Search

Elevated levels of reactive oxygen species and intracellular Ca2+ play a key role in endothelial barrier dysfunction in acute lung injury. We previously showed that H2O2-induced increases in intracellular calcium concentrations ([Ca2+]i) in lung microvascular endothelial cells (LMVECs) involve the membrane Ca2+ channel, transient receptor potential vanilloid-4 (TRPV4) and that inhibiting this channel attenuated H2O2-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to H2O2-induced Ca2+ influx in LMVEC. In endothelial cells, Fyn is tethered to the cell membrane by CD36, a fatty acid transporter. In this study, we assessed the effect of genetic loss or pharmacological inhibition of CD36 on Ca2+ responses to H2O2. H2O2-induced Ca2+ influx was attenuated in LMVEC isolated from mice lacking CD36 ( CD36−/−). TRPV4 expression and function was unchanged in LMVEC isolated from wild-type (WT) and CD36−/− mice, as well as mice with deficiency for Fyn ( Fyn−/−). TRPV4 immunoprecipitated with Fyn, but this interaction was decreased in CD36−/− LMVEC. The amount of phosphorylated TRPV4 was decreased in LMVEC from CD36−/− mice compared with WT controls. Loss of CD36 altered subcellular localization of Fyn, while inhibition of CD36 fatty acid transport with succinimidyl oleate did not attenuate H2O2-induced Ca2+ influx. Lastly, we found that CD36−/− mice were protected from ischemia-reperfusion injury in vivo. In conclusion, our data suggest that CD36 plays an important role in H2O2-mediated lung injury and that the mechanism may involve CD36-dependent scaffolding of Fyn to the cell membrane to facilitate TRPV4 phosphorylation.

In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca2+]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca2+]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca2+]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca2+]i through Ca2+ influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca2+ influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca2+ influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca2+ influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca2+]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.

Phosphodiesterase 2A (PDE2A) is stimulated by cGMP to hydrolyze cAMP, a potent endothelial barrier-protective molecule. We previously found that lung PDE2A contributed to a mouse model of ventilator-induced lung injury (VILI). The purpose of the present study was to determine the contribution of PDE2A in a two-hit mouse model of 1-day intratracheal (IT) LPS followed by 4 h of 20 ml/kg tidal volume ventilation. Compared with IT water controls, LPS alone (3.75 μg/g body wt) increased lung PDE2A mRNA and protein expression by 6 h with a persistent increase in protein through day 4 before decreasing to control levels on days 6 and 10. Similar to the PDE2A time course, the peak in bronchoalveolar lavage (BAL) neutrophils, lactate dehydrogenase (LDH), and protein concentration also occurred on day 4 post-LPS. IT LPS (1 day) and VILI caused a threefold increase in lung PDE2A and inducible nitric oxide synthase (iNOS) and a 24-fold increase in BAL neutrophilia. Compared with a control adenovirus, PDE2A knockdown with an adenovirus expressing a short hairpin RNA administered IT 3 days before LPS/VILI effectively decreased lung PDE2A expression and significantly attenuated BAL neutrophilia, LDH, protein, and chemokine levels. PDE2A knockdown also reduced lung iNOS expression by 53%, increased lung cAMP by nearly twofold, and improved survival from 47 to 100%. We conclude that in a mouse model of LPS/VILI, a synergistic increase in lung PDE2A expression increased lung iNOS and alveolar inflammation and contributed significantly to the ensuing acute lung injury.

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKGI). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKGIbarrier protection because VASP is a cytoskeletal phosphorylation target of PKGIexpressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKGI-mediated phosphorylation of Ser157and Ser239. To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H2O2in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKGIexpression introduced by adenoviral infection (Ad.PKG). In the absence of PKGIexpression, H2O2(100–250 μM) caused a transient increased permeability and pSer157-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser157phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H2O2. With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H2O2-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKGI. Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKGIto attenuate H2O2-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKGIon H2O2-induced endothelial barrier dysfunction in HPAEC.

Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)I, human pulmonary artery endothelial cells (HPAEC) lacking PKGIexpression were infected with a recombinant adenovirus encoding PKGIβ(Ad.PKG) and compared with uninfected and control-infected (Ad.βgal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H2O2(250 μM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.βgal, expressed PKGIprotein and demonstrated Ser239and Ser157phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.βgal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 μM) caused a PKGI-independent decrease in permeability (8.2 ± 0.6%). In all three groups, H2O2(250 μM) caused a similar ∼35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca2+concentration ([Ca2+]i). In uninfected and Ad.βgal-infected HPAEC, pretreatment with CPT-cGMP (100 μM) partially blocked the increased permeability induced by H2O2. In Ad.PKG-infected HPAEC, CPT-cGMP (50 μM) prevented the H2O2-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca2+]icaused by H2O2similarly (23%) in Ad.βgal- and Ad.PKG-infected HPAEC, indicating a PKGI-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKGI-independent process, whereas the ability of cGMP to prevent H2O2-induced barrier dysfunction was predominantly mediated by PKGIthrough a Ca2+-independent mechanism.