血管生成抑制因子K4K5 Cdna基因的克隆及其在毕赤酵母中的表达

应用PCR方法,扩增人纤溶酶原cDNA基因中K4K5 cDNA片段,与酵母表达载体pPIC9K重组,获得表达质凿p9kkk-18。该质粒转化毕赤酵母菌GS115,用G418－YPD筛选高拷贝表型,PCR筛选K4K5 cDNA与酵母染色体整全形成的阳性克隆,阳性克隆用甲醇诱导表达。表达产物r-K4K5分子量约21.5kD,占分泌总蛋白80％以上,产物浓度为150－250mg/L。初步纯化产物抑制牛毛细血管内皮（BCE）细胞增殖与鸡胚绒毛尿囊膜（CAM）新生血管生成。     

基质金属蛋白酶-2 C端片段PEX的原核表达及其对血管发生的抑制作用

为了克隆表达鸡的基质金属蛋白酶-2(MMP-2)的C端片段PEX,并探讨其对血管发生的抑制作用,利用RT-PCR从鸡胚成纤维细胞克隆MMP-2 C端片段PEX,构建原核表达载体pCal-n-PEX;转化大肠杆菌BL21(DE3)-pLys,异丙基β-D硫代半乳糖苷（IPTG）诱导产生PEX融合蛋白,包涵体蛋白用盐酸胍法变性、复性;生长曲线观察PEX融合蛋白对人脐静脉血管内皮细胞增殖的影响;鸡胚绒毛尿囊膜血管发生实验研究其对血管发生的抑制作用.结果表明融合蛋白CBP/PEX具有抑制人脐静脉血管内皮细胞的生长和鸡胚绒毛尿囊膜血管发生的作用.提示PEX是有待进一步开发的潜在抑制血管发生的药物.     

Inhibition of angiogenesis by a novel neutralizing antibody targeting human VEGFR-3

Vascular endothelial growth factor receptor 3 (VEGFR-3) is a receptor for the vascular endothelial growth factor C and D (VEGF-C and D) and plays a critical role in the development of embryonic vascular system and regulation of tumor lymphangiogenesis. In this report, we generated a novel panel of 17 monoclonal antibodies (mAbs) against human VEGFR-3 and determined their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. Furthermore, in chick embryo CAM angiogenesis experiments, the angiogenesis induced by recombinant glutathione-S-transferase-VEGF-D was decreased in the presence of antibody BDD073. These data suggest that this novel neutralizing antibody against human VEGFR-3 could be a tool for the investigations into the biology of VEGFR-3, and potentially a reagent for blocking VEGF-D-induced angiogenesis and lymphogenesis.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.     

Angiogenesis is associated with collagenous protein synthesis and degradation in the chick chorioallantoic membrane.

Extracts of the chick embryo chorioallantoic membrane (CAM) obtained from 7-20 day old embryos, contained enzyme activity that could degrade type IV collagen. Peak enzyme activity was observed on days 8-10 of embryogenesis, which coincides with the stage of maximum angiogenesis. This activity decreased to lowest values at days 13-15 and increased thereafter up to day 20. Maximum rate of collagen biosynthesis in CAM was observed between days 7 and 10, with a drastic decrease at day 12, when vascular density has reached a plateau. The type IV collagen-degrading activity of CAM was of the metalloprotease type, since it was inhibited by 1,10-phenanthroline and EDTA but was also partially inhibited by serine and thiol protease inhibitors.     

Enzyme induction and receptor-binding affinity of steroidal 20-carboxamides in rat hepatoma tissue culture cells.

The steroidal 20-carboxamides [(20R)- and (20S)-21-(N-substituted amino)-11 beta,17,20-trihydroxy-3,21-dioxo-1,4-pregnadiene] recently have been shown to possess anti-inflammatory activity in animal models of inflammation. These N-substituted methyl, ethyl, n-propyl, and benzyl derivatives also exhibited suppressive effects on plasma corticosterone and thymus function. Generally, the (20R)-hydroxy-20-carboxamides were more potent than the corresponding (20S)-epimers. In continuing investigations on the glucocorticoid effects of these compounds, we have studied their ability to induce tyrosine aminotransferase (TAT), inhibit uptake of [3H]thymidine into DNA, and complete with [3H] dexamethasone for binding to the hepatoma tissue culture glucocorticoid receptor. Results indicated that the N-substituted methyl, ethyl, and n-propyl derivatives were full glucocorticoid agonists in the three measurements. Receptor binding affinities of the N-substituted carboxamides correlated well with their ability to induce TAT activity and to inhibit thymocyte proliferation. Structure-activity relationships indicated that the larger the N-substituent, the weaker the agonist activity in this system, and 20R isomers exhibited higher glucocorticoid agonist activity than the corresponding 20S isomers. This investigation is part of our effort to elucidate structure-activity relationships of steroidal carboxamides synthesized on the basis of the antedrug concept.     

Rate of basement membrane biosynthesis as an index to angiogenesis

A method was developed for assessing collagenous protein biosynthesis from [U-14C]proline in relation to angiogenesis in the chick chorioallantoic membrane (CAM). The rate of collagenous protein biosynthesis both in vitro and in vivo was maximum between days 8 and 11 of chick embryo development. This was the stage of maximum angiogenesis as shown by morphological evaluation of the vascular density. At day 10 the rate of collagenous protein biosynthesis was 11-fold higher than that of day 15, when angiogenesis had reached a plateau. The collagenous protein formed by CAM co-elutes on SDS-agarose chromatography with the collagenous component of [3H]-acetylated-basement membrane (BM) from bovine lens capsule. 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide (GPA1734), which was shown previously to be a specific inhibitor of BM collagen biosynthesis, caused about 80% reduction in collagenous protein synthesis by CAM. These results indicate that most of the collagenous protein synthesized by CAM was BM collagen and this can be used as a biochemical index of angiogenesis.     

重组小鼠canstatin N端片段对鸡胚新生血管生成的抑制作用(英)

为了研究重组小鼠canstatin N端片段的体内抗血管生成活性, 通过PCR扩增小鼠canstatin N端片段cDNA,定向克隆于原核表达载体pET30a(+)中,构建小鼠canstatin N端片段重组表达载体pET-mCanN, 转化E.coli BL21(DE3), IPTG诱导表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹检测小鼠canstatin N端片段的表达. 结果表明,IPTG诱导原核表达载体pET-mCanN在大肠杆菌E.coli BL21(DE3)中高效表达, 小鼠canstatin N端片段表达量约占菌体总蛋白量的18%, 小鼠canstatin N端片段主要以包涵体形式存在,包涵体经过洗涤、裂解、Ni-spin column亲合柱层析以及蛋白质复性等步骤纯化后,获得了纯度约为92%的重组小鼠canstatin N端片段. 鸡胚绒毛尿囊膜(chicken embryo choriollantoic membrane,CAM)实验表明,原核表达的小鼠canstatin N端片段能有效地按剂量依赖的方式抑制鸡胚新生血管的形成.     

Lysyl hydroxylase. Further purification and characterization of the enzyme from chick embryos and chick embryo cartilage.

A purification of up to 4000-fold is reported for lysyl hydroxylase (EC 1.14.11.4) from extract of chick-embryo homogenate and one of about 300-fold from extract of chick-embryo cartilage. Multiple forms of the enzyme were observed during purification from whole chick embryos. In gel filtration the elution positions of the two main forms corresponded to average molecular weights of about 580000 and 220000. These two forms could also be clearly separated in hydroxyapatite chromatography. In addition, some enzyme activity was always eluted between the two main peaks both in gel filtration and in hydroxyapatite chromatography. The presence of the two main forms was also observed when purifying enzyme from chick embryo cartilage. Both forms of the enzyme hydroxylated lysine in arginine-rich histone, which does not contain any -X-Lys-Gly- sequence. No difference was found between the enzyme from whole chick embryos and from chick embryo cartilage in this respect. Lysyl hydroxylase was found to have affinity for concanavalin A, indicating the presence of some carbohydrate residues in the enzyme molecule. Lysyl and prolyl hydroxylase activities increased when the chick embryo homogenate was assayed in the presence of lysolecithin. Preincubation of the homogenate either with lysolecithin or with Triton X-100 increased lysyl hydroxylase activity in homogenate, and in the 1500 x g and 150000 x g supernatants, suggesting that the increase in the enzyme activity was due to liberation of the enzyme from the membranes. Divalent cations were found to inhibit the activity of lysyl and prolyl hydroxylases in vitro. An inhibition of about 50% was achieved with 15 mM calcium 60 muM copper and 3 muM zinc concentrations. The mode of inhibition was tested with Cu2+, and was found to be competitive with Fe2+.     

Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis

A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti-proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)-plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U-24067 and U-42129), heparin, suramin, interferon alpha-2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen-dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha-2a reduce urokinase-type PA (u-PA) and PA inhibitor-1 activity, while heparin and retinoic acid increase u-PA activity. Suramin reduces cell-associated u-PA activity and greatly increases PAI-1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti-angiogenesis, and that anti-angiogenesis and anti-proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti-angiogenesis is likely to be varied and complex. © 1994 Wiley-Liss, Inc.     

血管内皮细胞生长因子受体Flt—1胞外区基因的克隆及其在中国仓？…

朋原代培养的人脐静脉血管内皮细胞（ＨＵＶＥＣ）提取细胞总ＲＮＡ,采用逆转录ＰＣＲ（ＲＴ－ＰＣＲ）方法得到ＶＥＧＦ受体Ｆｌｔ－１胞外区前３个ＩｇＧ样区域ｃＤＮＡ片段（Ｆｌｔ－１ｎ３）。将获得的受体基因克隆到真核表达载体ｐｃＤ－ＮＡ３．１中,得到重组质粒ｐｃＤＮＡ３．１／Ｆｌｔ－１ｎ３,通过南体转染方法将其转入中国仓鼠卵巢细胞（ＣＨＯ）,用Ｇ４１８筛选得到稳定表达目的蛋白的细胞砍隆。经固相结合实验筛选     

人大肠癌鸡胚尿囊膜移植模型的建立及血管生成特征的观察

目的研究人大肠癌腺癌细胞株HT-29鸡胚尿囊膜移植模型建立的方法,观察和分析其血管生成的特征。方法将不同浓度的人大肠癌腺癌细胞株HT-29接种于鸡胚尿囊膜（chick embryo chorioallantoic membrane,CAM）,观察影响大肠癌鸡胚移植模型瘤体成活的因素、瘤体生长特征和血管生成情况。结果建立了人大肠癌CAM移植模型。移植模型瘤体易于生长,具有较强的血管生成作用。结论该模型易于复制,能动态观察大肠癌的血管生成过程,可用于大肠癌的生物学行为、药物筛选等领域的研究。     

Raddeanin A,a triterpenoid saponin isolated from Anemone raddeana,suppresses the angiogenesis and growth of human colorectal tumor by inhibiting VEGFR2 signaling

Raddeanin A (RA) is an active triterpenoid saponin from a traditional Chinese medicinal herb, Anemone raddeana Regel. It was previously reported that RA possessed attractive antitumor activity through inhibiting proliferation and inducing apoptosis of multiple cancer cells. However, whether RA can inhibit angiogenesis, an essential step in cancer development, remains unknown. In this study, we found that RA could significantly inhibit human umbilical vein endothelial cell (HUVEC) proliferation, motility, migration, and tube formation. RA also dramatically reduced angiogenesis in chick embryo chorioallantoic membrane (CAM), restrained the trunk angiogenesis in zebrafish, and suppressed angiogenesis and growth of human HCT-15 colorectal cancer xenograft in mice. Western blot assay showed that RA suppressed VEGF-induced phosphorylation of VEGFR2 and its downstream protein kinases including PLCγ1, JAK2, FAK, Src, and Akt. Molecular docking simulation indicated that RA formed hydrogen bonds and hydrophobic interactions within the ATP binding pocket of VEGFR2 kinase domain. Our study firstly provides the evidence that RA has high antiangiogenic potency and explores its molecular basis, demonstrating that RA is a potential agent or lead candidate for antiangiogenic cancer therapy.     

The inhibition of commitment of mouse erythroleukemia cells by steroids involves a glucocorticoid-receptor mediated process(es) acting at the nuclear level

Dexamethasone has been shown to inhibit dimethylsulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (or Friend) cells by blocking commitment to terminal erythroid maturation. In this study, we confirmed previous reports indicating the presence of glucocorticoid receptors in murine erythroleukemia cells and examined the mechanism(s) by which steroids block commitment. Untreated murine erythroleukemia cells contain dexamethasone receptors which decrease in number during DMSO-induced cell differentiation. When steroids of different classes (estrogenic, androgenic, glucocorticoid) were tested for inhibition of commitment and for displacement of [3H]dexamethasone from its receptors in DMSO-treated cells, we observed that the glucocorticoids dexamethasone, prednisolone and hydrocortisone, all blocked commitment and substantially displaced [3H]dexamethasone. In contrast, steroids other than glucocorticoids failed to inhibit commitment or displace [3H]dexamethasone. Analysis of kinetics of dexamethasone binding to chromatin revealed that dexamethasone binds to the nucleus via the receptor and preferentially interacts with active chromatin. Inhibition of commitment by dexamethasone persisted in cells released from this agent and reincubated with DMSO in the presence of another glucocorticoid of similar affinity to steroid receptors; inhibition of commitment, however, was not obtained when cells removed from dexamethasone were incubated in the presence of beta-estradiol, progesterone and testosterone. These data indicate that inhibition of commitment of mouse erythroleukemia cells by steroids is associated with binding to glucocorticoid receptors and may involve interactions of steroids and their receptors with regions of chromatin.     

Differences in steroidogenesis by the subcellular fractions of adrenocortical special zone and cortex proper of the female possum (Trichosurus vulpecula)

Steroidogenesis by subcellular fractions of adrenal cortex proper (C.P.) and special zone (S.Z.) of female possum (Trichosurus vulpecula) was studied. Mitochondrial, microsomal and cytosol cell fractions were incubated with appropriate substrates in the presence of an NADPH-generating system. The major products formed from [3H]progesterone and [3H]17 alpha-hydroxyprogesterone by the microsomal fraction of the C.P. were 11-deoxycortisol and 11-deoxycorticosterone and 3 alpha (beta)-hydroxy-5 alpha-androstan-17-one by the S.Z. The mitochondrial fraction converted [3H]11-deoxycortisol to cortisol in yields twenty times higher by the C.P. than by the S.Z. and to 17 alpha, 20 beta,21-trihydroxy-4-pregn-3-one thirty times higher by the S.Z. The conversion of [3H]androstenedione to 11 beta-hydroxyandrostenedione by the C.P. was approximately double that of the S.Z., while 18-hydroxyandrostenedione (tentatively identified) formed the highest yield in both zones. Incubation of the same substrates with cytosol formed two 5 beta-pregnane and two 5 beta-androstane derivatives in total yields less than 5% by C.P. and greater than 60% by S.Z. Aromatase activity, estimated by the release of [3H2O] from [1 beta 3H]testosterone, in the adrenals of 8 possums, was in each experiment negligibly low. Determination of total enzyme activities in the two zones revealed that 11 beta, 18 and 21-hydroxylases were higher in the C.P., while 17 alpha-hydroxylase was higher in the S.Z. Similar results were obtained when the rates of formation of hydroxylated products were estimated in the presence of saturating amounts of substrates. Active 5 alpha- and 5 beta-reductases, C17-20-lyase and 3 alpha (beta) and 20 beta-hydroxysteroid dehydrogenases were found almost exclusively in the S.Z. We conclude that the S.Z. at lower levels of activity than the C.P. could contribute to the basal secretion of corticosteroids. In addition, the S.Z. has a high capacity to form C19 steroids and 5 alpha- and 5 beta-reduced steroids. The possible role of the S.Z. in possum is discussed.     

Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K_d, are 0.89 and 0.18 μM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.     

Glucocorticoid potentiation of delta-aminolevulinic acid synthetase in chick embryo liver cells: a "permissive" effect

Glucocorticoids at physiologic concentrations do not induce δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the heme biosynthetic pathway, in cultured chick embryo hepatocytes. Etiocholanolone-mediated induction of this enzyme is however markedly potentiated by cortisol (2.6-fold vs. 4.4-fold). The order of effectiveness in this “permissive” effect is dexamethasone ≥ cortisol > corticosterone > progesterone. The addition of 17α-methyltestosterone, an “anti-glucocorticoid”, substantially decreased the action of cortisol. This “permissive” action is specific for steroid hormones possessing glucocorticoid activity and may play an important role in heme biosynthesis.     

Solid phase synthesis and biological evaluation of probestin as an angiogenesis inhibitor

Probestin is a potent aminopeptidase N (APN) inhibitor originally isolated from the bacterial culture broth. Here, we report probestin synthesis by solid phase peptide synthesis (SPPS) method and evaluated its activity to inhibit angiogenesis using a chicken embryo chorioallantoic membrane (CAM) assay and a CAM tumor xenograft model. Results from these studies demonstrate that probestin inhibits the angiogenic activity and tumor growth.     

Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene

Tumors require ongoing angiogenesis to support their growth. Inhibition of angiogenesis by production of angiostatic factors should be a viable approach for cancer gene therapy. Endostatin, a potent angiostatic factor, was expressed in mouse muscle and secreted into the bloodstream for up to 2 weeks after a single intramuscular administration of the endostatin gene. The biological activity of the expressed endostatin was demonstrated by its ability to inhibit systemic angiogenesis. Moreover, the sustained production of endostatin by intramuscular gene therapy inhibited both the growth of primary tumors and the development of metastatic lesions. These results demonstrate the potential utility of intramuscular delivery of an antiangiogenic gene for treatment of disseminated cancers.     

一种新的源自赤NFDA1的强血管生成抑制剂福安泰-03的分离和鉴定

从赤NFDA1软骨和去皮的软组织中分离并鉴定了一种新的强血管生成抑制剂福安泰-03(Fuantai-03, FAT-03). 利用组织匀浆、盐析、离子交换层析、疏水层析和反向层析等方法进行分离和纯化. 鸡胚绒毛尿囊膜（CAM）试验检测FAT-03对血管生成的影响. SDS-PAGE分析揭示,FAT-03为单一银染条带,分子量大约为43 000.层析洗脱实验证实,这一分子量蛋白质具有强抗血管生成活性. FAT-03的纯度进一步因其独特的N末端氨基酸序列（PFGNTHNKWKLNYSAEQEFP）而肯定.每日20、40和80μg FAT-03给药组（每胚给药3 d）对血管生成的抑制率分别为 23.6%、33.1%和50.8%. 本研究首次证实,赤NFDA1产生上述强血管生成抑制剂.