Mahendra Damarla

Xanthine oxidoreductase in respiratory and cardiovascular disorders

In addition to its critical role in purine metabolism, xanthine oxidoreductase (XOR) has been implicated in the development of tissue oxidative damage in a wide variety of respiratory and cardiovascular disorders such as acute lung injury, ischemia-reperfusion injury, atherosclerosis, heart failure, and arterial hypertension. Although much remains to be clarified about the regulation and signaling pathways of this enzyme, it is quite evident from abundant investigation in animal models and some human trials that XOR inhibition can favorably alter critical disease processes and impact outcomes. From promising bench-to-bedside data, a better understanding of this enigmatic enzyme is emerging. However, the positive findings related to XOR inhibition need to be confirmed in large-scale, well-designed clinical trials. This will hopefully provide new opportunities for therapeutic intervention. This article reviews the available evidence involving XOR in oxidative states with specific emphasis on respiratory and cardiovascular diseases.

Alveolar cell apoptosis is dependent on p38 MAP kinase-mediated activation of xanthine oxidoreductase in ventilator-induced lung injury

Signaling via p38 MAP kinase has been implicated in the mechanotransduction associated with mechanical stress and ventilator-induced lung injury (VILI). However, the critical downstream mediators of alveolar injury remain incompletely defined. We provide evidence that high-tidal volume mechanical ventilation (HVt MV) rapidly activates caspases within the lung, resulting in increased alveolar cell apoptosis. Antagonism of MV-induced p38 MAP kinase activity with SB-203580 suppresses both MV-induced caspase activity and alveolar apoptosis, placing p38 MAP kinase upstream of MV-induced caspase activation and programmed cell death. The reactive oxygen species (ROS)-producing enzyme xanthine oxidoreductase (XOR) is activated in a p38 MAP kinase-dependent manner following HVt MV. Allopurinol, a XOR inhibitor, also suppresses HVt MV-induced apoptosis, implicating HVt MV-induced ROS in the induction of alveolar cell apoptosis. Finally, systemic administration of the pan-caspase inhibitor, z-VAD-fmk, but not its inactive peptidyl analog, z-FA-fmk, blocks ventilator-induced apoptosis of alveolar cells and alveolar-capillary leak, indicating that caspase-dependent cell death is necessary for VILI-associated barrier dysfunction in vivo.

Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

p53 Mediates Cigarette Smoke–Induced Apoptosis of Pulmonary Endothelial Cells: Inhibitory Effects of Macrophage Migration Inhibitor Factor

Exposure to cigarette smoke (CS) is the most common cause of emphysema, a debilitating pulmonary disease histopathologically characterized by the irreversible destruction of lung architecture. Mounting evidence links enhanced endothelial apoptosis causally to the development of emphysema. However, the molecular determinants of human endothelial cell apoptosis and survival in response to CS are not fully defined. Such determinants could represent clinically relevant targets for intervention. We show here that CS extract (CSE) triggers the death of human pulmonary macrovascular endothelial cells (HPAECs) through a caspase 9-dependent apoptotic pathway. Exposure to CSE results in the increased expression of p53 in HPAECs. Using the p53 inhibitor, pifithrin-α (PFT-α), and RNA interference (RNAi) directed at p53, we demonstrate that p53 function and expression are required for CSE-mediated apoptosis. The expression of macrophage migration inhibitory factor (MIF), an antiapoptotic cytokine produced by HPAECs, also increases in response to CSE exposure. The addition of recombinant human MIF prevents cell death from exposure to CSE. Further, the suppression of MIF or its receptor/binding partner, Jun activation domain-binding protein 1 (Jab-1), with RNAi enhances the sensitivity of human pulmonary endothelial cells to CSE via a p53-dependent (PFT-α-inhibitable) pathway. Finally, we demonstrate that MIF is a negative regulator of p53 expression in response to CSE, placing MIF upstream of p53 as an antagonist of CSE-induced apoptosis. We conclude that MIF can protect human vascular endothelium from the toxic effects of CSE via the antagonism of p53-mediated apoptosis.

Activated protein C protects against ventilator-induced pulmonary capillary leak

The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothelial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high (HV(T); 20 ml/kg) or low (LV(T); 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HV(T) induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. HV(T) also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from HV(T)-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-beta(6)) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection.

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung

AbstractAim:To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling.Methods:Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS−/−) were exposed to mechanical ventilation (MV) with low (LVT, 7 mL/kg) and high tidal volume (HVT, 20 mL/kg) for 0−4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review.Results:MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS−/− mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38.Conclusion:Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

Moderate oxygen augments lipopolysaccharide-induced lung injury in mice

Despite the associated morbidity and mortality, underlying mechanisms leading to the development of acute lung injury (ALI) remain incompletely understood. Frequently, ALI develops in the hospital, coinciding with institution of various therapies, including the use of supplemental oxygen. Although pathological evidence of hyperoxia-induced ALI in humans has yet to be proven, animal studies involving high oxygen concentration reproducibly induce ALI. The potentially injurious role of lower and presumably safer oxygen concentrations has not been well characterized in any species. We hypothesized that in the setting of a preexisting insult to the lung, the addition of moderate-range oxygen can augment lung injury. Our model of low-dose intratracheal LPS (IT LPS) followed by 60% oxygen caused a significant increase in ALI compared with LPS or oxygen alone with increased alveolar neutrophils, histological injury, and epithelial barrier permeability. In the LPS plus oxygen group, regulatory T cell number was reduced, and macrophage activation markers were increased, compared with LPS alone. Antibody-mediated depletion of neutrophils significantly abrogated the observed lung injury for all measured factors. The enhanced presence of alveolar neutrophils in the setting of LPS and oxygen is due, at least in part, to elevated chemokine gradients signaling neutrophils to the alveolar space. We believe these results strongly support an effect of lower concentrations of oxygen to augment the severity of a mild preexisting lung injury and warrants further investigation in both animals and humans.

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.

Xanthine oxidoreductase is a critical mediator of cigarette smoke-induced endothelial cell DNA damage and apoptosis

Cigarette smoke (CS) exposure is unquestionably the most frequent cause of emphysema in the United States. Accelerated pulmonary endothelial cell (EC) apoptosis is an early determinant of lung destruction in emphysema. One of the pathogenic causes of emphysema is an alveolar oxidant and antioxidant imbalance. The enzyme xanthine oxidoreductase (XOR) has been shown to be a source of reactive oxygen species (ROS) in a multitude of diseases (S. Sakao et al., FASEB J.21, 3640-3652; 2007). The contribution of XOR to CS-induced apoptosis is not well defined. Here we demonstrate that C57/bl6 mice exposed to CS have increased pulmonary XOR activity and protein levels compared to filtered-air-exposed controls. In addition, we demonstrate that primary pulmonary human lung microvascular endothelial cells exposed to cigarette smoke extract undergo increased rates of caspase-dependent apoptosis that are reliant on XOR activity, ROS production, and p53 function/expression. We also demonstrate that exogenous XOR is sufficient to increase p53 expression and induce apoptosis, suggesting that XOR is an upstream mediator of p53 in CS-induced EC apoptosis. Furthermore, we show that XOR activation results in DNA double-strand breaks that activate the enzyme ataxia telangiectasia mutated, which phosphorylates histone H2AX and upregulates p53. In conclusion, CS increases XOR expression, and the enzyme is both sufficient and necessary for p53 induction and CS-induced EC apoptosis.

Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 Mediates Apoptosis during Lung Vascular Permeability by Regulating Movement of Cleaved Caspase 3

Apoptosis is a key pathologic feature in acute lung injury. Animal studies have demonstrated that pathways regulating apoptosis are necessary in the development of acute lung injury, and that activation of p38 mitogen-activated protein kinase (MAPK) is linked to the initiation of the apoptotic cascade. In this study, we assessed the role of the MAPK-activated protein kinase (MK) 2, one of p38 MAPKs immediate downstream effectors, in the development of apoptosis in an animal model of LPS-induced pulmonary vascular permeability. Our results indicate that wild-type (WT) mice exposed to LPS demonstrate increased apoptosis, as evidenced by cleavage of caspase 3 and poly (ADP-ribose) polymerase 1 and increased deoxynucleotidyl transferase-mediated dUDP nick-end labeling staining, which is accompanied by increases in markers of vascular permeability. In contrast, MK2(-/-) mice are protected from pulmonary vascular permeability and apoptosis in response to LPS. Although there was no difference in activation of caspase 3 in MK2(-/-) compared with WT mice, interestingly, cleaved caspase 3 translocated to the nucleus in WT mice while it remained in the cytosol of MK2(-/-) mice in response to LPS. In separate experiments, LPS-induced apoptosis in human lung microvascular endothelial cells was also associated with nuclear translocation of cleaved caspase 3 and apoptosis, which were both prevented by MK2 silencing. In conclusion, our data suggest that MK2 plays a critical role in the development of apoptosis and pulmonary vascular permeability, and its effects on apoptosis are in part related to its ability to regulate nuclear translocation of cleaved caspase 3.