Lactate produced by alveolar type II cells suppresses inflammatory alveolar macrophages in acute lung injury.

Alveolar inflammation is a hallmark of acute lung injury (ALI), and its clinical correlate is acute respiratory distress syndrome-and it is as a result of interactions between alveolar type II cells (ATII) and alveolar macrophages (AM). In the setting of acute injury, the microenvironment of the intra-alveolar space is determined in part by metabolites and cytokines and is known to shape the AM phenotype. In response to ALI, increased glycolysis is observed in AT II cells, mediated by the transcription factor hypoxia-inducible factor (HIF) 1α, which has been shown to decrease inflammation. We hypothesized that in acute lung injury, lactate, the end product of glycolysis, produced by ATII cells shifts AMs toward an anti-inflammatory phenotype, thus mitigating ALI. We found that local intratracheal delivery of lactate improved ALI in two different mouse models. Lactate shifted cytokine expression of murine AMs toward increased IL-10, while decreasing IL-1 and IL-6 expression. Mice with ATII-specific deletion of Hif1a and mice treated with an inhibitor of lactate dehydrogenase displayed exacerbated ALI and increased inflammation with decreased levels of lactate in the bronchoalveolar lavage fluid; however, all those parameters improved with intratracheal lactate. When exposed to LPS (to recapitulate an inflammatory stimulus as it occurs in ALI), human primary AMs co-cultured with alveolar epithelial cells had reduced inflammatory responses. Taken together, these studies reveal an innate protective pathway, in which lactate produced by ATII cells shifts AMs toward an anti-inflammatory phenotype and dampens excessive inflammation in ALI.

Release of extracellular superoxide dismutase into alveolar fluid protects against acute lung injury and inflammation in Staphylococcus aureus pneumonia.

Acute respiratory distress syndrome (ARDS) remains a significant cause of morbidity and mortality in critically ill patients. Oxidative stress and inflammation play a crucial role in the pathogenesis of ARDS. Extracellular superoxide dismutase (EC-SOD) is abundant in the lung and is an important enzymatic defense against superoxide. Human single-nucleotide polymorphism in matrix binding region of EC-SOD leads to the substitution of arginine to glycine at position 213 (R213G) and results in release of EC-SOD into alveolar fluid, without affecting enzyme activity. We hypothesized that R213G EC-SOD variant protects against lung injury and inflammation via the blockade of neutrophil recruitment in infectious model of methicillin-resistant S. aureus (MRSA) pneumonia. After inoculation with MRSA, wild-type (WT) mice had impaired integrity of alveolar-capillary barrier and increased levels of IL-1ß, IL-6, and TNF-α in the broncho-alveolar lavage fluid (BALF), while infected mice expressing R213G EC-SOD variant maintained the integrity of alveolar-capillary interface and had attenuated levels of proinflammatory cytokines. MRSA-infected mice expressing R213G EC-SOD variant also had attenuated neutrophil numbers in BALF and decreased expression of neutrophil chemoattractant CXCL1 by the alveolar epithelial ATII cells, compared with the infected WT group. The decreased neutrophil numbers in R213G mice were not due to increased rate of apoptosis. Mice expressing R213G variant had a differential effect on neutrophil functionality-the generation of neutrophil extracellular traps (NETs) but not myeloperoxidase (MPO) levels were attenuated in comparison with WT controls. Despite having the same bacterial load in the lung as WT controls, mice expressing R213G EC-SOD variant were protected from extrapulmonary dissemination of bacteria.

Isolation of vasa vasorum endothelial cells from pulmonary artery adventitia: Implementation to vascular biology research.

Isolated endothelial cells are valuable in vitro model for vascular research. At present, investigation of disease-relevant changes in vascular endothelium at the molecular level requires established endothelial cell cultures, preserving vascular bed-specific phenotypic characteristics. Vasa vasorum (VV) form a microvascular network around large blood vessels, in both the pulmonary and systemic circulations, that are critically important for maintaining the integrity and oxygen supply of the vascular wall. However, despite the pathophysiological significance of the VV, methods for the isolation and culture of vasa vasorum endothelial cells (VVEC) have not yet been reported. In our prior studies, we demonstrated the presence of hypoxia-induced angiogenic expansion of the VV in the pulmonary artery (PA) of neonatal calves; an observation which has been followed by a series of in vitro studies on isolated PA VVEC. Here we present a detailed protocol for reproducible isolation, purification, and culture of PA VVEC. We show these cells to express generic endothelial markers, (vWF, eNOS, VEGFR2, Tie1, and CD31), as well as progenitor markers (CD34 and CD133), bind lectin Lycopersicon Esculentum, and incorporate acetylated low-density lipoproteins labeled with acetylated LDL (DiI-Ac-LDL). qPCR analysis additionally revealed the expression of CD105, VCAM-1, ICAM-1, MCAM, and NCAM. Ultrastructural electron microscopy and immunofluorescence staining demonstrated that VVEC are morphologically characterized by a developed actin and microtubular cytoskeleton, mitochondrial network, abundant intracellular vacuolar/secretory system, and cell-surface filopodia. VVEC exhibit exponential growth in culture and can be mitogenically activated by multiple growth factors. Thus, our protocol provides the opportunity for VVEC isolation from the PA, and potentially from other large vessels, enabling advances in VV research.

Intense light-elicited alveolar type 2-specific circadian PER2 protects from bacterial lung injury via BPIFB1.

Circadian amplitude enhancement has the potential to be organ protective but has not been studied in acute lung injury (ALI). Consistent light and dark cycles are crucial for the amplitude regulation of the circadian rhythm protein Period2 (PER2). Housing mice under intense instead of ambient light for 1 wk (light: dark cycle:14h:10h), we demonstrated a robust increase of pulmonary PER2 trough and peak levels, which is consistent with circadian amplitude enhancement. A search for the affected lung cell type suggested alveolar type 2 (ATII) cells as strong candidates for light induction of PER2. A head-to-head comparison of mice with cell-type-specific deletion of Per2 in ATII, endothelial, or myeloid cells uncovered a dramatic phenotype in mice with an ATII-specific deletion of Per2. During Pseudomonas aeruginosa-induced ALI, mice with Per2 deletion in ATII cells showed 0% survival, whereas 85% of control mice survived. Subsequent studies demonstrated that intense light therapy dampened lung inflammation or improved the alveolar barrier function during P. aeruginosa-induced ALI, which was abolished in mice with an ATII-specific deletion of Per2. A genome-wide mRNA array uncovered bactericidal/permeability-increasing fold-containing family B member 1 (BPIFB1) as a downstream target of intense light-elicited ATII-PER2 mediated lung protection. Using the flavonoid and PER2 amplitude enhancer nobiletin, we recapitulated the lung-protective and anti-inflammatory effects of light and BPIFB1, respectively. Together, our studies demonstrate that light-elicited amplitude enhancement of ATII-specific PER2 is a critical control point of inflammatory pathways during bacterial ALI.

Targeting alveolar-specific succinate dehydrogenase A attenuates pulmonary inflammation during acute lung injury.

Acute lung injury (ALI) is an inflammatory lung disease, which manifests itself in patients as acute respiratory distress syndrome (ARDS). Previous studies have implicated alveolar-epithelial succinate in ALI protection. Therefore, we hypothesized that targeting alveolar succinate dehydrogenase SDH A would result in elevated succinate levels and concomitant lung protection. Wild-type (WT) mice or transgenic mice with targeted alveolar-epithelial Sdha or hypoxia-inducible transcription factor Hif1a deletion were exposed to ALI induced by mechanical ventilation. Succinate metabolism was assessed in alveolar-epithelial via mass spectrometry as well as redox measurements and evaluation of lung injury. In WT mice, ALI induced by mechanical ventilation decreased SDHA activity and increased succinate in alveolar-epithelial. In vitro, cell-permeable succinate decreased epithelial inflammation during stretch injury. Mice with inducible alveolar-epithelial Sdha deletion (Sdhaloxp/loxp SPC-CreER mice) revealed reduced lung inflammation, improved alveolar barrier function, and attenuated histologic injury. Consistent with a functional role of succinate to stabilize HIF, Sdhaloxp/loxp SPC-CreER experienced enhanced Hif1a levels during hypoxia or ALI. Conversely, Hif1aloxp/loxp SPC-CreER showed increased inflammation with ALI induced by mechanical ventilation. Finally, wild-type mice treated with intra-tracheal dimethlysuccinate were protected during ALI. These data suggest that targeting alveolar-epithelial SDHA dampens ALI via succinate-mediated stabilization of HIF1A. Translational extensions of our studies implicate succinate treatment in attenuating alveolar inflammation in patients suffering from ARDS.

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases.

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a "calm" or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a "activated" state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

Glycolysis and oxidative phosphorylation are essential for purinergic receptor-mediated angiogenic responses in vasa vasorum endothelial cells.

Angiogenesis is an energy-demanding process; however, the role of cellular energy pathways and their regulation by extracellular stimuli, especially extracellular nucleotides, remain largely unexplored. Using metabolic inhibitors of glycolysis (2-deoxyglucose) and oxidative phosphorylation (OXPHOS) (oligomycin, rotenone, and FCCP), we demonstrate that glycolysis and OXPHOS are both essential for angiogenic responses of vasa vasorum endothelial cell (VVEC). Treatment with P2R agonists, ATP, and 2-methylthioadenosine diphosphate trisodium salt (MeSADP), but not P1 receptor agonist, adenosine, increased glycolytic activity in VVEC (measured by extracellular acidification rate and lactate production). Stimulation of glycolysis was accompanied by increased levels of phospho-phosphofructokinase B3, hexokinase (HK), and GLUT-1, but not lactate dehydrogenase. Moreover, extracellular ATP and MeSADP, and to a lesser extent adenosine, increased basal and maximal oxygen consumption rates in VVEC. These effects were potentiated when the cells were cultured in 20 mM galactose and 5 mM glucose compared with 25 mM glucose. Treatment with P2R agonists decreased phosphorylation of pyruvate dehydrogenase (PDH)-E1α and increased succinate dehydrogenase (SDH), cytochrome oxidase IV, and ß-subunit of F1F0 ATP synthase expression. In addition, P2R stimulation transiently elevated mitochondrial Ca2+ concentration, implying involvement of mitochondria in VVEC angiogenic activation. We also demonstrated a critical role of phosphatidylinositol 3-kinase and Akt pathways in lactate production, PDH-E1α phosphorylation, and the expression of HK, SDH, and GLUT-1 in ATP-stimulated VVEC. Together, our findings suggest that purinergic and metabolic regulation of VVEC energy pathways is essential for VV angiogenesis and may contribute to pathologic vascular remodeling in pulmonary hypertension.

High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization.

Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

CNP-miR146a Decreases Inflammation in Murine Acute Infectious Lung Injury.

Acute respiratory distress syndrome (ARDS) has approximately 40% in-hospital mortality, and treatment is limited to supportive care. Pneumonia is the underlying etiology in many cases with unrestrained inflammation central to the pathophysiology. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA(miR)-146a, reduces bleomycin- and endotoxin-induced acute lung injury (ALI) by decreasing inflammation. We therefore hypothesized that CNP-miR146a would decrease inflammation in murine infectious ALI. Mice were injured with intratracheal (IT) MRSA or saline followed by treatment with IT CNP-miR146a or saline control. Twenty-four hours post-infection, bronchoalveolar lavage fluid (BALF) and whole lungs were analyzed for various markers of inflammation. Compared to controls, MRSA infection significantly increased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1ß; p < 0.05), BALF proinflammatory cytokines (IL-6, IL-8, TNFα, IL-1ß; p < 0.01), and inflammatory cell infiltrate (p = 0.03). CNP-miR146a treatment significantly decreased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1ß; p < 0.05), bronchoalveolar proinflammatory protein leak (IL-6, IL-8, TNFα; p < 0.05), and inflammatory infiltrate (p = 0.01). CNP-miR146a decreases inflammation and improves alveolar-capillary barrier integrity in the MRSA-infected lung and has significant promise as a potential therapeutic for ARDS.

Mitogen-activated protein kinase phosphatase-1 is a key regulator of hypoxia-induced vascular endothelial growth factor expression and vessel density in lung.

Although mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key deactivator of MAP kinases, known effectors of lung vessel formation, whether it plays a role in the expression of proangiogenic vascular endothelial growth factor (VEGF) in hypoxic lung is unknown. We therefore hypothesized that MKP-1 is a crucial modulator of hypoxia-stimulated vessel development by regulating lung VEGF levels. Wild-type MKP-1(+/+), heterozygous MKP-1(+/-), and deficient MKP-1(-/-) mice were exposed to sea level (SL), Denver altitude (DA) (1609 m [5280 feet]), and severe high altitude (HYP) (∼5182 m [∼17,000 feet]) for 6 weeks. Hypoxia enhanced phosphorylation of p38 MAP kinase, a substrate of MKP-1, as well as α smooth muscle actin (αSMA) expression in vessels, respiratory epithelium, and interstitium of phosphatase-deficient lung. αSMA-positive vessel (<50 µm outside diameter) densities were markedly reduced, whereas vessel wall thickness was increased in hypoxic MKP-1(-/-) lung. Mouse embryonic fibroblasts (MEFs) of all three genotypes were isolated to pinpoint the mechanism involved in hypoxia-induced vascular abnormalities of MKP-1(-/-) lung. Sustained phosphorylation of p38 MAP kinase was observed in MKP-1-null MEFs in response to hypoxia exposure. Although hypoxia up-regulated VEGF levels in MKP-1(+/+) MEFs eightfold, only a 70% increase in VEGF expression was observed in MKP-1-deficient cells. Therefore, our data strongly suggest that MKP-1 might be the key regulator of vascular densities through the regulation of VEGF levels in hypoxic lung.

HIF1A-dependent induction of alveolar epithelial PFKFB3 dampens acute lung injury.

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.

P2Y1 and P2Y13 purinergic receptors mediate Ca2+ signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells.

Extracellular ATP and ADP have been shown to exhibit potent angiogenic effects on pulmonary artery adventitial vasa vasorum endothelial cells (VVEC). However, the molecular signaling mechanisms of extracellular nucleotide-mediated angiogenesis remain not fully elucidated. Since elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is required for cell proliferation and occurs in response to extracellular nucleotides, this study was undertaken to delineate the purinergic receptor subtypes involved in Ca(2+) signaling and extracellular nucleotide-mediated mitogenic responses in VVEC. Our data indicate that stimulation of VVEC with extracellular ATP resulted in the elevation of [Ca(2+)](i) via Ca(2+) influx through plasma membrane channels as well as Ca(2+) mobilization from intracellular stores. Moreover, extracellular ATP induced simultaneous Ca(2+) responses in both cytosolic and nuclear compartments. An increase in [Ca(2+)](i) was observed in response to a wide range of purinergic receptor agonists, including ATP, ADP, ATPγS, ADPßS, UTP, UDP, 2-methylthio-ATP (MeSATP), 2-methylthio-ADP (MeSADP), and BzATP, but not adenosine, AMP, diadenosine tetraphosphate, αßMeATP, and ßγMeATP. Using RT-PCR, we identified mRNA for the P2Y1, P2Y2, P2Y4, P2Y13, P2Y14, P2X2, P2X5, P2X7, A1, A2b, and A3 purinergic receptors in VVEC. Preincubation of VVEC with the P2Y1 selective antagonist MRS2179 and the P2Y13 selective antagonist MRS2211, as well as with pertussis toxin, attenuated at varying degrees agonist-induced intracellular Ca(2+) responses and activation of ERK1/2, Akt, and S6 ribosomal protein, indicating that P2Y1 and P2Y13 receptors play a major role in VVEC growth responses. Considering the broad physiological implications of purinergic signaling in the regulation of angiogenesis and vascular homeostasis, our findings suggest that P2Y1 and P2Y13 receptors may represent novel and specific targets for treatment of pathological vascular remodeling involving vasa vasorum expansion.

Chronic hypoxia impairs extracellular nucleotide metabolism and barrier function in pulmonary artery vasa vasorum endothelial cells.

Vascular remodeling plays a pivotal role in a variety of pathophysiological conditions where hypoxia and inflammation are prominent features. Intravascular ATP, ADP and adenosine are known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signalling to endothelial cell growth and angiogenesis remains poorly understood. By using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitia of control and chronically hypoxic neonatal calves, these studies were aimed to evaluate the effect of hypoxia on biochemical and functional properties of microvascular endothelial network at the sites of angiogenesis. In comparison with normoxic controls, VVEC from hypoxic animals are characterized by (1) drastically impaired nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5'-nucleotidase/CD73 activities with respective increases in basal extracellular ATP and ADP levels (2) higher proliferative responses to low micromolar concentrations of ATP and ADP; and (3) enhanced permeability and disordered adenosinergic control of vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran). Together, these results suggest that unique pattern of purine-mediated angiogenic activation and enhanced leakiness of VVEC from chronically hypoxic vessels may be defined by disordered endothelial nucleotide homeostasis at sites of active neovascularization.

Immunomodulatory strategies prevent the development of autoimmune emphysema.

BACKGROUND: The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known. METHODS: Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed. RESULTS: The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vß16.1 in the lung tissue. CONCLUSIONS: Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.

Rho kinase-mediated vasoconstriction is important in severe occlusive pulmonary arterial hypertension in rats.

Vascular remodeling, rather than vasoconstriction, is believed to account for high vascular resistance in severe pulmonary arterial hypertension (PAH). We have found previously that acute Rho kinase inhibition nearly normalizes PAH in chronically hypoxic rats that have no occlusive neointimal lesions. Here we examined whether Rho kinase-mediated vasoconstriction was also important in a rat model of severe occlusive PAH. Adult rats were exposed to chronic hypoxia ( approximately 10% O(2)) after subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SUGEN 5416. Hemodynamic measurements were made in anesthetized rats after 2 weeks of hypoxia (early group) and 3 weeks of hypoxia plus 2 weeks of normoxia (late group). Both groups developed PAH, with greater severity in the late group. In the early group, intravenous fasudil was more effective than intravenous bradykinin, inhaled NO, or intravenous iloprost in reducing right ventricular systolic pressure. Despite more occlusive vascular lesions, fasudil also markedly reduced right ventricular systolic pressure in late-stage rats. Blood-perfused lungs from late-stage rats showed spontaneous vasoconstriction, which was reversed partially by the endothelin A receptor blocker BQ123 and completely by fasudil or Y-27632. Phosphorylation of MYPT1, a downstream target of Rho kinase, was increased in lungs from both groups of rats, and fasudil (intravenous) reversed the increased phosphorylation in the late group. Thus, in addition to structural occlusion, Rho kinase-mediated vasoconstriction is an important component of severe PAH in SUGEN 5416/hypoxia-exposed rats, and PAH can be significantly reduced in the setting of a severely remodeled lung circulation if an unconventional vasodilator is used.

The cancer paradigm of severe pulmonary arterial hypertension.

The plexiform lesions of severe pulmonary arterial hypertension (PAH) are similar in histologic appearance, whether the disease is idiopathic or secondary. Both forms of the disease show actively proliferating endothelial cells without evidence of apoptosis. Here, we discuss the pathobiology of the atypical, angioproliferative endothelial cells in severe PAH. The concept of the endothelial cell as a "quasi-malignant" cell provides a new framework for antiproliferative, antiangiogenic therapy in severe PAH.

Hypoxia exposure induces the emergence of fibroblasts lacking replication repressor signals of PKCzeta in the pulmonary artery adventitia.

AIMS: Cultured fibroblasts of hypoxia-stimulated remodelled pulmonary artery (PA) adventitia proliferate at a greater rate compared with those of normal adventitia. Since protein kinase C (PKC) zeta is a replication repressor of normal adventitial fibroblasts, we hypothesized that loss of the repressor activity of PKCzeta might contribute to increased rate of proliferation in adventitial cells of remodelled PA. METHODS AND RESULTS: Isolated PA adventitial fibroblasts of neonatal control (Fib-C) and chronic hypoxia-exposed (Fib-H) calves were used to test our hypothesis. For evaluation of the role of PKCzeta in hypoxia-induced vascular adventitial remodelling, expression and activation of PKCzeta were also examined in lung sections of Fib-C and Fib-H animals by immunoperoxidase staining. Although constitutively active PKCzeta expression attenuated DNA synthesis in Fib-C, it stimulated proliferation in Fib-H. PKCzeta-specific myristoylated pseudosubstrate peptide inhibitor (PKCzeta-PI) induced replication in Fib-C, whereas the inhibitor blocked DNA synthesis in Fib-H. Hypoxia stimulated PKCzeta as well as MAP kinase kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation in Fib-H cells. However, ERK1/2 activation was mediated by both MEK1/2-dependent and MEK1/2-independent PKCzeta-regulated mechanisms in hypoxia-exposed Fib-H. PKCzeta was selectively activated in the adventitial cells of the remodelled vascular wall, as demonstrated by strong immunoreactivity against the anti-phosphoPKCzeta antibody in the Fib-H lung sections. CONCLUSION: PKCzeta acts as a replication repressor in Fib-C cells; however, the same isozyme mediates Fib-H proliferation. Thus, chronic exposure to hypoxia leads to the emergence of cells lacking anti-replication activity of PKCzeta in the PA adventitia.

Capturing the multifactorial nature of ARDS - "Two-hit" approach to model murine acute lung injury.

Severe acute respiratory distress syndrome (ARDS) presents typically with an initializing event, followed by the need for mechanical ventilation. Most animal models of ALI are limited by the fact that they focus on a singular cause of acute lung injury (ALI) and therefore fail to mimic the complex, multifactorial pathobiology of ARDS. To better capture this scenario, we provide a comprehensive characterization of models of ALI combining two injuries: intra tracheal (i.t.) instillation of LPS or hypochloric acid (HCl) followed by ventilator-induced lung injury (VILI). We hypothesized, that mice pretreated with LPS or HCl prior to VILI and thus receiving a ("two-hit injury") will sustain a superadditive lung injury when compared to VILI. Mice were allocated to following treatment groups: control with i.t. NaCl, ventilation with low peak inspiratory pressure (PIP), i.t. HCl, i.t. LPS, VILI (high PIP), HCl i.t. followed by VILI and LPS i.t. followed by VILI. Severity of injury was determined by protein content and MPO activity in bronchoalveolar lavage (BAL), the expression of inflammatory cytokines and histopathology. Mice subjected to VILI after HCl or LPS instillation displayed augmented lung injury, compared to singular lung injury. However, mice that received i.t. LPS prior to VILI showed significantly increased inflammatory lung injury compared to animals that underwent i.t. HCl followed by VILI. The two-hit lung injury models described, resulting in additive but differential acute lung injury recaptures the clinical relevant multifactorial etiology of ALI and could be a valuable tool in translational research.

SIRT1 is required for mitochondrial biogenesis reprogramming in hypoxic human pulmonary arteriolar smooth muscle cells.

Although recent studies have reported that mitochondria are putative oxygen sensors underlying hypoxic pulmonary vasoconstriction, little is known concerning the sirtuin 1 (SIRT1)-mediated mitochondrial biogenesis regulatory program in pulmonary arteriolar smooth muscle cells (PASMCs) during hypoxia/reoxygenation (H/R). We investigated the epigenetic regulatory mechanism of mitochondrial biogenesis and function in human PASMCs during H/R. Human PASMCs were exposed to hypoxia of 24-48 h and reoxygenation of 24-48 h. The expression of SIRT1 was reduced in a time-dependent manner. Mitochondrial transcription factor A (TFAM) expression was increased during hypoxia and decreased during reoxygenation, while the release of TFAM was increased in a time-dependent manner. Lentiviral overexpression of SIRT1 preserved SIRT3 deacetylase activity in human PASMCs exposed to H/R. Knockdown of PGC-1α suppressed the effect of SIRT1 on SIRT3 activity. Knockdown of SIRT3 abrogated SIRT1-mediated deacetylation of cyclophilin D (CyPD). Notably, knockdown of SIRT3 or PGC-1α suppressed the incremental effect of SIRT1 on mitochondrial TFAM, mitochondrial DNA (mtDNA) content and cellular ATP levels. Importantly, polydatin restored SIRT1 levels in human PASMCs exposed to H/R. Knockdown of SIRT1 suppressed the effect of polydatin on mitochondrial TFAM, mtDNA content and cellular ATP levels. In conclusion, SIRT1 expression is decreased in human PASMCs during H/R. TFAM expression in mitochondria is reduced and the release of TFAM is increased by H/R. PGC-1α/SIRT3/CyPD mediates the protective effect of SIRT1 on expression and release of TFAM and mitochondrial biogenesis and function. Polydatin improves mitochondrial biogenesis and function by enhancing SIRT1 expression in hypoxic human PASMCs.

Detrimental ELAVL-1/HuR-dependent GSK3ß mRNA stabilization impairs resolution in acute respiratory distress syndrome.

A hallmark of acute respiratory distress syndrome (ARDS) is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK) 3ß during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3ß could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3ß messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3ß, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like) as a central component in a likely GSK3ß signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3ß mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3ß at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3ß. Together, our findings indicate a previously unknown interaction between GSK3ß and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3ß pathways as a novel ARDS treatment approach.