NF-κB-dependent repression of Sox18 transcription factor requires the epigenetic regulators histone deacetylases 1 and 2 in acute lung injury.

In acute lung injury (ALI), the NF-κB-mediated downregulation of Sox18 gene expression leads to the disruption of the pulmonary endothelial barrier. Previous studies have suggested that the action of NF-κB as a transcriptional repressor also requires the action of class I histone deacetylases (HDACs). Thus, the purpose of this study was to investigate and further delineate the mechanism of Sox18 repression during lipopolysaccharide (LPS) induced ALI. Using selective inhibitors and specific siRNA-driven depletion of HDACs 1-3 in human lung microvascular endothelial cells (HLMVEC) we were able to demonstrate a critical role for HDACs 1 and 2 in the LPS-mediated repression of Sox18 gene expression and the loss of endothelial monolayer integrity. Moreover, our data demonstrate that HDAC1 associates with a transcription-repressive complex within the NF-κB-binding site of Sox18 promoter. Further, we were able to show that the selective inhibitor of HDAC1, tacedinaline, significantly reduced the endothelial permeability and injury associated with LPS challenge in the mouse lung. Taken together, our data demonstrate, for the first time, that transcription repressors HDACs 1 and 2 are involved in pathological mechanism of ALI and can be considered as therapeutic targets.

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis.

BACKGROUND: MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. METHODS: We employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. RESULTS: As previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation. CONCLUSIONS: Altogether, our results indicate that Muc1 has anti-fibrotic properties in the mouse lung and suggest that elevated levels of MUC1 in patients with interstitial lung disease may serve a protective role, which aims to limit the severity of tissue remodeling in the lung.

Pseudomonas aeruginosa increases MUC1 expression in macrophages through the TLR4-p38 pathway.

Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.

Low anticoagulant heparin blocks thrombin-induced endothelial permeability in a PAR-dependent manner.

Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhance lung inflammation, activate endothelial cells and disrupt lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (ECs). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin was also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH can interfere with thrombin-activated signaling.

Protective effect of adenosine receptors against lipopolysaccharide-induced acute lung injury.

Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) affect 200,000 people a year in the USA. Pulmonary vascular and specifically endothelial cell (EC) barrier compromise is a hallmark of these diseases. We have recently shown that extracellular adenosine enhances human pulmonary (EC) barrier via activation of adenosine receptors (ARs) in cell cultures. On the basis of these data, we hypothesized that activation of ARs might exert barrier-protective effects in a model of ALI/ARDS in mice. To test this hypothesis, we examined the effects of pre- and posttreatment of adenosine and 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective stable AR agonist, on LPS-induced lung injury. Mice were given vehicle or LPS intratracheally followed by adenosine, NECA, or vehicle instilled via the internal jugular vein. Postexperiment cell counts, Evans Blue Dye albumin (EBDA) extravasation, levels of proteins, and inflammatory cytokines were analyzed. Harvested lungs were used for histology and myeloperoxidase studies. Mice challenged with LPS alone demonstrated an inflammatory response typical of ALI. Cell counts, EBDA extravasation, as well as levels of proteins and inflammatory cytokines were decreased in adenosine-treated mice. Histology displayed reduced infiltration of neutrophils. NECA had a similar effect on LPS-induced vascular barrier compromise. Importantly, posttreatment with adenosine or NECA recovers lung vascular barrier and reduces inflammation induced by LPS challenge. Furthermore, adenosine significantly attenuated protein degradation of A2A and A3 receptors induced by LPS. Collectively, our results demonstrate that activation of ARs protects and restores vascular barrier functions and reduces inflammation in LPS-induced ALI.

Taxol alleviates 2-methoxyestradiol-induced endothelial permeability.

We have previously shown that the anti-cancer agent 2-methoxyestradiol (2ME) induces hyperpermeability across endothelial monolayers. Here, we show that both microtubule disruptor, 2ME, and microtubule stabilizer, paclitaxel (taxol), increase vascular lung permeability in vitro and in vivo. Simultaneous application of 2ME and taxol alleviates 2ME-induced endothelial barrier dysfunction, which is evident by the decreased Evans Blue Dye accumulation in lung tissue and increased transendothelial resistance across monolayers. 2ME significantly increases the level of p38 and MLC phosphorylation in both endothelial monolayers and murine lungs; this increase is suppressed in the presence of taxol. Taxol treatment leads to an immediate and sustained increase in tubulin acetylation in human pulmonary artery endothelial cells (HPAEC). Surprisingly, 2ME treatment also increases tubulin acetylation; however, the onset of this process is delayed and coincides with the stage of a partial barrier restoration in HPAEC monolayer. Inhibition of histone deacetylase 6 (HDAC6) with tubacin increases tubulin acetylation level, suppresses 2ME-induced HSP27 and MLC phosphorylation, and decreases 2ME-induced barrier dysfunction, suggesting barrier-protective and/or barrier-restorative role for tubulin acetylation in vascular endothelium.

Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke.

BACKGROUND: Minocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans. METHODS: Five groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed. RESULTS: The model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P < 0.0001) and also improved neurologic score (P < 0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P = 0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P < 0.0001). CONCLUSION: In a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

TIMAP protects endothelial barrier from LPS-induced vascular leakage and is down-regulated by LPS.

TIMAP is a regulatory subunit of protein phosphatase 1, whose role remains largely unknown. Our recent data suggested that TIMAP is involved in the regulation of barrier function in cultured pulmonary endothelial monolayers [Csortos et al., 2008. Am. J. Physiol. Lung Cell. Mol. Physiol. 295, L440-L450]. Here we showed that TIMAP depletion exacerbates lipopolysaccharide (LPS)-induced vascular leakage in murine lung, suggesting that TIMAP has a barrier-protective role in vivo. Real-Time RT PCR analysis revealed that treatment with LPS significantly suppressed Timap mRNA level. This suppression was not achieved via the down-regulation of Timap promoter activity, suggesting that LPS decreased Timap mRNA stability. Pretreatment with protein kinase A (PKA) inhibitor H-89 reduced TIMAP mRNA level, whereas pretreatment with PKA activator, bnz-cAMP, increased this level and attenuated LPS-induced decrease in TIMAP mRNA. Altogether, these data confirmed the barrier-protective role of TIMAP and suggested that barrier-disruptive and barrier-protective agents may employ modulation of TIMAP expression as a mechanism affecting barrier permeability.

Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability.

We showed previously that microtubule disruptor 2-methoxyestradiol (2ME) induces hyperpermeability of the endothelial monolayer via mechanisms that include the activation of p38 and Rho kinase (ROCK) and rearrangement of the actin cytoskeleton. Using the protein kinase C (PKC) inhibitors Ro-31-7549 and Ro-32-0432, we show in vitro and in vivo that 2ME-induced barrier dysfunction is also PKC-dependent. The known PKC substrates ezrin, radixin, and moesin (ERM) were recently implicated in the regulation of endothelial permeability. This study tested the hypotheses that ERM proteins are phosphorylated in response to 2ME, and that this phosphorylation is involved in 2ME-induced barrier dysfunction. We show that the application of 2ME leads to a dramatic increase in the level of ERM phosphorylation. This increase is attenuated in cells pretreated with the microtubule stabilizer taxol. In human pulmonary artery endothelial cells (HPAECs), the phosphorylation of ERM occurs in a p38-dependent and PKC-dependent manner. The activation of p38 appears to occur upstream from the activation of PKC, in response to 2ME. Phosphorylated ERM are localized at the cell periphery during the early phase of response to 2ME (15 minutes), and colocalize with F-actin branching points during the later phase of response (60 minutes). Using the short interfering RNA approach, we also showed that individual ERM depletion significantly attenuates 2ME-induced hyperpermeability. HPAEC monolayers, depleted of ERM proteins and monolayers, overexpressing phosphorylation-deficient ERM mutants, exhibit less attenuation of 2ME-induced barrier disruption in response to the PKC inhibitor Ro-31-7549. These results suggest a critical role of PKC activation in response to microtubule-disrupting agents, and implicate the phosphorylation of ERM in the barrier dysfunction induced by 2ME.

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.

Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability.

Up to date, the nature of the sepsis-induced vascular leakage is understood only partially, which limits pharmacological approaches for its management. Here we studied the protective effect of cAMP using endotoxin-induced hyperpermeability as a model for barrier dysfunction observed in gram-negative sepsis. We demonstrated that the alleviation of lipopolysaccharide (LPS)-induced barrier compromise could be achieved by the specific activation of either protein kinase A (PKA) or Epac with cAMP analogs Bnz-cAMP or O-Me-cAMP, respectively. We next studied the involvement of PKA substrates VASP and filamin1 in barrier maintenance and LPS-induced barrier compromise. Depletion of both VASP and filamin1 with the specific siRNAs significantly exacerbated both the quiescent cells barrier and LPS-induced barrier dysfunction, suggesting barrier-protective role of these proteins. VASP depletion was associated with the more severe loss of ZO-1 peripheral staining in response to LPS, whereas filamin1-depleted cells reacted to LPS with more robust stress fiber induction and more profound changes in ZO-1 and VE-cadherin peripheral organization. Both VASP and filamin1 phosphorylation was significantly increased as a result of PKA activation. We next analyzed the effect of VASP and filamin1 depletion on the PKA-dependent alleviation of LPS-induced barrier compromise. We observed that Bnz-cAMP ability to counteract LPS-induced hyperpermeability was attenuated only by VASP, but not filamin1 depletion. Our data indicate that while PKA-dependent VASP phosphorylation contributes to the protective effect of cAMP elicited on LPS-compromised monolayers, filamin1 phosphorylation is unlikely to play a significant role in this process.