Joyce Gonzales

Extracellular β‐nicotinamide adenine dinucleotide (β‐NAD) promotes the endothelial cell barrier integrity via PKA‐ and EPAC1/Rac1‐dependent actin cytoskeleton rearrangement

Extracellular β‐NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5‐trisphate and cAMP. Recently, β‐NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β‐NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β‐NAD‐activated purinergic receptors upon β‐NAD stimulation. Our data demonstrate that extracellular β‐NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependent manner indicating endothelial barrier enhancement. Importantly, β‐NAD significantly attenuated thrombin‐induced EC permeability as well as the barrier‐compromising effects of Gram‐negative and Gram‐positive bacterial toxins representing the barrier‐protective function of β‐NAD. Immunofluorescence microscopy reveals more pronounced staining of cell–cell junctional protein VE‐cadherin at the cellular periphery signifying increased tightness of the cell‐cell contacts after β‐NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β‐NAD‐induced TER increase. β‐NAD‐treatment attenuates the lipopolysaccharide (LPS)‐induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP‐dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β‐NAD‐induced EC barrier enhancement. With these results, we conclude that β‐NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1‐ and MLCP‐ dependent signaling pathways. J. Cell. Physiol. 223: 215–223, 2010.

Potential role of A2A adenosine receptor in traumatic optic neuropathy.

In traumatic optic neuropathy (TON), apoptosis of retinal ganglion cells is closely related to the local production of reactive oxygen species and inflammatory mediators from activated microglial cells. Adenosine receptor A2A (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in TON. In the present study, we examined the role of A2AAR in retinal complications associated with TON. Initial studies in wild-type mice revealed that treatment with the A2AAR agonist resulted in marked decreases in the TON-induced microglial activation, retinal cell death and releases of reactive oxygen species and pro-inflammatory cytokines TNF-α and IL-6. To further assess the role of A2AAR in TON, we studied the effects of A2AAR ablation on the TON-induced retinal abnormalities. A2AAR-/- mice with TON showed a significantly higher mRNA level of TNF-α, Iba1-1 in retinal tissue, and ICAM-1 expression in retinal sections compared with wild-type mice with TON. To explore a potential mechanism by which A2AAR-signaling regulates inflammation in TON, we performed additional studies using hypoxia- or LPS-treated microglial cells as an in vitro model for TON. Activation of A2AAR attenuates hypoxia or LPS-induced TNF-α release and significantly repressed the inflammatory signaling, ERK in the activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in TON and suggests that the retinal protective effect of A2AAR is mediated by attenuating the inflammatory response that occurs in microglia via interaction with MAPKinase 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.

β-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury

ABSTRACT Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) occur in approximately 200,000 patients per year. Studies indicate that lung endothelium plays a significant role in ALI. The authors’ recent in vitro studies demonstrate a novel mechanism of β-nicotinamide adenine dinucleotide (β-NAD)–induced protection against gram-positive (pneumolysin, PLY) and gram-negative (lipopolysaccharide, LPS) toxin–induced lung endothelial cell (EC) barrier dysfunction. The objective of the current study was to evaluate the protective effect of β-NAD against LPS-induced ALI in mice. C57BL/6J mice were randomly divided into 4 groups: vehicle, β-NAD, LPS, and LPS/β-NAD. After surgery, mice were allowed to recover for 24 hours. Evans blue dye–albumin (EBA) was given through the internal jugular vein 2 hours prior to the termination of the experiments. Upon sacrificing the animals, bronchoalveolar lavage fluid (BALF) was collected and the lungs were harvested. β-NAD treatment significantly attenuated the inflammatory response by means of reducing the accumulation of cells and protein in BALF, blunting the parenchymal neutrophil infiltration, and preventing capillary leak. In addition, the histological examination demonstrated decreased interstitial edema in the LPS/β-NAD specimens, as compared to the LPS-only specimens. The mRNA levels of the anti-inflammatory cytokines were up-regulated in the LPS group treated with β-NAD compared to the LPS-only–treated group. β-NAD treatment down-regulated the mRNA levels of the proinflammatory cytokines. These findings suggest that β-NAD could be investigated as a therapeutic option against bacterial toxin–induced lung inflammation and ALI in mice.

Protective effect of Growth Hormone-Releasing Hormone agonist in bacterial toxin-induced pulmonary barrier dysfunction

Rationale: Antibiotic treatment of patients infected with G− or G+ bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in GHRH agonist-treated HL-MVEC, in the presence of PLY, by means of siRNA-mediated protein kinase A (PKA) depletion. Methods: Barrier function measurements were done in HL-MVEC monolayers using Electrical Cell substrate Impedance Sensing (ECIS) and VE-cadherin expression by Western blotting. Capillary leak was assessed by Evans Blue dye (EBD) incorporation. Cytokine generation in broncho-alveolar lavage fluid (BALF) was measured by multiplex analysis. PKA and PKC-α activity were assessed by Western blotting. Results: GHRH agonist JI-34 significantly blunts LPS-induced barrier dysfunction, at least in part by preserving VE-cadherin expression, while not affecting inflammation. In addition to activating PKA, GHRH agonist also increases PKC-α activity in PLY-treated HL-MVEC. Treatment with PLY significantly decreases resistance in control siRNA-treated HL-MVEC, but does so even more in PKA-depleted monolayers. Pretreatment with GHRH agonist blunts PLY-induced permeability in control siRNA-treated HL-MVEC, but fails to improve barrier function in PKA-depleted PLY-treated monolayers. Conclusions: GHRH signaling in HL-MVEC protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective PKA-mediated and a barrier-disruptive PKC-α-induced pathway in the presence of PLY, the former of which dominates the latter.

Epithelial sodium channel-α mediates the protective effect of the TNF-derived TIP peptide in pneumolysin-induced endothelial barrier dysfunction

Background Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar–capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The α subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. Methods The presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. Results HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. Conclusion Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

Pulmonary Platelet Thrombi and Vascular Pathology in Acute Chest Syndrome in Patients with Sickle Cell Disease

A growing body of evidence suggests a role for platelets in sickle cell disease (SCD). Despite the proinflammatory, occlusive nature of platelets, a role for platelets in acute chest syndrome (ACS), however, remains understudied. To provide evidence and potentially describe contributory factors for a putative link between ACS and platelets, we performed an autopsy study of 20 SCD cases—10 of whom died from ACS and 10 whose deaths were not ACS‐related. Pulmonary histopathology and case history were collected. We discovered that disseminated pulmonary platelet thrombi were present in 3 out of 10 of cases with ACS, but none of the matched cases without ACS. Those cases with detected thrombi were associated with significant deposition of endothelial vWF and detection of large vWF aggregates adhered to endothelium. Potential clinical risk factors were younger age and higher platelet count at presentation. However, we also noted a sharp and significant decline in platelet count prior to death in each case with platelet thrombi in the lungs. In this study, neither hydroxyurea use nor perimortem transfusion was associated with platelet thrombi. Surprisingly, in all cases, there was profound pulmonary artery remodeling with both thrombotic and proliferative pulmonary plexiform lesions. The severity of remodeling was not associated with a severe history of ACS, or hydroxyurea use, but was inversely correlated with age. We thus provide evidence of undocumented presence of platelet thrombi in cases of fatal ACS and describe clinical correlates. We also provide novel correlates of pulmonary remodeling in SCD. Am. J. Hematol. 91:173–178, 2016.

Impaired pulmonary endothelial barrier function in sickle cell mice

Acute and chronic pulmonary complications leading to significant morbidity and mortality occur in persons with sickle cell disease (SCD). One of the leading causes of death is acute chest syndrome (ACS),[1][1] diagnosed by a new infiltrate on chest x-ray often triggered by infection.[2][2] The