Idiopathic pulmonary fibrosis (IPF) is characterized by the sustained activation of interstitial fibroblasts leading to excessive collagen deposition and progressive organ failure. Epigenetic and metabolic abnormalities have been shown to contribute to the persistent activated state of scar-forming fibroblasts. However, how epigenetic changes regulate fibroblast metabolic responses to promote fibroblast activation and progressive fibrosis remains largely unknown. Here, we show that the epigenetic regulator CBX5 (chromobox protein homolog 5) is critical to the transition of quiescent fibroblasts to activated collagen-producing fibroblasts in response to bleomycin-induced lung injury. Loss of mesenchymal CBX5 attenuated fibrosis development, and this effect was accompanied by the downregulation of pathogenic fibroblast genes, including Cthrc1, Col1a1, and Spp1, and by the upregulation of metabolic genes with antifibrotic activity such as Ppara and Pparg. Single-cell RNA sequencing and immunohistochemistry analyses revealed that CBX5 expression was enriched in pathogenic fibroblasts and fibroblastic foci of IPF lungs. Bulk RNA-sequencing analysis combined with metabolic assessments demonstrated that CBX5 silencing in IPF fibroblasts potently inhibited transforming growth factor-stimulated glycolysis while enhancing AMPK signaling and mitochondrial metabolism. Finally, interruption of the CBX5 pathway in IPF fibroblasts in vitro and in IPF lung explants ex vivo synergistically potentiated the activation of metformin-induced AMP-activated protein kinase activation and inhibited collagen secretion. Collectively, our findings identify CBX5 as an epigenetic regulator linking metabolic maladaptation to the persistent activated state of lung fibroblasts during IPF progression.

Progressive lung fibrosis is associated with poorly understood aging-related endothelial cell dysfunction. To gain insight into endothelial cell alterations in lung fibrosis we performed single cell RNA-sequencing of bleomycin-injured lungs from young and aged mice. Analysis reveals activated cell states enriched for hypoxia, glycolysis and YAP/TAZ activity in ACKR1+ venous and TrkB+ capillary endothelial cells. Endothelial cell activation is prevalent in lungs of aged mice and can also be detected in human fibrotic lungs. Longitudinal single cell RNA-sequencing combined with lineage tracing demonstrate that endothelial activation resolves in young mouse lungs but persists in aged ones, indicating a failure of the aged vasculature to return to quiescence. Genes associated with activated lung endothelial cells states in vivo can be induced in vitro by activating YAP/TAZ. YAP/TAZ also cooperate with BDNF, a TrkB ligand that is reduced in fibrotic lungs, to promote capillary morphogenesis. These findings offer insights into aging-related lung endothelial cell dysfunction that may contribute to defective lung injury repair and persistent fibrosis. The regenerative capacity of the lung in response to injury deteriorates with aging. Here, Raslan et al. discover that aging-associated progressive lung fibrosis is accompanied by persistent activation of blood vessels. The authors identified the vascular YAP/TrkB axis as a putative driver of this process and potential therapeutic target.

Lung regeneration deteriorates with aging leading to increased susceptibility to pathologic conditions, including fibrosis. Here, we investigated bleomycin-induced lung injury responses in young and aged mice at single-cell resolution to gain insights into the cellular and molecular contributions of aging to fibrosis. Analysis of 52,542 cells in young (8 weeks) and aged (72 weeks) mice identified 15 cellular clusters, many of which exhibited distinct injury responses that associated with age. We identified Pdgfra + alveolar fibroblasts as a major source of collagen expression following bleomycin challenge, with those from aged lungs exhibiting a more persistent activation compared to young ones. We also observed age-associated transcriptional abnormalities affecting lung progenitor cells, including ATII pneumocytes and general capillary (gCap) endothelial cells (ECs). Transcriptional analysis combined with lineage tracing identified a sub-population of gCap ECs marked by the expression of Tropomyosin Receptor Kinase B (TrkB) that appeared in bleomycin-injured lungs and accumulated with aging. This newly emerged TrkB + EC population expressed common gCap EC markers but also exhibited a distinct gene expression signature associated with aberrant YAP/TAZ signaling, mitochondrial dysfunction, and hypoxia. Finally, we defined ACKR1 + venous ECs that exclusively emerged in injured lungs of aged animals and were closely associated with areas of collagen deposition and inflammation. Immunostaining and FACS analysis of human IPF lungs demonstrated that ACKR1 + venous ECs were dominant cells within the fibrotic regions and accumulated in areas of myofibroblast aggregation. Together, these data provide high-resolution insights into the impact of aging on lung cell adaptability to injury responses.

Understanding the dynamic pathogenesis and treatment response in pulmonary diseases requires probing the lung at cellular resolution in real time. Despite advances in intravital imaging, optical imaging of the lung during active respiration and circulation has remained challenging. Here, we introduce the crystal ribcage: a transparent ribcage that allows multiscale optical imaging of the functioning lung from whole-organ to single-cell level. It enables the modulation of lung biophysics and immunity through intravascular, intrapulmonary, intraparenchymal and optogenetic interventions, and it preserves the three-dimensional architecture, air-liquid interface, cellular diversity and respiratory-circulatory functions of the lung. Utilizing these capabilities on murine models of pulmonary pathologies we probed remodeling of respiratory-circulatory functions at the single-alveolus and capillary levels during disease progression. The crystal ribcage and its broad applications presented here will facilitate further studies of nearly any pulmonary disease as well as lead to the identification of new targets for treatment strategies.

Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

Transcriptional coactivator PGC-1α and its splice variant NT-PGC-1α regulate metabolic adaptation by modulating many gene programs. Selective ablation of PGC-1α attenuates diet-induced obesity through enhancing fatty acid oxidation and thermogenesis by upregulation of NT-PGC-1α in brown adipose tissue (BAT). Recently, we have shown that selective ablation of NT-PGC-1α reduces fatty acid oxidation in BAT. Thus, the objective of this study was to test our hypothesis that NT-PGC-1α −/− mice would be more prone to diet-induced obesity. Male and female NT-PGC-1α +/+ (WT) and NT-PGC-1α −/− mice were fed a regular chow or 60% high-fat (HF) diet for 16 weeks. Contrary to our expectations, both male and female NT-PGC-1α −/− mice fed HFD were protected from diet-induced obesity, with more pronounced effects in females. This lean phenotype was primarily driven by reduced dietary fat intake. Intriguingly, HFD-fed female, but not male, NT-PGC-1α −/− mice further exhibited decreased feed efficiency, which was closely associated with increased fecal fat excretion and decreased uptake of fatty acids by the intestinal enterocytes and adipocytes with a concomitant decrease in fatty acid transporter gene expression. Collectively, our results highlight the role for NT-PGC-1α in regulating whole body lipid homeostasis under HFD conditions.

Brown and beige adipocytes are specialized to dissipate energy as heat. Sgk2, encoding a serine/threonine kinase, has been identified as a brown and beige adipocyte-specific gene in rodents and humans; however, its function in brown/beige adipocytes remains unraveled. Here, we examined the regulation and role of Sgk2 in brown/beige adipose tissue thermogenesis. We found that transcriptional coactivators PGC-1α and NT-PGC-1α activated by the β3 adrenergic receptor-cAMP-PKA pathway are recruited to the Sgk2 promoter, triggering Sgk2 transcription in response to cold. SGK2 elevation was closely associated with increased serine/threonine phosphorylation of proteins carrying the consensus RxRxxS/T phosphorylation site. However, despite cold-dependent activation of SGK2, mice lacking Sgk2 exhibited normal cold tolerance at 4°C. In addition, Sgk2+/+ and Sgk2−/− mice induced comparable increases in energy expenditure during pharmacological activation of brown and beige adipose tissue with a β3AR agonist. In vitro loss- and gain-of-function studies further demonstrated that Sgk2 ablation or activation does not alter thermogenic gene expression and mitochondrial respiration in brown adipocytes. Collectively, our results reveal a new signaling component SGK2, although dispensable for cold-induced thermogenesis that adds an additional layer of complexity to the β3AR signaling network in brown/beige adipose tissue.

Abstract Background Brown adipose tissue (BAT) is specialized to dissipate energy in the form of heat. BAT-mediated heat production in rodents and humans is critical for effective temperature adaptation of newborns to the extrauterine environment immediately after birth. However, very little is known about whether and how fetal BAT development is modulated in-utero in response to changes in maternal thermal environment during pregnancy. Using BL6 mice, we evaluated the impact of different maternal environmental temperatures (28 °C and 18 °C) on the transcriptome of the placenta and fetal BAT to test if maternal cold exposure influences fetal BAT development via placental remodeling. Results Maternal weight gain during pregnancy, the average number of fetuses per pregnancy, and placental weight did not differ between the groups at 28 °C and 18 °C. However, the average fetal weight at E18.5 was 6% lower in the 18 °C-group compared to the 28 °C-group. In fetal BATs, cold exposure during pregnancy induced increased expression of genes involved in de novo lipogenesis and lipid metabolism while decreasing the expression of genes associated with muscle cell differentiation, thus suggesting that maternal cold exposure may promote fetal brown adipogenesis by suppressing the myogenic lineage in bidirectional progenitors. In placental tissues, maternal cold exposure was associated with upregulation of genes involved in complement activation and downregulation of genes related to muscle contraction and actin-myosin filament sliding. These changes may coordinate placental adaptation to maternal cold exposure, potentially by protecting against cold stress-induced inflammatory damage and modulating the vascular and extravascular contractile system in the placenta. Conclusions These findings provide evidence that environmental cold temperature sensed by the mother can modulate the transcriptome of placental and fetal BAT tissues. The ramifications of the observed gene expression changes warrant future investigation.

The central nervous system controls feeding behavior and energy expenditure in response to various internal and external stimuli to maintain energy balance. Here we report that the newly identified transcription factor zinc finger and BTB domain containing 16 (Zbtb16) is induced by energy deficit in the paraventricular (PVH) and arcuate (ARC) nuclei of the hypothalamus via glucocorticoid (GC) signaling. In the PVH, Zbtb16 is expressed in the anterior half of the PVH and co-expressed with many neuronal markers such as corticotropin-releasing hormone (Crh), thyrotropin-releasing hormone (Trh), oxytocin (Oxt), arginine vasopressin (Avp), and nitric oxide synthase 1 (Nos1). Knockdown (KD) of Zbtb16 in the PVH results in attenuated cold-induced thermogenesis and improved glucose tolerance without affecting food intake. In the meantime, Zbtb16 is predominantly expressed in agouti-related neuropeptide/neuropeptide Y (Agrp/Npy) neurons in the ARC and its KD in the ARC leads to reduced food intake. We further reveal that chemogenetic stimulation of PVH Zbtb16 neurons increases energy expenditure while that of ARC Zbtb16 neurons increases food intake. Taken together, we conclude that Zbtb16 is an important mediator that coordinates responses to energy deficit downstream of GCs by contributing to glycemic control through the PVH and feeding behavior regulation through the ARC, and additionally reveal its function in controlling energy expenditure during cold-evoked thermogenesis via the PVH. As a result, we hypothesize that Zbtb16 may be involved in promoting weight regain after weight loss.

Transcriptional coactivator PPAR γ coactivator (PGC)-1α and its splice variant N-terminal (NT)-PGC-1α mediate transcriptional regulation of brown adipose tissue (BAT) thermogenesis in response to changes in ambient temperature. PGC-1α is dispensable for cold-induced BAT thermogenesis as long as NT-PGC-1α is present. However, the functional significance of NT-PGC-1α in BAT has not been determined. In the present study, we generated NT-PGC-1α−/− mice to investigate the effect of NT-PGC-1α deficiency on adaptive BAT thermogenesis. At thermoneutrality, NT-PGC-1α−/− mice exhibited abnormal BAT phenotype with increased accumulation of large lipid droplets concomitant with marked downregulation of FA oxidation (FAO)-related genes. Consistent with transcriptional changes, mitochondrial FAO was significantly diminished in NT-PGC-1α−/− BAT. This alteration, in turn, enhanced glucose utilization within the NT-PGC-1α−/− BAT mitochondria. In line with this, NT-PGC-1α−/− mice had higher reliance on carbohydrates. In response to cold or β3-adrenergic receptor agonist, NT-PGC-1α−/− mice transiently exhibited lower thermogenesis but reached similar thermogenic capacities as their WT littermates. Collectively, these findings demonstrate that NT-PGC-1α is an important contributor to the maintenance of FAO capacity in BAT at thermoneutrality and provide deeper insights into the relative contributions of PGC-1α and NT-PGC-1α to temperature-regulated BAT remodeling.