key: cord-1033554-m5plpcda
authors: Kikuchi-Taura, Akie; Okinaka, Yuka; Saino, Orie; Takeuchi, Yukiko; Ogawa, Yuko; Kimura, Takafumi; Gul, Sheraz; Claussen, Carsten; Boltze, Johannes; Taguchi, Akihiko
title: Gap Junction-Mediated Cell-Cell Interaction Between Transplanted Mesenchymal Stem Cells and Vascular Endothelium in Stroke
date: 2021-03-11
journal: Stem Cells
DOI: 10.1002/stem.3360
sha: 2bf5a142d5b396c69ad2a59ed6584d933f463e61
doc_id: 1033554
cord_uid: m5plpcda

We have shown previously that transplanted bone marrow mononuclear cells (BM-MNC), which are a cell fraction rich in hematopoietic stem cells, can activate cerebral endothelial cells via gap junction-mediated cell-cell interaction. In the present study, we investigated such cell-cell interaction between mesenchymal stem cells (MSC) and cerebral endothelial cells. In contrast to BM-MNC, for MSC we observed suppression of vascular endothelial growth factor uptake into endothelial cells and transfer of glucose from endothelial cells to MSC in vitro. The transfer of such a small molecule from MSC to vascular endothelium was subsequently confirmed in vivo and was followed by suppressed activation of macrophage/microglia in stroke mice. The suppressive effect was absent by blockade of gap junction at MSC. Furthermore, gap junction-mediated cell-cell interaction was observed between circulating white blood cells and MSC. Our findings indicate that gap junction-mediated cell-cell interaction is one of the major pathways for MSC-mediated suppression of inflammation in the brain following stroke and provides a novel strategy to maintain the blood-brain barrier in injured brain. Furthermore, our current results have the potential to provide a novel insight for other ongoing clinical trials that make use of MSC transplantation aiming to suppress excess inflammation, as well as other diseases such as COVID-19 (coronavirus disease 2019).

Mesenchymal stem cells (MSC), bone marrow mononuclear cells (BM-MNC), and neural stem cells are the major cell sources in cell-based therapies for stroke. 1 However, the therapeutic mechanism of each therapy has been difficult to elucidate and this is likely to be a consequence of their complex composition. Therefore, significant research is under way to generate the next generation of cell-based therapies with improved characteristics. Recently, we demonstrated that direct cell-cell interaction between transplanted BM-MNC and cerebral endothelial cells via gap junction following cell transplantation is the prominent pathway for activation of regenerative processes after ischemia. 2 Our findings revealed that BM-MNC activate injured endothelial cells by providing glucose via gap junction and accelerate vascular endothelial growth factor (VEGF) uptake into endothelial cells followed by activation of angiogenesis at post stroke brain. 2 Similar to BM-MNC transplantation in stroke, a number of clinical trials of MSC transplantation in stroke are ongoing 3 and multiple therapeutic mechanisms have been proposed, including the acceleration of angiogenesis, 4 secretion of multiple cytokines, and immunomodulation. 5 However, the significance of the proposed mechanisms has largely been unclear. In this article, we report that MSC receive glucose from endothelial cells via gap junction and suppress VEGF uptake into endothelial cells followed by stabilization of the bloodbrain barrier in ischemic brain. This finding is essentially the converse of what was expected with regard to cell-cell interaction between BM-MNC and endothelial cell.

The present study was approved by the Animal Care and Use Committee 

Murine MSC obtained from C57BL/6 mice were purchased from Cyagen Biosciences (California). MSC were cultured with growth medium (OriCell Mouse MSC Growth Medium; Cyagen Biosciences) according to the manufacturer's protocol. After thawing the freezing ampule, cold growth medium was added, and the cell suspension was centrifuged and the supernatant removed. The resuspended cells in growth medium were seeded into a flask and incubated at 37 C and 5% CO 2 . After reaching 80% to 90% confluence, cells were dissociated with 0.25% Trypsin-EDTA (Thermo Fisher Scientific, Massachusetts) and expanded. The growth medium was changed every 3 days. Cells in passage 9 were used for in vitro and in vivo experiments.

Human umbilical vein endothelial cells (HUVEC, Kurabo, Osaka, Japan) were cultured with medium, serum and growth factors (HuMedia-EB2, Kurabo) according to manufacturer's protocol. HUVEC in passage 6 were used for all experiments. VEGF uptake was evaluated the methods as described elsewhere. 2 Biotin-conjugated human vascular endothelial growth factor (hVEGF, R&D Systems, Minneapolis, Minnesota) was incubated with streptavidin-conjugated APC (Thermo Fisher Scientific), at a 4:1 M ratio for 10 minutes at room temperature. 

The glucose concentrations in MSC and HUVEC were measured using a glucose assay kit (Biovision, California) according to manufacturer's protocol. Briefly, 2 × 10 5 MSC or 2 × 10 5 HUVEC were incubated with HuMedia-EB2 (0.1% glucose) for 1 hour and washed twice with PBS before cell lysis. The glucose and protein concentration in cell lysates was evaluated by glucose assay kit. 

The authors have demonstrated that gap junction-mediated direct cell-cell interaction between transplanted mesenchymal stem cells (MSC) and cells of a recipient, including endothelial cells and circulating lymphocyte/monocytes, is one of the prominent pathways that suppress excessive inflammation following MSC transplantation. The results of this study have the potential to provide novel insights in clinical trials that make use of MSC transplantation aiming to suppress excess inflammation. This is also relevant for other diseases, were characterized using FACS as described previously. 6 

A murine stroke model with excellent reproducibility that made use of 7-week-old male CB-17 mice (C.B-17/Icr− +/+Jcl: Oriental yeast, Tokyo, Japan) was utilized as described previously. 7 Briefly, permanent focal cerebral ischemia was induced by permanent ligation and disconnection of the distal portion of the left middle cerebral artery (MCA) using bipolar forceps under 3% halothane inhalation anesthesia. During surgery, rectal temperature was monitored and controlled at 37.0 ± 0.2 C by a feedback-regulated heating pad. Cerebral blood flow (CBF) in the MCA area was also monitored. Mice showing a ≥75% decrease in CBF immediately after MCA occlusion were used for in vivo experiments (success rate was 100%). The weight of animals was~20-25 g before surgery. Twenty-four hours after induction of stroke, 5 × 10 5 MSC or heparinized HBSS were injected via a tail vein.

Mice were anesthetized using sodium pentobarbital and perfused transcardially with saline followed by 4% paraformaldehyde (PFA).

The brain was carefully removed and cut into coronal sections (20 μm) 

VEGF is one of the most prominent pro-angiogenic factors. 2 Cerebral endothelial cells are known to uptake VEGF followed by activation of angiogenesis with increased permeability of the blood-brain barrier. 8 We previously showed that BM-MNC increase VEGF uptake into HUVEC via gap junction-mediated cell-cell interaction. 2 To investigate the analogous property of MSC, these were cocultured with HUVEC and any changes in VEGF uptake into HUVEC were assessed. Surprisingly, a significant reduction of VEGF uptake into HUVEC was observed when cocultured with MSC ( Figure 1A) . To evaluate the importance of gap junction channel of MSC, its 1-octanol or car- 

We previously showed that the glucose concentration in BM-MNC is significantly higher than in HUVEC and that the transfer of glucose 

MSC are known to suppress inflammatory reactions following stroke, including macrophage/microglia activation in the affected brain. 5 However, the mechanism of inflammatory suppression by MSC transplantation in ischemic brain has been disputed. 

Circulating WBC are also known to express gap junction. 10 To investigate direct cell-cell interaction between transplanted MSC and circulating WBC, transfer of small molecules from WBC to MSC was evaluated in vitro. As shown in Figure 4A , transfer of BCECF from WBC to MSC was observed and the transfer was inhibited by the blockade of gap junction. Analysis of WBC with or without coculture with MSC revealed the level of BCECF was significantly decreased in lymphocyte and monocyte by coculture with MSC ( Figure 4B ). These findings indicated the transplanted MSC affect circulating WBS along with injured endothelial cells in brain and major WBC that react with MSC are lymphocyte and monocyte.

We have demonstrated that MSC decrease VEGF uptake into endothelial cells in vitro and suppress inflammation in vivo through gap junction-mediated cell-cell interaction. Our findings indicate that gap junction-mediated signaling is one of the major pathways for MSCmediated suppression of inflammation in the brain following stroke.

A gap junction channel between two cells is composed of two connexin in each half of the cell pair and they allow the prompt movement of small molecules according to their concentration gradient between cells. 11 In our previous study, we had shown BM-MNC increases VEGF uptake into endothelial cells via gap junction-medi- F I G U R E 5 Schematic representation of our conclusions. A, We have previously demonstrated that BM-MNC, which have a higher glucose concentration than endothelial cells, supply glucose to endothelia cell via gap junction followed by increased uptake of VEGF into endothelial cell with activation of angiogenesis. B, In marked contrast, MSC have lower glucose concentration than endothelial cells. The reverse glucose flow suppresses VEGF uptake into endothelial cell followed by reducing the blood-brain barrier permeability. C, In addition to the interaction between MSC and endothelial cells, direct cell-cell interaction via gap junction was observed between MSC and circulating lymphocytes/monocytes the proposed therapeutic mechanisms of MSC transplantation for stroke had been acceleration of angiogenesis, 4 we expected a similar therapeutic mechanism in MSC with BM-MNC. However, the transfer of glucose from endothelial cells to MSC was observed in this study with decreased VEGF uptake into endothelial cells by gap junctionmediated cell-cell interaction. These findings indicate that the therapeutic mechanisms of MSC and BM-MNC would be significantly different ( Figure 5A,B) .

Uptake of VEGF is one of the key signals for endothelial cells to activate angiogenesis and increase permeability of barrier function following increased inflammation in ischemic brain. [12] [13] [14] In contrast, suppression of VEGF uptake attenuates blood-brain barrier disruption. 15 We have demonstrated that MSC suppressed VEGF uptake of endothelial cells in vitro and intravenous injection of MSC reduced inflammatory responses at peri-stroke area. Furthermore, inhibition of gap junction channel of MSC abolishes the effect of MSC in vitro and in vivo. These results indicate that transplanted MSC suppressed inflammatory response in brain by inhibiting VEGF uptake into cerebral endothelial cells via gap junction-mediated cell-cell interaction.

Pericytes are known to be one of the MSC 16 that are important for the stabilization of the blood-brain barrier. 17 Pericytes and cerebral endothelial cells are connected via gap junction and their dissociation after ischemia had been shown to increase permeability followed by activation of macrophages. 17 These findings suggest that transplanted MSC can substitute the function of dissociated pericytes.

We have demonstrated that small molecules can be transferred from circulating WBC to MSC via gap junction ( Figure 5C ). Our results also indicate that the major cell populations that react with MSC are lymphocytes and monocytes, but not granulocytes. Lymphocyte causes graft-vs-host disease (GvHD) after allogenic hematopoietic stem cell transplantation 18 and MSC transplantation is known to have a therapeutic effect in GvHD, although the mechanism is not fully understood. 19 Activation of lymphocytes are known to be related to increased glucose level in lymphocytes 20 and glucose is one of the major factors that are transferred via gap junction between cells. 2 Our current results relating to the small molecule outflow from lymphocytes to MSC via gap junction provides a novel insight of MSC therapy for GvHD. MSC transplantation is also known to have a therapeutic potential for COVID-19 (coronavirus disease 2019), although this mechanism is not fully understood either. 21 Fatal vascular deterioration caused by cytokine release syndrome has been shown to be critical COVID-19 patients 22 and the major players of cytokine release syndrome caused by COVID-19 are monocytes, lymphocytes and endothelial cells. 23 Our current data indicate that MSC have the potential to directly regulate monocyte, lymphocyte and endothelial cells via gap junction-mediated cell-cell interaction.

Although further studies are required to reveal the full linkage between MSC, monocytes, lymphocytes and endothelial cells via gap junction-mediated cell-cell interaction, extending our hypothesis to COVID-19, although speculative at present has the potential to provide a novel insight for new therapeutic strategy against the COVID-19 cytokine storm.

Our findings establish that gap junction-mediated direct cell-cell interaction between transplanted MSC and cells of recipient, including endothelial cells and circulating lymphocyte/monocyte is highly significant. Furthermore, our current results have potential to provide a novel insight to other clinical trials that make use of MSC transplantation aiming to suppress excess inflammation, which are ongoing for various diseases including COVID-19. 24 

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Akie Kikuchi-Taura https://orcid.org/0000-0001-7004-3071

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