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） 成为持续活化的肌成纤维细胞，这有助于病理性主动脉瓣叶硬化。AVS 通过瓣膜置换手术治疗，如果小分子药物组合可以 被鉴定为抑制持续的肌成纤维细胞活化。然而，调节的分子机制 持续性肌成纤维细胞活化是未知的，并且可能因患者和/或性别而异。我现在的 研究表明，在刚性聚乙二醇（PEG）上可以获得持久的肌成纤维细胞， 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， 个体AVS患者不影响工程水凝胶上的肌成纤维细胞活化。我提议的研究 旨在描述导致肌成纤维细胞持续活化的性别和患者特异性差异 在指导的K99阶段的AVS期间，并优化药物组合以抑制肌成纤维细胞活化， 患者特异性提示在独立R 00阶段的功能。我们假设（i）性连锁差异如何 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作出反应 血清有助于持续活化和随后的肌成纤维细胞对小分子药物的应答。在Aim中 1，我们将描述性连锁表观遗传修饰剂，调节男性肌成纤维细胞持久性途径， 使用染色质表征分析和转录组学， 分析。在目标2中，我们将在人AVS患者血清中产生持续活化的肌成纤维细胞， 确定由于患者特异性血清因子导致的维克表观基因组的改变（例如开放染色质区域） 使用测序的转座酶可降解染色质测定法（ATAC-seq）。在目标3中，我们将确定 小分子药物的最佳组合，以抑制存在下的持续肌成纤维细胞活化， 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 个性化组合药物剂量。在该奖项的K99阶段，Kristi Anseth教授将担任我的主要 mentor，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 指导团队，包括Leslie Leinwand教授（性别特异性心脏病）、Tim McKinsey教授 （纤维化过程中的表观遗传学），玛丽艾伦博士（短读测序）和何院长教授（计算 用于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 和表观遗传学特征技术，推动我开发精确的基于药物的治疗方法， 在独立研究者R 00阶段使用生物材料进行AVS。总之，拟议的研究将 解决了对性别特异性和精确医学方法的迫切需求， 肌成纤维细胞持久性的机制，这可能会提供一个桥梁非手术AVS治疗。

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.