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Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca]) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca] in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca], migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.

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.

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Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population-epithelial (CD326(+)CD31(-)CD45(-)), endothelial (CD326(-)CD31(+)CD45(-)), and hematopoietic lineage (CD326(-)CD31(-)CD45(+))-and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis.

(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 Kf caused by HVT 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 M 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 wetto-dry weight ratio in an anesthetized mouse model of HV T injury. Additional activation of sGC (1.5 M BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K f in lungs ventilated with 15 ml/kg V T. HVT endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 M IBMX) as well as an inhibitor (10 M BAY 60-7550) specific for the cGMPstimulated 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. nitric oxide; guanosine 3Ј,5Ј-cyclic monophosphate; phosphodiesterase 2A; adenosine 3Ј,5Ј-cyclic monophosphate; atrial natriuretic peptide; endothelial permeability MECHANICAL VENTILATION is necessary to support patients with the acute respiratory distress syndrome. However, mechanical ventilation also contributes to lung injury by a mechanism that directly correlates with tidal volume (V T ) (39). High V T (HV T ) ventilation causes pulmonary endothelial barrier dysfunction and edema, leading to ventilator-induced lung injury (VILI) (42). The mechanisms behind this injury are complex, but it is evident that HV T -induced changes in intracellular signaling play a significant role (12).Recent evidence from animal models of VILI implicates nitric oxide (NO) in the pathogenesis of HV T endothelial barrier dysfunction (4,6,29). NO is synthesized by three isoforms of nitric oxide synthase (NOS): endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS. Ventilatory lung stretch stimulates pulmonary microvascular NO production by an increase in eNOS expression (6), phosphorylation of eNOS by phosphatidylinositol 3-kinase-induced protein kinase B activation (22), or induction of iNOS expression (13,29). Once produced by NOS, NO reacts with reactive oxygen species (ROS) to generate peroxynitrite, a toxic free radical that contributes to VILI (29). NO can also stimulate endothelial soluble guanylyl cyclase (sGC) to generat...

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.

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.