Up-Regulation of Connective Tissue Growth Factor in Endothelial Cells by the Microtubule-Destabilizing Agent Combretastatin A-4

Abstract

Incubation of microvascular endothelial cells with combretastatin A-4 phosphate (CA-4P), a microtubule-destabilizing compound that preferentially targets tumor vessels, altered cell morphology and induced scattering of Golgi stacks. Concomitantly, CA-4P up-regulated connective tissue growth factor (CTGF/CCN2), a pleiotropic factor with antiangiogenic properties. In contrast to the effects of other microtubule-targeting agents such as colchicine or nocodazole, up-regulation of CTGF was only detectable in sparse cells, which were not embedded in a cell monolayer. Furthermore, CA-4P induced CTGF expression in endothelial cells, forming tube-like structures on basement membrane gels. Up-regulation of CTGF by CA-4P was dependent on Rho kinase signaling and was increased when p42/44 mitogen-activated protein kinase was inhibited. Additionally, FoxO transcription factors were identified as potent regulators of CTGF expression in endothelial cells. Activation of FoxO transcription factors by inhibition of phosphatidylinositol 3-kinase/AKT signaling resulted in a synergistic increase in CA-4P-mediated CTGF induction. CA-4P-mediated expression of CTGF was thus potentiated by the inhibition of kinase pathways, which are targets of novel antineoplastic drugs. Up-regulation of CTGF by low concentrations of CA-4P may thus occur in newly formed tumor vessels and contribute to the microvessel destabilization and antiangiogenic effects of CA-4P observed in vivo. (Mol Cancer Res 2009;7(2):180–8)

FoxO Proteins Mediate Hypoxic Induction of Connective Tissue Growth Factor in Endothelial Cells

“…In accordance with our results, microarray data showed that CTGF was down-regulated upon overexpression of constitutively active AKT, whereas active FoxO1 up-regulated CTGF in HUVEC (10). Furthermore, we observed a role for FoxO transcription factors in CTGF expression induced by TGF-␤, alterations of the cytoskeleton, or inhibition of histone deacetylases (7,8). These data suggested a central role for FoxO transcription factors in endothelial CTGF regulation.…”

“…In endothelial cells, inhibition of PI-3K/AKT signaling led to upregulation of CTGF expression (8). Moreover, knockdown of FoxO1 and FoxO3a by siRNA almost completely inhibited the induction of CTGF, providing evidence for a role of FoxOs in CTGF regulation (7). In accordance with our results, microarray data showed that CTGF was down-regulated upon overexpression of constitutively active AKT, whereas active FoxO1 up-regulated CTGF in HUVEC (10).…”

“…In recent studies, we provided evidence that CTGF is regulated by FoxO proteins in endothelial cells (8,7,27). Therefore, to investigate whether FoxOs were also relevant for the hypoxic induction of CTGF, FoxO1 and FoxO3a were silenced by small interfering RNA as described (7).…”

“…In recent studies we have shown that the expression of CTGF is modulated by proteins of the forkhead family of transcription factors FoxO (forkhead family of transcription factors group O) in endothelial cells but not in epithelial cells (7,8). FoxO1, FoxO3a, and FoxO4 have been implicated in the regulation of endothelial cell biology.…”

Combretastatins: More Than Just Vascular Targeting Agents?

“…The molecular mechanism of the antiangiogenic properties of TR-644 CA-4 analog was linked to the inhibition of vascular endothelial growth factor-induced phosphorylation of VE-cadherin (Porcù et al, 2013). It has also been suggested that CA-4 may exert its antiangiogenic properties by downregulation of connective tissue growth factor, a negative regulator of angiogenesis (Samarin et al, 2009). Preclinical studies combining the combretastatins with other antiangiogenic agents gave conflicting results.…”