Trans-3,4,5,4′-tetramethoxystilbene,a resveratrol analog,potently inhibits angiogenesis in vitro and in vivo

Aim:

Trans-3,4,5,4′-tetramethoxystilbene (DMU-212) has shown strong antiproliferative activities against a variety of cancer cells. The aim of this study was to investigate the anti-angiogenic effects of DMU-212 in vitro and in vivo.

Methods:

Human umbilical vein endothelial cells (HUVECs) were used in this study. Cell viability was studied with MTT assay, and cell apoptosis was evaluated using TUNEL assay and morphological observation. The expression of the related genes and proteins was analyzed with qRT-PCR and Western blot, respectively. Angiogenesis of HUVECs were studied using cell migration and capillary-like tube formation assays in vitro, and mouse Matrigel plug assay and chick chorioallantoic membrane (CAM) assay in vivo. The tyrosine kinase activities of VEGFR1 and VEGFR2 were measured using commercial kits.

Results:

DMU-212 (5–80 μmol/L) significantly inhibited VEGF-stimulated proliferation of HUVECs (IC₅₀ value was approximately 20 μmol/L), and induced apoptosis. Furthermore, DMU-212 concentration-dependently inhibited VEGF-induced migration of HUVECs and capillary-like structure formation in vitro. DMU-212 also inhibited VEGF-induced generation of new vasculature in Matrigel plugs in vivo with significantly decreased area of infiltrating CD31-positive endothelial cells, and inhibited newly formed microvessels in chick CAMs. Moreover, DMU-212 concentration-dependently suppressed VEGF-induced phosphorylation of VEGFR2, and inhibited phosphorylation of multiple downstream signaling components in the VEGFR2 pathway, including c-Src, FAK, Erk1/2, Akt, mTOR, and p70S6K in HUVECs. DMU-212 had no effect on VEGF-induced phosphorylation of VEGFR1.

Conclusion:

DMU-212 is a potent inhibitor of angiogenesis that exerts anti-angiogenic activity at least in part through the VEGFR2 signaling pathway.     

Anti-angiogenic effects of two cystine-knot miniproteins from tomato fruit

BACKGROUND AND PURPOSE

Cystine-knot miniproteins are characterized by a similar molecular structure. Some cystine-knot miniproteins display therapeutically useful biological activities, as antithrombotic agents or tumour growth inhibitors. A critical event in the progression of tumours is the formation of new blood vessels. The aim of this work was to test two tomato cystine-knot miniproteins for their effects on endothelial cell proliferation and angiogenesis in vitro.

EXPERIMENTAL APPROACH

Two tomato cystine-knot miniproteins (TCMPs) were expressed and purified either as recombinant or as native proteins from tomato fruits. The Matrigel assay was used to investigate the effects of TCMPs on in vitro angiogenesis. Viability and proliferation of endothelial cells were tested. Extracellular signal-regulated kinase (ERK)1/2 phosphorylation was assayed in either HUVEC or A431 epidermal growth factor receptor (EGFR)-overexpressing cells treated with TCMPs. EGFR phosphorylation was tested in A431 cells.

KEY RESULTS

Both recombinant and native TCMPs inhibited in vitro angiogenesis of HUVEC cells at concentrations of 15–100 nM. The anti-angiogenic effect of TCMPs was associated with the inhibition of ERK phosphorylation. The two miniproteins did not alter the viability and proliferation of the endothelial cells.

CONCLUSIONS AND IMPLICATIONS

The anti-angiogenetic properties of TCMPs are of potential pharmacological interest because they are common and natural components of the human diet, they possess low toxicity, they are active at submicromolar concentrations, they share a common molecular structure that can be used as a molecular platform for the design of molecules with enhanced biological activity.     

Isoindolone derivative QSN-IOc induces leukemic cell apoptosis and suppresses angiogenesis via PI3K/AKT signaling pathway inhibition

Aim： 2-（4,6-Dimethoxy-l,3-dioxoisoindolin-2-yl） ethyl 2-chloroacetate （QSN-IOc） is one of isoindolone derivatives with antiproliferative activity against human umbilical vein endothelial cells （HUVECs）. The aim of this study was to investigate its antitumor activity in vitro and anti-angiogenic effects in vitro and in vivo. Methods： K562 leukemic cells and HUVECs were used for in vitro studies. Cell viability was examined using MTT assay. Cell apoptosis and mitochondrial transmembrane potential （Δψm） were detected with flow cytometry. Tube formation and migration of HUVECs were studied using two-dimensional Matrigel assay and wound-healing migration assay, respectively. VEGF levels were analyzed with RT-PCR and Western blotting. A zebrafish embryo model was used for in vivo anti-angiogenic studies. The molecular mechanisms for apoptosis in K562 cells and antiangiogenesis were measured with Western blotting. Results： In antitumor activity studies, QSN-IOc suppressed the viability of K562 cells and induced apoptosis in dose- and time- dependent manners. Furthermore, QSN-IOc dose-dependently decreased the Δψm in K562 cells, increased the release of cytochrome c and the level of Bax, and decreased the level of Bcl-2, suggesting that QSN-10c-induced apoptosis of K562 cells was mediated via the mitochondrial apoptotic pathway. In anti-angiogenic activity studies, QSN-IOc suppressed the viability of HUVECs and induced apoptosis in dose dependent manners. QSN-IOc treatment did not alter the Δψm in HUVECs, but dose-dependently inhibited the expression of VEGF, inhibited the tube formation and cell migration in vitro, and significantly suppressed the number of ISVs in zebrafish embryos in vivo. Furthermore, QSN-IOc dose-dependently suppressed the phosphorylation of AKT and GSK313 in both HUVECs and K562 cells. Conclusion： QSN-IOc is a novel antitumor compound that exerts both antitumor and anti-angiogenic effects via inhibiting the PI3K/ AKT/GSK313 signaling pathway.     

Combined treatment with bexarotene and rosuvastatin reduces angiotensin-II-induced abdominal aortic aneurysm in apoE-/- mice and angiogenesis

Background and Purpose

Abdominal aortic aneurysm (AAA) is a degenerative vascular disease associated with angiogenesis. Bexarotene is a retinoid X receptor (RXR) ligand with anti-angiogenic activity. Statins also exert anti-angiogenic activity and activate PPARs. Because RXR ligands form permissive heterodimers with PPARs and a single anti-angiogenic drug may not be sufficient to combat the wide array of angiogenic factors produced during AAA, we evaluated the effect of combined low doses of bexarotene and rosuvastatin in a mouse model of AAA.

Experimental Approach

The effect of the combined treatment was investigated in a murine model of angiotensin II-induced AAA in apoE^−/− mice. This combination therapy was also evaluated in in vivo (Matrigel plug assay) and in vitro (endothelial cell differentiation assay) models of angiogenesis as well as the underlying mechanisms involved.

Key Results

Co-treatment with bexarotene plus rosuvastatin reduced aneurysm formation, inflammation and neovascularization compared with each single treatment. In HUVEC, the combination of suboptimal concentrations of bexarotene and rosuvastatin inhibited angiotensin II-induced morphogenesis, proliferation and migration. These effects were accompanied by diminished production of pro-angiogenic chemokines (CXCL1, CCL2 or CCL5) and VEGF, and seemed to be mediated by RXRα/PPARα and RXRα/PPARγ activation. This combined therapy reduced the activation of members of the downstream PI3K pathway (Akt/mTOR and p70S6K1) in vivo and in vitro.

Conclusions and Implications

The combination of RXR agonists with statins at low doses synergistically interferes with the signalling pathways that modulate inflammation and angiogenesis and may constitute a new and safer therapeutic treatment for the control of AAA.     

Acurhagin-C, an ECD disintegrin, inhibits integrin ��v��3-mediated human endothelial cell functions by inducing apoptosis via caspase-3 activation

Background and purpose:

Acurhagin, a member of versatile metalloproteinase disintegrins from Agkistrodon acutus venom, has been identified as a platelet aggregation inhibitor, previously. Here, acurhagin-C, the C-terminal Glu-Cys-Asp (ECD)-containing fragment of acurhagin, was evaluated for its biological activities and potential applications in anti-angiogenic therapy.

Experimental approach:

Human umbilical vein endothelial cells (HUVECs) were treated with acurhagin-C to assay effects on viability, apoptosis, adhesion, migration, invasion, proliferation and angiogenesis. The recognition site and signalling involved for the interactions of acurhagin-C with HUVEC were determined using flow cytometric, electrophoresis and immunoblotting analyses.

Key results:

Acurhagin-C decreased viability and induced apoptosis in HUVEC. It also dose-dependently inhibited HUVEC adhesion to immobilized extracellular matrices fibronectin, collagen I and vitronectin with respective IC₅₀ values of approximately 0.6, 0.3 and 0.1 µM. Acurhagin-C prevented migration and invasion of HUVEC through vitronectin- and Matrigel-coated barriers respectively. Furthermore, acurhagin-C attenuated fibroblast growth factor-2-primed angiogenesis both in vitro and in vivo, and specifically blocked the binding of anti-αvβ3 monoclonal antibody 23C6 to HUVEC in an ECD-dependent manner. However, purified αvβ3 also dose-dependently bound to immobilized acurhagin and acurhagin-C with a saturable pattern. Interference with integrin αvβ3-mediated functions and promotion of caspase-3 activation by acurhagin-C affected morphology of HUVEC and induced apoptosis.

Conclusions and implications:

Acurhagin-C elicited endothelial anoikis via disruption of αvβ3/focal adhesion kinase/phosphatidylinositol 3-kinase/Akt survival cascade and subsequent initiation of the procaspase-3 apoptotic signalling pathway.     

Anti-angiogenic effects of the tubulysin precursor pretubulysin and of simplified pretubulysin derivatives

BACKGROUND AND PURPOSE

The use of tubulin-binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti-angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA).

EXPERIMENTAL APPROACH

The compounds were tested in cellular angiogenesis assays (proliferation, cytotoxicity, cell cycle, migration, chemotaxis, tube formation) and in vitro (tubulin polymerization). The efficacy of Prt was also tested in vivo in a murine subcutaneous tumour model induced with HUH7 cells; tumour size and vascularization were measured.

KEY RESULTS

The anti-angiogenic potency of all the compounds tested ran parallel to their inhibition of tubulin polymerization in vitro. Prt showed nearly the same efficacy as TubA (EC₅₀ in low nanomolar range in all cellular assays). Some modifications in the Prt molecule caused only a moderate drop in potency, while others resulted in a dramatic loss of action, providing initial insight into structure–activity relations. In vivo, Prt completely prevented tumour growth and reduced vascular density to 30%.

CONCLUSIONS AND IMPLICATIONS

Prt, a chemically accessible precursor of some tubulysins is a highly attractive anti-angiogenic compound both in vitro and in vivo. Even more simplified derivatives of this compound still retain high anti-angiogenic efficacy.     

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody

BACKGROUND AND PURPOSE

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect.

EXPERIMENTAL APPROACH

Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice.

KEY RESULTS

Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody.

CONCLUSION AND IMPLICATIONS

These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL.     

Galbanic Acid Isolated from Ferula assafoetida Exerts In Vivo Anti-tumor Activity in Association with Anti-angiogenesis and Anti-proliferation

Purpose

To investigate whether galbanic acid (GBA) exerts anti-angiogenic and anti-cancer activities.

Methods

Using human umbilical vein endothelial cell (HUVEC) model, we analyzed effects of GBA on cellular and molecular events related to angiogenesis. We tested its direct anti-proliferative action on mouse Lewis lung cancer (LLC) cells and established its in vivo anti-angiogenic and anti-tumor efficacy using LLC model.

Results

GBA significantly decreased vascular endothelial growth factor (VEGF)-induced proliferation and inhibited VEGF-induced migration and tube formation of HUVECs. These effects were accompanied by decreased phosphorylation of p38-mitogen-activated protein kinase (MAPK), c-jun N-terminal kinase (JNK), and AKT, and decreased expression of VEGFR targets endothelial nitric oxide synthase (eNOS) and cyclin D1 in VEGF-treated HUVECs. GBA also decreased LLC proliferation with an apparent G2/M arrest, but did not induce apoptosis. In vivo, inclusion of GBA in Matrigel plugs reduced VEGF-induced angiogenesis in mice. Galbanic acid given by daily i.p. injection (1 mg/kg) inhibited LLC-induced angiogenesis in an intradermal inoculation model and inhibited the growth of s.c. inoculated LLC allograft in syngenic mice. Immunohistochemistry revealed decreased CD34 microvessel density index and Ki-67 proliferative index in GBA-treated tumors.

Conclusions

GBA exerts anti-cancer activity in association with anti-angiogenic and anti-proliferative actions.     

Anti-angiogenic and vascular disrupting effects of C9, a new microtubule-depolymerizing agent

Background and purpose:

The critical role of blood supply in the growth of solid tumours makes blood vessels an ideal target for anti-tumour drug discovery. The anti-angiogenic and vascular disrupting activities of C9, a newly synthesized microtubule-depolymerizing agent, were investigated with several in vitro and in vivo models. Possible mechanisms involved in its activity were also assessed.

Experimental approach:

Microtubule-depolymerizing actions were assessed by surface plasmon resonance binding, competitive inhibition and cytoskeleton immunofluorescence. Anti-angiogenic and vascular disrupting activities were tested on proliferation, migration, tube formation with human umbilical vein endothelial cells, and in rat aortic ring, chick chorioallantoic membrane and Matrigel plug assays. Western blots and Rho activation assays were employed to examine the role of Raf-MEK-ERK (mitogen-activated ERK kinase, extracellular signal-regulated kinase) and Rho/Rho kinase signalling.

Key results:

C9 inhibited proliferation, migration and tube formation of endothelial cells and inhibited angiogenesis in aortic ring and chick chorioallantoic membrane assays. C9 induced disassembly of microtubules in endothelial cells and down-regulated Raf-MEK-ERK signalling activated by pro-angiogenic factors. In addition, C9 disrupted capillary-like networks and newly formed vessels in vitro and rapidly decreased perfusion of neovasculature in vivo. Endothelial cell contraction and membrane blebbing induced by C9 in neovasculature was dependent on the Rho/Rho kinase pathway.

Conclusions and implications:

Anti-angiogenic and vascular disruption by C9 was associated with changes in morphology and function of endothelial cells, involving the Raf-MEK-ERK and Rho/Rho kinase signalling pathways. These findings strongly suggest that C9 is a new microtubule-binding agent that could effectively target tumour vasculature.     

Microvesicles secreted from human multiple myeloma cells promote angiogenesis

Aim： To investigate whether human multiple myeloma （MM） cells secrete microvesicles （MVs） and whether the MVs secreted from MM cells （MM-MVs） promote angiogenesis. Methods： RPMI8226 human MM cells and EA.hy926 human umbilical vein cells were used. MVs isolated from RPMI 8226 cells were characterized under laser confocal microscopy, electron microscopy and with flow cytometry. The fusion of MM-MVs and EA.hy926 cells was studied under confocal microscopy, and the transfer of CD138 to EA.hy926 cells was demonstrated with flow cytometry. The proliferation, invasion and tube formation of EA.hy926 cells in vitro were evaluated using M]-r, transwell migration and tube formation assays, respectively. The vasculization of EA.hy926 cells in vivo was studied using Matrigel plug assay. The expression of IL-6 and VEGF was analyzed with PCR and ELISA. Results： MM-MVs from the RPMI 8226 cells had the characteristic cup-shape with diameter of 100-1000 nm. Most of the MM-MVs expressed phosphatidylserine and the myeloma cell marker CD138, confirming that they were derived from myeloma cells. After added to EA.hy926 cells, the MM-MVs transferred CD138 to the endothelial cells and significantly stimulated the endothelial cells to proliferate, invade, secrete IL-6 and VEGF, two key angiogenic factors of myeloma, and form tubes in vitro and in vivo. Conclusion： Our results confirm the presence of MVs in MM cells and support the idea that MM-MVs are newfound mediators for myeloma angiogenesis and may serve as a therapeutic target to treat MM.     

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

Cyclopeptide RA-V inhibits angiogenesis by down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-1 endothelial cells

BACKGROUND AND PURPOSE

Anti-angiogenic agents have recently become one of the major adjuvants for cancer therapy. A cyclopeptide, RA-V, has been shown to have anti-tumour activities. Its in vitro anti-angiogenic activities were evaluated in the present study, and the underlying mechanisms were also assessed.

EXPERIMENTAL APPROACH

Two endothelial cell lines, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1), were used. The effects of RA-V on the proliferation, cell cycle phase distribution, migration, tube formation and adhesion were assessed. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant molecules.

KEY RESULTS

RA-V inhibited HUVEC and HMEC-1 proliferation dose-dependently with IC₅₀ values of 1.42 and 4.0 nM respectively. RA-V inhibited migration and tube formation of endothelial cells as well as adhesion to extracellular matrix proteins. RA-V treatment down-regulated the protein and mRNA expression of matrix metalloproteinase-2. Regarding intracellular signal transduction, RA-V interfered with the activation of ERK1/2 in both cell lines. Furthermore, RA-V significantly decreased the phosphorylation of JNK in HUVEC whereas, in HMEC-1, p38 MAPK was decreased.

CONCLUSIONS AND IMPLICATIONS

RA-V exhibited anti-angiogenic activities in HUVEC and HMEC-1 cell lines with changes in function of these endothelial cells. The underlying mechanisms of action involved the ERK1/2 signalling pathway. However, RA-V may regulate different signalling pathways in different endothelial cells. These findings suggest that RA-V has the potential to be further developed as an anti-angiogenic agent.     

A novel angiopoietin-derived peptide displays anti-angiogenic activity and inhibits tumour-induced and retinal neovascularization

BACKGROUND AND PURPOSE

Pathological angiogenesis is associated with various human diseases, such as cancer, autoimmune diseases and retinopathy. The angiopoietin (Ang)–Tie2 system plays critical roles in several steps of angiogenic remodelling. Here, we have investigated the anti-angiogenic effect of a novel angiopoietin-derived peptide.

EXPERIMENTAL APPROACH

Using computational methods, we identified peptides from helical segments within angiopoietins, which were predicted to inhibit their activity. These peptides were tested using biochemical methods and models of angiogenesis. The peptide with best efficacy, A11, was selected for further characterization as an anti-angiogenic compound.

KEY RESULTS

The potent anti-angiogenic activity of A11 was demonstrated in a multicellular assay of angiogenesis and in the chorioallantoic membrane model. A11 bound to angiopoietins and reduced the binding of Ang-2 to Tie2. A11 was also significantly reduced vascular density in a model of tumour-induced angiogenesis. Its ability to inhibit Ang-2 but not Ang-1-induced endothelial cell migration, and to down-regulate Tie2 levels in tumour microvessels, suggests that A11 targets the Ang–Tie2 pathway. In a rat model of oxygen-induced retinopathy, A11 strongly inhibited retinal angiogenesis. Moreover, combination of A11 with an anti-VEGF antibody showed a trend for further inhibition of angiogenesis, suggesting an additive effect.

CONCLUSIONS AND IMPLICATIONS

Our results indicate that A11 is a potent anti-angiogenic compound, through modulation of the Ang–Tie2 system, underlining its potential as a therapeutic agent for the treatment of ocular and tumour neovascularization, as well as other pathological conditions that are dependent on angiogenesis.     

Azaindole derivatives are inhibitors of microtubule dynamics,with anti-cancer and anti-angiogenic activities

Background and Purpose

Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents.

Experimental Approach

Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro.

Key Results

CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities.

Conclusions and Implications

CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations.     

The GPR 55 agonist,L-α-lysophosphatidylinositol,mediates ovarian carcinoma cell-induced angiogenesis

Background and Purpose

Highly vascularized ovarian carcinoma secretes the putative endocannabinoid and GPR55 agonist, L-α-lysophosphatidylinositol (LPI), into the circulation. We aimed to assess the involvement of this agonist and its receptor in ovarian cancer angiogenesis.

Experimental Approach

Secretion of LPI by three ovarian cancer cell lines (OVCAR-3, OVCAR-5 and COV-362) was tested by mass spectrometry. Involvement of cancer cell-derived LPI on angiogenesis was tested in the in vivo chicken chorioallantoic membrane (CAM) assay along with the assessment of the effect of LPI on proliferation, network formation, and migration of neonatal and adult human endothelial colony-forming cells (ECFCs). Engagement of GPR55 was verified by using its pharmacological inhibitor CID16020046 and diminution of GPR55 expression by four different target-specific siRNAs. To study underlying signal transduction, Western blot analysis was performed.

Key Results

Ovarian carcinoma cell-derived LPI stimulated angiogenesis in the CAM assay. Applied LPI stimulated proliferation, network formation, and migration of neonatal ECFCs in vitro and angiogenesis in the in vivo CAM. The pharmacological GPR55 inhibitor CID16020046 inhibited LPI-stimulated ECFC proliferation, network formation and migration in vitro as well as ovarian carcinoma cell- and LPI-induced angiogenesis in vivo. Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis. These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

Conclusions and Implications

We conclude that inhibiting the pro-angiogenic LPI/GPR55 pathway appears a promising target against angiogenesis in ovarian carcinoma.     

Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase

Background and purpose:

Nitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targets.

Experimental approach:

Cell-based angiogenesis assays (wound healing of monolayers and tube formation in ECV304, EAhy926 and bovine arterial endothelial cells), along with ex vivo and in vivo angiogenesis assays, were used to explore interactions between thalidomide and NO. We also carried out in silico homology modelling and docking studies to elucidate possible molecular interactions of thalidomide and soluble guanylyl cyclase (sGC).

Key results:

Thalidomide inhibited pro-angiogenic functions in endothelial cell cultures, whereas 8-bromo-cGMP, sildenafil (a phosphodiesterase inhibitor) or a NO donor [sodium nitroprusside (SNP)] increased these functions. The inhibitory effects of thalidomide were reversed by adding 8-bromo-cGMP or sildenafil, but not by SNP. Immunoassays showed a concentration-dependent decrease of cGMP in endothelial cells with thalidomide, without affecting the expression level of sGC protein. These results suggested that thalidomide inhibited the activity of sGC. Molecular modelling and docking experiments revealed that thalidomide could interact with the catalytic domain of sGC, which would explain the inhibitory effects of thalidomide on NO-dependent angiogenesis.

Conclusion and implications:

Our results showed that thalidomide interacted with sGC, suppressing cGMP levels in endothelial cells, thus exerting its anti-angiogenic effects. These results could lead to the formulation of thalidomide-based drugs to curb angiogenesis by targeting sGC.     

Endocannabinoid-like N-arachidonoyl serine is a novel pro-angiogenic mediator

BACKGROUND AND PURPOSE

N-arachidonoyl serine (ARA-S) is a recently identified endocannabinoid-like lipid with weak affinity for the fully characterized cannabinoid receptors (CB₁ and CB₂) and the transient receptor potential vanilloid receptor 1 (TRPV-1). ARA-S induces vasodilatation and shows vasoprotective potential via activation of key signalling pathways in endothelial cells. Based on these findings, the effect of ARA-S on endothelial functions was further studied.

EXPERIMENTAL APPROACH

Primary human dermal microvascular endothelial cells (HMVEC) were used to investigate effects of ARA-S (0–10 µM) on certain endothelial functions, using cell proliferation, migration and wound repair models in vitro, and angiogenesis assays in vitro and ex vivo. Selective CB receptor antagonists and specific siRNAs were deployed to block individual CB receptors.

KEY RESULTS

We found that ARA-S stimulated angiogenesis and endothelial wound healing through induction of vascular endothelial growth factor C and its cognate receptor expression in primary HMVEC. Moreover, knock-down of G protein-coupled receptor 55 (GPR55) partly inhibited ARA-S-induced signal transduction and endothelial functions.

CONCLUSIONS AND IMPLICATIONS

Our results indicate that ARA-S is a pro-angiogenic factor in addition to a vessel dilator. The GPR55 receptor may serve as one target of ARA-S.     

In vitro and in vivo evaluation of the anti-angiogenic actions of 4-hydroxybenzyl alcohol

BACKGROUND AND PURPOSE

4-Hydroxybenzyl alcohol (HBA) is a phenolic plant compound, which has been shown to influence many cellular mechanisms. In the present study, we analysed in vitro and in vivo the anti-angiogenic actions of this pleiotropic agent.

EXPERIMENTAL APPROACH

Migration and protein expression of HBA- and vehicle-treated endothelial-like eEND2 cells was assessed by cell migration assay and Western blot analyses. HBA action on vascular sprouting was analysed in an aortic ring assay. In vivo anti-angiogenic actions of HBA were studied in the dorsal skinfold chamber model of endometriosis in mice.

KEY RESULTS

Western blot analyses demonstrated that HBA inhibited proliferation of eEND2 cells, as indicated by down-regulation of proliferating cell nuclear antigen expression, and reduced expression of vascular endothelial growth factor and matrix metalloproteinase 9. HBA suppressed the migration of eEND2 cells, accompanied by inhibition of actin filament reorganization, revealed by fluorescence staining of the cytoskeleton. In addition, HBA reduced vascular sprouting in the aortic ring assay. Finally, we found, in the dorsal skinfold chamber model in vivo using intravital fluorescence microscopy, that HBA inhibited the vascularization of developing endometriotic lesions, as indicated by a decreased functional capillary density of lesions in HBA-treated mice and a reduced lesion size, compared with control animals.

CONCLUSIONS AND IMPLICATIONS

HBA targets several angiogenic mechanisms and therefore represents a promising anti-angiogenic agent for the treatment of angiogenic diseases, such as endometriosis.     

UniPR129 is a competitive small molecule Eph-ephrin antagonist blocking in vitro angiogenesis at low micromolar concentrations

Background and Purpose

The Eph receptor tyrosine kinases and their ephrin ligands are key players in tumorigenesis and many reports have correlated changes in their expression with a poor clinical prognosis in many solid tumours. Agents targeting the Eph-ephrin system might emerge as new tools useful for the inhibition of different components of cancer progression. Even if different classes of small molecules targeting Eph-ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are crucial to achieve robust pharmacological performance.

Experimental Approach

UniPR129 (the L-homo-Trp conjugate of lithocholic acid) was designed by means of computational methods, synthetized and tested for its ability to inhibit the interaction between the EphA2 receptor and the ephrin-A1 ligand in an elisa binding study. The ability of UniPR129 to disrupt EphA2-ephrin-A1 interaction was functionally evaluated in a prostate adenocarcinoma cell line and its anti-angiogenic effect was tested in vitro using cultures of HUVECs.

Key Results

UniPR129 disrupted EphA2-ephrin-A1 interaction with K_i = 370 nM in an elisa binding assay and with low micromolar potency in cellular functional assays, including inhibition of EphA2 activation, inhibition of PC3 cell rounding and disruption of in vitro angiogenesis, without cytotoxic effects.

Conclusions and Implications

The discovery of UniPR129 represents not only a major advance in potency compared with the existing Eph-ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound.     

Angiogenic effect of the aqueous extract of Cynodon dactylon on human umbilical vein endothelial cells and granulation tissue in rat

Background

Cynodon dactylon, a valuable medicinal plant, is widely used in Iranian folk medicine for the treatment of various cardiovascular diseases such as heart failure and atherosclerosis. Moreover, its anti-diabetic, anti-cancer and anti-microbial properties have been also reported. Concerning the critical role of angiogenesis in the incidence and progression of tumors and also its protective role in cardiovascular diseases, we investigated the effects of the aqueous extract prepared from the rhizomes of C. dactylon on vascular endothelial growth factor (VEGF) expressions in Human Umbilical Vein Endothelial Cells (HUVECs) and also on angiogenesis in carrageenan induced air-pouch model in rats.

Methods

In the air-pouch model, carrageenan was injected into an air-pouch on the back of the rats and following an IV injection of carmine red dye on day 6, granulation tissue was processed for the assessment of the dye content. Furthermore, in an in vitro study, angiogenic property of the extract was assessed through its effect on VEGF expression in HUVECs.

Results

Oral administration of 400 mg/kg/day of the extract significantly increased angiogenesis (p < 0.05) and markedly decreased neutrophil (p < 0.05) and total leukocyte infiltration (p < 0.001) into the granulation tissues. Moreover, the extract increased the expression of total VEGF in HUVECs at a concentration of (100 μl/ml).

Conclusion

The present study showed that the aqueous extract of C. dactylon promotes angiogenesis probably through stimulating VEGF expression.