Rationale: Adipose-derived stem cells express multiple growth factors that inhibit endothelial cell apoptosis, and demonstrate substantial pulmonary trapping after intravascular delivery. Objectives: We hypothesized that adipose stem cells would ameliorate chronic lung injury associated with endothelial cell apoptosis, such as that occurring in emphysema. Methods: Therapeutic effects of systemically delivered human or mouse adult adipose stem cells were evaluated in murine models of emphysema induced by chronic exposure to cigarette smoke or by inhibition of vascular endothelial growth factor receptors. Measurements and Main Results: Adipose stem cells were detectable in the parenchyma and large airways of lungs up to 21 days after injection. Adipose stem cell treatment was associated with reduced inflammatory infiltration in response to cigarette smoke exposure, and markedly decreased lung cell death and airspace enlargement in both models of emphysema. Remarkably, therapeutic results of adipose stem cells extended beyond lung protection by rescuing the suppressive effects of cigarette smoke on bone marrow hematopoietic progenitor cell function, and by restoring weight loss sustained by mice during cigarette smoke exposure. Pulmonary vascular protective effects of adipose stem cells were recapitulated by application of cell-free conditioned medium, which improved lung endothelial cell repair and recovery in a wound injury repair model and antagonized effects of cigarette smoke in vitro. Conclusions: These results suggest a useful therapeutic effect of adipose stem cells on both lung and systemic injury induced by cigarette smoke, and implicate a lung vascular protective function of adipose stem cell derived paracrine factors.

Rationale: Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. Objectives: The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. Methods: Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. Measurements and Main Results: Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. Conclusions: Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.

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.

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.

EMAP II is an endothelial cell and monocyte activating proinflammatory cytokine, which has been demonstrated to induce endothelial cell apoptosis. In order to analyze its role in disease models linked to inflammation and endothelial cell death, we aimed to develop a neutralizing antibody against mouse EMAP II. Therefore, we generated rat monoclonal anti-mouse EMAP II antibodies by immunization with recombinant full length, mouse pro-EMAP II protein. From fusion with mouse myeloma SP2/0 we could identify by ELISA hybridoma clones, which produce antibodies recognizing both full length and processed EMAP II. We further characterized one antibody, M7/1 and demonstrated its ability to detect both EMAP II forms by Western blotting and to neutralize EMAP II directed migration of human peripheral blood monocytes as well as EMAP II induced tumor and endothelial cell apoptosis. We conclude that this antibody can be useful to both target and analyze murine disease models, in which EMAP II may be involved.

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Recent studies uncovered non‐cannonical functions of A1AT that lead to cellular protection from apoptosis and inflammation, including suppression of cigarette smoke (CS)‐induced increases in TNFα. We set out to investigate the mechanisms by which A1AT antagonizes TNFα?in vivo and in primary lung endothelial cell cultures. Mice were exposed to CS for up to 6 months and total lung RNA or protein was analyzed by real‐time PCR or Western blot, respectively. Primary rat lung microvascular cells were pretreated with A1AT, followed by studies of signaling and inflammatory responses in response to TNFα. Despite a decrease in the total lung TNFα message level (21 % at 16 weeks), both IκBα and MCP1 levels increased in CS‐exposed mice (1.7 fold + sem=.075; p=.05 and 1.4 fold +sem=.029; p<0.01, respectively). A1AT treatment increased basal IκBα?and delayed the IκBα?degradation induced by TNFα (10 ng/ml) in endothelial cells by 15 min. In conclusion, although TNFα message levels are not increased in smoked animals, the increased IκBα and MCP1 expression suggest that TNFα protein is activated by cigarette smoke. At the endothelial cell level, A1AT may alter TNFα signaling by regulating IκBα degradation. Understanding the anti‐inflammatory mechanisms of A1AT is important for developing therapeutic strategies against CS‐induced emphysema.Funded by: 1P50 HL084945