Differential changes in energy metabolism in response to mechanical tension give rise to human scaring heterogeneity

项目总结 皮肤损伤会导致纤维化和疤痕形成，这可能是发病率的一个重要来源。有趣的是， 人类对相同的皮肤损伤的反应是不同程度的疤痕形成，从低到高不等 吓人的表型。了解导致不同类型疤痕形成的机制将使我们 设计策略，引导伤口愈合，促进再生，减少疤痕形成。生物力学力 都会影响皮肤的愈合方式。生物力学张力信号传递成纤维细胞的增殖、迁移、 炎症功能和细胞外基质(ECM)的产生。这些回应与 伤口中的氧化应激，对成纤维细胞有很高的能量需求。通常，新陈代谢需求 细胞通过线粒体氧化磷酸化(OXPHOS)在稳态条件下或通过 氧气受限时的糖酵解。最近的研究表明，机械信号的增加可以改变能量 通过促进糖酵解来代谢。值得一提的是，代谢转向“有氧糖酵解”的现象 (Warburg效应)主要在纤维化疾病的进展中被描述，但我们的数据显示成纤维细胞 来自健康患者未损伤皮肤的高疤痕表型(HS)具有更高的OXPHOS和糖酵解 并显示线粒体功能的变化，表明能量更高。 基准线的状态。有氧糖酵解的关键限速酶PKM2在HS中的表达也较高 成纤维细胞，随着PKM2磷酸化/二聚化增加，代谢产物向增加的ATP分流 在转化生长因子-β刺激下产生和促进有氧糖酵解和促纤维化途径。HS成纤维细胞 对机械张力也有夸大的反应，总的和磷酸化的PKM2增加。 这些数据支持PKM2介导的成纤维细胞在张力下的有氧糖酵解可能 影响纤维化的程度。一贯地，我们也注意到磷酸化的夸大增加。 HSP27在HS成纤维细胞中的表达。据我们所知，这是差异有氧糖酵解的第一个证据。 而生物力学张力反应与生理性伤口中相反的疤痕结果有关，这 可以解释伤口愈合的异质性。我们假设患者特有的疤痕反应是由于 创伤生物力学影响成纤维细胞有氧糖酵解中依赖PKM2/Hsp27的变化 力量。在目标1中，我们设计了低瘢痕和高瘢痕患者成纤维细胞的体外和体内实验。 为了研究PKM2和Hsp27磷酸化/激活的差异及其对代谢途径的影响， 能量代谢和细胞外基质的产生。在目标2中，我们将利用体外和人类皮肤移植伤口 生物力学张力如何改变PKM2/Hsp27介导的能量代谢以驱动患者的模型 疤痕反应，然后开发和验证一种新的预测个体疤痕倾向的模型 (低或高)基于成纤维细胞的生物能量特征。这将导致抗纤维化的发展 基于个人代谢特征的治疗，这可能对其他纤维化疾病有影响。

PROJECT SUMMARY Dermal injury leads to fibrosis and scar formation, which can be a significant source of morbidity. Interestingly, humans respond to identical skin injuries with different degrees of scar formation that range from low to high scaring phenotypes. Understanding the mechanisms that drive heterogeneous scarring outcomes will allow us to design strategies to direct wound healing toward regeneration and reduced scarring. Biomechanical forces are known to influence how the skin heals. Biomechanical tension signals fibroblast proliferation, migration, inflammatory functions and production of extracellular matrix (ECM). These responses, in conjunction with oxidative stress in the wound, place a high energy demand on fibroblasts. Typically, metabolic requirements of cells are met via mitochondrial oxidative phosphorylation (OXPHOS) under homeostatic conditions or via glycolysis when oxygen is limited. Recent studies have shown that increase in mechanical cues can alter energy metabolism by promoting glycolysis. Notably, the phenomenon of a metabolic shift towards ‘aerobic glycolysis’ (Warburg effect) was mainly described in progression of fibrotic diseases, but our data showed that fibroblasts from uninjured skin of healthy patients with high scarring phenotype (HS) have higher OXPHOS and glycolysis than those from low scarrers, and demonstrated changes in mitochondrial function that suggest a higher energy state at baseline. Expression of PKM2, a key rate-limiting enzyme of aerobic glycolysis, was also higher in HS fibroblasts, with increased PKM2 phosphorylation/dimerization shunting metabolites toward increased ATP production and promoting aerobic glycolysis and pro-fibrotic pathways under TGF-β stimulation. HS fibroblasts also had an exaggerated response to mechanical tension, with an increase in total and phosphorylated PKM2. These data support the concept that PKM2-mediated aerobic glycolysis in fibroblasts under tension may influence the magnitude of fibrosis. Consistently, we also noted an exaggerated increase in phosphorylation of Hsp27 in HS fibroblast under tension. To our knowledge, this is the first evidence of differential aerobic glycolysis and biomechanical tension responses being linked to opposing scar outcomes in physiologic wounds, which could explain wound healing heterogeneity. We hypothesize that patient-specific scarring responses are due to PKM2/Hsp27-dependent alterations in fibroblast aerobic glycolysis that are influenced by wound biomechanical forces. In Aim 1, we designed in vitro and in vivo experiments with low and high scar-derived patient fibroblasts to investigate differences in PKM2 and Hsp27 phosphorylation/activation and their effect on metabolic pathways, energy metabolism, and ECM production. In Aim 2, we will utilize in vitro and human skin xenotransplant wound models to examine how biomechanical tension alters PKM2/Hsp27 mediated energy metabolism to drive patient scarring responses and then develop and validate a novel predictive model for individual scarring propensity (low or high) based on fibroblast bioenergetic signatures. This will lead to the development of anti-fibrotic therapies based on an individual’s metabolic profile, which could have implications for other fibrotic diseases.