Otgonchimeg Rentsendorj

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice.

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.

Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG(I)). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG(I) barrier protection because VASP is a cytoskeletal phosphorylation target of PKG(I) expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG(I)-mediated phosphorylation of Ser(157) and Ser(239). To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H(2)O(2) in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKG(I) expression introduced by adenoviral infection (Ad.PKG). In the absence of PKG(I) expression, H(2)O(2) (100-250 microM) caused a transient increased permeability and pSer(157)-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser(157) phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H(2)O(2). With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H(2)O(2)-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 PKG(I). Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG(I) to attenuate H(2)O(2)-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG(I) on H(2)O(2)-induced endothelial barrier dysfunction in HPAEC.

Knockdown of lung phosphodiesterase 2A attenuates alveolar inflammation and protein leak in a two-hit mouse model of acute lung injury

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.

Protein kinase G increases antioxidant function in lung microvascular endothelial cells by inhibiting the c-Abl tyrosine kinase

Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKGI) posttranscriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKGI is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKGI with the cGMP analog 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild-type but not PKGI(-/-) MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a posttranscriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKGI, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKGI may be the downstream mechanism underlying PKGI-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.

Phosphodiesterase 2A is a major negative regulator of iNOS expression in lipopolysaccharide‐treated mouse alveolar macrophages

PDE2A is a dual‐function PDE that is stimulated by cGMP to hydrolyze cAMP preferentially. In a two‐hit model of ALI, we found previously that PDE2A decreased lung cAMP, up‐regulated lung iNOS, and exacerbated ALI. Recent data suggest that macrophage iNOS expression contributes to ALI but later, promotes lung‐injury resolution. However, macrophage iNOS is increased by cAMP, suggesting that PDE2A could negatively regulate macrophage iNOS expression. To test this, we examined the effects of manipulating PDE2A expression and function on LPS‐induced iNOS expression in a mouse AM cell line (MH‐S) and primary mouse AMs. In MH‐S cells, LPS (100 ng/ml) increased PDE2A expression by 15% at 15 min and 50% at 6 h before decreasing at 24 h and 48 h. iNOS expression appeared at 6 h and remained increased 48 h post‐LPS. Compared with control Ad, Ad.PDE2A‐shRNA enhanced LPS‐induced iNOS expression further by fourfold, an effect mimicked by the PDE2A inhibitor BAY 60–7550. Adenoviral PDE2A overexpression or treatment with ANP decreased LPS‐induced iNOS expression. ANP‐induced inhibition of iNOS was lost by knocking down PDE2A and was not mimicked by 8‐pCPT‐cGMP, a cGMP analog that does not stimulate PDE2A activity. Finally, we found that in primary AMs from LPS‐treated mice, PDE2A knockdown also increased iNOS expression, consistent with the MH‐S cell data. We conclude that increased AM PDE2A is an important negative regulator of macrophage iNOS expression.

CD36 mediates H2O2-induced calcium influx in lung microvascular endothelial cells

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.

Nitric Oxide Synthase Promotes Distension-Induced Tracheal Venular Leukocyte Adherence

The process of leukocyte recruitment to the airways in real time has not been extensively studied, yet airway inflammation persists as a major contributor to lung pathology. We showed previously in vivo, that neutrophils are recruited acutely to the large airways after periods of airway distension imposed by the application of positive end-expiratory pressure (PEEP). Given extensive literature implicating products of nitric oxide synthase (NOS) in lung injury after ventilatory over-distension, we questioned whether similar mechanisms exist in airway post-capillary venules. Yet, endothelial nitric oxide has been shown to be largely anti-inflammatory in other systemic venules. Using intravital microscopy to visualize post-capillary tracheal venules in anesthetized, ventilated mice, the number of adherent leukocytes was significantly decreased in eNOS-/- mice under baseline conditions (2±1 cell/60 min observation) vs wild type (WT) C57BL/6 mice (7±2 cells). After exposure to PEEP (8 cmH2O for 1 min; 5 times), adherent cells increased significantly (29±5 cells) in WT mice while eNOS-/- mice demonstrated a significantly decreased number of adherent cells (11±4 cells) after PEEP. A similar response was seen when thrombin was used as the pro-inflammatory stimulus. In addition, mouse tracheal venular endothelial cells studied in vitro after exposure to cyclic stretch (18% elongation) or thrombin both demonstrated increased p-selectin expression that was significantly attenuated by NG-nitro-L-arginine methyl ester, N-acetylcysteine amide (NACA) and excess BH4. In vivo treatment with the ROS inhibitor NACA or co-factor BH4 abolished completely the PEEP-induced leukocyte adherence. These results suggest that pro-inflammatory stimuli cause leukocyte recruitment to tracheal endothelium in part due to eNOS uncoupling.

Janus-faced signaling of cGMP in acute lung injury

The effect of increasing pulmonary endothelial cGMP concentration on endothelial function in acute lung injury appears to depend on 1) the presence of specific cGMP targets, 2) intracellular cGMP compartmentalization and 3) the timing of the increase in cGMP relative to the injury onset [1-4]. For example, we recently showed that pretreatment of pulmonary artery endothelial monolayers with 8pCPT-cGMP attenuated oxidant-induced barrier dysfunction by a cGMP-dependent kinase-1 (cGKI)-dependent mechanism [1,2]. More recently, however, we found that the increase in endogenous lung cGMP resulting from increased NO production in a ventilator-induced lung injury (VILI) mouse model caused lung endothelial barrier dysfunction [4]. The injurious effect of sGC-derived cGMP in VILI was mediated by the simultaneous generation of phosphodiesterase 2A (PDE2A), which was stimulated by cGMP to hydrolyze cAMP. Interestingly, in the same model, pretreatment with BAY 41-2272 (1.5 μM) to stimulate sGC before injurious tidal volume ventilation attenuated VILI. Recent evidence suggests that endothelial apoptosis may contribute to VILI [5] so we wondered if the protective effect of increasing lung endothelial cGMP before injury could be mediated by an anti-apoptotic effect of cGMP signalling. Mouse lung microvascular endothelial cells (MLMVEC) were isolated and purified by flow cytometry and shown to express cGK1 by Western blot and phosphorylation of VASP Ser235. A 6 hr pretreatment with 8pCPT-cGMP (50 μM), significantly attenuated H2O2-induced cell death assessed by flow cytometry (Annexin-, 7AAD-) and nuclear condensation. A similar protection was not observed in human pulmonary artery endothelial cells (HPAEC) which lack cGK1 expression in vitro. Restoration of cGK1 expression in HPAEC resulted in cGMP-mediated protection against oxidant cell death suggesting a cGK1-mediated effect. To determine if this protective effect was upstream of apoptotic signaling, MLMVEC from C57BL6 mice were treated with 8p-CPT-cGMP (50 uM) before exposure to increasing concentrations of H2O2. The extracellular H2O2 concentration ([H2O2]ext) was continuously measured with a H2O2 electrode. Compared with untreated cells, wildtype MLMVEC pre-treated with 8p-CPT-cGMP for 2 or 4 hrs (but not 30 min) significantly decreased the maximal Δ[H2O2]ext by 33 ± 11, 32 ± 10 and 25 ± 10% in cells exposed to 20, 50, and 100 μM H2O2, respectively (N = 8, P < 0.01). Consistent with this effect, 8pCPT-cGMP pretreatment attenuated H2O2-induced H2DCF fluorescence as well as p38MAPK and Akt phosphorylation suggesting that intracellular H2O2 concentration was also decreased. MLMVEC isolated from cGK1-/- mice failed to enhance H2O2 uptake suggesting cGK1-mediated signaling was responsible. An assessment of the major H2O2 degrading enzyme systems revealed a significant cGMP-mediated increase in catalase expression without an increase in catalase mRNA suggesting a post-translational effect. We conclude that the effects of cGMP signalling on lung endothelial function in acute lung injury are complex and include both injurious and protective mechanisms depending on the specific downstream signalling pathways that are present. Activation of lung microvascular endothelial cell cGK1 by cGMP protects against oxidant-mediated cell death possibly through an increase in endothelial antioxidant function.