Myeloid Nrf2 Protects against Neonatal Oxidant-Stress-Induced Lung Inflammation and Alveolar Simplification in Mice.

Bronchopulmonary dysplasia (BPD) is a chronic condition affecting preterm infants, characterized by lung alveolar simplification/hypoalveolarization and vascular remodeling. The nuclear factor erythroid 2 like 2 (Nfe2l2, or Nrf2) plays a critical role in the cytoprotective response to neonatal hyperoxia, and its global deficiency exacerbates hypoalveolarization in mice. The abnormal recruitment and activation of myeloid cells are associated with the pathogenesis of BPD. Therefore, we employed a genetic approach to investigate the role of myeloid Nrf2 in regulating hyperoxia-induced hypoalveolarization. Pups, both wild-type (Nrf2f/f) and those with a myeloid Nrf2 deletion (abbreviated as Nrf2∆/∆mye), were exposed to hyperoxia for 72 h at postnatal day 1 (Pnd1), and then sacrificed at either Pnd4 or Pnd18 following a two-week recovery period. We analyzed the hypoalveolarization, inflammation, and gene expression related to cytoprotective and inflammatory responses in the lungs of these pups. The hypoalveolarization induced by hyperoxia was significantly greater in Nrf2∆/∆mye pups compared to their Nrf2f/f counterparts (35.88% vs. 21.01%, respectively) and was accompanied by increased levels of inflammatory cells and IL-1ß activation in the lungs. Antioxidant gene expression in response to neonatal hyperoxia was lower in Nrf2∆/∆mye pups compared to their Nrf2f/f counterparts. Furthermore, Nrf2-deficient macrophages exposed to hyperoxia exhibited markedly decreased cytoprotective gene expression and increased IL-1ß levels compared to Nrf2-sufficient cells. Our findings demonstrate the crucial role of myeloid Nrf2 in mitigating hyperoxia-induced lung hypoalveolarization and inflammatory responses in neonatal mice.

Endothelial FoxM1 reactivates aging-impaired endothelial regeneration for vascular repair and resolution of inflammatory lung injury.

Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS). Here, we demonstrated that persistent lung injury and high mortality in aged mice after sepsis challenge were attributable to impaired endothelial regeneration and vascular repair. Genetic lineage tracing study showed that endothelial regeneration after sepsis-induced vascular injury was mediated by lung resident endothelial proliferation in young adult mice, whereas this intrinsic regenerative program was impaired in aged mice. Expression of forkhead box M1 (FoxM1), an important mediator of endothelial regeneration in young mice, was not induced in lungs of aged mice. Transgenic FOXM1 expression or in vivo endothelium-targeted nanoparticle delivery of the FOXM1 gene driven by an endothelial cell (EC)-specific promoter reactivated endothelial regeneration, normalized vascular repair and resolution of inflammation, and promoted survival in aged mice after sepsis challenge. In addition, treatment with the FDA-approved DNA demethylating agent decitabine was sufficient to reactivate FoxM1-dependent endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. Mechanistically, aging-induced Foxm1 promoter hypermethylation in mice, which could be inhibited by decitabine treatment, inhibited Foxm1 induction after sepsis challenge. In COVID-19 lung autopsy samples, FOXM1 was not induced in vascular ECs of elderly patients in their 80s, in contrast with middle-aged patients (aged 50 to 60 years). Thus, reactivation of FoxM1-mediated endothelial regeneration and vascular repair may represent a potential therapy for elderly patients with ARDS.

The Unexpected Protective Role of Thrombosis in Sepsis-Induced Inflammatory Lung Injury Via Endothelial Alox15.

Background: Patients with sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) commonly suffer from severe pulmonary thrombosis, but clinical trials of anti-coagulant therapies in sepsis and ARDS patients have failed. ARDS patients with thrombocytopenia also exhibit increased mortality, and widespread pulmonary thrombosis is often seen in coronavirus disease 2019 (COVID-19) ARDS patients. Methods: Employing different amounts of microbeads to induce various levels of pulmonary thrombosis. Acute lung injury was induced by either lipopolysaccharide i.p. or cecal ligation and puncture. Endothelial cell (EC)-targeted nanoparticle coupled with CDH5 promoter was employed to delivery plasmid DNA expressing the CRISPR/Cas9 system for EC-specific gene knockout or expressing Alox15 for EC-specific overexpression. Additionally, thrombocytopenia was induced by genetic depletion of platelets using DTR Pf4Cre mice by breeding Pf4 Cre mice into the genetic background of DTR mice. Results: We show that while severe pulmonary thrombosis or thrombocytopenia augments sepsis-induced ALI, the induction of mild pulmonary thrombosis conversely reduces endothelial cell (EC) apoptosis, ALI, and mortality via sustained expression of endothelial arachidonate 15-lipoxygenase (Alox15). Endothelial Alox15 knockout via EC-targeted nanoparticle delivery of CRISPR/Cas9 plasmid DNA in adult mice abolished the protective impact of mild lung thrombosis. Conversely, overexpression of endothelial Alox15 inhibited the increases in ALI caused by severe pulmonary thrombosis. The clinical relevance of the findings was validated by the observation of reduced ALOX15-expressing ECs in lung autopsy samples of ARDS patients. Additionally, restoration of pulmonary thrombosis in thrombocytopenic mice also normalized endotoxemia-induced ALI. Conclusion: We have demonstrated that moderate levels of thrombosis protect against sepsis-induced inflammatory lung injury via endothelial Alox15. Overexpression of Alox5 inhibits severe pulmonary thrombosis-induced increase of ALI. Thus, activation of ALOX15 signaling represents a promising therapeutic strategy for treatment of ARDS, especially in sub-populations of patients with thrombocytopenia and/or severe pulmonary thrombosis.

Endothelial PHD2 deficiency induces nitrative stress via suppression of caveolin-1 in pulmonary hypertension.

BACKGROUND: Nitrative stress is a characteristic feature of the pathology of human pulmonary arterial hypertension. However, the role of nitrative stress in the pathogenesis of obliterative vascular remodelling and severe pulmonary arterial hypertension remains largely unclear. METHOD: Our recently identified novel mouse model (Egln1Tie2Cre, Egln1 encoding prolyl hydroxylase 2 (PHD2)) has obliterative vascular remodelling and right heart failure, making it an excellent model to use in this study to examine the role of nitrative stress in obliterative vascular remodelling. RESULTS: Nitrative stress was markedly elevated whereas endothelial caveolin-1 (Cav1) expression was suppressed in the lungs of Egln1Tie2Cre mice. Treatment with a superoxide dismutase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride or endothelial Nos3 knockdown using endothelial cell-targeted nanoparticle delivery of CRISPR-Cas9/guide RNA plasmid DNA inhibited obliterative pulmonary vascular remodelling and attenuated severe pulmonary hypertension in Egln1Tie2Cre mice. Genetic restoration of Cav1 expression in Egln1Tie2Cre mice normalised nitrative stress, reduced pulmonary hypertension and improved right heart function. CONCLUSION: These data suggest that suppression of Cav1 expression secondary to PHD2 deficiency augments nitrative stress through endothelial nitric oxide synthase activation, which contributes to obliterative vascular remodelling and severe pulmonary hypertension. Thus, a reactive oxygen/nitrogen species scavenger might have therapeutic potential for the inhibition of obliterative vascular remodelling and severe pulmonary arterial hypertension.

Therapeutic Targeting of Vascular Remodeling and Right Heart Failure in Pulmonary Arterial Hypertension with a HIF-2α Inhibitor.

RATIONALE: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling that leads to right heart failure (RHF) and death. Current therapies do not target vascular remodeling and RHF, and result in only modest improvement of morbidity and mortality. OBJECTIVES: To determine whether targeting HIF-2α (hypoxia-inducible factor-2α) with a HIF-2α-selective inhibitor could reverse PAH and RHF in various rodent PAH models. METHODS: HIF-2α and its downstream genes were evaluated in lung samples and pulmonary arterial endothelial cells and smooth muscle cells from patients with idiopathic PAH as well as various rodent PAH models. A HIF-2α-selective inhibitor was used in human lung microvascular endothelial cells and in Egln1Tie2Cre mice, and in Sugen 5416/hypoxia- or monocrotaline-exposed rats. MEASUREMENTS AND MAIN RESULTS: Upregulation of HIF-2α and its target genes was observed in lung tissues and isolated pulmonary arterial endothelial cells from patients with idiopathic PAH and three distinct rodent PAH models. Pharmacological inhibition of HIF-2α by the HIF-2α translation inhibitor C76 (compound 76) reduced right ventricular systolic pressure and right ventricular hypertrophy and inhibited RHF and fibrosis as well as obliterative pulmonary vascular remodeling in Egln1Tie2Cre mice and Sugen 5416/hypoxia PAH rats. Treatment of monocrotaline-exposed PAH rats with C76 also reversed right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling; prevented RHF; and promoted survival. CONCLUSIONS: These findings demonstrate that pharmacological inhibition of HIF-2α is a promising novel therapeutic strategy for the treatment of severe vascular remodeling and right heart failure in patients with PAH.

Endothelial and Smooth Muscle Cell Interaction via FoxM1 Signaling Mediates Vascular Remodeling and Pulmonary Hypertension.

RATIONALE: Angioproliferative vasculopathy is a hallmark of pulmonary arterial hypertension (PAH). However, little is known about how endothelial cell (EC) and smooth muscle cell (SMC) crosstalk regulates the angioproliferative vascular remodeling. OBJECTIVES: To investigate the role of EC and SMC interaction and underlying signaling pathways in pulmonary hypertension (PH) development. METHODS: SMC-specific Foxm1 (forkhead box M1) or Cxcr4 knockout mice, EC-specific Foxm1 or Egln1 knockout mice, and EC-specific Egln1/Cxcl12 double knockout mice were used to assess the role of FoxM1 on SMC proliferation and PH. Lung tissues and cells from patients with PAH were used to validate clinical relevance. FoxM1 inhibitor thiostrepton was used in Sugen 5416/hypoxia- and monocrotaline-challenged rats. MEASUREMENTS AND MAIN RESULTS: FoxM1 expression was markedly upregulated in lungs and pulmonary arterial SMCs of patients with idiopathic PAH and four discrete PH rodent models. Mice with SMC- (but not EC-) specific deletion of Foxm1 were protected from hypoxia- or Sugen 5416/hypoxia-induced PH. The upregulation of FoxM1 in SMCs induced by multiple EC-derived factors (PDGF-B, CXCL12, ET-1, and MIF) mediated SMC proliferation. Genetic deletion of endothelial Cxcl12 in Egln1Tie2Cre mice or loss of its cognate receptor Cxcr4 in SMCs in hypoxia-treated mice inhibited FoxM1 expression, SMC proliferation, and PH. Accordingly, pharmacologic inhibition of FoxM1 inhibited severe PH in both Sugen 5416/hypoxia and monocrotaline-challenged rats. CONCLUSIONS: Multiple factors derived from dysfunctional ECs induced FoxM1 expression in SMCs and activated FoxM1-dependent SMC proliferation, which contributes to pulmonary vascular remodeling and PH. Thus, targeting FoxM1 signaling represents a novel strategy for treatment of idiopathic PAH.

Oxidant-induced cell death and Nrf2-dependent antioxidative response are controlled by Fra-1/AP-1.

AP-1 (Jun/Fos) transcription factors play key roles in various biological processes, including cell death. Here we report a novel role for Fra-1 in oxidant-induced cell death controlled by modulating antioxidant gene expression. Fra-1-deficient (Fra-1(Δ/Δ)) mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts (PLFs) were remarkably resistant to H(2)O(2)- and diquat-induced cell death, compared to their wild-type (Fra-1(+/+)) counterparts. Fra-1 deficiency ablated oxidant-induced mitochondrion-dependent apoptosis. Fra-1(Δ/Δ) cells had elevated basal levels of antioxidant enzymes and intracellular glutathione (GSH), which were further stimulated by oxidants. Loss of Fra-1 led to an increased half-life of transcription factor Nrf2 and increased recruitment of this protein to the promoters of antioxidant genes and increased their expression. Depletion of intracellular GSH or RNA interference (RNAi)-mediated knockdown of Nqo1, Hmox1, and Nrf2 restored oxidant-induced cell death in Fra-1(Δ/Δ) cells. Thus, Fra-1 appears to increase susceptibility to oxidants and promotes cell death by attenuating Nrf2-driven antioxidant responses.

A Fra-1-dependent, matrix metalloproteinase driven EGFR activation promotes human lung epithelial cell motility and invasion.

We and others have shown a persistently high induction of Fra-1 transcription factor (a dimeric partner of AP-1) levels by respiratory carcinogens in pulmonary epithelial cells. Fra-1 is frequently overexpressed in various human tumors and cancer cells. We have recently shown that Fra-1 significantly promotes growth, motility, and invasion of human pulmonary epithelial cells, the precise molecular mechanisms by which this enhancement occurs are unclear. Because matrix metalloproteinases (MMPs) play key roles in wound healing and lung tumor metastasis, we tested the hypothesis that Fra-1 promotes lung epithelial cell motility and invasion via MMP activation. We show here that MMP-9 and MMP-2 activated signaling plays a critical role in regulating Fra-1-induced lung epithelial cell growth and invasion. Ectopic Fra-1 markedly stimulates MMP-2 and MMP-9 mRNA expression. Inhibition of MMP-2 and MMP-9 activity significantly attenuated Fra-1-driven cell motility and invasion. Furthermore, Fra-1 induced EGFR phosphorylation in an MMP-dependent manner, and an EGFR-specific inhibitor was able to block Fra-1-enhanced cell motility and invasion. Taken together, our data suggest that Fra-1 enhances lung cancer epithelial cell motility and invasion by inducing the activity of MMPs, in particular MMP-2 and MMP-9, and EGFR-activated signaling.