Importance of co-expression and co-activation of AT1, thromboxane A2 and EGF(HER1) receptors for angiotensin II-mediated signaling in vascular cells: analysis of molecular and functional interactions - part 2

血管紧张素II（Ang II）通过激活AT 1受体（AT 1 R）诱导内皮功能障碍、血管重塑和动脉粥样硬化以及腹主动脉瘤形成。关于血管紧张素II在血管细胞中的病理作用，越来越清楚的是，只有额外受体类型的平行激活才能触发全面的血管紧张素II介导的信号转导。血管平滑肌细胞中表达的EGF受体（EGFR）或血栓素A2受体（TP）促进高脂饮食-引起的血管变化和动脉粥样硬化。此外，我们的初步工作揭示了AT 1 R，TP和EGFR的分子和功能相互作用与信号转导，基因表达和血管细胞稳态的潜在相关性。因此，该项目的第二阶段将进一步研究受体AT 1 R，TP和EGFR的相互作用如何影响血管紧张素II诱导的细胞信号转导，随后的基因表达控制和血管细胞中的细胞生理后果。为了检验这一假设，工作包（WP）将在（i）生物物理受体相互作用，（ii）下游信号传导途径的细胞相互作用，和（iii）受体相互作用的细胞生理后果在体外和体内的水平。继续我们的细胞生物学方法，并使用经验证的FRET显微镜方法，受体-受体相互作用的机制将被调查，以及是否AT 1 R-EGFR-TP三重复合物在活细胞中形成。此外，血管细胞的血管紧张素II诱导的信号体将进行分析，特别强调受体-受体相互作用，使用质谱方法，然后验证蛋白质印迹分析（WP 1）。此外，将继续表征与SRF转录控制的细胞内信号传导途径相关的受体相互作用（WP 2），分析人血管细胞中转录组调控的受体相互作用（WP 3），并对人血管细胞中（病理）生理变化的生物信息学预测进行实验验证（WP 4和WP 5）。最终，将在体内测试EGFR和TP的药理学阻断对ApoE敲除小鼠中Ang II诱导的主动脉粥样硬化形成和动脉瘤形成、心脏肥大和死亡的治疗潜力（AP 6）。我们预计，该项目将有助于澄清AT 1 R，TP和EGFR之间的功能和分子相互作用的重要性，血管紧张素II介导的血管细胞的影响。通过提高机制的理解，它有望支持血管疾病的合理药理学治疗策略的发展。

Angiotensin II (Ang II) induces endothelial dysfunction, vascular remodeling, and atherosclerosis as well as abdominal aortic aneurysm formation through activation of the AT1 receptor (AT1R). With regard to the pathological effects of Ang II in vascular cells, it is becoming increasingly clear that only the parallel activation of additional receptor types triggers full-scale Ang II-mediated signal transduction.In the first phase of the project, we demonstrated, for example, that EGF receptors (EGFR) or thromboxane A2 receptors (TP) expressed in vascular smooth muscle cells promote high-fat diet-induced vascular changes and atherosclerosis. In addition, our preliminary work reveals molecular and functional interactions of AT1R, TP, and EGFR with potential relevance to signal transduction, gene expression, and vascular cell homeostasis. Therefore, the proposed second phase of the project will further investigate how the interactions of the receptors AT1R, TP, and EGFR affect Ang II-induced cellular signal transduction, subsequent control of gene expression, and cell physiological consequences in vascular cells. To test this hypothesis, work packages (WP) will be addressed at the levels of (i) biophysical receptor interaction, (ii) cellular interaction of downstream signaling pathways, and (iii) cell physiological consequences of receptor interaction in vitro and in vivo. Continuing our cell biological approach and using validated FRET microscopy methods, mechanisms of receptor-receptor interaction will be investigated as well as whether AT1R-EGFR-TP triple complexes form in living cells. In addition, the Ang II-induced signalosome of vascular cells will be analyzed with particular emphasis on receptor-receptor interactions using mass spectrometric methods followed by validating Western Blot analyses (WP1). In addition, the characterization of receptor interaction with respect to intracellular signaling pathways of SRF transcriptional control will be continued (WP2), an analysis of receptor interaction for transcriptome regulation in human vascular cells will be pursued (WP3), and an experimental validation of bioinformatic predictions of (patho)physiological changes in human vascular cells will be performed (WP4 and WP5). Ultimately, the therapeutic potential of pharmacological blockade of EGFR and TP for Ang II-induced aortic atherogenesis and aneurysm formation, cardiac hypertrophy and mortality in ApoE knockout mice will be tested in vivo (AP6). We anticipate that the project will help clarify the importance of functional and molecular interactions between AT1R, TP, and EGFR for Ang II-mediated effects on vascular cells. By improving mechanistic understanding, it is expected to support the development of rational pharmacological therapeutic strategies for vascular diseases.