Conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury

Abstract

Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1β, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.

Keywords: growth factors; hypoxia; inflammation; membrane transport; mesenchymal stem cells; sodium channels.

Fig. 1.

Effects of cytomix and hypoxia on apoptosis and necrosis of human mesenchymal stromal cells (MSCs). The MSCs were exposed to either normoxia (21% O₂) or to cytomix (20 ng/ml) plus hypoxia (3% O₂) for 12 h. Apoptosis and necrosis were determined using annexin-V7AAD kit and expressed as the percentage of total cells. Experiments were repeated 3 times. SSC, side-scatter angle.

Fig. 2.

Effect of cytomix plus hypoxia on the release of soluble mediators by MSCs. The MSC were cultured in DMEM in the absence or presence of cytomix (20 ng/ml) plus hypoxia (3% O₂). After 12 h, the media of nonexposed cells (MSC-M) or the media of cells exposed to cytomix (20 ng/ml) plus hypoxia (3% O₂; HCYT-MSC-M) were used for the determination of IL-1 receptor antagonist (IL-1ra), PGE₂, and keratinocyte growth factor (KGF) concentrations. The values represent the means ± SD of 4–6 experiments in each group. *P < 0.01 by unpaired Student's t-test.

Fig. 3.

Effect of MSC medium on cytomix-hypoxia-induced decrease of epithelial permeability to albumin in cultured alveolar epithelial cells. Alveolar epithelial cells (AECs) were seeded on Transwell filters. AECs were exposed or not to cytomix (20 ng/ml) plus hypoxia (3% O₂) for 6 h. Then, the media were replaced for additional 12 h by 1) DMEM alone and AECs were exposed to normoxia (21% O₂); 2) DMEM plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); 3) media of nonexposed MSC (MSC-M) plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); and 4) medium of MSC exposed to cytomix plus hypoxia (HCYT-MSC-M) and cells were exposed to hypoxia (3% O₂). Epithelial protein permeability was then assessed by adding labeled ¹²⁵I-albumin only to the upper compartment, at the time of replacement of the media, and the unidirectional flux to the lower compartment was measured over 12 h. The values are the means ± SD of 3 experiments in each group. *P < 0.01, compared with control conditions [one-way ANOVA followed by Fisher's protected least significant difference (PLSD)].

Fig. 4.

Effect of MSC-conditioned media on cytomix plus hypoxia-induced decrease of Na transport in alveolar epithelial cells. AECs grown on Snapwell filters were exposed to either normoxia or cytomix (20 ng/ml) plus hypoxia (3% O₂) for 6 h. Then, the media were replaced for additional 12 h by 1) DMEM alone and AECs were exposed to normoxia (21% O₂); 2) DMEM plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); 3) media of nonexposed MSC (MSC-M) plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); and 4) medium of MSC exposed to cytomix plus hypoxia (HCYT-MSC-M) and cells were exposed to hypoxia (3% O₂). AECs were mounted into a voltage-clamp system, and short-circuit current (I_sc) was measured at baseline (black bars) and after addition of amiloride (10⁻⁵ M) into the apical bath (A; gray bars). Amiloride-sensitive I_sc (B) represents the difference of I_sc before and after addition of amiloride. The values are the means ± SD of 4–6 filters for each condition. *P < 0.05, **P < 0.01, and ***P < 0.001, significantly different from corresponding control value (one-way ANOVA followed by Fisher's PLSD).

Fig. 5.

AECs grown on Snapwell filters were exposed to either normoxia or cytomix (20 ng/ml) plus hypoxia (3% O₂) for 6 h. Then, the media were replaced for additional 12 h by 1) DMEM alone and AECs were exposed to normoxia (21% O₂); 2) DMEM plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); and 3) media of nonexposed MSC (MSC-M) plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂). A: AECs were mounted into a voltage-clamp system in the presence of a Na⁺ concentration gradient (mucosal to serosal, 140:10 mM). Addition of nystatin into the basolateral bath increased the I_sc to a peak value before amiloride (10 μM) was added into the apical bath. Apical amiloride-sensitive current is the difference between I_sc peak value and I_sc value after amiloride addition and reflects epithelial sodium channel (ENaC) activity; B: AECs were then immediately mounted into a voltage-clamp system. After stabilization, nystatin into the apical bath evoked a slowly developing rise in I_sc and ouabain (1 mM) was added to the basolateral solution once this response had reached its peak value. Basolateral ouabain-sensitive current representing the fall in I_sc evoked by this application of ouabain was measured to estimate the extrusion capacity of the basolateral Na⁺ pump. The values are the means ± SD of 5–8 filters for each condition. *P < 0.01, compared with control conditions (one-way ANOVA followed by Fisher's PLSD).

Fig. 6.

Effect of cytomix plus hypoxia in the presence or absence of MSC-conditioned media on total protein levels of the α- and β-subunits of ENaC in alveolar epithelial cells. AECs grown on Transwell filters were exposed to either normoxia or cytomix (20 ng/ml) plus hypoxia (3% O₂). After 6 h, the media were replaced for additional 12 h by 1) DMEM alone and AECs were exposed to normoxia (21% O₂); and 2) DMEM plus cytomix (20 ng/ml) and presence of cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂). Western blot experiments were performed on whole cells extracts using rabbit polyclonal antibodies raised against rat α-ENaC (A, left) and rat β-ENaC (A, right). Quantification of α-ENaC (85 and 65 kDa) and β-ENaC levels was obtained using Scion Image software, and the data were normalized for the corresponding actin signal in each lane (B). Results are expressed as the ratios of α- and β-ENaC/actin. The values are the means ± SD of 5 separate experiments (one-way ANOVA followed by Fisher's PLSD).

Fig. 7.

Effect of cytomix plus hypoxia on apical cell surface expression of α- and β-ENaC subunits in alveolar epithelial cells in the presence or absence of MSC-conditioned media. AEC grown on Transwell filters were exposed to either normoxia or cytomix (20 ng/ml) plus hypoxia (3% O₂). After 6 h, the media were replaced for additional 12 h by 1) DMEM alone and AECs were exposed to normoxia (21% O₂); 2) DMEM plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); and 3) the medium of nonexposed MSC (MSC-M) in the presence of cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂). Then, AECs were immediately processed for apical biotinylation experiments as described in Experimental Protocols before immunoblotting. A: representative immunoblots of α-ENaC (left) and β-ENaC (right) cell surface, and of intracellular protein expression of β-actin. The validity of surface biotinylation approach in control and treated cells is attested by the cell surface expression of α- and β-ENaC but not of the intracellular protein β-actin. The quantification of biotinylated surface proteins for α-ENaC (65 kDa) (left) and β-ENaC (right) is shown in B. Quantification of biotinylated α- and β-ENaC signals was obtained using Scion Image software, and the data were normalized for the actin signal in corresponding intracellular extracts. The values are the means ± SD of 4 separate experiments. *P < 0.01, compared with control conditions (one-way ANOVA followed by Fisher's PLSD).

Fig. 8.

Effect of MSC-conditioned media depleted of KGF and of recombinant human KGF (rhKGF) on the cytomix plus hypoxia-induced decrease in Na transport. A: AECs grown on Transwell filters were exposed to either normoxia or cytomix (20 ng/ml) plus hypoxia (3% O₂). After 6 h, the media were replaced for additional 12 h by 1) DMEM alone and AECs were exposed to normoxia (21% O₂); 2) DMEM plus cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂); 3) the media of nonexposed MSC (MSC-M) in the presence of cytomix (20 ng/ml) plus IgG and cells were exposed to hypoxia (3% O₂); and 4) the media of nonexposed MSC (MSC-M) depleted of KGF as described in Experimental Procedures in the presence of cytomix (20 ng/ml) and cells were exposed to hypoxia (3% O₂). I_eq was measured before and after addition of amiloride (10 μM) was added into the apical bath. Amiloride-sensitive I_eq represents the difference of I_sc before and after addition of amiloride. The values are the means ± SD of 8–14 filters for each condition. **P < 0.01, compared with control conditions; §P < 0.05, compared with cytomix plus hypoxia; +P < 0.05, compared with cytomix plus hypoxia in presence of MSC-M plus IgG (one-way ANOVA followed by Fisher's PLSD). B: after a 6 h exposure to normoxia (white bars) or cytomix plus hypoxia (black bars), AECs were treated with rhKGF (200 or 400 pg/ml) or vehicle for additional 12 h and amiloride-sensitive I_eq was calculated. Values are means ± SD of 8–17 filters for each condition. ***P < 0.001, compared with normoxic corresponding value (one-way ANOVA followed by Fisher's PLSD).