Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain.

RATIONALE: Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD). OBJECTIVES: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. METHODS: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. MEASUREMENTS AND MAIN RESULTS: We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. CONCLUSIONS: The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor.

Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high-fat diet (HFD) in some inbred mouse strains. We conducted a genome-wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD-induced PH. Mice from 36 inbred and wild-derived strains were fed with regular diet or HFD for 20 weeks beginning at 6-12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end-systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest-path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH-related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high-fat feeding. Network-based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest-path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high-fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network-based approach adds evidence for a role for Egfr in murine PH.

Effects of intra-abdominal sepsis on atherosclerosis in mice.

INTRODUCTION: Sepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis. METHODS: We performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE-/-) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE-/- mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation. RESULTS: ApoE-/- mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (-6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05). CONCLUSIONS: Using a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.

RIP3-dependent necroptosis contributes to the pathogenesis of chronic obstructive pulmonary disease.

Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking-induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow-derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK'872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.

KIAA0317 regulates pulmonary inflammation through SOCS2 degradation.

Dysregulated proinflammatory cytokine release has been implicated in the pathogenesis of several life-threatening acute lung illnesses such as pneumonia, sepsis, and acute respiratory distress syndrome. Suppressors of cytokine signaling proteins, particularly SOCS2, have recently been described as antiinflammatory mediators. However, the regulation of SOCS2 protein has not been described. Here we describe a mechanism of SOCS2 regulation by the action of the ubiquitin E3 ligase KIAA0317. KIAA0317-mediated degradation of SOCS2 exacerbated inflammation in vitro, and depletion of KIAA0317 in vivo ameliorated pulmonary inflammation. KIAA0317-knockout mice exhibited resistance to LPS-induced pulmonary inflammation, while KIAA03017 reexpression mitigated this effect. We uncovered a small molecule inhibitor of KIAA0317 protein (BC-1365) that prevented SOCS2 degradation and attenuated LPS- and P. aeruginosa-induced lung inflammation in vivo. These studies show KIAA0317 to be a critical mediator of pulmonary inflammation through its degradation of SOCS2 and a potential candidate target for therapeutic inhibition.