C-X-C Chemokine Receptor Type 4 Axis Research Articles

Extracellular Hsp70 Is Involved in the CXCL12/CXCR4 Pathway in Primary Human Nasal Epithelial Cells: A Preliminary Study

Background and Objectives: To date, no studies have been conducted on the interaction between extracellular heat shock protein 70 (Hsp70) and C-X-C chemokine receptor type 4 (CXCR4) in the upper airway. We aimed to evaluate the relationship between extracellular Hsp70 and CXCR4 and their role in the primary human nasal epithelium.Methods: We cultured primary human nasal epithelial (HNE) cells in an air–liquid interface. Macrogen performed single-cell quantitative polymerase chain reaction and sequencing. We conducted western blot analysis for the CXCR4 and mitogen-activated protein kinase (MAPK) pathways.Results: Extracellular Hsp70 treatment significantly increased the genetic expression and protein levels of CXCR4 in primary HNE cells. Phospho-ERK expression was increased by cotreatment with Hsp70 and CXCL12, but inhibited by pretreatment with AMD3100, a CXCR4 inhibitor. Pretreatment with an anti-Hsp70 antibody reduced phospho-ERK expression upregulation induced by cotreatment with Hsp70 and CXCL12.Conclusion: Extracellular Hsp70 participates in the activation of the CXCR4-dependent downstream signaling pathway in HNE cells. Further studies should evaluate the extracellular Hsp70-CXCL12/CXCR4 axis and the role of its components in the development of inflammatory diseases.

Enterovirus 71 induces pyroptosis of human neuroblastoma SH-SY5Y cells through miR-146a/ CXCR4 axis

Enterovirus 71 (EV71) is a predominant causative pathogen of hand-foot-and-mouth disease (HFMD) in children. Compared with other HFMD-associated viruses, EV71 tends to induce more severe neurological complications and even death. However, the detailed mechanism of EV71 causes nervous system disorder is still unclear. In this study, we found that EV71 induced the GSDMD/NLRP3-mediated pyroptosis of SH-SY5Y cells through up-regulated miR-146a. Through bioinformatic analysis, we identified C-X-C chemokine receptor type 4 (CXCR4) as the potential target of miR-146a. We noticed that the expression of CXCR4 was regulated by miR-146a during EV71 infection. Moreover, our results show that over-expression of CXCR4 attenuated EV71-induced pyroptosis of SY-SY5Y cells. These results reveal a previously unrecognized mechanism in which EV71 induces nervous system cells damage through regulating miR-146a/CXCR4 mediated pyroptosis.

Breast tumor metastasis following filgrastim administration due to the SDF-1/CXCR4 pathway.

Filgrastim, a recombinant type of granulocyte-colony stimulating factor (G-CSF), has a high potential to manage chemotherapy-induced leukopenia. It can increase stromal cell-derived factor 1 (SDF-1) which may stimulate C-X-C chemokine receptor type 4 (CXCR4) to migrate bone marrow-derived stem/progenitor cells to the bloodstream. Here, we aimed to investigate in vitro and in vivo effects of filgrastim on cell migration, invasion, and metastasis. A lentivirus vector of the anti-CXCR4 receptor was first used for the CXCR4 knockout. Effects of filgrastim on cell proliferation and migration were then investigated on 4T1 cells by Transwell migration and wound healing assay. At last, the effects of filgrastim on cell metastasis and the possible involved mechanisms have been investigated in a metastatic murine breast tumor. The knockout of the CXCR4 receptor could lead to a decrease in cell proliferation, migration, and invasion of the 4T1 cells. Filgrastim could directly target SDF-1 and upregulate the expression of the CXCR4 receptor. The knockout of the CXCR4 receptor reduced cell metastasis in an animal model of breast cancer. CXCR4 receptor stimulation by the filgrastim-affected pathways is a conserved evolutionary response that could increase cancer cell proliferation and consequent cell metastasis. Our results suggest that the activation of the CXCR4 receptor is a conserved evolutionary response that can increase cell proliferation, migration, and consequent metastasis. It seems that filgrastim may increase the chance of cancer cell metastasis in people continuously receiving it to increase the number of neutrophils. Filgrastim induces the SDF-1/CXCR4 axis on tumor cell growth. SDF-1 and its receptor CXCR4 are vital targets for filgrastim. The CXCR4 can stimulate the PI3K/AKT, NF-κB, and JAK/STAT signaling pathways. The SDF-1/CXCR4 pathway promotes cell chemotaxis and proliferation via MAPKs signaling. It also enhances cell survival, proliferation, and angiogenesis, increasing tumor cell metastasis. The STAT3-mediated inflammation is essential for tumorigenesis processes, and Akt, Wnt, STAT3, and CXCR4 signaling pathways are all correlated. CXCR4 = C-X-C chemokine receptor type 4, SDF-1 = stromal-derived-factor-1, MAPK = mitogen activated protein kinase; NF-κB = nuclear factor-κB, PI3K = phosphoinositide 3-kinase, JAK = Janus kinase, STAT = signal transducer and activator of transcription, PLC = phospholipase C, PKC = Protein kinase C, GRK = G protein-coupled receptor kinase.

CD44 potentiates hepatocellular carcinoma migration and extrahepatic metastases via the AKT/ERK signaling CXCR4 axis.

BackgroundCell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM).MethodsThe expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot.ResultsIn this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs.ConclusionsCD44 could represent a future therapeutic target for EHM.

Ginsenoside Rb1 Modulates the Migration of Bone-Derived Mesenchymal Stem Cells through the SDF-1/CXCR4 Axis and PI3K/Akt Pathway.

Methods Wound-healing assay and Transwell assay were utilized to evaluate the effect of ginsenoside Rb1 on the migration of BMSCs. RT-PCR and Western blotting were performed to evaluate the expression of stromal-derived factor 1 (SDF-1), C-X-C chemokine receptor type 4 (CXCR4), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (PKB; AKT). Results Ginsenoside Rb1 significantly enhanced the migration of BMSCs through the activation of SDF-1, CXCR4, p-PI3K/PI3K, and p-Akt/Akt relative expression. Furthermore, this stimulus was blocked by the pretreatment with AMD3100 and LY294002. Conclusions Ginsenoside Rb1 facilitated the migration of BMSCs through the activation of the SDF-1/CXCR4 axis and PI3K/Akt pathway.

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies.

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12–CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12–CXCR4/CXCR7 axis as a treatment strategy for CRC.

Up-regulated circBACH2 contributes to cell proliferation, invasion, and migration of triple-negative breast cancer

An increasing amount of evidence has proven the vital role of circular RNAs (circRNAs) in cancer progression. However, there remains a dearth of knowledge on the function of circRNAs in triple-negative breast cancer (TNBC). Utilizing a circRNA microarray dataset, four circRNAs were identified to be abnormally expressed in TNBC. Among them, circBACH2 was most significantly elevated in TNBC cancerous tissues and its high expression was positively correlated to the malignant progression of TNBC patients. In normal human mammary gland cell line, the overexpression of circBACH2 facilitated epithelial to mesenchymal transition and cell proliferation. In TNBC cell lines, circBACH2 knockdown suppressed the malignant progression of TNBC cells. Mechanistically, circBACH2 sponged miR-186-5p and miR-548c-3p, thus releasing the C-X-C chemokine receptor type 4 (CXCR4) expression. The interference of miR-186-5p/miR-548c-3p efficiently promoted the cell proliferation, migration, and invasion suppressed by circBACH2 knockdown in the TNBC cell lines. Finally, circBACH2 knockdown repressed the growth and lung metastasis of TNBC xenografts in nude mice. In summary, circBACH2 functions as an oncogenic circRNA in TNBC through a novel miR-186-5p/miR-548c-3p/CXCR4 axis.

Role and Therapeutic Targeting of SDF-1α/CXCR4 Axis in Multiple Myeloma.

Simple SummaryThe SDF-1α/CXCR4 axis plays crucial roles in proliferation, survival, invasion, dissemination, and drug resistance in multiple myeloma. This review summarizes the pleiotropic role of the SDF-1α/CXCR4 axis in multiple myeloma and introduces the SDF-1α/CXCR4 axis-targeted therapies in multiple myeloma.The C-X-C chemokine receptor type 4 (CXCR4) is a pleiotropic chemokine receptor that is expressed in not only normal hematopoietic cells but also multiple myeloma cells. Its ligand, stromal cell-derived factor 1α (SDF-1α) is produced in the bone marrow microenvironment. The SDF-1α/CXCR4 axis plays a pivotal role in the major physiological processes associated with tumor proliferation, survival, invasion, dissemination, and drug resistance in myeloma cells. This review summarizes the pleiotropic role of the SDF-1α/CXCR4 axis in multiple myeloma and discusses the future perspective in the SDF-1α/CXCR4 axis-targeted therapies in multiple myeloma.

SDF1α/CXCR4 axis may be associated with the malignant progression of gastric cancer in the hypoxic tumor microenvironment.

Stromal cell-derived factor 1α (SDF1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) have been reported to form an important chemokine signaling pathway. Our previous study reported that SDF1α from tumor stromal cells may stimulate the proliferation of gastric cancer (GC) cells through the CXCR4 axis in a hypoxic microenvironment. However, a limited number of studies have addressed the clinicopathological significance of the expression of SDF1α and CXCR4 in GC, particularly at hypoxic regions. Immunohistochemistry was used to investigate the expression levels of SDF1α, CXCR4 and the hypoxic marker carbonic anhydrase 9 (CA9) in 185 patients with stage II and III GC. The results demonstrated that CA9 was expressed on cancer and stromal cells in hypoxic lesions, CXCR4 was mainly expressed in cancer cells, and SDFα was mainly expressed in stromal cells. CXCR4 expression in cancer cells and SDFα expression in stromal cells were associated with the hypoxic regions with CA9 expression. The CA9 and CXCR4 expression in the cancer cells, and the SDF1α expression in the stromal cells (CA9/CXCR4/SDF1α) was significantly associated with macroscopic type 4 tumor (P=0.012) and the pattern of tumor infiltration into the surrounding tissue (P<0.001). The prognosis of the all CA9/CXCR4/SDF1α-positive patients was significantly poorer compared with that of patients with CA9-, CXCR4- or SDF1α-negative GC at Stage III (P=0.041). These results indicated that hypoxia may upregulate SDFα production in stromal cells and CXCR4 expression in cancer cells. The SDF1α/CXCR4 axis may serve an important role in the progression of GC.

Abstract C129: MicroRNA-155 targets AGTR1/NF-κB /CXCR4 axis and attenuates oncogenesis in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive malignancy of all primary brain tumors. Despite the recent advances in GBM treatment, the average survival time for a patient is less than 15 months. Therefore, we sought to investigate the molecular mechanisms underlying GBM initiation, progression and identify novel drug targets for this deadly disease. Higher levels of Angiotensin II Receptor Type 1 (AGTR1) and Angiotensin II Receptor Type 2 (AGTR2) have been associated with poor patient prognosis in astrocytomas. Additionally, contribution of these receptors in proliferative and infiltrative properties of glioma cells has already been established. Moreover, overexpression of AGTR1 has been known to play a critical role in multiple human malignancies including brain. However, the underlying mechanism involved in AGTR1 upregulation in GBM is not clearly understood and remains a matter of conjecture. Here, we analysed online available RNA-seq data from two independent GBM patients’ cohorts for AGTR1 expression and observed significant overexpression of AGTR1 in glioma patients compared to normal adjacent tissue. Importantly, the overall survival probability of GBM patients with high AGTR1 expression was low as compared to patients with low AGTR1 levels. To understand the post-transcriptional regulation of AGTR1 in GBM, we employed miRNA prediction algorithms namely, TargetScan, miRanda (microRNA.org), MicroT4 (DIANA tools), RNA22 along with experimentally supported database (TarBase), and examined miRNAs that bind to 3’UTR of AGTR1. Interestingly, conserved binding of miRNA-155 was predicted by all algorithms. Further, AGTR1-3’UTR-luciferase reporter assay in HEK-293T cells also confirmed the miRNA-155 mediated post-transcriptional regulation of AGTR1. Moreover, ectopic expression of miRNA-155 in GBM cells abrogate AGTR1-mediated proliferation, invasion, foci formation and anchorage independent growth. Intriguingly, immunodeficient mice implanted with stable miRNA-155 overexpressing SNB19 cells showed a significant reduction (~95%) in tumor burden as compared to the control group. Mechanistically, we showed that miRNA-155 attenuates AGTR1-mediated epithelial-to-mesenchymal transition, angiogenesis, ERK/MAPK signaling and promotes apoptosis. Importantly, stimulation with Angiotensin II (Ang II), a bonafide ligand of AGTR1, results in activation of NF-κB signaling downstream of AGTR1, followed by an increase in expression of C-X-C chemokine receptor type 4 (CXCR4) and AGTR1. Conversely, miRNA-155 overexpression in GBM cells attenuate NF-κB signalling, leading to a notable decrease in the expression of CXCR4 and its downstream signalling accompanied with reduction in AGTR1 levels. Alternately, we treated GBM cells with IKK-16, an inhibitor of IKK complex, to abrogate the NF-κB signalling downstream of AGTR1. Interestingly, we observed a significant reduction in the AGTR1 expression along with a marked decrease in CXCR4 expression. Taken together, we demonstrate that miRNA-155 is involved in post-transcriptional regulation of AGTR1 and disrupts AGTR1/NF-κB /CXCR4 signaling axis via anticancer pleiotropic effects. The present study opens new avenues to use IKK inhibitors and possible use of miRNA-155 replacement therapy for AGTR1-positive malignancies. Citation Format: Anukriti Singh, Nidhi Srivastava, Bushra Ateeq. MicroRNA-155 targets AGTR1/NF-κB /CXCR4 axis and attenuates oncogenesis in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C129. doi:10.1158/1535-7163.TARG-19-C129

Role of Acute Myeloid Leukemia Derived-Extracellular Vesicles in Dysregulating Normal Hematopoiesis

Acute myeloid leukemia (AML) is an aggressive and heterogeneous clonal disorder of hematopoietic stem/progenitor cells (HSPCs). Leukemogenesis is characterized by rapid growth and impaired differentiation of leukemic cells, which progressively occupy and likely alter bone marrow (BM) niche where normal HSPCs reside [Kumar B., et al. Leukemia. 2018]. The homing and adhesion of AML cells to the BM niche is critical for its pathogenesis and involves many adhesion molecules, chemokine receptors, their ligands and signaling cascades. In the healthy context, the CXC chemokine receptor type 4 (CXCR4) expressed on HSPCs and its ligand, stromal derived factor-1 alpha (SDF-1α) produced by BM niche, are key mediators of HSPC-BM niche interaction inducing the homing, quiescence and survival of HSPCs, and the regulation of the HSC pool amount [Sánchez-Aguilera A., et al. Cell Mol Life Sci. 2017]. In AML context, where CXCR4/SDF-1α axis is deregulated, growth, BM niche-homing and adhesion of leukemic cells are altered also inducing AML relapse/resistance [Sánchez-Aguilera A., et al. Cell Mol Life Sci. 2017]. Despite recent improvements in therapeutic approaches, only a fraction of AML patients is cured. Therefore, it is crucial to identify new molecular mechanisms driving the AML pathogenesis. Extracellular vesicles (EVs) are lipidic bi-layer membrane particles released by normal and neoplastic cells. They shuttle a complex molecular cargo which reflects that of the origin cell [Caivano A., et al. Tumor Biol 2015; Caivano A., et al. Cell Oncol 2017] and which can be transferred to target cells altering their biological functions [De Luca L., et al. Oncotarget 2016; De Luca L., et al. Int J Mol Sci. 2017]. In particular, neoplasm derived EVs promote a tumor-supporting environment in non-malignant cells favoring cancer proliferation [Laurenzana I., et al. Stem Cells Int. 2018; De Luca L., et al. Expert Rev Mol Diagn. 2019]. We studied the effect of leukemia derived-EVs (LEVs) on healthy umbilical cord blood HSPCs to explore their potential role in dysregulating normal hematopoiesis. Specifically, the proliferation and differentiation, the quantitative and qualitative colony-forming features and the SDF-1α /CXCR4 mediated-migration of HSPCs induced by LEVs were investigated. EVs were isolated from AML cell lines by centrifugation steps and analyzed for AML surface markers by flow cytometry. Next, they were co-incubated with healthy CD34+ sorted cells for 24 hours. After treatment, cell count, apoptosis, HSPC-specific cluster of differentiation marker (CD10, CD34, CD38, CD45RA, CD90, CD123) expression [Karamitros D., et al. Nat Immunol. 2018], in vitro colony-forming unit cell assay, CXCR4 levels and SDF-1α mediated-migration assay were assessed. Our data showed: i) LEVs displaying specific AML surface markers; ii) a similar number of CD34+ cells in LEV treated-HSPCs, including HSCs, compared with controls; iii) no differences in apoptosis between the two groups; iv) an increase of multipotent progenitor (MPP, p=0.01) associated with a reduction of lymphoid-primed multipotent progenitors (LMPP, p=0.01) in treated cells; v) an increase of common myeloid progenitors (CMP, p=0.01), together with reduction of granulocyte macrophage progenitors (GMP, p=0.01) and megakaryocyte erythroid progenitors (MEP) in treated group; vi) a reduction of colony-forming ability, including erythroid, granulocytic and megakaryocytic colonies (p=0.03), in treated cells; vii) decreased levels of CXCR4 in LEV treated-HSPCs, that were accompanied by reduced HSPC migration mediated by SDF-1α. Altogether, this study suggests that AML derived-EVs can dysregulate the normal hematopoiesis promoting an increase of less differentiated precursors (MPP and CMP) which is associated with a reduction of more committed myeloid progenitors (GMP and MEP) (Figure 1). Furthermore, AML derived-EVs seem to induce less attraction of HSPCs in BM niche by interfering with SDF-1α/CXCR4 axis. Strategies to block EV production and secretion, as well as EV induced-reprogramming, could be novel exciting therapeutic approaches in AML. Disclosures Musto: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

LMP1–miR-146a–CXCR4 axis regulates cell proliferation, apoptosis and metastasis

Epstein–Barr virus (EBV), the first human tumor virus to have been discovered, sustains an asymptomatic lifelong infection in ∼95% of the world’s population. Reportedly, EBV infection induces the expression of specific cellular microRNAs (miRNAs), such as miR-155, miR-146a, miR-21, which can contribute to the persistence of latently infected cells. In this study, we investigated whether C-X-C chemokine receptor type 4 (CXCR4) is a cellular target of human miR-146a. We also investigated the role of miR-146a and CXCR4 in EBV-associated cells. The results indicate that miR-146a is more abundantly expressed in EBV-positive than in EBV-negative cells. MiR-146a down-regulated CXCR4 expression in a dose- and time-dependent manner. Phenotypic experiments detected miR-146a mimics that could suppress cell proliferation and cell migration and promote cell apoptosis by targeting CXCR4. In addition, miR-146a mimics suppressed cell survival by decreasing the population of G0/G1 phase cells. Latent membrane protein (LMP)1, an importance oncoprotein, can stimulate miR-146a and inhibit the CXCR4 expression. Our findings indicate that LMP1–miR-146a–CXCR4 axis functions as a regulator in the EBV-associated cells.

SDF-1/CXCR4 axis coordinates crosstalk between subchondral bone and articular cartilage in osteoarthritis pathogenesis.

Crosstalk between subchondral bone and articular cartilage is considered a central feature of osteoarthritis (OA) initiation and progression, but its underlying molecular mechanism remains elusive. Meanwhile, specific administration of drugs in subchondral bone is also a great challenge during investigation of the process. We here explore the role of stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis in the crosstalk between subchondral bone and articular cartilage in OA pathogenesis, using osmotic infusion pumps implanted in tibial subchondral bone directly to ensure quantitative, continuous and steady drug delivery over the entire experiment. We found that increased SDF-1 in subchondral bone firstly induced subchondral bone deterioration by erroneous Mesenchymal Stem Cells (MSCs) recruitment and excessive bone resorption in anterior cruciate ligament transection (ACLT) mice. Deterioration of subchondral bone then led to the traverse of SDF-1 from subchondral bone to overlying cartilage. Finally, SDF-1 from underlying subchondral bone combined with CXCR4 in chondrocytes to induce articular cartilage degradation by promoting the shift of transforming growth factor-β receptor type I (TβRI) in chondrocytes from activin receptor-like kinase 5 (ALK5) to activin receptor-like kinase 1 (ALK1). More importantly, specific inhibition of SDF-1/CXCR4 axis in ACLT rats attenuated OA by stabilizing subchondral bone microarchitecture, reducing SDF-1 in cartilage and abrogating the shift of TβRI in chondrocytes. Our data demonstrate that the SDF-1/CXCR4 axis may coordinate the crosstalk between subchondral bone and articular cartilage in OA pathogenesis. Therefore, specific inhibition of SDF-1/CXCR4 axis in subchondral bone or intervention in SDF-1 traverse may be therapeutic targets for OA.

EPO enhances the protective effects of MSCs in experimental hyperoxia-induced neonatal mice by promoting angiogenesis.

Bronchopulmonary dysplasia (BPD) is the most common type of chronic lung disease in infancy; however, there is no effective treatment for it. In the present study, a neonatal mouse BPD model was established by continuous exposure to high oxygen (HO) levels. Mice were divided randomly into 5 groups: control, BPD, EPO, MSCs, and MSCs+EPO. At 2 weeks post-treatment, vessel density and the expression levels of endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1), and its receptor C-X-C chemokine receptor type 4 (CXCR4) were significantly increased in the MSC+EPO group compared with the EPO or MSCs group alone; moreover, EPO significantly enhanced MSCs proliferation, migration, and anti-apoptosis ability in vitro. Furthermore, the MSCs could differentiate into cells that were positive for the type II alveolar epithelial cell (AECII)-specific marker surfactant protein-C, but not positive for the AECI-specific marker aquaporin 5. Our present results suggested that MSCs in combination with EPO could significantly attenuate lung injury in a neonatal mouse model of BPD. The mechanism may be by the indirect promotion of angiogenesis, which may involve the SDF-1/CXCR4 axis.

Stromal cell-derived factor-1α promotes recruitment and differentiation of nucleus pulposus-derived stem cells.

BACKGROUNDIntervertebral disc (IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus (NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIMTo investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells (NPSCs).METHODSWe performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups. Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTSSDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Real-time RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1α not only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement. Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSIONThese findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

CCR2+ Monocyte-Derived Ly6C-F4/80+ Fibrocytes Inhibit Collagen Degradation and Contribute to the Development of Colon Fibrosis Via Production of TIMP-1

Introduction: Fibrocytes are derived from a subset of monocytes and express mesenchymal markers such as collagen type I (Col I) and hematopoietic markers such as CD45 and CD11b. They also express several chemokine receptors such as CC chemokine receptor (CCR)-1, CCR2, CCR5, CCR7, and CXC chemokine receptor type 4 (CXCR4). They circulate in the peripheral blood (PB) and can be isolated from many fibrotic tissues. Fibrocytes participate in both physiological wound healing and various pathological fibrosis including myelofibrosis, hypertrophic scar, systemic sclerosis, idiopathic pulmonary fibrosis, liver cirrhosis, and progressive kidney disease. In murine and human colitis, fibrocytes are also reported to be associated with the colon fibrosis. It has been described that migration of fibrocytes to the injured sites involves CC chemokine ligand 2 (CCL2)/CCR2 axis in the liver and kidney and CXC chemokine ligand 12 (CXCL12)/CXCR4 axis in the lung. However, there are few reports concerning the role of fibrocytes and their expression of chemokine receptors related to the induction of colon fibrosis.Methods: We generated bone marrow (BM) chimeric mice by transplantation of BM total-nucleated cells, which were isolated from enhanced green fluorescent protein (EGFP)-transgenic mice or CCR2 knockout (KO) mice, into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, BM chimeric mice were treated with a single intraperitoneal injection of azoxymethane (10 mg/kg body weight) followed by 3 cycles of 1% dextran sulfate sodium (DSS) in the drinking water. We assessed the level of fibrosis in the colon using Sirius red staining and analyzed the presence of BM-derived CD45+CD11b+Col I+ fibrocytes in the colon lamina propria (LP) using immunofluorescence staining and flow cytometry. Furthermore, we investigated the expressions of Col I, transforming growth factor-ß (TGF- ß), matrix metalloproteinases (MMPs), and tissue inhibitor of MMPs (TIMP)-1 in the colon tissues and fibrocytes sorted from colon LP cells after chronic DSS treatment using quantitative real-time RT-PCR.Results: During chronic inflammation, infiltration of CCR2+ BM-derived monocytes and fibrocytes and production of CCL2 in the colon were particularly increased and colon fibrosis was developed in EGFP BM chimeric mice. Two types of fibrocytes, CCR2+CXCR4+Ly6C-F4/80+ fibrocytes and CCR2-CXCR4+Ly6ChighF4/80- fibrocytes, were identified in the colon LP, whereas only the latter fibrocytes were detected in the PB. Adoptive transferred CCR2+Ly6ChighCol I- monocytes migrated to the injured colon and a part of them differentiated into CCR2+Col I+ fibrocytes. In CCR2KO BM chimeric mice, the numbers of monocytes and fibrocytes in the colon LP were significantly decreased and colon fibrosis was attenuated. However, there was no difference in the mRNA expressions of Col I, TGF-ß, and MMPs (MMP-1a, MMP-8, and MMP-13, known as collagenases) in colon tissues between EGFP BM chimeric mice and CCR2KO BM chimeric mice. Improvement of colon fibrosis in CCR2KO BM chimeric mice was associated with the decreased expression of Timp1 mRNA in colon tissues. We analyzed the expression of Timp1 mRNA in CCR2+ cells and CCR2- cells sorted from colon LP cells and found a high expression of Timp1 in CCR2+ monocytes/macrophages and fibrocytes.Conclusions: Circulating CCR2+ monocytes migrate into the inflamed colon via CCL2/CCR2 axis and differentiate into CCR2+Ly6C-F4/80+ fibrocytes, which inhibit collagen degradation and contribute to the development of colon fibrosis by the production of TIMP-1. DisclosuresMasuya:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Katayama:Ono Pharmaceutical: Research Funding; Novo Nordisk: Honoraria, Research Funding; Chugai Pharma: Honoraria, Research Funding; Toyama Chemical Co: Research Funding; Sysmex: Honoraria; Mochida Pharmaceutical Co. Ltd.,: Research Funding; Bristol-Myers Squibb: Honoraria; Astellas Pharma: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Takeda: Honoraria, Research Funding; Teijin Pharma: Research Funding; Eisai: Research Funding; Sumitomo Group: Honoraria, Research Funding; Nippon Shinyaku: Honoraria, Research Funding; Shire: Honoraria; Alexion Pharmaceuticals: Honoraria; Celgene: Honoraria; Taisho Toyama Pharma: Honoraria; Pfizer: Honoraria, Research Funding; Shionogi Pharmaceutical: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding.

Abnormal expression of fibrosis markers, estrogen receptor α and stromal derived factor‑1/chemokine (C‑X‑C motif) receptor‑4 axis in intrauterine adhesions.

Intrauterine adhesions (IUAs) are mainly derived from fibrous tissue formation following endometrial damage. The aim of the present study was to assess whether fibrosis markers, estrogen receptor (ER)α and the stromal derived factor (SDF)-1/C-X-C chemokine receptor type 4 (CXCR-4) axis are abnormally expressed in IUA endometrium. A total of 76 human endometrial biopsy samples (normal, n=20; mild-to-moderate IUAs, n=40; and severe IUAs, n=16) were employed, and Sprague-Dawley rat IUA models at different time points were constructed. Subsequently, the expression of transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-9, ERα and the SDF-1/CXCR-4 axis was evaluated in human and rat IUAs using histology, immunohistochemistry, reverse transcription quantitative polymerase chain reaction and western blotting. In patients and rats with IUA formation, the expression of TGF-β1, MMP-9 and ERα was significantly higher compared with the control group at the mRNA and protein levels (P<0.05); in addition, in patients, the TGF-β1, MMP-9 and ERα levels were significantly higher in severe IUAs compared with those in mild-to-moderate IUA endometrium (P<0.05). Although the chemokine SDF-1 level in rats increased significantly during the early postoperative phase (reaching a peak at the second estrus phase) in rat endometrium (P<0.05), its special receptor CXCR-4 expression did not differ significantly compared with the control group in rats or patients (P>0.05). Our findings indicated that aberrant activation of fibrosis and expression of ERα may be involved in the pathology of IUA formation. The role of the SDF-1/CXCR-4 axis in IUAs as inflammatory medium in the short-term or special homing factors for bone marrow mesenchymal stem cells requires further verification in in vivo animal models.

In vitro cell behaviors of bone mesenchymal stem cells derived from normal and postmenopausal osteoporotic rats.

Postmenopausal osteoporosis (PMO) increases bone fragility and the risk of fractures, and impairs the healing procedure of bone defects in aged women. The stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor type 4 (CXCR4) axis helps to maintain the biological and physiological functions of bone marrow mesenchymal stem cells (BMSCs) and increase the homing efficiency of BMSCs. The present study aimed to provide insights into the possible association between migration and osteogenic ability and the SDF-1α/CXCR4 axis in BMSCs derived from a rat model of PMO. In order to do this, the general and SDF-1α/CXCR4-associated biological characteristics as well as associated molecular mechanisms in BMSCs isolated from a PMO rat model (OVX-BMSCs) and normal rats (Sham-BMSCs) were investigated and compared. In comparison with Sham-BMSCs, OVX-BMSCs exhibited an impaired osteogenic ability, but a stronger adipogenic activity as well as a higher proliferative ability. In addition, OVX-BMSCs presented a lower chemotactic activity towards SDF-1α, lower expression levels of CXCR4 and reduced levels of phosphorylated AKT (p-AKT). Therefore, the lower expression levels of CXCR4 and p-AKT may be responsible for the impaired osteogenic ability and lower chemotactic activity towards SDF-1α of OVX-BMSCs.

The chemokine receptor type 4 antagonist, AMD3100, interrupts experimental tooth movement in rats

ObjectiveThe aim of this study was to clarify the role of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis in osteoclast accumulation, and the influence of orthodontic tooth movement (OTM) under mechanical force application to periodontal tissues, by administration of the CXCR4 antagonist AMD3100. DesignThe upper right first molar (M1) of rats was moved mesially with a 10-g force titanium-nickel closed coil spring. Rats were treated with phosphate-buffered saline or AMD3100 (5mg/kg), which is a SDF-1 antagonist. After 0, 1, 3, and 7days, alveolar bones in all groups were examined at each time point by micro-computed tomography and histological analysis. ResultsTooth movement was decreased significantly in the AMD3100-treated group at 1, 3, and 7days after beginning OTM. The numbers of tartrate-resistant acid phosphatase-positive multinucleated cells in the periodontal ligament around the maxillary M1 were decreased significantly in the treated as compared to the control group on Days 1 and 3. ConclusionAdministration of AMD3100 decreases OTM and osteoclast accumulation in rat molars under orthodontic force application. These findings suggest that the SDF-1/CXCR4 axis plays an important role in alveolar bone metabolism during OTM.

Danshensu accelerates angiogenesis after myocardial infarction in rats and promotes the functions of endothelial progenitor cells through SDF-1α/CXCR4 axis

The present study was performed to investigate the potential role of Danshensu in therapeutic angiogenesis in ischemic myocardium and endothelial progenitor cells (EPCs) function. The rat model of myocardial infarction (MI) injury was induced by left anterior descending coronary artery ligation for 14 days. Danshensu significantly alleviated myocardial ischemia injury by ameliorating left ventricular function and reducing infarct size. Furthermore, Danshensu potentiated post-ischemia neovascularization as evidenced by increased microvessel density in infarction boundary zone, as well as the expression of marker proteins vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Moreover, Danshensu notably promoted stromal cell-derived factor-1α (SDF-1α) level in plasma and C-X-C chemokine receptor type 4 (CXCR4) expression in peri-infarction myocardium, and AMD3100 (CXCR4 antagonist) could reverse the angiogenic and cardioprotective effects of Danshensu. For in vitro study, EPCs were isolated from bone marrow of rats. On the one hand, Danshensu provided significant cytoprotection against hypoxia insult by boosting EPCs viability and inhibiting apoptosis, and upregulated Akt phosphorylation. On the other hand, Danshensu enhanced proangiogenic functions of EPCs on cell migration and tube formation, and increased SDF-1α and CXCR4 expression. Likewise, the cytoprotection and proangiogenic functions of Danshensu on EPCs were partly negated by LY294002 (PI3K antagonist) and CXCR4 siRNA, respectively. Taken together, our results suggested that the cardioprotection of Danshensu in MI rats may be related to promoting myocardial neovascularization. The possible mechanisms may involve improving EPCs survival in hypoxia condition through Akt phosphorylation, and accelerating EPCs proangiogenic functions through SDF-1α/CXCR4 axis.