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Cited by 258Mesenchymal stem cell researchRespiratory Support and MechanismsCancer Cells and Metastasis

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Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1*

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Fang, Xiaohui, Neyrinck, Arne P., Matthay, Michael A., Lee, Jae W.·2010·Journal of Biological Chemistry
DOI 10.1074/jbc.m110.119917PMC PMC2924032
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Abstract

Acute lung injury is characterized by injury to the lung epithelium that leads to impaired resolution of pulmonary edema and also facilitates accumulation of protein-rich edema fluid and inflammatory cells in the distal airspaces of the lung. Recent in vivo and in vitro studies suggest that mesenchymal stem cells (MSC) may have therapeutic value for the treatment of acute lung injury. Here we tested the ability of human allogeneic mesenchymal stem cells to restore epithelial permeability to protein across primary cultures of polarized human alveolar epithelial type II cells after an inflammatory insult. Alveolar epithelial type II cells were grown on a Transwell plate with an air-liquid interface and injured by cytomix, a combination of IL-1beta, TNFalpha, and IFNgamma. Protein permeability measured by (131)I-labeled albumin flux was increased by 5-fold over 24 h after cytokine-induced injury. Co-culture of human MSC restored type II cell epithelial permeability to protein to control levels. Using siRNA knockdown of potential paracrine soluble factors, we found that angiopoietin-1 secretion was responsible for this beneficial effect in part by preventing actin stress fiber formation and claudin 18 disorganization through suppression of NFkappaB activity. This study provides novel evidence for a beneficial effect of MSC on alveolar epithelial permeability to protein.

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Crossref · 10.1074/jbc.m110.119917

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FIGURE 1. Transwell co-culture system. A lateral view of the Transwell system is shown. Primary cultures of human alveolar epithelial type II cells were seeded at a density of 1 × 106 cells/well in collagen I-coated Transwell plates (0.4-μm pore size and a surface area of 0.33 cm2). Human allogeneic mesenchymal stem cells or normal human lung fibroblasts were added simultaneously to the lower compartment. There was no direct cell-contact between cell types. Cytomix was added to both compartments according to the experimental protocol. Alveolar protein permeability was assessed by adding labeled 131I-albumin only to the upper compartment and measuring its unidirectional flux to the lower compartment. There was no hydrostatic pressure gradient between compartments.
FIGURE 1. Transwell co-culture system. A lateral view of the Transwell system is shown. Primary cultures of human alveolar epithelial type II cells were seeded at a density of 1 × 106 cells/well in collagen I-coated Transwell plates (0.4-μm pore size and a surface area of 0.33 cm2). Human allogeneic mesenchymal stem cells or normal human lung fibroblasts were added simultaneously to the lower compartment. There was no direct cell-contact between cell types. Cytomix was added to both compartments according to the experimental protocol. Alveolar protein permeability was assessed by adding labeled 131I-albumin only to the upper compartment and measuring its unidirectional flux to the lower compartment. There was no hydrostatic pressure gradient between compartments.CC BY-NC · PubMed Central
FIGURE 2. Effect of MSC on protein permeability across human alveolar epithelial type II cells injured by cytomix. Epithelial permeability to protein was measured by the unidirectional flux of labeled 131I-albumin from the upper to the lower compartment of the transwell plate over 24 h. The addition of cytomix (50 ng/ml) increased epithelial protein permeability among ATII cells. The simultaneous addition of MSC to the bottom chamber restored alveolar epithelial barrier integrity. In contrast, the addition of normal human lung fibroblasts did not significantly restore protein permeability. Epithelial protein permeability is expressed as the mean ± S.D. (% change/24 h, n = 15; *, p < 0.02 versus control; √, p < 0.03 versus cytomix-injured).
FIGURE 2. Effect of MSC on protein permeability across human alveolar epithelial type II cells injured by cytomix. Epithelial permeability to protein was measured by the unidirectional flux of labeled 131I-albumin from the upper to the lower compartment of the transwell plate over 24 h. The addition of cytomix (50 ng/ml) increased epithelial protein permeability among ATII cells. The simultaneous addition of MSC to the bottom chamber restored alveolar epithelial barrier integrity. In contrast, the addition of normal human lung fibroblasts did not significantly restore protein permeability. Epithelial protein permeability is expressed as the mean ± S.D. (% change/24 h, n = 15; *, p < 0.02 versus control; √, p < 0.03 versus cytomix-injured).CC BY-NC · PubMed Central
FIGURE 3. Angiopoietin-1 secretion by human allogeneic mesenchymal stem sells. A, protein levels of Ang1 were measured in the medium of the cell culture system (Transwell) under different experimental conditions. Allogeneic human MSC secreted significant amounts of Ang1 that increased further after exposure to cytomix. Production of Ang1 by ATII cells alone and in the presence of cytomix was almost undetectable. Data are expressed as the mean ± S.D. (pg/ml, n = 3; *, p < 0.05 versus control; √, p < 0.05 versus cytomix-injured for ATII cells). B, lipid-based transfection of MSC with two different Ang1 siRNAs (#121280 and #121281) significantly reduced Ang1 secretion by 48 h. Transfection of MSC with negative control siRNA did not affect Ang1 production. Cells were plated in 24-well plates at a density of 50,000 cells/well. Data are expressed as mean ± S.D. (pg/ml, n = 3; *, p < 0.05 versus control).
FIGURE 3. Angiopoietin-1 secretion by human allogeneic mesenchymal stem sells. A, protein levels of Ang1 were measured in the medium of the cell culture system (Transwell) under different experimental conditions. Allogeneic human MSC secreted significant amounts of Ang1 that increased further after exposure to cytomix. Production of Ang1 by ATII cells alone and in the presence of cytomix was almost undetectable. Data are expressed as the mean ± S.D. (pg/ml, n = 3; *, p < 0.05 versus control; √, p < 0.05 versus cytomix-injured for ATII cells). B, lipid-based transfection of MSC with two different Ang1 siRNAs (#121280 and #121281) significantly reduced Ang1 secretion by 48 h. Transfection of MSC with negative control siRNA did not affect Ang1 production. Cells were plated in 24-well plates at a density of 50,000 cells/well. Data are expressed as mean ± S.D. (pg/ml, n = 3; *, p < 0.05 versus control).CC BY-NC · PubMed Central
FIGURE 4. Effect of Ang1 secretion by human allogeneic mesenchymal stem cells on epithelial protein permeability of human alveolar epithelial type II cells injured by cytomix. Pretreatment of MSC with Ang1 siRNA (#121280) abolished the therapeutic effect of MSC on epithelial permeability to protein across ATII cells after cytomix exposure. Simultaneous addition of rhAng1 (100 ng/ml) alone on injured ATII monolayers had a similar effect on restoring epithelial permeability as compared with MSC. Epithelial protein permeability is expressed as the mean ± S.D. (% change/24h, n = 3 – 11; *, p < 0.03 versus control; √, p < 0.05 versus cytomix-injured).
FIGURE 4. Effect of Ang1 secretion by human allogeneic mesenchymal stem cells on epithelial protein permeability of human alveolar epithelial type II cells injured by cytomix. Pretreatment of MSC with Ang1 siRNA (#121280) abolished the therapeutic effect of MSC on epithelial permeability to protein across ATII cells after cytomix exposure. Simultaneous addition of rhAng1 (100 ng/ml) alone on injured ATII monolayers had a similar effect on restoring epithelial permeability as compared with MSC. Epithelial protein permeability is expressed as the mean ± S.D. (% change/24h, n = 3 – 11; *, p < 0.03 versus control; √, p < 0.05 versus cytomix-injured).CC BY-NC · PubMed Central
FIGURE 5. Effect of MSC or recombinant Ang1 on the phosphorylation of Tie2 among primary cultures of human alveolar epithelial type II cells injured by cytomix. Levels of Tie2 phosphorylation were measured in protein lysates of ATII. Base-line expression of the phosphorylated Tie2 receptor among human ATII cells decreased at 24 h after exposure to cytomix. The addition of MSC or rhAng1 partially restored the decrease in phosphorylated Tie2 receptor levels. Pretreatment of MSC with Ang1 siRNA abolished the therapeutic effect of MSC on receptor phosphorylation. Data are expressed as the mean ± S.D. (% of control, n = 3; *, p < 0.008 versus control; √, p < 0.05 versus cytomix-injured). Control is arbitrarily set at 100%.
FIGURE 5. Effect of MSC or recombinant Ang1 on the phosphorylation of Tie2 among primary cultures of human alveolar epithelial type II cells injured by cytomix. Levels of Tie2 phosphorylation were measured in protein lysates of ATII. Base-line expression of the phosphorylated Tie2 receptor among human ATII cells decreased at 24 h after exposure to cytomix. The addition of MSC or rhAng1 partially restored the decrease in phosphorylated Tie2 receptor levels. Pretreatment of MSC with Ang1 siRNA abolished the therapeutic effect of MSC on receptor phosphorylation. Data are expressed as the mean ± S.D. (% of control, n = 3; *, p < 0.008 versus control; √, p < 0.05 versus cytomix-injured). Control is arbitrarily set at 100%.CC BY-NC · PubMed Central
FIGURE 6. RhoA activation in human ATII cells exposed to cytomix. RhoA activation signals (GTP-bound) were measured in the protein lysates of ATII cells. Levels were assessed at 10, 30, and 60 min after cytomix exposure. The addition of cytomix immediately increased the activated form of RhoA as early as 10 min after exposure. However, when rhAng1 was added to the cell culture system, this peak activation was significantly reduced and comparable with control levels. Data are presented as the mean ± S.D. (% of control, n = 3; *, p < 0.02 versus control; √, p < 0.05 versus cytomix-injured at 10 min). Control is arbitrarily set at 100%.
FIGURE 6. RhoA activation in human ATII cells exposed to cytomix. RhoA activation signals (GTP-bound) were measured in the protein lysates of ATII cells. Levels were assessed at 10, 30, and 60 min after cytomix exposure. The addition of cytomix immediately increased the activated form of RhoA as early as 10 min after exposure. However, when rhAng1 was added to the cell culture system, this peak activation was significantly reduced and comparable with control levels. Data are presented as the mean ± S.D. (% of control, n = 3; *, p < 0.02 versus control; √, p < 0.05 versus cytomix-injured at 10 min). Control is arbitrarily set at 100%.CC BY-NC · PubMed Central
FIGURE 7. Rac1/2/3 activation in human ATII cells exposed to cytomix treated with and without MSC. Rac1/2/3 activation signals (GTP-bound) were measured in the protein lysates of ATII cells at 24 h. The addition of MSC to the bottom chamber significantly increased the expression of Rac1/2/3 among human ATII cells exposed to cytomix. Data are presented as the mean ± S.D. (% of control, n = 3; *, p < 0.0001 versus control; √, p < 0.0001 versus cytomix-injured). Control is arbitrarily set at 100%.
FIGURE 7. Rac1/2/3 activation in human ATII cells exposed to cytomix treated with and without MSC. Rac1/2/3 activation signals (GTP-bound) were measured in the protein lysates of ATII cells at 24 h. The addition of MSC to the bottom chamber significantly increased the expression of Rac1/2/3 among human ATII cells exposed to cytomix. Data are presented as the mean ± S.D. (% of control, n = 3; *, p < 0.0001 versus control; √, p < 0.0001 versus cytomix-injured). Control is arbitrarily set at 100%.CC BY-NC · PubMed Central
FIGURE 8. Immunofluorescence staining of human ATII cells exposed to cytomix treated with and without MSC or rhAng1. Alveolar type II cells grown on glass-slides were stained with phalloidin (green) for actin and rhodamine (red) for phosphorylated myosin light chain-2. Base-line staining for actin (green) in control human ATII showed a typical peripheral distribution. There was minimal staining for phosphorylated myosin light chain 2 (P-MLC-2) (red). Exposure to cytomix for 24 h increased the total amount of phosphorylated MLC-2 levels. More significantly, total cellular actin was re-distributed toward the center of the cells along with the phosphorylated MLC-2 to form actin stress fibers. The overlap of actin and phosphorylated MLC-2 is shown by the orange/yellow staining. The addition of MSC-conditioned medium or recombinant human Ang1 to human ATII cells exposed to cytomix partially restored the peripheral distribution of actin and reduced the activated form of phosphorylated MLC-2. Images are representative for each condition run in triplicates.
FIGURE 8. Immunofluorescence staining of human ATII cells exposed to cytomix treated with and without MSC or rhAng1. Alveolar type II cells grown on glass-slides were stained with phalloidin (green) for actin and rhodamine (red) for phosphorylated myosin light chain-2. Base-line staining for actin (green) in control human ATII showed a typical peripheral distribution. There was minimal staining for phosphorylated myosin light chain 2 (P-MLC-2) (red). Exposure to cytomix for 24 h increased the total amount of phosphorylated MLC-2 levels. More significantly, total cellular actin was re-distributed toward the center of the cells along with the phosphorylated MLC-2 to form actin stress fibers. The overlap of actin and phosphorylated MLC-2 is shown by the orange/yellow staining. The addition of MSC-conditioned medium or recombinant human Ang1 to human ATII cells exposed to cytomix partially restored the peripheral distribution of actin and reduced the activated form of phosphorylated MLC-2. Images are representative for each condition run in triplicates.CC BY-NC · PubMed Central
FIGURE 9. Effect of ROCK inhibitor, myosin light chain kinase inhibitor, or NFκB pathway inhibitor on protein permeability induced by cytomix. A, treatment with Y-27632, a Rho kinase inhibitor, had no beneficial effect on protein permeability across human AT II cell monolayer. B, ML-7, a myosin light chain kinase inhibitor, significantly restored epithelial protein permeability induced by cytomix. NFκB pathway inhibitor BMS-345541 (C) or a NF-κB p50 (NLS) inhibitory peptide (D) significantly restored the increase in protein permeability induced by cytomix as well. Epithelial protein permeability is expressed as the mean ± S.D. (% change/24 h, n = 3; *, p < 0.02 versus Control; √, p < 0.05 versus cytomix-injured).
FIGURE 9. Effect of ROCK inhibitor, myosin light chain kinase inhibitor, or NFκB pathway inhibitor on protein permeability induced by cytomix. A, treatment with Y-27632, a Rho kinase inhibitor, had no beneficial effect on protein permeability across human AT II cell monolayer. B, ML-7, a myosin light chain kinase inhibitor, significantly restored epithelial protein permeability induced by cytomix. NFκB pathway inhibitor BMS-345541 (C) or a NF-κB p50 (NLS) inhibitory peptide (D) significantly restored the increase in protein permeability induced by cytomix as well. Epithelial protein permeability is expressed as the mean ± S.D. (% change/24 h, n = 3; *, p < 0.02 versus Control; √, p < 0.05 versus cytomix-injured).CC BY-NC · PubMed Central
FIGURE 10. Effect of cytomix on total protein levels of the major tight junction proteins. Shown is a representative Western blot analyses of tight junctional protein expressions in human AT II cells injured by cytomix. There are no significant differences in expression of ZO-1, occludin, claudin 3, claudin 4, claudin 5, and JAM-A between control and cytomix exposed groups. Blots were normalized to GAPDH protein expression. Blots are representative for each condition run in triplicates.
FIGURE 10. Effect of cytomix on total protein levels of the major tight junction proteins. Shown is a representative Western blot analyses of tight junctional protein expressions in human AT II cells injured by cytomix. There are no significant differences in expression of ZO-1, occludin, claudin 3, claudin 4, claudin 5, and JAM-A between control and cytomix exposed groups. Blots were normalized to GAPDH protein expression. Blots are representative for each condition run in triplicates.CC BY-NC · PubMed Central
FIGURE 11. Effect of cytomix on the distribution of tight junction proteins. Immunostaining of tight and adherent junctional proteins in human AT II cells injured by cytomix is shown. A, immunostaining of ZO-1, occludin, and claudin 4 in human AT II cells is shown. There are no significant differences in the distribution pattern of ZO-1, occludin and claudin 4. B, immunostaining of adherent junctional protein E-cadherin and β-catenin is shown. There are no significant differences in the distribution pattern of E-cadherin and β-catenin. Images are representative for each condition run in triplicates.
FIGURE 11. Effect of cytomix on the distribution of tight junction proteins. Immunostaining of tight and adherent junctional proteins in human AT II cells injured by cytomix is shown. A, immunostaining of ZO-1, occludin, and claudin 4 in human AT II cells is shown. There are no significant differences in the distribution pattern of ZO-1, occludin and claudin 4. B, immunostaining of adherent junctional protein E-cadherin and β-catenin is shown. There are no significant differences in the distribution pattern of E-cadherin and β-catenin. Images are representative for each condition run in triplicates.CC BY-NC · PubMed Central
FIGURE 12. Effect of NFκB pathway inhibitor BMS-345541, rhAng-1, or MSC on claudin 18 protein distribution among human AT II cells injured by cytomix. A, immunofluorescent staining of claudin 18 expression in human AT II cells injured by cytomix demonstrated a re-distribution of the tight junction protein away from the periphery of the cell. The simultaneous addition of BMS-345541, rhAng-1, or MSC restored the re-distribution of claudin 18 expression caused by cytomix. B, Western blot analysis of claudin 18 expression in human AT II cells injured by cytomix demonstrated a decrease in total protein levels. The addition of BMS-345541 or MSC partially restored the protein expression of claudin 18. RhAng-1 did not show a significant effect on protein expression of claudin 18. All experiments were run in triplicates. Claudin 18 expression were normalized to GAPDH expression. A representative Western blot is depicted above each graph. Data are expressed as the mean ± S.D. (% of control, n = 3; *, p < 0.01 versus control; √, p < 0.05 versus cytomix-injured). Control is arbitrarily set at 100%.
FIGURE 12. Effect of NFκB pathway inhibitor BMS-345541, rhAng-1, or MSC on claudin 18 protein distribution among human AT II cells injured by cytomix. A, immunofluorescent staining of claudin 18 expression in human AT II cells injured by cytomix demonstrated a re-distribution of the tight junction protein away from the periphery of the cell. The simultaneous addition of BMS-345541, rhAng-1, or MSC restored the re-distribution of claudin 18 expression caused by cytomix. B, Western blot analysis of claudin 18 expression in human AT II cells injured by cytomix demonstrated a decrease in total protein levels. The addition of BMS-345541 or MSC partially restored the protein expression of claudin 18. RhAng-1 did not show a significant effect on protein expression of claudin 18. All experiments were run in triplicates. Claudin 18 expression were normalized to GAPDH expression. A representative Western blot is depicted above each graph. Data are expressed as the mean ± S.D. (% of control, n = 3; *, p < 0.01 versus control; √, p < 0.05 versus cytomix-injured). Control is arbitrarily set at 100%.CC BY-NC · PubMed Central

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Metadata

Authors
Fang, X., Neyrinck, A. P., Matthay, M. A., & Lee, J. W.
Affiliations
University of California, San Francisco (US)
Corresponding author
Lee, Jae W.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2010-08
Volume
285
Issue
34
Pages
26211-26222
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m110.119917
PMC
PMC2924032
OpenAlex
W2044217893
Cited by
258
References
55
Citations by year
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FWCI
8.68
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • University of Pittsburgh
  • National Institutes of Health (HL51856)
  • National Heart, Lung, and Blood Institute (093026, HL51854, K08HL093026, HL-51856, R01HL051856, HL-093026, 51856, R01HL051854, R37HL051856)
  • National Center for Research Resources (P40RR017447)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Respiratory Support and Mechanisms
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
Keywords
allogeneic mesenchymal stem cells, angiopoietin-1, alveolar type II cells, acute lung injury, paracrine signaling, stress fibers, claudin-18, NF-κB, pulmonary edema
MeSH terms
Cells, Cultured, Epithelium, Humans, Models, Biological, Permeability, Pulmonary Alveoli, Cytokines, Coculture Techniques, Protein Transport, Angiopoietin-1, Acute Lung Injury, Mesenchymal Stem Cells

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