Human mesenchymal stem cells inhibit vascular permeability by modulating vascular endothelial cadherin/β-catenin signaling

Abstract

The barrier formed by endothelial cells (ECs) plays an important role in tissue homeostasis by restricting passage of circulating molecules and inflammatory cells. Disruption of the endothelial barrier in pathologic conditions often leads to uncontrolled inflammation and tissue damage. An important component of this barrier is adherens junctions, which restrict paracellular permeability. The transmembrane protein vascular endothelial (VE)-cadherin and its cytoplasmic binding partner β-catenin are major components of functional adherens junctions. We show that mesenchymal stem cells (MSCs) significantly reduce endothelial permeability in cocultured human umbilical vascular endothelial cells (HUVECs) and following exposure to vascular endothelial growth factor, a potent barrier permeability-enhancing agent. When grown in cocultures with HUVECs, MSCs increased VE-cadherin levels and enhanced recruitment of both VE-cadherin and β-catenin to the plasma membrane. Enhanced membrane localization of β-catenin was associated with a decrease in β-catenin-driven gene transcription. Disruption of the VE-cadherin/β-catenin interaction by overexpressing a truncated VE-cadherin lacking the β-catenin interacting domain blocked the permeability-stabilizing effect of MSCs. Interestingly, a conditioned medium from HUVEC-MSC cocultures, but not from HUVEC or MSC cells cultured alone, significantly reduced endothelial permeability. In addition, intravenous administration of MSCs to brain-injured rodents reduced injury-induced enhanced blood-brain barrier permeability. Similar to the effect on in vitro cultures, this stabilizing effect on blood-brain barrier function was associated with increased expression of VE-cadherin. Taken together, these results identify a putative mechanism by which MSCs can modulate vascular EC permeability. Further, our results suggest that the mediator(s) of these vascular protective effects is a secreted factor(s) released as a result of direct MSC-EC interaction.

FIG. 1.

Mesenchymal stem cells (MSCs) reduce paracellular permeability of endothelial cell (EC) monolayers in vitro. (A) Flow cytometry staining of isotype control (red), MSCs (blue), or human umbilical vascular endothelial cells (HUVECs) (green) for CD44, the marker used for negative selection to obtain a pure HUVEC population for in vitro study. (B) Representative flow cytometry data for CD44 staining of HUVEC-MSC cocultures before (left panel) and after (right panel) magnetic cell sorting (MACS) separation using negative selection for CD44⁺ cells. CD44–flouriscein isothiocyanate (FITC) signal is shown on the Y-axis, and forward scatter (FSC) is shown on the X-axis. (C) Schematic of experiments used to determine the effects of MSCs on EC monolayer permeability. (D) Permeability to 40-kDa dextran-FITC is reduced in HUVECs cocultured with MSCs. HUVECs were cultured with or without MSCs and separated as described in C. CD44⁻ HUVECs were subsequently seeded in transwell chambers (0.4-μm pore size) and allowed to adhere for 8 h. Permeability of the HUVEC monolayer was assessed by diffusion of 40-kDa dextran-FITC into the bottom chamber. Stimulation with vascular endothelial growth factor (VEGF)-A (10 ng/mL; 60 min) increased permeability in control HUVEC cultures, but was significantly reduced in HUVECs cocultured with MSCs. Data shown are the average ± SEM of 3 independent experiments.

FIG. 2.

MSCs enhance vascular endothelial (VE)-cadherin/β-catenin interaction at the cell membrane. After coculture with MSCs or culture alone, HUVECs were separated using CD44-based MACS and replated for experiments displayed in A–C. Representative 100 × fields from images captured under identical conditions are shown. Immunoflourescence staining of β-catenin (A) and VE-cadherin (B) in HUVECs after coculture (+MSCs) or alone (−MSCs). Coculture with MSCs resulted in clustering of HUVECs and increased staining of β-catenin and VE-cadherin at the membrane as compared to HUVECs alone. (C) Immunofluorescence staining of activated (de-phosphorylated) β-catenin in HUVECs after culture alone or together with MSCs. (D) Immunoprecipitation of VE-cadherin from lysates of HUVECs cultured alone (−) or together with MSCs (+) and then magnetically sorted, demonstrating that for a fixed amount of HUVEC lysate (25 μg) increased expression of VE-cadherin is observed along with increased β-catenin binding.

FIG. 3.

Coculture with MSCs inhibits β-catenin-dependent transcriptional activity in ECs. (A) Firefly luciferase activity normalized to Renilla–luciferase activity (relative luciferase units) from HUVECs cotransfected with a T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter construct (TOPFLASH), a constitutively active Renilla–luciferase construct, and wild-type β-catenin, and then cultured alone or with MSCs overnight. Data from 3 independent experiments are shown as mean (B) luciferase activity, normalized to Renilla, from HUVECs cotransfected with a mutant TCF/LEF reporter construct lacking β-catenin binding sites (FOPFLASH), a constitutively active Renilla–luciferase construct, and wild-type β-catenin and then cultured alone or with MSCs overnight. Group data from 3 independent experiments are shown as mean ± SEM. (C) Western blots of HUVECs that were cultured with (H + M) or without MSCs (H), MACS separated, lysed, and probed with antibodies directed against the indicated antigens. (D) Luciferase activity, normalized to Renilla, from HUVECs cotransfected with a TCF/LEF reporter construct (TOPFLASH), a constitutively active Renilla–luciferase construct, and wild-type β-catenin, and then cultured alone or with MSCs overnight followed by culture in the absence or presence of lithium chloride (LiCl) (10 mM) for 6 h.

FIG. 4.

Effects of MSCs on HUVEC paracellular permeability requires VE-cadherin/β-catenin interaction. Transfection of HUVECs with a truncated expression construct for a mutant VE-cadherin (truncated VE-cadherin [TVCad]) results in reduced VE-cadherin membrane staining (A, lower panel) and reduced β-catenin membrane staining (B, lower panel). Control, vector-only transfected HUVECs (pECE-HUVECs) exhibit robust membrane expression of VE-Cadherin (A, upper panel) and β-catenin (B, upper panel) upon replating after coculture with MSCs and magnetic separation, similar to findings seen in Fig. 2 using untransfected HUVECs (Fig. 2A, B). Images are representative of at least 10 independent high-powered fields from 2 independent experiments. Images shown here were acquired under identical conditions as those shown in Fig. 2. (C) Coculture with MSCs has no effect on permeability of HUVECs overexpressing TVCad compared with permeability of unmodified HUVECs or HUVECs transfected with TVCad and cultured alone.

FIG. 5.

Conditioned medium from MSC + HUVEC coculture recapitulates the effects of MSCs on EC permeability. (A) Experimental schematic of transwell cocultures with MSCs ± HUVECs to determine if a secreted factor(s) from MSCs cultured alone or cultured with HUVECs is able to reproduce the effects on EC permeability seen when HUVECs were directly cocultured with MSCs. (B) Permeability of HUVEC monolayers to 40-kDa FITC-Dextran when cultured in the conditioned medium from HUVEC alone (2 × 10⁵ cells) MSC alone (2 × 10⁵ cells), or HUVECs cocultured with increasing numbers of MSCs (total number of cells in coculture was held constant at 2 × 10⁵). Results represent data from 3 independent experiments. (C) Western blot probed with antibodies directed against de-phosphorylated β-catenin, VE-cadherin, or glyceraldehyde 3-phophate dehydrogenase (GAPDH) (loading control) of EC lysates after overnight treatment with the conditioned medium from HUVECs (EC), MSCs (MSC), or HUVEC/MSC cocultures (MSC + EC, 1:1 cell ratio).

FIG. 6.

MSCs inhibit blood–brain barrier (BBB) permeability in mice with traumatic brain injury (TBI). (A) Schematic depiction of in vivo experiments to determine the effects of MSCs on blood–brain barrier permeability after TBI (B). Representative images of the site of cortical impact injury from sham injured mice, injured mice treated with vehicle, MSC-treated injured mice, or mice treated with control (HUVEC) cells. Blue represents Evans Blue dye extravasation at the site of injury. (C) Quantification of absorbance at 610 nm/g of tissue (proportional to the amount of Evans Blue extravasation) from ipsilateral cerebral hemisphere tissue of mice from the indicated treatment groups. At least 6 mice were studied in each group. (D) Brain sections from MSC-treated mice (3 days post-TBI) reveal MSCs in the brain. Immunofluorescence imaging demonstrates the presence of Q-dot-labeled MSCs (indicated by arrows) in the brain. The MSCs present in the brain were typically located in the perivascular niche adjacent to blood vessel lumens (red = MSC, blue = DAPI nuclear staining).

FIG. 7.

Enhanced expression of junctional proteins in the penumbra after TBI in MSC-treated mice. Immunoflourescent staining of brain sections from the penumbra region of MSC-treated mice with antibodies reveals preservation of VE-cadherin immunoreactivity (A), preservation of occludin immunoreactivity (B), and increased claudin-5 immunoreactivity (C) as compared to vehicle-treated injured mice (middle panels) or shams (left panels); 100 × images that are representative of multiple fields examined from each experimental group are shown. (D) Western Blot analysis of ipsilateral hemispheric brain homogenates from these mice revealed that MSCs modestly increased occludin and VE-cadherin immunoreactivity after injury. Densitometric (normalized to glyceraldehyde 3-phophate dehydrogenase) quantification of the increased immunoreactivity of VE-cadherin (E) and occludin (F) in brain homogenates from injured, MSC-treated mice as compared with vehicle-control-treated injured mice or sham injured mice (n = 3 animals/group).