Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are characterized by excessive intra-alveolar 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.

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. Hypothesis 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 (IT) injection and studied at 24 hours. Results Contrary to our hypothesis, LTF mice had increased lung inflammation and injury as measured by bronchoalveolar lavage cell count (3.4 × 105 WT LPS versus 3.3 × 105 LTF LPS, p=0.947) and protein (493 μg/ml WT LPS versus 1014 μg/ml LTF LPS, p=0.006), proinflammatory cytokines (TNF-α, IL-10, IL-12, p<0.035 WT LPS versus LTF LPS) and histology compared to wild type mice. LTF mice also had increased hemorrhage and free hemoglobin 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 hemorrhage following LPS leading to increased lipid peroxidation. Strategies to globally inhibit tissue factor may be deleterious in patients with ALI.

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The role of the mitochondrial Ca2+ uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during cell cycling. During the G1/S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption and Ca2+ uptake increased in wild-type cells, but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser616. The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit whereby mitochondrial Ca2+ uptake affects cell proliferation through Drp1.

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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.

Asthma is a common lung disease affecting over 300 million people worldwide and is associated with increased reactive oxygen species (ROS), 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. 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.

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 Ca uniporter (MCU) resides in the inner mitochondrial membrane and controls mitochondrial Ca 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 Ca 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 Ca uptake through MCU as a key controller of epithelial cell viability in acute allergic asthma.