Bone Marrow Derived Mesenchymal Stem Cells Inhibit Inflammation and Preserve Vascular Endothelial Integrity in the Lungs after Hemorrhagic Shock

Hemorrhagic shock (HS) and trauma is currently the leading cause of death in young adults worldwide. Morbidity and mortality after HS and trauma is often the result of multi-organ failure such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), conditions with few therapeutic options. Bone marrow derived mesenchymal stem cells (MSCs) are a multipotent stem cell population that has shown therapeutic promise in numerous pre-clinical and clinical models of disease. In this paper, in vitro studies with pulmonary endothelial cells (PECs) reveal that conditioned media (CM) from MSCs and MSC-PEC co-cultures inhibits PEC permeability by preserving adherens junctions (VE-cadherin and β-catenin). Leukocyte adhesion and adhesion molecule expression (VCAM-1 and ICAM-1) are inhibited in PECs treated with CM from MSC-PEC co-cultures. Further support for the modulatory effects of MSCs on pulmonary endothelial function and inflammation is demonstrated in our in vivo studies on HS in the rat. In a rat “fixed volume” model of mild HS, we show that MSCs administered IV potently inhibit systemic levels of inflammatory cytokines and chemokines in the serum of treated animals. In vivo MSCs also inhibit pulmonary endothelial permeability and lung edema with concurrent preservation of the vascular endothelial barrier proteins: VE-cadherin, Claudin-1, and Occludin-1. Leukocyte infiltrates (CD68 and MPO positive cells) are also decreased in lungs with MSC treatment. Taken together, these data suggest that MSCs, acting directly and through soluble factors, are potent stabilizers of the vascular endothelium and inflammation. These data are the first to demonstrate the therapeutic potential of MSCs in HS and have implications for the potential use of MSCs as a cellular therapy in HS-induced lung injury

TNFα, MIP-1α, and MCP-1 are decreased and anti-inflammatory cytokine IL-10 is increased by MSC treatment.

<p>Blood was drawn at two hours post-hemorrhage and analyzed for serum levels of TNFα, MCP-1, IL-10 and MIP-1α. As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g005" target="_blank">Figures 5A–C</a>, bioplex analysis of serum shows that MCP-1, TNFα, and MIP-1α all significantly increase after HS and HS+LR. This rise is significantly decreased by MSC administration (* indicates p<0.05 for HS vs. LR+MSC and LR vs. LR+MSC). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g005" target="_blank">Figure 5D</a> shows that anti-inflammatory cytokine IL-10 is significantly increased by MSC (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g005" target="_blank">Figure 5D</a>).</p

MSCs in the lung vasculature.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figures 6A-C</a> show, IV MSCs localize to the lung vasculature and preserve integrity of AJs and tight junctions (TJs). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figure 6A</a>, MSCs (see white arrow) were detected around blood vessels in the lungs (BVL = blood vessel lumen) using a human mitochondrial antibody (green) showing staining in lung tissue from MSC treated rats. (red = VE-Cadherin and DAPI staining nuclei -blue). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figure 6B:</a> Staining of blood vessels in lungs (BVL = blood vessel lumen) for VE-Cadherin (red) and β-catenin (green) shows that in the LR group, HS and resuscitation compromise the continuity of the AJs (see white arrow). This continuity is preserved in MSC treated animals. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figure 6C:</a> Similar findings are found for TJs staining of Occludin-1 (green) and Claudin-1 (red). MSCs preserve compromise of TJs in the lungs (see white arrows). Morphological changes (thickening) in AJs and TJs are noted in MSC treated lung vessels (long white arrows). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figures 6D–H</a> show MSCs preserve PDGFRβ positive pericytes on lung microvasculature after HS. Sectioned lung tissue was stained for PDGFRβ (green) to identify pericytes or smooth muscle (SM) cells and vWF (red) to identify blood vessels. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figures 6E and 6F</a> shows that HS and LR groups show diminished or compromised PDGFRβ staining (small white arrows), indicating decreased pericyte/SM cell coverage on microvasculature. This is increased above Sham (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figure 6D</a>) in MSC treated animals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figure 6G</a>-see large white arrow). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g006" target="_blank">Figure 6H</a> shows that MSCs (red) contribute to some, but not all, of the increased PDGFRβ (green) staining found in treated lungs.</p

CM inhibits adhesion of U937s and adhesion molecule expression in PECs.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g002" target="_blank">Figures 2A and 2B</a>, CM from MSC and MSC+PEC co-cultures inhibits adhesion of U937 to PECs. Calcein labeled U937 cells were allowed to adhere to PECs that had been pre-treated with CM from PEC and MSC-PEC co-cultures. Cultures were stimulated with TNFα and U937s were added to the wells. After gentle washing, bound cells were quantitatively and qualitatively assessed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g002" target="_blank">Figure 2A</a> shows decreased binding of U937 (green dots) in CM treated cultures. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g002" target="_blank">Figure 2B</a> shows quantitative significant decreases in CM treated cultures as indicated by (*), which signifies p<0.05 between control (TNFα treated cells) and MSC CM and control and CM-CO (MSC-PEC CM). “Unstim” represents the group not treated with TNFα. Eight wells were included in each group (n = 8). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g002" target="_blank">Figures 2C–F</a>, CM from MSC+PEC co-cultures inhibits PECs expression of adhesion molecules VCAM-1 and ICAM-1. Conditioned media was made in three groups: (1) PECs alone, (2) MSCs alone, (3) PECs/MSCs co-culture. EGM-2 (Lonza) was added to wells as culture medium. After 48 hours of culture the media was removed and stored at −80°C. PECs grown to confluence on 6 well plates and growth media was replaced with the conditioned media in the above groups. The experiment was performed in replicates of four. After 12 hour incubation, TNFα (50 ng/mL) was added to the wells and incubated for 4 hours. The cells were then collected and stained with fluorophore conjugated antibodies to I-CAM, V-CAM, E-selectin, and P-selectin. The cells treated with PEC/MSC conditioned media demonstrated decreased I-CAM and V-CAM expression compared to both those treated with PEC or MSC conditioned media(p<0.01, ANOVA). This difference was not seen in E-selectin or P-selectin.</p

Conditioned media (CM) inhibits permeability and restores adherens junctions (AJs).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g001" target="_blank">Figure 1A</a>, CM from MSCs and MSC-PEC co-cultures inhibits PEC permeability <i>in vitro</i>. A schematic depicting how CM is prepared is shown. MSCs and PECs are cultured separately and together in co-culture. CM is collected 24 hours later and used in a transwell assay of PEC permeability to 40 kD FITC-Dextran. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g001" target="_blank">Figure 1A</a> shows that CM from both MSCs and MSC-PEC co-cultures inhibits permeability induced by VEGF-A (10 ng/ml). (*) signs indicate significance p<0.05 between MSC and VEGF and MSC-PEC and VEGF groups. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g001" target="_blank">Figures 1B–E</a>, CM from MSCs and MSC-PEC co-cultures restore AJs. PECs were cultured overnight. After 24 hours, breakdown of AJs (β-catenin and VE-Cadherin) is induced by treatment with VEGF-A (10 ng/ml). Cells were subsequently treated with CM. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g001" target="_blank">Figures 1B–E</a> show that MSC and MSC-PEC CM restored AJ breakdown as determined by confocal microscopy imaging of cultures stained with antibodies to VE-Cadherin (red) and β-Catenin (green).</p

<i>In Vivo</i> IV MSCs do not alter mean arterial pressure (MAP) in rat model of hemorrhagic shock and resuscitation.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g003" target="_blank">Figure 3A</a> shows a schematic of the <i>in vivo</i> rat model. Animals were pre-instrumented three days prior to hemorrhage. Hemorrhage of fixed volume at a rate of 2 ml/100 g/10 minutes was performed. One hour later, resuscitation of Lactated Ringer's (LR) (3× shed blood) was administered. MSCs at a dose of 2×10⁶ were administered with LR at 1 and 24 hours post hemorrhage. Blood was drawn at 0, 2 and 96 hours. Tissues were harvested on day four. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g003" target="_blank">Figures 3B and 3C</a> show that hemorrhage volume and weight of rats is similar in all groups. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g003" target="_blank">Figure 3D</a> shows that MAP drops respectively in all hemorrhaged groups and returns to normal by 2 hours post-hemorrhage. MSC administration does not affect MAP.</p

Soluble factors play a role in the effects of IV MSCs in vivo.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025171#pone-0025171-g007" target="_blank">Figure 7</a> shows a working biological model of how MSCs may function biologically when delivered IV after HS. MSC attach to pulmonary vascular endothelial cells in the lungs where they produce soluble factor(s) that affect vascular stability in the lungs through modulation of AJs, TJs and checking inflammation. We hypothesize that the soluble factor(s) produced promote local and systemic vascular stability.</p