Pharmacologic PTEN upregulation: A novel therapeutic approach to prevent pathological vascular remodeling and fibrosis

项目摘要/摘要 心血管疾病是美国的主要死亡原因，其特点是 导致疾病进展的病理性血管重塑。对受伤或病理性的反应 在刺激下，驻留的血管平滑肌细胞(SMCs)通过破坏血管内皮细胞的功能参与这些重塑过程。 从静止的、收缩的表型向增殖的、促炎的、促纤维化的表型分化 能够招募免疫细胞的表型，如巨噬细胞和T细胞。初步数据支持 这一建议表明，强大的肿瘤抑制因子PTEN维持着SMC的遗传性、系统性上调 分化，减少血管周围免疫细胞募集并钝化多种临床前动物的重塑 心血管疾病的模型。这表明药理学上的PTEN上调可能具有显著的意义 通过抑制血管重塑治疗心血管疾病的治疗潜力。目前， PTEN的治疗潜力没有得到充分发挥，因为几乎没有已知的能增加PTEN的化合物 表情。为了填补这一知识空白，最近完成的高通量复合筛选(HTS)超过了 使用基于荧光的PTEN启动子-报告系统的2500个化合物被用于鉴定11个HIT 具有剂量反应能力的化合物可诱导PTEN启动子活性。最近，JQ1，一种血管紧张素转换酶的抑制剂 已知具有抗重构作用的表观遗传阅读器蛋白Brd4被证明上调PTEN。 然而，这项提案中提出的初步数据表明，JQ1的抗重构作用是钝化的。 在体内外PTEN耗竭的SMC中，提示JQ1的保护作用是通过介导的 通过PTEN上调。对JQ1的靶标Brd4在调节PTEN水平中的作用知之甚少 在SMCS中。然而，Brd4已被证明直接与组蛋白乙酰转移酶p300和 促炎症转录因子NFkB的p65亚基，这两个亚基都已知会影响PTEN 表情。本申请提出了一种新的PTEN调节模型，其中对有限数量的竞争 P300通过启动子乙酰化驱动PTEN的表达，或通过 Brd4介导的P300-NFkB相互作用。具体目标1将在初步数据的基础上进行扩展，并将在 体外和体内PTEN耗竭以确定JQ1的血管保护作用是否由于增加 PTEN启动子p300活性增强和Brd4-p300-NFkB抑制介导的PTEN表达 复杂的队形。特定目标2将扩展完成的HTS的结果，以在体外测试HIT化合物 以及体内诱导PTEN表达和减少血管重塑的能力，特别关注 PTEN上调如何影响SMC和招募的巨噬细胞/T细胞之间的串扰。总而言之， 这些研究将探索一种潜在的新的调节PTEN表达的机制，它可能发挥一种 JQ1在调节已知的抗重构作用中的重要作用，并将确定新的靶向化合物 PTEN可能被开发为治疗心血管疾病的新的翻译疗法。

Project Summary/Abstract Cardiovascular disease is the leading cause of death in the United States and is marked by the development of pathological vascular remodeling which contributes to disease progression. In response to injury or pathological stimuli, resident vascular smooth muscle cells (SMCs) contribute to these remodeling processes by de- differentiating from a quiescent, contractile phenotype towards a proliferative, pro-inflammatory, pro-fibrotic phenotype capable of recruiting immune cells like macrophages and T-cells. The preliminary data supporting this proposal indicates genetic, systemic upregulation of the potent tumor suppressor PTEN maintains SMC differentiation, reduces perivascular immune cell recruitment and blunts remodeling in multiple pre-clinical animal models of cardiovascular disease. This suggests that pharmacologic PTEN upregulation could hold significant therapeutic potential in treating cardiovascular disease by inhibiting vascular remodeling. Currently, the therapeutic potential of PTEN is not fully exploited as there are few compounds known to increase PTEN expression. To fill this knowledge gap, a recently completed high-throughput compound screen (HTS) of over 2,500 compounds using a fluorescence-based PTEN promoter-reporter system was used to identify 11 hit compounds with dose-responsive ability to induce PTEN promoter activity. Recently, JQ1, an inhibitor of the epigenetic reader protein Brd4 that is known to have anti-remodeling effects, was shown to upregulate PTEN. However, preliminary data presented in this proposal suggest that the anti-remodeling effects of JQ1 are blunted in PTEN depleted SMCs both in vitro and in vivo, suggesting that the protective effects of JQ1 are mediated through PTEN upregulation. Little is known about the role of Brd4, the target of JQ1, in regulating PTEN levels in SMCs. However, Brd4 has been shown to interact directly with the histone acetyltransferase p300 and the p65 subunit of the pro-inflammatory transcription factor NFkB, both of which are known to affect PTEN expression. This application presents a novel model of PTEN regulation where competition for limited amounts of p300 drives either PTEN expression through promoter acetylation, or inflammatory gene expression through p300-NFkB interaction mediated by Brd4. Specific Aim 1 will expand upon the preliminary data and will use in vitro and in vivo PTEN depletion to determine whether the vasculoprotective effects of JQ1 are due to increased PTEN expression mediated by increased p300 activity at the PTEN promoter and inhibition of Brd4-p300-NFkB complex formation. Specific Aim 2 will extend the results of the completed HTS to test hit compounds in vitro and in vivo for their ability to induce PTEN expression and reduce vascular remodeling, focusing specifically on how PTEN upregulation affects crosstalk between SMCs and recruited macrophages/T-cells. To summarize, these studies will explore a potentially novel mechanism regulating PTEN expression which may play an essential role in mediating the known anti-remodeling effects of JQ1 and will identify novel compounds targeting PTEN that could be developed as new translational therapies for cardiovascular disease.