Molecular Mechanisms of Anti-Angiogenic Effects of 3-Methylcholanthrene, an Aryl-Hydrocarbon Receptor Agonist, in Relation to Cell Proliferation, Adhesion and Migration of Endothelial Cells, and Its Therapeutic Intervention Using Resveratrol

Biochemical and genetic evidence has indicated that the biochemical mechanisms of action of xenobiotics such as benzepyrene, dioxin, and policylic aromatic hydrocarbons, are mediated by the aryl hydrocarbon receptor (AhR), an intracellular receptor, to which these chemicals bind with high affinity. Mechanistically, xenobiotics bind to the AhR complex present in the cytoplasmic compartment. Liganded AhR complexes subsequently translocated into nucleus and following their association with the nuclear Arnt (AhR nuclear translocator) protein, the AhR complex is converted into its high affinity DNA binding form. The binding of the transformed heteromeric xenobiotics:AhR complex to its specific DNA recognition sequence, the xenobiotics responsive element (XRE), upstream of the CYP1A1 gene (as well as other responsive genes) increased promoter accessibility and CYP1A1 gene transcription. Angiogenesis, the formation of new blood vessels from the preexisting vessels, occurs extensively during wound healing, menstrual cycle, and embryonic development. Our recent two manuscripts indicated that 3-methylcholanthrene (3MC), an AhR agonist, has anti-angiogenic effects in HUVECs, accompanied with anti-proliferation, anti-adhesion, anti-migration, and decrease in focal kinase activity (FAK). The anti-proliferative effects of 3MC in HUVECs are associated with AhR-related transcriptional upregulation of p21 and 27. Importantly, the aforementioned effects of 3MC were reversed by resveratrol, an AhR antagonist. Furthermore, our preliminary data have demonstrated that molecular mechanisms of anti-angiogenic effects of 3MC include to decrease protein levels or active forms of FAK, Skp2, Akt and eNOS, but to increase p21, p27, PTEN protein levels, and transient RhoA activation. Resveratrol, an important component of red wine, has been shown to act as an AhR antagonist and antioxidant, and to reverse oxLDL-mediated decrease in Akt/eNOS. We set out to investigate the molecular mechanisms and the adverse effects of 3MC on angiogenesis in relation to proliferation, migration, and adhesion and to clarify the molecular actions of resveratrol to rescue the adverse effects of 3MC in HUVECs. Anti-angiogenic effect of 3MC is a multifaceted phenomenon interwoven with anti-proliferation, anti-adhesion, and anti-chemotaxis. Accordingly, the aim of this research in year 1 is to characterize the role of increased cytosolic levels of p27 by 3MC on their anti-migrative effects, except for our previous findings of their anti-proliferative effects on HUVECs. We also aim to clarify whether the decreased pFAK and pAKT/eNOS/NO by 3MC are AhR-dependent and whether they are attributable to the anti-proliferation of 3MC on HUVECs. In year 2, we aim to delineate the linkage among the 3MC-mediated alterations in pFAK/RhoA/Rock and PTEN/pAKT/eNOS/NO, p27/RhoA, p27 subcellular localization and anti-angiogenic effect of 3MC, with particular focus on adhesion, chemotaxis, and permeability of endothelial cells and even vascular vessels. In year 3, after solving effects of each individual signal transduction pathway on the anti-angiogenic effect of 3MC in HUVECs, the effects of in vitro finding will be confirmed in vivo studies including tube formation, Matrigel plug assay, microvessel permeability, in vivo angiogenesis, and CAM assay. These pathways should provide fruitful targets for intervention in cardiovascular diseases. We aim to develop the therapeutic basis to maintain the integrity of vascular endothelial cells in prevention of cardiovascular diseases or stoke by toxicities of aryl hydrocarbon-related environmental hormones. Abbreviations: Akt/PKB, protein kinase B; PTEN, phosphatase and tensin homologue; FAK; focal adhesion kinase; NOS; nitric oxide synthase; PI3K, phosphatidylinositol 3-kinase