Stromal cell-derived factor-1α regulates chondrogenic differentiation via activation of the Wnt/β-catenin pathway in mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have been applied to treat degenerative articular diseases,and stromal cell-derived factor-1α(SDF-1α)may enhance their therapeutic efficacy.However,the regulatory effects of SDF-1αon cartilage differentiation remain largely unknown.Identifying the specific regulatory effects of SDF-1αon MSCs will provide a useful target for the treatment of degenerative articular diseases.AIM To explore the role and mechanism of SDF-1αin cartilage differentiation of MSCs and primary chondrocytes.METHODS The expression level of C-X-C chemokine receptor 4(CXCR4)in MSCs was assessed by immunofluorescence.MSCs treated with SDF-1αwere stained for alkaline phosphatase(ALP)and with Alcian blue to observe differentiation.Western blot analysis was used to examine the expression of SRY-box transcription factor 9,aggrecan,collagen II,runt-related transcription factor 2,collagen X,and matrix metalloproteinase(MMP)13 in untreated MSCs,of aggrecan,collagen II,collagen X,and MMP13 in SDF-1α-treated primary chondrocytes,of glycogen synthase kinase 3β(GSK3β)p-GSK3βandβ-catenin expression in SDF-1α-treated MSCs,and of aggrecan,collagen X,and MMP13 in SDF-1α-treated MSCs in the presence or absence of ICG-001(SDF-1αinhibitor).RESULTS Immunofluorescence showed CXCR4 expression in the membranes of MSCs.ALP stain was intensified in MSCs treated with SDF-1αfor 14 d.The SDF-1αtreatment promoted expression of collagen X and MMP13 during cartilage differentiation,whereas it had no effect on the expression of collagen II or aggrecan nor on the formation of cartilage matrix in MSCs.Further,those SDF-1α-mediated effects on MSCs were validated in primary chondrocytes.SDF-1αpromoted the expression of p-GSK3βandβ-catenin in MSCs.And,finally,inhibition of this pathway by ICG-001(5μmol/L)neutralized the SDF-1α-mediated up-regulation of collagen X and MMP13 expression in MSCs.CONCLUSION SDF-1αmay promote hypertrophic cartilage differentiation in MSCs by activating the Wnt/β-catenin pathway.These findings provide further evidence for the use of MSCs and SDF-1αin the treatment of cartilage degeneration and osteoarthritis.

C-X-C chemokine receptor type 7 antibody enhances neural plasticity after ischemic stroke

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4(CXCR4) have been shown to regulate neural regeneration after stroke.Howeve r,whether stromal cell-derived factor-1 receptor CXCR7,which is widely distributed in the develo ping and adult central nervous system,participates in neural regeneration remains poorly unde rstood.In this study,we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral co rtex and striatum.Starting on day 7 after injury,CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days.Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting co rticospinal tra ct fibers in rats with cerebral ischemia,increased the expression of vesicular glutamate transporter 1 and growth-related protein 43,marke rs of the denervated spinal cord synapses,and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum.In addition,CXCR7 antibody increased the expression of CXCR4 in the striatum,increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway,and im proved rat motor function.These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration,synaptogenesis,and myelin regeneration,which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

CXCR4/SDF-1 axis is involved in lymph node metastasis of gastric carcinoma

AIM:To investigate the role of CXC chemokine receptor-4 (CXCR4) and stromal cell-derived factor-1 (SDF-1) in lymph node metastasis of gastric carcinoma.METHODS:In 40 cases of gastric cancer,expression of CXCR4 mRNA in cancer and normal mucous membrane and SDF-1 mRNA in lymph nodes around the stomach was detected using quantitative polymerase chain reaction (PCR) (TaqMan) and immunohistochemistric assay.SGC-7901 and MGC80-3 cancer cells were used to investigate the effect of SDF-1 on cell proliferation and migration.RESULTS:Quantitative reverse transcription PCR and immunohistochemistry revealed that the expression level of CXCR4 in gastric cancer was significantly higher than that in normal mucous membrane (1.6244 ± 1.3801 vs 1.0715 ± 0.5243,P < 0.05).The expression level of CXCR4 mRNA in gastric cancer with lymph node metastasis was also significantly higher than that without lymph node metastasis (0.823 ± 0.551 vs 0.392 ± 0.338,P < 0.05).CXCR4 expression was significantly related to poorly differentiated,high tumor stage and lymph node metastasis.Significant differences in the expression level of SDF-1 mRNA were found between lymph nodes in metastatic gastric cancer and normal nodes (0.5432 ± 0.4907 vs 0.2640 ± 0.2601,P < 0.05).The positive expression of SDF-1 mRNA in lymph nodes of metastatic gastric cancer was consistent with the positive expression of CXCR4 mRNA in gastric cancer (r=0.776,P < 0.01).Additionally,human gastric cancer cell lines expressed CXCR4 and showed vigorous proliferation and migratory responses to SDF-1.AMD3100 (a specific CXCR4 antagonist) was also found to effectively reduce the migration of gastric cancer cells.CONCLUSION:The CXCR4/SDF-1 axis is involved in the lymph node metastasis of gastric cancer.CXCR4 is considered as a potential therapeutic target in the treatment of gastric cancer.

CXCR7/CXCL12 axis is involved in lymph node and liver metastasis of gastric carcinoma

AIM To investigate the role of CXC chemokine receptor (CXCR)-7 and CXCL12 in lymph node and liver metastasis of gastric carcinoma. METHODS In 160 cases of gastric cancer, the expression of CXCR7 and CXCL12 in tumor and matched tumoradjacent non-cancer tissues, in the lymph nodes around the stomach and in the liver was detected using immunohistochemistry to analyze the relationship between CXCR7/CXCL12 expression and clinicopathological features and to determine whether CXCR7 and CXCL12 constitute a biological axis to promote lymph node and liver metastasis of gastric cancer. Furthermore, the CXCR7 gene was silenced and overexpressed in human gastric cancer SGC-7901 cells, and cell proliferation, migration and invasiveness were measured by the MTT, wound healing and Transwell assays, respectively. RESULTS CXCR7 expression was up-regulated in gastric cancer tissues (P = 0.011). CXCR7/CXCL12 expression was significantly related to high tumor stage and lymph node (r = 0.338, P = 0.000) and liver metastasis (r = 0.629, P = 0.000). The expression of CXCL12 in lymph node and liver metastasis was higher than that in primary gastric cancer tissues (chi(2) = 6.669, P = 0.010; chi(2) = 25379, P = 0.000), and the expression of CXCL12 in lymph node and liver metastasis of gastric cancer was consistent with the positive expression of CXCR7 in primary gastric cancer (r = 0.338, P = 0.000; r = 0.629, P = 0.000). Overexpression of the CXCR7 gene promoted cell proliferation, migration and invasion. Silencing of the CXCR7 gene suppressed SGC-7901 cell proliferation, migration and invasion. Human gastric cancer cell lines expressed CXCR7 and showed vigorous proliferation and migratory responses to CXCL12. CONCLUSION The CXCR7/CXCL12 axis is involved in lymph node and liver metastasis of gastric cancer. CXCR7 is considered a potential therapeutic target for the treatment of gastric cancer.

CXCR4 antagonist AMD3100 attenuates colonic damage in mice with experimental colitis

AIM:To investigate the effects of the chemokine stromal cell-derived factor-1(CXCL12) receptor(CXCR4) antagonist AMD3100 on colonic inflammation and epithelial barrier in dextran sulfate sodium(DSS)-induced colitis in mice.METHODS:Experimental colitis was induced by administration of 5% DSS for 7 d,and assays performed on intestinal segments from the ileocecal valve to the anus.Colonic morphology was examined by hematoxylin and eosin staining.Colonic cytokines were determined by enzyme-linked immunosorbent assay.Myeloperoxidase(MPO) activity(indicator of inflammatory infiltration) was observed spectrophotometrically.Gut permeability was assessed by mucosal-to-serosal clearance of fluorescein isothiocyanate-conjugated dextran 4000(FD4) in everted gut sacs.The apoptosis of colonic epithelium was assessed by Hoechst-33342 staining.To further elucidate the role of CXCR4 in colonic inflammation,we also investigated the effect of AMD3100 on migration and cytokine production of isolated peripheral blood mononuclear cells(PBMCs).RESULTS:DSS-induced colitis was characterized by morphologic changes,as well as increased colonic cytokines,inflammatory infiltration,epithelial apoptosis,and intestinal permeability in mice.In AMD3100-treated mice,epithelial destruction,inflammatory infiltration,and submucosal edema were markedly reduced;colonic tumor necrosis factor-α(TNF-α),interleukin-6(IL-6) and interferon-γ(IFN-γ) levels,as well as MPO activity were significantly decreased.Increased intestinal permeability in DSS-treated mice was signif icantly reduced by AMD3100.The number of apoptotic cells in colitis mice was markedly increased after DSS administration,and decreased when treated with the CXCR4 antagonist AMD3100.In pre-activated PBMCs,CXCL12 stimulation signif icantly increased the migration of PBMCs,and was inhibited by AMD3100.Moderately increased TNF-α,IL-6,and IFN-γ from CXCL12-treated PBMCs were also reduced by AMD3100.CONCLUSION:The CXCR4 antagonist AMD3100 exerts therapeutic effects on experimental colitis by inhibiting colonic inflammation and enhancing epithelial barrier integrity.

Functions and mechanisms of chemokine receptor 7 in tumors of the digestive system

Chemokine(C-X-C motif)receptor 7(CXCR7),recently termed ACKR3,belongs to the G protein-coupled cell surface receptor family,binds to stromal cellderived factor-1[SDF-1,or chemokine(C-X-C motif)ligand 12]or chemokine(CX-C motif)ligand 11,and is the most common chemokine receptor expressed in a variety of cancer cells.SDF-1 binds to its receptor chemokine(C-X-C motif)receptor 4(CXCR4)and regulates cell proliferation,survival,angiogenesis and migration.In recent years,another new receptor for SDF-1,CXCR7,has been discovered,and CXCR7 has also been found to be expressed in a variety of tumor cells and tumor-related vascular endothelial cells.Many studies have shown that CXCR7 can promote the growth and metastasis of a variety of malignant tumor cells.Unlike CXCR4,CXCR7 exhibits a slight modification in the DRYLAIV motif and does not induce intracellular Ca^2+release following ligand binding,which is essential for recruiting and activating G proteins.CXCR7 is generally thought to work in three ways:(1)Recruitingβ-arrestin 2;(2)Heterodimerizing with CXCR4;and(3)Acting as a“scavenger”of SDF-1,thus lowering the level of SDF-1 to weaken the activity of CXCR4.In the present review,the expression and role of CXCR7,as well as its prognosis in cancers of the digestive system,were investigated.

Expression of CXCL12-CXCR4 in osteosarcoma and its correlation with angiogenesis

Objective The expression of CXCL12(stromal cell-derived factor-1)-CXCR4(chemokine receptors-4) in osteosarcoma and its role in angiogenesis were examined.Methods The expression of CXCR4 and CXCL12 in 40 cases of osteosarcoma was detected by immunohistochemistry and real-time fluorescence quantitative PCR.The expression of CD34 in osteosarcoma was detected by immunohistochemistry.Morphometric image analysis was performed to measure microvessel density(MVD).Additionally,the relationship between CXCL12 and CXCR4 expression and MVD of osteosarcoma and pulmonary metastasis were analyzed.Results The positive rates of CXCL12 and CXCR4 protein expression in osteosarcoma were 40.0%(16/40) and 60.0%(24/40),respectively.Fluorescence quantitative real-time PCR indicated that the expression level of CXCR4 m RNA in pulmonary metastatic osteosarcoma was higher than that in nonpulmonary metastatic osteosarcoma(P < 0.01).The level of MVD in pulmonary metastatic osteosarcoma was higher than that in non-pulmonary metastatic osteosarcoma(P < 0.01).Conclusion The expression level of CXCR4 was significantly associated with pulmonary metastasis and angiogenesis of osteosarcoma.

SDF-1/CXCR4 expression in bladder cancer tissue and the correlation with negative costimulatory molecule PD-L1, cell apoptosis and invasion

Objective:To study the SDF-1/CXCR4 expression in bladder cancer tissue and the correlation with negative costimulatory molecule PD-L1, cell apoptosis and invasion.Methods: A total of 118 cases of bladder cancer tissue and para-carcinoma tissue surgically removed in our hospital between May 2014 and May 2016 were selected as the research samples, the RNA was extracted and then reverse-transcribed into cDNA, and the expression levels of SDF-1/CXCR4, PD-L1/PD-1, cell apoptosis-related molecules and cell invasion-related molecules were detected.Results: SDF-1 and CXCR4 mRNA expression in bladder cancer tissue were significantly higher than those in para-carcinoma tissue;PD-L1, PD-1, Rec1, Survivin, MRPS5, Nanog, BCAPP2Ac, TRPM8, TRPV2, ILK,β-catenin and GUGBP1 mRNA expression in bladder cancer tissue were significantly higher than those in para-carcinoma tissue and positively correlated with SDF-1 and CXCR4 mRNA expression.Conclusion:Highly expressed SDF-1/CXCR4 in bladder cancer tissue are closely related to the high expression of negative costimulatory molecule PD-L1, pro-proliferation molecules and pro-invasion molecules, and SDF-1/CXCR4 can promote the immune escape, proliferation and invasion of bladder cancer cells.

Hyperbaric oxygen improves functional recovery of rats after spinal cord injury via activating stromal cell-derived factor-1/CXC chemokine receptor 4 axis and promoting brain-derived neurothrophic factor expression

Background: Spinal cord injury (SCI) is a worldwide medical concern. This study aimed to elucidate the mechanism underlying protective effect of hyperbaric oxygen (HBO) against SCI-induced neurologic defects in rats via exploring the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) axis and expression of brain-derived neurotrophic factor (BDNF). Methods: An acute SCI rat model was established in Sprague-Dawley rats using the Allen method. Sixty rats were divided into four groups (w = 15 in each group): sham-operated, SCI, SCI treated with HBO (SCI + HBO), and SCI treated with both HBO and AMD3100 (an antagonist of CXCR4;SCI + HBO + AMD) groups. The rats were treated with HBO twice a day for 3 days and thereafter once a day after the surgery for up to 28 days. Following the surgery, neurologic assessments were performed with the Basso-Bettie-Bresnahan (BBB) scoring system on postoperative day (POD) 7, 14, 21, and 28. Spinal cord tissues were harvested to assess the expression of SDF-1, CXCR4, and BDNF at mRNA and protein levels, using quantitative real-time polymerase chain reaction, Western blot analysis, and histopathologic analysis. Results: HBO treatment recovered SCI-induced descent of BBB scores on POD 14,(1.25±0.75 vs. 1.03 ±0.66, P< 0.05), 21 (5.27± 0.89 vs. 2.56± 1.24, P< 0.05), and 28 (11.35±0.56 vs. 4.23± 1.20, P<0.05) compared with the SCI group. Significant differences were found in the mRNA levels of SDF-1 (mRNA: day 21, SCI + HBO vs. SCI + HBO + AMD, 2.89± 1.60 vs. 1.56±0.98, P<0.05), CXCR4 (mRNA: day 7, SCI + HBO vs. SCI, 2.99± 1.60 vs. 1.31 ±0.98, P<0.05;day 14, SCI + HBO vs. SCI + HBO + AMD, 4.18± 1.60 vs. 0.80±0.34, P<0.05;day 21, SCI + HBO vs. SCI, 2.10±1.01 vs.1.15±0.03, P<0.05), and BDNF (mRNA: day 7, SCI + HBO vs. SCI, 3.04±0.41 vs. 2.75±0.31, P<0.05;day 14, SCI + HBO vs. SCI, 3.88± 1.59 vs. 1.11 ±0.40, P<0.05), indicating the involvement of SDF-1/CXCR4 axis in the protective effect of HBO. Conclusions: HBO might promote the recovery of neurologic function after SCI in rats via activating the SDF-1/CXCR4 axis and promoting BDNF expression.

Maraviroc promotes recovery from traumatic brain injury in mice by suppression of neuroinflammation and activation of neurotoxic reactive astrocytes

Neuroinflammation and the NACHT,LRR,and PYD domains-containing protein 3 inflammasome play crucial roles in secondary tissue damage following an initial insult in patients with traumatic brain injury(TBI).Maraviroc,a C-C chemokine receptor type 5 antagonist,has been viewed as a new therapeutic strategy for many neuroinflammatory diseases.We studied the effect of maraviroc on TBI-induced neuroinflammation.A moderate-TBI mouse model was subjected to a controlled cortical impact device.Maraviroc or vehicle was injected intraperitoneally 1 hour after TBI and then once per day for 3 consecutive days.Western blot,immunohistochemistry,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)analyses were performed to evaluate the molecular mechanisms of maraviroc at 3 days post-TBI.Our results suggest that maraviroc administration reduced NACHT,LRR,and PYD domains-containing protein 3 inflammasome activation,modulated microglial polarization from M1 to M2,decreased neutrophil and macrophage infiltration,and inhibited the release of inflammatory factors after TBI.Moreover,maraviroc treatment decreased the activation of neurotoxic reactive astrocytes,which,in turn,exacerbated neuronal cell death.Additionally,we confirmed the neuroprotective effect of maraviroc using the modified neurological severity score,rotarod test,Morris water maze test,and lesion volume measurements.In summary,our findings indicate that maraviroc might be a desirable pharmacotherapeutic strategy for TBI,and C-C chemokine receptor type 5 might be a promising pharmacotherapeutic target to improve recovery after TBI.

Effects of insulin-like growth factor-1 on the properties of mesenchymal stem cells in vitro

Objective:To explore the effects of insulin-like growth factor-1(IGF-1) on migration,proliferation and differentiation of mesenchymal stem cells(MSCs).Methods:MSCs were obtained from Sprague-Dawley rats by a combination of gradient centrifugation and cell culture techniques and treated with IGF-1 at concentrations of 5-20 ng/ml.Proliferation of MSCs was determined as the mean doubling time.Expression of CXC chemokine receptor 4(CXCR4) and migration property were determined by flow cytometry and transwell migration essay,respectively.mRNA expression of GATA-4 and collagen II was determined by reverse transcription-polymerase chain reaction(RT-PCR).Results:The mean doubling time of MSC proliferation was decreased,and the expression of CXCR4 on MSCs and migration of MSCs were increased by IGF-1,all in a dose-dependent manner,while the optimal concentration of IGF-1 on proliferation and migration was different.IGF-1 did not affect the expression of GATA-4 or collagen II mRNA.Conclusions:IGF-1 dose-dependently stimulated the proliferation of MSCs,upregulated the expression of CXCR4,and accelerated migration.There was no apparent differentiation of MSCs to cardiomyocytes or chondrocytes after culturing with IGF-1 alone.

Mobilization of hematopoietic progenitor cells in patients with liver cirrhosis

AIM:To test the hypothesis that liver cirrhosis is associated with mobilization of hematopoietic progenitor cells. METHODS:Peripheral blood samples from 72 patients with liver cirrhosis of varying etiology were analyzed by flow cytometry.Identified progenitor cell subsets were immunoselected and used for functional assays in vitro. Plasma levels of stromal cell-derived factor-1(SDF-1) were measured using an enzyme linked immunosorbent assay.RESULTS:Progenitor cells with a CD133 + /CD45 + CD14 + phenotype were observed in 61%of th patients.Between 1%and 26%of the peripheral bloo mononuclear cells(MNCs)displayed this phenotype Furthermore,a distinct population of c-kit + progenito cells(between 1%and 38%of the MNCs)could b detected in 91%of the patients.Additionally,18% of the patients showed a population of progenito cells(between 1%and 68%of the MNCs)that wa characterized by expression of breast cancer resistanc protein-1.Further phenotypic analysis disclosed tha the circulating precursors expressed CXC chemokin receptor 4,the receptor for SDF-1.In line with thi finding,elevated plasma levels of SDF-1 were presen in all patients and were found to correlate with th number of mobilized CD133 + progenitor cells.

Interleukin-1β-induced inflammatory signaling in C20 human microglial cells

Aim: Increased inflammatory signaling in microglia is implicated in the pathogenesis of neurodegenerative diseases, trauma, psychiatric disorders, and anxiety/depression. Understanding inflammatory signaling in microglia is critical for advancing treatment options. Studying rodent-derived microglia has yielded substantial information, yet, much remains to better understand inflammatory signaling in human microglia. Hence, there is great interest in developing immortalized human microglial cell lines. The C20 human microglial cell line was recently developed and our primary objective was to advance our knowledge of inflammatory signaling in these cells. Methods: Expression of the microglia specific marker transmembrane protein 119 (TMEM119) was assessed by western blot analysis. Lipopolysaccharide (LPS)- and interleukin-1β (IL-1β)-induced cytokine/chemokine expression was determined by ELISA. Phosphorylation of inhibitory kappa B alpha (IκBα), nuclear factor (NF)-κB p65, and p38 mitogen-activated protein kinase (p38 MAPK) was measured by western blot analysis. Results: TMEM119 was expressed in unstimulated C20 cells, and to a greater extent in IL-1β-stimulated cells. IL-1βsignificantly induced IL-6, monocyte chemoattractant protein-1/CCL2, and interferon-γ inducible protein 10/CXCL10 expression. LPS induced CCL2 expression, but not IL-6 or CXCL10 expression. IL-1β induced inflammatory signaling as indicated by increased phosphorylation of IκBα, NF-κB p65 and p38 MAPK. Conclusion: We provide the first evidence that C20 microglia express TMEM119. This is the initial report of IL-1β-induced activation of IκBα, NF-κB p65, and p38 MAPK and subsequent CXCL10, CCL2 and IL-6 secretion in C20cells. These findings advance our understanding of inflammatory signaling in C20 cells and support the value of this cell line as a research tool.