- Open Access
Human decidua basalis mesenchymal stem/stromal cells protect endothelial cell functions from oxidative stress induced by hydrogen peroxide and monocytes
© The Author(s). 2018
- Received: 28 May 2018
- Accepted: 28 September 2018
- Published: 25 October 2018
Human decidua basalis mesenchymal stem/multipotent stromal cells (DBMSCs) inhibit endothelial cell activation by inflammation induced by monocytes. This property makes them a promising candidate for cell-based therapy to treat inflammatory diseases, such as atherosclerosis. This study was performed to examine the ability of DBMSCs to protect endothelial cell functions from the damaging effects resulting from exposure to oxidatively stress environment induced by H2O2 and monocytes.
DBMSCs were co-cultured with endothelial cells isolated from human umbilical cord veins in the presence of H2O2 and monocytes, and various functions of endothelial cell were then determined. The effect of DBMSCs on monocyte adhesion to endothelial cells in the presence of H2O2 was also examined. In addition, the effect of DBMSCs on HUVEC gene expression under the influence of H2O2 was also determined.
DBMSCs reversed the effect of H2O2 on endothelial cell functions. In addition, DBMSCs reduced monocyte adhesion to endothelial cells and also reduced the stimulatory effect of monocytes on endothelial cell proliferation in the presence of H2O2. Moreover, DBMSCs modified the expression of many genes mediating important endothelial cell functions. Finally, DBMSCs increased the activities of glutathione and thioredoxin reductases in H2O2-treated endothelial cells.
We conclude that DBMSCs have potential for therapeutic application in inflammatory diseases, such as atherosclerosis by protecting endothelial cells from oxidative stress damage. However, more studies are needed to elucidate this further.
- Decidua basalis mesenchymal stem cells
- Endothelial cells
Mesenchymal stem cells (MSCs) are adult multipotent stromal cells that can be isolated from many tissues, such as human placenta . Recently, we isolated MSCs from the maternal decidua basalis tissue (DBMSCs) of human term placenta . The tissue of decidua basalis is a main source of oxidative stress molecules, which are found in the maternal circulation due to pregnancy . Therefore, DBMSCs in their niche (vascular microenvironment) are in direct contact with the maternal circulation, and therefore, they are exposed to high levels of inflammation and oxidative stress mediators . In addition, we also isolated MSCs from the fetal tissue (chorionic villi) of the placenta . These fetal chorionic MSCs are in direct contact with the fetal circulation and therefore exposed to lower levels of inflammation and oxidative stress molecules as compared to DBMSCs [5–7].
MSCs from placenta and other sources can differentiate into multiple cell lineages including adipocyte, osteoblast, and chondrocyte . In addition, MSCs show low immunogenicity and anti-inflammatory properties . Therefore, MSCs have been investigated as promising therapeutic agents in many inflammatory diseases, such as atherosclerosis .
Atherosclerosis is characterized by endothelial activation due to the accumulation of high amounts of low-density lipoprotein (LDL) and immune cells that lead to the production of high levels of oxidative stress mediators, such as hydrogen peroxide (H2O2) [9, 10].
H2O2 has several important effects on endothelial cell functions in physiological homeostasis and in inflammatory diseases [9, 10]. H2O2 alters the functional activities of proteins that cause the generation of more toxic radicals (i.e., peroxynitrite (ONOO−) and hydroxyl (·OH)), which induce oxidative damage in the cellular DNA and proteins [9, 10]. In addition, H2O2 can rapidly inactivate nitric oxide (NO) and this causes endothelial cell damage [9, 10].
Endothelial cell damage is usually associated with phenotypic changes (i.e., increased expression of inflammatory molecules), dysfunctional activities [i.e., increased endothelial cell proliferation, adhesion, migration, permeability, angiogenesis (blood vessel formational)], and also enhanced endothelial cell interaction with immune cells (i.e., enhanced monocyte adhesion to the endothelium and their infiltration into the tissues); these events are the typical characteristics of atherosclerosis . In atherosclerosis, an inflammatory response is initiated at the injury site of endothelium that increases the expression of adhesion molecules (i.e., VCAM-1), which activates the recruitment and adhesion of immune cells (i.e., monocytes) to the injured site of endothelium . This interaction between monocytes and endothelial cells will loosen up the tight junction between endothelial cells that increases the permeability of endothelium and subsequently monocytes and LDL will pass through the intima, where LDL undergoes oxidation while monocytes differentiate into macrophages, which take up oxidized LDL . This lipid laden macrophages are known as “foam cells”, which eventually die by apoptosis, but the lipid content will accumulate in the intimal area leading to the formation of plaque .
Recently, we reported that DBMSCs can protect endothelial cells from activation by inflammation triggered by monocyte adhesion and increased endothelial cell proliferation . These events are manifest in inflammatory diseases, such as atherosclerosis. These data make DBMSCs as a useful candidate to be employed in a therapeutic strategy for treating atherosclerosis. We performed this study to examine the ability of DBMSCs to protect endothelial cell functions from the damaging effects resulting from exposure to oxidatively stress environment induced by H2O2 and monocytes. We investigated the ability of DBMSCs to protect endothelial cell functions (adhesion, proliferation, and migration) from oxidative stress induced by H2O2. The effect of DBMSCs on the adhesion of monocytes to endothelial cells in oxidative stress environment was also examined. Finally, we investigated the effect of DBMSCs on endothelial cell expression of many genes under oxidative stress, and the mechanism underlying DBMSC protection of endothelial cells from oxidative stress was also determined. Our data suggest that DBMSCs have a protective effect on endothelial cells in oxidative stress environment and suggest that DBMSCs have the potential to treat inflammatory diseases, such as atherosclerosis by protecting endothelial cells from injury induced by oxidative stress and inflammatory cells. However, future studies are necessary to elucidate this further in vitro and in vivo.
Ethics and collection of human placentae and umbilical cords
The study was approved by the institutional review board (reference number IRBC/246/13) of KAIMRC (King Abdulla International Medical Research Centre, Saudi Arabia). Samples (placentae and umbilical cords of uncomplicated human pregnancies, 38–40 gestational weeks) were obtained and used immediately after signing consent forms. All clinical and experimental procedures were performed in compliance with KAIMRC research guidelines and regulations.
Isolation and culture of DBMSCs
Monoclonal antibodies used in this study
Endothelial Cell Marker
Isolation and culture of human umbilical vein endothelial cells (HUVEC)
HUVEC were isolated from umbilical cord veins using our previously published method . Following rinsing the cannulated umbilical veins with PBS for several times, veins were filled with a digestion PBS solution containing 6 mg/ml collagenase type II (catalogue number 17101-015, Life Technologies) and then incubated at 37 °C in a cell culture incubator. After 25 min, HUVEC were collected and then resuspended in a complete endothelial cell growth medium (catalogue number PCS-100-041™, ATCC, USA) and cultured at 37 °C in a cell culture incubator as previously described . Prior to using HUVEC in subsequent experiments, they were characterized by flow cytometry using a CD31 endothelial cell marker (R and D Systems, Abingdon, UK) as previously described . HUVEC (> 95% purity) from passages 3 to 5 of 30 umbilical cords were used in this study.
HUVEC proliferation in response to DBMSCs and H2O2
HUVEC (5 × 103) were seeded in wells of 96-well culture plates containing a complete endothelial cell growth medium and cultured at 37 °C in a cell culture incubator. Following 24 h, adherent HUVEC were incubated with different concentrations [1%, 5% and 25% (v/v) conditioned medium (CM) harvested from DBMSC culture (CMDBMSC) diluted in a complete DBMSC growth medium] of CMDBMSC and different ratios of 1:1, 5:1, and 10:1 HUVEC to DBMSC. Cells were then cultured in a complete endothelial cell growth medium with or without 100 μM H2O2 for 72 h at 37 °C in a cell culture incubator.
HUVEC proliferation was then evaluated after each indicated culture time points (24, 48, and 72 h) by a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] kit (catalogue number G5421, CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Promega, Germany), as previously described . CMDBMSC was produced as previously described . Before adding DBMSCs to HUVEC culture, DBMSCs were treated with 25 μg/ml Mitomycin C to inhibit their proliferation as previously described . The blank was cells incubated in MTS solution in a complete endothelial cell growth medium alone. Results were presented as means (± standard errors). Each experiment was performed in triplicate and repeated with five independent HUVEC (passages 3–5) and DBMSC (passage 3) preparations.
Culture of HUVEC with different treatments of DBMSCs (conditioned medium, supernatant, and intercellular direct contact) and H2O2
HUVEC adhesion and proliferation using xCELLigence system
The xCELLigence system (RTCA-DP version; Roche Diagnostics, Mannheim, Germany) was used as we previously described [12, 13] to evaluate the adhesion and proliferation of HUVEC. The xCELLigence system is a real-time cell analyzer that constantly monitors and records the changes in electrical impedance, because of cellular events, and these changes are reported as an arbitrary cell index [12, 13]. Briefly, 100-μL complete endothelial cell growth medium was added to well in 16-well culture plates (catalogue number 05469813001, E-Plate 16, Roche Diagnostics), and the background impedance was then achieved as previously described [12, 13]. Then, 20 × 104 HUVEC (HUVEC were initially co-cultured with DBMSCs and 100 μM H2O2 or cultured alone as described above) were seeded in 100 μL of complete endothelial cell growth medium in quadruplicate wells, and equilibrium was achieved by leaving the culture plates for 30 min at RT before data recording. To record data, culture plates were placed in the xCELLigence system at 37 °C in a cell culture incubator. HUVEC cell index was then automatically monitored for 72 h. For data analysis, the xCELLigence software (version 1.2.1) was used. For cell adhesion, data was measured after 2 h and the value of cell index was then expressed as mean ± standard errors of the cell index. For cell proliferation, data was expressed as mean ± standard errors of the cell index normalized to the cell index recorded after 2 h (adhesion time point). The rate of cell proliferation was determined by calculating the normalized cell index at 24, 48, and 72 h. Each experiment was performed and repeated as described above.
HUVEC migration using xCELLigence system
The migration of HUVEC was evaluated using CIM migration plates (catalogue number 05665825001, Roche Diagnostics) in the xCELLigence system as previously described by us [12, 13]. The CIM plates have 16-migration wells that each consists of two chambers (upper and lower) separated by a membrane (polyethylene terephthalate) with a porous of 8 μm in size. The membrane is in contact with microelectrodes. Following the addition of 50-μl pre-warmed media to the wells of the upper chamber and 160-μl endothelial cell growth medium containing 30% FBS to the lower chamber, the plates were then locked in the RTCA DP device at 37 °C in a cell culture incubator for 1 h to obtain equilibrium, and a measurement step was then performed as previously described [12, 13]. The migration experiments were then initiated by seeding 20 × 103 HUVEC [HUVEC were initially co-cultured with DBMSCs and 100 μM H2O2 or cultured with DBMSCs (CMDBMSC, SFDBMSC and ICDBMSC) or cultured alone as described above] in the upper chamber containing 100-μL endothelial cell serum free medium and the plates were then incubated for 30 min at RT to allow the cells to settle onto the membrane as previously described [12, 13]. Experiments were performed in quadruplicate, and after equilibration, the impedance value of each well was automatically monitored every 15 min for 24 h by the xCELLigence system and then expressed as a cell index value. HUVEC migration observed in the presence and absence of 30% FBS served as positive and negative controls, respectively. Each experiment was performed and repeated as described above.
HUVEC proliferation in response to monocytes pretreated with DBMSCs and H2O2
Next, endothelial cell proliferation in response to monocytes pre-cultured with DBMSCs and H2O2 at the indicated ratios (below) in the presence of 100 μM H2O2 was examined (Fig. 2). After 24-h culture with DBMSCs in the presence of 100 μM H2O2, THP-1 [THP-1 alone, THP-1+ H2O2 (THP-1 pretreated with H2O2), and THP-1/DBMSC+ H2O2 (THP-1 pretreated with DBMSCs and H2O2)] were harvested and then added to HUVEC at different THP-1:HUVEC ratios (2.5:1, 5:1, 10:1, and 20:1 THP-1:HUVEC) in the presence of 100 μM H2O2. Briefly, THP-1 were added to HUVEC that were initially seeded at a density of 5 × 103 per well in 96-well tissue culture plates. After 24-h culture in a complete HUVEC culture medium at 37 °C in a cell culture incubator, HUVEC proliferation was examined using the MTS assay as previously described . Before using DBMSCs and THP-1 in the proliferation assays, cells were treated with 25 μg/ml Mitomycin C to inhibit their proliferation as previously described . Results were presented as means (± standard errors). Each experiment was performed in triplicate and repeated for five times with five independent preparations of DBMSCs and HUVEC. DBMSCs and THP-1 cultured alone were included as negative controls.
Adhesion of monocyte to HUVEC
DBMSC effect on THP-1 adhesion to HUVECs was examined using our previously published method . Briefly, H2O2-untreated THP-1 or pretreated with 100 μM H2O2 (TTHP-1) for 24 h were cocultured with H2O2-untreated DBMSCs (TTHP-1/UDBMSC) or with H2O2-treated DBMSCs (TTHP-1/TDBMSC) for 24 h at 5:1 THP-1:DBMSC ratio in a physical contact experiment by adding THP-1 to DBMSCs that were initially cultured on a plastic surface of 6-well culture plate for 24 h to allow cells to be fully adhered (Fig. 2). After 24-h incubation in a complete RPMI-1640 culture medium (above), THP-1 were harvested and then labelled with 5 μM green fluorescent cell tracker stain (5-chloromethylfluorescin diacetate; CMFDA; Molecular Probes, Life Technologies) for 4 h as previously described . Following washing THP-1 with fresh RPMI-1640 culture medium, they were added to a monolayer layer of HUVEC at a ratio of 5THP-1:1HUVEC (HUVEC were initially cultured alone or with 100 μM H2O2 for 24 h). After incubation for 30 min, non-adherent THP-1 were gently removed by washing with PBS, and the fluorescence intensity of the THP-1 that had adhered to the monolayer of HUVEC was then measured at excitation 485 nm and emission 528 nm using a fluorescence microplate reader (Glomax Multi Detection System, Promega, Germany). Results were expressed as relative fluorescence intensity (RFI). Different ratios of HUVEC to THP-1 were evaluated. Experiments were performed in triplicate using HUVEC prepared from independent umbilical cord tissue and repeated three times.
Measurement of glutathione reductase activity
HUVEC (HUVEC were initially co-cultured with DBMSCs and 100 μM H2O2 or cultured alone as described above) were washed twice with cold PBS, and they were then lysed as previously described [12, 13]. Total protein in the supernatant was then determined by Bradford method [12, 13].
The activity of glutathione reductase was measured using OxiSelect™ Glutathione Reductase Assay Kit (catalogue number STA-812, Cell Biolabs, San Diego, USA) as previously described by us . This assay is based on the reduction of glutathione disulfide (oxidized glutathione) (GSSG) to reduced glutathione (GSH) by glutathione reductase, using NADPH as a donor for H. Subsequently, the chromogen reacts with the thiol group of GSH to produce a colored compound that absorbs at 405 nm. The glutathione reductase content in HUVEC samples is determined by comparison with the predetermined glutathione reductase standard curve. The assay was performed using 100-μl aliquots of HUVEC supernatant protein immediately after preparation (30 μg protein) added to phosphate buffer containing excess GSSG and NADPH. The level of change was determined at 405 nm using a standard curve performed. Three experiments were performed in triplicate using HUVEC and DBMSCs as indicated above.
Measurement of thioredoxin reductase activity
Total protein was extracted from HUVEC (prepared as described above), and thioredoxin reductase (TrxR) activity (catalogue number 10007892, Cayman, Michigan, USA) was then evaluated as per the manufacturer’s instructions. This assay is based on the reduction of 5,5′-dithiobis (2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid, which generates a strong yellow color that can be measured at 412 nm. In the crude biological sample, glutathione reductase and glutathione peroxidase can also be reduced by DTNB. Therefore, TrxR specific inhibitor is used to determine the specific activity of TrxR. Therefore, the total DTNB reduction by the sample is initially estimated and the DTNB reduction by the sample in the presence of the TrxR specific inhibitor will then be estimated. The difference between the two results is the DTNB reduction due to TrxR activity. Three experiments were performed in triplicate using HUVEC and DBMSCs as indicated above.
RNA isolation, cDNA synthesis, and real-time polymerase chain reaction (RT-PCR) analysis
The expression of 84 genes related to endothelial cell biology (catalogue number PAHS-015ZD-24, Qiagen, Hilden, Germany) by HUVEC was determined using QuantiTect Primer Assay (Qiagen, Hilden, Germany) in a real-time polymerase chain reaction (RT-PCR) as previously published . Briefly, total RNA from HUVEC pretreated with DBMSCs and 100 μM H2O2 for 48 h was isolated, and cDNA was then synthesized using FastLane Cell cDNA kit and RT Primer Mix (Qiagen) as previously published . After quantifying mRNA using QuantiTect SYBR Green PCR Kit (Qiagen), the real-time PCR reaction was performed in triplicate on the CFX96 real-time PCR detection system (BIO-RAD) as previously published . To analyze the data, the CFX manager software (Bio-Rad, CA, USA) was used. The results were exported to Microsoft Excel for further analysis. The results were expressed as fold change by calculating the ΔΔ−2 values. The relative expression of an internal house-keeping gene as a loading control was used as provided in the kit. Experiments were performed in triplicate using HUVEC prepared from independent umbilical cord tissue and repeated three times.
Cells were characterized by flow cytometry as previously described . Briefly, cells (1 × 105) were stained with monoclonal antibodies (Table 1) for 30 min. Cells were then washed twice by adding cold PBS and centrifuged at 150×g for 5 min at 8 °C. Unstained and isotype controls were used. Immunoreactivity to cell surface antibody markers or intracellular proteins was assayed by a BD FACS CANTO II (Becton Dickinson, NJ, USA) flow cytometer.
Data were analyzed using the t test (unpaired t test, two tailed). These analyses were performed using GraphPad Prism 5. Results were considered to be statistically significant if P < 0.05.
Isolation and characterization of DBMSCs
MSCs from decidua basalis of human term placenta were previously isolated and characterized by us . DBMSCs at passage 3 were positive (> 95%) for MSC markers and negative for hematopoietic markers and were able to differentiate into adipocytes, chondrocytes, and osteocytes as previously report . Subsequently, DBMSCs at passage 3 were used in all experiments.
DBMSCs and H2O2 modulated the proliferation of HUVEC
To evaluate the effects of DBMSCs on endothelial cell functions, the proliferation of HUVEC cultured with DBMSCs in the presence or absence of 100 μM H2O2 was examined using the MTS assay. The viability HUVEC exposed to 100 μM H2O2 was more than 90% at all culture time points (24, 48, and 72 h). This was consistent with our previous report . The exposure of HUVEC to concentrations higher than 100 μM H2O2 reduced their viability to less than 50%, as we previously reported . Consequently, 100 μM H2O2 was used in this study.
The effects of DBMSCs and H2O2 on HUVEC proliferation are reversible
DBMSCs and H2O2 modulated HUVEC adhesion
DBMSCs and H2O2 modulated HUVEC migration
DBMSCs reduced the stimulatory effect of monocytes and H2O2 on HUVEC proliferation
After 24 h, the proliferation of HUVEC cultured in H2O2 significantly increased after the addition of a high ratio of monocytes pretreated with H2O2 (THP-1 + H2O2) to HUVEC (1HUVEC:10THP-1 + H2O2), and this stimulatory effect of monocytes pretreated with H2O2 on HUVEC proliferation was significantly reduced (P < 0.05) by monocytes pretreated with DBMSCs and H2O2 (THP-1/DBMSC + H2O2), P < 0.05 (Fig. 7b). This stimulatory effect of monocytes pretreated with H2O2 (THP-1 + H2O2) on the proliferation of HUVEC cultured in H2O2 and the inhibitory effect of DBMSCs on monocytes pretreated with H2O2 (THP-1/DBMSC + H2O2) inducing the proliferation of HUVEC cultured in H2O2 are reversible and time dependent (data not shown). The effects of DBMSCs on the proliferative responses of HUVEC cultured with or without H2O2 and monocytes pretreated with or without H2O2 were not significantly changed, P > 0.05 (data not shown).
DBMSCs reduced the adhesion of H2O2-treated monocytes to HUVEC
DBMSCs increased the activities of glutathione and thioredoxin reductases in H2O2-treated HUVEC
Cells are protected from injury induced by oxidative stress by employing several antioxidant defense mechanisms . To address the possibility that DBMSCs can protect endothelial cells from injury induced by H2O2, we examined the effect of DBMSCs on the activities of glutathione and thioredoxin reductases (an antioxidant enzyme) in H2O2-treated endothelial cells.
Glutathione reductase activity in H2O2-treated HUVEC cultured with CMDMSC, SFDBMSC, and ICDBMSC is significantly higher than H2O2-treated HUVEC (P < 0.05). At baseline, the level of glutathione reductase in HUVEC was 77.5 mU/mL ± 4.87 mU/mL. Exposure of HUVEC to 100 μM H2O2 for 48 h, the level of glutathione reductase was 29.50 ± 2.55. The level of glutathione reductase levels in H2O2-treated endothelial cell cultured with CMDBMSC, SFDBMSC, and ICDBMSC were 56.90 mU/mL ± 3.11 mU/mL, 62.38 mU/mL ± 3.01 mU/mL, and 65.49 mU/mL ± 3.95 mU/mL, respectively. As compared to H2O2-treated HUVEC, there were an approximately 1.92-, 2.11-, and 2.22-fold increase in the levels of glutathione reductase in H2O2-treated HUVEC cultured with CMDMSC, SFDBMSC, and ICDBMSC, respectively, P < 0.05.
Thioredoxin reductase activity in H2O2-treated HUVEC cultured with CMDMSC, SFDBMSC, and ICDBMSC is significantly higher than H2O2-treated HUVEC (P < 0.05). At baseline, the activity of thioredoxin reductase was 91.67 ± 10.14 mU/106 cell. Exposure of HUVEC to 100 μM H2O2 for 48 h, the activity of the enzyme was reduced to 43.33 ± 10.73 mU/106 cell. The activity of thioredoxin reductase in H2O2-treated endothelial cell cultured with CMDBMSC, SFDBMSC, and ICDBMSC was 79.33 ± 7.21 mU/106 cell, 88.33 ± 9.28 mU/106 cell, and 83.33 ± 9.28 mU/106 cell, respectively. As compared to H2O2-treated HUVEC, there were an approximately 1.83-, 2.03-, and 1.92-fold increase in the activity of thioredoxin reductase in H2O2-treated HUVEC cultured with CMDBMSC, SFDBMSC, and ICDBMSC, respectively. These data suggest that culturing H2O2-treated HUVEC with DBMSCs can protect endothelial cells from oxidative stress induced by H2O2.
DBMSCs modulated the effect of H2O2 on the expression of genes important in endothelial cell functions
DBMSCs modulate the expression of genes involved in endothelial cell (EC) survival, apoptosis, injury, fibrosis formation, and inflammation. THUVEC (HUVEC were cultured with 100 μM H2O2 for 48 h). TDBMSC (HUVEC were cultured with DBMSC and 100 μM H2O2 for 48 h)
Gene full name
THUVEC mean ΔΔ−2 values
TDBMSC Mean ΔΔ−2 values
Fold change (TDBMSC Vs. THUVEC)
P < 0.05
B-cell Lymphoma 2
Induce EC survival
> 6.64-fold ↑
Endothelin-1 (ET-1) Receptor A
Vascular Endothelial Growth Factor Receptor 3 (VEGFR3)
Matrix Metallopeptidase 2
Sphingosine Kinase 1
Matrix Metallopeptidase 9
Platelet endothelial cell adhesion molecule
TNF-related apoptosis-inducing ligand (TRAIL)
> 4.20-fold ↑
> 5-fold ↑
Fas cell surface death receptor
> 3-fold ↓
Induce EC apoptosis
Platelet Factor 4
> 30-fold ↓
> 14-fold ↓
Induce EC injury
Type 1 plasminogen activator inhibitor
TIMP Metallopeptidase Inhibitor 1
Angiotensin I Converting Enzyme
Coagulation Factor II Thrombin Receptor
Induce EC inflammation
A disintegrin and Metalloprotease 17
Vascular Endothelial Growth Factor Receptor 1
Tumor Necrosis Factor-α
> 295-fold ↓
PTK2 protein tyrosine kinase 2 (PTK2) or focal adhesion kinase (FAK),
Inhibits inflammation and fibrosis
> 11-fold ↑
Inhibits inflammation and, promotes fibrin clearance
Coagulation Factor III, Tissue Factor
Induces EC injury by induction of fibrin and thrombus formation
Thrombospondin 1 (TSP-1)
> 365-fold ↓
Induces EC inflammation and injury
Tissue Factor Pathway Inhibitor
~ 2-fold ↑
Inhibits EC injury by reducing thrombus formation
DBMSCs modulate the expression of genes mediating endothelial cell (EC) angiogenesis and migration. THUVEC (HUVEC were cultured with 100 μM H2O2 for 48 h). TDBMSC (HUVEC were cultured with DBMSC and 100 μM H2O2 for 48 h)
Gene full name
THUVEC mean ΔΔ−2 values
TDBMSC mean ΔΔ−2 values
Fold change (TDBMSC Vs. THUVEC)
P < 0.05
> 122-fold ↑
Inhibit EC angiogenesis
von Willebrand Factor
> 173-fold ↑
> 7-fold ↑
Coagulation Factor II Thrombin Receptor
Induce EC angiogenesis
Fibroblast Growth Factor 1
Fibroblast Growth Factor 2
Tyrosine Protein Kinase Kit or CD117
> 165568-fold ↓
P-selectin glycoprotein ligand-1
EK Receptor Tyrosine Kinase (TIE-2)
Vascular Endothelial Growth Factor A
> 5613-fold ↓
CASP8 and FADD like apoptosis regulator
> 6-fold ↓
Induce EC migration
Sphingosine Kinase 1
DBMSCs modulate the expression of genes mediating endothelial cell (EC) permeability. THUVEC (HUVEC were cultured with 100 μM H2O2 for 48 h). TDBMSC (HUVEC were cultured with DBMSC and 100 μM H2O2 for 48 h)
Gene full name
THUVEC Mean ΔΔ−2 values
TDBMSC Mean ΔΔ−2 values
(TDBMSC Vs. THUVEC)
P < 0.05
Angiotensin I Converting Enzyme
Induce EC permeability
A disintegrin and Metalloprotease 17
Interleukin 1 beta
> 3-fold ↓
Vascular Endothelial Growth Factor A
> 5613-fold ↓
> 122-fold ↑
Inhibit EC permeability
Natriuretic Peptide Receptor A/ Guanylate Cyclase A (Atrionatriuretic Peptide Receptor A)
DBMSCs modulate the expression of genes mediating leukocyte infiltration of endothelial cells (EC), adhesion of inflammatory cells and monocyte adhesion and transmigration. THUVEC (HUVEC were cultured with 100 μM H2O2 for 48 h). TDBMSC (HUVEC were cultured with DBMSC and 100 μM H2O2 for 48 h)
Gene full name
THUVEC mean ΔΔ−2 values
TDBMSC mean ΔΔ−2 values
Fold change (TDBMSC Vs. THUVEC)
P < 0.05
VE-Cadherin (Vascular Endothelial Cadherin)
~ 20-fold ↓
Induce leukocyte infiltration
Vascular Cell Adhesion Molecule 1
~ 11-fold ↑
Inhibits adhesion of inflammatory cells
Thrombospondin 1 (TSP-1)
> 365-fold ↓
Induces monocyte adhesion and transmigration
DBMSCs effects on genes involved in endothelial cell (EC) biology. THUVEC (HUVEC were cultured with 100 μM H2O2 for 48 h). TDBMSC (HUVEC were cultured with DBMSC and 100 μM H2O2 for 48 h)
Gene full name
THUVEC mean ΔΔ−2 values
TDBMSC mean ΔΔ−2 values
Fold change (TDBMSC Vs. THUVEC)
P < 0.05
Fold change is not statically significant, P > 0.05
Angiotensin II Receptor Type 1
Calcitonin Related Polypeptide Alpha
C-C motif chemokine ligand 2
C-X3-C Motif Chemokine Ligand 1
Intercellular Adhesion Molecule 1
Kallikrein Related Peptidase 3
Matrix Metallopeptidase 1
Nitric Oxide Synthase 3
Natriuretic Peptide B
Platelet Derived Growth Factor Receptor Alpha
Placental Growth Factor
Plasminogen Activator, Tissue Type
Plasminogen Activator, Urokinase
Superoxide Dismutase 1
Transforming Growth Factor Beta 1
We previously reported that DBMSCs protect endothelial cell activation by reducing the adhesion of monocytes to endothelial cells and their stimulatory effect on the proliferation of endothelial cells [2, 12]. These two events are the basis of endothelial cell injury in inflammatory diseases, such as atherosclerosis . Inflammatory diseases are also associated with high level of oxidative stress mediators, such as H2O2 [15–19]. Recently, we reported the ability of DBMSCs to survive and function under the stress of H2O2 . In addition, DBMSCs inhibit the angiogenesis of endothelial cells in H2O2 environment . Therefore, DBMSCs have the potential to be used as a cell-based therapy for the treatment of inflammatory diseases. In this study, we investigated the ability of DBMSCs to protect endothelial cell functions from stress induced by both H2O2 and monocytes.
First, we determined the effect of DBMSCs on endothelial cell function under H2O2. DBMSCs significantly induced the stimulatory effect of H2O2 on endothelial cell proliferation (Fig. 3a, b). This contrasts with our recent finding that MSCs from the chorionic villi of human placentae (pMSCs) reverse the proliferative effect of H2O2 on endothelial cells . This discrepancy can be attributed to the niche of both DBMSCs and pMSCs. During normal pregnancy, DBMSCs are located in the decidua where they are in a continuous exposure to high levels of oxidative stress mediators, because they are in a closed proximity to the maternal vessels [21, 22] while pMSCs are usually exposed to a lower levels of oxidative stress mediators because they are in a continuous contact with the fetal circulation [6, 7]. Therefore, DBMSCs have possibly acquired characteristics similar to H2O2 on the functions of endothelial cells.
Next, we demonstrated that the stimulatory effects of DBMSCs and H2O2 on endothelial cell proliferation are reversible (Fig. 4). However, the paracrine communication between DBMSCs and endothelial cells in the presence of H2O2 showed more stimulatory effect on endothelial cell proliferation than CMDBMSC (molecules produced by unstimulated DBMSCs) and ICDBMSC (intercellular direct contact), Fig. 4b, c. We also showed that DBMSCs may protect endothelial cells from oxidative stress by the finding that DBMSCs can induce the expression of many genes mediating the survival of endothelial cells and can also reduce the expression of genes that trigger apoptosis, injury, and inflammation in endothelial cells (Table 2) [23–48]. This protective role for DBMSCs on endothelial cells from stress induced by H2O2 is further confirmed by the ability of DBMSCs to increase the activities of glutathione and thioredoxin reductases (antioxidant enzymes) in H2O2-treated endothelial cells. Therefore, DBMSCs can protect endothelial cells from stress induced by H2O2, therefore suggesting a therapeutic potential for DBMSCs in inflammatory diseases.
We also found that ICDBMSC can reduce the stimulatory effect of H2O2 on endothelial cell adhesion (Fig. 5). In contrast, CMDBMSC and SFDBMSC could not reverse the stimulatory effect of H2O2 on endothelial cell adhesion (Fig. 5). Instead, SFDBMSC induced the stimulatory effect of H2O2 on the adhesiveness of endothelial cells. H2O2 is known to increase the adhesiveness property of endothelial cells through ICAM-1 and VCAM-1 [49–52]. DBMSCs secrete IL-1β and IL-10 . IL-1β is a proinflammatory cytokine that induces endothelial cell production of H2O2 while IL-10 is an anti-inflammatory cytokine that reduces endothelial cell production of H2O2 [53, 54]. This may explain why DBMSCs exert dual functions on endothelial cell adhesion depending on the nature of DBMSC treatment. Therefore, paracrine communication with endothelial cells may stimulate DBMSC production of IL1-β while the intercellular direct contact with endothelial cells may stimulate DBMSC production of IL-10. The interaction with DBMSCs (ICDBMSC) reduced endothelial cell expression of VCAM (Table 5), thus suggesting that VCAM may mediate the anti-adhesive effect of DBMSC on endothelial cells. Our data highlight that DBMSCs have dual effects “a double-edged sword” on endothelial cells as it was previously reported for the immunomodulatory properties of bone marrow-derived MSCs . However, a future study is essential to reveal this mechanism.
DBMSCs show also dual effects on endothelial cell migration. DBMSCs (SFDBMSC and ICDBMSC) reversed the inhibitory effect of H2O2 on endothelial cell migration while CMDBMSC enhanced the inhibitory effect of H2O2 on the migration of endothelial cells (Fig. 6). In this study, we found that DBMSCs induced the expression of a number of genes (e.g., endothelin-1, sphingosine kinase 1, and heme oxygenase-1) by H2O2-treated endothelial cells. These genes mediate the migration of endothelial cells [56–58], thus suggesting that these genes may mediate the stimulatory effect of DBMSCs on endothelial cell migration.
Adhesion and migration are the early steps towards endothelial cell angiogenesis . Recently, we showed that DBMSCs inhibit H2O2-treated endothelial cell angiogenesis . In this study, we found that DBMSCs increased and decreased H2O2-treated endothelial cell expression of various antiangiogenic [25, 59–63] and proangiogenic [24, 39, 64–70] genes, respectively (Table 3). These data suggest that these genes may mediate DBMSC inhibitory effect on endothelial cell angiogenesis. However, future functional studies are essential to elucidate the roles of these genes in the antiangiogenic properties of DBMSCs.
We previously reported that DBMSCs have an inhibitory effect on monocyte induction of endothelial cell proliferation and on their adhesion to endothelial cells . In this study, we also show that in the presence of H2O2, DBMSCs inhibit the adhesion of monocytes to endothelial cells (Fig. 8) and also inhibit endothelial cell proliferation (Fig. 7b). This further confirms the protective role of DBMSCs on endothelial cells proliferation (discussed above) from oxidative stress. These data indicate that DBMSCs have the ability to reduced endothelial cell proliferation, a pathological phenomenon that is known to contribute to the formation of atheroma plaque in atherosclerosis .
DBMSCs reduced monocyte expression of VCAM-1 (Fig. 9b), thus indicating that this adhesion molecule may mediate monocyte adhesion to endothelial cells. We also found that DBMSCs modulated H2O2-treated endothelial cell expression of various genes mediating endothelial cell proliferation, adhesion [44, 45], and permeability [38, 40, 59, 69, 72–74] as well as monocyte infiltration of endothelial cells [44, 75, 76]. Together, these data demonstrate the protective roles that DBMSCs may exert on endothelial cells via mechanisms may involve genes listed in Tables 2, 3, 4, and 5. However, functional studies are necessary to elucidate the effects of these genes in mediating DBMCs protective activities on endothelial cells.
This is the first comprehensive study to demonstrate the protective role of DBMSCs on endothelial cells in harsh oxidative stress environment. DBMSCs can protect endothelial cells from injury induced by oxidative stress or immune cells. Endothelial cell injury is a hallmark of inflammatory diseases, such as atherosclerosis where endothelial cells show increased functional activities (proliferation, adhesion, migration, angiogenesis, and permeability) and increased fibrin and thrombus formation as well as increased adhesion to immune cells, such as monocytes and their infiltration. These functional activities could be therapeutical targets for DBMSCs to repair endothelial cell injury and treat atherosclerosis.
We appreciate the staff and patients of the Delivery Unit, King Abdul Aziz Medical City for giving us placentae.
This study was supported by grants from KAIMRC (Grant No. RC12/133).
Availability of data and materials
All data generated during this study are included in this published article.
MHA proposed and supervised the project. MHA designed the experiments. MAA performed the experiments. MHA, MAA, and TK analyzed the data. MHA wrote the manuscript. MHA, FMA, TK, BK, SAM, RK, AOA, and ASA contributed to the data analysis and interpretation of results. All authors reviewed the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The institutional review board (IRB) at King Abdulla International Medical Research Centre (KAIMRC), Saudi Arabia, approved this study. Samples (placentae and umbilical cords of uncomplicated human pregnancies, 38–40 gestational weeks) were obtained and used immediately after signing consent forms.
Consent for publication
“Not applicable”. All authors agree to publish this manuscript.
The authors declare that they have no competing interests.
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