Investigating sex differences in persistent valvular myofibroblast activation using hydrogel culture substrates

项目概要/摘要 主动脉瓣狭窄 (AVS) 是一种进行性疾病，其中成纤维细胞样瓣膜间质细胞 (VIC) 成为持续激活的肌成纤维细胞，导致病理性主动脉瓣叶硬化。 AVS 通过瓣膜置换手术进行治疗，如果小分子药物组合可以避免这种情况 被鉴定可抑制持续的肌成纤维细胞活化。然而，调节的分子机制 持续的肌成纤维细胞激活尚不清楚，并且可能因患者和/或性别而异。我现在的 研究表明，可以在硬质聚乙二醇 (PEG) 上获得持久的肌成纤维细胞 水凝胶再现了纤维化瓣膜硬度，我们的初步数据表明性别特异性差异 男性和女性 VIC 如何随着时间的推移获得持久性。我的研究还表明血清因子 个别 AVS 患者对工程水凝胶的肌成纤维细胞活化有不同的影响。我提出的研究 试图描述导致肌成纤维细胞持续激活的性别和患者特异性差异 在 AVS 指导的 K99 阶段，优化药物组合以抑制肌成纤维细胞活化 独立 R00 阶段患者特定线索的功能。我们假设（i）性别相关的差异 男性和女性 VIC 对机械线索和 (ii) 在 AVS 患者中发现的患者特异性生化线索做出反应 血清有助于持久激活和随后的肌成纤维细胞对小分子药物的反应。瞄准 1，我们将表征调节男性肌成纤维细胞持久性途径的性别相关表观遗传修饰剂 使用染色质表征分析和转录组学将雌性 VIC 接种在 PEG 水凝胶上 分析。在目标 2 中，我们将在人类 AVS 患者血清中产生持续活化的肌成纤维细胞，并 确定由于患者特异性血清因素导致的 VIC 表观基因组变化（例如开放染色质区域） 使用转座酶可及染色质测定和测序 (ATAC-seq)。在目标 3 中，我们将确定 在存在肌成纤维细胞的情况下抑制持续肌成纤维细胞活化的小分子药物的最佳组合 AVS 患者血清使用差分进化算法，将肌成纤维细胞抑制与肌成纤维细胞抑制相关联 个性化组合药物剂量。在K99阶段的奖项中，Kristi Anseth教授将作为我的主要负责人 导师，他是使用 PEG 水凝胶材料操纵细胞表型的先驱。我会咨询我的 导师团队，包括 Leslie Leinwand 教授（性别特异性心脏病）、Tim McKinsey 教授 （纤维化期间的表观遗传学）、Mary Allen 博士（短读长测序）和 Dean Ho 教授（计算 优化药物治疗的算法）。我的 K99 培训将包括学习关键的短读测序 和表观遗传学表征技术推动我开发基于精准医学的治疗方法 在独立研究者 R00 阶段使用生物材料进行 AVS。总之，拟议的研究将 解决对性别特异性和精准医学方法识别分子的迫切、未满足的需求 肌成纤维细胞持久性的机制，这可能为非手术 AVS 治疗提供桥梁。

PROJECT SUMMARY / ABSTRACT Aortic valve stenosis (AVS) is a progressive disease where fibroblast-like valvular interstitial cells (VICs) become persistently activated myofibroblasts, which contribute to pathologic aortic valve leaflet stiffening. AVS is treated with valve replacement surgeries, which may be avoided if small molecule drug combinations could be identified to inhibit persistent myofibroblast activation. However, the molecular mechanisms regulating persistent myofibroblast activation are unknown and may vary from patient-to-patient and/or by sex. My current research has revealed that persistent myofibroblasts can be obtained on stiff poly(ethylene glycol) (PEG) hydrogels that recapitulate fibrotic valve stiffness, and our preliminary data suggest sex-specific differences in how male and female VICs obtain persistence over time. My research also suggests serum factors from individual AVS patients variably impact myofibroblast activation on engineered hydrogels. My proposed research seeks to characterize the sex- and patient-specific differences that lead to persistent myofibroblast activation during AVS in the mentored K99 phase and optimize drug combinations to inhibit myofibroblast activation as a function of patient-specific cues in the independent R00 phase. We hypothesize (i) sex-linked differences in how male and female VICs respond to mechanical cues and (ii) patient-specific biochemical cues found in AVS patient sera contribute to persistent activation and subsequent myofibroblast response to small molecule drugs. In Aim 1, we will characterize sex-linked epigenetic modifiers that regulate myofibroblast persistence pathways in male and female VICs seeded on PEG hydrogels using chromatin characterization assays and transcriptomics analyses. In Aim 2, we will generate persistently activated myofibroblasts in human AVS patient sera and determine alterations (e.g. open chromatin regions) in the VIC epigenome due to patient-specific serum factors using Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq). In Aim 3, we will identify optimal combinations of small molecule drugs to inhibit persistent myofibroblast activation in the presence of AVS patient serum using a differential evolution algorithm that correlates myofibroblast inhibition with a personalized combinatorial drug dose. In the K99 phase of the award, Prof. Kristi Anseth will serve as my main mentor, who is a pioneer in using PEG hydrogel materials for manipulating cellular phenotypes. I will consult my mentoring team, including Prof. Leslie Leinwand (sex-specific cardiac diseases), Prof. Tim McKinsey (epigenetics during fibrosis), Dr. Mary Allen (short-read sequencing), and Prof. Dean Ho (computational algorithms for optimizing drug treatments). My K99 training will consist of learning key short-read sequencing and epigenetic characterization techniques to propel me toward developing precision medicine-based treatments for AVS using biomaterials during the independent investigator R00 phase. In sum, the proposed research will address an urgent, unmet need for sex-specific and precision medicine approaches for identifying molecular mechanisms of myofibroblast persistence, which may provide a bridge toward non-surgical AVS therapies.