Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos.

ETHNOPHARMACOLOGICAL RELEVANCE: The sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities - providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. AIM OF THE STUDY: This study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: The toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1-10 µg/embryo (6.7-670 µg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. RESULTS: The cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2-37.7 µg/mL) while the hot-water extracts are non-toxic up to 670 µg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. CONCLUSIONS: The hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

The potential of Paeonia lactiflora pall seeds oil as a pure natural cosmetics raw material: In Vitro findings.

BACKGROUND: As a traditional Chinese herbal medicine, Paeonia lactiflora Pall is rich in various active ingredients such as polysaccharides and total flavonoids while having ornamental value. It has potential application value in the development of food and cosmetics. OBJECTIVE: To study the in vitro efficacy of Paeonia lactiflora Pall seeds oil. METHODS: Firstly, the levels of linolenic acid and linoleic acid in Paeonia lactiflora Pall seeds oil were quantified using gas chromatography. The impact of Paeonia lactiflora Pall seeds oil on the proliferation rate of B16F10 cells was assessed through the CCK-8 method, while the melanin content of B16F10 cells was determined using the sodium hydroxide lysis method. The inhibitory effects of Paeonia lactiflora Pall seeds oil on elastase, collagenase and hyaluronidase were evaluated by biochemical techniques in vitro. Lastly, the hen's egg chorioallantoic membrane test (HET-CAM) was conducted to confirm the absence of eye irritation caused by Paeonia lactiflora Pall seeds oil. RESULTS: Paeonia lactiflora Pall seeds oil within a certain volume concentration range (0.5%-4%) had no effect on the proliferation of B16F10 cells. Paeonia lactiflora Pall seeds oil showed significant inhibition of elastase, collagenase and hyaluronidase. Notably, the highest concentration tested, 4% Paeonia lactiflora Pall seed oil, yielded the most pronounced outcomes without causing any irritation. CONCLUSION: A certain concentration of Paeonia lactiflora Pall seeds oil has a significant effect on decreasing the melanin content in B16F10 cells and inhibiting the activities of elastase, collagenase, and hyaluronidase, which can provide a reference for the development of pure natural cosmetics raw materials.

Intra-allantoic injection of calcium promotes hatching of chick embryos grown in shell-less culture.

The hatch rate of chick embryos cultured outside of the eggshell with 350 mg calcium l-lactate hydrate (CaL) and 3.5 mL water is fourfold greater in cultures in which the chorioallantoic membrane (CAM) surrounds the egg contents by incubation day 17.5 (E17.5) an event which occurs in ovo by E13. It was first investigated whether decreasing the volume of water added with 350 mg CaL would promote CAM expansion due to the smaller volume to enclose. When 350 mg CaL was present, the CAM did not surround the egg contents by E13. By E17.5, the CAM surrounded the egg contents in 53%-74% of cultures; however, CAM expansion was not significantly different when 0, 1, 2, or 3.5 mL water was present. The hatch rate with 2 or 3.5 mL water was greater than 50% but was not improved with less water. Second, it was investigated whether CaL or water inhibits CAM expansion. In the absence of CaL, the CAM surrounded the egg contents in up to two-thirds of cultures by E13, whether 2 mL water was present or not. Thus CaL, but not water, inhibits expansion of the CAM by E13, even though CaL promotes hatching. Finally, it was investigated whether injection of aqueous CaL into the allantoic fluid, in conjunction with not adding CaL to culture hammocks, would promote CAM expansion. Allantoic injection of CaL starting at E13 did not promote CAM expansion at E17.5 but resulted in hatch rates of approximately 30%. Allantoic injection is a novel route for supplementation of calcium in cultured chick embryos.

Calcium Electroporation versus Electrochemotherapy with Bleomycin in an In Vivo CAM-Based Uveal Melanoma Xenograft Model.

Despite recent advancements in the diagnosis and treatment of uveal melanoma (UM), its metastatic rate remains high and is accompanied by a highly dismal prognosis, constituting an unmet need for the development of novel adjuvant therapeutic strategies. We established an in vivo chick chorioallantoic membrane (CAM)-based UM xenograft model from UPMD2 and UPMM3 cell lines to examine its feasibility for the improvement of selection of drug candidates. The efficacy of calcium electroporation (CaEP) with 5 or 10 mM calcium chloride (Ca) and electrochemotherapy (ECT) with 1 or 2.5 µg/mL bleomycin in comparison to monotherapy with the tested drug or electroporation (EP) alone was investigated on the generated UM tumors. CaEP and ECT showed a similar reduction of proliferation and melanocytic expansion with a dose-dependent effect for bleomycin, whereas CaEP induced a significant increase of the apoptosis and a reduction of vascularization with varying sensitivity for the two xenograft types. Our in vivo results suggest that CaEP and ECT may facilitate the adequate local tumor control and contribute to the preservation of the bulbus, potentially opening new horizons in the adjuvant treatment of advanced UM.

Testing the effects of photobiomodulation on angiogenesis in a newly established CAM burn wound model.

Burn wounds are a common challenge for medical professionals. Current burn wound models hold several limitations, including a lack of comparability due to the heterogeneity of wounds and differences in individual wound healing. Hence, there is a need for reproducible in vivo models. In this study, we established a new burn wound model using the chorioallantoic membrane assay (CAM) as a surrogate model for animal experiments. The new experimental setup was tested by investigating the effects of the auspicious biophysical therapy, photobiomodulation (PBM), on the wound healing of an induced CAM burn wound with a metal stamp. PBM has been shown to positively influence wound healing through vascular proliferative effects and the increased secretion of chemotactic substances. The easily accessible burn wounds can be treated with various therapies. The model enables the analysis of ingrowing blood vessels (angiogenesis) and diameter and area of the wounds. The established model was used to test the effects of PBM on burn wound healing. PBM promoted angiogenesis in burn wounds on day 4 (p = 0.005). Furthermore, there was a not significant trend toward a higher number of vessels for day 6 (p = 0.065) in the irradiated group. Changes in diameter (p = 0.129) and the burn area (p = 0.131) were not significant. Our results suggest that CAM can be a suitable model for studying burn wounds. The novel experimental design enables reproducible and comparable studies on burn wound treatment.

PLGA- corosolic acid implants for potential application in ocular neovascularization diseases

Angiogenesis is the formation of new blood vessels from preexisting vasculature. Uncontrolled angiogenesis is associated with progression of several ocular pathologies, such as diabetic retinopathy and macular degeneration. Thus, the inhibition of this process consists in an interesting therapeutic target. Corosolic acid (CA) is a natural derivative of ursolic acid, found in many medicinal herbs and exhibits numerous biological properties, including the antiangiogenic activity. The present study reports the production of CA-loaded poly d,l-lactidecoglycolide acid (PLGA) devices by melt technique. HPLC-UV method was developed and validated to evaluate the uniformity and the release profile of the developed systems. The devices were also characterized by Fourier transform infrared spectroscopy, thermal analysis, and scanning electron morphology. It was studied the antiangiogenic activity of the CA-polymer system, using an in vivo model, the chorioallantoic membrane assay (CAM). CA was dispersed uniformly in the polymer matrix and no chemical interaction between the components of the formulation was verified. The implants presented a sustained release of the drug, which was confirmed by the morphological study and demonstrated an antiangiogenic activity. Therefore, the developed delivery system is a promising therapeutic tool for the treatment of ocular diseases associated with neovascularization or others related to the angiogenic process.

Chemical constituents of stems and leaves of Salvia yunnanensis and their anti-angiogeneic activities / 中国中药杂志

Present study was focused on the chemical constituents of the stems and leaves of Salvia yunnanensis C . H. Wright and their anti-angiogeneic activities. The compounds were isolated by column chromatography over silica gel and Sephadex LH-20, and other isolation techniques. Their structures were elucidated on the basis of spectral analysis and chemical evidences. Their anti-angiogeneic activities were evaluated by the chicken chorioallantoic membrane (CAM) neovascularisation model. Seven compounds were separated and identified as ( + ) -spathulenol( 1), 5,7,4'-trihydroxyflavanone(2) , beta-amyrin(3), 3 beta-hydroxy-12-ursene(4), 2alpha,3 beta-dihydroxyursa-12-en-28-oic acid(5), ursolic acid (6) and 3-oxo-12-ursen-28-oic acid (7). Compounds 1, 2, 5 and 6 were obtained from this plant for the first time. Compounds 5 (an oleanane compound) and 6 (an ursane compound) could inhibit angiogenesis significantly in a dose-dependent manner.

Synthesis and characterization of protocatechuic acid derivants / 中国中药杂志

To explore the effects of protocatechuic acid (PCA) and its derivants on angiogenesis of the chick embryo chorioallantoic membrane (CAM) and scavenging DPPH radical in vitro. The protection of benzyl and alkaline hydrolysis of benzyl ester were employed. The structures of PCA-1, PCA-2 and PCA-3, the derivates of PCA, were elucidated by 1H, 13C-NMR and MS data The bioactivity of PCA and its derivants was evaluated on the models of DPPH radical and chick embryo chorioallantoic membrane (CAM), respectively. PCA and PCA-1 showed the best activity of scavenging DPPH radical among all the compounds. In contrast to PCA-2, PCA and PCA-3 displayed inhibition to angiogenesis (P < 0.001). Pyrocatechol hydroxyl is the active site of PCA on scavenging DPPH radical in vitro. PCA with carboxyl and without pyrocatechol hydroxyl seems to show promotion to angiogenesis, but it needs more evidences.

Anti-Inflammatory, Antioxidant, Anti-Angiogenic and Skin Whitening Activities of Phryma leptostachya var. asiatica Hara Extract

This work aimed to assess some pharmacological activities of P. leptostachya var. asiatica Hara. The dried roots of P. leptostachya var. asiatica Hara were extracted with 70% ethanol to generate the powdered extract, named PLE. Anti-angiogenic activity was detected using chick chorioallantoic membrane (CAM) assay. In vitro anti-inflammatory activity was evaluated via analyzing nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and reactive oxygen species (ROS) level in the stimulated macrophage cells. Matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) activities in the culture media were detected using zymography. PLE exhibits an anti-angiogenic activity in the CAM assay, and displays an inhibitory action on the generation of NO in the LPS-stimulated macrophage cells. In the stimulated macrophage cells, it is able to diminish the enhanced ROS level. It can potently scavenge the stable DPPH free radical. It suppresses the induction of iNOS and COX-2 and the enhanced MMP-9 activity in the stimulated macrophage cells. Both monooxygenase and oxidase activities of tyrosinase were strongly inhibited by PLE. Taken together, the dried roots of P. leptostachya var. asiatica Hara possess anti-angiogenic, anti-inflammatory, antioxidant and skin whitening activities, which might partly provide its therapeutic efficacy in traditional medicine.

Angio-suppressive triterpenoids from Ardisia cf. elliptica (subgenus Tinus) on duck (Anas platyrynchos L.) chorioallantoic membrane / 中国天然药物

The dichloromethane extract of the air-dried leaves of Ardisia cf. elliptica (subgenus Tinus) afforded a mixture of bauerenol (1a), α-amyrin (1b) and β-amyrin (1c). Their structures were identified by NMR spectroscopy. Mixtures of the triterpenes (1a-1c) at ratios of 2 : 2 : 1, 2 : 2 : 3 and 1 : 1 : 1 were tested for their angio-suppressive effects on duck chorioallantoic membrane (CAM). All three ratios were found to be effective in restricting inter-capillary length, while 1a-1c (2 : 2 : 1) was most effective in reducing branch point density with 100% CAM viability and embryo survivability, suggesting a high impact angio-suppressive potential of 1a-1c (2 : 2 : 1).

Effects of Pien Tze Huang on angiogenesis in vivo and in vitro / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the anti-angiogenic effects of Pien Tze Huang in vivo and in vitro.</p><p><b>METHODS</b>Human umbilical vein endothelial cells (HUVECs) were treated with 0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL of PZH for 24 h, 48 h and 72 h, respectively. Chicken embryo chorioallantoic membrane (CAM) model was used to evaluate in vivo angiogenesis. An ECMatrix gel system was used to evaluate in vitro angiogenesis by examining the tube formation of HUVECs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine HUVEC viability. Cell density of HUVECs was observed by phase-contrast microscopy. HUVEC migration was determined by wound healing method. The mRNA and protein expression of vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF) in both HUVEC and human colon adenocarcinoma cells (HT-29) was examined by reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immune sorbent assay (ELISA), respectively.</p><p><b>RESULTS</b>PZH treatment significantly reduced the total number of blood vessels compared with the untreated control in the chicken embryos and resulted in a significant decrease in capillary tube formation and cell density of HUVECs (P<0.05). In addition, treatment with 0.25-1 mg/mL of PZH for 24 h, 48 h, and 72 h respectively reduced cell viability by 9%-52%, 24%-87% or 25%-87%, compared with the untreated control cells (P<0.05). Moreover, PZH treatment decreased the migration of HUVECs. Furthermore, PZH dose-dependently suppressed the expression of VEGF-A and bFGF on both mRNA and protein levels (P<0.05).</p><p><b>CONCLUSION</b>PZH could inhibit angiogenesis in vivo in CAM model and in vitro on HUVECs, suggesting that inhibiting tumor angiogenesis might be one of the mechanisms by which PZH treats cancer.</p>

Angiogenic activity of Calendula officinalis flowers L. in rats / Atividade angiogênica das flores da Calendula officinalis L. em ratos

Purpose: In this work, angiogenic activity of Calendula officinalis L. (Asteraceae) ethanolic extract and dichloromethane and hexanic fractions were evaluated, considering medicinal properties, especially healing activity, are attributed to this plant. Methods: Models using 36 rats and 90 embryonated eggs were used to evaluate healing and angiogenic activities of extracts and fractions of the plant, through the induction of skin wounds and the chorioallantoic membrane, respectively. The effect of vascular proliferation was also tested from the study to verify the intensity of expression of vascular endothelial growth factor (VEGF) in cutaneous wounds in rats. Results: The angiogenic activity of the extract and the fractions was evidenced in both experimental models. It was verified that this effect is not directly related to the expression of VEGF and it could be associated to other pro-angiogenic factors. Conclusion: The healing activity referred to C. officinalis is related, among other factors, to its positive effect on angiogenesis, characterized by the induction of neovascularization.

Objetivo: Neste trabalho a atividade sobre a angiogênese do extrato etanólico (EEC) e das frações diclorometano e hexânica das flores de Calendula officinalis L. (Asteraceae) cultivada no Brasil foram avaliados, visto que propriedades medicinais têm sido atribuídas às flores da planta, destacando-se a atividade cicatrizante. Métodos: Modelos utilizando 36 ratos e 90 ovos embrionados foram usados para avaliar as atividades cicatrizante e angiogênica dos extratos e frações da planta, por meio da indução de feridas cutâneas e da membrana corioalantóide, respectivamente. O efeito proliferativo vascular foi também testado a partir do estudo imunoistoquímico, realizado para verificar a intensidade da expressão do fator de crescimento endotelial vascular (VEGF) na derme de ratos. Resultados: A atividade angiogênica do extrato e das frações foi evidenciada nos dois modelos experimentais empregados. Foi evidenciado que este efeito não estava diretamente relacionado à expressão do VEGF, podendo estar associado a outros fatores pró-angiogênicos. Conclusão: A atividade cicatrizante referida a C. officinalis está relacionada ao seu efeito positivo sobre a angiogênese, e este foi caracterizado pela indução de neovascularização.

Angiogenic activity of Synadenium umbellatum Pax latex / Atividade angiogênica do látex de Synadenium umbellatum Pax

Synadenium umbellatum Pax, popularly known as "cola-nota", is a medicinal plant that grows in tropical regions. Latex of this plant is used to treat various diseases such as diabetes mellitus, Hansen´s disease, tripanosomiases, leukemia and several malignant tumors. In the present study, the angiogenic activity of S. umbellatum latex was evaluated using the chick embryo chorioallantoic membrane (CAM) assay. Results showed significant increase of the vascular net (p < 0.05) compared to the negative control (H2O). The histological analysis was in accordance with the results obtained. In conclusion, our data indicate that S. umbellatum latex, under the conditions of this research, presented angiogenic effect.

Synadenium umbellatum Pax, popularmente conhecida como "cola-nota", é uma planta medicinal que cresce em regiões tropicais. O látex desta planta tem sido utilizado no tratamento de várias doenças, como diabetes mellitus, hanseníase, tripanossomíases, leucemia e vários tumores malignos. No presente estudo, a atividade angiogênica do látex de S. umbellatum foi avaliada pelo ensaio da membrana corio-alantóide (MCA) de ovo embrionado de galinha. Os resultados mostraram aumento significativo da rede vascular (p < 0.05) em relação ao controle negativo (H2O). A análise histológica está em concordância com os resultados obtidos. Em conclusão, os dados indicaram que, nas condições experimentais deste estudo, o látex de S. umbellatum exibiu efeito angiogênico.

Angiogenesis promoting effect of Morinda officinalis oligosaccharides on chicken embryo chorioallantoic membrane / 中国中药杂志

<p><b>OBJECTIVE</b>To study the angiogenesis promoting effect of Morinda officinalis oligosaccharides(MOO) on chick embryo chorioallantoic membrane (CAM).</p><p><b>METHOD</b>Rats blood serum containing low, medium and high doses of MOO was prepared using Chinese herbs serum pharmacology method. 60 chick embryoes were randomly divided into low, medium and high doses of MOO groups, as well as NS group, blank serum group and bFGF group. Each group included 10 embryoes. CAM model was prepared after 7 days incubation. Then NS, blank serum, bFGF (2500 U x mL(-1)), three doses of serum containing MOO were added respectively onto the carriers on the CAM. CAM sample was prepared after 3 days incubation. The state of angiogenesis was observed and the number of new blood vessels was counted.</p><p><b>RESULT</b>Compared with blank serum and NS group, a more specific CAM angiogenesis appearance could be observed in each MOO group and bFGF group. Compared with blank serum group, the number of new blood vessels in each MOO group increased significantly (P < 0.05). But the drug had a lower efficacy than bFGF (P < 0.05). Compared with low dose group, the number of new blood vessels increased significantly in medium and high doses groups (P < 0.05). But there was no significant difference between the latter two groups. The number of new blood vessels showed no significant difference between NS group and blank serum group.</p><p><b>CONCLUSION</b>MOO can obviously promote angiogenesis of CAM.</p>

Experimental study of Solanum nigrum on inhibiting angiogenesis in chick chorioallantoic membrane / 中国中药杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of Solanum nigrum on angiogenesis.</p><p><b>METHOD</b>We examined the effects of S. nigrum on angiogenesis in the chick chorioallantoic membrane (CAM) model. On day 7 of chick embryo incubation, three concentrations of S. nigrum aqueous extracts were applied to CAMs, and their effects were evaluated on day 9.</p><p><b>RESULT</b>The angiogenesis area was significantly smaller in the CAM treated with S. nigrum than that in the control group (P < 0.001). Pathology analysis indicated that less angiogenesis occurred in the tissue of CAM under the filter paper treated with S. nigrum and the structure of large arteries was destroyed. The surrounding CAM showed a few angiogenesis formation. However, in the control group, a number of angiogenesis were observed.</p><p><b>CONCLUSION</b>S. nigrum could inhibit the angiogenesis on CAM.</p>

Inhibitory effect of oxaliplatin in combination with hyperthermia on angiogenesis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of oxaliplatin in combination with hyperthermia on angiogenesis in vitro and in vivo.</p><p><b>METHODS</b>MTT method was used to observe the influence of oxaliplatin on the proliferation of human umbilical vein endothelial cells (HUVEC) or human colon cancer cells (LOVO). The influence of oxaliplatin on HUVEC migration was evaluated by Transwell. Chick embryo chorioallantoic membrane (CAM) model was used to check whether the neovascularization of CAM could be suppressed in vivo.</p><p><b>RESULTS</b>The survival rate of HUVEC was 80.1% - 42.5% within a range of 0.5 - 16 microg/ml and was negatively correlated with the concentration (correlation coefficient was - 0. 943, P = 0.005). The survival rate of LOVO cells within those doses was more than that of HUVEC. There was a synergistic antiangiogenic effect when a combination of oxaliplatin (0.5 microg/ml, 1 microg/ml and 16 microg/ml) with hyperthermia was used while additional effect was shown by the combinatioin of oxaliplatin (2 microg/ml, 4 microg/ml and 8 microg/ml) and hyperthermia in vitro. Oxaliplatin inhibited migration of HUVEC in vitro at low doses (0.25 - 2 microg/ml), and also suppressed angiogenesis of CAM in vivo at doses of 1 -4 microg/ml.</p><p><b>CONCLUSION</b>The results of this experiment showed that low dose of oxaliplatin has anti-angiogenic effect in vitro, while in combination with hyperthermia has additional effect both in vivo and in vitro.</p>

Angiogenesis promoting effects of Chinese herbal medicine for activating blood circulation to remove stasis on chick embryo chorio-allantoic membrane / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the angiogenesis promoting effects of clinical common used Chinese herbal medicines (CHM) for activating blood circulation to remove stasis on chick embryo chorio-allantoic membrane (CAM).</p><p><b>METHODS</b>Chicken CAM model was established and mice blood serum containing different kinds of medicines, including Radix Peaoniae rubra, Radix Angelicae sinensis, Flos Carthami, Rhizoma Chuanxiong, Radix Salviae miltiorrhizae, Astragalus membranaceus, and their complex prescriptions, Danggui Buxue Decoction, Xuefu Zhuyu Decoction, Xiongshao Capsule, was applied on it respectively to observe the condition of angiogenesis 72 hrs after incubation. Besides, the normal saline group, blank serum group, blank group and basic fibroblast growth factor (bFGF) group were set up for control.</p><p><b>RESULTS</b>All the CHM applied and bFGF had the CAM angiogenetic promoting effect, among them, Radix Salviae Miltiorrhizae and the three complex prescriptions showed better effects than the three negative control groups in capillary formation and count, with the efficacy similar to that of bFGF. The effect of complex prescriptions was superior to that of single herb except Radix Salviae miltiorrhizae.</p><p><b>CONCLUSION</b>Radix Salviae miltiorrhizae, Danggui Buxue Decoction, Xuefu Zhuyu Decoction and Xiongshao Capsule have good angiogenesis promoting effect on CAM. This study elucidated, from a certain aspect, the mechanism of action of CHM on ischemic diseases, and unfolded the scientific evidence of applying complex prescription.</p>

cDNA Cloning and Expression of Angiostatin, an Angiogenesis Inhibitor , from Human Liver Tissue mRNA / 대한암학회지

PURPOSE: Angiostatin, a 38 kDa internal fragment of plasminogen, is a potent inhibitor of angiogenesis. It blocks neovascularization and growth of primary and metastatic tumors in mice. To produce recombinant angiostatin protem comprising kringle 1-4 of plasminogen, we cloned the angiostatin cDNA from human liver tissue mRNA and expressed it in E. coli. MATERIALS AND METHODS: We cloned angiostatin cDNA from human liver tissue mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) method. Cloned cDNA was ligated to pET22b (+) expression vector, transformed into E. coli stram BL21 (DE3) and expressed by IPTG induction. Recombinant human angiostatin protein was purified from the inclusion bodies of lysated bacterial pellet with 8 M urea solubilization, refolding, single step Lysine-Sepharose 4B affinity chromatography and 0.2 M E-aminocarproic acid elution. The anti-angiogenic activity of purified recombinant angiostatin was assayed with endothelial cell proliferation assay and chorioallantoic membrane assay (CAM). RESULTS: The identification of cloned angiostatin cDNA was confirmed by Southern hybridization and Pst I restriction enzyme digestion pattern. Angiostatin cDNA was expressed in E. coli, refolded in vitro and purified by Lysine Sepharose 4B affinity chromatography. The molecular weight of purified recombinant angiostatin was about 55 kDa on the SDS-PAGE. It inhibited the proliferation of bovine capillary endothelial (BCE) cells in vitro with a half-maximal inhibition concentration (ED50) of approximately 500 ng/mL. It also suppressed neovasculrization on the CAM assay. CONCLUSION: These results demonstrated that recombinant human angiostatin has similar function and biological activity compared with human angiostatin which is purified from porcine elastase digested human plasminogen fragment.