Abstract
Angiogenesis has key roles in development and in the progression of human diseases such as cancer. Consequently, identifying the novel markers and regulators of angiogenesis is a critical task. The dioxin receptor (AhR) contributes to vascular homeostasis and to the endothelial response to toxins, although the mechanisms involved are largely uncharacterized. Here, we show that AhR-null mice (AhR(-/-)) have impaired angiogenesis in vivo that compromises tumor xenograft growth. Aortic rings emigration experiments and RNA interference indicated that AhR(-/-) endothelial cells failed to branch and to form tube-like structures. Such a phenotype was found to be vascular endothelial growth factor (VEGF)-dependent, as AhR(-/-) aortic endothelial cells (MAECs) secreted lower amounts of active VEGF-A and their treatment with VEGF-A rescued angiogenesis in culture and in vivo. Further, the addition of anti-VEGF antibody to AhR(+/+) MAECs reduced angiogenesis. Treatment under hypoxic conditions with 2-methoxyestradiol suggested that HIF-1alpha modulates endothelial VEGF expression in an AhR-dependent manner. Importantly, AhR-null stromal myofibroblasts produced increased transforming growth factor-beta (TGFbeta) activity, which inhibited angiogenesis in human endothelial cells (HMECs) and AhR(-/-) mice, whereas the co-culture of HMECs with AhR(-/-) myofibroblasts or with their conditioned medium inhibited branching, which was restored by an anti-TGFbeta antibody. Moreover, VEGF and TGFbeta activities cooperated in modulating angiogenesis, as the addition of TGFbeta to AhR(-/-) MAECs further reduced their low basal VEGF-A activity. Thus, AhR modulates angiogenesis through a mechanism requiring VEGF activation in the endothelium and TGFbeta inactivation in the stroma. These data highlight the role of AhR in cardiovascular homeostasis and suggest that this receptor can be a novel regulator of angiogenesis during tumor development.
Highlights
Angiogenesis, on the other hand, depends on the characteristics of the endothelial cells and on the interactions that they establish with other stromal cells, in particular with fibroblasts and pericytes [13]
Despite the fact that fibroblasts have different properties depending on their tissue of origin, the analysis of ␣-smooth muscle actin-expressing myofibroblasts isolated from a panel of breast carcinomas revealed common patterns of gene expression [14], which supports their conserved role in the synthesis and maintenance of extracellular matrix (ECM) components
5-m steps using a Zeiss LSM 510 confocal microscope. of aryl hydrocarbon (dioxin) receptor (AhR)-null transformed fibroblasts to induce tumors in mice, Wound closure experiments were performed using human endothelial cells (HMECs)-1 in we suggested that lack of AhR expression could compromise the absence or presence of AhR small interfering RNA (siRNA) essentially as described tumor development by reducing cell migration and/or angiogenesis [33]
Summary
Reagents and Cell Lines—Matrigel and FITC-labeled antiCD102 were purchased from BD Biosciences and mouse recombinant VEGF-A164 from Calbiochem. RNA was purified and used to analyze VEGF mRNA levels as indicated below This concentration of CoCl2 has been shown to efficiently mimic hypoxia in cell culture [41]. The contribution of hypoxia inducible factor-1␣ (HIF-1␣) in maintaining VEGF levels was determined by adding 5 M 2-Me E2 to CoCl2-treated MAECs or to AhR small interfering RNA (siRNA)-transfected HMEC-1 cells. This concentration of 2-Me E2 was shown to induce degradation of HIF-1␣ in cultured cells [42, 43]. Tumor Xenografts—Aliquots of 5 ϫ 105 B16F10 cells (in 100 l of phosphate-buffered saline) were injected subcutaneously
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