trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

The angiocrine Rspondin3 instructs interstitial macrophage transition via metabolic-epigenetic reprogramming and resolves inflammatory injury.

Macrophages demonstrate remarkable plasticity that is essential for host defense and tissue repair. The tissue niche imprints macrophage identity, phenotype and function. The role of vascular endothelial signals in tailoring the phenotype and function of tissue macrophages remains unknown. The lung is a highly vascularized organ and replete with a large population of resident macrophages. We found that, in response to inflammatory injury, lung endothelial cells release the Wnt signaling modulator Rspondin3, which activates ß-catenin signaling in lung interstitial macrophages and increases mitochondrial respiration by glutaminolysis. The generated tricarboxylic acid cycle intermediate α-ketoglutarate, in turn, serves as the cofactor for the epigenetic regulator TET2 to catalyze DNA hydroxymethylation. Notably, endothelial-specific deletion of Rspondin3 prevented the formation of anti-inflammatory interstitial macrophages in endotoxemic mice and induced unchecked severe inflammatory injury. Thus, the angiocrine-metabolic-epigenetic signaling axis specified by the endothelium is essential for reprogramming interstitial macrophages and dampening inflammatory injury.

mtDNA Activates cGAS Signaling and Suppresses the YAP-Mediated Endothelial Cell Proliferation Program to Promote Inflammatory Injury.

Cytosolic DNA acts as a universal danger-associated molecular pattern (DAMP) signal; however, the mechanisms of self-DNA release into the cytosol and its role in inflammatory tissue injury are not well understood. We found that the internalized bacterial endotoxin lipopolysaccharide (LPS) activated the pore-forming protein Gasdermin D, which formed mitochondrial pores and induced mitochondrial DNA (mtDNA) release into the cytosol of endothelial cells. mtDNA was recognized by the DNA sensor cGAS and generated the second messenger cGAMP, which suppressed endothelial cell proliferation by downregulating YAP1 signaling. This indicated that the surviving endothelial cells in the penumbrium of the inflammatory injury were compromised in their regenerative capacity. In an experimental model of inflammatory lung injury, deletion of cGas in mice restored endothelial regeneration. The results suggest that targeting the endothelial Gasdermin D activated cGAS-YAP signaling pathway could serve as a potential strategy for restoring endothelial function after inflammatory injury.

The TWIK2 Potassium Efflux Channel in Macrophages Mediates NLRP3 Inflammasome-Induced Inflammation.

Potassium (K+) efflux across the plasma membrane is thought to be an essential mechanism for ATP-induced NLRP3 inflammasome activation, yet the identity of the efflux channel has remained elusive. Here we identified the two-pore domain K+ channel (K2P) TWIK2 as the K+ efflux channel triggering NLRP3 inflammasome activation. Deletion of Kcnk6 (encoding TWIK2) prevented NLRP3 activation in macrophages and suppressed sepsis-induced lung inflammation. Adoptive transfer of Kcnk6-/- macrophages into mouse airways after macrophage depletion also prevented inflammatory lung injury. The K+ efflux channel TWIK2 in macrophages has a fundamental role in activating the NLRP3 inflammasome and consequently mediates inflammation, pointing to TWIK2 as a potential target for anti-inflammatory therapies.

Hematopoietic Jagged1 is a fetal liver niche factor required for functional maturation and engraftment of fetal hematopoietic stem cells.

Notch signaling is essential for the emergence of definitive hematopoietic stem cells (HSCs) in the embryo and their development in the fetal liver niche. However, how Notch signaling is activated and which fetal liver cell type provides the ligand for receptor activation in HSCs is unknown. Here we provide evidence that endothelial Jagged1 (Jag1) has a critical early role in fetal liver vascular development but is not required for hematopoietic function during fetal HSC expansion. We demonstrate that Jag1 is expressed in many hematopoietic cells in the fetal liver, including HSCs, and that its expression is lost in adult bone marrow HSCs. Deletion of hematopoietic Jag1 does not affect fetal liver development; however, Jag1-deficient fetal liver HSCs exhibit a significant transplantation defect. Bulk and single-cell transcriptomic analysis of HSCs during peak expansion in the fetal liver indicates that loss of hematopoietic Jag1 leads to the downregulation of critical hematopoietic factors such as GATA2, Mllt3, and HoxA7, but does not perturb Notch receptor expression. Ex vivo activation of Notch signaling in Jag1-deficient fetal HSCs partially rescues the functional defect in a transplant setting. These findings indicate a new fetal-specific niche that is based on juxtracrine hematopoietic Notch signaling and reveal Jag1 as a fetal-specific niche factor essential for HSC function.

Engineered Wnt7a ligands rescue blood-brain barrier and cognitive deficits in a COVID-19 mouse model.

Respiratory infection with SARS-CoV-2 causes systemic vascular inflammation and cognitive impairment. We sought to identify the underlying mechanisms mediating cerebrovascular dysfunction and inflammation following mild respiratory SARS-CoV-2 infection. To this end, we performed unbiased transcriptional analysis to identify brain endothelial cell signalling pathways dysregulated by mouse adapted SARS-CoV-2 MA10 in aged immunocompetent C57Bl/6 mice in vivo. This analysis revealed significant suppression of Wnt/ß-catenin signalling, a critical regulator of blood-brain barrier (BBB) integrity. We therefore hypothesized that enhancing cerebrovascular Wnt/ß-catenin activity would offer protection against BBB permeability, neuroinflammation, and neurological signs in acute infection. Indeed, we found that delivery of cerebrovascular-targeted, engineered Wnt7a ligands protected BBB integrity, reduced T-cell infiltration of the brain, and reduced microglial activation in SARS-CoV-2 infection. Importantly, this strategy also mitigated SARS-CoV-2 induced deficits in the novel object recognition assay for learning and memory and the pole descent task for bradykinesia. These observations suggest that enhancement of Wnt/ß-catenin signalling or its downstream effectors could be potential interventional strategies for restoring cognitive health following viral infections.

Notch transcriptional target tmtc1 maintains vascular homeostasis.

Proper lung function requires the maintenance of a tight endothelial barrier while simultaneously permitting the exchange of macromolecules and fluids to underlying tissue. Disruption of this barrier results in an increased vascular permeability in the lungs, leading to acute lung injury. In this study, we set out to determine whether transcriptional targets of Notch signaling function to preserve vascular integrity. We tested the in vivo requirement for Notch transcriptional signaling in maintaining the pulmonary endothelial barrier by using two complementary endothelial-specific Notch loss-of-function murine transgenic models. Notch signaling was blocked using endothelial-specific activation of an inhibitor of Notch transcriptional activation, Dominant Negative Mastermindlike (DNMAML; CDH5CreERT2), or endothelial-specific loss of Notch1 (Notch1f/f; CDH5CreERT2). Both Notch mutants increased vascular permeability with pan-Notch inhibition by DNMAML showing a more severe phenotype in the lungs and in purified endothelial cells. RNA sequencing of primary lung endothelial cells (ECs) identified novel Notch targets, one of which was transmembrane O-mannosyltransferase targeting cadherins 1 (tmtc1). We show that tmtc1 interacts with vascular endothelial cadherin (VE-cadherin) and regulates VE-cadherin egress from the endoplasmic reticulum through direct interaction. Our findings demonstrate that Notch signaling maintains endothelial adherens junctions and vascular homeostasis by a transcriptional mechanism that drives expression of critical factors important for processing and transport of VE-cadherin.

Nanoparticle targeting of de novo profibrotic macrophages mitigates lung fibrosis.

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)ß1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFß1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFß-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

A Bayesian inference transcription factor activity model for the analysis of single-cell transcriptomes.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful experimental approach to study cellular heterogeneity. One of the challenges in scRNA-seq data analysis is integrating different types of biological data to consistently recognize discrete biological functions and regulatory mechanisms of cells, such as transcription factor activities and gene regulatory networks in distinct cell populations. We have developed an approach to infer transcription factor activities from scRNA-seq data that leverages existing biological data on transcription factor binding sites. The Bayesian inference transcription factor activity model (BITFAM) integrates ChIP-seq transcription factor binding information into scRNA-seq data analysis. We show that the inferred transcription factor activities for key cell types identify regulatory transcription factors that are known to mechanistically control cell function and cell fate. The BITFAM approach not only identifies biologically meaningful transcription factor activities, but also provides valuable insights into underlying transcription factor regulatory mechanisms.

Engineered ACE2 decoy mitigates lung injury and death induced by SARS-CoV-2 variants.

Vaccine hesitancy and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) escaping vaccine-induced immune responses highlight the urgency for new COVID-19 therapeutics. Engineered angiotensin-converting enzyme 2 (ACE2) proteins with augmented binding affinities for SARS-CoV-2 spike (S) protein may prove to be especially efficacious against multiple variants. Using molecular dynamics simulations and functional assays, we show that three amino acid substitutions in an engineered soluble ACE2 protein markedly augmented the affinity for the S protein of the SARS-CoV-2 WA-1/2020 isolate and multiple VOCs: B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). In humanized K18-hACE2 mice infected with the SARS-CoV-2 WA-1/2020 or P.1 variant, prophylactic and therapeutic injections of soluble ACE22.v2.4-IgG1 prevented lung vascular injury and edema formation, essential features of CoV-2-induced SARS, and above all improved survival. These studies demonstrate broad efficacy in vivo of an engineered ACE2 decoy against SARS-CoV-2 variants in mice and point to its therapeutic potential.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Complement C1q essential for aeroallergen sensitization via CSF1R+ conventional dendritic cells type 2.

BACKGROUND: Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens. OBJECTIVE: It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization. METHODS: We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q. RESULTS: We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s. CONCLUSIONS: C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1).

La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.

Mechanisms of Lung Injury Induced by SARS-CoV-2 Infection.

The lung is the major target organ of SARS-CoV-2 infection, which causes COVID-19. Here, we outline the multistep mechanisms of lung epithelial and endothelial injury induced by SARS-CoV-2: direct viral infection, chemokine/cytokine-mediated damage, and immune cell-mediated lung injury. Finally, we discuss the recent progress in terms of antiviral therapeutics as well as the development of anti-inflammatory or immunomodulatory therapeutic approaches. This review also provides a systematic overview of the models for studying SARS-CoV-2 infection and discusses how an understanding of mechanisms of lung injury will help identify potential targets for future drug development to mitigate lung injury.

Inhibition of IRAK4 dysregulates SARS-CoV-2 spike protein-induced macrophage inflammatory and glycolytic reprogramming.

Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (MÏ´) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human MÏ´s exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated MÏ´s nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of MÏ´s by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated MÏ´s, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated MÏ´s was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human MÏ´s as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated MÏ´ hyperactivation. IRAK4i therapy counteracts MÏ´ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human MÏ´s. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.

Increased mitochondrial function downstream from KDM5A histone demethylase rescues differentiation in pRB-deficient cells.

The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype.

Quantitative Pulmonary Neutrophil Dynamics Using Computer-Vision Stabilized Intravital Imaging.

In vivo intravital imaging in animal models in the lung remains challenging owing to respiratory motion artifacts. Here we describe a novel intravital imaging approach based on the computer-vision stabilization algorithm, Computer-Vision Stabilized Intravital Imaging. This method corrects lung movements and deformations at submicron precision in respiring mouse lungs. The precision enables high-throughput quantitative analysis of intravital pulmonary polymorphonuclear neutrophil (PMN) dynamics in lungs. We quantified real-time PMN patrolling dynamics of microvessels in the basal state and PMN recruitment resulting from sequestration in a model of endotoxemia in mice. We focused on determining the marginated pool of PMNs in the lung. Direct visualization of marginated PMNs revealed that they are not static but highly dynamic and undergo repeated cycles of "catch and release." PMNs briefly arrest in larger diameter capillary junction (∼10 µm) and then squeeze into narrower, approximately 5-µm diameter vessels through PMN deformation. We also observed that the sequestered PMNs in lung microvessels lost their migratory capabilities in association with cell morphological change following prolonged endotoxemia. These observations underscore the value of direct visualization and quantitative analysis of PMN dynamics in lungs to study PMN physiology and pathophysiology and role in inflammatory lung injury.

Notch1 promotes ordered revascularization through Semaphorin 3g modulation of downstream vascular patterning signalling factors.

Here we genetically and functionally addressed potential pathways of Notch signalling in mediating vascular regeneration in mouse models. We first used transgenic adult mice with either gain- or loss-of-function Notch signalling in vascular endothelial cells and monitored perfusion in the hindlimb following ischaemia induced by femoral artery ligation. Mice deficient in Notch signalling showed defective perfusion recovery and expansion of collateral arteries. Transcriptomics analysis of arterial endothelial cells in the Notch mutants identified the guidance factor Sema3g as a candidate gene mediating reperfusion downstream of Notch. Studies in the retinal circulation showed the central role of SEMA3G downstream of Notch signalling in the orderly regulation of vascular patterning. These studies in multiple vascular beds show the primacy of Notch signalling and downstream generation of guidance peptides such as SEMA3G in promoting well-ordered vascular regeneration. KEY POINTS: Notch signalling is a critical mediator of revascularization. Yet the cellular processes activated during recovery following vascular injury are incompletely understood. Here we used genetic and cellular approaches in two different vascular beds and cultured endothelial cells to address the generalizability of mechanisms. By utilizing a highly reproducible murine model of hindlimb ischaemia in transgenic mice in which Notch signalling was inhibited at the transcriptional level, we demonstrated the centrality of Notch signalling in perfusion recovery and revascularization. RNA-sequencing of Notch mutants identified class 3 Semaphorins regulated by Notch signalling as downstream targets. Studies in retinal vessels and endothelial cells showed an essential role of guidance peptide Sema3g as a modulator of angiogenesis and orderly vascular patterning. The Notch to Sema3g signalling axis functions as a feedback mechanism to sculpt the growing vasculature in multiple beds.

Mitophagy mediates metabolic reprogramming of induced pluripotent stem cells undergoing endothelial differentiation.

Pluripotent stem cells are known to shift their mitochondrial metabolism upon differentiation, but the mechanisms underlying such metabolic rewiring are not fully understood. We hypothesized that during differentiation of human induced pluripotent stem cells (hiPSCs), mitochondria undergo mitophagy and are then replenished by the biogenesis of new mitochondria adapted to the metabolic needs of the differentiated cell. To evaluate mitophagy during iPSC differentiation, we performed live cell imaging of mitochondria and lysosomes in hiPSCs differentiating into vascular endothelial cells using confocal microscopy. We observed a burst of mitophagy during the initial phases of hiPSC differentiation into the endothelial lineage, followed by subsequent mitochondrial biogenesis as assessed by the mitochondrial biogenesis biosensor MitoTimer. Furthermore, hiPSCs undergoing differentiation showed greater mitochondrial oxidation of fatty acids and an increase in ATP levels as assessed by an ATP biosensor. We also found that during mitophagy, the mitochondrial phosphatase PGAM5 is cleaved in hiPSC-derived endothelial progenitor cells and in turn activates ß-catenin-mediated transcription of the transcriptional coactivator PGC-1α, which upregulates mitochondrial biogenesis. These data suggest that mitophagy itself initiates the increase in mitochondrial biogenesis and oxidative metabolism through transcriptional changes during endothelial cell differentiation. In summary, these findings reveal a mitophagy-mediated mechanism for metabolic rewiring and maturation of differentiating cells via the ß-catenin signaling pathway. We propose that such mitochondrial-nuclear cross talk during hiPSC differentiation could be leveraged to enhance the metabolic maturation of differentiated cells.

Interleukin-1RA Mitigates SARS-CoV-2-Induced Inflammatory Lung Vascular Leakage and Mortality in Humanized K18-hACE-2 Mice.

[Figure: see text].