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Conditioned media from adipose stromal cells limit lipopolysaccharide-induced lung injury, endothelial hyperpermeability and apoptosis.

Lu H, Poirier C, Cook T, Traktuev DO, Merfeld-Clauss S, Lease B, Petrache I, March KL, Bogatcheva NV - J Transl Med (2015)

Bottom Line: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6.ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3.ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Indiana University, Indianapolis, IN, USA. honlu@iu.edu.

ABSTRACT

Background: Acute Respiratory Distress Syndrome (ARDS) is a condition that contributes to morbidity and mortality of critically ill patients. We investigated whether factors secreted by adipose stromal cells (ASC) into conditioned media (ASC-CM) will effectively decrease lung injury in the model of lipopolysaccharide (LPS)-induced ARDS.

Methods: To assess the effect of ASC-CM on ARDS indices, intravenous delivery of ASC and ASC-CM to C57Bl/6 mice was carried out 4 h after LPS oropharyngeal aspiration; Evans Blue Dye (EBD) was injected intravenously 1 h prior to animal sacrifice (48 h post-LPS). Lungs were either fixed for histopathology, or used to extract bronchoalveolar lavage fluid (BALF) or EBD. To assess the effect of ASC-CM on endothelial barrier function and apoptosis, human pulmonary artery endothelial cells were treated with ASC-CM for 48-72 h.

Results: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6. White Blood Cells (WBC) from BALF of LPS-challenged mice receiving ASC-CM had decreased reactive oxygen species (ROS) generation compared to WBC from LPS-challenged mice receiving control media injection. Treatment of pulmonary endothelial monolayers with ASC-CM significantly suppressed H2O2-induced leakage of FITC dextran and changes in transendothelial resistance, as well as gap formation in endothelial monolayer. ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3. ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation.

Conclusions: Factors secreted by ASC efficiently reduce ARDS indices, endothelial barrier hyperpermeability, and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.

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Related in: MedlinePlus

mASC and mASC-CM effect on neutrophil numbers and activity. Total WBC (T, grey) and PMN (N, white) counts in BALF were significantly reduced by mASC (A), but not mASC-CM (B). The ratio between macrophages (M), lymphocytes (L) and neutrophils (N) in BALF of LPS-treated mice was not significantly affected by either experimental treatment. N = 5-6 animals per group. (C) WBC from BALF of LPS-challenged mice (N = 6) were subjected to vehicle (LPC cn) or 1 μg/ml LPS stimulation (LPS st). In parallel, WBC from BALF of LPS/ASC-CM-treated mice (N = 6) were subjected to vehicle control (LPS/CM cn) or LPS stimulation (LPS/CM st). LPS-induced ROS generation by WBC from BALF of LPS/ASC-CM mice was significantly lower comparing to WBC from BALF of LPS/media mice. T-test was used to analyze the significance of differences between groups.
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Fig5: mASC and mASC-CM effect on neutrophil numbers and activity. Total WBC (T, grey) and PMN (N, white) counts in BALF were significantly reduced by mASC (A), but not mASC-CM (B). The ratio between macrophages (M), lymphocytes (L) and neutrophils (N) in BALF of LPS-treated mice was not significantly affected by either experimental treatment. N = 5-6 animals per group. (C) WBC from BALF of LPS-challenged mice (N = 6) were subjected to vehicle (LPC cn) or 1 μg/ml LPS stimulation (LPS st). In parallel, WBC from BALF of LPS/ASC-CM-treated mice (N = 6) were subjected to vehicle control (LPS/CM cn) or LPS stimulation (LPS/CM st). LPS-induced ROS generation by WBC from BALF of LPS/ASC-CM mice was significantly lower comparing to WBC from BALF of LPS/media mice. T-test was used to analyze the significance of differences between groups.

Mentions: Assessment of total WBC count in BALF showed that the number of cells in airspaces increased dramatically in response to LPS stimulation (Figure 5A,B). Whereas resident lung macrophages comprised the majority of cells in BALF from saline lungs, neutrophils were the dominant cell type in BALF from LPS-challenged lungs. Although ASC or ASC-CM treatment did not shift the overall neutrophil/macrophage balance (Figure 5A,B), a significant suppression of total WBC count was evident in LPS mice receiving ASC injection (Figure 5A). A similar trend was observed in mice receiving ASC-CM, but did not reach significance at 48 h post-injection (Figure 5B). To further characterize possible mechanisms of ASC-CM-mediated lung injury limitation, we compared the ability of BALF WBC, obtained from LPS- or LPS/ASC-CM-receiving mice, to generate ROS. The method for the measurement of neutrophils oxidative activity was based on ROS-dependent oxidation of DCFH-DA (non-fluorescent) to highly fluorescent DCF. Isolated BALF WBC were tested for their abilities to oxidize DCFH-DA in response to LPS stimulation in vitro. We observed that WBC from mice receiving LPS can be further induced by LPS stimulation, whereas WBC from mice receiving LPS/ASC-CM no longer respond to LPS stimulation (Figure 5C), suggesting that mice exposure to ASC-CM leads to lung recruitment of neutrophils with a reduced potential for oxidative response.Figure 5


Conditioned media from adipose stromal cells limit lipopolysaccharide-induced lung injury, endothelial hyperpermeability and apoptosis.

Lu H, Poirier C, Cook T, Traktuev DO, Merfeld-Clauss S, Lease B, Petrache I, March KL, Bogatcheva NV - J Transl Med (2015)

mASC and mASC-CM effect on neutrophil numbers and activity. Total WBC (T, grey) and PMN (N, white) counts in BALF were significantly reduced by mASC (A), but not mASC-CM (B). The ratio between macrophages (M), lymphocytes (L) and neutrophils (N) in BALF of LPS-treated mice was not significantly affected by either experimental treatment. N = 5-6 animals per group. (C) WBC from BALF of LPS-challenged mice (N = 6) were subjected to vehicle (LPC cn) or 1 μg/ml LPS stimulation (LPS st). In parallel, WBC from BALF of LPS/ASC-CM-treated mice (N = 6) were subjected to vehicle control (LPS/CM cn) or LPS stimulation (LPS/CM st). LPS-induced ROS generation by WBC from BALF of LPS/ASC-CM mice was significantly lower comparing to WBC from BALF of LPS/media mice. T-test was used to analyze the significance of differences between groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358867&req=5

Fig5: mASC and mASC-CM effect on neutrophil numbers and activity. Total WBC (T, grey) and PMN (N, white) counts in BALF were significantly reduced by mASC (A), but not mASC-CM (B). The ratio between macrophages (M), lymphocytes (L) and neutrophils (N) in BALF of LPS-treated mice was not significantly affected by either experimental treatment. N = 5-6 animals per group. (C) WBC from BALF of LPS-challenged mice (N = 6) were subjected to vehicle (LPC cn) or 1 μg/ml LPS stimulation (LPS st). In parallel, WBC from BALF of LPS/ASC-CM-treated mice (N = 6) were subjected to vehicle control (LPS/CM cn) or LPS stimulation (LPS/CM st). LPS-induced ROS generation by WBC from BALF of LPS/ASC-CM mice was significantly lower comparing to WBC from BALF of LPS/media mice. T-test was used to analyze the significance of differences between groups.
Mentions: Assessment of total WBC count in BALF showed that the number of cells in airspaces increased dramatically in response to LPS stimulation (Figure 5A,B). Whereas resident lung macrophages comprised the majority of cells in BALF from saline lungs, neutrophils were the dominant cell type in BALF from LPS-challenged lungs. Although ASC or ASC-CM treatment did not shift the overall neutrophil/macrophage balance (Figure 5A,B), a significant suppression of total WBC count was evident in LPS mice receiving ASC injection (Figure 5A). A similar trend was observed in mice receiving ASC-CM, but did not reach significance at 48 h post-injection (Figure 5B). To further characterize possible mechanisms of ASC-CM-mediated lung injury limitation, we compared the ability of BALF WBC, obtained from LPS- or LPS/ASC-CM-receiving mice, to generate ROS. The method for the measurement of neutrophils oxidative activity was based on ROS-dependent oxidation of DCFH-DA (non-fluorescent) to highly fluorescent DCF. Isolated BALF WBC were tested for their abilities to oxidize DCFH-DA in response to LPS stimulation in vitro. We observed that WBC from mice receiving LPS can be further induced by LPS stimulation, whereas WBC from mice receiving LPS/ASC-CM no longer respond to LPS stimulation (Figure 5C), suggesting that mice exposure to ASC-CM leads to lung recruitment of neutrophils with a reduced potential for oxidative response.Figure 5

Bottom Line: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6.ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3.ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Indiana University, Indianapolis, IN, USA. honlu@iu.edu.

ABSTRACT

Background: Acute Respiratory Distress Syndrome (ARDS) is a condition that contributes to morbidity and mortality of critically ill patients. We investigated whether factors secreted by adipose stromal cells (ASC) into conditioned media (ASC-CM) will effectively decrease lung injury in the model of lipopolysaccharide (LPS)-induced ARDS.

Methods: To assess the effect of ASC-CM on ARDS indices, intravenous delivery of ASC and ASC-CM to C57Bl/6 mice was carried out 4 h after LPS oropharyngeal aspiration; Evans Blue Dye (EBD) was injected intravenously 1 h prior to animal sacrifice (48 h post-LPS). Lungs were either fixed for histopathology, or used to extract bronchoalveolar lavage fluid (BALF) or EBD. To assess the effect of ASC-CM on endothelial barrier function and apoptosis, human pulmonary artery endothelial cells were treated with ASC-CM for 48-72 h.

Results: ASC-CM markedly reduced LPS-induced histopathologic changes of lung, protein extravasation into BALF, and suppressed the secretion of proinflammatory cytokines TNFα and IL6. White Blood Cells (WBC) from BALF of LPS-challenged mice receiving ASC-CM had decreased reactive oxygen species (ROS) generation compared to WBC from LPS-challenged mice receiving control media injection. Treatment of pulmonary endothelial monolayers with ASC-CM significantly suppressed H2O2-induced leakage of FITC dextran and changes in transendothelial resistance, as well as gap formation in endothelial monolayer. ASC-CM exposure reduced the percentage of endothelial cells expressing ICAM-1, and suppressed TNFα-induced expression of E-selectin and cleavage of caspase-3. ASC-CM reduced the endothelial level of pro-apoptotic protein Bim, but did not affect the level of Bcl-2, Bad, or Bad phosphorylation.

Conclusions: Factors secreted by ASC efficiently reduce ARDS indices, endothelial barrier hyperpermeability, and activation of pro-inflammatory and pro-apoptotic pathways in endothelium.

Show MeSH
Related in: MedlinePlus