Netrin-4 regulates angiogenic responses and tumor cell growth

Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas.Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.     

Antiangiogenic Activity of the Lipophilic Antimicrobial Peptides from an Endophytic Bacterial Strain Isolated from Red Pepper Leaf

The induction of angiogenesis is a crucial step in tumor progression, and therefore, efficient inhibition of angiogenesis is considered a powerful strategy for the treatment of cancer. In the present study, we report that the lipophilic antimicrobial peptides from EML-CAP3, a new endophytic bacterial strain isolated from red pepper leaf (Capsicum annuum L.), exhibit potent antiangiogenic activity both in vitro and in vivo. The newly obtained antimicrobial peptides effectively inhibited the proliferation of human umbilical vein endothelial cells at subtoxic doses. Furthermore, the peptides suppressed the in vitro characteristics of angiogenesis such as endothelial cell invasion and tube formation stimulated by vascular endothelial growth factor, as well as neovascularization of the chorioallantoic membrane of growing chick embryos in vivo without showing cytotoxicity. Notably, the angiostatic peptides blocked tumor cell-induced angiogenesis by suppressing the expression levels of hypoxia-inducible factor-1α and its target gene, vascular endothelial growth factor (VEGF). To our knowledge, our findings demonstrate for the first time that the antimicrobial peptides from EML-CAP3 possess antiangiogenic potential and may thus be used for the treatment of hypervascularized tumors.     

CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development

Angiogenesis, a process that newly-formed blood vessels sprout from pre-existing ones, is vital for vertebrate development and adult homeostasis. Previous studies have demonstrated that the neuronal guidance molecule netrin-1 participates in angiogenesis and morphogenesis of the vascular system. Netrin-1 exhibits dual activities in angiogenesis: either promoting or inhibiting angiogenesis. The anti-angiogenic activity of netrin-1 is mediated by UNC5B receptor. However, how netrin-1 promotes angiogenesis remained unclear. Here we report that CD146, an endothelial transmembrane protein of the immunoglobulin superfamily, is a receptor for netrin-1. Netrin-1 binds to CD146 with high affinity, inducing endothelial cell activation and downstream signaling in a CD146-dependent manner. Conditional knockout of the cd146 gene in the murine endothelium or disruption of netrin-CD146 interaction by a specific anti-CD146 antibody blocks or reduces netrin-1-induced angiogenesis. In zebrafish embryos, downregulating either netrin-1a or CD146 results in vascular defects with striking similarity. Moreover, knocking down CD146 blocks ectopic vascular sprouting induced by netrin-1 overexpression. Together, our data uncover CD146 as a previously unknown receptor for netrin-1 and also reveal a functional ligand for CD146 in angiogenesis, demonstrating the involvement of netrin-CD146 signaling in angiogenesis during vertebrate development.     

NSK-01105, a Novel Sorafenib Derivative,Inhibits Human Prostate Tumor Growth via Suppression of VEGFR2/EGFR-Mediated Angiogenesis

The purpose of this study is to investigate the anti-angiogenic activities of NSK-01105, a novel sorafenib derivative, in in vitro, ex vivo and in vivo models, and explore the potential mechanisms. NSK-01105 significantly inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation of human umbilical vein endothelial cells at non-cytotoxic concentrations as shown by wound-healing, transwell migration and endothelial cell tube formation assays, respectively. Cell viability and invasion of LNCaP and PC-3 cells were significantly inhibited by cytotoxicity assay and matrigel invasion assay. Furthermore, NSK-01105 also inhibited ex vivo angiogenesis in matrigel plug assay. Western blot analysis showed that NSK-01105 down-regulated VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2) and the activation of epidermal growth factor receptor (EGFR). Tumor volumes were significantly reduced by NSK-01105 at 60 mg/kg/day in both xenograft models. Immunohistochemical staining demonstrated a close association between inhibition of tumor growth and neovascularization. Collectively, our results suggest a role of NSK-01105 in treatment for human prostate tumors, and one of the potential mechanisms may be attributed to anti-angiogenic activities.     

Ferulic acid augments angiogenesis via VEGF,PDGF and HIF-1α

Therapeutic angiogenesis is critical to wound healing and ischemic diseases such as myocardial infarction and stroke. For development of therapeutic agents, a search for new angiogenic agents is the key. Ferulic acid, a phytochemical found in many fruits and vegetables, exhibits a broad range of therapeutic effects on human diseases, including diabetes and cancer. This study investigated the augmenting effect of ferulic acid on angiogenesis through functional modulation of endothelial cells. Through endothelial cell migration and tube formation assays, ferulic acid (10^?6–10^?4 M) was found to induce significant angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro without cytotoxicity. With chorioallantoic membrane assay, ferulic acid (10^?6–10^?5 M) was also found to promote neovascularization in vivo. Using Western blot analysis and quantitative real-time polymerase chain reaction, we found that ferulic acid increased vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression in HUVECs. Furthermore, the amounts of hypoxic-induced factor (HIF) 1α mRNA and protein, the major regulator of VEGF and PDGF, also showed up-regulation by ferulic acid. Electrophoretic migration shift assay showed that the binding activity of HIF-1α was also enhanced with ferulic acid treatment of HUVECs. Moreover, inhibitors of extracellular-signal-regulated kinase 1/2 and phosphoinositide-3 kinase (PI3K) abolished the binding activity of HIF-1α and the subsequent activation of VEGF and PDGF production by ferulic acid. Thus, both mitogen-activated protein kinase and PI3K pathways were involved in the angiogenic effects of ferulic acid. Taken together, ferulic acid serves as an angiogenic agent to augment angiogenesis both in vitro and in vivo. This effect might be observed through the modulation of VEGF, PDGF and HIF-1α.     

Netrin-4 acts as a pro-angiogenic factor during zebrafish development

Netrins form a heterogeneous family of laminin-related molecules with multifunctional activities. Netrin-4, the most distant member of this family, is related to the laminin β chain and has recently been proposed to play an important role in embryonic and pathological angiogenesis. However, the data reported so far lead to the apparently contradictory conclusions supporting Netrin-4 as either a pro- or an anti-angiogenic factor. To elucidate this controversy, Netrin-4 was analyzed for a vascular activity in both cell-based models (human umbilical vein endothelial cells and human umbilical artery endothelial cells) and two zebrafish models: the wild-type AB/Tü strain and the transgenic Tg(fli1a:EGFP)(y1) strain. We show that Netrin-4 is expressed in endothelial cells and in the zebrafish vascular system. We also show evidence that Netrin-4 activates various kinases and induces various biological effects directly linked to angiogenesis in vitro. Using a morpholinos strategy, we demonstrate that Netrin-4 expression is crucial for zebrafish vessel formation and that a blood vessel formation defect induced by netrin-4 morpholinos can be partially rescued through drug delivery leading to protein kinase activation. Together these data underscore the crucial role of Netrin-4 in blood vessel formation and the involvement of protein kinases activation in Netrin-4-induced biological effects related to vascular development.     

CD146: the unveiling of a pro-angiogenic netrin receptor

Netrin-1, a classic neuronal guidance cue, can promote angiogenesis under certain developmental and pathological conditions, but key receptors on vascular endothelium have remained elusive. A recent study published in Cell Research by Tu et al. reveals that CD146, an endothelial receptor of the immunoglobulin superfamily, binds netrin-1 with high affinity and may play an important role in regulating angiogenesis.The anatomical similarity of the nervous and vascular systems has long been recognized, and it is now known that many of the neuronal guidance cues, such as netrins, slits, semaphorins and ephrins, also regulate angiogenesis — the sprouting of new blood vessels. During neurogenesis axonal growth cones play a key navigational role, sensing local cues in the environment and relaying signals that culminate in axon extension or retraction. Similarly, endothelial tip cells direct new blood vessel sprouts during angiogenesis. Netrins, derived from the Sanskrit word ''netr'' meaning ''one who guides'', were first shown to promote the outgrowth of commissural axons in 1994 and are comprised of a family that includes three secreted members (netrins 1, 3, and 4) and two GPI-linked members (netrins G1 and G2). Netrin-1, the prototypical netrin, attracts or repulses growth cones depending on receptor status. Beginning in 2004, several published studies have revealed that netrin-1 also has pro-angiogenic or anti-angiogenic activity, the balance depending on ligand concentration and presumably the receptors expressed on the endothelium lining the vessels, although the identity of the pro-angiogenic netrin receptor has remained elusive^1,2,3,4.To stimulate neuronal outgrowth, netrin-1 binds Deleted in Colorectal Cancer (DCC), a type I cell surface receptor of the immunoglobulin superfamily critical for neurogenesis (Figure 1). A related DCC family member, neogenin, also promotes axon guidance, whereas members of the uncoordinated 5 family (UNC5A-D) predominantly inhibit neurogenesis. Recent structural studies demonstrate that netrin-1 is able to crosslink its receptors, with DCC/DCC homodimers promoting chemoattraction and DCC/UNC5 heterodimers promoting chemorepulsion⁵. In the vascular system UNC5B is highly expressed in endothelial cells during embryonic development and pathological angiogenesis and, like the role of UNC5s in repression of neurogenesis, may mediate netrin-1 antiangiogenic activity³. However, it has been unclear how the pro-angiogenic activity of netrin-1 is mediated because DCC and neogenin are not detected in most endothelium. The previous lack of a clear angiogenesis-promoting netrin receptor on proliferating vascular endothelium led Mehlen''s group to propose that UNC5B could act as a dual purpose “dependence receptor” either stimulating or blocking endothelial apoptosis depending on ligand availability⁶.Open in a separate windowFigure 1Receptors of the immunoglobulin superfamily that bind Netrin-1. Schematic representation of netrin-1 and its receptors. Netrin-1 contains an N-terminal laminin type VI domain, a V region containing three laminin-like EGF domains, followed by a C-terminal netrin-like domain (NTR). The VI and V domains play an important role in receptor binding. The receptor binding sites for netrin-1 have been mapped to the regions shown in yellow. DSCAM, Down''s syndrome cell adhesion molecule; DCC, Deleted in Colorectal Cancer; DD, death domain; FNIII, fibronectin type III domain; Ig, immunoglobulin domain; TSP, thrombospondin type-1 domain; P1-3, conserved regions of the DCC cytoplasmic domain; UPA, UNC5, PIDD and ankyrin domain; ZU5, zona occludens 5 domain.In an elegant study recently published in Cell Research, Tao Tu and coworkers from Xiyun Yan''s laboratory provide compelling evidence that CD146, an endothelial cell surface receptor of the immunoglobulin superfamily (Figure 1) can stimulate angiogenesis in response to netrin-1⁷. CD146 was first described in 1987 based on its elevated expression in malignant versus normal melanocytes, and is also called Melanoma Cell Adhesion Molecule (MCAM or Mel-CAM). Soon after its discovery, CD146 was also found to be widely expressed on vascular endothelium and smooth muscle. CD146 is elevated in some tumor types, including melanoma, prostate and breast cancers, and recent studies demonstrate that it can promote epithelial to mesenchymal transition. In cultured endothelial cells CD146 has been shown to promote migration, proliferation and tube formation⁷. Although CD146-null mice are viable, impaired tumor angiogenesis and tumor growth in these mice suggests that this receptor may play a critical role in pathological angiogenesis⁸.Tu et al.⁷ show that netrin-1 directly binds CD146 with high affinity (Kd = 1.3 nM), somewhat higher than its affinity for the repulsive netrin receptor UNC5B (Kd = 5.1 nM). Through deletion analysis they show that the V domain of netrin-1, which contains three laminin-like EGF domains, plays an important role in binding. By analogy with data from the crystal structure of netrin-1 in complex with DCC⁵, regions in both the V domain and the N-terminal VI domain of netrin-1 may bind CD146, potentially explaining the residual binding the authors observe with their V domain mutant. Netrin-1 treatment of endothelial cells resulted in a biphasic response with low doses (50-200 ng/mL) inducing proliferation, migration and tube formation and high doses (1 000-2 000 ng/mL) inhibiting these effects. Knockdown of CD146 inhibited the pro-angiogenic phenotypes observed at low doses, whereas knockdown of UNC5B prevented the anti-angiogenic effects at high doses. Importantly, the authors demonstrate the importance of CD146 for angiogenesis in vivo using a CD146 neutralizing antibody and conditional knockout mice that lack CD146 in endothelium. Furthermore, the authors demonstrate that knockdown of either CD146 or netrin-1 leads to a similar defect in parachodal vessel formation during zebrafish development.Tu et al. find that addition of netrin-1 to endothelial cells is able to induce the formation of an unusually stable CD146 dimer that remains intact following exposure to the anionic detergent SDS. The authors also find that VEGFR2, the main VEGF receptor involved in VEGF-mediated angiogenesis, is rapidly phosphorylated upon addition of netrin-1 to serum-starved endothelial cells, which in turn activates signaling through ERK and p38 kinase, while CD146 knockdown prevents these effects. Netrin-1 was unable to bind VEGFR2 directly, and further studies are needed to determine exactly how these pathways intersect and the biological significance of these interactions in vivo. Like any landmark study, the work raises many new questions. For example, is the pro-angiogenic role of netrin-1 through CD146 completely dependent on the increased VEGFR2 signaling, or can netrin-1 also stimulate angiogenesis independent of VEGFR2? Is VEGF required for the rapid VEGFR2 phosphorylation in response to netrin-1 — for example, can netrin-1 mobilize matrix-bound VEGF or promote intracrine signaling, or does the purified recombinant netrin-1 contain trace amounts of VEGF? Can CD146 form heterodimers with other netrin receptors, like UNC5B? Can netrin signal through CD146 in non-endothelial cells, such as pericytes and smooth muscle cells that are also CD146 positive?Another important area concerns the role of netrin/CD146 signaling in pathological angiogenesis. While CD146 has been found to be widely expressed in adult normal vascular endothelium through gene expression profiling and using various anti-CD146 antibodies, for example the s-Endo-1 or the P1H12 antibodies, the AA98 mouse anti-human CD146 antibody developed in Yan''s laboratory appears to preferentially label CD146 on tumor-associated endothelium compared to normal endothelium^9,10. AA98, which binds the same region of CD146 as netrin-1, prevents dimerization of the receptor^7,11. If particular regions of CD146 are uniquely exposed during pathological angiogenesis, for example surfaces on the newly produced monomeric receptor that are normally masked upon ligand binding and dimerization, then this could potentially be exploited for specific targeting of tumor angiogenesis.Although the knockdown studies in zebrafish and cultured endothelial cells provide important evidence for the role of this receptor in angiogenesis, so far no clear developmental angiogenesis defect in CD146 and netrin-1 knockout mice has been reported. One possible explanation is ligand or receptor redundancy. In that regard, netrin-3 and netrin-4, which have not yet been tested for CD146 binding, have also been found to have angiogenic activity in vivo. Integrins, which are widely expressed on endothelium, have also been implicated as netrin-binding receptors¹². Another possibility is that the netrin/CD146 interactions are more important for angiogenesis associated with wound repair, ischemia and other pathological conditions than for development. If so, then targeting the netrin/CD146 axis could have important therapeutic implications for various angiogenesis-dependent diseases.     

Netrin-1 Promotes Glioblastoma Cell Invasiveness and Angiogenesis by Multiple Pathways Including Activation of RhoA,Cathepsin B,and cAMP-response Element-binding Protein

Glioblastomas are very difficult tumors to treat because they are highly invasive and disseminate within the normal brain, resulting in newly growing tumors. We have identified netrin-1 as a molecule that promotes glioblastoma invasiveness. As evidence, netrin-1 stimulates glioblastoma cell invasion directly through Matrigel-coated transwells, promotes tumor cell sprouting and enhances metastasis to lymph nodes in vivo. Furthermore, netrin-1 regulates angiogenesis as shown in specific angiogenesis assays such as enhanced capillary endothelial cells (EC) sprouting and by increased EC infiltration into Matrigel plugs in vivo, as does VEGF-A. This netrin-1 signaling pathway in glioblastoma cells includes activation of RhoA and cyclic AMP response element-binding protein (CREB). A novel finding is that netrin-1-induced glioblastoma invasiveness and angiogenesis are mediated by activated cathepsin B (CatB), a cysteine protease that translocates to the cell surface as an active enzyme and co-localizes with cell surface annexin A2 (ANXA2). The specific CatB inhibitor CA-074Me inhibits netrin-1-induced cell invasion, sprouting, and Matrigel plug angiogenesis. Silencing of CREB suppresses netrin-1-induced glioblastoma cell invasion, sprouting, and CatB expression. It is concluded that netrin-1 plays an important dual role in glioblastoma progression by promoting both glioblastoma cell invasiveness and angiogenesis in a RhoA-, CREB-, and CatB-dependent manner. Targeting netrin-1 pathways may be a promising strategy for brain cancer therapy.     

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy.     

Anti-Angiogenic Effects of a Mutant Endostatin: A New Prospect for Treating Retinal and Choroidal Neovascularization

Purpose

Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES).

Methods

M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection.

Results

The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models.

Conclusion

The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.     

Antiangiogenic activity of 2-deoxy-D-glucose

Background

During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated.

Methodology/Principal Findings

In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG''s ability to interfere with endothelial N-linked glycosylation. 2-DG''s effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG''s effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH_BETAT_AG transgenic retinoblastoma model.

Conclusions/Significance

In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.     

Angiogenic activity of sesamin through the activation of multiple signal pathways

The natural product sesamin has been known to act as a potent antioxidant and prevent endothelial dysfunction. We here found that sesamin increased in vitro angiogenic processes, such as endothelial cell proliferation, migration, and tube formation, as well as neovascularization in an animal model. This compound elicited the activation of multiple angiogenic signal modulators, such as ERK, Akt, endothelial nitric oxide synthase (eNOS), NO production, FAK, and p38 MAPK, but not Src. The MEK inhibitor PD98059 and the PI3K inhibitor Wortmannin specifically inhibited sesamin-induced activation of the ERK and Akt/eNOS pathways. These inhibitors reduced angiogenic events, with high specificity for MEK/ERK-dependent cell proliferation and migration and PI3K/Akt-mediated tube formation. Moreover, inhibition of p38 MAPK effectively inhibited sesamin-induced cell migration. The angiogenic activity of sesamin was not associated with VEGF expression. Furthermore, this compound did not induce vascular permeability and upregulated ICAM-1 and VCAM-1 expression, which are hallmarks of vascular inflammation. These results suggest that sesamin stimulates angiogenesis in vitro and in vivo through the activation of MEK/ERK-, PI3K/Akt/eNOS-, p125^FAK-, and p38 MAPK-dependent pathways, without increasing vascular inflammation, and may be used for treating ischemic diseases and tissue regeneration.     

Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model

The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenesis strategy to treat corneal neovascularization in a mouse model was evaluated. Subconjunctival injection of recombinant endostatin-AAV was used to examine the inhibition of corneal neovascularization induced by silver nitrate cauterization in mice. The results showed that gene expression in corneal tissue was observed as early as 4 days after gene transfer and stably lasted for over 8 months with minimal immune reaction. Subconjunctival injection of a high-titer rAAV-endostatin successfully inhibited neovascularization. Immunohistchemistry staining of CD 31 and endostatin showed that the treatment significantly inhibits angiogenesis in cornea. We concluded that the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained, high levels of gene expression in vivo to inhibit angiogenesis.     

Luteolin Inhibits Human Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.     

Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis

The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. Fas ligand is critical to the homeostatic regulation of the immune response, but its role in the angiogenic process of RA remains to be defined. In this study, we investigated whether soluble Fas ligand (sFasL) induces synoviocyte apoptosis and regulates angiogenesis of endothelial cells in RA. The levels of sFasL were elevated in the synovial fluids of RA patients when compared to those of osteoarthritis (OA) patients, and they correlated inversely with vascular endothelial growth factor₁₆₅ (VEGF₁₆₅) concentrations. sFasL, ranging from 10 to 100 ng/ml, induced the apoptosis of RA fibroblast-like synoviocytes (FLS) in vitro, and thereby decreased VEGF₁₆₅ production. In addition, sFasL inhibited VEGF₁₆₅-induced migration and chemotaxis of endothelial cells to basal levels in a manner independent of the Fas-mediated cell death. sFasL dose-dependently suppressed the VEGF₁₆₅-stimulated increase in pAkt expression in endothelial cells, which might be associated with its anti-migratory effect on endothelial cells. Moreover, sFasL strongly inhibited neovascularization in the Matrigel plug in vivo. Our data suggest that sFasL shows anti-angiogenic activity within RA joints not only by inducing apoptosis of VEGF₁₆₅-producing cells but also by blocking VEGF₁₆₅-induced migration of endothelial cells, independent of Fas-mediated apoptosis.     

Targeting CD9 produces stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis: a novel antiangiogenic therapy

The precise roles of tetraspanin CD9 are unclear. Here we show that CD9 plays a stimulus-independent role in angiogenesis and that inhibiting CD9 expression or function is a potential antiangiogenic therapy. Knocking down CD9 expression significantly inhibited in vitro endothelial cell migration and invasion induced by vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF). Injecting CD9-specific small interfering RNA (siRNA-CD9) markedly inhibited HGF- or VEGF-induced subconjunctival angiogenesis in vivo. Both results revealed potent and stimulus-independent antiangiogenic effects of targeting CD9. Furthermore, intravitreous injections of siRNA-CD9 or anti-CD9 antibodies were therapeutically effective for laser-induced retinal and choroidal neovascularization in mice, a representative ocular angiogenic disease model. In terms of the mechanism, growth factor receptor and downstream signaling activation were not affected, whereas abnormal localization of integrins and membrane type-1 matrix metalloproteinase was observed during angiogenesis, by knocking down CD9 expression. Notably, knocking down CD9 expression did not induce death and mildly inhibited proliferation of quiescent endothelial cells under conditions without an angiogenic stimulus. Thus, CD9 does not directly affect growth factor-induced signal transduction, which is required in angiogenesis and normal vasculature, but is part of the angiogenesis machinery in endothelial cells during angiogenesis. In conclusion, targeting CD9 produced stimulus-independent antiangiogenic effects predominantly in activated endothelial cells during angiogenesis, and appears to be an effective and safe antiangiogenic approach. These results shed light on the biological roles of CD9 and may lead to novel antiangiogenic therapies.     

Different effects of catechin on angiogenesis and inflammation depending on VEGF levels

Although physiological and pathological angiogenesis develop through similar processes, during pathological angiogenesis, proangiogenic factors are exacerbated. Polyphenols have been considered therapeutic tools for conditions exhibiting enhanced angiogenesis. However, the possibility that these compounds may also prevent vascularization in physiological situations is a major drawback for their use. The purpose of the current study was to investigate the effects of 0.1–100 μM catechin on endothelial cells (EC) and vascular smooth muscle cells (VSMC) regarding angiogenic and inflammatory processes. Catechin modulation of angiogenesis and inflammation was also evaluated in vivo using different models of angiogenesis: one physiological (skin wound-healing assay) and another one resembling pathological angiogenesis, exhibiting higher vascular endothelial growth factor (VEGF)-A stimulation (Matrigel plug assay). The in vitro results showed that 100 μM catechin increased viability (to 165.58% and to 165.34%) and decreased apoptosis (53.45% and 92.65%) and proliferation (33.19% and 23.36%) of EC and VSMC, respectively. Catechin affected migration and invasion, tending to increase both in EC and decreasing them in VSMC; however, it did not change sprouting angiogenesis. Nevertheless, catechin diminished in vitro inflammatory modulators such as tumor necrosis factor α (58.66% for human umbilical vein endothelial cells and 85.46% for human aortic smooth muscle cells) and nuclear factor kappa-B (38.43% for VSMC). The in vivo results demonstrated that catechin did not change angiogenesis and inflammation in skin wound-healing model and substantially decreased these processes in Matrigel plug assay. Altogether, the current study showed that catechin has different effects in angiogenesis and inflammation depending on VEGF-A levels. The absence of adverse effects in mature vasculature favors catechin potential use against pathological situations where angiogenesis is stimulated.     

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2^−/− than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2^−/− mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2^−/− mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2^−/− EPC intramyocardially into mice with induced MI. Per2^−/− reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2^−/− EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.