Pulmonary arterial hypertension (PAH) is a progressive disease characterized by pulmonary arterial wall remodeling, right ventricular (RV) hypertrophy, and interstitial fibrosis, ultimately culminating in RV failure. The latter is the strong predictor of mortality in PAH. While current therapies primarily target the pulmonary vasculature and reduce RV afterload, they do not directly address the metabolic and structural maladaptation of the RV. Emerging evidence indicates that cardiometabolic reprogramming occurs in the RV of PAH patients, encompassing dysregulated glucose, lipid, and amino acid metabolism. These metabolic dysregulations are mirrored in preclinical models, where cardiomyocytes, endothelial cells, and fibroblasts exhibit reduced fatty acid oxidation, enhanced glycolysis, and altered mitochondrial function. Such alterations may promote cardiomyocyte lipotoxicity, impair contractile efficiency, disrupt endothelial barrier integrity, facilitate monocyte recruitment, and drive fibroblast proliferation and activation, collectively contributing to RV inflammation, fibrosis, and functional decline. Understanding these cell-specific metabolic reprogramming is critical, as they may represent both compensatory and pathogenic mechanisms during the progression from RV adaptation to maladaptation. This review provides a comprehensive overview of recent advances in elucidating metabolic dysregulation in the RV of PAH patients and relevant in vivo and in vitro models. The therapeutic potential of targeting these pathways using metabolic modulators, small molecules, and natural products is also discussed, with the goal of developing RV-directed therapies that complement existing PAH treatments and improve patient outcomes.

RATIONALE:Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants exposed to ventilatory support and hyperoxia, involves alveolar and vascular simplification and is linked to cellular senescence. We previously found senescence predominantly in alveolar macrophages (AMs) after neonatal hyperoxic exposure, but the specific mechanisms driving this and its role in neonatal lung injury remain poorly understood. OBJECTIVES:This study investigated the mechanisms driving hyperoxia-induced AM senescence, the effects of senescence/hyperoxia on AM function and how the resulting secretome contributes to lung injury. METHODS:scRNA-seq datasets from hyperoxia-exposed neonatal mice were used to score AM senescence and characterize senescent/hyperoxia-induced AM clusters. Hyperoxia-induced metabolic shifts were analyzed alongside AM motility and phagocytosis. Proteomics characterized the senescent AM secretome. Finally, the effects of senescent and hyperoxic secretomes on lung injury were assessed with and without p38MAPK inhibition. RESULTS:scRNA-seq analysis confirmed that hyperoxia significantly increases senescence markers in AMs. This induction occurs via increased glycolysis and leads to reduced AM motility and phagocytosis. Proteomics identified p38MAPK-regulated proteins in the AM secretome including upregulated pro-fibrotic factors and downregulated structural regulators. Intranasal administration of senescent and/or hyperoxia-conditioned media caused alveolar and vascular simplification in neonatal mice, which was attenuated by p38MAPK inhibition. CONCLUSIONS:Hyperoxia causes glycolytic reprogramming in AMs, increasing senescence markers and impairing function. This also leads to a secretome that drives lung injury. Inhibiting macrophage glycolysis and p38MAPK pathways represent novel, promising therapeutic approaches to prevent lung injury.

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants. Neonatal hyperoxia induces a BPD-like phenotype and lung cell senescence in rodents. In our 3-day hyperoxia model, senescent cells were predominantly lung macrophages, with their abundance peaking at postnatal day 7 (pnd7). However, the molecular and functional characteristics of these senescent macrophages remain undefined. Methods: We reanalyzed a scRNA-seq dataset (GSE207866) generated from senescent lung cells isolated at pnd7 (SD7) following neonatal hyperoxia. Hierarchical clustering combined with manual annotation was used to compare transcriptional profiles with age-matched air-exposed controls (AirD7) and hyperoxia-exposed mice without senescent-cell enrichment (O2D7). Key molecular findings were validated by immunofluorescence. In vivo, neonatal mice received daily injections of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate (DCA) from pnd4 to pnd6, and a senolytic cocktail consisting of quercetin and dasatinib from pnd4 to pnd14, following 3 days of hyperoxia exposure. Results: Macrophages accounted for 65.90% of senescent cells in the SD7 group. Seven macrophage clusters were identified, enriched in M1-like and alveolar macrophage phenotypes. Two major clusters (clusters 0 and 1), together representing nearly half of all senescent macrophages, exhibited strong expression of genes associated with innate immunity, inflammation, and DNA damage responses. These clusters also showed a shift toward glycolysis, the pentose phosphate pathway, and glutamine metabolism, with reduced reliance on β-oxidation. Administration of DCA activated pyruvate dehydrogenase and attenuated hyperoxia-induced macrophage senescence and lung injury. Pathway enrichment analyses revealed enhanced metal-handling pathways, immune and stress signaling (including p38 mitogen-activated kinase, ataxia-telangiectasia mutated, and mechanistic target of rapamycin), apoptosis, and RNA regulatory processes. Conversely, genes involved in reactive oxygen species detoxification, DNA repair, phagocytosis, cytoskeletal organization, and cell adhesion were downregulated. Notably, reducing senescent cells by a senolytic cocktail during the alveolar stage mitigated hyperoxia-induced persistent lung injury. Conclusion: Neonatal hyperoxia drives the emergence of a heterogeneous population of senescent macrophages characterized by metabolic reprogramming and dysregulated signaling pathways, which contribute to the development and persistence of lung injury.

Pulmonary hypertension (PH) increases the mortality of preterm infants with bronchopulmonary dysplasia (BPD). There are no curative therapies for this disease. Lung endothelial carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme of the carnitine shuttle system, is reduced in a rodent model of BPD. It is unknown whether endothelial Cpt1a reduction causes pulmonary vascular (PV) remodeling. The latter can be the result of endothelial-mesenchymal transition (EndoMT). Here, endothelial cell (EC)-specific Cpt1a KO and WT mice (<12 h old) are exposed to hyperoxia (70% O2) for 14 days and allow them to recover in normoxia until postnatal day 28. Hyperoxia causes PH, which is aggravated in EC-specific Cpt1a KO mice. Upregulating endothelial Cpt1a expression inhibits hyperoxia-induced PV remodeling. Hyperoxia causes lung EndoMT, detected by immunofluorescence, scRNA-sequencing, and EC lineage tracing, which is further increased in EC-specific Cpt1a KO mice. Blocking EndoMT inhibits hyperoxia-induced PV remodeling. Male mice under the same high oxygen conditions develop a higher degree of PH than females, which is associated with reduced endothelial Cpt1a expression. Conclusively, neonatal hyperoxia causes PH by decreasing endothelial Cpt1a expression and upregulating EndoMT. This provides a valuable strategy for developing targeted therapies by upregulating endothelial Cpt1a levels or inhibiting EndoMT to treat BPD-associated PH.

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from mechanical ventilation and hyperoxia amongst other factors. We and others have shown that neonatal hyperoxia, known to lead to a BPD-like phenotype in rodent models, causes lung cellular senescence. In our 3-day hyperoxia model, the majority of senescent cells were lung macrophages, and these peaked at postnatal day (pnd) 7. The features of these senescent macrophages are not characterized. Here, we reanalyzed scRNA-seq datasets (GSE207866) of senescent lung cells from mice exposed to hyperoxia as neonates at pnd7 (SD7), and characterized their gene express profiling and compared them with air control (AirD7) and hyperoxia-exposed group without isolation of senescent cells at pnd7 (O2D7). We first classified the cells into epithelial, endothelial, immune, and mesenchymal cells to extract immune cells. By employing the workflow to the immune clusters, mixed populations of macrophages, monocytes, and dendritic cells and pure population of macrophages, we finally identified seven clusters of macrophages. In SD7 group, 65.9% senescent cells were macrophages, and comprised M1 (64%) and alveolar (62.8%) macrophages. Clusters 0 and 1 were M1 and alveolar macrophages, which were composed of 49.2% of senescent macrophages. These two clusters highly expressed genes involved in innate immunity, inflammation, DNA repair response and phagocytosis. Metabolic switch from mitochondrial respiration to glycolysis and pentose phosphate pathway was observed in these two clusters. Conclusively, senescent macrophages are heterogenous with distinct tissue compartments and metabolic dysregulation.

Background: Pulmonary hypertension (PH) is a common complication that increases the mortality of preterm infants with bronchopulmonary dysplasia (BPD). There are no curative therapies for this disease which results from exposure to assisted ventilation when the lung is still developing. We reported that lung endothelial carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme of the carnitine shuttle system for fatty acid β-oxidation, is reduced in a rodent model of BPD. It is unknown whether endothelial Cpt1a reduction contributes to pulmonary vascular remodeling. The latter could be the result of endothelial-mesenchymal transition (EndoMT). Objective: To test the hypothesis that endothelial Cpt1a reduction causes pulmonary vascular remodeling by upregulating EndoMT, leading to BPD-associated PH. Methods: Endothelial cell (EC)-specific Cpt1a KO mice and their WT littermates as well as Rosa26-tdTomato EC lineage traced mice (< 12 h old) were exposed to hyperoxia (70% O2) for 14 days and recovered in normoxia until postnatal day 28. Reanalysis of publicly available scRNA-seq datasets in the lung of mice exposed to hyperoxia (85% O2) for 7 days as neonates (GSE151974). Echocardiography was performed, and pulmonary arterial wall thickness and Fulton index were evaluated. Lung ECs were isolated from hyperoxia-exposed mice for evaluating gene and protein expression. Results: Hyperoxia caused pulmonary vascular remodeling, PH, and right ventricular hypertrophy. These effects were aggravated in EC-specific Cpt1a KO mice. Therapeutic upregulation of Cpt1a by L-carnitine and nanoparticle-mediated endothelial gene delivery inhibited hyperoxia-induced pulmonary vascular remodeling and PH. Hyperoxia caused lung EndoMT as detected by immunofluorescence, single-cell RNA sequencing, and EC lineage tracing. This was increased in EC-specific Cpt1a KO mice exposed to hyperoxia as neonates. Blocking EndoMT inhibited hyperoxia-induced pulmonary vascular remodeling and PH in both EC-specific Cpt1a KO mice and WT littermates. Conclusion: Neonatal hyperoxia causes PH by decreasing endothelial Cpt1a expression and upregulating EndoMT. This provides a valuable strategy to developing targeted therapies based on upregulating endothelial Cpt1a levels or inhibiting EndoMT to treat BPD-associated PH.

The morbidity and mortality of gastrointestinal tumours remain high worldwide. Surgical resection is currently the most critical radical therapeutic schedule, while postoperative complications and sentinel lymph node (SLN) identification are closely related to the outcome. Indocyanine green (ICG)-mediated fluorescence imaging is increasingly being used in gastrointestinal surgery. It has been embraced by various surgical disciplines as a potential method to improve lymph node detection and enhance surgical field visualization. ICG can passively concentrate in SLN because of enhanced permeation and retention effects. After excitation by near-infrared light devices, SLN can display higher intensity fluorescence, helping visualization for better lymph node dissection. In addition, visual assessment of intestinal blood flow through ICG may reduce the incidence of anastomotic leakage. Although it has good clinical application, ICG-imaging still faces some problems, such as a higher false-negative rate, poorly targeted biodistribution, and lower fluorescence contrast, due to the lack of active tumour targeting. Thus, different ICG-coupled nanoparticles with inherent characteristics or functional modification-enhanced SLN identification features for gastrointestinal cancers bring benefit through active tumour targeting, superior tumour-background ratio, and high resolution. Nano-ICG combined with potential substances, including enhanced imaging contrast and/or combination therapy (chemotherapy, targeted therapy, immunotherapy, etc.), have been packaged and accumulated in the tumour area through active targeting for multimodal imaging and treatment. In this review, we outline the intraoperative application and possible future nanodirections of ICG in gastrointestinal cancer. The prospects and challenges of nano-ICG diagnostic and therapeutic methods in clinical applications are also discussed.

Cellular senescence is a status of irreversible growth arrest, which can be triggered by the p53/p21cip1 and p16INK4/Rb pathways via intrinsic and external factors. Senescent cells are typically enlarged and flattened, and characterized by numerous molecular features. The latter consists of increased surfaceome, increased residual lysosomal activity at pH 6.0 (manifested by increased activity of senescence-associated beta-galactosidase [SA-β-gal]), senescence-associated mitochondrial dysfunction, cytoplasmic chromatin fragment, nuclear lamin b1 exclusion, telomere-associated foci, and the senescence-associated secretory phenotype. These features vary depending on the stressor leading to senescence and the type of senescence. Cellular senescence plays pivotal roles in organismal aging and in the pathogenesis of aging-related diseases. Interestingly, senescence can also both promote and inhibit wound healing processes. We recently report that senescence as a programmed process contributes to normal lung development. Lung senescence is also observed in Down Syndrome, as well as in premature infants with bronchopulmonary dysplasia and in a hyperoxia-induced rodent model of this disease. Furthermore, this senescence results in neonatal lung injury. In this review, we briefly discuss the molecular features of senescence. We then focus on the emerging role of senescence in normal lung development and in the pathogenesis of bronchopulmonary dysplasia as well as putative signaling pathways driving senescence. Finally, we discuss potential therapeutic approaches targeting senescent cells to prevent perinatal lung diseases.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants characterized by alveolar dysplasia, vascular simplification and dysmorphic vascular development. Supplemental oxygen and mechanical ventilation commonly used as life-saving measures in premature infants may cause BPD. microRNAs (miRNAs), a class of small, non-coding RNAs, regulate target gene expression mainly through post-transcriptional repression. miRNAs play important roles in modulating oxidative stress, proliferation, apoptosis, senescence, inflammatory responses, and angiogenesis. These cellular processes play pivotal roles in the pathogenesis of BPD. Accumulating evidence demonstrates that miRNAs are dysregulated in the lung of premature infants with BPD, and in animal models of this disease, suggesting contributing roles of dysregulated miRNAs in the development of BPD. Therefore, miRNAs are considered promising biomarker candidates and therapeutic agents for this disease. In this review, we discuss how dysregulated miRNAs and their modulation alter cellular processes involved in BPD. We then focus on therapeutic approaches targeting miRNAs for BPD. This review provides an overview of miRNAs as biomarkers, and highlights potential pathogenic roles, and therapeutic strategies for BPD using miRNAs.

Senescence causes age-related diseases and stress-related injury. Paradoxically, it is also essential for organismal development. Whether senescence contributes to lung development or injury in early life remains unclear. Here, we show that lung senescence occurred at birth and decreased throughout the saccular stage in mice. Reducing senescent cells at this stage disrupted lung development. In mice (<12 h old) exposed to hyperoxia during the saccular stage followed by air recovery until adulthood, lung senescence increased particularly in type II cells and secondary crest myofibroblasts. This peaked during the alveolar stage and was mediated by the p53/p21 pathway. Decreasing senescent cells during the alveolar stage attenuated hyperoxia-induced alveolar and vascular simplification. Conclusively, early programmed senescence orchestrates postnatal lung development whereas later hyperoxia-induced senescence causes lung injury through different mechanisms. This defines the ontogeny of lung senescence and provides an optimal therapeutic window for mitigating neonatal hyperoxic lung injury by inhibiting senescence.

Supplemental oxygen is a lifesaving measure in infants born premature to facilitate oxygenation. Unfortunately, it may lead to alveolar simplification and loss of proximal airway epithelial cilia. Little is known about the mechanism by which hyperoxia causes ciliary dysfunction in the proximal respiratory tract. We hypothesized that hyperoxia causes intraflagellar transport (IFT) dysfunction with resultant decreased cilia length. Differentiated basal human airway epithelial cells (HAEC) were exposed to hyperoxia or air for up to 48 h. Neonatal mice (<12 h old) were exposed to hyperoxia for 72 h and recovered in room air until postnatal day (PND) 60. Cilia length was measured from scanning electron microscopy images using a MATLAB-derived program. Proteomics and metabolomics were carried out in cells after hyperoxia. After hyperoxia, there was a significant time-dependent reduction in cilia length after hyperoxia in HAEC. Proteomic analysis showed decreased abundance of multiple proteins related to IFT including dynein motor proteins. In neonatal mice exposed to hyperoxia, there was a significant decrease in acetylated α tubulin at PND10 followed by recovery to normal levels at PND60. In HAEC, hyperoxia decreased the abundance of multiple proteins associated with complex I of the electron transport chain. In HAEC, hyperoxia increased levels of malate, fumarate, and citrate, and reduced the ATP/ADP ratio at 24 h with a subsequent increase at 36 h. Exposure to hyperoxia reduced cilia length, and this was associated with aberrant IFT protein expression and dysregulated metabolism. This suggests that hyperoxic exposure leads to aberrant IFT protein expression in the respiratory epithelium resulting in shortened cilia.

Background Premature infants, subjected to supplemental oxygen and mechanical ventilation, may develop bronchopulmonary dysplasia, a chronic lung disease characterized by alveolar dysplasia and impaired vascularization. We and others have shown that hyperoxia causes senescence in cultured lung epithelial cells and fibroblasts. Although miR-34a modulates senescence, it is unclear whether it contributes to hyperoxia-induced senescence. We hypothesized that hyperoxia increases miR-34a levels, leading to cellular senescence. Methods We exposed mouse lung epithelial (MLE-12) cells and primary human small airway epithelial cells to hyperoxia (95% O 2 /5% CO 2 ) or air (21% O 2 /5% CO 2 ) for 24 h. Newborn mice (< 12 h old) were exposed to hyperoxia (> 95% O 2 ) for 3 days and allowed to recover in room air until postnatal day 7. Lung samples from premature human infants requiring mechanical ventilation and control subjects who were not mechanically ventilated were employed. Results Hyperoxia caused senescence as indicated by loss of nuclear lamin B1, increased p21 gene expression, and senescence-associated secretory phenotype factors. Expression of miR-34a-5p was increased in epithelial cells and newborn mice exposed to hyperoxia, and in premature infants requiring mechanical ventilation. Transfection with a miR-34a-5p inhibitor reduced hyperoxia-induced senescence in MLE-12 cells. Additionally, hyperoxia increased protein levels of the oncogene and tumor-suppressor Krüppel-like factor 4 (KLF4), which were inhibited by a miR-34a-5p inhibitor. Furthermore, KLF4 knockdown by siRNA transfection reduced hyperoxia-induced senescence. Conclusion Hyperoxia increases miR-34a-5p, leading to senescence in lung epithelial cells. This is dictated in part by upregulation of KLF4 signaling. Therefore, inhibiting hyperoxia-induced senescence via miR-34a-5p or KLF4 suppression may provide a novel therapeutic strategy to mitigate the detrimental consequences of hyperoxia in the neonatal lung.

Background Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants that may cause long-term lung dysfunction. Accumulating evidence supports the vascular hypothesis of BPD, in which lung endothelial cell dysfunction drives this disease. We recently reported that endothelial carnitine palmitoyltransferase 1a (Cpt1a) is reduced by hyperoxia, and that endothelial cell-specific Cpt1a knockout mice are more susceptible to developing hyperoxia-induced injury than wild type mice. Whether Cpt1a upregulation attenuates hyperoxia-induced endothelial cell dysfunction and lung injury remains unknown. We hypothesized that upregulation of Cpt1a by baicalin or l- carnitine ameliorates hyperoxia-induced endothelial cell dysfunction and persistent lung injury. Methods Lung endothelial cells or newborn mice (< 12 h old) were treated with baicalin or l -carnitine after hyperoxia (50% and 95% O 2 ) followed by air recovery. Results We found that incubation with l -carnitine (40 and 80 mg/L) and baicalin (22.5 and 45 mg/L) reduced hyperoxia-induced apoptosis, impaired cell migration and angiogenesis in cultured lung endothelial cells. This was associated with increased Cpt1a gene expression. In mice, neonatal hyperoxia caused persistent alveolar and vascular simplification in a concentration-dependent manner. Treatment with l -carnitine (150 and 300 mg/kg) and baicalin (50 and 100 mg/kg) attenuated neonatal hyperoxia-induced alveolar and vascular simplification in adult mice. These effects were diminished in endothelial cell-specific Cpt1a knockout mice. Conclusions Upregulating Cpt1a by baicalin or l -carnitine ameliorates hyperoxia-induced lung endothelial cell dysfunction, and persistent alveolar and vascular simplification. These findings provide potential therapeutic avenues for using l -carnitine and baicalin as Cpt1a upregulators to prevent persistent lung injury in premature infants with BPD.

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in degrading heme into biliverdin and iron. HO-1 can also enter the nucleus and regulate gene transcription independent of its enzymatic activity. Whether HO-1 can alter gene expression through direct binding to target DNA remains unclear. Here, we performed HO-1 CHIP-seq and then employed 3D structural modeling to reveal putative HO-1 DNA binding domains. We identified three probable DNA binding domains on HO-1. Using the Proteinarium, we identified several genes as the most highly connected nodes in the interactome among the HO-1 gene binding targets. We further demonstrated that HO-1 modulates the expression of these key genes using Hmox1 deficient cells. Finally, mutation of four conserved amino acids (E215, I211, E201, and Q27) within HO-1 DNA binding domain 1 significantly increased expression of Gtpbp3 and Eif1 genes that were identified within the top 10 binding hits normalized by gene length predicted to bind this domain. Based on these data, we conclude that HO-1 protein is a putative DNA binding protein, and regulates targeted gene expression. This provides the foundation for developing specific inhibitors or activators targeting HO-1 DNA binding domains to modulate targeted gene expression and corresponding cellular function.