MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.

Fluid management in acute respiratory distress syndrome.

PURPOSE OF REVIEW: Fluid management is one of the most important measures shown to impact acute respiratory distress syndrome (ARDS) outcomes. This review summarizes the current strategies aimed at evaluating and modulating lung fluid balance. RECENT FINDINGS: Multiple recent studies have shown that a conservative fluid management in ARDS patients had beneficial effects on morbidity and mortality. These findings were replicated also in different patient populations assumed to have potential deleterious effects from this approach. So far, only one retrospective study raised the possibility of impaired cognitive function in ARDS patients managed with a conservative fluid strategy. Thermodilution methods and serum biomarkers can be used to monitor lung fluid balance and guide therapy. Recent evidence has indicated significant detrimental effects associated with beta-2 agonists use in ARDS, despite a putative beneficial role in the resolution of alveolar edema seen in preliminary studies. SUMMARY: Dynamic monitoring of lung fluid balance needs to be implemented to guide fluid therapy in ARDS patients. A conservative fluid strategy seems safe and yields overall good clinical outcomes, but its impact on cognitive function needs to be evaluated in further studies. The role of colloids and other pharmacological agents deserves further investigation.

Vasodilator-stimulated phosphoprotein deficiency potentiates PAR-1-induced increase in endothelial permeability in mouse lungs.

Vasodilator-stimulated phosphoprotein (VASP) is implicated in the protection of the endothelial barrier in vitro and in vivo. The function of VASP in thrombin signaling in the endothelial cells (ECs) is not known. For the first time we studied the effects of VASP deficiency on EC permeability and pulmonary vascular permeability in response to thrombin receptor stimulation. We provided the evidence that VASP deficiency potentiates the increase in endothelial permeability induced by activation of thrombin receptor in cultured human umbilical vein endothelial cells (HUVECs) and isolated mouse lungs. Using transendothelial resistance measurement, we showed that siRNA-mediated VASP downregulation in HUVECs leads to a potentiation of thrombin- and protease-activated receptor 1 (PAR-1) agonist-induced increase in endothelial permeability. Compared to control cells, VASP-deficient HUVECs had delayed endothelial junctional reassembly and abrogated VE-cadherin cytoskeletal anchoring in the recovery phase after thrombin stimulation, as demonstrated by immunofluorescence studies and cell fractionation analysis, respectively. Measurement of the capillary filtration coefficient in isolated mouse lungs demonstrated that VASP(-/-) mice have increased microvascular permeability in response to infusion with PAR-1 agonist compared to wild type mice. Lack of VASP led to decreased Rac1 activation both in VASP-deficient HUVECs after thrombin stimulation and VASP(-/-) mouse lungs after PAR-1 agonist infusion, indicating that VASP effects on thrombin signaling may be correlated with changes in Rac1 activity. This study demonstrates that VASP may play critical and complex role in the regulation of thrombin-dependent disruption of the endothelial barrier function.

Tiam1 and Rac1 are required for platelet-activating factor-induced endothelial junctional disassembly and increase in vascular permeability.

It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases.

Cdc42 regulates adherens junction stability and endothelial permeability by inducing alpha-catenin interaction with the vascular endothelial cadherin complex.

The endothelial adherens junctions (AJs) consist of trans-oligomers of membrane spanning vascular endothelial (VE)-cadherin proteins, which bind beta-catenin through their cytoplasmic domain. beta-Catenin in turn binds alpha-catenin and connects the AJ complex with the actin cytoskeleton. We addressed the in vivo effects of loss of VE-cadherin interactions on lung vascular endothelial permeability and the role of specific Rho GTPase effectors in regulating the increase in permeability induced by AJ destabilization. We used cationic liposomes encapsulating the mutant of VE-cadherin lacking the extracellular domain (DeltaEXD) to interfere with AJ assembly in mouse lung endothelial cells. We observed that lung vascular permeability (quantified as microvessel filtration coefficient [K(f,c)]) was increased 5-fold in lungs expressing DeltaEXD. This did not occur to the same degree on expression of the VE-cadherin mutant, DeltaEXDDeltabeta, lacking the beta-catenin-binding site. The increased vascular permeability was the result of destabilization of VE-cadherin homotypic interaction induced by a shift in the binding of beta-catenin from wild-type VE-cadherin to the expressed DeltaEXD mutant. Because DeltaEXD expression in endothelial cells activated the Rho GTPase Cdc42, we addressed its role in the mechanism of increased endothelial permeability induced by AJ destabilization. Coexpression of dominant-negative Cdc42 (N17Cdc42) prevented the increase in K(f,c) induced by DeltaEXD. This was attributed to inhibition of the association of alpha-catenin with the DeltaEXD-beta-catenin complex. The results demonstrate that Cdc42 regulates AJ permeability by controlling the binding of alpha-catenin with beta-catenin and the consequent interaction of the VE-cadherin/catenin complex with the actin cytoskeleton.