Strain-dependent lung transcriptomic differences in cigarette smoke and LPS models of lung injury in mice.

Cigarette smoking increases the risk of acute respiratory distress syndrome (ARDS; Calfee CS, Matthay MA, Eisner MD, Benowitz N, Call M, Pittet J-F, Cohen MJ. Am J Respir Crit Care Med 183: 1660-1665, 2011; Calfee CS, Matthay MA, Kangelaris KN, Siew ED, Janz DR, Bernard GR, May AK, Jacob P, Havel C, Benowitz NL, Ware LB. Crit Care Med 43: 1790-1797, 2015; Toy P, Gajic O, Bacchetti P, Looney MR, Gropper MA, Hubmayr R, Lowell CA, Norris PJ, Murphy EL, Weiskopf RB, Wilson G, Koenigsberg M, Lee D, Schuller R, Wu P, Grimes B, Gandhi MJ, Winters JL, Mair D, Hirschler N, Sanchez Rosen R, Matthay MA, TRALI Study Group. Blood 119: 1757-1767, 2012) and causes emphysema. However, it is not known why some individuals develop disease, whereas others do not. We found that smoke-exposed AKR mice were more susceptible to lipopolysaccharides (LPS)-induced acute lung injury (ALI) than C57BL/6 mice (Sakhatskyy P, Wang Z, Borgas D, Lomas-Neira J, Chen Y, Ayala A, Rounds S, Lu Q. Am J Physiol Lung Cell Mol Physiol 312: L56-L67, 2017); thus, we investigated strain-dependent lung transcriptomic responses to cigarette smoke (CS). Eight-week-old male AKR and C57BL/6 mice were exposed to 3 wk of room air (RA) or cigarette smoke (CS) for 6 h/day, 4 days/wk, followed by intratracheal instillation of LPS or normal saline (NS) and microarray analysis of lung homogenate gene expression. Other groups of AKR and C57 mice were exposed to RA or CS for 6 wk, followed by evaluation of static lung compliance and tissue elastance, morphometric evaluation for emphysema, or microarray analysis of lung gene expression. Transcriptomic analyses of lung homogenates show distinct strain-dependent lung transcriptional responses to CS and LPS, with AKR mice having larger numbers of genes affected than similarly treated C57 mice, congruent with strain differences in physiologic and inflammatory parameters previously observed in LPS-induced ALI after CS priming. These results suggest that genetic differences may underlie differing susceptibility of smokers to ARDS and emphysema. Strain-based differences in gene transcription contribute to CS and LPS-induced lung injury. There may be a genetic basis for smoking-related lung injury. Clinicians should consider cigarette smoke exposure as a risk factor for ALI and ARDS.NEW & NOTEWORTHY We demonstrate that transcriptomes expressed in lung homogenates also differ between the mouse strains and after acute (3 wk) exposure of animals to cigarette smoke (CS) and/or to lipopolysaccharide. Mouse strains also differed in physiologic, pathologic, and transcriptomic, responses to more prolonged (6 wk) exposure to CS. These data support a genetic basis for enhanced susceptibility to acute and chronic lung injury among humans who smoke cigarettes.

CIGARETTE SMOKING INCREASES THE RISK OF ACUTE RESPIRATORY DISTRESS SYNDROME.

Cigarette smoking (CS) remains a cause of considerable morbidity and mortality, despite recent progress in smoking cessation in the United States. Epidemiologic studies in humans have reported associations between CS and development of acute respiratory distress syndrome (ARDS) after a number of inciting risk factors. We have assessed the effects of CS exposure on lung vascular permeability and inflammation in mice and found that both acute and sustained CS exposure increased the severity of acute lung injury caused by subsequent intrapulmonary instillation of lipopolysaccharide. In addition to enhanced inflammation, CS exposure directly impaired lung endothelial cell barrier function. Our results indicate that mouse strains differ in susceptibility to CS exacerbation of acute lung injury and that there are differences in transcriptomic effects of CS. These results demonstrate the biologic basis for the association of CS with development of ARDS. We propose that CS be considered a cause of heterogeneity of ARDS phenotypes and that this be recorded as a risk factor in the design of clinical trials.

Blockade of equilibrative nucleoside transporter 1/2 protects against Pseudomonas aeruginosa-induced acute lung injury and NLRP3 inflammasome activation.

Pseudomonas aeruginosa infections are increasingly multidrug resistant and cause healthcare-associated pneumonia, a major risk factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Adenosine is a signaling nucleoside with potential opposing effects; adenosine can either protect against acute lung injury via adenosine receptors or cause lung injury via adenosine receptors or equilibrative nucleoside transporter (ENT)-dependent intracellular adenosine uptake. We hypothesized that blockade of intracellular adenosine uptake by inhibition of ENT1/2 would increase adenosine receptor signaling and protect against P. aeruginosa-induced acute lung injury. We observed that P. aeruginosa (strain: PA103) infection induced acute lung injury in C57BL/6 mice in a dose- and time-dependent manner. Using ENT1/2 pharmacological inhibitor, nitrobenzylthioinosine (NBTI), and ENT1-null mice, we demonstrated that ENT blockade elevated lung adenosine levels and significantly attenuated P. aeruginosa-induced acute lung injury, as assessed by lung wet-to-dry weight ratio, BAL protein levels, BAL inflammatory cell counts, pro-inflammatory cytokines, and pulmonary function (total lung volume, static lung compliance, tissue damping, and tissue elastance). Using both agonists and antagonists directed against adenosine receptors A2AR and A2BR, we further demonstrated that ENT1/2 blockade protected against P. aeruginosa -induced acute lung injury via activation of A2AR and A2BR. Additionally, ENT1/2 chemical inhibition and ENT1 knockout prevented P. aeruginosa-induced lung NLRP3 inflammasome activation. Finally, inhibition of inflammasome prevented P. aeruginosa-induced acute lung injury. Our results suggest that targeting ENT1/2 and NLRP3 inflammasome may be novel strategies for prevention and treatment of P. aeruginosa-induced pneumonia and subsequent ARDS.

Mitochondrial Fission Mediated Cigarette Smoke-induced Pulmonary Endothelial Injury.

Cigarette smoke (CS) exposure increases the risk for acute respiratory distress syndrome in humans and promotes alveolar-capillary barrier permeability and acute lung injury in animal models. However, the underlying mechanisms are not well understood. Mitochondrial fusion and fission are essential for mitochondrial homeostasis in health and disease. In this study, we hypothesized that CS caused endothelial injury via an imbalance of mitochondrial fusion and fission and resultant mitochondrial oxidative stress and dysfunction. We noted that CS altered mitochondrial morphology by shortening mitochondrial networks and causing perinuclear accumulation of damaged mitochondria in primary rat lung microvascular endothelial cells. We also found that CS increased mitochondrial fission likely by decreasing Drp1-S637 and increasing FIS1, Drp1-S616 phosphorylation, mitochondrial translocation, and tetramerization and reduced mitochondrial fusion likely by decreasing Mfn2 in lung microvascular endothelial cells and mouse lungs. CS also caused aberrant mitophagy, increased mitochondrial oxidative stress, and reduced mitochondrial respiration. An inhibitor of mitochondrial fission and a mitochondria-specific antioxidant prevented CS-induced increased endothelial barrier dysfunction and apoptosis. Our data suggest that excessive mitochondrial fission and resultant oxidative stress are essential mediators of CS-induced endothelial injury and that inhibition of mitochondrial fission and mitochondria-specific antioxidants may be useful therapeutic strategies for CS-induced endothelial injury and associated pulmonary diseases.

Effects of cigarette smoke on pulmonary endothelial cells.

Cigarette smoking is the leading cause of preventable disease and death in the United States. Cardiovascular comorbidities associated with both active and secondhand cigarette smoking indicate the vascular toxicity of smoke exposure. Growing evidence supports the injurious effect of cigarette smoke on pulmonary endothelial cells and the roles of endothelial cell injury in development of acute respiratory distress syndrome (ARDS), emphysema, and pulmonary hypertension. This review summarizes results from studies of humans, preclinical animal models, and cultured endothelial cells that document toxicities of cigarette smoke exposure on pulmonary endothelial cell functions, including barrier dysfunction, endothelial activation and inflammation, apoptosis, and vasoactive mediator production. The discussion is focused on effects of cigarette smoke-induced endothelial injury in the development of ARDS, emphysema, and vascular remodeling in chronic obstructive pulmonary disease.

Pulmonary Endothelial Cell Apoptosis in Emphysema and Acute Lung Injury.

Apoptosis plays an essential role in homeostasis and pathogenesis of a variety of human diseases. Endothelial cells are exposed to various environmental and internal stress and endothelial apoptosis is a pathophysiological consequence of these stimuli. Pulmonary endothelial cell apoptosis initiates or contributes to progression of a number of lung diseases. This chapter will focus on the current understanding of the role of pulmonary endothelial cell apoptosis in the development of emphysema and acute lung injury (ALI) and the factors controlling pulmonary endothelial life and death.

Enhancing Insights into Pulmonary Vascular Disease through a Precision Medicine Approach. A Joint NHLBI-Cardiovascular Medical Research and Education Fund Workshop Report.

The Division of Lung Diseases of the NHLBI and the Cardiovascular Medical Education and Research Fund held a workshop to discuss how to leverage the anticipated scientific output from the recently launched "Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics" (PVDOMICS) program to develop newer approaches to pulmonary vascular disease. PVDOMICS is a collaborative, protocol-driven network to analyze all patient populations with pulmonary hypertension to define novel pulmonary vascular disease (PVD) phenotypes. Stakeholders, including basic, translational, and clinical investigators; clinicians; patient advocacy organizations; regulatory agencies; and pharmaceutical industry experts, joined to discuss the application of precision medicine to PVD clinical trials. Recommendations were generated for discussion of research priorities in line with NHLBI Strategic Vision Goals that include: (1) A national effort, involving all the stakeholders, should seek to coordinate biosamples and biodata from all funded programs to a web-based repository so that information can be shared and correlated with other research projects. Example programs sponsored by NHLBI include PVDOMICS, Pulmonary Hypertension Breakthrough Initiative, the National Biological Sample and Data Repository for PAH, and the National Precision Medicine Initiative. (2) A task force to develop a master clinical trials protocol for PVD to apply precision medicine principles to future clinical trials. Specific features include: (a) adoption of smaller clinical trials that incorporate biomarker-guided enrichment strategies, using adaptive and innovative statistical designs; and (b) development of newer endpoints that reflect well-defined and clinically meaningful changes. (3) Development of updated and systematic variables in imaging, hemodynamic, cellular, genomic, and metabolic tests that will help precisely identify individual and shared features of PVD and serve as the basis of novel phenotypes for therapeutic interventions.

Alda-1 Protects Against Acrolein-Induced Acute Lung Injury and Endothelial Barrier Dysfunction.

Inhalation of acrolein, a highly reactive aldehyde, causes lung edema. The underlying mechanism is poorly understood and there is no effective treatment. In this study, we demonstrated that acrolein not only dose-dependently induced lung edema but also promoted LPS-induced acute lung injury. Importantly, acrolein-induced lung injury was prevented and rescued by Alda-1, an activator of mitochondrial aldehyde dehydrogenase 2. Acrolein also dose-dependently increased monolayer permeability, disrupted adherens junctions and focal adhesion complexes, and caused intercellular gap formation in primary cultured lung microvascular endothelial cells (LMVECs). These effects were attenuated by Alda-1 and the antioxidant N-acetylcysteine, but not by the NADPH inhibitor apocynin. Furthermore, acrolein inhibited AMP-activated protein kinase (AMPK) and increased mitochondrial reactive oxygen species levels in LMVECs-effects that were associated with impaired mitochondrial respiration. AMPK total protein levels were also reduced in lung tissue of mice and LMVECs exposed to acrolein. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside blunted an acrolein-induced increase in endothelial monolayer permeability, but not mitochondrial oxidative stress or inhibition of mitochondrial respiration. Our results suggest that acrolein-induced mitochondrial dysfunction may not contribute to endothelial barrier dysfunction. We speculate that detoxification of acrolein by Alda-1 and activation of AMPK may be novel approaches to prevent and treat acrolein-associated acute lung injury, which may occur after smoke inhalation.

Double-hit mouse model of cigarette smoke priming for acute lung injury.

Epidemiological studies indicate that cigarette smoking (CS) increases the risk and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The mechanism is not understood, at least in part because of lack of animal models that reproduce the key features of the CS priming process. In this study, using two strains of mice, we characterized a double-hit mouse model of ALI induced by CS priming of injury caused by lipopolysaccharide (LPS). C57BL/6 and AKR mice were preexposed to CS briefly (3 h) or subacutely (3 wk) before intratracheal instillation of LPS and ALI was assessed 18 h after LPS administration by measuring lung static compliance, lung edema, vascular permeability, inflammation, and alveolar apoptosis. We found that as little as 3 h of exposure to CS enhanced LPS-induced ALI in both strains of mice. Similar exacerbating effects were observed after 3 wk of preexposure to CS. However, there was a strain difference in susceptibility to CS priming for ALI, with a greater effect in AKR mice. The key features we observed suggest that 3 wk of CS preexposure of AKR mice is a reproducible, clinically relevant animal model that is useful for studying mechanisms and treatment of CS priming for a second-hit-induced ALI. Our data also support the concept that increased susceptibility to ALI/ARDS is an important adverse health consequence of CS exposure that needs to be taken into consideration when treating critically ill individuals.

Effect of α7 nicotinic acetylcholine receptor activation on cardiac fibroblasts: a mechanism underlying RV fibrosis associated with cigarette smoke exposure.

Right ventricular (RV) dysfunction is associated with numerous smoking-related illnesses, including chronic obstructive pulmonary disease (COPD), in which it is present even in the absence of pulmonary hypertension. It is unknown whether exposure to cigarette smoke (CS) has direct effects on RV function and cardiac fibroblast (CF) proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to CS and determined mechanisms of smoke-induced changes in CF signaling and fibrosis. AKR mice were exposed to CS for 6 wk followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat CFs exposed to CS extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. Mice exposed to CS had significantly decreased RV function, as determined by tricuspid annular plane systolic excursion. There were no changes in left ventricular parameters. RV collagen content was significantly elevated, but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased CF proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-δ, and reduced p38-MAPK phosphorylation. CS and nicotine have direct effects on CFs to induce proliferation and fibrosis, which may negatively affect right heart function.

Cigarette Smoke Disrupted Lung Endothelial Barrier Integrity and Increased Susceptibility to Acute Lung Injury via Histone Deacetylase 6.

Epidemiologic evidence indicates that cigarette smoke (CS) is associated with the development of acute lung injury (ALI). We have previously shown that brief CS exposure exacerbates lipopolysaccharide (LPS)-induced ALI in vivo and endothelial barrier dysfunction in vitro. In this study, we found that CS also exacerbated Pseudomonas-induced ALI in mice. We demonstrated that lung microvascular endothelial cells (ECs) isolated from mice exposed to CS had a greater permeability or incomplete recovery after challenges by LPS and thrombin. Histone deacetylase (HDAC) 6 deacetylates proteins essential for maintenance of endothelial barrier function. We found that HDAC6 phosphorylation at serine-22 was increased in lung tissues of mice exposed to CS and in lung ECs exposed to cigarette smoke extract (CSE). Inhibition of HDAC6 attenuated CSE-induced increases in EC permeability and CS priming of ALI. Similar barrier protection was provided by the microtubule stabilizer taxol, which preserved α-tubulin acetylation. CSE decreased α-tubulin acetylation and caused microtubule depolymerization. In coordination with increased HDAC6 phosphorylation, CSE inhibited Akt and activated glycogen synthase kinase (GSK)-3ß; these effects were ameliorated by the antioxidant N-acetyl cysteine. Our results suggest that CS increases lung EC permeability, thereby enhancing susceptibility to ALI, likely through oxidative stress-induced Akt inactivation and subsequent GSK-3ß activation. Activated GSK-3ß may activate HDAC6 via phosphorylation of serine-22, leading to α-tubulin deacetylation and microtubule disassembly. Inhibition of HDAC6 may be a novel therapeutic option for ALI in cigarette smokers.

An official American Thoracic Society Statement: pulmonary hypertension phenotypes.

BACKGROUND: Current classification of pulmonary hypertension (PH) is based on a relatively simple combination of patient characteristics and hemodynamics. This limits customization of treatment, and lacks the clarity of a more granular identification based on individual patient phenotypes. Rapid advances in mechanistic understanding of the disease, improved imaging methods, and innovative biomarkers now provide an opportunity to define PH phenotypes on the basis of biomarkers, advanced imaging, and pathobiology. This document organizes our current understanding of PH phenotypes and identifies gaps in our knowledge. METHODS: A multidisciplinary committee with expertise in clinical care (pulmonary, cardiology, pediatrics, and pathology), clinical research, and/or basic science in the areas of PH identified important questions and reviewed and synthesized the literature. RESULTS: This document describes selected PH phenotypes and serves as an initial platform to define additional relevant phenotypes as new knowledge is generated. The biggest gaps in our knowledge stem from the fact that our present understanding of PH phenotypes has not come from any particularly organized effort to identify such phenotypes, but rather from reinterpreting studies and reports that were designed and performed for other purposes. CONCLUSIONS: Accurate phenotyping of PH can be used in research studies to increase the homogeneity of study cohorts. Once the ability of the phenotypes to predict outcomes has been validated, phenotyping may also be useful for determining prognosis and guiding treatment. This important next step in PH patient care can optimally be addressed through a consortium of study sites with well-defined goals, tasks, and structure. Planning and support for this could include the National Institutes of Health and the U.S. Food and Drug Administration, with industry and foundation partnerships.

Cigarette smoke-induced lung endothelial apoptosis and emphysema are associated with impairment of FAK and eIF2α.

Lung endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. However, the mechanism underlying cigarette smoke (CS)-induced lung EC apoptosis and emphysema is not well defined. We have previously shown that cigarette smoke extract (CSE) decreased focal adhesion kinase (FAK) activity via oxidative stress in cultured lung EC. In this study, we compared FAK activation in the lungs of highly susceptible AKR mice and mildly susceptible C57BL/6 mice after exposure to CS for three weeks. We found that three weeks of CS exposure caused mild emphysema and increased lung EC apoptosis in AKR mice (room air: 12.8±5.6%; CS: 30.7±3.7%), but not in C57BL/6 mice (room air: 0±0%; CS: 3.5±1.7%). Correlated with increased lung EC apoptosis and early onset of emphysema, FAK activity was reduced in the lungs of AKR mice, but not of C57BL/6 mice. Additionally, inhibition of FAK caused lung EC apoptosis, whereas over-expression of FAK prevented CSE-induced lung EC apoptosis. These results suggest that FAK inhibition may contribute to CS-induced lung EC apoptosis and emphysema. Unfolded protein response (UPR) and autophagy have been shown to be activated by CS exposure in lung epithelial cells. In this study, we noted that CSE activated UPR and autophagy in cultured lung EC, as indicated by enhanced eIF2α phosphorylation and elevated levels of GRP78 and LC3B-II. However, eIF2α phosphorylation was significantly reduced by three-weeks of CS exposure in the lungs of AKR mice, but not of C57BL/6 mice. Markers for autophagy activation were not significantly altered in the lungs of either AKR or C57BL/6 mice. These results suggest that CS-induced impairment of eIF2α signaling may increase the susceptibility to lung EC apoptosis and emphysema. Taken together, our data suggest that inhibition of eIF2α and FAK signaling may play an important role in CS-induced lung EC apoptosis and emphysema.

Sustained adenosine exposure causes lung endothelial apoptosis: a possible contributor to cigarette smoke-induced endothelial apoptosis and lung injury.

Pulmonary endothelial cell (EC) apoptosis has been implicated in the pathogenesis of emphysema. Cigarette smoke (CS) causes lung EC apoptosis and emphysema. In this study, we show that CS exposure increased lung tissue adenosine levels in mice, an effect associated with increased lung EC apoptosis and the development of emphysema. Adenosine has a protective effect against apoptosis via adenosine receptor-mediated signaling. However, sustained elevated adenosine increases alveolar cell apoptosis in adenosine deaminase-deficient mice. We established an in vitro model of sustained adenosine exposure by incubating lung EC with adenosine in the presence of an adenosine deaminase inhibitor, deoxycoformicin. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter-facilitated intracellular adenosine uptake, subsequent activation of p38 and JNK in mitochondria, and ultimately mitochondrial defects and activation of the mitochondria-mediated intrinsic pathway of apoptosis. Our results suggest that sustained elevated adenosine may contribute to CS-induced lung EC apoptosis and emphysema. Our data also reconcile the paradoxical effects of adenosine on apoptosis, demonstrating that prolonged exposure causes apoptosis via nucleoside transporter-mediated intracellular adenosine signaling, whereas acute exposure protects against apoptosis via activation of adenosine receptors. Inhibition of adenosine uptake may become a new therapeutic target in treatment of CS-induced lung diseases.

Bad blood, bad endothelium: ill fate?

In this issue of Blood, Farha and colleagues have identified a myeloproliferative process that is intrinsic to human pulmonary arterial hypertension (PAH), but appears coupled to pathologic vascular remodeling in the lung.

IL-1ß-mediated adaptive reprogramming of endogenous human cardiac fibroblasts to cells with immune features during fibrotic remodeling.

The source and roles of fibroblasts and T-cells during maladaptive remodeling and myocardial fibrosis in the setting of pulmonary arterial hypertension (PAH) have been long debated. We demonstrate, using single-cell mass cytometry, a subpopulation of endogenous human cardiac fibroblasts expressing increased levels of CD4, a helper T-cell marker, in addition to myofibroblast markers distributed in human fibrotic RV tissue, interstitial and perivascular lesions in SUGEN/Hypoxia (SuHx) rats, and fibroblasts labeled with pdgfrα CreERt2/+ in R26R-tdTomato mice. Recombinant IL-1ß increases IL-1R, CCR2 receptor expression, modifies the secretome, and differentiates cardiac fibroblasts to form CD68-positive cell clusters. IL-1ß also activates stemness markers, such as NANOG and SOX2, and genes involved in dedifferentiation, lymphoid cell function and metabolic reprogramming. IL-1ß induction of lineage traced primary mouse cardiac fibroblasts causes these cells to lose their fibroblast identity and acquire an immune phenotype. Our results identify IL-1ß induced immune-competency in human cardiac fibroblasts and suggest that fibroblast secretome modulation may constitute a therapeutic approach to PAH and other diseases typified by inflammation and fibrotic remodeling.

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress.

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild-type (PKCδ(+/+)) and null (PKCδ(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKCδ(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ(-/-) hyperoxic mice, compared to lungs of PKCδ(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase-mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKCδ or the catalytically active PKCδ cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKCδ→p38 signaling, and we describe a PKCδ-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.

Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury.

The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ(-/-)) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients (k(f)) or wet-to-dry weight ratios between PKCδ(+/+) and PKCδ(-/-) mice. However, at 24 h after exposure to LPS, the k(f) values were significantly higher in lungs isolated from PKCδ(+/+) than PKCδ(-/-) mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ(+/+) mice displayed increased protein and cell content compared with LPS-exposed PKCδ(-/-) mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ(+/+) mouse lung parenchyma relative to PKCδ(-/-) mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased k(f) lung values in PKCδ(+/+) mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ(-/-) mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.

Focal adhesion kinase and endothelial cell apoptosis.

Focal adhesion kinase (FAK) is a key component of cell-substratum adhesions, known as focal adhesion complexes. Growing evidence indicates that FAK is important in maintenance of normal cell survival and that disruption of FAK signaling results in loss of substrate adhesion and anoikis (apoptosis) of anchorage-dependent cells, such as endothelial cells. Basal FAK activity in non-stimulated endothelial cells is important in maintaining cell adhesion to integrins via PI3 kinase/Akt signaling. FAK activity is dependent upon small GTPase signaling. FAK also appears to be important in cardiomyocyte hypertrophy and hypoxia/reoxygenation-induced cell death. This review summarizes the signaling pathways of FAK in prevention of apoptosis and the role of FAK in mediating adenosine and homocysteine-induced endothelial cell apoptosis and in cardiovascular diseases.