Secreted human apolipoprotein(a) kringle IV-10 and kringle V inhibit angiogenesis and xenografted tumor growth

Abstract Angiogenesis plays an important role in normal physiology of blood vessel growth, but can contribute to the pathogenesis of diseases, such as cancer. A new anti-angiogenic recombinant kringle protein, composed of the fused domains of human apolipoprotein(a) carboxyl-terminal kringle IV-10 and kringle V, was expressed in Pichia pastoris and human colorectal carcinoma (HCT 116) cells to investigate its influence on angiogenesis and tumor growth. The mature recombinant protein exhibited the characteristic features of kringle-containing proteins (glycosylation and disulfide bond formation) and, when added to cultures of human umbilical vein endothelial cell, resulted in a 31% decrease in proliferation relative to untreated controls (p<0.05). The neo-angiogenesis was diminished by 63% in chick embryos treated with 10 mug recombinant protein compared with 7% for phosphate buffer solution-treated embryos (p<0.01). Transfection of a kringle IV-10-kringle V fusion protein construct into HCT 116 cells decreased tumorigenesis and inhibited tumor growth in vivo without affecting tumor cell proliferation. HCT 116 cells that expressed recombinant protein displayed a much lower relative growth ratio of 8% (p<0.01) against the control tumor cells. From these results, we conclude that human apolipoprotein(a) carboxyl-terminal kringle IV-10-kringle V fusion protein is an effective inhibitor of angiogenesis and angiogenesis-dependent tumor growth.     

Disruption of interkringle disulfide bond of plasminogen kringle 1-3 changes the lysine binding capability of kringle 2, but not its antiangiogenic activity

Kringle 1-3 of human plasminogen is a potent inhibitor of endothelial cell proliferation. To understand a possible role for the unique cystine bridge between kringle 2 and kringle 3, we disrupted the interkringle disulfide bond by mutating Cys(169) and Cys(297) to serine residues. The yield of the mutant during the refolding process was decreased significantly. Anti-endothelial cell proliferative activity of the mutant was similar to that of the wild type. There was no significant difference in in vivo antiangiogenic activity between the wild type and the mutant in chorioallantoic membrane assay. However, in the mutant, the weak lysine binding capability of kringle 2 was not detected and its mobility in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis is different from that of the wild type. These results support the notion that the overall antiangiogenic function of angiostatin is mediated by individual kringles, and suggest that the lysine binding capability of kringle 2 is likely not important for the antiangiogenic activity of kringle 1-3.     

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.     

Generation of biologically active angiostatin kringle 1-3 by activated human neutrophils

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1-3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1-3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.     

Inhibition of endothelial cell proliferation by the recombinant kringle domain of tissue-type plasminogen activator

Tissue-type plasminogen activator (tPA) is a multidomain serine protease that converts the zymogen plasminogen to plasmin. tPA contains two kringle domains which display considerable sequence identity with those of angiostatin, an angiogenesis inhibitor. TK1-2, a recombinant kringle domain composed of t-PA kringles 1 and 2 (Ala(90)-Thr(263)), was produced by both bacterial and yeast expression systems. In vitro, TK1-2 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, and epidermal growth factor. It did not inhibit proliferation of non-endothelial cells. TK1-2 also inhibited in vivo angiogenesis in the chick embryo chorioallantoic membrane model. These results suggest that the recombinant kringle domain of t-PA is a selective inhibitor of endothelial cell growth and identifies this molecule as a novel anti-angiogenic agent.     

人血纤维蛋白溶酶原K5环的基因合成、表达、纯化与活性鉴定

 根据大肠杆菌遗传密码的偏爱性 ,人工合成人血纤维蛋白溶酶原 K5全基因 ,并在原核系统中以硫氧还蛋白融合蛋白的形式实现了高效表达 .重组蛋白通过 Ni2 +金属螯合层析得到初步纯化 ,通过肠激酶切割去除了融合标签 .应用鸡胚尿囊膜实验检测切割后的 rh K5的生物学活性 ,发现与对照组相比 ,rh K5能明显地降低血管管径、血管总面积以及血管总面积与视野面积的比值 ,表明切割后的产物具有显著抑制新生血管生成的生物学活性 .为进一步研究和开发抗血管生成药物奠定了基础 .     

DGDA, a local sequence of the kringle 2 domain, is a functional motif of the tissue-type plasminogen activator’s antiangiogenic kringle domain

Antiangiogenic activity can be elicited by the kringle domains 1 and 2 of tissue-type plasminogen activator (TK1-2), or the kringle 2 domain alone. In a previous report, we showed that the anti-migratory effect of TK1-2 is mediated in part by its interference with integrin α2β1. Since integrin α2β1 interacts with collagen type I through the DGEA (Asp-Gly-Glu-Ala) amino acid sequence, and a similar sequence, DGDA (Asp-Gly-Asp-Ala), exists in the kringle 2 domain, we investigated whether the DGDA sequence has a role in antiangiogenic activity of TK1-2. In an adhesion assay, the DGDA peptide inhibited adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized TK1-2. Pretreatment of the DGDA peptide also blocked anti-migratory activity of TK1-2. When the DGDA peptide alone was tested for antiangiogenic activity, it effectively inhibited VEGF-induced migration of HUVECs and tube formation on Matrigel. In addition, the DGDA peptide decreased differentiation of endothelial progenitor cells on collagen type I matrix. These data suggest that the DGDA sequence presents a functional epitope of TK1-2 and that it can be used as a potential novel antiangiogenic peptide.     

Antiangiogenic kringles derived from human plasminogen and apolipoprotein(a) inhibit fibrinolysis through a mechanism that requires a functional lysine-binding site

Many proteins in the fibrinolysis pathway contain antiangiogenic kringle domains. Owing to the high degree of homology between kringle domains, there has been a safety concern that antiangiogenic kringles could interact with common kringle proteins during fibrinolysis leading to adverse effects in vivo. To address this issue, we investigated the effects of several antiangiogenic kringle proteins including angiostatin, apolipoprotein(a) kringles IV(9)-IV(10)-V (LK68), apolipoprotein(a) kringle V (rhLK8) and a derivative of rhLK8 mutated to produce a functional lysine-binding site (Lys-rhLK8) on the entire fibrinolytic process in vitro and analyzed the role of lysine binding. Angiostatin, LK68 and Lys-rhLK8 increased clot lysis time in a dose-dependent manner, inhibited tissue-type plasminogen activator-mediated plasminogen activation on a thrombin-modified fibrinogen (TMF) surface, showed binding to TMF and significantly decreased the amount of plasminogen bound to TMF. The inhibition of fibrinolysis by these proteins appears to be dependent on their functional lysine-binding sites. However, rhLK8 had no effect on these processes owing to an inability to bind lysine. Collectively, these results indicate that antiangiogenic kringles without lysine binding sites might be safer with respect to physiological fibrinolysis than lysine-binding antiangiogenic kringles. However, the clinical significance of these findings will require further validation in vivo.     

High efficacy and minimal peptide required for the anti‐angiogenic and anti‐hepatocarcinoma activities of plasminogen K5

Kringle 5(K5) is the fifth kringle domain of human plasminogen and its anti‐angiogenic activity is more potent than angiostatin that includes the first four kringle fragment of plasminogen. Our recent study demonstrated that K5 suppressed hepatocarcinoma growth by anti‐angiogenesis. To find high efficacy and minimal peptide sequence required for the anti‐angiogenic and anti‐tumour activities of K5, two deletion mutants of K5 were generated. The amino acid residues outside kringle domain of intact K5 (Pro452‐Ala542) were deleted to form K5mut1(Cys462‐Cys541). The residue Cys462 was deleted again to form K5mut2(Met463‐Cys541). K5mut1 specifically inhibited proliferation, migration and induced apoptosis of endothelial cells, with an apparent two‐fold enhanced activity than K5. Intraperitoneal injection of K5mut1 resulted in more potent tumour growth inhibition and microvessel density reduction than K5 both in HepA‐grafted and Bel7402‐xenografted hepatocarcinoma mouse models. These results suggested that K5mut1 has more potent anti‐angiogenic activity than intact K5. K5mut2, which lacks only the amino terminal cysteine of K5mut1, completely lost the activity, suggesting that the kringle domain is essential for the activity of K5. The activity was enhanced to K5mut1 level when five acidic amino acids of K5 in NH₂ terminal outside kringle domain were replaced by five serine residues (K5mut3). The shielding effect of acidic amino acids may explain why K5mut1 has higher activity. K5, K5mut1 and K5mut3 held characteristic β‐sheet spectrum while K5mut2 adopted random coil structure. These results suggest that K5mut1 with high efficacy is the minimal active peptide sequence of K5 and may have therapeutic potential in liver cancer.     

纤溶酶原K5抗血管增生活性依赖其完整Kringle结构域

根据K5蛋白(Pro451—Ala541)的结构特征和二硫键分布特点,设计K5的两个缺失突变体K5 mut1(Cys461—Cys540,保留K5 kringle环3个完整二硫键但去除N端和C端多余氨基酸)和K5 mut2 (Cys482—Cys535,打开kringle环,只保留2个二硫键).以野生型人纤溶酶原K5 cDNA为模板,用PCR方法得到编码缺失突变体的DNA片段,定向克隆入pET22b(+)质粒载体,重组体转化进大肠杆菌BL21（DE3）,诱导表达,产物经亲和层析和高浓度甘油透析纯化后进行鉴定和生物活性测定.K5 mut1蛋白特异性抑制人视网膜微血管内皮细胞增殖,且活性强度是完整的K5蛋白2倍;K5 mut2对人视网膜微血管内皮细胞无显著抑制作用.结果提示,完整的Kringle结构（包含3个二硫键）是维持人纤溶酶原K5抗血管增生活性的必需结构域,而K5分子中Kringle结构域外的N端和C端氨基酸臂则并非其活性所必需.     

Angiostatin antagonizes the action of VEGF-A in human endothelial cells via two distinct pathways

Angiostatin consisting of the first four-kringle domains of the plasminogen potently inhibits angiogenesis in vitro and in vivo. However, the molecular mechanism of action whereby angiostatin mediates its inhibitory effect on proliferating endothelial cells remains elusive. We therefore used the proliferating cultured human umbilical vein endothelial cells (HUVECs) promoted by vascular endothelial growth factor A to identify the endogenous signaling elements that mediate the antiangiogenic effect of angiostatin. Treatment of HUVEC with angiostatin at a concentration known to inhibit cell proliferation and induce apoptosis resulted in induction of p53-, Bax-, and tBid-mediated release of cytochrome c into the cytosol. In addition, angiostatin also activated the Fas-mediated apoptotic pathway in part via up-regulation of FasL mRNA, down-regulation of c-Flip, and activation of caspase 3. These results suggest that the anti-angiogenic action of angiostatin is likely mediated by two distinct signaling pathways, one intrinsic mediated by p53 while the other extrinsic involved in FasL engagement and mitochondria dysfunction.     

The recombinant kringle domain of urokinase plasminogen activator inhibits VEGF165-induced angiogenesis of HUVECs by suppressing VEGFR2 dimerization and subsequent signal transduction

The recombinant kringle domain (UK1) of urokinase plasminogen activator was previously reported to exert antiangiogenic activity against Vascular Endothelial Growth Factor (VEGF)-induced angiogenesis in both in vitro and in vivo models. In this study, we explored the molecular signaling mechanisms involved in the antiangiogenic activity of UK1 by examining VEGF signaling proteins. VEGF165 stimulates the phosphorylation of VEGF signaling molecules, and pretreatment with UK1 blocked VEGF-induced signal transduction associated with proliferation, survival, and migration. UK1 also suppressed VEGF165-induced activation of MMP-2. Moreover, UK1 suppressed the phosphorylation and activation of VEGFR2 in VEGF-stimulated human umbilical cord vein endothelial cells (HUVECs) by blocking the dimerization of VEGFR2. Overall, our findings suggest that UK1 inhibits VEGF-induced proliferation, migration, and matrix metalloproteinase activity of HUVECs by suppressing VEGFR2 dimerization and subsequent angiogenic signals.     

Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments

Human tissue-type plasminogen activator (t-PA) consists of five domains designated (starting from the N-terminus) finger, growth factor, kringle 1, kringle 2, and protease. The binding of t-PA to lysine-Sepharose and aminohexyl-Sepharose was found to require kringle 2. The affinity for binding the lysine derivatives 6-aminohexanoic acid and N-acetyllysine methyl ester was about equal, suggesting that t-PA does not prefer C-terminal lysine residues for binding. Intact t-PA and a variant consisting only of kringle 2 and protease domains were found to bind to fibrin fragment FCB-2, the very fragment that also binds plasminogen and acts as a stimulator of t-PA-catalyzed plasminogen activation. In both cases, binding could completely be inhibited by 6-aminohexanoic acid, pointing to the involvement of a lysine binding site in this interaction. Furthermore, the second site in t-PA involved in interaction with fibrin, presumably the finger, appears to interact with a part of fibrin, different from FCB-2.     

Identification of annexin II heterotetramer as a plasmin reductase.

Annexin II heterotetramer (AIIt) is a Ca(2+)- and phospholipid-binding protein that consists of two copies of a p36 and p11 subunit. AIIt regulates the production and autoproteolysis of plasmin at the cell surface. In addition to its role as a key cellular protease, plasmin also plays a role in angiogenesis as the precursor for antiangiogenic proteins. Recently we demonstrated that the primary antiangiogenic plasmin fragment, called A(61) (Lys(78)-Lys(468)) was released from cultured cells. In the present study we report for the first time that AIIt possesses an intrinsic plasmin reductase activity. AIIt stimulated the reduction of the plasmin Cys(462)-Cys(541) bond in a time- and concentration-dependent manner, which resulted in the release of A(61) from plasmin. Mutagenesis of p36 C334S and either p11 C61S or p11 C82S inactivated the plasmin reductase activity of the isolated subunits, suggesting that specific cysteinyl residues participated in the plasmin reductase activity of each subunit. Furthermore, we demonstrated that the loss of AIIt from the cell surface of HT1080 cells transduced with a retroviral vector encoding p11 antisense dramatically reduced the cellular production of A(61) from plasminogen. This is the first demonstration that AIIt regulates the cellular production of the antiangiogenic plasminogen fragment, A(61).     

含人纤溶酶原K5基因的腺病毒载体制备和功能研究

应用PCR将人纤溶酶原信号肽序列引入K5cDNA基因 ,与真核表达载体pcDNA3重组 ,形成重组质粒pcDNA3K5 ,与穿梭质粒pShuttle重组得pShuttleK5 ,经与腺病毒DNA重组 ,PCR鉴定正确 ,即为pAd K5。脂质体法将其转染 2 93细胞后 ,制备细胞裂解液 ;噬斑分析法测定病毒滴度为 5× 10 8pfu mL。将病毒以不同的感染系数 (MOI)感染人脐静脉内皮细胞株ECV30 4和人乳腺癌细胞株MDA MB 2 31,MTT法检测两者的增殖情况 :ECV30 4细胞增殖受抑制 ,而MDA MB 2 31细胞增殖未受明显影响。将感染病毒的ECV30 4细胞接种于ECMatrixTM胶 ,显示内皮细胞分化和毛细血管管腔形成受抑制。表明所构建的含人纤溶酶原K5基因的重组复制缺陷型腺病毒具有抑制ECV30 4细胞增殖、分化和管腔形成的作用而对MDA MB 2 31细胞的生长则无影响。     

Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a)

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.     

Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells.

Angiostatin which contains the first four kringle domains of plasminogen has been documented to be a potent inhibitor of angiogenesis. More recently, another kringle structure within plasminogen but outside angiostatin, known as kringle 5 (K5), was found to inhibit endothelial cell proliferation and migration. Here, we report the cloning and expression of mouse kringle 5 (rK5) in a bacterial expression system. The protein was purified to homogeneity using a Ni-NTA column. rK5 inhibited both proliferation and migration of endothelial cells with ED50's of 10 nM and < 500 nM, respectively. In addition, we show for the first time that rK5 causes cell cycle arrest and apoptosis, shedding further insight into rK5's mechanism of action. Finally, we show that these actions are endothelial cell specific.     

Organization of the human hepatocyte growth factor-encoding gene.

Human genomic phage libraries were screened for the human hepatocyte growth factor (HGF)-encoding gene (HGF) using a cDNA encoding the human protein as a probe. Characterization of the clones revealed that this gene is composed of 18 exons interrupted by 17 introns spanning approx. 70 kb. The first exon contains the 5'-untranslated region and the signal peptide. The next ten exons encode the alpha-chain which contains four kringle structures. Each kringle domain is encoded by two exons as observed in other kringle-containing proteins. The twelfth exon contains the short spacer region between the alpha- and beta-chains and the remaining six exons comprise the beta-chain. The beta-chain is structurally similar to the catalytic domains of serine proteases; amino acid substitutions in the active site were found. The organization of the HGF gene is highly homologous to those of the serine proteases involved in blood coagulation and fibrinolysis, especially with that of plasminogen. This suggests that the human HGF gene is evolutionally related to these genes.     

Mechanisms and significance of bifunctional NK4 in cancer treatment

Based on the background that hepatocyte growth factor (HGF) and c-Met/HGF receptor tyrosine kinase play a definite role in tumor invasion and metastasis, NK4, four-kringles containing intramolecular fragment of HGF, was isolated as a competitive antagonist for the HGF-c-Met system. Independent of its HGF-antagonist action, NK4 inhibited angiogenesis induced by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF, indicating that NK4 is a bifunctional molecule that acts as an HGF-antagonist and angiogenesis inhibitor. Interestingly, kringle domains in distinct types of proteins, e.g., plasminogen, prothrombin, plasminogen activators, apolipoprotein(a), and HGF, share angioinhibitory actions. In experimental models of distinct types of cancers, NK4 protein administration or NK4 gene therapy inhibited tumor invasion, metastasis, and angiogenesis-dependent tumor growth. Cancer treatment with NK4 may prove to suppress malignant tumors to be 'static' in both tumor growth and spreading, as based on biological characteristics of malignant tumors.     

Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro

Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes.