Lung injury-induced endothelial cell states persist in aging-associated non-resolving fibrosis

The capacity of lung tissue to heal without scarring deteriorates with aging. The pulmonary vasculature has emerged as a central regulator of lung repair, and aging-associated endothelial cell dysfunction has been associated with defective lung response to injury and sustained fibrosis. To shed light into aging-associated cellular and transcriptional responses of lung endothelial cells during lung fibrogenesis, we carried out a single cell RNA-seq analysis of bleomycin-treated young (2 months) and aged (72 months) lungs during the early phase of fibrosis resolution (30 days post bleomycin). This analysis revealed that lung injury was associated with the appearance of distinct populations of endothelial cells that exhibit “activated” transcriptional states, originating from virtually all vascular beds. Activated endothelial cells deriving from venous and capillary beds were the most represented in aged compared to young lungs. Longitudinal single-cell analysis together with lineage tracing revealed that activated endothelial cells appeared transiently during the peak of collagen production in young lungs and disappeared during the resolution phase. Conversely, activated endothelial cell states persisted in aged lungs around fibrotic areas, indicating a failure of the aged vascular tissue to return to quiescence. We provide evidence that an axis between YAP and TrkB signaling acts as a node that directs injury-induced capillary endothelial cell activation in vivo and lung capillary morphogenesis ex vivo. Together, these data provide insights into the aging-related dynamics of lung endothelial cells in response to injury. Male young (2 months old) and aged (18 months old) C57BL/6J mice were administered with Bleomycin (1 U/kg) intratracheally. Thirty days after bleomycin challenge, whole lungs were isolated and digested using a mixture of collagenase and dispase to create a single-cell suspension used for single-cell sequencing.

肺组织无瘢痕愈合的能力随衰老逐渐下降。肺血管系统已被证实是肺修复的核心调控因子，而衰老相关的内皮细胞功能障碍与肺损伤应答缺陷及持续性纤维化密切相关。为阐明肺纤维化过程中肺内皮细胞的衰老相关细胞与转录应答机制，我们在纤维化消退早期（博莱霉素给药后30天），对经博莱霉素处理的年轻（2月龄）与衰老（72月龄）小鼠肺组织开展了单细胞RNA测序（single cell RNA-seq）分析。该分析显示，肺损伤会诱导出现呈现"活化"转录状态的独特内皮细胞群，这些细胞几乎全部源自各类血管床。与年轻小鼠肺组织相比，衰老小鼠肺组织中源自静脉与毛细血管床的活化内皮细胞占比最高。纵向单细胞分析结合谱系示踪结果显示，在年轻小鼠肺组织中，活化内皮细胞会在胶原产生高峰期暂时性出现，并在消退期消失。与之相反，衰老小鼠肺组织纤维化区域周围的活化内皮细胞状态会持续存在，表明衰老血管组织无法恢复至静息状态。本研究证实，YAP与TrkB信号通路构成的轴可作为核心节点，在体内调控损伤诱导的毛细血管内皮细胞活化，并在离体水平调控肺毛细血管形态发生。综上，这些数据为揭示衰老状态下肺内皮细胞对损伤的应答动态提供了新见解。本研究选取2月龄与18月龄的雄性C57BL/6J小鼠，经气管内给予博莱霉素（1 U/kg）。博莱霉素攻毒30天后，分离全肺并使用胶原酶与分散酶混合液进行消化，制备单细胞悬液用于单细胞测序。