Homocysteine inhibits angiogenesis in vitro and in vivo

Homocysteine has been reported to inhibit endothelial cell proliferation, which is closely related to angiogenesis. However, the relationship between homocysteine and angiogenesis is unknown. To clarify whether homocysteine would inhibit angiogenesis in vitro and in vivo, we examined the effect of homocysteine on tube formation by bovine aortic endothelial cells (BAECs) and by human microvessel endothelial cell-1 (HMEC-1) in vitro, and on angiogenesis in vivo using the chorioallantoic membrane (CAM) assay, as well as on BAEC proliferation and migration. Homocysteine, but not cysteine, inhibited BAEC proliferation, migration, and tube formation in a dose-dependent manner at concentrations from 0 to 10 mM. Homocysteine also inhibited tube formation by HMEC-1s. In these assay, 50% inhibition was induced by about 1 mM homocysteine. In the in vivo CAM assay, 0, 10, 100, 500, and 1000 microgram homocysteine induced an avascular zone by 0, 0, 16.7, 53.3 and 76.5%, respectively, also showing a dose-dependent effect. It was suggested that homocysteine inhibited angiogenesis by preventing proliferation and migration of endothelial cells.     

cAMP inhibits the in vitro assembly of microtubules.

A new method for assaying microtubule assembly is described. The method utilizes the colchicine binding property of tubulin. This technique was used to study the effect of cyclic AMP on tubulin assembly using 100,000g supernatant and cycle-purified tubulin prepared from porcine brain. Cyclic AMP, in the presence of NaF, inhibited tubulin assembly from 100,000g supernatant but had no effect on cycle-purified tubulin.     

Suramin inhibits C6 glioma-induced angiogenesis in vitro

Aspects of tumor-induced angiogenesis in vitro were examined using an assay involving collagen gel invasion by a surface monolayer of bovine endothelial cells under the influence of serum free conditioned medium produced by C6 cells, an experimentally derived rat glial tumor cell line. The effects of the polyanionic compound suramin, known to interfere with growth factor/cell signaling on this process were evaluated. Collagen gel invasion was quantified by adding C6 conditioned medium with or without various doses of suramin to monolayers of bovine aortic endothelial cells grown on type I collagen gels in transwell inserts. Cultures were monitored with phase-contrast microscopy. After various periods of incubation collagen gels were fixed, embedded in epoxy resin, and 1-μm thick sections were stained with toluidine blue. Additional cultures were used to evaluate the effects of C6 conditioned medium and suramin on endothelial cell proliferation, and on chemotaxis through 8-μm pores. C6 glioma cell conditioned medium induced large vessel endothelial cells to sprout into the underlying collagen matrix and subsequently from networks of capillary like tubes. Conditioned medium was also chemotactic and mitogenic for these cells. The addition of suramin to C6 glioma conditioned medium prevents tube formation in collagen gels, and inhibits both endothelial cell proliferation and chemotaxis in a dose dependent manner. These results suggest that glial tumor cell conditioned medium induces angiongenesis in large vessel endothelial cells in vitro via mechanisms which are disrupted by suramin, most likely involving tumor-derived growth factor release and/or endothelium-mediated matrix proteolysis.     

Evodiamine inhibits in vitro angiogenesis: Implication for antitumorgenicity

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.     

A novel RGDS-analog inhibits angiogenesis in vitro and in vivo

In this study the anti-angiogenic action of a novel non-peptide RGDS-analog named RAM was tested in vitro and in vivo. RAM inhibited FGF-2-induced chemotaxis by 80% in an adhesion-independent way. Further, it induced HUVEC-apoptosis in collagen-seeded HUVEC, indicating that such pro-apoptotic effect was adhesion-independent. In vivo studies revealed that RAM inhibited FGF-2 induced angiogenesis by 60% in the mouse Matrigel-assay and in the chicken-egg chorion-allantoic membrane assay. Finally, RAM was markedly more stable in serum as compared to the template RGDS and after 24 h incubation in 100% serum was significantly more active than RGDS. Taken together these results show that RAM exerts anti-chemotactic and pro-apoptotic effects, by an unexpected adhesion-independent mechanism, as we have recently shown for the template RGDS molecule [Blood 103 (2004) 4180], and has in vivo relevant anti-angiogenic properties, with marked stability in serum; therefore, RAM represents a novel promising anti-angiogenic molecule.     

Dihydrotanshinone I inhibits angiogenesis both in vitro and in vivo

Dihydrotanshinone I （DI）, a naturally occurring compound extracted from Salvia miltiorrhiza Bunge, has been reported to have cytotoxicity to a variety of tumor cells. In this study, we investigated its anti-angiogenic capacity in human umbilical vein endothelial cells. DI induced a potent cytotoxicity to human umbilical vein endothelial cells, with an IC50 value of approximately 1.28 μg/ml.At 0.25-1μg/ml, DI dose-dependently suppressed human umbilical vein endothelial cell migration, invasion, and tube formation detected by wound healing, Transwell invasion and Matrigel tube formation assays, respectively. Moreover, DI showed significant in vivo anti-angiogenic activity in chick embryo chorioailantoic membrane assay. DI induced a 61.1% inhibitory rate of microvessel density at 0.2 μg/egg. Taken together, our results showed that DI could inhibit angio-genesis through suppressing endothelial cell proliferation, migration, invasion and tube formation, indicating that DI has a potential to be developed as a novel anti-angiogenic agent.     

Incadronate disodium inhibits advanced glycation end products-induced angiogenesis in vitro

We report here a genetic assay suitable for detecting site-specific proteolysis in secretory pathways. The yeast enzyme invertase is linked to the truncated lumenal region of the yeast Golgi membrane protein STE13 via a protease substrate domain in a Saccharomyces cerevisiae strain lacking invertase. When the substrate is cleaved by a specific protease, the invertase moiety is released into the periplasmic space where it degrades sucrose to glucose and fructose. Therefore, site-specific proteolysis can be detected by monitoring the growth of yeast cells on selective media containing sucrose as the sole carbon source. We confirmed the validity of this assay with yeast Kex2 and human TMPRSS2 proteases. Our data suggest that this in vivo assay is an efficient method for the determination of substrate specificity and mutational analysis of secreted or membrane proteases.     

TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA

TGFbeta is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFbeta1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFbeta1 on the activation state of members of the RhoGTPase family. TGFbeta1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFbeta1-mediated change of phenotype. Although TGFbeta1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFbeta1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFbeta1 in the process of endothelial tube formation in collagen I gels.     

cAMP mediates IL-1-induced lymphocyte penetration through endothelial monolayers

Endothelial cell incubated with IL-1 have been shown adhere more lymphocytes than nontreated endothelial cells. Here we demonstrate that IL-1 can also increase lymphocyte penetration through endothelial monolayers in vitro. IL-1 induced a transient increase in the number of lymphocytes penetrated through the endothelial monolayer into a filter in a time- and dose-dependent manner. This effect could be mimicked by increasing the cytosolic cAMP levels in the endothelial cells either by forskolin or dibutyryl-cAMP. Concomitantly we were able to show that IL-1 increased the cytosolic cAMP levels in endothelial cells. An inhibitor of adenylate cyclase, ddAdo, decreased both the IL-1-induced cAMP elevation and lymphocyte penetration. A protein kinase A inhibitor HA 1004 could inhibit the IL-1-induced lymphocyte penetration, where as protein kinase C (N-(2-guamidino-ethyl)-5-isoquinolinesyl foamide hydrocloride) and calcium-calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalensulfanamide) inhibitors had no effect. Adding dibutyryl-cGMP or calcium ionophore to the endothelial cells could not mimic IL-1-induced penetration and finally IL-1 did not induce PKC translocation in endothelial cells. These data support the view that IL-1 acts via cAMP as a second messenger in regard to lymphocyte penetration through endothelial cells. The above data demonstrate that IL-1-induced lymphocyte penetration through endothelial cells and that this IL-1-induced signal is transduced via cAMP in endothelial cells.     

Netrin-1 inhibits sprouting angiogenesis in developing avian embryos

Netrin-1 is a bifunctional axonal guidance cue, capable of attracting or repelling developing axons via activation of receptors of the deleted in colorectal cancer (DCC) and uncoordinated 5 (UNC5) families, respectively. In addition to its role in axon guidance, Netrin-1 has been implicated in angiogenesis, where it may also act as a bifunctional cue. Attractive effects of Netrin-1 on endothelial cells appear to be mediated by an as yet unknown receptor, while repulsion of developing blood vessels in mouse embryos is mediated by the UNC5B receptor. To explore evolutionary conservation of vascular UNC5B expression and function, we have cloned the chick unc5b homologue. Chick and quail embryos showed unc5b expression in arterial EC and sprouting angiogenic capillaries. To test if Netrin-1 displayed pro- or anti-angiogenic activities in the avian embryo, we grafted cell lines expressing recombinant chick or human Netrin-1 at different stages of development. Netrin-1 expressing cells inhibited angiogenic sprouting of unc5b expressing blood vessels, but had no pro-angiogenic activity at any stage of development examined. Netrin-1 also had no effect on the recruitment of circulating endothelial precursor cells. Taken together, these data indicate that vascular unc5b expression and function is conserved between chick and mice.     

Mitochondrial BCL-2 inhibits AMBRA1-induced autophagy

BECLIN 1 is a central player in macroautophagy. AMBRA1, a BECLIN 1-interacting protein, positively regulates the BECLIN 1-dependent programme of autophagy. In this study, we show that AMBRA1 binds preferentially the mitochondrial pool of the antiapoptotic factor BCL-2, and that this interaction is disrupted following autophagy induction. Further, AMBRA1 can compete with both mitochondrial and endoplasmic reticulum-resident BCL-2 (mito-BCL-2 and ER-BCL-2, respectively) to bind BECLIN 1. Moreover, after autophagy induction, AMBRA1 is recruited to BECLIN 1. Altogether, these results indicate that, in normal conditions, a pool of AMBRA1 binds preferentially mito-BCL-2; after autophagy induction, AMBRA1 is released from BCL-2, consistent with its ability to promote BECLIN 1 activity. In addition, we found that the binding between AMBRA1 and mito-BCL-2 is reduced during apoptosis. Thus, a dynamic interaction exists between AMBRA1 and BCL-2 at the mitochondria that could regulate both BECLIN 1-dependent autophagy and apoptosis.     

Elevated TGFbeta signaling inhibits ocular vascular development

Alterations in the ocular vasculature are associated with retinal diseases such as retinopathy of prematurity and diabetic retinopathy. Vascular endothelial growth factor (VEGF) as a potent stimulator for normal and abnormal vascular growth has been extensively studied. However, little is known about secreted factors that negatively regulate vascular growth in ocular tissues. We now report that expression of a self-activating TGFbeta1 in the ocular lens of transgenic mice results in inhibition of retinal angiogenesis followed by retinal degeneration. Transgenic TGFbeta1 can rescue the hyperplasic hyaloid tissue and reverse the corneal deficiency in TGFbeta2-null embryos. These results demonstrate that TGFbeta signaling modulates development of ocular vasculature and cornea in a dosage-dependent manner and that TGFbeta1 can substitute for TGFbeta2 in ocular tissues.     

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.     

Hyperthermia inhibits angiogenesis

Since in vitro studies have demonstrated that capillary endothelial cells are thermosensitive, experiments were performed to determine the (in vivo) heat sensitivity of blood capillaries and their endothelial cells. Angiogenesis discs were implanted subcutaneously in mice, and vascular growth was stimulated by slow release of epidermal growth factor placed in the center of each disc. After 5 days of growth the discs were subjected to radiofrequency-induced hyperthermia. Heat exposures were 41, 42, 43, and 44 degrees C for 30 min. Control discs were sham treated. Seven days after heating the discs were extracted and paraffin embedded. Centripetal (radial) vessel growth was measured in magnified medial planar sections. An inverse relationship was demonstrated between vessel growth and exposure temperature. The extent of the fibroblastic growth was also inversely proportional to temperature. Thus, at least in this system, the microvasculature shows dose-dependent damage by hyperthermia, consistent with preceding in vitro observations. This inhibition of angiogenesis may result from endothelial cell killing, interference with cell replication, inhibition of cell migration, or a combination of these mechanisms.