Concentration- and schedule-dependent effects of chemotherapy on the angiogenic potential and drug sensitivity of vascular endothelial cells.

The anti-angiogenic activity of chemotherapy is both dose- and schedule-dependent. While conventional maximum tolerated dose (MTD) chemotherapy exerts only mild and reversible anti-angiogenic effects, low-dose metronomic (LDM) chemotherapy was developed to specifically target tumour angiogenesis. However, the long-term effects of either MTD or LDM chemotherapy on vascular endothelial cells have never been investigated. Here, we demonstrated that repeated exposure to MTD and LDM chemotherapy differentially impact on the angiogenic potential and chemosensitivity of immortalized endothelial cells. Repeated MTD vinblastine treatment of vascular endothelial cells led to an increased proliferation rate and resistance to paclitaxel. In contrast, repeated LDM treatment with vinblastine or etoposide impaired the angiogenic potential of endothelial cells and increased their chemosensitivity. This effect was associated with a significant decrease in ßII- and ßIII-tubulin expression. Functional analysis using siRNA showed that silencing the expression of ßIII-tubulin in endothelial cells significantly decreased their capacity to form vascular structures and increased their sensitivity to the anti-angiogenic and vascular-disrupting effects of chemotherapy, whereas silencing ßII-tubulin expression had no effect. Collectively our results show that LDM chemotherapy impairs the angiogenic potential of endothelial cells while increasing their chemosensitivity-an effect at least in part mediated by the down-regulation of ßIII-tubulin expression. Furthermore, our study suggests that ßIII-tubulin represents an attractive therapeutic target to increase the anti-angiogenic effects of chemotherapy and overall anti-tumour efficacy.

γ-Actin plays a key role in endothelial cell motility and neovessel maintenance.

BACKGROUND: Angiogenesis plays a crucial role in development, wound healing as well as tumour growth and metastasis. Although the general implication of the cytoskeleton in angiogenesis has been partially unravelled, little is known about the specific role of actin isoforms in this process. Herein, we aimed at deciphering the function of γ-actin in angiogenesis. METHODS: Localization of ß- and γ-actin in vascular endothelial cells was investigated by co-immunofluorescence staining using monoclonal antibodies, followed by the functional analysis of γ-actin using siRNA. The impact of γ-actin knockdown on the random motility and morphological differentiation of endothelial cells into vascular networks was investigated by timelapse videomicroscopy while the effect on chemotaxis was assessed using modified Boyden chambers. The implication of VE-cadherin, VEGFR-2 and ROCK signalling was then examined by Western blotting and using pharmacological inhibitors. RESULTS: The two main cytoplasmic isoforms of actin strongly co-localized in vascular endothelial cells, albeit with some degree of spatial preference. While ß-actin knockdown was not achievable without major cytotoxicity, γ-actin knockdown did not alter the viability of endothelial cells. Timelapse videomicroscopy experiments revealed that γ-actin knockdown cells were able to initiate morphological differentiation into capillary-like tubes but were unable to maintain these structures, which rapidly regressed. This vascular regression was associated with altered regulation of VE-cadherin expression. Interestingly, knocking down γ-actin expression had no effect on endothelial cell adhesion to various substrates but significantly decreased their motility and migration. This anti-migratory effect was associated with an accumulation of thick actin stress fibres, large focal adhesions and increased phosphorylation of myosin regulatory light chain, suggesting activation of the ROCK signalling pathway. Incubation with ROCK inhibitors, H-1152 and Y-27632, completely rescued the motility phenotype induced by γ-actin knockdown but only partially restored the angiogenic potential of endothelial cells. CONCLUSIONS: Our study thus demonstrates for the first time that ß-actin is essential for endothelial cell survival and γ-actin plays a crucial role in angiogenesis, through both ROCK-dependent and -independent mechanisms. This provides new insights into the role of the actin cytoskeleton in angiogenesis and may open new therapeutic avenues for the treatment of angiogenesis-related disorders.

ENMD-1198, a new analogue of 2-methoxyestradiol, displays both antiangiogenic and vascular-disrupting properties.

The formation of a new vascular network by angiogenesis is a key driver in tumor growth and metastasis, making this an attractive therapeutic target. Different strategies are being developed to either prevent tumor angiogenesis or disrupt the tumor vasculature already in place. In this in vitro study, we investigated the antivascular properties of ENMD-1198, a new anticancer drug currently in clinical trials. ENMD-1198 is a new analogue of 2-methoxyestradiol, a microtubule-targeting agent that has shown promising results in the treatment of multiple myeloma and hormone-refractory prostate cancer. Using both bone marrow-derived and dermal microvascular endothelial cell lines, we analyzed the effect of ENMD-1198 on the different functions of endothelial cells involved in angiogenesis. In both cell lines, ENMD-1198 was more potent than 2-methoxyestradiol at inhibiting endothelial cell proliferation, motility, migration, and morphogenesis. In addition, ENMD-1198 induced a significant decrease in vascular endothelial growth factor receptor-2 protein expression in endothelial cells. Furthermore, videomicroscopy experiments showed that ENMD-1198 was able to completely disrupt preformed vascular structures within 2 hours. This vascular-disrupting activity was associated with extensive depolymerization of the microtubule network and accumulation of actin stress fibers and large focal adhesions in vascular endothelial cells. Collectively, our results show that this new compound displays potent antivascular properties, and this study provides important insights into the mechanism of action of this promising new anticancer drug.