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

项目摘要 皮肤损伤导致纤维化和瘢痕形成，这可能是发病率的重要来源。有趣的是， 人类对相同的皮肤损伤有不同程度的疤痕形成， 吓人的表型了解驱动异质性瘢痕结局的机制将使我们能够 设计策略来引导伤口愈合朝向再生和减少疤痕。生物机械力 会影响皮肤的愈合生物力学张力信号成纤维细胞增殖，迁移， 炎症功能和细胞外基质（ECM）的产生。这些反应，连同 伤口中的氧化应激对成纤维细胞产生高能量需求。一般来说， 细胞在稳态条件下通过线粒体氧化磷酸化（OXPHOS）或通过 糖酵解时氧气是有限的。最近的研究表明，增加机械线索可以改变能源 通过促进糖酵解进行新陈代谢。值得注意的是，代谢向“有氧糖酵解”转变的现象 （瓦尔堡效应）主要描述在纤维化疾病的进展，但我们的数据表明，成纤维细胞， 来自具有高瘢痕形成表型（HS）的健康患者的未损伤皮肤的OXPHOS和糖酵解较高， 比那些低疤痕，并证明了线粒体功能的变化，表明更高的能量 基线状态。有氧糖酵解的关键限速酶PKM 2的表达在HS中也较高 成纤维细胞，PKM 2磷酸化/二聚化增加，使代谢物向ATP增加分流 产生和促进有氧糖酵解和TGF-β刺激下的促纤维化途径。HS成纤维细胞 也有一个夸张的反应，机械张力，增加总和磷酸化PKM 2。 这些数据支持PKM 2介导的张力下成纤维细胞有氧糖酵解可能 影响纤维化的程度。同样，我们也注意到，在细胞内， 张力作用下HS成纤维细胞中的Hsp 27。据我们所知，这是第一个差异有氧糖酵解的证据 和生物力学张力反应与生理性伤口中的相反疤痕结果有关， 可以解释伤口愈合的异质性我们假设患者特异性瘢痕形成反应是由于 创伤生物力学影响成纤维细胞有氧糖酵解的PKM 2/Hsp 27依赖性改变 力.在目标1中，我们设计了低和高瘢痕来源的患者成纤维细胞的体外和体内实验， 为了研究PKM 2和Hsp 27磷酸化/活化的差异及其对代谢途径的影响， 能量代谢和ECM产生。在目标2中，我们将利用体外和人皮肤异种移植伤口， 模型来研究生物力学张力如何改变PKM 2/Hsp 27介导的能量代谢， 疤痕反应，然后开发并验证个人疤痕倾向的新型预测模型 (low或高）。这将导致抗纤维化的发展 基于个体代谢特征的治疗，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.