Somatic stem cells are characterized by their low overall protein-synthesis rates, a feature implicated in driving their stemness. However, how aging reshapes the translational landscape of stem cells remains poorly understood. Here, we present an in vivo single-cell ribosome profiling strategy to monitor tissue-wide translational landscapes of the epidermis during aging. By implementing ribosomal elongation-inhibited cell isolation and switching to RNase I, we expand the applicability of single-cell ribosome profiling to in vivo systems and facilitate the evaluation of triplet periodicity, a hallmark of high-quality data. Leveraging this strategy, we document the in vivo translational landscapes of the major epidermal cell types, outline cell-type-specific translational efficiencies, and identify a pronounced translational reprogramming of AP-1 subunits specifically in aged epidermal stem cells. Our study illustrates the power of in vivo single-cell ribosome profiling to map cell-type-specific translational programs and offers a scalable strategy for tissue-wide interrogation of translational landscapes.

BackgroundChronic respiratory diseases represent a large group of non-communicable diseases that are a leading cause of mortality and morbidity globally. Many of the methods utilized to assess airway simplification in experimental models of the conditions are overly time-consuming and are sensitive to inter-operator biases, necessitating the need for unbiased and efficient tools to supplement analyses.MethodsWe propose a semi-automated method to quantitate the characteristics of large terminal respiratory airways and alveoli that uses free image-processing software (Fiji). We aimed to develop and test this method in a mouse model of bronchopulmonary dysplasia (BPD), a disease of blunted airway and pulmonary vascular development that remains a leading cause of mortality among preterm infants. Optimal macro parameters were determined with a test set of images from postnatal day 14 (P14) mice exposed to acute postnatal hyperoxia by determining which area and circularity values best correlated with mean linear intercept (LM). Validation was performed on a separate set of images from P7 mice subjected to the same hyperoxic model of BPD.ResultsBoth alveolar duct (r: 0.7866, p = 0.0359) and alveolar (r: 0.9475, p = 0.0012) area correlated with LM measurements from the test set. Using our method on a validation dataset, we demonstrate that hyperoxia-exposed mice possess fewer, enlarged alveoli that occupy less total area, as well as enlarged alveolar ducts that occupy a greater proportion of the parenchyma.ConclusionsWe report a semi-automated method of quantitating the characteristics of large and small terminal respiratory airways. This tool expedites analysis and removes operator bias relative to existing methods. We also demonstrate that LM changes in an acute model of hyperoxia-induced BPD result from both alveolar simplification and inadequate primary septation at the level of the alveolar ducts.

The BMPR2 gene encodes the BMPR-II (bone morphogenetic protein receptor type-II) and is a known regulator of endothelial proliferation, apoptosis, and translational stress responses. While these effects are generally attributed to the actions of BMPR-II protein, we used circular RNA profiling to identify circ3218 and circ5078 as new BMPR2-derived functional RNAs. Circular RNAs were profiled by ultradeep RNA sequencing of human pulmonary artery endothelial cells. Novel BMPR2-derived circular RNAs were assessed for their effects on endothelial proliferation, apoptosis, and translational stress responses in human pulmonary artery endothelial cells and endothelial cells from patients with pulmonary arterial hypertension with heterozygous loss-of-function BMPR2 mutations. circ5078 to linear BMPR2 mRNA ratios were quantified in cultured lymphocytes from patients with pulmonary arterial hypertension with BMPR2 mutations versus unaffected mutation carriers. Depletion of circ3218 enhanced human pulmonary artery endothelial cell apoptosis, whereas circ5078 silencing increased proliferation. Enhanced proliferation with circ5078 depletion was eliminated by cosilencing of circ5078 with either linear BMPR2 mRNA or the stress granule protein, Caprin-1, indicating a potential interdependence of BMPR2 transcripts in the regulation of endothelial function. Patients with pulmonary arterial hypertension with BMPR2 mutations exhibited increased circ5078 to linear BMPR2 mRNA ratios, alongside impaired stress responses in patient endothelial cells that were deficient in linear BMPR2 transcripts but not circ5078. circ5078 depletion enhanced stress responses in human pulmonary artery endothelial cells and rescued stress granule formation in patient endothelial cells, independent of BMPR-II protein levels. Assessment of translational regulation by polysome profiling did not identify any impact of linear or circular BMPR2 transcript loss on global protein synthesis or stress-induced eIF2α (eukaryotic initiation factor 2α) phosphorylation but did identify the enhanced translational efficiency of select nuclear-encoded mitochondrial ribosome proteins with circ5078 depletion, offering a link between mitochondrial function and the circ5078-deficient endothelial phenotype. The identification of circ3218 and circ5078 as novel BMPR2-derived gene products reveals interdependent roles for coding and noncoding BMPR2 transcripts as regulators of endothelial function.

Background & Aims We recently showed that a bacterial infection can break oral tolerance to food and lead to immunoglobulin E (IgE)-dependent mast cell activation and food-induced abdominal pain, which could constitute an important pathogenic mechanism in postinfectious irritable bowel syndrome (IBS). Here, we investigated whether similar immune mechanisms in response to psychological stress lead to food-evoked pain signaling, and thus potentially explain the pathophysiology in a larger group of patients with IBS. Methods Mice were exposed to ovalbumin (OVA) during water avoidance stress (WAS) and re-exposed to OVA 5 weeks later. Nociception was evaluated by visceromotor responses and afferent nerve recordings to intestinal distension, and patch-clamp recordings of sensory neurons incubated with intestinal supernatants. The role of IgE and type 2 immunity was evaluated using pharmacologic and genetic approaches. Results Re-exposure to OVA increased pain signaling in the colon and small intestine only in mice exposed to OVA during WAS, in the absence of systemic allergy. OVA-induced increases in pain responses depended on mast cells, IgE, and signal transducer and activator of transcription 6 signaling. Notably, incubation of sensory neurons with ileum and colon supernatants from WAS/OVA+OVA mice lowered their threshold of excitability. Finally, treatment with histamine receptor H1 antagonist pyrilamine blocked the increased sensory neuron excitability, and reduced ileal afferent nerve firing to distension in WAS/OVA+OVA mice. Conclusions Psychological stress induces a type 2 immune response to food antigens, with IgE-mediated mast cell activation and increased pain signaling in the small intestine and colon in response to food. These findings may explain the potential role of psychological stress in food-induced symptoms in IBS.

Bone morphogenetic protein-9 (BMP9) has been implicated as a regulator of metastasis and tumor angiogenesis, with contrasting studies demonstrating both pro- and antiangiogenic roles for BMP9 across different cancer cell lines and animal models. However, these works have yet to define the contribution of the type-II BMP receptor (BMPR-II) to these processes, or assess whether the effects of BMP9 are mediated via actions on the endothelium, the tumor, or its microenvironment. Here, we demonstrate that the heterozygous (Bmpr2EC+/-) or homozygous (Bmpr2EC-/-) deletion of BMPR-II in the pulmonary endothelium is associated with increased overall burden and vascularization of metastases in the lungs of mice subjected to the EO771 orthotopic engraftment model of metastatic breast cancer. These increases, relative to Bmpr2EC+/+ littermates, were observed despite equivalent primary mammary tumor growth across mice of all genotypes. In vitro, secreted factors or extracellular matrix components from BMPR-II-silenced human pulmonary arterial endothelial cells (HPAECs) did not alter EO771 proliferation relative to controls. However, endothelial BMPR-II depletion did eliminate the capacity of BMP9 to suppress both HPAEC migration to VEGF165 and EO771 transmigration across an HPAEC monolayer. In a tail vein injection model, the short-term establishment of EO771 cell metastatic lesions was equivalent in the lungs of female Bmpr2EC+/- and Bmpr2EC-/- mice, relative to Bmpr2EC+/+ controls, suggesting that the enhanced lung tumor burden observed in orthotopically implanted mice with endothelial Bmpr2 deletion is likely a consequence of enhanced tumor vascularization, rather than altered lung retention and engraftment. Our findings identify an important role for endothelial BMPR-II signaling in regulating the vascularization of metastatic lesions in the lungs.

Background Bronchopulmonary dysplasia (BPD) is a disease of neonatal lung development that is linked to impaired pulmonary vascularization, dysregulated transforming growth factor-β (TGF-β) signaling and the accumulation of senescent cells. Despite the established role for TGF-β signaling in promoting vascular remodeling and suppressing the senolytic activity of natural killer (NK) cells, the contribution of NK cell TGF-β signaling to postnatal lung patterning and the pathogenesis of BPD remains unclear. Methods Mice bearing an NK cell-selective deletion of the type-II TGF-β receptor ( Tgfbr2NK-/- ) were analyzed for vascular and alveolar structure, lung NK cell infiltration, senescence markers and lung function testing across neonatal and adult timepoints. Single-cell RNA sequencing of lung tissue from both neonatal mice and human infants with BPD was performed. The effect of enhanced NK cell activity in a hyperoxia-induced model of BPD was assessed in Tgfbr2NK-/- neonates, as well as pharmacologically, using the TGF-β ligand trap/IL-15 superagonist, HCW9218. Results Neonatal Tgfbr2NK-/- mice exhibited a baseline reduction in distal arteriolar density, impaired alveolarization, and sex-specific deficits in long-term lung function. Single-cell RNA sequencing identified the excessive clearance of senescent endothelial cells by TGF-β insensitive NK cells in the lungs of Tgfbr2NK-/- neonates, which served as a contributor of the BPD-like phenotype observed in naïve animals. Tgfbr2NK-/- mice were protected from impaired lung development in the hyperoxia model. Sequencing from lung tissue from infants with BPD confirmed excessive TGF-β signaling and cytotoxic impairment in NK cells. Treatment with HCW9218 prevented senescent cell accumulation and rescued lung development in the hyperoxia mouse model. Conclusions These findings identify TGF-β as a tunable regulator of NK cell senolytic activity that is essential to normal postnatal lung development. Excessive NK cell TGF-β signaling contributes to impaired lung development following exposure to neonatal hyperoxia and may serve as a viable therapeutic target for human BPD. ### Competing Interest Statement Hing Wong and Niraj Shrestha are employees of HCW Biologics.

The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues(1-3), the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes.We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.

Germline loss-of-function BMPR2 mutations are the leading genetic cause of pulmonary arterial hypertension (PAH) and are strongly linked to aberrant endothelial proliferation and impaired translational stress responses. While these effects are generally attributed to a loss of the type-II bone morphogenetic protein receptor (BMPR-II), we used circular RNA profiling to identify circ5078 , a BMPR2 -derived circular RNA that regulates endothelial translation and cellular phenotype. circ5078 and linear BMPR2 mRNA exert opposing effects on endothelial proliferation and stress granule formation, while influencing the translational efficiency of multiple genes by regulating ribosome assembly and translational initiation. In PAH patient-derived endothelial cells lacking linear BMPR2 mRNA, circ5078 depletion rescued impaired translational stress responses by rebalancing circular to linear transcript levels, independent of BMPR-II protein. By identifying circ5078 as a functional BMPR2 gene product, this work reveals interdependent roles for both linear and circular BMPR2 transcripts as regulators endothelial translation and proliferation.### Competing Interest StatementThe authors have declared no competing interest.

BACKGROUND:Drp1 (dynamin-related protein 1), a large GTPase, mediates the increased mitochondrial fission, which contributes to hyperproliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension (PAH). We developed a potent Drp1 GTPase inhibitor, Drpitor1a, but its specificity, pharmacokinetics, and efficacy in PAH are unknown.METHODS:Drpitor1a's ability to inhibit recombinant and endogenous Drp1 GTPase was assessed. Drpitor1a's effects on fission were studied in control and PAH human pulmonary artery smooth muscle cells (hPASMC) and blood outgrowth endothelial cells (BOEC). Cell proliferation and apoptosis were studied in hPASMC. Pharmacokinetics and tissue concentrations were measured following intravenous and oral drug administration. Drpitor1a's efficacy in regressing monocrotaline-PAH was assessed in rats. In a pilot study, Drpitor1a reduced PA remodeling only in females. Subsequently, we compared Drpitor1a to vehicles in control and monocrotaline-PAH females.RESULTS:Drp1 GTPase activity was increased in PAH hPASMC. Drpitor1a inhibited the GTPase activity of recombinant and endogenous Drp1 and reversed the increased fission, seen in PAH hPASMC and PAH BOEC. Drpitor1a inhibited proliferation and induced apoptosis in PAH hPASMC without affecting electron transport chain activity, respiration, fission/fusion mediator expression, or mitochondrial Drp1 translocation. Drpitor1a did not inhibit proliferation or alter mitochondrial dynamics in normal hPASMC. Drpitor1a regressed monocrotaline-PAH without systemic vascular effects or toxicity.CONCLUSIONS:Drpitor1a is a specific Drp1 GTPase inhibitor that reduces mitochondrial fission in PAH hPASMC and PAH BOEC. Drpitor1a reduces proliferation and induces apoptosis in PAH hPASMC and regresses monocrotaline-PAH. Drp1 is a therapeutic target in PAH, and Drpitor1a is a potential therapy with an interesting therapeutic sexual dimorphism.

Natural killer (NK) cell phenotype and function are altered in patients with prostate cancer, and increased NK cell activity is associated with a better prognosis in patients with disease. For patients with advanced stage prostate cancer, immunotherapies are a promising approach when standard treatment options have been exhausted. With the rapid emergence of NK cell-based therapies, it is important to understand the mechanisms by which NK cells can be triggered to kill cancer cells that have developed immune-evasive strategies. Altering the cytokine profiles of advanced prostate cancer cells may be an area to explore when considering ways in which NK cell activation can be modulated. We have previously demonstrated that combining the cytokine, IL-27, with TLR3 agonist, poly(I:C), changes cytokine secretion in the advanced prostate cancer models, PC3 and DU145 cells. Herein, we extend our previous work to study the effect of primary human NK cells on prostate cancer cell death in an in vitro co-culture model. Stimulating PC3 and DU145 cells with IL-27 and poly(I:C) induced IFN-β secretion, which was required for activation of primary human NK cells to kill these stimulated prostate cancer cells. PC3 cells were more sensitized to NK cell-mediated killing when compared to DU145 cells, which was attributed to differential levels of IFN-β produced in response to stimulation with IL-27 and poly(I:C). IFN-β increased granzyme B secretion and membrane-bound TRAIL expression by co-cultured NK cells. We further demonstrated that these NK cells killed PC3 cells in a partially TRAIL-dependent manner. This work provides mechanistic insight into how the cytotoxic function of NK cells can be improved to target cancer cells.

The tumor evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions occur frequently in human epithelia and can remodel almost entire tissues, the mechanisms behind why only a small number of clones transform into malignant tumors remain enigmatic. Here, we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing, guide capture and spatial transcriptomics to longitudinally monitor cell type-specific clonal expansions, document their underlying gene programs and contrast clonal expansions from tumor initiation. We uncover a TNF-α signaling module that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF-α signaling module is downregulated, and instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF-α gene program. By analyzing clonally expanded perturbations and their frequency in tumors, we demonstrate that the autocrine TNF-α gene program is associated with epithelial-mesenchymal transition (EMT) and is preexistent in a subpopulation of expanded epidermal stem cells, contributing to the predisposition for tumor initiation. Finally, we provide in vivo evidence that the epithelial TNF-α gene program is sufficient to mediate invasive properties of epidermal stem cells and show that the TNF-α signature correlates with shorter overall survival in human squamous cell carcinoma patients. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues and unveils distinct TNF-α programs in tumor evolution. Understanding the biology of clonal expansions in phenotypically normal epithelia and the mechanisms governing their transformation will guide the development of novel strategies for early cancer detection and therapy.

AbstractGermline loss-of-functionBMPR2mutations are the leading genetic cause of pulmonary arterial hypertension (PAH) and are strongly linked to aberrant endothelial proliferation and impaired translational stress responses. While these effects are generally attributed to a loss of the type-II bone morphogenetic protein receptor (BMPR-II), we used circular RNA profiling to identifycirc5078as a new functional RNA derived from exon 12 of theBMPR2gene.circ5078and linearBMPR2mRNA exert opposing effects on endothelial proliferation and stress granule formation, driving impaired stress responses in PAH patient-derived endothelial cells that are deficient in linearBMPR2transcripts, but notcirc5078. Rebalancing circular to linear transcript abundance bycirc5078depletion rescued stress granule formation in patient-derived endothelial cells, independent of BMPR-II protein levels. Polysome analysis demonstrated a reduction in free ribosomal subunits with the depletion of either linear or circularBMPR2transcripts, and an accumulation of 80S monosomes exclusively with linearBMPR2mRNA loss. These effects did not impact global protein synthesis or stress-induced eIF2α phosphorylation, but did alter the translational efficiency of multiple genes, including a group of nuclear encoded, mitochondrial ribosome proteins that were translationally enhanced withcirc5078silencing. Endothelialcirc5078depletion increased the efficiency of oxidative metabolism while reducing mitochondrial spare capacity, providing a potential link between mitochondrial function, proliferation and translational stress responses. Together, these findings reveal interdependent roles for linear and circularBMPR2transcripts as functional contributors to the endothelial phenotype of PAH.

Natural killer (NK) cells are cytotoxic group 1 innate lymphoid cells (ILC), known for their role as killers of stressed, cancerous, and virally infected cells. Beyond this cytotoxic function, NK cell subsets can influence broader immune responses through cytokine production and have been linked to central roles in non-immune processes, such as the regulation of vascular remodeling in pregnancy and cancer. Attempts to exploit the anti-tumor functions of NK cells have driven the development of various NK cell-based therapies, which have shown promise in both pre-clinical disease models and early clinical trials. However, certain elements of the tumor microenvironment, such as elevated transforming growth factor (TGF)-β, hypoxia, and indoalemine-2,3-dioxygenase (IDO), are known to suppress NK cell function, potentially limiting the longevity and activity of these approaches. Recent studies have also identified these factors as contributors to NK cell plasticity, defined by the conversion of classical cytotoxic NK cells into poorly cytotoxic, tissue-resident, or ILC1-like phenotypes. This review summarizes the current approaches for NK cell-based cancer therapies and examines the challenges presented by tumor-linked NK cell suppression and plasticity. Ongoing efforts to overcome these challenges are discussed, along with the potential utility of NK cell therapies to applications outside cancer.

The role for endothelial nitric oxide synthase (eNOS) in the pathogenesis of pulmonary arterial hypertension (PAH) is complex and multifaceted. Initially, it was reported that eNOS expression was profoundly reduced in the lung vasculature of patients with PAH [1]. The pairing of this reduction with a marked upregulation of endothelin-1 [2] evoked a straightforward and attractive paradigm of endothelial dysfunction in PAH in which an imbalance in the production of vasodilator and vasoconstrictor factors led to abnormal pulmonary vasoconstriction and arterial remodelling. However, subsequent studies were unable to confirm downregulation of lung vascular eNOS in similar PAH patient populations, and instead showed no change or a robust upregulation [3, 4], suggesting that increased nitric oxide (NO) production may in fact be involved in arterial remodelling in this disease via some unknown mechanism. The PHD2 deficient mouse model of nitrative stress provides an excellent opportunity to explore these important relationships, potentially providing critical insight into the pathogenesis of PAH https://bit.ly/3r66aBF

Pulmonary arterial hypertension (PAH) is a deadly disease, characterized by increased vascular resistance, pulmonary arteriolar loss, and occlusive arterial remodeling, leading to eventual right heart failure. Evidence increasingly points to the pulmonary endothelium as a central actor in PAH. Endothelial cell apoptosis can result directly in distal lung arteriolar pruning and indirectly in the formation of complex and occlusive arterial lesions, reflecting an imbalance between endothelial injury and repair in the development and progression of PAH. Many of the mutations implicated in PAH are in genes, which are predominantly, or solely, expressed in endothelial cells, and the endothelium is a major target for therapeutic interventions to restore BMP signaling. We explore how arterial pruning can promote the emergence of occlusive arterial remodeling mediated by ongoing endothelial injury secondary to hemodynamic perturbation and pathological increases in luminal shear stress. The emerging role of endothelial cell senescence is discussed in the transition from reversible to irreversible arterial remodeling in advanced PAH, and we review the sometimes conflicting evidence that female sex hormones can both protect or promote vascular changes in disease. Finally, we explore the contribution of the endothelium to metabolic changes and the altered inflammatory and immune state in the PAH lung, focusing on the role of excessive TGFβ signaling. Given the complexity of the endothelial pathobiology of PAH, we anticipate that emerging technologies that allow the study of molecular events at a single cell level will provide answers to many of the questions raised in this review.