A novel link between angiogenesis and natural products: Anti-angiogenic effects of Opuntia dillenii

Angiogenesis is the formation of new blood vessels from the existing blood vessels and is involved in both physiological and pathological events. Pathological angiogenesis provokes in several important diseases as in inflammatory diseases, ischemic conditions, diabetic neuropathy and cancer. The discovery of new drugs from phyto-chemicals has a long history and anti-angiogenic agents from plant sources provide a platform for the development of phyto-medicines to treat/control pathological angiogenesis. The current project was designed to determine the efficacy of different concentrations of aqueous extract from Opuntia dillenii (OD) on angiogenesis by employing chicken chorioallantoic membrane (CAM) assay. Computer based 3D image probing was utilized to quantify anti-angiogenic effects of OD extract. Extremely aggravated dose dependent anti-angiogenic response was recorded in all groups treated with OD extract with significant reduction (P<0.01) in total area, diameter of the primary, secondary, tertiary blood vessels and capillary plexuses. Analysis of 3D parameters of OD treated CAMs revealed deteriorated angular spectrum and reduction in the height of Abbott curves. In addition, histological data revealed thinning of ectodermal layer and damaged extracellular matrix. The results spotlight that OD extract possess considerable anti-angiogenic potential and further characterization/isolation of diverse phyto-chemicals would turn up with the discovery of novel phyto-agents to control/mitigate pathological angiogenesis.     

Inhibition of virus-induced fusion of Ehrlich ascites tumor cells by cytochalasin B and D.

Cytochalasin B and D were found to inhibit HVJ (Sendai virus)-induced fusion of Ehrlich ascites tumor cells. Nearly complete inhibition was attained by 4 uM (2 μg/ml) cytochalasin D, whereas cytochalasin B was a less effective inhibitor. The inhibition was largely reversible. Since the transport of 2-deoxy-glucose into the tumor cells was not affected by cytochalasin D (though inhibited by cytochalasin B), the observed inhibition was not related to the effect of the drugs on sugar transport. Instead, it was suggested that the inhibition was due to the action of the drugs on microfilaments. The requirement of ATP for the cell fusion could be explained at least partly by the involvement of microfilaments in the cell fusion process.     

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.     

Entamoeba invadens: enhancement of excystation and metacystic development by cytochalasin D

Effects of three actin-modifying drugs, cytochalasin D, latrunculin A, and jasplakinolide, on the excystation and metacystic development in vitro of Entamoeba invadens were examined by transfer of the cysts to growth medium with the drugs. Cytochalasin D unexpectedly increased the number of metacystic amoebae of E. invadens strain IP-1 during incubation. Metacystic development, which was determined by the number of nuclei of metacystic amoebae, was faster in the culture with cytochalasin D than in the culture without the drug. These results suggest that cytochalasin D enhances the excystation and metacystic development. In contrast, latrunculin A and jasplakinolide inhibited these process. No excystation occurred in encystation medium even in the presence of cytochalasin D, suggesting that growth medium is essential for excystation. Excystation was further enhanced when the cysts were incubated with cytochalasin D before culture in growth medium with the drug. The enhancing effect of cytochalasin D on the excystation and metacystic development was abrogated by jasplakinolide. Thus, the results indicate that cytochalasin D, unlike latrunculin A and jasplakinolide, caused enhancement of the excystation and metacystic development of this parasite.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis.     

The anti-angiogenic potential of (±) gossypol in comparison to suramin

Cotton, a staple fiber that grows around the seeds of the cotton plants (Gossypium), is produced throughout the world, and its by products, such as cotton fibers, cotton-seed oil, and cottonseed proteins, have a variety of applications. Cotton-seed contains gossypol, a natural phenol compound. (±)-Gossypol is a yellowish polyphenol that is derived from different parts of the cotton plant and contains potent anticancer properties. Tumor growth and metastasis are mainly related to angiogenesis; therefore, anti-angiogenic therapy targets the new blood vessels that provide oxygen and nutrients to actively proliferating tumor cells. The aim of the present study was to evaluate the anti-angiogenic potential of (±)-gossypol in vitro. (±)-Gossypol has anti-proliferative effects on cancer cell lines; however, its anti-angiogenic effects on normal cells have not been studied. Anti-proliferative activities of gossypol assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, anti-angiogenic activities using tube formation assay, and cell migration inhibition capability using a wound-healing assay on human umbilical vein endothelial cells (HUVECs) were revealed. (±)-Gossypol displayed the following potent anti-angiogenic activities in vitro: it inhibited the cell viability of HUVECs, it inhibited the migration of HUVECs, and disrupted endothelial tube formation in a dose-dependent manner. In addition, the anti-angiogenic effects of (±)-gossypol were investigated in ovo in a model using a chick chorioallantoic membrane (CAM). Decreases in capillary density were assessed and scored. (±)-Gossypol showed dose-dependent anti-angiogenic effects on CAM. These findings suggest that (±)-gossypol can be used as a new anti-angiogenic agent.     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block     

Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo

Prospective skin ectoderm is underlaid by a relatively thick (100 +/- 20 micrometer) avascular zone of mesoderm in most regions of the early embryo. To determine whether or not the ectoderm exercises a role in the establishment and maintenance of the avascular zone, trypsin-isolated pieces of backskin ectoderm from chick or quail embryos were implanted as a sheet into a slit cut deep into the capillary bed of the wing bud of host chick embryos of stages 19-23. In sham operations, slits were cut at various anteroposterior levels, and the wing was allowed to heal. At intervals of 3-48 hr after these operations, embryos were injected with India ink, fixed, and cleared. Implants formed flattened vesicles, usually in continuity with host ectoderm, but sometimes completely internalized. Periderm cells from each side of the vesicle faced each other, and the cells of the cuboidal layer faced an avascular mesodermal layer at least 100 micrometer thick at all points. The implantation of prospective skin ectoderm resulted in the formation of an avascular zone in normally vascularized mesoderm of the wing bud. In contrast, the vascular bed of the limb bud abutted directly on implants of Millipore filters or of Silastic silicone (Dow Corning). Likewise, the capillary bed came in direct contact with implants of retinal pigment epithelium, an ectodermal derivative normally in close contact with the vascular choroid coat of the eye. These results, taken in conjunction with earlier experiments that show the necessity of the apical ectodermal ridge for the formation of the marginal veins of the limb bud, suggest that epithelial-mesenchymal interactions are involved in important aspects of vasculogenesis in early embryos.     

Gametogenesis in Plasmodium; the inhibitory effects of anticytoskeletal agents

Gametocytes of Plasmodium yoelii were incubated with colchicine, vinblastine sulphate and cytochalasin B and then induced to undergo gametogenesis. All the drugs inhibited gamete formation in a dose and time dependent manner. Electron microscopy revealed that colchicine and vinblastine sulphate inhibited the polymerisation of cytoplasmic- axonemal- and intranuclear mitotic- microtubules. Cytochalasin B did not inhibit microtubule assembly but blocked spindle pole separation and axoneme distribution into the microgamete. All drugs prevented escape of the gametocyte from the erythrocyte. The mechanism of mitotie spindle action is discussed.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurrs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis. (Mol Cell Biochem 264: 51–61, 2004)     

Receptor-mediated gonadotropin action in the ovary. Action of cytoskeletal element-disrupting agents on gonadotropin-induced steroidogenesis in rat luteal cells.

The role of the cellular cytoskeletal system of microtubules and microfilaments on gonadotropin-stimulated progesterone production by isolated rat luteal cells has been investigated. Exposure of luteal cells to human choriogonadotropin resulted in a stimulation of cyclic AMP (4-7-fold) and progesterone (3-4-fold) responses.l Incubation of cells with the microfilament modifier cytochalasin B inhibited the gonadotropin-induced steroidogenesis in a dose- and time-dependent manner. The effect of cytochalasin B on basal production of steroid was less pronounced. Cytochalasin B also inhibited the accumulation of progesterone in response to lutropin, cholera enterotoxin, dibutyryl cyclic AMP and 8-bromo cyclic AMP. The inhibition of steroidogenesis by cytochalasin B was not due to (a) inhibition of 125I-labelled human choriogonadotropin binding to luteal cells, (b) inhibition of gonadotropin-stimulated cyclic AMP formation or (c) a general cytotoxic effect and/or inhibition of protein biosynthesis. Cytochalasin D, like cytochalasin B, inhibited gonadotropin- and 8-bromo cyclic AMP-stimulated steroidogenesis. Although cytochalasin B also blocked the transport of 3-O-methyl-glucose into luteal cells, cytochalasin D was without such an effect. Increasing glucose concentration in the medium, or using pyruvate as an alternative energy source, failed to reverse the inhibitory effect of cytochalasin B. The anti-microtubular agent colchicine failed to modulate synthesis and release of progesterone by luteal cells in response to human choriogonadotropin. These studies suggest that the cellular microfilaments may be involved in the regulation of gonadotropin-induced steroidogenesis. In contrast, microtubules appear to be not directly involved in this process.     

Embryonic and adult vasculogenesis

Two mechanisms account for the formation of blood vessels, vasculogenesis and angiogenesis. Unfortunately, the terms vasculogenesis and angiogenesis literally have the same meaning, i.e., the genesis of blood vessels, and thus do little to distinguish between the two processes. Despite the nomenclature, the two processes are clearly distinct. Vasculogenesis, the de novo formation of blood vessels from mesoderm, is driven by the recruitment of undifferentiated mesodermal cells to the endothelial lineage and the de novo assembly of such cells into blood vessels. Angiogenesis is the generation of new blood vessels from endothelial cells of existing blood vessels, a process driven by endothelial cell proliferation. Recent years have seen dramatic changes in our understanding of the process of vasculogenesis, expanding the scope of its occurrence beyond the earliest stages of development to include involvement in neovascular processes throughout development as well as in the adult. In this review, emphasis is placed on discussion of emerging perspectives on the process of vasculogenesis in both the embryo and the adult.     

Pericytes in experimental MDA-MB231 tumor angiogenesis

The role of pericytes (PCs) during embryonic or tumor angiogenesis is a matter of debate. We studied the expression of cytoskeletal, membrane, and matrix markers in experimental tumors of the human mammary ductal adenoma MDA-MB231 cell line that were grown on avian chorioallantoic membranes (CAMs) from incubation day 10 to 18 (chick) or 8 to 15 (quail). The expression patterns of alpha-smooth muscle actin (alphaSMA) and desmin, of adhesion molecules beta1 integrin and neurothelin, and of fibronectin and laminin were analyzed with conventional and confocal laser scanning microscopy. The CAM arterial wall showed strong alphaSMA signal in all smooth muscle cell layers but the innermost layer, which was desmin positive. Ramified alphaSMA-negative cells with delicate desmin staining accompanied most minor vessels and were also seen basal to the capillary plexus indicating the presence of PCs. In the tumor nodules, a diffuse alphaSMA signal without definite relationship to vascular structures was detected. Strongly desmin-positive, alphaSMA-negative cells were frequent in the zone of contact to the CAM in small nodules, and were scattered in larger tumors. In some regions they were associated with microvessels, and in others appeared detaching from endothelial cells (ECs) or as single migrating cells. We conclude that: (a) the CAM tumor angiogenesis assay is useful for studying PC/EC interactions, (b) PCs are recruited from the CAM into experimental tumor nodules, (c) variability of vasculature in MDA-MB231 tumors may be due to variable PC/EC interactions, and (d) alphaSMA should be used with caution as a general PC marker.     

Comparative effects of cytochalasin D on the protein discharge induced by α- and β-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via α-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2·10^?6 M cytochalasin D when the discharge resulted from cholinergic, α- or β-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.     

Comparative effects of cytochalasin D on the protein discharge induced by alpha- and beta-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via alpha-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2 X 10(-6) M cytochalasin D when the discharge resulted from cholinergic, alpha- or beta-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.     

Vascular remodeling by intussusceptive angiogenesis

Intussusception (growth within itself) is an alternative to the sprouting mode of angiogenesis. The protrusion of opposing microvascular walls into the capillary lumen creates a contact zone between endothelial cells. The endothelial bilayer is perforated, intercellular contacts are reorganized, and a transluminal pillar with an interstitial core is formed, which is soon invaded by myofibroblasts and pericytes leading to its rapid enlargement by the deposition of collagen fibrils. Intussusception has been implicated in three processes of vascular growth and remodeling. (1) Intussusceptive microvascular growth permits rapid expansion of the capillary plexus, furnishing a large endothelial surface for metabolic exchange. (2) Intussusceptive arborization causes changes in the size, position, and form of preferentially perfused capillary segments, creating a hierarchical tree. (3) Intussusceptive branching remodeling (IBR) leads to modification of the branching geometry of supplying vessels, optimizing pre- and postcapillary flow properties. IBR can also lead to the removal of branches by pruning in response to changes in metabolic needs. None of the three modes requires the immediate proliferation of endothelial cells but rather the rearrangement and plastic remodeling of existing ones. Intussusception appears to be triggered immediately after the formation of the primitive capillary plexus by vasculogenesis or sprouting. The advantage of this mechanism of growth over sprouting is that blood vessels are generated more rapidly in an energetically and metabolically more economic manner, as extensive cell proliferation, basement membrane degradation, and invasion of the surrounding tissue are not required; the capillaries thereby formed are less leaky. This process occurs without disrupting organ function. Improvements in our understanding of the process should enable the development of novel pro- and anti-angiogenic therapeutic treatments.Electronic Supplementary Material Supplementary material is available in the online version of this article at This research was supported by the Swiss National Science Foundation (grant no. 3100-055895.98/2) and the Bernese Cancer League     

Distribution of hyaluronic acid and sulfated glycosaminoglycans during blood-vessel development in the chick chorioallantoic membrane

This study uses histochemical methods to determine the ultrastructural distribution of specific glycosaminoglycans (GAGs) during the development of blood vessels in the chick chorioallantoic membrane (CAM) and to correlate changes in GAG composition with the significant structural events in the development of these vessels. Tissues were stained with tannic acid, ruthenium red, and high iron diamine and digested in various GAG-degrading enzymes to identify specific GAGs. The results are consistent with a role for hyaluronic acid in the formation, alignment, or migration of the capillary plexus of the CAM and a role for sulfated GAGs (heparan sulfate, chondroitin sulfate, dermatan sulfate) in the differentiation and development of arterial and venous vessels of the chorioallantoic membrane.     

Anti-angiogenic effects of the water extract of HangAmDan (WEHAD), a Korean traditional medicine

We investigated the anti-angiogenic effects of the water extract of HangAmDan (WEHAD), which is a crude extract of nine Korean medicinal substances of animal and plant origin. In human umbilical vein endothelial cells, WEHAD significantly inhibited bFGF-induced proliferation, adhesion, migration, and capillary tube formation. We used an antibody array to perform an analysis of signaling proteins, which showed up-regulated expression of various proteins including RAD51, RAD52, and p73, and down-regulated expression of pFAK. Blood vessel formation in a chick chorioallantoic membrane (CAM) treated with WEHAD was markedly reduced in length compared with a PBS-treated control group. These results suggest that inhibition of angiogenesis by WEHAD may be the mechanism of action for the anti-cancer effects of HAD.     

The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.