Peroxynitrite (ONOO⁻) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO⁻ interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO⁻ scavenger, iron-(III)-tetrakis(N-methyl-49pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO⁻ reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO⁻-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose–exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO⁻ reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO⁻ selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients.

Rationale: Alveolar liquid clearance is regulated by Na⁺ uptake through the apically expressed epithelial sodium channel (ENaC) and basolaterally localized Na⁺-K⁺-ATPase in type II alveolar epithelial cells. Dysfunction of these Na⁺ transporters during pulmonary inflammation can contribute to pulmonary edema. Objectives: In this study, we sought to determine the precise mechanism by which the TIP peptide, mimicking the lectin-like domain of tumor necrosis factor (TNF), stimulates Na⁺ uptake in a homologous cell system in the presence or absence of the bacterial toxin pneumolysin (PLY). Methods: We used a combined biochemical, electrophysiological, and molecular biological in vitro approach and assessed the physiological relevance of the lectin-like domain of TNF in alveolar liquid clearance in vivo by generating triple-mutant TNF knock-in mice that express a mutant TNF with deficient Na⁺ uptake stimulatory activity. Measurements and Main Results: TIP peptide directly activates ENaC, but not the Na⁺-K⁺-ATPase, upon binding to the carboxy-terminal domain of the α subunit of the channel. In the presence of PLY, a mediator of pneumococcal-induced pulmonary edema, this binding stabilizes the ENaC-PIP2-MARCKS complex, which is necessary for the open probability conformation of the channel and preserves ENaC-α protein expression, by means of blunting the protein kinase C-α pathway. Triple-mutant TNF knock-in mice are more prone than wild-type mice to develop edema with low-dose intratracheal PLY, correlating with reduced pulmonary ENaC-α subunit expression. Conclusions: These results demonstrate a novel TNF-mediated mechanism of direct ENaC activation and indicate a physiological role for the lectin-like domain of TNF in the resolution of alveolar edema during inflammation.

Background: Autolysis and antibiotic-mediated lysis of S. pneumoniae induces release of the pore-forming toxin, pneumolysin (PLY), which is a prominent cause of acute lung injury in pneumonia. PLY inhibits alveolar liquid clearance (ALC) and severely compromises alveolar-capillary barrier function, leading to permeability edema. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The alpha  subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-alpha and whether this subunit is involved in the regulation of capillary barrier function. Methods: The presence of alpha, beta and gamma subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in Western blotting. The role of ENaC-alpha in endothelial monolayer resistance was examined by depletion of the subunit using specific siRNA and by employing the TNF-derived TIP peptide, a specific activator, which directly binds to ENaC-alpha. Results: HL-MVEC express all three subunits of ENaC, as well as ASIC1a, which has the capacity to form hybrid non-selective cation channels with ENaC-alpha. Both TIP peptide, which specifically binds to ENaC-alpha and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-alpha depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-alpha and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin dependent Kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-alpha and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-alpha in both barrier function and liquid clearance. Conclusions: Strategies aiming to activate endothelial NSC channels that contain ENaC-alpha should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

TGF-β-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the β-isoform of the catalytic subunit of PP1 (PP1cβ) from pulmonary artery EC. As PP1cβ, but not PP1cα, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

The integrity of epithelial and endothelial barriers in the lower airspaces of the lungs has to be tightly regulated, in order to prevent leakage and to assure efficient gas exchange between the alveoli and capillaries. Both G⁻ and G⁺ bacterial toxins, such as lipopolysaccharide and pneumolysin, respectively, can be released in high concentrations within the pulmonary compartments upon antibiotic treatment of patients suffering from acute respiratory distress syndrome (ARDS) or severe pneumonia. These toxins are able to impair endothelial barrier function, either directly, or indirectly, by induction of pro-inflammatory mediators and neutrophil sequestration. Toxin-induced endothelial hyperpermeability can involve myosin light chain phosphorylation and/or microtubule rearrangement. Endothelial nitric oxide synthase (eNOS) was proposed to be a guardian of basal barrier function, since eNOS knock-out mice display an impaired expression of inter-endothelial junction proteins and as such an increased vascular permeability, as compared to wild type mice. The enzyme arginase, the activity of which can be regulated by the redox status of the cell, exists in two isoforms – arginase 1 (cytosolic) and arginase 2 (mitochondrial) – both of which can be expressed in lung microvascular endothelial cells. Upon activation, arginase competes with eNOS for the substrate l-arginine, as such impairing eNOS-dependent NO generation and promoting reactive oxygen species generation by the enzyme. This mini-review will discuss recent findings regarding the interaction between bacterial toxins and arginase during acute lung injury and will as such address the role of arginase in bacterial toxin-induced pulmonary endothelial barrier dysfunction.