Krzysztof Kamocki

Adipose Stem Cell Treatment in Mice Attenuates Lung and Systemic Injury Induced by Cigarette Smoking

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.

Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide

The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer(12:0) or Cer(8:0); 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramides proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.

Bioactive Nanofibrous Scaffolds for Regenerative Endodontics

Here we report the synthesis, materials characterization, antimicrobial capacity, and cytocompatibility of novel antibiotic-containing scaffolds. Metronidazole (MET) or Ciprofloxacin/(CIP) was mixed with a polydioxanone (PDS)polymer solution at 5 and 25 wt% and processed into fibers. PDS fibers served as a control. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), tensile testing, and high-performance liquid chromatography (HPLC) were used to assess fiber morphology, chemical structure, mechanical properties, and drug release, respectively. Antimicrobial properties were evaluated against those of Porphyromonas gingivalis/Pg and Enterococcus faecalis/Ef. Cytotoxicity was assessed in human dental pulp stem cells (hDPSCs). Statistics were performed, and significance was set at the 5% level. SEM imaging revealed a submicron fiber diameter. FTIR confirmed antibiotic incorporation. The tensile values of hydrated 25 wt% CIP scaffold were significantly lower than those of all other groups. Analysis of HPLC data confirmed gradual, sustained drug release from the scaffolds over 48 hrs. CIP-containing scaffolds significantly (p < .00001) inhibited biofilm growth of both bacteria. Conversely, MET-containing scaffolds inhibited only Pg growth. Agar diffusion confirmed the antimicrobial properties against specific bacteria for the antibiotic-containing scaffolds. Only the 25 wt% CIP-containing scaffolds were cytotoxic. Collectively, this study suggests that polymer-based antibiotic-containing electrospun scaffolds could function as a biologically safe antimicrobial drug delivery system for regenerative endodontics.

Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema.

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.

Apoptotic sphingolipid signaling by ceramides in lung endothelial cells.

The de novo pathway of ceramide synthesis has been implicated in the pathogenesis of excessive lung apoptosis and murine emphysema. Intracellular and paracellular-generated ceramides may trigger apoptosis and propagate the death signals to neighboring cells, respectively. In this study we compared the sphingolipid signaling pathways triggered by the paracellular- versus intracellular-generated ceramides as they induce lung endothelial cell apoptosis, a process important in emphysema development. Intermediate-chain length (C(8:0)) extracellular ceramides, used as a surrogate of paracellular ceramides, triggered caspase-3 activation in primary mouse lung endothelial cells, similar to TNF-alpha-generated endogenous ceramides. Inhibitory siRNA against serine palmitoyl transferase subunit 1 but not acid sphingomyelinase inhibited both C(8:0) ceramide- and TNF-alpha (plus cycloheximide)-induced apoptosis, consistent with the requirement for activation of the de novo pathway of sphingolipid synthesis. Tandem mass spectrometry analysis detected increases in both relative and absolute levels of C(16:0) ceramide in response to C(8:0) and TNF-alpha treatments. These results implicate the de novo pathway of ceramide synthesis in the apoptotic effects of both paracellular ceramides and TNF-alpha-stimulated intracellular ceramides in primary lung endothelial cells. The serine palmitoyl synthase-regulated ceramides synthesis may contribute to the amplification of pulmonary vascular injury induced by excessive ceramides.

Lung endothelial monocyte-activating protein 2 is a mediator of cigarette smoke-induced emphysema in mice

Pulmonary emphysema is a disease characterized by alveolar cellular loss and inflammation. Recently, excessive apoptosis of structural alveolar cells has emerged as a major mechanism in the development of emphysema. Here, we investigated the proapoptotic and monocyte chemoattractant cytokine endothelial monocyte-activating protein 2 (EMAPII). Lung-specific overexpression of EMAPII in mice caused simplification of alveolar structures, apoptosis, and macrophage accumulation, compared with that in control transgenic mice. Additionally, in a mouse model of cigarette smoke-induced (CS-induced) emphysema, EMAPII levels were significantly increased in murine lungs. This upregulation was necessary for emphysema development, as neutralizing antibodies to EMAPII resulted in reduced alveolar cell apoptosis, inflammation, and emphysema-associated structural changes in alveoli and small airways and improved lung function. The mechanism of EMAPII upregulation involved an apoptosis-dependent feed-forward loop, since caspase-3 instillation in the lung markedly increased EMAPII expression, while caspase inhibition decreased its production, even in transgenic EMAPII mice. These findings may have clinical significance, as both current smokers and ex-smoker chronic obstructive pulmonary disease (COPD) patients had increased levels of secreted EMAPII in the bronchoalveolar lavage fluid compared with that of nonsmokers. In conclusion, we suggest that EMAPII perpetuates the mechanism of CS-induced lung emphysema in mice and, given its secretory nature, is a suitable target for neutralization antibody therapy.

The Development and Maintenance of Paclitaxel-induced Neuropathic Pain Require Activation of the Sphingosine 1-Phosphate Receptor Subtype 1

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a critical dose-limiting side effect of many chemotherapeutic agents, including paclitaxel. Results: Spinal activation of the S1P-to-S1PR1 axis contributes to the development and maintenance of paclitaxel-induced neuropathic pain through enhanced neuroinflammatory processes. Conclusion: Inhibition of S1PR1 blocks and reverses paclitaxel-induced neuropathic pain without interfering with anticancer effects. Significance: Targeting the S1PR1 signaling pathway could be an effective approach for the treatment of CIPN. The ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy-induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents. We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR1)-dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration-approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.

Mechanism of α-1 antitrypsin endocytosis by lung endothelium

The integrity of lung alveoli is maintained by proper circulating levels of α‐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 A1ATs 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 serpins protection against developing cigarette smoke‐induced emphysema. Sohrab, S., Petrusca, D. N., Lockett, A. D., Schweitzer, K. S., Rush, N. I., Gu, Y., Kamocki, K., Garrison, J., Petrache, I. Mechanism of α‐1 antitrypsin endocytosis by lung endothelium. FASEB J. 23, 3149–3158 (2009). www.fasebj.org

Ceramide Synthases Expression and Role of Ceramide Synthase-2 in the Lung: Insight from Human Lung Cells and Mouse Models

Increases in ceramide levels have been implicated in the pathogenesis of both acute or chronic lung injury models. However, the role of individual ceramide species, or of the enzymes that are responsible for their synthesis, in lung health and disease has not been clarified. We now show that C24- and C16-ceramides are the most abundant lung ceramide species, paralleled by high expression of their synthetic enzymes, ceramide synthase 2 (CerS2) and CerS5, respectively. Furthermore, the ceramide species synthesis in the lung is homeostatically regulated, since mice lacking very long acyl chain C24-ceramides due to genetic deficiency of CerS2 displayed a ten-fold increase in C16-ceramides and C16-dihydroceramides along with elevation of acid sphingomyelinase and CerS5 activities. Despite relatively preserved total lung ceramide levels, inhibition of de novo sphingolipid synthesis at the level of CerS2 was associated with significant airflow obstruction, airway inflammation, and increased lung volumes. Our results suggest that ceramide species homeostasis is crucial for lung health and that CerS2 dysfunction may predispose to inflammatory airway and airspace diseases.

Involvement of Ceramide in Cell Death Responses in the Pulmonary Circulation

Ceramides are signaling sphingolipids involved in cellular homeostasis but also in pathological processes such as unwanted apoptosis, growth arrest, oxidative stress, or senescence. Several enzymatic pathways are responsible for the synthesis of ceramides, which can be activated in response to exogenous stimuli such as cytokines, radiation, or oxidative stress. Endothelial cells are particularly rich in acid sphingomyelinases, which can be rapidly activated to produce ceramides, both intracellular and at the plasma membrane. In addition, neutral sphingomyelinases, the de novo pathway and the ceramide recycling pathway, may generate excessive ceramides involved in endothelial cell responses. When up-regulated, ceramides trigger signaling pathways that culminate in endothelial cell death, which in murine lungs has been linked to the development of emphysema-like disease. Furthermore, ceramides may be released paracellularly where they are believed to exert paracrine activities. Such effects, along with ceramides released by inflammatory mediators, may contribute to lung inflammation and pulmonary edema, because ceramide-challenged pulmonary endothelial cells exhibit decreased barrier function, independent of apoptosis. Reestablishing the sphingolipid homeostasis, either by modulating ceramide synthesis or by opposing its biological effects through augmentation of the prosurvival sphingosine-1 phosphate, may alleviate acute or chronic pulmonary conditions characterized by vascular endothelial cell death or dysfunction.