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.
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•Activated Gasdermin D forms mitochondrial pores in the endothelium•Mitochondrial pore formation releases mitochondrial DNA into the cytosol•Released mtDNA activates cGAS signaling and suppresses vascular regeneration•Deletion of cGAS in an experimental model of polymicrobial sepsis reduces tissue injury
Sepsis induces profound vascular injury by promoting endothelial cell death, but the role of mitochondria in vascular endothelial injury is not well understood. Huang et al. demonstrate that the pore-forming molecule Gasdermin D releases mitochondrial DNA into the cytosol, which sets into motion an amplified response resulting in suppression of endothelial regeneration mediated by the cytosolic DNA sensor cGAS.
Increased pulmonary microvessel pressure experienced in left heart failure, head trauma, or high altitude can lead to endothelial barrier disruption referred to as capillary “stress failure” that ...causes leakage of protein-rich plasma and pulmonary edema. However, little is known about vascular endothelial sensing and transduction of mechanical stimuli inducing endothelial barrier disruption. Piezo1, a mechanosensing ion channel expressed in endothelial cells (ECs), is activated by elevated pressure and other mechanical stimuli. Here, we demonstrate the involvement of Piezo1 in sensing increased lung microvessel pressure and mediating endothelial barrier disruption. Studies were made in mice in which Piezo1 was deleted conditionally in ECs (Piezo1iΔEC), and lung microvessel pressure was increased either by raising left atrial pressure or by aortic constriction. We observed that lung endothelial barrier leakiness and edema induced by raising pulmonary microvessel pressure were abrogated in Piezo1iΔEC mice. Piezo1 signaled lung vascular hyperpermeability by promoting the internalization and degradation of the endothelial adherens junction (AJ) protein VE-cadherin. Breakdown of AJs was the result of activation of the calcium-dependent protease calpain and degradation of the AJ proteins VE-cadherin, β-catenin, and p120-catenin. Deletion of Piezo1 in ECs or inhibition of calpain similarly prevented reduction in the AJ proteins. Thus, Piezo1 activation in ECs induced by elevated lung microvessel pressure mediates capillary stress failure and edema formation secondary to calpain-induced disruption of VE-cadherin adhesion. Inhibiting Piezo1 signaling may be a useful strategy to limit lung capillary stress failure injury in response to elevated vascular pressures.
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.
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•Macrophage NLRP3 inflammasome activation is inhibited by potassium channel inhibition•TWIK2 is a potassium efflux channel required for NLRP3 inflammasome activation•Genetic deletion of TWIK2 prevents endotoxemia-induced inflammatory lung injury•P2X7 receptor and TWIK2 act in cooperation to regulate NLRP3 inflammasome activation
Potassium efflux is required for NLRP3 inflammasome activation, but the channel mediating the efflux has remained elusive. Di et al. identify the potassium channel TWIK2 as a mediator of potassium efflux and NLRP3 activation in macrophages. Targeting TWIK2 could form the basis for therapeutic approaches in inflammatory injury.
Acute lung injury is a leading cause of death in bacterial sepsis due to the wholesale destruction of the lung endothelial barrier, which results in protein-rich lung edema, influx of proinflammatory ...leukocytes, and intractable hypoxemia. Pyroptosis is a form of programmed lytic cell death that is triggered by inflammatory caspases, but little is known about its role in EC death and acute lung injury. Here, we show that systemic exposure to the bacterial endotoxin lipopolysaccharide (LPS) causes severe endothelial pyroptosis that is mediated by the inflammatory caspases, human caspases 4/5 in human ECs, or the murine homolog caspase-11 in mice in vivo. In caspase-11-deficient mice, BM transplantation with WT hematopoietic cells did not abrogate endotoxemia-induced acute lung injury, indicating a central role for nonhematopoietic caspase-11 in endotoxemia. Additionally, conditional deletion of caspase-11 in ECs reduced endotoxemia-induced lung edema, neutrophil accumulation, and death. These results establish the requisite role of endothelial pyroptosis in endotoxemic tissue injury and suggest that endothelial inflammatory caspases are an important therapeutic target for acute lung injury.
Blood vessels are lined by endothelial cells engaged in distinct organ-specific functions but little is known about their characteristic gene expression profiles. RNA-Sequencing of the brain, lung, ...and heart endothelial translatome identified specific pathways, transporters and cell-surface markers expressed in the endothelium of each organ, which can be visualized at http://www.rehmanlab.org/ribo. We found that endothelial cells express genes typically found in the surrounding tissues such as synaptic vesicle genes in the brain endothelium and cardiac contractile genes in the heart endothelium. Complementary analysis of endothelial single cell RNA-Seq data identified the molecular signatures shared across the endothelial translatome and single cell transcriptomes. The tissue-specific heterogeneity of the endothelium is maintained during systemic in vivo inflammatory injury as evidenced by the distinct responses to inflammatory stimulation. Our study defines endothelial heterogeneity and plasticity and provides a molecular framework to understand organ-specific vascular disease mechanisms and therapeutic targeting of individual vascular beds.
Mitochondrial fission, regulated by dynamin‐related protein‐1 (Drp1), is a newly recognized determinant of mitochondrial function, but its contribution to left ventricular (LV) impairment following ...ischemia‐reperfusion (IR) injury is unknown. We report that Drp1 activation during IR results in LV dysfunction and that Drp1 inhibition is beneficial. In both isolated neonatal murine cardiomyocytes and adult rat hearts (Langendorff preparation) mitochondrial fragmentation and swelling occurred within 30 min of IR Drp1‐S637 (serine 637) dephosphorylation resulted in Drp1 mitochondrial translocation and increased mitochondrial fission. The Drp1 inhibitor Mdivi‐1 preserved mitochondrial morphology, reduced cytosolic calcium, and prevented cell death. Drp1 siRNA similarly preserved mitochondrial morphology. In Langendorff hearts, Mdivi‐1 reduced mitochondrial reactive oxygen species, improved LV developed pressure (92±5 vs. 28±10 mmHg, P<0.001), and lowered LV end diastolic pressure (10±1 vs. 86±13 mmHg, P<0.001) following IR Mdivi‐1 was protective if administered prior to or following ischemia. Because Drp1‐S637 dephosphorylation is calcineurin sensitive, we assessed the effects of a calcineurin inhibitor, FK506. FK506 treatment prior to IR prevented Drp1‐S637 dephosphorylation and preserved cardiac function. Likewise, therapeutic hypothermia (30°C) inhibited Drp1‐S637 dephosphorylation and preserved mitochondrial morphology and myocardial function. Drp1 inhibition is a novel strategy to improve myocardial function following IR.—Sharp, W. S., Fang, Y. H., Han, M., Zhang, H. J., Hong, Z., Banathy, A., Morrow, E., Ryan, J. J., Archer, S. L. Dynamin‐related protein 1 (Drp1)‐mediated diastolic dysfunction in myocardial ischemia‐reperfusion injury: therapeutic benefits of Drp1 inhibition to reduce mitochondrial fission. FASEB J. 28, 316–326 (2014). www.fasebj.org
Mitochondria exist in dynamic networks that undergo fusion and fission. Mitochondrial fusion and fission are mediated by several GTPases in the outer mitochondrial membrane, notably mitofusin‐2 ...(Mfn‐2), which promotes fusion, and dynamin‐related protein (Drp‐1), which promotes fission. We report that human lung cancer cell lines exhibit an imbalance of Drp‐1/Mfn‐2 expression, which promotes a state of mitochondrial fission. Lung tumor tissue samples from patients demonstrated a similar increase in Drp‐1 and decrease in Mfn‐2 when compared to adjacent healthy lung. Complementary approaches to restore mitochondrial network formation in lung cancer cells by overexpression of Mfn‐2, Drp‐1 inhibition, or Drp‐1 knockdown resulted in a marked reduction of cancer cell proliferation and an increase in spontaneous apoptosis. The number of cancer cells in S phase decreased from 32.4 ± 0.6 to 6.4 ± 0.3% with Drp‐1 inhibition (P< 0.001). In a xenotransplantation model, Mfn‐2 gene therapy or Drp‐1 inhibition could regress tumor growth. The tumor volume decreased from 205.6 ± 59 to 70.6 ± 15 mm3 (P<0.05) with Mfn‐2 overexpression and from 186.0 ± 19 to 87.0 ± 6 mm3 (P<0.01) with therapeutic Drp‐1 inhibition. Impaired fusion and enhanced fission contribute fundamentally to the proliferation/apoptosis imbalance in cancer and constitute promising novel therapeutic targets.—Rehman, J., Zhang, H. J., Toth, P. T., Zhang, Y., Marsboom, G., Hong, Z., Salgia, R., Husain, A. N., Wietholt, C., Archer, S. L. Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer. FASEB J. 26, 2175‐2186 (2012). www.fasebj.org
Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This ...cancer-like phenotype is promoted by increased cytosolic calcium (Ca
), aerobic glycolysis, and mitochondrial fission.
To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH's cancer-like phenotype.
PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore-forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid-mediated up-regulation, as well as through modulation of the upstream microRNAs (miRs) miR-138 and miR-25. In vivo, nebulized anti-miRs were administered to rats with monocrotaline-induced PAH.
Impaired MCUC function, resulting from down-regulation of MCU and up-regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAH's pathogenesis. MCUC dysfunction decreases intramitochondrial calcium (Ca
), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing Ca
, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering Ca
, raising Ca
, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR-138) down-regulate MCU directly and also by decreasing MCU's transcriptional regulator cAMP response element-binding protein 1. Nebulized anti-miRs against miR-25 and miR-138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model.
These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted.
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 β-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.
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