Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

Signal Transduction during Metabolic and Inflammatory Reprogramming in Pulmonary Vascular Remodeling.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by (mal)adaptive remodeling of the pulmonary vasculature, which is associated with inflammation, fibrosis, thrombosis, and neovascularization. Vascular remodeling in PAH is associated with cellular metabolic and inflammatory reprogramming that induce profound endothelial and smooth muscle cell phenotypic changes. Multiple signaling pathways and regulatory loops act on metabolic and inflammatory mediators which influence cellular behavior and trigger pulmonary vascular remodeling in vivo. This review discusses the role of bioenergetic and inflammatory impairments in PAH development.

Sphingosine Kinase 1 Regulates the Pulmonary Vascular Immune Response.

The aberrant proliferation of pulmonary artery smooth muscle (PASMCs) cells is a defining characteristic of pulmonary arterial hypertension (PAH) and leads to increased vascular resistance, elevated pulmonary pressure, and right heart failure. The sphingosine kinase 1 (SPHK1)/sphingosine-1 phosphate/sphingosine-1 phosphate receptor 2 pathway promotes vascular remodeling and induces PAH. The aim of this study was to identify genes and cellular processes that are modulated by over-expression of SPHK1 in human PASMCs (hPASMCs). RNA was purified and submitted for RNA sequencing to identify differentially expressed genes. Using a corrected p-value threshold of <0.05, there were 294 genes significantly up-regulated while 179 were significantly down-regulated. Predicted effects of these differentially expressed genes were evaluated using the freeware tool Enrichr to assess general gene set over-representation (enrichment) and ingenuity pathway analysis (IPA™) for upstream regulator predictions. We found a strong change in genes that regulated the cellular immune response. IL6, STAT1, and PARP9 were elevated in response to SPHK1 over-expression in hPASMCs. The gene set enrichment mapped to a few immune-modulatory signaling networks, including IFNG. Furthermore, PARP9 and STAT1 protein were elevated in primary hPASMCs isolated from PAH patients. In conclusion, these data suggest a role of Sphk1 regulates pulmonary vascular immune response in PAH.

Elastase alters contractility and promotes an inflammatory synthetic phenotype in airway smooth muscle tissues.

Neutrophil elastase is secreted by inflammatory cells during airway inflammation and can elicit airway hyperreactivity in vivo. Elastase can degrade multiple components of the extracellular matrix. We hypothesized that elastase might disrupt the connections between airway smooth muscle (ASM) cells and the extracellular matrix and that this might have direct effects on ASM tissue responsiveness and inflammation. The effect of elastase treatment on ASM contractility was assessed in vitro in isolated strips of canine tracheal smooth muscle by stimulation of tissues with cumulatively increasing concentrations of acetylcholine (ACh) and measurement of contractile force. Elastase treatment potentiated contractile responses to ACh at low concentrations but suppressed the maximal contractile force generated by the tissues without affecting the phosphorylation of myosin regulatory light chain (RLC). Elastase also promoted the secretion of eotaxin and the activation of Akt in ASM tissues and decreased expression of smooth muscle myosin heavy chain, consistent with promotion of a synthetic inflammatory phenotype. As the degradation of matrix proteins can alter integrin engagement, we evaluated the effect of elastase on the assembly and activation of integrin-associated adhesion junction complexes in ASM tissues. Elastase led to talin cleavage, reduced talin binding to vinculin, and suppressed activation of the adhesome proteins paxillin, focal adhesion kinase, and vinculin, indicating that elastase causes the disassembly of adhesion junction complexes and the inactivation of adhesome signaling proteins. We conclude that elastase promotes an inflammatory phenotype and increased sensitivity to ACh in ASM tissues by disrupting signaling pathways mediated by integrin-associated adhesion complexes.

Alpha-1 Antitrypsin Transcytosis and Secretion.

Protective levels of Alpha-1 antitrypsin (A1AT) are achieved in the lung through the uptake of the pulmonary vasculature of hepatocyte-secreted A1AT. The anti-inflammatory, anti-apoptotic, and anti-protease properties of A1AT are critical toward maintaining the function of pulmonary endothelial and epithelial cells and for the structural integrity of the pulmonary interstitium. To perform these functions A1AT must cross the pulmonary-endothelial barrier. Using transwell inserts, we have demonstrated that the endocytosis of A1AT at the apical surface of endothelial cells, followed by the transcytosis and secretion at the basolateral surface, is a mechanism through which A1AT is transported into the lung epithelium and interstitium.

Alpha-1 antitrypsin supplementation improves alveolar macrophages efferocytosis and phagocytosis following cigarette smoke exposure.

Cigarette smoking (CS), the main risk factor for COPD (chronic obstructive pulmonary disease) in developed countries, decreases alveolar macrophages (AM) clearance of both apoptotic cells and bacterial pathogens. This global deficit of AM engulfment may explain why active smokers have worse outcomes of COPD exacerbations, episodes characterized by airway infection and inflammation that carry high morbidity and healthcare cost. When administered as intravenous supplementation, the acute phase-reactant alpha-1 antitrypsin (A1AT) reduces the severity of COPD exacerbations in A1AT deficient (AATD) individuals and of bacterial pneumonia in murine models, but the effect of A1AT on AM scavenging functions has not been reported. Apoptotic cell clearance (efferocytosis) was measured in human AM isolated from patients with COPD, in primary rat AM or differentiated monocytes exposed to CS ex vivo, and in AM recovered from mice exposed to CS. A1AT (100 µg/mL, 16 h) significantly ameliorated efferocytosis (by ~50%) in AM of active smokers or AM exposed ex vivo to CS. A1AT significantly improved AM global engulfment, including phagocytosis, even when cells were simultaneously challenged with apoptotic and Fc-coated (bacteria-like) targets. The improved efferocytosis in A1AT-treated macrophages was associated with inhibition of tumor necrosis factor-α converting enzyme (TACE) activity, decreased mannose receptor shedding, and markedly increased abundance of efferocytosis receptors (mannose- and phosphatidyl serine receptors and the scavenger receptor B2) on AM plasma membrane. Directed airway A1AT treatment (via inhalation of a nebulized solution) restored in situ airway AM efferocytosis after CS exposure in mice. The amelioration of CS-exposed AM global engulfment may render A1AT as a potential therapy for COPD exacerbations.

National Heart, Lung, and Blood Institute Workshop Summary: Enhancing Opportunities for Training and Retention of a Diverse Biomedical Workforce.

RATIONALE: Committed to its mission of conducting and supporting research that addresses the health needs of all sectors of the nation's population, the Division of Lung Diseases, National Heart, Lung, and Blood Institute of the National Institutes of Health (NHLBI/NIH) seeks to identify issues that impact the training and retention of underrepresented individuals in the biomedical research workforce. OBJECTIVES: Early-stage investigators who received grant support through the NIH Research Supplements to Promote Diversity in Health Related Research Program were invited to a workshop held in Bethesda, Maryland in June, 2015, in order to (1) assess the effectiveness of the current NHLBI diversity program, (2) improve its strategies towards achieving its goal, and (3) provide guidance to assist the transition of diversity supplement recipients to independent NIH grant support. METHODS: Workshop participants participated in five independent focus groups to discuss specific topics affecting underrepresented individuals in the biomedical sciences: (1) Socioeconomic barriers to success for diverse research scientists; (2) role of the academic research community in promoting diversity; (3) life beyond a research project grant: non-primary investigator career paths in research; (4) facilitating career development of diverse independent research scientists through NHLBI diversity programs; and (5) effectiveness of current NHLBI programs for promoting diversity of the biomedical workforce. MEASUREMENTS AND MAIN RESULTS: Several key issues experienced by young, underrepresented biomedical scientists were identified, and solutions were proposed to improve on training and career development for diverse students, from the high school to postdoctoral trainee level, and address limitations of currently available diversity programs. Although some of the challenges mentioned, such as cost of living, limited parental leave, and insecure extramural funding, are also likely faced by nonminority scientists, these issues are magnified among diversity scientists and are complicated by unique circumstances in this group, such as limited exposure to science at a young age, absence of role models and mentors from underrepresented backgrounds, and social norms that relegate their career endeavors, particularly among women, to being subordinate to their expected cultural role. CONCLUSIONS: The factors influencing the participation of underrepresented minorities in the biomedical workforce are complex and span several continuous or overlapping stages in the professional development of scientists from these groups. Therefore, a multipronged approach is needed to enable the professional development and retention of underrepresented minorities in biomedical research. This approach should address both individual and social factors and should involve funding agencies, academic institutions, mentoring teams, professional societies, and peer collaboration. Implementation of some of the recommendations, such as access to child care, institutional support and health benefits for trainees, teaching and entrepreneurial opportunities, grant-writing webinars, and pre-NIH career development (Pre-K) pilot programs would not only benefit biomedical scientists from underrepresented groups but also improve the situation of nondiverse junior scientists. However, other issues, such as opportunities for early exposure to science of disadvantaged/minority groups, and identifying mentors/life coaches/peer mentors who come from similar cultural backgrounds and vantage points, are unique to this group.

Scavenger receptor class B, type I-mediated uptake of A1AT by pulmonary endothelial cells.

In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30-50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.

Selective endothelin-A receptor blockade attenuates endotoxin-induced pulmonary hypertension and pulmonary vascular dysfunction.

Endothelin-1 is a potent mediator of sepsis-induced pulmonary hypertension (PH). The pulmonary vascular effects of selective blockade of endothelin receptor subtype A (ETAR) during endotoxemia remain unknown. We hypothesized that selective ETAR antagonism attenuates endotoxin-induced PH and improves pulmonary artery (PA) vasoreactivity. Adult male Sprague-Dawley rats (250-450 g) received lipopolysaccharide (LPS; Salmonella typhimurium; 20 mg/kg intraperitoneally) or vehicle 6 hours before hemodynamic assessment and tissue harvest. The selective ETAR antagonist sitaxsentan (10 or 20 mg/kg) or vehicle was injected intravenously 3 hours after receipt of LPS. Right ventricular systolic pressure, mean arterial pressure (MAP), cardiac output (CO), oxygenation (P/F ratio), and serum bicarbonate were measured. Bronchoalveolar lavage (BAL) cell differential and lung wet-to-dry ratios were obtained. Endothelium-dependent and endothelium-independent vasorelaxations were determined in isolated PA rings. PA interleukin (IL)-1ß, IL-6, tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) were measured. LPS caused PH, decreased MAP, CO, and serum bicarbonate, and increased PA IL-1ß, IL-6, TNF-α, and iNOS mRNA. Sitaxsentan attenuated sepsis-induced PH and increased MAP. The P/F ratio, CO, serum bicarbonate, and BAL neutrophilia were not affected by sitaxsentan. In isolated PA rings, while not affecting phenylephrine-induced vasocontraction or endothelium-dependent relaxation, sitaxsentan dose-dependently attenuated LPS-induced alterations in endothelium-independent relaxation. PA cytokine mRNA levels were not significantly attenuated by ETAR blockade. We conclude that ETAR blockade attenuates endotoxin-induced alterations in systemic and PA pressures without negatively affecting oxygenation. This protective effect appears to be mediated not by attenuation of sepsis-induced cardiac dysfunction, acidosis, or alveolar inflammation but rather by improved endothelium-independent vasorelaxation.

Active trafficking of alpha 1 antitrypsin across the lung endothelium.

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.

α1-Antitrypsin modulates lung endothelial cell inflammatory responses to TNF-α.

α1-Antitrypsin (A1AT) is an acute-phase reactant, but also a major protective factor against the development of chronic obstructive pulmonary disease, a complex disease with sustained chronic inflammation. The lung-protective effects of A1AT have been attributed to the inhibition of proteases involved in lung matrix fragmentation, macrophage activation, and endothelial-cell apoptosis. More recently, A1AT has been shown to directly interact with or modulate the actions of cytokines such as TNF-α or IL-1 in inflammatory cells, but its effect on the lung endothelium, an active participant in the amplification and resolution of inflammation, has received little attention. An important role of A1AT in modulating lung endothelial inflammatory responses is expected, given the high concentrations of circulating A1AT during inflammation and its active uptake by endothelial cells. We investigated the role of A1AT in primary lung microvascular endothelial cell activation by relevant cytokines such as TNF-α or IL-1ß. Despite an initial marked augmentation of TNF-α self-induced transcription, A1AT inhibited TNF-α receptor 1 up-regulation and significantly reduced TNF-α secretion, effects that were associated with inhibition of TNF-α-converting enzyme activity. Furthermore, A1AT inhibited calpain activity, whose activation by TNF-α contributed to decreased intracellular A1AT concentrations. These data indicate that A1AT initially facilitates acute responses of the endothelium to TNF-α, followed by selective inhibition of TNF-α-induced-self amplification, which may assist the vasculature in the resolution of chronic inflammation.

Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases.

α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.

Mechanism of alpha-1 antitrypsin endocytosis by lung endothelium.

The integrity of lung alveoli is maintained by proper circulating levels of alpha-1 antitrypsin (A1AT). Next to cigarette smoking, A1AT deficiency is a major risk factor for lung emphysema development. We recently reported that in addition to neutralizing neutrophil elastases in the extracellular compartment, A1AT is internalized by lung endothelial cells and inhibits apoptosis. We hypothesized that the intracellular uptake of A1AT by endothelial cells may be required for its protective function; therefore, we studied the mechanisms of A1AT internalization by primary rat lung microvascular endothelial cells and the effect of cigarette smoke on this process both in vitro and in vivo (in mice). Purified A1AT was taken up intracellularly by endothelial cells in a time-dependent, dose-dependent, and conformer-specific manner and was detected in the cytoplasm of endothelial cells of nondiseased human lung sections. Despite a critical role for caveoli in endothelial cell endocytosis in general, specific inhibition of clathrin-mediated, but not caveoli-mediated, endocytosis profoundly decreased A1AT internalization and reversed the A1AT's antiapoptotic action. Further more, A1AT associated with clathrin heavy chains, but not with caveolin-1 in the plasma membrane fraction of endothelial cells. Interestingly, cigarette smoke exposure significantly inhibited A1AT uptake both in endothelial cells and in the mouse lung and altered the intracellular distribution of clathrin heavy chains. Our results suggest that clathrin-mediated endocytosis regulates A1AT intracellular function in the lung endothelium and may be an important determinant of the serpin's protection against developing cigarette smoke-induced emphysema.

Transient membrane recruitment of IRAK-1 in response to LPS and IL-1beta requires TNF R1.

The transcription factor NF-kappaB is an essential regulator of the innate immune response that functions as the first line of defense against infections. Activation of the innate immune response by bacterial lipopolysaccharide (LPS) triggers production of tumor necrosis factor-alpha (TNF-alpha) followed by interleukin-1 (IL-1). The IL-1 receptor associated kinase-1 (IRAK-1) is an integral component of the LPS, TNF-alpha, and IL-1 signaling pathways that regulate NF-kappaB. Thus we hypothesized that IRAK-1 coordinates cellular NF-kappaB responses to LPS, TNF-alpha, and IL-1. In contrast to TNF-alpha where IRAK-1 subcellular localization does not change, treatment with LPS or IL-1 leads to a loss in cytoplasmic IRAK-1 with a coordinate increase in plasma membrane associated modified IRAK-1. In fibroblasts lacking the type 1 TNF-alpha receptor (TNF R1), IRAK-1 turnover is altered and modification of IRAK-1 in the plasma membrane is decreased in response to LPS and IL-1, respectively. When NF-kappaB controlled gene expression is measured, fibroblasts lacking TNF R1 are hyperresponsive to LPS, whereas a more variable response to IL-1 is seen. Further analysis of the LPS response revealed that plasma membrane-associated IRAK-1 is found in Toll 4, IL-1, and TNF R1-containing complexes. The data presented herein suggest a model whereby the TNF R1-IRAK-1 interaction integrates the cellular response to LPS, TNF-alpha, and IL-1, culminating in a cell poised to activate TNF-alpha-dependent NF-kappaB controlled gene expression. In the absence of TNF R1-dependent events, exposure to LPS or IL-1 leads to hyperactivation of the inflammatory response.