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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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hASC-CM preserves microscopic HPAEC monolayer integrity. Preincubation of endothelial monolayers with hASC-CM suppressed H2O2-induced rearrangement of F-actin cytoskeleton and disintegration of peripheral adherens junctions. Arrows indicate the gaps appeared in previously intact monolayer in response to H2O2.
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Fig9: hASC-CM preserves microscopic HPAEC monolayer integrity. Preincubation of endothelial monolayers with hASC-CM suppressed H2O2-induced rearrangement of F-actin cytoskeleton and disintegration of peripheral adherens junctions. Arrows indicate the gaps appeared in previously intact monolayer in response to H2O2.

Mentions: To further analyze mechanisms underlying ASC and ASC-CM effects on lung permeability, we directly assessed ASC/ASC-CM effects on monolayer barrier function of well-characterized human pulmonary endothelial cell line HPAEC. Since recently it was suggested that syngeneic ASC therapy has more potent effect than xenogeneic therapy [20], we tested the effect of hASC/hASC-CM on the permeability of human pulmonary endothelium. First, HPAEC monolayers were grown on collagen-coated polyester inserts in the presence of hASC or NHDF (as a non-stem control) in the lower chamber. Prior to analysis, inserts were transferred to fresh wells to avoid the possibility of direct ROS scavenging by hASC/NHDF. The top chamber was loaded with FITC-dextran, and monolayers were stimulated with H2O2, the edemagenic product of the neutrophil oxidative burst. We observed marked HPAEC barrier dysfunction in response to H2O2, which was significantly attenuated in monolayers grown in the presence of hASC (Figure 8A). We next pretreated HPAEC monolayers with either hASC-CM or NHDF-CM. Similar to HPAEC grown in the presence of hASC, HPAEC grown in the presence of hASC-CM responded to H2O2 with less barrier dysfunction (Figure 8B); this stabilizing effect was not seen with NHDF-CM. To further characterize the hASC-CM-mediated stabilization of HPAEC barrier, we assessed transendothelial electrical resistance (TER). HPAEC grown on gold electrodes were pretreated with hASC-CM or NHDF-CM; then stimulated with H2O2. Control monolayers responded to H2O2 with marked reduction of TER, which was restored within 3 h. NHDF-CM-pretreated monolayers displayed a similar response curve, whereas hASC-CM-pretreated monolayers had a significantly attenuated barrier dysfunction in response to H2O2 (Figure 8C). Immunofluorescent microscopy of NHDF-CM-pretreated HPAEC showed that H2O2 caused marked rearrangement of the actin cytoskeleton along with distortion of junctional organization and gap formation in endothelial monolayers (Figure 9, arrowheads). hASC-CM-pretreated HPAEC displayed less severe changes in response to H2O2, with attenuated gap formation.Figure 8


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)

hASC-CM preserves microscopic HPAEC monolayer integrity. Preincubation of endothelial monolayers with hASC-CM suppressed H2O2-induced rearrangement of F-actin cytoskeleton and disintegration of peripheral adherens junctions. Arrows indicate the gaps appeared in previously intact monolayer in response to H2O2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig9: hASC-CM preserves microscopic HPAEC monolayer integrity. Preincubation of endothelial monolayers with hASC-CM suppressed H2O2-induced rearrangement of F-actin cytoskeleton and disintegration of peripheral adherens junctions. Arrows indicate the gaps appeared in previously intact monolayer in response to H2O2.
Mentions: To further analyze mechanisms underlying ASC and ASC-CM effects on lung permeability, we directly assessed ASC/ASC-CM effects on monolayer barrier function of well-characterized human pulmonary endothelial cell line HPAEC. Since recently it was suggested that syngeneic ASC therapy has more potent effect than xenogeneic therapy [20], we tested the effect of hASC/hASC-CM on the permeability of human pulmonary endothelium. First, HPAEC monolayers were grown on collagen-coated polyester inserts in the presence of hASC or NHDF (as a non-stem control) in the lower chamber. Prior to analysis, inserts were transferred to fresh wells to avoid the possibility of direct ROS scavenging by hASC/NHDF. The top chamber was loaded with FITC-dextran, and monolayers were stimulated with H2O2, the edemagenic product of the neutrophil oxidative burst. We observed marked HPAEC barrier dysfunction in response to H2O2, which was significantly attenuated in monolayers grown in the presence of hASC (Figure 8A). We next pretreated HPAEC monolayers with either hASC-CM or NHDF-CM. Similar to HPAEC grown in the presence of hASC, HPAEC grown in the presence of hASC-CM responded to H2O2 with less barrier dysfunction (Figure 8B); this stabilizing effect was not seen with NHDF-CM. To further characterize the hASC-CM-mediated stabilization of HPAEC barrier, we assessed transendothelial electrical resistance (TER). HPAEC grown on gold electrodes were pretreated with hASC-CM or NHDF-CM; then stimulated with H2O2. Control monolayers responded to H2O2 with marked reduction of TER, which was restored within 3 h. NHDF-CM-pretreated monolayers displayed a similar response curve, whereas hASC-CM-pretreated monolayers had a significantly attenuated barrier dysfunction in response to H2O2 (Figure 8C). Immunofluorescent microscopy of NHDF-CM-pretreated HPAEC showed that H2O2 caused marked rearrangement of the actin cytoskeleton along with distortion of junctional organization and gap formation in endothelial monolayers (Figure 9, arrowheads). hASC-CM-pretreated HPAEC displayed less severe changes in response to H2O2, with attenuated gap formation.Figure 8

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