AEBP1 Promotes the Occurrence and Development of Abdominal Aortic Aneurysm by Modulating Inflammation via the NF-κB Pathway.

AIM: Inflammation plays a significant role in the pathogenesis of human abdominal aortic aneurysm (AAA). AEBP1 can promote activation of the NF-κB pathway, subsequently affecting the expression of NF-κB target genes, including inflammatory cytokines and matrix metalloproteinases (MMPs). Our objective was to examine the role of AEBP1 in the development of AAA and characterize the underlying mechanism. METHODS: ITRAQ, RT-PCR, western blot, immunohistochemistry, and ELISA were used to compare different experimental groups with the controls and to determine the differentially expressed genes. We generated an AAA model using porcine pancreatic elastase in Sprague-Dawley rats and silenced their AEBP1 in vivo by adenoviruses injected intra-adventitially. We also silenced or overexpressed AEBP1 in human vascular smooth muscle cells in vitro in the presence and in the absence of NF-κB inhibitor BAY 11-7082. RESULTS: Proteome iTRAQ revealed a high expression of AEBP1 in AAA patients, which was verified by qRT-PCR, western blot, immunohistochemistry, and ELISA. The mean expression level of AEBP1 in AAA patients was higher than that in controls. Along with AEBP1 upregulation, we also verified mis-activation of NF-κB in human AAA samples. The in vivo studies indicated that AEBP1 knockdown suppressed AAA progression. Finally, the in vitro studies illustrated that AEBP1 promotes activation of the NF-κB pathway, subsequently upregulating pro-inflammatory factors and MMPs. CONCLUSIONS: Our results indicate a role of AEBP1 in the pathogenesis of AAA and provide a novel insight into how AEBP1 causes the development of AAA by activating the NF-κB pathway.

Sertoli cells promote proliferation of bone marrow-derived mesenchymal stem cells in co-culture.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a major source for cell transplantation. The proliferative ability of BMSCs is an important determinant of the efficiency of transplant therapy. Sertoli cells are "nurse" cells for development of sperm cells. Our recent study showed that Sertoli cells promoted proliferation of human umbilical cord mesenchymal stem cells (hUCMSCs) in co-culture. Studies by other groups also showed that Sertoli cells promoted growth of endothelial cells and neural stem cells. In this study, we investigated the effect of Sertoli cells on proliferation of BMSCs. Our results showed that Sertoli cells in co-culture significantly enhanced proliferation of BMSCs (P < 0.01). Moreover, co-culture with Sertoli cells also markedly increased mRNA and/or protein expressions of Mdm2, p-Akt and Cyclin D1, and decreased p53 expression in BMSCs (P < 0.01 or < 0.05). These findings indicate that Sertoli cells have the potential to enhance proliferation of BMSCs.

TGF-ß1 induces apoptosis of bone marrow-derived mesenchymal stem cells via regulation of mitochondrial reactive oxygen species production.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells in regenerative medicine. Our previous study demonstrated that transforming growth factor (TGF)-ß1 induced BMSC senescence in vitro. Whether TGF-ß1 affects the apoptosis of BMSCs has not been examined; therefore the aim of the present study was to investigate this effect. BMSCs were isolated from mouse bone marrow, and the third-passage cells were exposed to 0, 10 and 20 ng/ml TGF-ß1 for 24 h. Cell proliferation was measured by MTT assay; apoptosis was assessed using DAPI staining; and the apoptotic signals Annexin V, B-cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax) were measured using western blotting. Mitochondrial reactive oxygen species (ROS) were measured by flow cytometry following staining with MitoSOX™ Red mitochondrial superoxide indicator. The MTT assay showed that 10 and 20 ng/ml TGF-ß1 inhibited BMSC proliferation. DAPI staining demonstrated that 10 and 20 ng/ml TGF-ß1 promoted BMSC apoptosis, which was further confirmed by a western blotting assay showing a significant increase in the pro-apoptotic signals Annexin V and Bax but a decrease in the anti-apoptotic signal Bcl-2. It was also found that TGF-ß1 markedly increased the mitochondrial ROS levels in BMSCs. It is well known that mitochondrial ROS are strong stimulators of cell apoptosis. These findings indicate that TGF-ß1 can induce BMSC apoptosis, and the mechanism may involve mitochondrial ROS generation.

Small concentrations of TGF-ß1 promote proliferation of bone marrow-derived mesenchymal stem cells via activation of Wnt/ß-catenin pathway.

Bone marrow-derived mesenchymal stem cells (BMSCs) are the most promising seed cells for cell transplant. The proliferation of BMSCs is one of the most important determinants of the efficiency of MSC-based transplant therapy. It has been reported that transforming growth factor-ß1 (TGF-ß1) activates Wnt/ß-catenin signaling and regulates cell proliferation. In this study, we investigated the effect of low concentrations of TGF-ß1 on proliferation of BMSCs and the related mechanisms. BMSCs were treated with 0, 1, 5 and 10 ng/L recombinant mouse TGF-ß1 for 12 h. Cell proliferation was measured by cell counting and MTT assay, and the proliferation-related signals p53, Mdm2, Aktl, Wnt3, phospho-Akt and ß-catenin were measured by quantitative polymerase chain reaction (qPCR) and/or Western blot. Our results showed that TGF-ß1 at low concentrations induced BMSC proliferation and expression of Mdm2, Aktl, phospho-Akt, Wnt3 and ß-catenin, and inhibited p53 expression in dose dependent manner. Importantly, ß-catenin siRNA significantly inhibited TGF-ß1-induced BMSC proliferation. These findings suggest that low concentrations of TGF-ß1 can stimulate proliferation of BMSCs, which is at least partially dependent on the activation of Wnt/ß-catenin pathway.

Downregulation of ß-catenin and Akt signaling is responsible for poor proliferation of the late passage of bone marrow mesenchymal stem cells.

It is well established that mesenchymal stem cells (MSCs) will partially lose their proliferative ability with continuous expansion. However, the specific mechanisms underlying this effect remain unclear. In the present study, it was identified that ß-catenin was downregulated in the late passage (passage 8) of bone marrow mesenchymal stem cells (bmMSCs). Following ß-catenin expression, the expression of phospho-Akt was also significantly decreased in the late passage of bmMSCs. More notably, overexpression of ß-catenin in passage 8 of bmMSCs by transfection with pMXs-ß-catenin plasmids, significantly increased cell proliferation and Akt expression. These results indicate that the downregulation of ß-catenin and Akt signaling may be a critical factor for the proliferation of the late passage of bmMSCs.

MicroRNA-10b promotes the migration of mouse bone marrow-derived mesenchymal stem cells and downregulates the expression of E-cadherin.

The ability of mesenchymal stem cells (MSCs) to migrate is an important determinant of the efficiency of MSC transplant therapy. MicroRNA-10b (miR-10b) has been positively involved in the migration of a number of tumor cells lineages. To date, it remains unknown whether miR-10b affects the migration of MSCs. In the current study, the effect of miR-10b on the migration of mouse bone marrow-derived MSCs (bmMSCs) was investigated. Third-passage bmMSCs were transfected with miR-10b mimic and negative control precursor miRNA using Lipofectamine™ 2000. miR-10b and E-cadherin expression and bmMSC migration were determined. The present results showed that primary bmMSCs exhibit a spindled or triangular morphology and that third­passage bmMSCs present a typical fibroblast-like morphology, exhibiting CD90-positive and CD45-negative expression. Compared with the transfection of negative control miRNA, transfection of miR-10b mimic markedly upregulated miR-10b expression in bmMSCs, increased their migration and downregulated E-cadherin expression. The current observations indicate that the upregulation of miR-10b increases bmMSC migration ability, which may be involved in the downregulation of E-cadherin.

Lectin-like oxidized LDL receptor-1 expresses in mouse bone marrow-derived mesenchymal stem cells and stimulates their proliferation.

The bone marrow-derived mesenchymal stem cells (bmMSCs) have been widely used in cell transplant therapy, and the proliferative ability of bmMSCs is one of the determinants of the therapy efficiency. Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) as a transmembrane protein is responsible for binding, internalizing and degrading oxidized low density lipoprotein (ox-LDL). It has been identified that LOX-1 is expressed in endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts and monocytes. In these cells, low concentration of ox-LDL (<40 µg/mL) stimulates their proliferation via LOX-1 activation. However, it is poor understood that whether LOX-1 is expressed in bmMSCs and which role it plays. In this study, we investigated the status of LOX-1 expression in bmMSCs and its function on bmMSC proliferation. Our results showed that primary bmMSCs exhibiting a typical fibroblast-like morphology are positive for CD44 and CD90, but negative for CD34 and CD45. LOX-1 in both mRNA and protein levels is highly expressed in bmMSCs. Meanwhile, bmMSCs exhibit a strong potential to take up ox-LDL. Moreover, LOX-1 expression in bmMSCs is upregulated by ox-LDL with a dose- and time-dependent manner. Presence of ox-LDL also enhances the proliferation of bmMSCs. Knockdown of LOX-1 expression significantly inhibits ox-LDL-induced bmMSC proliferation. These findings indicate that LOX-1 plays a role in bmMSC proliferation.

Aspirin attenuates angiotensin II-induced inflammation in bone marrow mesenchymal stem cells via the inhibition of ERK1/2 and NF-κB activation.

Angiotensin II (Ang II) is a peptide hormone that plays a critical role in numerous physiological and pathophysiological processes. It is also commonly used as an inducer for the directional differentiation of bone marrow mesenchymal stem cells (bmMSCs). Previous studies demonstrated that Ang II induces inflammatory responses in endothelial cells, smooth muscle cells and fibroblasts. Aspirin is generally used as analgesic, antipyretic and occasionally anti-inflammatory medication. Whether aspirin suppresses inflammatory responses in bmMSCs has not been elucidated. In this study, we investigated the effect of aspirin on Ang II-induced inflammation in bmMSCs. Our results demonstrated that Ang II (10 nM-10 µM) increased the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6 from bmMSCs in a dose-dependent manner. This result was further confirmed by a reverse transcription-polymerase chain reaction (RT-PCR) assay, which demonstrated a dose-dependent increase in the mRNA expression of TNF-α, IL-6, IL-1ß and monocyte chemotactic protein-1 (MCP-1) in bmMSCs following exposure to Ang II. Furthermore, it was also observed that Ang II increased the expression of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-p65 in bmMSCs. The application of aspirin (0.1 mM) significantly inhibited the activation of ERK1/2 and NF-κB, the expression of TNF-α, IL-6, IL-1ß and MCP-1 genes and the secretion of TNF-α and IL-6. Our findings indicated that aspirin may attenuate Ang II-induced inflammation in bmMSCs via the inhibition of ERK1/2 and NF-κB activation.

Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells.

Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of "nurse" cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neural stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P<0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.