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For clinically low-risk stage III colorectal cancer, the decision on cycles of adjuvant chemotherapy after surgery is disputed. The present study investigates the use of additional biomarkers of ploidy and stroma-ratio(PS) to stratify patients with low-risk stage III colorectal cancer, providing a basis for individualized treatment in the future.This study retrospectively enrolled 198 patients with clinical-low-risk stage III colorectal cancer (T1-3N1M0) and analyzed the DNA ploidy and stroma ratio of FFPE tumor tissues. The patients were divided into PS-low-risk group (Diploidy or Low-stroma) and PS-high-risk group (Non-diploid and High-stroma). For survival analyses, Kaplan-Meier and Cox regression models were used.The results showed that the 5-year DFS of the PS-high-risk group was significantly lower than that in the PS-low-risk group (78.6 vs. 91.2%, HR = 2.606 [95% CI: 1.011-6.717], P = 0.039). Besides, in the PS-low-risk group, the 5 year OS (98.2 vs. 86.7%, P = 0.022; HR = 5.762 [95% CI: 1.281-25.920]) and DFS (95.6, vs 79.9%, P = 0.019; HR = 3.7 [95% CI: 1.24-11.04]) of patients received adjuvant chemotherapy for 3 months were significantly higher than those received adjuvant chemotherapy for 3 months. We also found that the PS could stratify the prognosis of patients with dMMR tumors. The 5-year OS (96.3 vs 71.4%, P = 0.037) and DFS (92.6 vs 57.1%, P = 0.015) were higher in the PS-low-risk dMMR patients than those in the PS-high-risk dMMR patients.In this study, we found that PS can predict the prognosis of patients with stage III low-risk CRC. Besides, it may guide the decision on postoperative adjuvant chemotherapy.

The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (∼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils

Endothelial adherens junctions (AJs) are critical for the regulation of vascular barrier integrity and undergo dis-assembly during inflammatory injury, thus causing vascular leakiness. AJ re-assembly is thus necessary for restoration of the endothelial barrier following the initial injury. Here we examine the metabolic underpinnings that drive restoration of vascular integrity. In response to inflammatory stimuli, the glycolysis regulatory enzyme PFKFB3 is activated, resulting in a rapid and sustained increase of intracellular glycolytic ATP, especially in the proximity of AJs at the plasma membrane. We engineered a novel chemo-genetic construct (RapT) which allowed for precise temporal control of PFKFB3 recruitment to the plasma membrane. Activation of RapT by rapamycin during the barrier restoration phase increased regional ATP and accelerated AJ re-assembly. Mechanistically, we observed that PFKFB3 is activated through two modes. Src-mediated post-translational phosphorylation rapidly increases PFKFB3 activity. Using another chemo-genetic approach to temporally control Src activity, we found that Src activates PFKFB3 by binding to and phosphorylating it at residues Y175, Y334, and Y363. Tyrosine-phospho-deficient mutants of PFKFB3 at these residues block the glycolytic activation upon inflammatory stimuli. In parallel, elevated reactive oxygen species generated during inflammatory stimulation create pockets of regional hypoxia and allow for increased Hif1α-mediated transcription of PFKFB3, leading to sustained glycolytic activation. Moreover, inhibition of PFKFB3 delays AJ reassembly and restoration of vascular integrity both in vitro and in vivo. In conclusion, we show that while inflammatory activation acutely compromises the endothelial barrier, inflammatory signaling also concomitantly generates a metabolic milieu in anticipation of the subsequent re-assembly of AJs and restoration of the vascular barrier.

Rationale: Right ventricular (RV) fibrosis in pulmonary arterial hypertension contributes to RV failure. While RV fibrosis reflects changes in the function of resident RV fibroblasts (RVfib), these cells are understudied. Objective: Examine the role of mitochondrial metabolism of RVfib in RV fibrosis in human and experimental pulmonary arterial hypertension. Methods and Results: Male Sprague-Dawley rats received monocrotaline (MCT; 60 mg/kg) or saline. Drinking water containing no supplement or the PDK (pyruvate dehydrogenase kinase) inhibitor dichloroacetate was started 7 days post-MCT. At week 4, treadmill testing, echocardiography, and right heart catheterization were performed. The effects of PDK activation on mitochondrial dynamics and metabolism, RVfib proliferation, and collagen production were studied in RVfib in cell culture. Epigenetic mechanisms for persistence of the profibrotic RVfib phenotype in culture were evaluated. PDK expression was also studied in the RVfib of patients with decompensated RV failure (n=11) versus control (n=7). MCT rats developed pulmonary arterial hypertension, RV fibrosis, and RV failure. MCT-RVfib (but not left ventricular fibroblasts) displayed excess mitochondrial fission and had increased expression of PDK isoforms 1 and 3 that persisted for >5 passages in culture. PDK-mediated decreases in pyruvate dehydrogenase activity and oxygen consumption rate were reversed by dichloroacetate (in RVfib and in vivo) or siRNA targeting PDK 1 and 3 (in RVfib). These interventions restored mitochondrial superoxide and hydrogen peroxide production and inactivated HIF (hypoxia-inducible factor)-1α, which was pathologically activated in normoxic MCT-RVfib. Redox-mediated HIF-1α inactivation also decreased the expression of TGF-β1 (transforming growth factor-beta-1) and CTGF (connective tissue growth factor), reduced fibroblast proliferation, and decreased collagen production. HIF-1α activation in MCT-RVfib reflected increased DNMT (DNA methyltransferase) 1 expression, which was associated with a decrease in its regulatory microRNA, miR-148b-3p. In MCT rats, dichloroacetate, at therapeutic levels in the RV, reduced phospho-pyruvate dehydrogenase expression, RV fibrosis, and hypertrophy and improved RV function. In patients with pulmonary arterial hypertension and RV failure, RVfib had increased PDK1 expression. Conclusions: MCT-RVfib manifest a DNMT1-HIF-1α-PDK–mediated, chamber-specific, metabolic memory that promotes collagen production and RV fibrosis. This epigenetic mitochondrial-metabolic pathway is a potential antifibrotic therapeutic target.

Disruption of alveolar-capillary barriers is a major complication of high-volume mechanical ventilation referred to as "ventilator-induced lung injury." The stretching force in alveoli is transmitted to endothelial cells, increasing the tension on underlying endothelial plasma membrane. The mechanosensor Piezo1, a plasma membrane cation channel, was inducibly deleted in endothelial cells of mice (Piezo1iEC-/-), which allowed us to study its role in regulating the endothelial barrier response to alveolar stretch. We observed significant increase in lung vascular permeability in Piezo1iEC-/- mice as compared with control Piezo1fl/fl mice in response to high-volume mechanical ventilation. We also observed that human lung endothelial monolayers depleted of Piezo1 and exposed to cyclic stretch had increased permeability. We identified the calcium-dependent cysteine protease calpain as a downstream target of Piezo1. Furthermore, we showed that calpain maintained stability of the endothelial barrier in response to mechanical stretch by cleaving Src kinase, which was responsible for disassembling endothelial adherens junctions. Pharmacological activation of calpain caused Src cleavage and thereby its inactivation, and it restored the disrupted lung endothelial barrier seen in Piezo1iEC-/- mice undergoing high-volume mechanical ventilation. Our data demonstrate that downregulation of Piezo1 signaling in endothelium is a critical factor in the pathogenesis of ventilator-induced lung injury, and thus augmenting Piezo1 expression or pharmacologically activating Piezo1 signaling may be an effective therapeutic strategy.

Significance Increased hydrostatic pressure in lung capillaries experienced during high altitude, head trauma, and left heart failure can lead to disruption of lung endothelial barrier and edema formation. We identified Piezo1 as a mechanical sensor responsible for endothelial barrier breakdown (barotrauma) secondary to reduced expression of the endothelial adherens junction proteins VE-cadherin, β-catenin, and p120-catenin. Endothelial-specific deletion or pharmacological inhibition of Piezo1 prevented lung capillary leakage, suggesting a therapeutic approach for preventing edema and associated lung failure., 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.

Polymorphonuclear neutrophils (PMN) are highly dynamic innate immune cells which are essential for lung host defense. However, in vivo intravital imaging in moving organs such as the lung remains challenging due to motion artifacts. Here we describe a novel intravital imaging method with high-throughput analytical capability based on a computer vision stabilization algorithm, Computer-vision-Assisted STabilized intravital imaging (CASTii). The sub-micron precision of this approach enables analysis of compartmentalized intravital PMN dynamics. We quantified in real-time a novel patrolling function of lung intracapillary circulating PMN. We also describe the dynamics of intracapillary PMN pooling (marginated PMN pool) using direct imaging of PMNs. The pool was formed by repeated catch-and-release kinetics involving PMN deformation inside microvessels during the passage of PMNs in vessels. We observed rapid PMN recruitment into the lung tissue compartments from pooled PMNs in response to alveolar chemoattract exposure. In contrast, endotoxemia-induced intracapillary sequestration of PMN impaired PMN transmigration into the alveolar space and defective phagocytosis of live bacteria. Intravital imaging of PMN dynamics with CASTii provides fundamental insights into host-defense functions of lung capillary PMN.

As a kind of secondary tumor of the ovary, ovarian metastasis from colorectal cancer (OMCRC) happens rarely. Prognostic factors of OMCRC are still undetermined. This study was conducted to analyze clinical characteristics and prognostic factors of OMCRC patients. Data of patients with OMCRC were collected retrospectively from four large-capacity hospitals in China. Kaplan-Meier method was applied to estimate disease-specific overall survival (OS), and multivariate Cox regression analysis was used to identify prognostic factors. A novel nomogram was developed to estimate individual survival probability, whose performance was internally validated using concordance index (C-index) and calibration curve. Totally, 162 cases were eligible, with a median age at diagnosis of 49 years old. The median size of ovarian metastases was 9.0 cm (95% CI: 8.5–10.4 cm). 93.8% of patients received surgery of ovarian metastases. Median time from CRC diagnosis to metachronous ovarian metastasis was 13.0 months (95% CI: 13.5–17.7 months). Median OS after ovarian metastasis diagnosis was 26.0 months (95% CI: 22.3–29.7 months). Integrating univariate and multivariate analyses, eight factors (including age, menopausal status, primary tumor location, N stage of primary tumor, surgery of primary tumor, differentiation grade, bilateral metastasis, and systemic chemotherapy) were used to develop a novel nomogram, with a C-index of 0.65 (95% CI: 0.595–0.705). Calibration curves indicated relatively good agreement between predicted and actual survival. This nomogram could be a promising tool to help clinicians to estimate individual survival outcome of patients with OMCRC. Further study is warranted to validate the practicality of this model.

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. The angiocrine Rspondin3 is produced by endothelial cells (ECs) and controls growth and development. Malik and colleagues show that lung ECs produce Rspondin3 following injury and specifically direct interstitial macrophages into an anti-inflammatory and wound-healing program.

Inefficient delivery is a major obstacle to the development of peptide-based drugs targeting the intracellular compartment. We recently showed that selectively inhibiting integrin outside-in signaling using a peptide (mP6) derived from the Gα13-binding ExE motif within the integrin β3 cytoplasmic domain had antithrombotic effects. Here, we engineered lipid-stabilized, high-loading peptide nanoparticles (HLPN), in which a redesigned ExE peptide (M3mP6) constituted up to 70% of the total nanoparticle molarity, allowing efficient in vivo delivery. We observed that M3mP6 HLPN inhibited occlusive thrombosis more potently than a clopidogrel/aspirin combination without adverse effects on hemostasis in rodents. Furthermore, M3mP6 HLPN synergized with P2Y12 receptor inhibitors or the clopidogrel/aspirin combination in preventing thrombosis, without exacerbating hemorrhage. M3mP6 HLPN also inhibited intravascular coagulation more potently than the P2Y12 inhibitor cangrelor. Postischemia injection of M3mP6 HLPN protected the heart from myocardial ischemia-reperfusion injury in a mouse model. This study demonstrates an efficient in vivo peptide delivery strategy for a therapeutic that not only efficaciously prevented thrombosis with minimal bleeding risk but also protected from myocardial ischemia-reperfusion injury in mice.

BRAF and MLH1 promoter methylation testings have been proven effective prescreens for Lynch Syndrome. We aimed to compare different screening strategies for Lynch Syndrome in patients with MLH1(-) CRC. Patients with MLH1(-) CRC who had been tested for BRAF mutation and germline variants of DNA mismatch repair genes were included. We compared the sensitivities and specificities for identifying Lynch Syndrome and the cost-effectiveness of four screening approaches that used the following tests as prescreens: BRAF testing, MLH1 methylation testing, MLH1 methylation & BRAF testing, and MLH1 methylation testing & Revised Bethesda Criteria. Of 109 patients included, 23 (21.1%) were Lynch Syndrome patients. BRAF mutation and MLH1 methylation occurred in 6 (5.5%) and 40 (36.7%) patients, respectively. The sensitivity for identifying Lynch syndrome of BRAF testing was 100%, but the specificity was only 7%. MLH1 methylation testing had a lower sensitivity than BRAF testing (97.5% vs 100%), but had a markedly higher specificity (45.3% vs 7%). The combination of the two testings had a slightly higher specificity than MLH1 methylation testing alone (47.7% vs 45.3%). The MLH1 methylation testing approach had a 10% lower cost of identifying MLH1(-) Lynch syndrome carriers per case than universal genetic testing, but it missed 4.5% of patients. BRAF and MLH1 promoter methylation testings as prescreens for Lynch syndrome are less effective in Chinese patients with MLH1(-) CRC than in their Western counterparts. Universal genetic testing could be considered an up-front option for this population.

Inflammatory tissue injury such as acute lung injury (ALI) is a disorder that leads to respiratory failure, a major cause of morbidity and mortality worldwide. Excessive neutrophil influx is a critical pathogenic factor in the development of ALI. Here, we identify the subset of neutrophils that is responsible for ALI and lethality in polymicrobial sepsis. The pro-inflammatory neutrophil subpopulation was characterized by its unique ability to endocytose albumin nanoparticles (ANP), upregulation of pro-inflammatory cytokines and chemokines as well as the excessive production of reactive oxygen species (ROS) in models of endotoxemia and septicemia. ANP delivery of the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, to the susceptible subset of neutrophils, prevented ALI and mortality in mice subjected to polymicrobial infection. Targeted inhibition of Syk in ANP-susceptible neutrophils had no detrimental effect on neutrophil-dependent host defense because the subset of ANPlowneutrophils effectively controlled polymicrobial infection. The results show that neutrophil heterogeneity can be leveraged therapeutically to prevent ALI without compromising host defense.

Unchecked inflammation is a hallmark of inflammatory tissue injury in diseases such as acute respiratory distress syndrome (ARDS). Yet the mechanisms of inflammatory lung injury remain largely unknown. Here we showed that bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation and puncture-induced (CLP-induced) polymicrobial sepsis decreased the expression of transcription factor cAMP response element binding (CREB) in lung endothelial cells. We demonstrated that endothelial CREB was crucial for VE-cadherin transcription and the formation of the normal restrictive endothelial adherens junctions. The inflammatory cytokine IL-1β reduced cAMP generation and CREB-mediated transcription of VE-cadherin. Furthermore, endothelial cell-specific deletion of CREB induced lung vascular injury whereas ectopic expression of CREB in the endothelium prevented the injury. We also observed that rolipram, which inhibits type 4 cyclic nucleotide phosphodiesterase-mediated (PDE4-mediated) hydrolysis of cAMP, prevented endotoxemia-induced lung vascular injury since it preserved CREB-mediated VE-cadherin expression. These data demonstrate the fundamental role of the endothelial cAMP-CREB axis in promoting lung vascular integrity and suppressing inflammatory injury. Therefore, strategies aimed at enhancing endothelial CREB-mediated VE-cadherin transcription are potentially useful in preventing sepsis-induced lung vascular injury in ARDS.

How Can Green Tax Laws Spur Green Behavior? An Analysis of the Influence of Chinese Tax Policy on Green Behavior

Whole exome sequencing (WES) was used in the research of familial pulmonary arterial hypertension (FPAH). CAV1 and KCNK3 were found as two novel candidate genes of FPAH. However, few pathogenic genes were identified in idiopathic pulmonary arterial hypertension (IPAH). We conducted WES in 20 unrelated IPAH patients who did not carry the known PAH-pathogenic variants among BMPR2, CAV1, KCNK3, SMAD9, ALK1, and ENG. We found a total of 4,950 variants in 3,534 genes, including 4,444 single-nucleotide polymorphisms and 506 insertions/deletions (InDels). Through the comprehensive and multilevel analysis, we disclosed several novel signaling cascades significantly connected to IPAH, including variants related to cadherin signaling pathway, dilated cardiomyopathy, glucose metabolism, immune response, mucin-type O-glycosylation, phospholipase C (PLC)-activating G protein-coupled receptor (GPCR) signaling pathway, vascular contraction and generation, and voltage-dependent Ca2+ channels. We also conducted validation studies in five mutant genes related to PLC-activating GPCR signaling pathway potentially involved in intracellular calcium regulation through Sanger sequencing for mutation accuracy, qRT-PCR for mRNA stability, immunofluorescence for subcellular localization, Western blotting for protein level, Fura-2 imaging for intracellular calcium, and proliferation analysis for cell function. The validation experiments showed that those variants in CCR5 and C3AR1 significantly increased the rise of intracellular calcium and the variant in CCR5 profoundly enhanced proliferative capacity of human pulmonary artery smooth muscle cells. Thus, our study suggests that multiple genetically affected signaling pathways take effect together to cause the formation of IPAH and the development of right heart failure and may further provide new therapy targets or putative clues for the present treatments such as limited therapeutic effectiveness of Ca2+ channel blockers.

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., eLife digest Blood vessels supply nutrients, oxygen and other key molecules to all of the organs in the body. Cells lining the blood vessels, called endothelial cells, regulate which molecules pass from the blood to the organs they supply. For example, brain endothelial cells prevent toxic molecules from getting into the brain, and lung endothelial cells allow immune cells into the lungs to fight off bacteria or viruses. Determining which genes are switched on in the endothelial cells of major organs might allow scientists to determine what endothelial cells do in the brain, heart, and lung, and how they differ; or help scientists deliver drugs to a particular organ. If endothelial cells from different organs switch on different groups of genes, each of these groups of genes can be thought of as a ‘genetic signature’ that identifies endothelial cells from a specific organ. Now, Jambusaria et al. show that brain, heart, and lung endothelial cells have distinct genetic signatures. The experiments used mice that had been genetically modified to have tags on their endothelial cells. These tags made it possible to isolate RNA – a molecule similar to DNA that contains the information about which genes are active – from endothelial cells without separating the cells from their tissue of origin. Next, RNA from endothelial cells in the heart, brain and lung was sequenced and analyzed. The results show that each endothelial cell type has a distinct genetic signature under normal conditions and infection-like conditions. Unexpectedly, the experiments also showed that genes that were thought to only be switched on in the cells of specific tissues are also on in the endothelial cells lining the blood vessels of the tissue. For example, genes switched on in brain cells are also active in brain endothelial cells, and genes allowing heart muscle cells to pump are also on in the endothelial cells of the heart blood vessels. The endothelial cell genetic signatures identified by Jambusaria et al. can be used as “postal codes” to target drugs to a specific organ via the endothelial cells that feed it. It might also be possible to use these genetic signatures to build organ-specific blood vessels from stem cells in the laboratory. Future work will try to answer why endothelial cells serving the heart and brain use genes from these organs.

This work describes a role for endothelial N-cadherin in the regulation of endothelial permeability in the brain and lung. N-cadherin adhesions formed between endothelial cells and pericytes increase the abundance of VE-cadherin at adherens junctions through the RhoGEF Trio-dependent activation of RhoA and Rac1., Vascular endothelial (VE)–cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

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.

Background: The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. Methods: CD34 + progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34 + cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. Results: Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34 + progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. Conclusions: Dedifferentiation of fibroblasts to intermediate CD34 + progenitors gives rise to endothelial cells and erythroblasts in a SOX17-dependent manner. These findings identify the intermediate CD34 + progenitor state as a critical bifurcation point, which can be tuned to generate functional blood vessels or erythrocytes and salvage ischemic tissue.

Rationale: 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 ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH’s cancer-like phenotype. Methods: 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. Measurements and Main Results: 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 ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, 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. Conclusions: These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted.

Inefficient delivery is a major obstacle to the development of peptide-based drugs targeting the intracellular compartment. We recently showed that selectively inhibiting integrin outside-in signaling using a peptide (mP6) derived from the Gα(13)-binding ExE motif within the integrin β(3) cytoplasmic domain had anti-thrombotic effects. Here, we engineered lipid-stabilized high loading peptide nanoparticles (HLPN), in which a redesigned ExE peptide (M3mP6) constituted up to 70% of the total nanoparticle molarity, allowing efficient in vivo delivery. We observed that M3mP6 HLPNs inhibited occlusive thrombosis more potently than a clopidogrel/aspirin combination without adverse effects on hemostasis in rodents. Furthermore, M3mP6 HLPN synergized with P2Y12 receptor inhibitors or the clopidogrel/aspirin combination in preventing thrombosis, without exacerbating hemorrhage. M3mP6 HLPN also inhibited intravascular coagulation more potently than the P2Y12 inhibitor cangrelor. Post-ischemia injection of M3mP6 HLPN protected the heart from myocardial ischemia-reperfusion injury in a mouse model. This study demonstrates an efficient in vivo peptide delivery strategy for a therapeutic that not only efficaciously prevented thrombosis with minimal bleeding risk but also protected from myocardial ischemia/reperfusion injury in mice.

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 following inflammatory injury.

Under the current situation of ultra-low emission, it is of great significance to study the formation and removal of SO3 in various equipments of coal-fired power plants. At present, most researches on SO3 in coal-fired power plant focus on the influence of single equipment on flue gas SO3 under a certain load condition. In this research, a 300MW generating unit is tested under variable load conditions to explore the variation law of SO3 in various equipments under variable load conditions. The results show that SO3 is mainly generated in the boiler furnace and SCR denitrification device, and the total removal rate of SO3 is 84.6%. However, the removal efficiency of SO3 in electrostatic precipitators and the wet desulfurization systems shows great differences. Under full load condition, the removal rate of SO3 is 46.8% - 69.8%. When the load changes, the SO3 removal rate changes with the change of load, and the ESP is affected by the load most. The SO3 content in the ash increases from 11.267mg/g under 50% load to 14.763mg/g under 100% load, and such changes are mainly determined by flue gas temperature and flue gas flow.

Under the general background of population aging, suitable products of the old have been developed, among which furniture of the old has also received people’s attention. Combining with the product service system (PSS), this paper studies the sitting-type furniture suitable for the elderly according to the psychological and physiological particularity of the elderly, and discusses the mode of PSS applied to the sitting-type furniture suitable for the aged, so as to promote the development of the sitting-type furniture suitable for the aged and better service for the old.

Angiogenesis is initiated in response to a variety of external cues, including mechanical and biochemical stimuli; however, the underlying signaling mechanisms remain unclear. Here, we investigated the proangiogenic role of the endothelial mechanosensor Piezo1. Genetic deletion and pharmacological inhibition of Piezo1 reduced endothelial sprouting and lumen formation induced by wall shear stress and proangiogenic mediator sphingosine 1-phosphate, whereas Piezo1 activation by selective Piezo1 activator Yoda1 enhanced sprouting angiogenesis. Similarly to wall shear stress, sphingosine 1-phosphate functioned by activating the Ca2+ gating function of Piezo1, which in turn signaled the activation of the matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase during sprouting angiogenesis. Studies in mice in which Piezo1 was conditionally deleted in endothelial cells demonstrated the requisite role of sphingosine 1-phosphate-dependent activation of Piezo1 in mediating angiogenesis in vivo. These results taken together suggest that both mechanical and biochemical stimuli trigger Piezo1-mediated Ca2+ influx and thereby activate matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase and synergistically facilitate sprouting angiogenesis.

Figure S1. A) Hierarchical clustering of Endothelial cells from 7 mouse organs Intra- and inter-tissue heterogeneity. Tree plot generated via hierarchical clustering of 500 most variable genes across all distinct tissue endothelial cell samples B) Hierarchical clustering of Neuronal cells from 5 different regions of the mouse forebrain Intra- and inter-tissue heterogeneity. Tree plot generated via hierarchical clustering of 500 most variable genes across all distinct tissue neuronal cell samples. Figure S2. Comparison of statistical power and type-I error rate between HeteroPath, GSEA, and PGSEA for DE Gene Set size of 50 genes. The averaged results of 500 simulations are depicted as function of the sample size on the x-axis, for each of the methods. On the y-axis either the statistical power or the empirical type-I error rate is shown. GSE scores were calculated with each method with respect to two gene sets, one of them differentially expressed (DE) and the other one not. Statistical power and empirical type-I error rates were estimated by performing an ANOVA on the DE and non-DE gene sets, respectively, at a significance level of α = 0.05. Figure S3. Comparison of statistical power and type-I error rate between HeteroPath, GSEA, and PGSEA for DE Gene Set size of 150 genes. The averaged results of 500 simulations are depicted as function of the sample size on the x-axis, for each of the methods. On the y-axis either the statistical power or the empirical type-I error rate is shown. GSE scores were calculated with each method with respect to two gene sets, one of them differentially expressed (DE) and the other one not. Statistical power and empirical type-I error rates were estimated by performing an ANOVA on the DE and non-DE gene sets, respectively, at a significance level of α = 0.05. Figure S4. A) Enriched Wnt Signaling Motifs from Brain endothelial cells The table shows the five most enriched motifs in ChIP-seq peaks and the associated transcription factors. Significance values and significant p-values (p ≤ 0.05) are shown. B) Enriched Oxidative Phosphorylation Motifs from Hippocampal Neurons The table shows the five most enriched motifs in ChIP-seq peaks and the associated transcription factors. Significance values and significant p-values (p ≤ 0.05) are shown. (PPTX 1265 kb)

Background The heterogeneity of cells across tissue types represents a major challenge for studying biological mechanisms as well as for therapeutic targeting of distinct tissues. Computational prediction of tissue-specific gene regulatory networks may provide important insights into the mechanisms underlying the cellular heterogeneity of cells in distinct organs and tissues. Results Using three pathway analysis techniques, gene set enrichment analysis (GSEA), parametric analysis of gene set enrichment (PGSEA), alongside our novel model (HeteroPath), which assesses heterogeneously upregulated and downregulated genes within the context of pathways, we generated distinct tissue-specific gene regulatory networks. We analyzed gene expression data derived from freshly isolated heart, brain, and lung endothelial cells and populations of neurons in the hippocampus, cingulate cortex, and amygdala. In both datasets, we found that HeteroPath segregated the distinct cellular populations by identifying regulatory pathways that were not identified by GSEA or PGSEA. Using simulated datasets, HeteroPath demonstrated robustness that was comparable to what was seen using existing gene set enrichment methods. Furthermore, we generated tissue-specific gene regulatory networks involved in vascular heterogeneity and neuronal heterogeneity by performing motif enrichment of the heterogeneous genes identified by HeteroPath and linking the enriched motifs to regulatory transcription factors in the ENCODE database. Conclusions HeteroPath assesses contextual bidirectional gene expression within pathways and thus allows for transcriptomic assessment of cellular heterogeneity. Unraveling tissue-specific heterogeneity of gene expression can lead to a better understanding of the molecular underpinnings of tissue-specific phenotypes. Electronic supplementary material The online version of this article (10.1186/s12859-018-2190-6) contains supplementary material, which is available to authorized users.

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 (K(2P)) TWIK2 as the K(+) efflux channel triggering NLRP3 inflammasome activation. Deletion of the Kcnk6 gene (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.

Right ventricular (RV) function determines prognosis in pulmonary arterial hypertension (PAH). We hypothesize that ischemia causes RV dysfunction in PAH by triggering dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. RV function was compared in control rats (n = 50) versus rats with monocrotaline-induced PAH (MCT-PAH; n = 60) both in vivo (echocardiography) and ex vivo (RV Langendorff). Mitochondrial membrane potential and morphology and RV function were assessed before or after 2 cycles of ischemia-reperfusion injury challenge (RV-IR). The effects of Mdivi-1 (25 μM), a Drp1 GTPase inhibitor, and P110 (1 μM), a peptide inhibitor of Drp1-Fis1 interaction, were studied. We found that MCT caused RV hypertrophy, RV vascular rarefaction, and RV dysfunction. Prior to IR, the mitochondria in MCT-PAH RV were depolarized and swollen with increased Drp1 content and reduced aconitase activity. RV-IR increased RV end diastolic pressure (RVEDP) and mitochondrial Drp1 expression in both control and MCT-PAH RVs. IR depolarized mitochondria in control RV but did not exacerbate the basally depolarized MCT-PAH RV mitochondria. During RV IR mdivi-1 and P110 reduced Drp1 translocation to mitochondria, improved mitochondrial structure and function, and reduced RVEDP. In conclusion, RV ischemia occurs in PAH and causes Drp1-Fis1-mediated fission leading to diastolic dysfunction. Inhibition of mitochondrial fission preserves RV function in RV-IR.Right ventricular ischemia reperfusion (RV-IR) causes RV diastolic dysfunction. IR-induced mitochondrial fission causes RV diastolic dysfunction. In RV-IR Drp1 translocates to mitochondria, binds Fis1 and causes fission and injury. A baseline RV mitochondriopathy in MCT PAH resembles IR-induced mitochondrial injury. Drp1 inhibitors (Mdivi-1 and P110) preserve RV diastolic function post RV-IR.