Detrimental Effects of an Aryl-Hydrocarbon Receptor Agonist, 3mc, on Brain Vascular Integrity and Permeability, and the Therapeutic Intervention by Statin Derivatives.

The interplays of vascular endothelial cells (VEC) and extracellular matrix proteins (ECM) at adheren junctions (AJs) contribute to the maintenance of the integrity of vascular endothelial barrier. Our previous publication showed that an aryl-hydrocarbon receptor (AhR) agonist, 3MC, increased RhoA expression/activity via an AhR-dependent transcriptional upregulation and suppression of a negative feedback pathway of FAK/p190RhoGAP. Increased RhoA activity causes stress fiber and focal adhesion complex formation in HUVEC. Herein, we extend our previous work to investigate the effects of RhoA activation by 3MC in the pathways of 1) PKCδ/GSK3/-catenin/TCF/LEF, 2) PI3K/AKT/subcellular localization of p21/p27 or eNOS activity, and 3) RB/HDAC1/E2F1 and to evaluate these outcome impacts on endothelial integrity and plasticity. Our pilot study showed that 3MC-mediated RhoA activation increased PKCδactivity leading to suppression of ECM/receptors via increased PTEN activity, but RhoA activation conversely decreased PI3K/AKT pathway leading to retain p27 in nuclei and decrease eNOS activity. Therefore, we hypothesized that 3MC affects vascular endothelial function and plasticity, which will be examined by both in vitro co-culture system of CMVEC-astrocytes and in vivo for the assessment of blood-brain barrier (BBB) integrity. Additionally, 3MC also increased retinoblastoma protein (RB) and HDAC1 expression/activity/nuclear translocation, accompanied by the observed decreases in histone H3/H4 acetylation. We aim to examine the molecular mechanisms of RB upregulation/activation and consequent reduction of downstream molecules such as CDK2 and CDK4 or other anti-angiogenic molecules. According, in Year 1, we aim to examine the causal relationship of the observed increased -catenin proteasomal degradation and decreases in ECM receptors via a decrease in T-cell factor/lymphocyte enhancer factor (TCF/LEF)-dependent transcriptional activity, possibly resulting from RhoA/PTEN-mediated PKCδactivation in 3MC-treated CMVEC. In Year 2, the effects of the decreased ECM receptors in endothelial plasticity and permeability will be evaluated both in vitro and in vivo models of BBB. In Year 3, we intend to examine the molecular mechanisms of the decreased PI3K/AKT pathway in altered p21/p27 subcellular localization and eNOS dimerization/activity in relation to 3MC-mediated RhoA activation. Additionally, their roles in the anti-proliferative and anti-migratory effects of 3MC in CMVEC will be evaluated by both gain- or loss-of-function of RhoA activity (i.e., constitutive active (CA)RhoA, dominant negative (DN)RhoA or an inhibitor). In Year 4, we will focus on 3MC-mediated increases in RB activity and HDAC1 recruitment to E2F1 complex for epigenetic histone modifications. This might lead to the modulation of gene expression involved in anti-proliferative and anti-migratory effects of 3MC in CMVEC. Based on our pilot study, these above-mentioned phenomena are mediated either by AhR-dependent mechanism or RhoA activation. Therefore, therapeutic approaches using RhoA inactivators, simvastatin and pravastatin, will be evaluated in 3MC-mediated endothelial dysfunction. RhoA inactivation by statin derivatives is to inhibit the conversion of mevalonate and geranylgeranyl pyrophosphate (GGPP) from HMG-CoA, by which they might exert their protective effects in maintaining the integrity and normal function of VEC. Taken together, the underlying molecular actions of AhR agonist will not only shed light on understanding the physiological role of AhR, but also provide a novel molecular basis to develop therapeutic targets in eliminating AhR-mediated injury by xenobiotics in CMVEC.