Julie Newton

Isoprenylcysteine Carboxyl Methyltransferase Modulates Endothelial Monolayer Permeability: Involvement of RhoA Carboxyl Methylation

Abstract— RhoA and Rac1 regulate formation of stress fibers and intercellular junctions, thus modulating endothelial monolayer permeability. Posttranslational modifications of RhoA and Rac1 regulate enzyme activity and subcellular localization, resulting in altered cellular function. The role of RhoA and Rac1 carboxyl methylation in modulating endothelial monolayer permeability is not known. In this study, we found that inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT) with adenosine plus homocysteine or N-acetyl-S-geranylgeranyl-l-cysteine decreased RhoA carboxyl methylation, RhoA activity, and endothelial monolayer permeability, suggesting that RhoA carboxyl methylation may play a role in the ICMT-modulated monolayer permeability. Similar studies showed no effect of ICMT inhibition on Rac1 carboxyl methylation or localization. Bovine pulmonary artery endothelial cells (PAECs) stably overexpressing ICMT-GFP cDNA were established to determine if increased ICMT expression could alter RhoA or Rac1 carboxyl methylation, activation, and endothelial monolayer permeability. PAECs stably overexpressing ICMT demonstrated increased RhoA carboxyl methylation, membrane-bound RhoA, and RhoA activity. Additionally, PAECs stably overexpressing ICMT had diminished VE-cadherin and &bgr;-catenin at intercellular junctions, with resultant intercellular gap formation, as well as enhanced monolayer permeability. These effects were blunted by adenosine plus homocysteine and by inhibition of RhoA, but not by inhibition of Rac1. These results indicate that ICMT modulates endothelial monolayer permeability by altering RhoA carboxyl methylation and activation, thus changing the organization of intercellular junctions. Therefore, carboxyl methylation of RhoA may modulate endothelial barrier function.

Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A(1) or A(3) antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A(2A) and A(2B), respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A(2A) and A(2B) receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A(2A)/A(2B) receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A(2A) and A(2B) agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A(2A) and A(2B) receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by alpha-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.

Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury

Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases.

Cigarette smoke-induced lung endothelial apoptosis and emphysema are associated with impairment of FAK and eIF2α.

Lung endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. However, the mechanism underlying cigarette smoke (CS)-induced lung EC apoptosis and emphysema is not well defined. We have previously shown that cigarette smoke extract (CSE) decreased focal adhesion kinase (FAK) activity via oxidative stress in cultured lung EC. In this study, we compared FAK activation in the lungs of highly susceptible AKR mice and mildly susceptible C57BL/6 mice after exposure to CS for three weeks. We found that three weeks of CS exposure caused mild emphysema and increased lung EC apoptosis in AKR mice (room air: 12.8±5.6%; CS: 30.7±3.7%), but not in C57BL/6 mice (room air: 0±0%; CS: 3.5±1.7%). Correlated with increased lung EC apoptosis and early onset of emphysema, FAK activity was reduced in the lungs of AKR mice, but not of C57BL/6 mice. Additionally, inhibition of FAK caused lung EC apoptosis, whereas over-expression of FAK prevented CSE-induced lung EC apoptosis. These results suggest that FAK inhibition may contribute to CS-induced lung EC apoptosis and emphysema. Unfolded protein response (UPR) and autophagy have been shown to be activated by CS exposure in lung epithelial cells. In this study, we noted that CSE activated UPR and autophagy in cultured lung EC, as indicated by enhanced eIF2α phosphorylation and elevated levels of GRP78 and LC3B-II. However, eIF2α phosphorylation was significantly reduced by three-weeks of CS exposure in the lungs of AKR mice, but not of C57BL/6 mice. Markers for autophagy activation were not significantly altered in the lungs of either AKR or C57BL/6 mice. These results suggest that CS-induced impairment of eIF2α signaling may increase the susceptibility to lung EC apoptosis and emphysema. Taken together, our data suggest that inhibition of eIF2α and FAK signaling may play an important role in CS-induced lung EC apoptosis and emphysema.

Release of soluble E-selectin from activated endothelial cells upon apoptosis.

Circulating soluble E-selectin is increased in diseases associated with endothelial apoptosis such as sepsis and acute respiratory distress syndrome. We investigated the mechanism by which endothelial cell (EC) apoptosis may promote soluble E-selectin release. We found that serum deprivation of EC caused apoptosis, yet it did not induce E-selectin EC surface expression. Tumor necrosis factor-α (TNFα) significantly increased EC E-selectin surface expression. Soluble E-selectin was noted, however, only in the medium of TNFα-activated, apoptotic EC. Preincubation of the EC with the caspase inhibitor z-VAD-fmk significantly attenuated soluble E-selectin levels in the culture medium of TNFα-activated, apoptotic EC, but it had no effect on E-selectin surface expression. These results indicate that TNFα activation, but not apoptosis, is necessary for E-selectin surface expression in EC. Furthermore, E-selectin release from EC requires caspase-3 activation. Thus, increased concentrations of circulating E-selectin in serum may serve as a marker for endothelial apoptosis in certain disease states.

Sustained Adenosine Exposure Causes Lung Endothelial Barrier Dysfunction via Nucleoside Transporter–Mediated Signaling

Previous studies by our group as well as others have shown that acute adenosine exposure enhances lung vascular endothelial barrier integrity and protects against increased permeability lung edema. In contrast, there is growing evidence that sustained adenosine exposure has detrimental effects on the lungs, including lung edema. It is well established that adenosine modulates lung inflammation. However, little is known concerning the effect of sustained adenosine exposure on lung endothelial cells (ECs), which are critical to the maintenance of the alveolar-capillary barrier. We show that exogenous adenosine plus adenosine deaminase inhibitor caused sustained elevation of adenosine in lung ECs. This sustained adenosine exposure decreased EC barrier function, elevated cellular reactive oxygen species levels, and activated p38, JNK, and RhoA. Inhibition of equilibrative nucleoside transporters (ENTs) prevented sustained adenosine-induced p38 and JNK activation and EC barrier dysfunction. Inhibition of p38, JNK, or RhoA also partially attenuated sustained adenosine-induced EC barrier dysfunction. These data indicate that sustained adenosine exposure causes lung EC barrier dysfunction via ENT-dependent intracellular adenosine uptake and subsequent activation of p38, JNK, and RhoA. The antioxidant N-acetylcysteine and the NADPH inhibitor partially blunted sustained adenosine-induced JNK activation but were ineffective in attenuation of p38 activation or barrier dysfunction. p38 was activated exclusively in mitochondria, whereas JNK was activated in mitochondria and cytoplasm by sustained adenosine exposure. Our data further suggest that sustained adenosine exposure may cause mitochondrial oxidative stress, leading to activation of p38, JNK, and RhoA in mitochondria and resulting in EC barrier dysfunction.

Cigarette Smoke Disrupted Lung Endothelial Barrier Integrity and Increased Susceptibility to Acute Lung Injury via Histone Deacetylase 6.

Epidemiologic evidence indicates that cigarette smoke (CS) is associated with the development of acute lung injury (ALI). We have previously shown that brief CS exposure exacerbates lipopolysaccharide (LPS)-induced ALI in vivo and endothelial barrier dysfunction in vitro. In this study, we found that CS also exacerbated Pseudomonas-induced ALI in mice. We demonstrated that lung microvascular endothelial cells (ECs) isolated from mice exposed to CS had a greater permeability or incomplete recovery after challenges by LPS and thrombin. Histone deacetylase (HDAC) 6 deacetylates proteins essential for maintenance of endothelial barrier function. We found that HDAC6 phosphorylation at serine-22 was increased in lung tissues of mice exposed to CS and in lung ECs exposed to cigarette smoke extract (CSE). Inhibition of HDAC6 attenuated CSE-induced increases in EC permeability and CS priming of ALI. Similar barrier protection was provided by the microtubule stabilizer taxol, which preserved α-tubulin acetylation. CSE decreased α-tubulin acetylation and caused microtubule depolymerization. In coordination with increased HDAC6 phosphorylation, CSE inhibited Akt and activated glycogen synthase kinase (GSK)-3β; these effects were ameliorated by the antioxidant N-acetyl cysteine. Our results suggest that CS increases lung EC permeability, thereby enhancing susceptibility to ALI, likely through oxidative stress-induced Akt inactivation and subsequent GSK-3β activation. Activated GSK-3β may activate HDAC6 via phosphorylation of serine-22, leading to α-tubulin deacetylation and microtubule disassembly. Inhibition of HDAC6 may be a novel therapeutic option for ALI in cigarette smokers.

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild‐type (PKCδ+/+) and null (PKCδ−/−) mice housed under normoxia (21% O2) or hyperoxia (∼95% O2). We noted a significantly greater number of TUNEL‐positive cells in lungs of hyperoxic PKCδ+/+ mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ+/+ mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ−/− hyperoxic mice, compared to lungs of PKCδ+/+ hyperoxic mice. In vitro, both hyperoxia and H2O2 promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H2O2 treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H2O2‐induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase‐mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H2O2‐induced LMVEC p38 activation. Conversely, overexpression of wild‐type PKCδ or the catalytically active PKCδ cleavage product greatly increased H2O2‐induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant‐induced apoptosis is due to increased PKCδ → p38 signaling, and we describe a PKCδ‐centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress. J. Cell. Physiol. 227: 1899–1910, 2012.

Alda-1 Protects Against Acrolein-Induced Acute Lung Injury and Endothelial Barrier Dysfunction

&NA; Inhalation of acrolein, a highly reactive aldehyde, causes lung edema. The underlying mechanism is poorly understood and there is no effective treatment. In this study, we demonstrated that acrolein not only dose‐dependently induced lung edema but also promoted LPS‐induced acute lung injury. Importantly, acrolein‐induced lung injury was prevented and rescued by Alda‐1, an activator of mitochondrial aldehyde dehydrogenase 2. Acrolein also dose‐dependently increased monolayer permeability, disrupted adherens junctions and focal adhesion complexes, and caused intercellular gap formation in primary cultured lung microvascular endothelial cells (LMVECs). These effects were attenuated by Alda‐1 and the antioxidant N‐acetylcysteine, but not by the NADPH inhibitor apocynin. Furthermore, acrolein inhibited AMP‐activated protein kinase (AMPK) and increased mitochondrial reactive oxygen species levels in LMVECs—effects that were associated with impaired mitochondrial respiration. AMPK total protein levels were also reduced in lung tissue of mice and LMVECs exposed to acrolein. Activation of AMPK with 5‐aminoimidazole‐4‐carboxamide‐1‐&bgr;‐4‐ribofuranoside blunted an acrolein‐induced increase in endothelial monolayer permeability, but not mitochondrial oxidative stress or inhibition of mitochondrial respiration. Our results suggest that acrolein‐induced mitochondrial dysfunction may not contribute to endothelial barrier dysfunction. We speculate that detoxification of acrolein by Alda‐1 and activation of AMPK may be novel approaches to prevent and treat acrolein‐associated acute lung injury, which may occur after smoke inhalation.