Bone Marrow Progenitor Cells Induce Endothelial Adherens Junction Integrity by Sphingosine-1-Phosphate–Mediated Rac1 and Cdc42 Signaling
Rationale: Little is known about the contribution of bone marrow–derived progenitor cells (BMPCs) in the regulation endothelial barrier function as defined by microvascular permeability alterations at the level of adherens junctions (AJs).
Objective: We investigated the role of BMPCs in annealing AJs and thereby in preventing lung edema formation induced by endotoxin (LPS).
Methods and Results: We observed that BMPCs enhanced basal endothelial barrier function and prevented the increase in pulmonary microvascular permeability and edema formation in mice after LPS challenge. Coculture of BMPCs with endothelial cells induced Rac1 and Cdc42 activation and AJ assembly in endothelial cells. However, transplantation of BMPCs isolated from sphingosine kinase-1–null mice (SPHK1−/−), having impaired S1P production, failed to activate Rac1 and Cdc42 or protect the endothelial barrier.
Conclusions: These results demonstrate that BMPCs have the ability to reanneal endothelial AJs by paracrine S1P release in the inflammatory milieu and the consequent activation of Rac-1 and Cdc42 in endothelial cells.
The endothelial cell monolayer lining blood vessels forms a semipermeable barrier that performs the vital tasks of regulating tissue fluid homeostasis and transmigration of blood cells. Bone marrow–derived endothelial progenitor cells induce angiogenesis and have been touted for therapeutic angiogenesis in the treatment of myocardial ischemia and other diseases requiring revascularization.1 Recent studies showed that transplantation of bone marrow–derived mesenchymal cells can also limit lung inflammation in experimental models.2,3 As disruption of the endothelial barrier in vascular inflammation leads to tissue edema and injury and there are no current means to mitigate vascular leakage, we addressed the possible role of bone marrow–derived progenitor cells (BMPCs) in regulating endothelial barrier function.
The phospholipid sphingosine-1-phosphatase (S1P), generated by platelets, red blood cells (RBCs), and endothelial cells, has an endothelial barrier protective function.4,5 Sphingosine kinases phosphorylate sphingosine leading to formation of S1P.6 Platelets lack the S1P degradation enzyme sphingosine-1-lyase and thus release S1P in the circulation.7 In addition, RBCs and endothelial cells produce abundant amounts of S1P.8–10 The endothelial barrier promoting effect of S1P is concentration-dependent and characterized by a rapid increase in transendothelial electric resistance.4,11 S1P signaling on the basis of its effects on immune and endothelial cells is also a modulator of immune and inflammatory responses.12 In the murine model of LPS-mediated acute lung injury, S1P was shown to attenuate pulmonary vascular leakage and inflammation.12,13 The S1P effects depend on ligation of Gi-coupled Edg-1 and Edg-3 receptors.14 In the present study, we surmised that BMPCs are an important source of S1P and thereby contribute to BMPC-mediated effects on the endothelium.
We previously showed that reestablishing endothelial contact and junction integrity after injury requires the activation of the RhoGTPases Cdc42 and Rac1 in endothelial cells.15 Thus, we also investigated their involvement in mediating the underlying effects of BMPCs on endothelial junctions. We demonstrate here that homing of BMPCs to lungs profoundly enhanced endothelial barrier function at the level of adherens junctions (AJs) and prevented the LPS-induced increase in endothelial permeability by the generation of S1P and consequent activation of Rac1 and Cdc42 signaling.
All mice were bred and maintained according to NIH guidelines, and experiments were approved by the Animal Care and Use Committee.
Isolation of Mouse BMPCs
Mouse BMPCs were isolated using modifications of methods based on published markers and methods used for human and rat BMPC isolation (see online Material and Methods, available online at http://circres.ahajournals.org).16–18 The phenotype of confluent cell population was assessed by determining expression of protein markers by FACS analysis.17
Mouse Lung Vascular Endothelial Cells
Mouse lung vascular endothelial cells were isolated as described.19
Pulmonary Uptake of BMPCs
BMPCs labeled with rhodamine fluorophore chloromethyl tetramethyl rhodamine (CMTMR, Molecular Probes) were injected into mice (3×105 cells per mouse) through the external jugular vein. The animals were then euthanized at different times to determine the location of BMPCs within mouse lung microvasculature by confocal microscopy. In other studies, we labeled 3×105 BMPCs with 111Indium oxine20 to determine organ uptake of BMPCs after i.v. injection.
Cells were detached using 1 mmol/L EDTA in PBS, and incubated with the following antibodies for 30 minutes before FACS analysis: phycoerythrin-labeled antimurine Sca1 (BD PharMingen), and antimurine CD31 (Becton-Dickinson); allophycocyanin-labeled antimurine c-Kit and CD34 antibodies (BD PharMingen); mouse anti-human CD133 (BD PharMingen), rabbit anti-human VE-cadherin (Santa Cruz), and rat anti-mouse CD45 (BD PharMingen). Rabbit anti-mouse or goat anti-mouse FITC (Vector) or Alexa Fluor-labeled anti-goat 594 was used as the secondary antibody (Invitrogen).
Mice in all cases were injected with 3×105 BMPCs (in 200 μL of EBM-2MV medium) through the external jugular vein.17
All mice (except those used for mortality studies) received a single sublethal dose of 10 mg/kg bw LPS (E coli 0111:B4, Sigma L-2630) i.p. Animals were euthanized at 6 hours after LPS administration for microvessel filtration coefficient (Kf,c) determinations (see below) and at 12 hours for final lung extravascular lung water measurements.21
Rho GTPase Activity Assays
Rac1, Cdc42, and RhoA activities were measured using the GSTrhotekin-Rho binding domain that precipitates activated Rac1 and Cdc42.22,23 Mouse endothelial cell monolayers were cocultured with BMPCs for different times. Cell lysates were clarified by centrifugation at 4°C and 1000g for 5 minutes, and equal volumes of lysates were incubated with PAK-GST protein beads (Cytoskeleton Inc) at 4°C for 2 hours. Beads were washed 3×, and bound Rac1 or Cdc42 was eluted by boiling each sample in Laemmli sample buffer to elute samples. Total cell lysates were electrophoresed on 12.5% SDS-polyacrylamide gels and immunoblotted with either mouse anti-Rac1 or rabbit anti-Cdc42 antibody.
siRNA constructs were transduced in cells by electroporation. Mouse endothelial cells, 60% confluent, were trypsinized, mixed with 2.5 μg siRNA for Rac1, 10 μg for Cdc42, or 3.0 μg for nonsilencing siRNA along with 100 μL of transfection solution (Thermo Scientific Dharmacon siGENOME). Cells were electroporated using a nucleofector device with manufacturer’s protocol (VPI-1001, Amaxa). Cells were removed, mixed in EBM-MV2, and plated on either 100-mm dishes, or Transwell chamber filters, or gold-plated 10-well electrodes for functional analyses.
Data are presented as mean±SEM. Differences were tested using analysis of variance (ANOVA with posthoc comparisons using unpaired t test or Mann–Whitney tests as appropriate). Survival data were analyzed using Peto-Peto-Wilcoxon test. P<0.05 denotes significant difference.
Cultured wild-type (wt) BMPCs were positive for various stem/progenitor cell markers CD133 (92%), Sca1 (83%), CD34 (75%; Figure 1A). BMPCs based on their high expression of these markers are therefore referred to as progenitor cells. Only a small number of BPMCs expressed the mature endothelial markers CD31 (3%) and VE-cadherin (12%), and generic myeloid cell marker CD45 (0.3%; Figure 1A). Quantitative real-time RT-PCR analysis also showed minimal expression of CD45 in BMPCs (Online Figure IC).
In comparing wt and SPHK1 KO BMPCs, we found similar CD133 expression (92% for wt and 90% for SPHK1 KO BMPCs). Although Sca1 and CD34 expression was elevated in both cell types, their expression was greater in wt BMPCs (for Sca1, 83% in wt BMPCs and 40% in SPHK1 KO BMPCs; for CD34, 75% in wt BMPCs and 40% for SPHK1 KO BMPCs). There were also differences in CD31, VE-cadherin, and CD45 expression in wt and SPHK1 KO BMPCs (for CD31, 2.6% in wt BMPCs and 66% in SPHK1 KO BMPCs; for VE-cadherin, 12% in wt BMPCs and 40% for SPHK1 KO BMPCs; for CD45, 0.3% in wt BMPCs and 15% for SPHK1 KO BMPCs; Online Figure IA).
Localization of BMPCs in Lungs
We next determined the uptake of transplanted BMPCs in lungs. At day 3 after i.v. injection of 111Indium oxine–labeled BMPCs, lungs contained 80% of the injected BMPCs compared to other organs (Online Figure IB). To visualize BMPCs, they were incubated with the fluorophore CMTMR (25 μmol/L) for 40 minutes, then washed and injected into the external jugular vein. We found similar 60% of the wt and SPHK1−/− BMPCs were retained at 20 minutes (with most in lung microvessels) after injection (Figure 1B and 1C). By 24 hours, total number of fluorescent cells was decreased in both groups with a greater decrease in SPHK1−/− BMPCs (24% in SPHK1−/− BMPCs and 47% in wt BMPCs (P<0.05). The location of cells in both groups was the same, with most cells detected in the pulmonary parenchyma or lodged extramurally in vessels (Figure 1B). At 3 days after injection, there was a further decrease in accumulation of both wt and SPHK1−/− BMPCs (34% and 20%, respectively, P<0.05 between 2 groups), with most cells localized in the parenchyma (Figure 1B).
BMPCs Prevent Increase in Lung Vascular Permeability
We quantified alterations in pulmonary microvascular permeability by determining the Kf,c in mice receiving either BMPCs or mouse lung microvessel endothelial cells (ECs; in each case 3×105 cells were injected i.v.). The mice were challenged at day 3 after BMPC injection with a sublethal LPS dose (10 mg/kg i.p.). BMPC transplantation significantly prevented the increase in Kf,c induced by LPS whereas injection of ECs afforded no barrier protection (Figure 2A). Lung extravascular water content increased in control mice (7.0±1.8g/g) and EC-injected group (5.9±1.1) in response to LPS, whereas it was normal in LPS-challenged mice transplanted with BMPCs (4.3±1.0; Figure 2B).
We also determined the effects of BMPC transplantation on survival post-LPS insult (Figure 2C). Mice in this case were challenged with a lethal dose of LPS (22 mg/kg, i.p.) at day 3 after BMPC injection or either EC injection or PBS injection as controls. In PBS-injected mice, only 11% of mice survived 3 days post-LPS challenge. However, mice receiving 3×105 BMPCs i.v. exhibited 83% survival. In contrast, LPS-challenged mice receiving 3×105 ECs (Figure 2C) or 3×105 blood monocytes (not shown) showed no reductions in mortality.
BMPCs Induce Rac1 and Cdc42 Activation in Endothelial Cells
To address mechanisms of BMPC-induced endothelial barrier protection, we first studied alterations in endothelial junctions that are crucial for maintenance of barrier integrity. Transendothelial electric resistance (TER) was measured in confluent endothelial cell monolayers to provide assessment of junction alterations.19 BMPCs added to cultured EC monolayers (≈15×103 ECs in the confluent monolayer) in increasing numbers (from 0 to 5×103 cells) increased TER (Figure 3A). The response peaked at 30 minutes, then decreased but remained elevated above baseline during the experiment period (Figure 3A). In contrast, ECs added in same numbers had no effect on TER (Figure 3B). The maximum increase in TER induced by BMPCs was 4-fold greater than that by ECs (Figure 3C).
Because monomeric RhoGTPases Rac1 and Cdc42 regulate AJ assembly,11,15 we next investigated their role in signaling junction barrier enhancement observed with BMPCs. BMPC addition to EC monolayers induced activation of Rac1 and Cdc42 in ECs within 30 minutes of BMPC addition, and activities of both GTPases remained elevated for the study duration (Figure 3D and 3E). Using siRNA to suppress the expression of Rac1 or Cdc42 in ECs (Figure 3F), we addressed the role Rac1 and Cdc42 in signaling the barrier enhancement. Reduction in the expression of either Rac1 or Cdc42 in ECs prevented the BMPC-induced increases in TER (Figure 3G through 3I), suggesting that BMPCs induce endothelial barrier enhancement by the activation of Rac1 and Cdc42. Specificity of siRNA-mediated silencing was verified by Western blot using Rac1 siRNA–treated cells. Rac1 siRNA reduced Rac1 activity without affecting Cdc42 (not shown).
As VE-cadherin localization at junctions is required for AJ barrier integrity,15 we next determined the effects of BMPCs on VE-cadherin assembly. VE-cadherin immunostaining showed increased membrane localization of VE-cadherin in ECs in the presence of BMPCs (Online Figure IIA). However, knockdown of Rac1 or Cdc42 in ECs inhibited BMPC-induced AJ annealing (Online Figure IIA).
We quantified transendothelial 125I-albumin permeability using Transwell microporous filters coated with a confluent endothelial monolayer to determine the functional effects of BMPCs on barrier function (Figure 4A). Permeability of 125I-albumin increased after LPS exposure (Figure 4B), and addition of 5×104 BMPCs in the lower chamber not only prevented the increase in endothelial permeability but also BMPCs reduced basal endothelial permeability to a value below baseline. This experiment also demonstrated that endothelial barrier protection induced by BMPCs did not require direct contact with the EC monolayer, indicating the involvement of a paracrine mechanism. Depletion of either Rac1 or Cdc42 in EC monolayers prevented the BMPC-induced reduction in transendothelial 125I-albumin permeability that was seen in control cells (Figure 4B). Neither Rac1 nor Cdc42 silencing in ECs significantly altered the basal endothelial permeability values relative to that observed in control EC monolayers (Figure 4B).
As paracrine mediators of progenitor-like cells from a monocyte/myeloid lineage could also mediate endothelial repair,24 we addressed the possibility that barrier protection induced by BMPCs was the result of a CD45+ myeloid cell subpopulation. However, addition of an enriched population of CD45+ cells (50×103) did not increase endothelial barrier function (Figure 4C).
BMPC Expression of SPHK1 Is Required for Endothelial Barrier Annealing
We next addressed the possibility that endothelial barrier protection was the result of production of S1P by BMPCs because of the importance of S1P in regulating endothelial AJ barrier integrity.11 We thus isolated BMPCs from sphingosine kinase-1–null mice (SPHK1−/−) in which S1P production is severely impaired, and measured TER in the presence of SPHK1−/− BMPCs added in the lower chamber. Addition of SPHK1−/− BMPCs failed to increase TER in contrast to wt BMPCs (Figure 5A through 5C). The SPHK inhibitor, SKI-II, significantly decreased the BMPC-induced enhancement of endothelial junction barrier (Online Figure ID). We also determined Kf,c in mice injected with either wt BMPCs or SPHK1−/− BMPCs. Administration of LPS to SPHK1−/− mice resulted in similar increase in Kf,c as in wt mice challenged with LPS (Figure 5D). Transplantation of BMPCs from SPHK1−/− mice into either wt mice or SPHK1−/− mice failed to prevent the LPS-induced increase in lung vascular permeability. However, transplantation of wt BMPCs into SPHK1−/− mice prevented the increase in lung vascular permeability induced by LPS (Figure 5D). Pulmonary edema formation followed the same pattern as alterations in lung vascular permeability; pulmonary edema did not develop after transplantation of wt BMPCs into lungs of SPHK1−/− mice (Figure 5E).
Using the Transwell microporous filter described above, we next determined the activities of Rac1 and Cdc42 in ECs in the presence of wt or SPHK1−/− BMPCs cultured on the filter while wt ECs were cultured in the lower chamber. Activities of Rac1 and Cdc42 were markedly reduced in LPS-challenged ECs with the addition of either SPHK1−/− BMPCs or with conditioned medium from SPHK1−/− BMPCs compared to ECs with the addition of either wt BMPCs or medium from wt BMPCs (Figure 5F). We measured generation of S1P in the presence of LPS by either wt BMPCs or SPHK1−/− BMPCs cultured with ECs in the Transwell chamber (Figure 5G). S1P production increased with addition of wt BMPCs but not with SPHK1−/− BMPCs (Online Figure WS-IIB), indicating the paracrine role of BMPC-derived S1P in mediating endothelial barrier protection.
Here we have addressed the role of BMPCs in regulating endothelial barrier function and defined the crucial underlying signaling mechanisms mediating this response. We demonstrate that transplantation of BMPCs prevents the increase in lung vascular permeability and edema formation and significantly reduces mortality in mice in response to LPS challenge, and does so primarily through a S1P-mediated paracrine mechanism. The studies were made using BMPCs expressing the progenitor/stem cell markers Sca-1 and CD133 as well as hematopoietic stem/progenitor cell marker CD34. These cells importantly did not express the pan-leukocytic marker CD45 or a battery of mature endothelial cell markers. The study was designed specifically to test the function of BMPCs in the mechanism of endothelial barrier repair. We carried out the studies in cells, which we defined a BMPCs, to avoid the implication that they were a uniform population. In a sense this was fortuitous because a mixed cell population of progenitor cell can act synergistically to induce a high degree of revascularization and tissue repair.24
Analysis of uptake of i.v.-injected 111indium oxine–labeled BMPCs showed the sequestration of cells persisting up to 3 days after injection with relatively few cells localizing in other organs. This was also evident by immunostainng of BMPCs in lungs on day 3 after transplantation when BMPCs were localized in the lung parenchyma. The basis of preferential lung uptake is not clear. The uptake is likely the result of BMPCs being trapped in the first vascular bed encountered after their i.v. injection.17
Based on both cell culture and lung vascular permeability results, it is evident that BMPC transplantation marked endothelial barrier protection. However, mature mouse endothelial cells, CD45+ leukocytes, or blood monocytes afforded no protection. We also observed that transplantation of BMPCs induced greater than 7-fold increase in survival compared to wt mice after a lethal dose of LPS challenge. Such a marked reduction in mortality may be the result of multiple factors besides the reannealing of endothelial junctions. It is possible that BMPCs also have a direct antiinflammatory effect that could improve survival.2,25
Addition of BMPCs induced the activation of Cdc42 and Rac1 in endothelial cells, whereas siRNA-mediated suppression of Cdc42 or Rac1 expression in endothelial cells abolished BMPC-mediated endothelial barrier protection. These intriguing findings suggest a cross-talk between BMPCs and endothelial cells that resulted in the Rac1 and Cdc42 activation in endothelial cells. Both Cdc42 and Rac1 have been shown to induce the formation of membrane protrusions, resulting in the formation of the junction adhesion complexes that thereby enhances barrier integrity.26 We determined alterations in VE-cadherin, the AJ protein regulating endothelial permeability,27 to address whether BMPCs mediated their barrier protective effect by promoting junctional integrity. We observed that BMPCs induced Rac1- and Cdc42-dependent assembly of AJs in endothelial cells, indicating that BMPC-induced activation of Rac1 and Cdc42 was required for AJ assembly and thus the repair of endothelial barrier.
To identify mechanisms of BMPC-mediated endothelial barrier protection, we focused on the endothelial barrier annealing mediator S1P, which promotes endothelial junction integrity via Rac1 and Cdc42 signaling.11 Our results here demonstrated that S1P generation by BMPCs and the activation of Rac1 and Cdc42 in endothelial cells plays a crucial role in restoring the integrity of the endothelial barrier. We observed that BMPCs not only released S1P to protect the endothelial barrier but also S1P generation exerted its maximal effects in the presence of LPS. This latter finding is consistent with studies showing that LPS induces the activation of SPHK1 in macrophages and hepatic cells, which can serve to dampen the hyper-immune response induced by Gram-negative bacteria.28 Our studies made using BMPCs isolated from SPHK1−/− mice,29 the key enzyme responsible for S1P production in endothelial cells,9 showed that SPHK1−/− BMPCs failed to prevent the increase in lung vascular permeability induced by LPS whereas wt BMPCs were protective. In addition, an SK inhibitor SKI-II prevented BMPC-induced endothelial barrier protective effect, indicating a causal relationship between BMPC SPHK functional activity and endothelial barrier protection. Thus, these findings demonstrate the fundamental importance of the SPHK1-generated S1P in the mechanism of endothelial barrier protection. Although we have identified a novel mechanism, the present findings do not preclude the possibility that other mediators such as IL-10 released by BMPCs may also be involved in the endothelial barrier protection and dampening the inflammatory response in vivo.
Other factors should also be considered to explain the role of SPHK in the mechanism of endothelial barrier protection. We observed that some antigen markers in SPHK1−/− BMPCs were different from wt BMPCs and also that fewer SPHK1−/− BMPCs were sequestered in lungs after LPS challenge than in wt BMPCs. Although we cannot the rule out contribution of these factors in the reduced endothelial barrier protection in lungs seen with SPHK1−/− BMPCs, we observed in the cell culture experiments that when the same number of wt and SPHK1−/− BMPCs were added to endothelial monolayer only the SPHK1−/− BMPCs failed to prevent increased endothelial permeability. Thus, although SPHK1-generated S1P is an important paracrine mediator responsible for BMPC-induced endothelial barrier protection, other factors in the whole lung such as the reduced uptake of SPHK1−/− BMPCs could also explain our results.
In conclusion, BMPC sensing of LPS and the consequent activation of SPHK1 and generation of S1P is a critical factor preventing increased vascular permeability. The S1P release function of BMPCs stabilizes endothelial junction barrier by a paracrine mechanism that activates Cdc42 and Rac1 signaling in endothelial cells. The present results provide a rationale for the therapeutic use of BMPCs in inflammatory diseases such as acute lung injury.
The authors acknowledge the outstanding technical assistance of Qiyan Zhou and Nanjia Yu.
Sources of Funding
The study was supported by research grants P01 HL090152 and P01 HL077806 and the Illinois Stem Cell Research Program. H.O. was supported, in part, by the Banyu Fellowship Program of Banyu Life Science Foundation International.
Original received January 27, 2009; resubmission received April 24, 2009; revised resubmission received August 5, 2009; accepted August 7, 2009.
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