NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.

Radiotherapy suppresses angiogenesis in mice through TGF-betaRI/ALK5-dependent inhibition of endothelial cell sprouting.

BACKGROUND: Radiotherapy is widely used to treat cancer. While rapidly dividing cancer cells are naturally considered the main target of radiotherapy, emerging evidence indicates that radiotherapy also affects endothelial cell functions, and possibly also their angiogenic capacity. In spite of its clinical relevance, such putative anti-angiogenic effect of radiotherapy has not been thoroughly characterized. We have investigated the effect of ionizing radiation on angiogenesis using in vivo, ex vivo and in vitro experimental models in combination with genetic and pharmacological interventions. PRINCIPAL FINDINGS: Here we show that high doses ionizing radiation locally suppressed VEGF- and FGF-2-induced Matrigel plug angiogenesis in mice in vivo and prevented endothelial cell sprouting from mouse aortic rings following in vivo or ex vivo irradiation. Quiescent human endothelial cells exposed to ionizing radiation in vitro resisted apoptosis, demonstrated reduced sprouting, migration and proliferation capacities, showed enhanced adhesion to matrix proteins, and underwent premature senescence. Irradiation induced the expression of P53 and P21 proteins in endothelial cells, but p53 or p21 deficiency and P21 silencing did not prevent radiation-induced inhibition of sprouting or proliferation. Radiation induced Smad-2 phosphorylation in skin in vivo and in endothelial cells in vitro. Inhibition of the TGF-beta type I receptor ALK5 rescued deficient endothelial cell sprouting and migration but not proliferation in vitro and restored defective Matrigel plug angiogenesis in irradiated mice in vivo. ALK5 inhibition, however, did not rescue deficient proliferation. Notch signaling, known to hinder angiogenesis, was activated by radiation but its inhibition, alone or in combination with ALK5 inhibition, did not rescue suppressed proliferation. CONCLUSIONS: These results demonstrate that irradiation of quiescent endothelial cells suppresses subsequent angiogenesis and that ALK5 is a critical mediator of this suppression. These results extend our understanding of radiotherapy-induced endothelial dysfunctions, relevant to both therapeutic and unwanted effects of radiotherapy.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.

Reduced choroidal neovascularization by AAV-anti-VEGF shRNA delivery

VEGF plays an essential role in ocular angiogenic diseases including the late-stage form of AMD, the primary cause of vision loss in the western world. Over-expression of VEGF leads to development of vasculature emanating from the choroid, invading the subretinal space through breaks in Bruch's membrane. Strategies leading to long-term suppression of inappropriate ocular angiogenesis are required. A panel of 10 shRNAs targeting the coding region of human VEGF165 was tested in HEK293 cells and in the human retinal pigment epithelial cell line, ARPE-19. VEGF knock-down up to 92% was achieved by co-transfecting shRNAexpressing constructs with plasmid encoding the Renilla luciferase gene fused to the VEGF165 sequence. For in vivo delivery of the most potent shRNA cassette, both single-stranded and self-complementary rAAV vectors were packaged in serotype 8 capsids. Intramuscular administration in mice led to localized expression and 96% knock-down of endogenous VEGF. Using eGFP as a marker, efficient gene transfer of retinal pigment epithelial cells, the cells thought to be responsible for the abnormal VEGF production, was obtained by subretinal delivery of rAAV2.8 vectors. The capacity of rAAV-encoded shRNAs to silence endogenous VEGF gene expression was evaluated in the laser-induced murine model of choroidal neovascularization (CNV). In this mouse model of AMD, sizes of the CNV were found to be significantly reduced following rAAV-shRNA subretinal delivery. Thus, our results indicate that gene transfer combining AAV-mediated delivery with triggering of the endogenous RNAi pathway can be used for anti-VEGF therapy and holds great promise for the treatment of AMD.

Traitements antiangiogéniques des cancers métastatiques du côlon et du rectum, du sein et du poumon: bénéfices et risques [Anti-angiogenic therapies for metastatic colorectal, breast and lung cancer: benefits and risks]

Anti-angiogenic therapies have recently enriched the therapeutic armentarium against the most common cancers. Among these, bevacizumab, a monoclonal antibody against vascular endothelial growth factor, is currently used most frequently. While the addition of bevacizumab to chemotherapy improves overall survival in first and second line treatment of metastatic colorectal cancer, its effect in metastatic breast cancer is limited to improvements in tumor response and progression-free-survival. In non-small-cell lung cancer, the positive results of a first American phase III study have not been confirmed by a second European study and are subject to controversies. A summary of the data concerning anti-angiogenic therapies in these three cancers is presented including safety information.

Vascular integrins in tumor angiogenesis: mediators and therapeutic targets.

The notion that tumor angiogenesis may have therapeutic implications in the control of tumor growth was introduced by Dr. Judah Folkman in 1971. The approval of Avastin in 2004 as the first antiangiogenic systemic drug to treat cancer patients came as a validation of this visionary concept and opened new perspectives to the treatment of cancer. In addition, this success boosted the field to the quest for new therapeutic targets and antiangiogenic drugs. Preclinical and clinical evidence indicate that vascular integrins may be valid therapeutic targets. In preclinical studies, pharmacological inhibition of integrin function efficiently suppressed angiogenesis and inhibited tumor progression. alphaVbeta3 and alphaVbeta5 were the first vascular integrins targeted to suppress tumor angiogenesis. Subsequent experiments revealed that at least four additional integrins (i.e., alpha1beta1, alpha2beta1, alpha5beta1, and alpha6beta4) might be potential therapeutic targets. In clinical studies low-molecular-weight integrin inhibitors and anti-integrin function-blocking antibodies demonstrated low toxicity and good tolerability and are now being tested in combination with radiotherapy and chemotherapy for anticancer activity in patients. In this article the authors review the role of integrins in angiogenesis, present recent development in the use of alphaVbeta3 and alpha5beta1 integrin antagonists as potential therapeutics in cancer, and discuss future perspectives.

Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA.

BACKGROUND: Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. METHODS: We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. RESULTS: Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. CONCLUSIONS: Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Complex molecular mechanisms cooperate to mediate histone deacetylase inhibitors anti-tumour activity in neuroblastoma cells

BACKGROUND: Histone deacetylase inhibitors (HDACi) are a new class of promising anti-tumour agent inhibiting cell proliferation and survival in tumour cells with very low toxicity toward normal cells. Neuroblastoma (NB) is the second most common solid tumour in children still associated with poor outcome in higher stages and, thus NB strongly requires novel treatment modalities. RESULTS: We show here that the HDACi Sodium Butyrate (NaB), suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA) strongly reduce NB cells viability. The anti-tumour activity of these HDACi involved the induction of cell cycle arrest in the G2/M phase, followed by the activation of the intrinsic apoptotic pathway, via the activation of the caspases cascade. Moreover, HDACi mediated the activation of the pro-apoptotic proteins Bid and BimEL and the inactivation of the anti-apoptotic proteins XIAP, Bcl-xL, RIP and survivin, that further enhanced the apoptotic signal. Interestingly, the activity of these apoptosis regulators was modulated by several different mechanisms, either by caspases dependent proteolytic cleavage or by degradation via the proteasome pathway. In addition, HDACi strongly impaired the hypoxia-induced secretion of VEGF by NB cells. CONCLUSION: HDACi are therefore interesting new anti-tumour agents for targeting highly malignant tumours such as NB, as these agents display a strong toxicity toward aggressive NB cells and they may possibly reduce angiogenesis by decreasing VEGF production by NB cells.

Targeting vascular NADPH oxidase 1 blocks tumor angiogenesis through a PPARα mediated mechanism.

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.

The role of integrins in glioma biology and anti-glioma therapies.

The tumor environment is critical for tumor maintenance and progression. Integrins are a large family of cell surface receptors mediating the interaction of tumor cells with their microenvironment and play important roles in glioma biology, including migration, invasion, angiogenesis and tumor stem cell anchorage. Here, we review preclinical and clinical data on integrin inhibition in malignant gliomas. Various pharmacological approaches to the modulation of integrin signaling have been explored including antibodies and peptide-based agents. Cilengitide, a cyclic RGD-mimetic peptide of αvβ3 and αvβ5 integrins is in advanced clinical development in glioblastoma. Cilengitide had only limited activity as a single agent in glioblastoma, but, when added to standard radiochemotherapy, appeared to prolong progression-free and overall survival in patients with newly diagnosed glioblastomas and methylation of the promoter of the O⁶ methylguanine methyltransferase (MGMT) gene. MGMT gene promoter methylation in turn predicts benefit from alkylating chemotherapy. A phase III randomized clinical trial in conjunction with standard radiochemotherapy in newly diagnosed glioblastoma patients with MGMT gene promoter methylation has recently completed accrual (EORTC 26071-22072). A companion trial explores a dose-escalated regimen of cilengitide added to radiotherapy plus temozolomide in patients without MGMT gene promoter methylation. Promising results in these trials would probably result in a broader interest in integrins as targets for glioma therapy and hopefully the development of a broader panel of anti-integrin agents.

Ophtalmologie. Thérapies anti-vasoprolifératives

The most important recent advance in the treatment of neovascular age-related macular degeneration (AMD) is the development of antivascular endothelial growth factor (anti-VEGF) therapeutic agents that preserve and improve visual acuity by arresting choroidal neovascular growth and reducing vascular permeability. Two anti-VEGF agents, ranibizumab and pegaptanib sodium, are currently approved by Swissmedic for the treatment of neovascular AMD. A third anti-VEGF agent, bevacizumab, is currently used as an off label treatment option for exsudative AMD. Other anti-VEGF agent strategies that have shown efficacy include among others, small interfering RNA agents to silence the VEGF gene and receptor and the fusion protein VEGF trap. Anti-VEGF therapies have been used successfully in the clinic, encouraging their use in the treatment of other neovascular and exudative eye diseases.

Frequent nitric oxide synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression.

An increased expression of nitric oxide synthase (NOS) has been observed in human colon carcinoma cell lines as well as in human gynecological, breast, and central nervous system tumors. This observation suggests a pathobiological role of tumor-associated NO production. Hence, we investigated NOS expression in human colon cancer in respect to tumor staging, NOS-expressing cell type(s), nitrotyrosine formation, inflammation, and vascular endothelial growth factor expression. Ca2+-dependent NOS activity was found in normal colon and in tumors but was significantly decreased in adenomas (P < 0.001) and carcinomas (Dukes' stages A-D: P < 0.002). Ca2+-independent NOS activity, indicating inducible NOS (NOS2), is markedly expressed in approximately 60% of human colon adenomas (P < 0.001 versus normal tissues) and in 20-25% of colon carcinomas (P < 0.01 versus normal tissues). Only low levels were found in the surrounding normal tissue. NOS2 activity decreased with increasing tumor stage (Dukes' A-D) and was lowest in colon metastases to liver and lung. NOS2 was detected in tissue mononuclear cells (TMCs), endothelium, and tumor epithelium. There was a statistically significant correlation between NOS2 enzymatic activity and the level of NOS2 protein detected by immunohistochemistry (P < 0.01). Western blot analysis of tumor extracts with Ca2+-independent NOS activity showed up to three distinct NOS2 protein bands at Mr 125,000-Mr 138,000. The same protein bands were heavily tyrosine-phosphorylated in some tumor tissues. TMCs, but not the tumor epithelium, were immunopositive using a polyclonal anti-nitrotyrosine antibody. However, only a subset of the NOS2-expressing TMCs stained positively for 3-nitrotyrosine, which is a marker for peroxynitrite formation. Furthermore, vascular endothelial growth factor expression was detected in adenomas expressing NOS2. These data are consistent with the hypothesis that excessive NO production by NOS2 may contribute to the pathogenesis of colon cancer progression at the transition of colon adenoma to carcinoma in situ.

A role for angiogenesis in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic debilitating disease characterized by distinct autoimmune, inflammatory and fibrovascular components which lead to synovial proliferation and joint destruction. However, existing treatments specifically target only autoimmune and inflammatory components despite the fact that neovascularization of the inflamed synovium is a hallmark of rheumatoid arthritis. Angiogenesis may contribute to synovial growth, leukocyte recruitment and tissue remodeling, thus potentiating disease progression. Although no therapies currently target angiogenesis, several existing therapies have anti-angiogenic activity. Recent advances in anti-angiogenic strategies in oncology, including the identification of integrin avß3 as a crucial effector of angiogenesis, suggest a means to assess the role of angiogenesis in rheumatoid arthritis. Synovial endothelial cells have been shown to express integrin avß3, suggesting that these cells may be targeted for angiogenesis inhibition. Prior studies in rat arthritis models have shown benefit after the addition of broad spectrum integrin antagonists. However, formal assessment of integrin-targeted anti-angiogenic activity is now underway. These controlled studies will be important in assessing the efficacy of therapies which target angiogenesis in RA.

Étude de l'activité anti-cancéreuse du PCK3145, un peptide dérivé de PSP-94, sur les cancers hématologiques

Le PCK3145 est un peptide de 15 acides aminés inhibant la sécrétion de MMP-9 et démontrant une activité anti-tumorale contre le cancer de la prostate. Comme les cancers hématologiques sécrètent MMP-9, nous avons donc évalué l’effet du PCK3145 sur ces cancers. Nous avons démontré que les lignées humaines de lymphome non- Hodgkinien (LNH) SR et de myélome multiple RPMI-8226 ainsi que la lignée murine de mastocytome P815 ont une prolifération réduite suite à une exposition au PCK3145. Ce peptide diminue également la clonogénicité de ces cellules. In vivo, le PCK3145 diminue significativement la croissance des tumeurs sous-cutanées P815 comparativement au PBS (p<0.001) et aux peptides contrôles (« scrambled peptide » (p<0.05) et PCK5266 (p<0.01)). De plus, le traitement au PCK3145 diminue le nombre de métastases au niveau du foie par rapports aux contrôles (p<0.05). Les niveaux de MMP-9 dans le sang des souris traitées au PCK3145 sont similaires à ceux dans le sang des souris sans tumeur. Par contre, chez les souris recevant le PBS ou le « scrambled peptide », les niveaux de MMP-9 étaient significativement plus élevés que dans les souris sans tumeur et les souris traitées au PCK3145 (p<0.05). De surcroît, dans un modèle de xénogreffe, le PCK3145 diminue significativement la croissance des lymphomes SR par rapport au PBS (p<0.01) et au « scrambled peptide » (p<0.001). Ces résultats indiquent que le PCK3145 possède une activité anti-tumorale et pourrait représenter un agent intéressant pour le traitement de plusieurs cancers hématologiques.

Anti-VEGFA therapy reduces tumor growth and extends survival in a murine model of ovarian granulosa cell tumor

Les tumeurs des cellules de la granulosa (GCTs) sont des tumeurs avec un potentiel malin ayant tendance à récidiver, provoquant ainsi la mort dans 80% des cas de stade avancé consécutif à une rechute. Bien que les GCTs représentent 5% des tumeurs ovariennes, peu d’études ont évalué les protocoles de traitement adjuvant pour la maladie avancée ou récurrente. Notre but était d’évaluer l’efficacité de la voie de signalisation du facteur de croissance de l’endothélium vasculaire A (VEGFA) comme cible pour le traitement de la GCT utilisant le modèle murin transgénique Ptentm1Hwu/tm1Hwu; Ctnnb1tm1Mmt/+; Amhr2tm3(cre)Bhr/+ (PCA) qui reproduit le stade avancé de la maladie humaine. Un anticorps anti-VEGFA a été administré une fois par semaine par voie intrapéritonéale (IP) à partir de 3 semaines d’âge. La thérapie anti-VEGFA a permis une réduction de la taille des tumeurs à 6 semaines d’âge (p<0.05) et une prolongation de la survie des animaux traités, lorsque comparé aux animaux contrôles. L’analyse des GCTs a montré une réduction significative de la prolifération cellulaire (p<0.05) et de la densité microvasculaire (p<0.01) mais aucune différence significative n’a été détectée dans l’apoptose cellulaire. p44/p42 MAPK, un effecteur de la signalisation pour le récepteur 2 de VEGFA (VEGFR2) associé à la prolifération cellulaire, était moins activé dans les tumeurs traitées (p<0.05). Par contre, l’activation d’AKT, un effecteur impliqué dans la survie cellulaire, était similaire d’un groupe à l’autre. Ces résultats suggèrent que l’anticorps anti-VEGFA réduit la prolifération cellulaire et la densité microvasculaire chez les souris PCA par inhibition de la voie de signalisation VEGFR2-MAPK, inhibant ainsi la croissance tumorale. En conclusion, l’efficacité de la thérapie anti- VEGFA mérite d’être évaluée en essais contrôlés randomisés pour le traitement des GCTs chez l’homme.