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

项目摘要/摘要 主动脉瓣狭窄(AVS)是一种进展性疾病，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，这有助于病理性的主动脉瓣叶僵硬。AVS 用瓣膜置换手术治疗，如果小分子药物组合可以避免 被证实可以抑制持续性肌成纤维细胞的激活。然而，调控的分子机制 持续性肌成纤维细胞激活尚不清楚，可能因患者和/或性别的不同而不同。我目前的情况 研究表明，在硬质聚乙二醇膜上可以获得持续性肌成纤维细胞。 重现纤维性瓣膜僵硬的水凝胶，以及我们的初步数据表明， 男性和女性受害者如何随着时间的推移获得毅力。我的研究还表明，血清因素来自 不同的AVS患者对工程水凝胶上肌成纤维细胞的激活有不同程度的影响。我提议的研究 寻求表征导致肌成纤维细胞持续激活的性别和患者特定的差异 在指导的K99期的AVS期间，并优化药物组合以抑制肌成纤维细胞的激活 患者特定提示在独立R00阶段的作用。我们假设(I)性别相关的差异是如何 男性和女性受害者对机械提示和(Ii)在AVS患者中发现的患者特定的生化提示做出反应 血清有助于对小分子药物的持续激活和随后的肌成纤维细胞反应。在AIM 1，我们将表征调节男性肌成纤维细胞持续通路的性连锁表观遗传修饰物 和接种在聚乙二醇水凝胶上的雌性VIC进行染色质特征分析和转录 分析。在目标2中，我们将在人AVS患者血清中产生持续激活的肌成纤维细胞，并 确定VIC表观基因组因患者特定的血清因素而发生的改变(例如开放染色质区域) 使用转座酶可及染色质测序分析(ATAC-SEQ)。在目标3中，我们将确定 小分子药物的最佳组合在存在的情况下抑制持续的肌成纤维细胞激活 使用差异进化算法的AVS患者血清，该算法将肌成纤维细胞抑制与 个性化组合用药剂量。在K99奖项阶段，Kristi Anseth教授将担任我的主要 他是使用聚乙二醇水凝胶材料操纵细胞表型的先驱。我会咨询我的 指导团队，包括Leslie Leinwand教授(特定性别的心脏病)、Tim McKinsey教授 (纤维化过程中的表观遗传学)、Mary Allen博士(短读测序)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篇阅读顺序 和表观遗传学表征技术，推动我开发基于精确医学的治疗方法 对于在独立调查者R00阶段使用生物材料的AVS。总而言之，拟议的研究将 满足对识别分子的特定性别和精确医学方法的迫切、未得到满足的需求 肌成纤维细胞持续存在的机制，这可能为非手术AVS治疗提供一座桥梁。

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.