Sara C. Sebag

Low levels of tissue factor lead to alveolar haemorrhage, potentiating murine acute lung injury and oxidative stress

Background Systemic blockade of tissue factor (TF) attenuates acute lung injury (ALI) in animal models of sepsis but the effects of global TF deficiency are unknown. We used mice with complete knockout of mouse TF and low levels (∼1%) of human TF (LTF mice) to test the hypothesis that global TF deficiency attenuates lung inflammation in direct lung injury. Methods LTF mice were treated with 10 μg of lipopolysaccharide (LPS) or vehicle administered by direct intratracheal injection and studied at 24 h. Results Contrary to our hypothesis, LTF mice had increased lung inflammation and injury as measured by bronchoalveolar lavage cell count (3.4×105 wild-type (WT) LPS vs 3.3×105 LTF LPS, p=0.947) and protein (493 μg/ml WT LPS vs 1014 μg/ml LTF LPS, p=0.006), proinflammatory cytokines (TNF-α, IL-10, IL-12, p<0.035 WT LPS vs LTF LPS) and histology compared with WT mice. LTF mice also had increased haemorrhage and free haemoglobin in the airspace accompanied by increased oxidant stress as measured by lipid peroxidation products (F2 isoprostanes and isofurans). Conclusions These findings indicate that global TF deficiency does not confer protection in a direct lung injury model. Rather, TF deficiency causes increased intra-alveolar haemorrhage following LPS leading to increased lipid peroxidation. Strategies to globally inhibit TF may be deleterious in patients with ALI.

Therapeutic Modulation of Coagulation and Fibrinolysis in Acute Lung Injury and the Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are characterized by excessive intraalveolar fibrin deposition, driven, at least in part by inflammation. The imbalance between activation of coagulation and inhibition of fibrinolysis in patients with ALI/ARDS favors fibrin formation and appears to occur both systemically and in the lung and airspace. Tissue factor (TF), a key mediator of the activation of coagulation in the lung, has been implicated in the pathogenesis of ALI/ARDS. As such, there have been numerous investigations modulating TF activity in a variety of experimental systems in order to develop new therapeutic strategies for ALI/ARDS. This review will summarize current understanding of the role of TF and other proteins of the coagulation cascade as well the fibrinolysis pathway in the development of ALI/ARDS with an emphasis on the pathways that are potential therapeutic targets. These include the TF inhibitor pathway, the protein C pathway, antithrombin, heparin, and modulation of fibrinolysis through plasminogen activator- 1 (PAI-1) or plasminogen activators (PA). Although experimental studies show promising results, clinical trials to date have proven unsuccessful in improving patient outcomes. Modulation of coagulation and fibrinolysis has complex effects on both hemostasis and inflammatory pathways and further studies are needed to develop new treatment strategies for patients with ALI/ARDS.

Interferon-γ and tumor necrosis factor-α act synergistically to up-regulate tissue factor in alveolar epithelial cells

ABSTRACT Fibrin deposition mediated through activation of tissue factor (TF) in the airspace is central to the pathogenesis of acute lung injury. Defining the mechanisms of TF regulation in the lung is critical to understanding pulmonary fibrin formation. Tumor necrosis factor-α (TNF-α) up-regulates TF in the injured lung, and there is emerging evidence that another cytokine, interferon-γ (IFN-γ), also modulates expression. The effects of TNF-α and IFN-γ on regulation of TF were studied in alveolar epithelial A549 cells. In addition, potential mechanisms of modulation of TF expression by the 2 cytokines were analyzed with the hypothesis that IFN-γ acts synergistically with TNF-α to up-regulate alveolar epithelial TF through modulation of TNF receptor (TNFR) expression. TNF-α but not IFN-γ treatment increased TF mRNA, protein, and cell surface TF activity. The combination of IFN-γ and TNF-α treatment augmented the effects of TNF-α on TF up-regulation and also increased release of procoagulant microparticles (MPs) from A549 cells. IFN-γ modulated expression of both TNF-α receptors. Studies utilizing neutralizing antibodies against the two TNF receptors showed that the TF effects were mediated primarily through augmentation of TNFR1-dependent cellular responses. These findings have important implications for regulation of fibrin formation in the lung in the setting of acute inflammation.

Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma

Excessive ROS promote allergic asthma, a condition characterized by airway inflammation, eosinophilic inflammation, and increased airway hyperreactivity (AHR). The mechanisms by which airway ROS are increased and the relationship between increased airway ROS and disease phenotypes are incompletely defined. Mitochondria are an important source of cellular ROS production, and our group discovered that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is present in mitochondria and activated by oxidation. Furthermore, mitochondrial-targeted antioxidant therapy reduced the severity of allergic asthma in a mouse model. Based on these findings, we developed a mouse model of CaMKII inhibition targeted to mitochondria in airway epithelium. We challenged these mice with OVA or Aspergillus fumigatus. Mitochondrial CaMKII inhibition abrogated AHR, inflammation, and eosinophilia following OVA and A. fumigatus challenge. Mitochondrial ROS were decreased after agonist stimulation in the presence of mitochondrial CaMKII inhibition. This correlated with blunted induction of NF-κB, the NLRP3 inflammasome, and eosinophilia in transgenic mice. These findings demonstrate a pivotal role for mitochondrial CaMKII in airway epithelium in mitochondrial ROS generation, eosinophilic inflammation, and AHR, providing insights into how mitochondrial ROS mediate features of allergic asthma.

CaMKII inhibition in type II pneumocytes protects from bleomycin-induced pulmonary fibrosis by preventing Ca2+-dependent apoptosis

The calcium and calmodulin-dependent kinase II (CaMKII) translates increases in intracellular Ca(2+) into downstream signaling events. Its function in pulmonary pathologies remains largely unknown. CaMKII is a well-known mediator of apoptosis and regulator of endoplasmic reticulum (ER) Ca(2+). ER stress and apoptosis of type II pneumocytes lead to aberrant tissue repair and progressive collagen deposition in pulmonary fibrosis. Thus we hypothesized that CaMKII inhibition alleviates fibrosis in response to bleomycin by attenuating apoptosis and ER stress of type II pneumocytes. We first established that CaMKII was strongly expressed in the distal respiratory epithelium, in particular in surfactant protein-C-positive type II pneumocytes, and activated after bleomycin instillation. We generated a novel transgenic model of inducible expression of the CaMKII inhibitor peptide AC3-I limited to type II pneumocytes (Tg SPC-AC3-I). Tg SPC-AC3-I mice were protected from development of pulmonary fibrosis after bleomycin exposure compared with wild-type mice. CaMKII inhibition also provided protection from apoptosis in type II pneumocytes in vitro and in vivo. Moreover, intracellular Ca(2+) levels and ER stress were increased by bleomycin and significantly blunted with CaMKII inhibition in vitro. These data demonstrate that CaMKII inhibition prevents type II pneumocyte apoptosis and development of pulmonary fibrosis in response to bleomycin. CaMKII inhibition may therefore be a promising approach to prevent or ameliorate the progression of pulmonary fibrosis.

Mechanical Stretch Inhibits Lipopolysaccharide-induced Keratinocyte-derived Chemokine and Tissue Factor Expression While Increasing Procoagulant Activity in Murine Lung Epithelial Cells

Background: The influence of mechanical stretch on LPS-induced alveolar epithelial cell inflammatory (keratinocyte-derived chemokine (KC)) and coagulant (tissue factor (TF)) responses is unclear. Results: Stretch down-regulates LPS-mediated KC and TF expression while enhancing TF activity. Conclusion: Different signaling pathways regulate stretch- and LPS-induced TF and KC. Significance: Lung stretch during mechanical ventilation may alter the innate immune response of the lung epithelium. Previous studies have shown that the innate immune stimulant LPS augments mechanical ventilation-induced pulmonary coagulation and inflammation. Whether these effects are mediated by alveolar epithelial cells is unclear. The alveolar epithelium is a key regulator of the innate immune reaction to pathogens and can modulate both intra-alveolar inflammation and coagulation through up-regulation of proinflammatory cytokines and tissue factor (TF), the principal initiator of the extrinsic coagulation pathway. We hypothesized that cyclic mechanical stretch (MS) potentiates LPS-mediated alveolar epithelial cell (MLE-12) expression of the chemokine keratinocyte-derived cytokine (KC) and TF. Contrary to our hypothesis, MS significantly decreased LPS-induced KC and TF mRNA and protein expression. Investigation into potential mechanisms showed that stretch significantly reduced LPS-induced surface expression of TLR4 that was not a result of increased degradation. Decreased cell surface TLR4 expression was concomitant with reduced LPS-mediated NF-κB activation. Immunofluorescence staining showed that cyclic MS markedly altered LPS-induced organization of actin filaments. In contrast to expression, MS significantly increased LPS-induced cell surface TF activity independent of calcium signaling. These findings suggest that cyclic MS of lung epithelial cells down-regulates LPS-mediated inflammatory and procoagulant expression by modulating actin organization and reducing cell surface TLR4 expression and signaling. However, because LPS-induced surface TF activity was enhanced by stretch, these data demonstrate differential pathways regulating TF expression and activity. Ultimately, loss of LPS responsiveness in the epithelium induced by MS could result in increased susceptibility of the lung to bacterial infections in the setting of mechanical ventilation.

Cationic CaMKII Inhibiting Nanoparticles Prevent Allergic Asthma

Asthma is a common lung disease affecting over 300 million people worldwide and is associated with increased reactive oxygen species, eosinophilic airway inflammation, bronchoconstriction, and mucus production. Targeting of novel therapeutic agents to the lungs of patients with asthma may improve efficacy of treatments and minimize side effects. We previously demonstrated that Ca2+/calmodulin-dependent protein kinase (CaMKII) is expressed and activated in the bronchial epithelium of asthmatic patients. CaMKII inhibition in murine models of allergic asthma reduces key disease phenotypes, providing the rationale for targeted CaMKII inhibition as a potential therapeutic approach for asthma. Herein we developed a novel cationic nanoparticle (NP)-based system for delivery of the potent and specific CaMKII inhibitor peptide, CaMKIIN, to airways.1 CaMKIIN-loaded NPs abrogated the severity of allergic asthma in a murine model. These findings provide the basis for development of innovative, site-specific drug delivery therapies, particularly for treatment of pulmonary diseases such as asthma.

Alterations in sarcomere function modify the hyperplastic to hypertrophic transition phase of mammalian cardiomyocyte development

It remains unclear how perturbations in cardiomyocyte sarcomere function alter postnatal heart development. We utilized murine models that allowed manipulation of cardiac myosin-binding protein C (MYBPC3) expression at critical stages of cardiac ontogeny to study the response of the postnatal heart to disrupted sarcomere function. We discovered that the hyperplastic to hypertrophic transition phase of mammalian heart development was altered in mice lacking MYBPC3 and this was the critical period for subsequent development of cardiomyopathy. Specifically, MYBPC3-null hearts developed evidence of increased cardiomyocyte endoreplication, which was accompanied by enhanced expression of cell cycle stimulatory cyclins and increased phosphorylation of retinoblastoma protein. Interestingly, this response was self-limited at later developmental time points by an upregulation of the cyclin-dependent kinase inhibitor p21. These results provide valuable insights into how alterations in sarcomere protein function modify postnatal heart development and highlight the potential for targeting cell cycle regulatory pathways to counteract cardiomyopathic stimuli.

Nuclear localized Raf1 isoform alters DNA-dependent protein kinase activity and the DNA damage response

Raf1/c‐Raf is a well‐characterized serine/threonine–protein kinase that links Ras family members with the MAPK/ERK signaling cascade. We have identified a novel splice isoform of human Raf1 that causes protein truncation and loss of the C‐terminal kinase domain (Raf1‐tr). We found that Raf1‐tr has increased nuclear localization compared with full‐length Raf1, and this finding was secondary to reduced binding of Raf1‐tr to the cytoplasmic chaperone FK506 binding protein 5. We show that Raf1‐tr has increased binding to DNA‐dependent protein kinase (DNA‐PK), which inhibits DNA‐PK function and causes amplification of irradiation‐ and bleomycin‐induced DNA damage. We found that the human colorectal cancer cell line, HCT‐116, displayed reduced expression of Raf1‐tr, and reintroduction of Raf1‐tr sensitized the cells to bleomycin‐induced apoptosis. Furthermore, we identified differential Raf1‐tr expression in breast cancer cell lines and showed that breast cancer cells with increased Raf1‐tr expression become sensitized to bleomycin‐induced apoptosis. Collectively, these results demonstrate a novel Raf1 isoform in humans that has a unique noncanonical role in regulating the double‐stranded DNA damage response pathway through modulation of DNA‐PK function.—Nixon, B. R., Sebag, S. C., Glennon, M. S., Hall, E. J., Kounlavong, E. S., Freeman, M. L., Becker, J. R. Nuclear localized Raf1 isoform alters DNA‐dependent protein kinase activity and the DNA damage response. FASEB J. 33, 1138–1150 (2019). www.fasebj.org

Inhibition of the mitochondrial calcium uniporter prevents IL-13 and allergen-mediated airway epithelial apoptosis and loss of barrier function

&NA; Mitochondria are increasingly recognized as key mediators of acute cellular stress responses in asthma. However, the distinct roles of regulators of mitochondrial physiology on allergic asthma phenotypes are currently unknown. The mitochondrial Ca2+ uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca2+ uptake into the mitochondrial matrix. To understand the function of MCU in models of allergic asthma, in vitro and in vivo studies were performed using models of functional deficiency or knockout of MCU. In primary human respiratory epithelial cells, MCU inhibition abrogated mitochondrial Ca2+ uptake and reactive oxygen species (ROS) production, preserved the mitochondrial membrane potential and protected from apoptosis in response to the pleiotropic Th2 cytokine IL‐13. Consequently, epithelial barrier function was maintained with MCU inhibition. Similarly, the endothelial barrier was preserved in respiratory epithelium isolated from MCU‐/‐ mice after exposure to IL‐13. In the ovalbumin‐model of allergic airway disease, MCU deficiency resulted in decreased apoptosis within the large airway epithelial cells. Concordantly, expression of the tight junction protein ZO‐1 was preserved, indicative of maintenance of epithelial barrier function. These data implicate mitochondrial Ca2+ uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.