tumors exhibit marked and aberrant blood vessel formation indicating angiogenic endothelial cells as a potential target for brain tumor treatment. The brain tumor blood vessels are used for nutrient delivery, and possibly for cancer cell migration. The process of angiogenesis is complex and involves multiple players. The current angiogenesis inhibitors used in clinical trials mostly target single angiogenic proteins and so far show limited effects on tumor growth. Besides the conventional angiogenesis inhibitors, RNA-based inhibitors such as small-interfering RNAs (siRNAs) are being analyzed for their capacity to silence the message of proteins involved in neovascularization. More recently, a new family of non-coding RNAs, named angiomirs [microRNAs (miRNAs) involved in angiogenesis] has emerged. These small RNAs have the advantage over siRNAs in that they have the potential of silencing multiple messages at the same time and therefore they might become therapeutically relevant in a “one-hit multiple-target” context against brain tumor angiogenesis. In this review we will discuss the emerging technologies in anti-angiogenesis emphasizing on RNA-based therapeutics.     

Anti-angiogenic gene therapy of cancer: current status and future prospects

The discovery of endogenous inhibitors of angiogenesis has made it possible to test the hypothesis that blocking the angiogenic switch may keep tumor growth in check, and has added a new investigational arm to the field of cancer gene therapy. Angiogenesis inhibitors are heterogeneous in origin and potency, and their growing list includes proteolysis products of larger molecules with a different function, such as angiostatin, endostatin and vasostatin, modulators of vascular endothelial growth factor activity, such as sFLT-1, and some cytokines/chemokines with marked anti-endothelial activity, such as IL-12, IFN-alpha, and CXCL10. Pre-clinical studies have clearly indicated that these factors are essentially cytostatic and that they need long-term administration in order to obtain prolonged anti-tumor effects, representing a rational basis for their delivery by a gene therapy approach. The experimental approaches attempted to date, reviewed herein, indicate overall that anti-angiogenic gene therapy has efficacy mainly as an early intervention strategy and that a better understanding of the biological mechanisms underlying resistance to angiogenesis inhibition, as well as appropriate combined treatments, are required to generate a conceptual advancement which could drive the field towards successful management of established tumors.     

Inhibition of tumor growth in mice by endostatin derived from abdominal transplanted encapsulated cells

Endostatin,a C-terminal fragment of collagen 18a,inhibits the growth of established tumorsand metastases in vivo by inhibiting angiogenesis.However,the purification procedures required for large-scale production and the attendant cost of these processes,together with the low effectiveness in clinicaltests,suggest that alternative delivery methods might be required for efficient therapeutic use of endostatin.In the present study,we transfected Chinese hamster ovary(CHO)cells with a human endostatin geneexpression vector and encapsulated the CHO cells in alginate-poly-L-lysine microcapsules.The release ofbiologically active endostatin was confirmed using the chicken chorioallantoic membrane assay.The encap-sulated endostatin-expressing CHO cells can inhibit the growth of primary tumors in a subcutaneous B 16tumor model when injected into the abdominal cavity of mouse.These results widen the clinical applicationof the microencapsulated cell endostatin delivery system in cancer treatment.     

Detection and characterization of an acid-induced folding intermediate of endostatin

Endostatin, an important angiogenesis inhibitor, is very acid resistant. We are particularly interested in knowing that whether or not endostatin can form a folding intermediate during acid titration. 1H-NMR, CD spectrum, and ANS binding assay show that endostatin at pH 2.0 contains little tertiary structure, but retains substantial secondary structure with strong ANS binding, and Na2SO4 or TFE is found to strongly stabilize endostatin at pH 2.0. All these observations are consistent with the formation of a folding intermediate at pH 2.0. Kinetics studies show that sulfate anions significantly slow down the process for endostatin to reach its equilibrium state at pH 2.0. A regular increase in the amount of alpha-helix content of the intermediate of endostatin at pH 2.0 is found when the concentration of TFE is increased in the range of 0-40%, suggesting that endostatin has an intrinsic alpha-helical propensity.     

Endothelial cell integrins and COX-2: mediators and therapeutic targets of tumor angiogenesis

Vascular integrins are essential regulators and mediators of physiological and pathological angiogenesis, including tumor angiogenesis. Integrins provide the physical interaction with the extracellular matrix (ECM) necessary for cell adhesion, migration and positioning, and induce signaling events essential for cell survival, proliferation and differentiation. Integrins preferentially expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, are considered as relevant targets for anti-angiogenic therapies. Anti-integrin antibodies and small molecular integrin inhibitors suppress angiogenesis and tumor progression in many animal models, and are currently tested in clinical trials as anti-angiogenic agents. Cyclooxygense-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxans, is highly up-regulated in tumor cells, stromal cells and angiogenic endothelial cells during tumor progression. Recent experiments have demonstrated that COX-2 promotes tumor angiogenesis. Chronic intake of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors significantly reduces the risk of cancer development, and this effect may be due, at least in part, to the inhibition of tumor angiogenesis. Endothelial cell COX-2 promotes integrin alphaVbeta3-mediated endothelial cell adhesion, spreading, migration and angiogenesis through the prostaglandin-cAMP-PKA-dependent activation of the small GTPase Rac. In this article, we review the role of integrins and COX-2 in angiogenesis, their cross talk, and discuss implications relevant to their targeting to suppress tumor angiogenesis.     

Sphingosine 1-phosphate and cell migration: resistance to angiogenesis inhibitors

Naturally occurring angiogenesis inhibitors can inhibit different steps of the angiogenic process, such as endothelial cell migration. However, the mechanisms underlying this inhibition have not been elucidated. We demonstrate that migration of human umbilical vein endothelial cells induced by the potent endothelial cell chemoattractant sphingosine 1-phosphate is refractory to inhibition by well-characterized angiogenesis inhibitors such as endostatin and plasminogen-related protein-B. Our data support the contention that for effective blockage of tumor-induced angiogenesis, antagonists of both G protein-coupled receptor signaling and receptor tyrosine kinase signaling must be combined.     

Biosynthesis of d-Erythroascorbic Acid by Candida

Epigallocatechin 3-gallate (EGCG) has cytotoxic effects in many cancer cells. It has been reported that A549 lung cancer cells are markedly resistant to cell death induced by EGCG. In the present study, the effects of EGCG on A549 lung cancer cell growth and angiogenesis were studied. We found that EGCG dose-dependently suppressed A549 cell growth, while A549 cells were markedly resistant to cell death in vitro. Next we found that EGCG increased endostatin expression and suppressed vascular endothelial growth factor (VEGF) expression. We further studied to determine whether EGCG would suppress A549 tumor growth in nude mouse and angiogenesis. EGCG in drinking water significantly suppressed A549 tumor growth in nude mice. Histological analysis revealed that the number of CD34 positive vessels had a tendency to decrease in the tumor. In sum, EGCG had anti-proliferative effects of A549 on tumor growth and showed a tendency to suppress angiogenesis.