AMD3100 Research Articles

Distribution of neutrophil and monocyte/macrophage populations induced by the CXCR4 inhibitor AMD3100 in blood and periodontal tissue early after periodontitis induction.

CXCR4, a CXCL12 receptor, is expressed on epithelial cells, fibroblasts, and inflammatory cells. The CXCR4-CXCL12 interaction is related to the migration of neutrophils and monocytes/macrophages. Periodontitis, an inflammatory disease mainly caused by gram-negative bacteria, is characterized by infiltration of circulating inflammatory cells and alveolar bone (AB) loss. To investigate whether CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages early after periodontitis induction, we examined the effects of AMD3100 (AMD), a CXCR4 antagonist, in ligature-induced periodontitis mice and LPS-injected air pouch mice. The periodontitis study was accomplished in control (C), periodontitis (P), and P+AMD groups. Periodontitis was induced by ligation of the mandibular first molar. AMD was intraperitoneally administered daily beginning the day before ligation until sacrifice on the third day after ligation. The air pouch study was accomplished in C, lipopolysaccharide (LPS), and LPS+AMD groups. Air pouches on mice backs were formed by subcutaneous injection of sterilized air. AMD was administered and then LPS was injected into the air pouch. For the detection of neutrophils and monocytes/macrophages in blood and air pouch exudates, flow cytometry was performed with anti-Ly6G/anti-CD11b antibodies (Abs) and anti-CD115 Ab, respectively. In periodontal tissue, Ly6G+ cells and CD115+ cells were counted by immunohistological analysis. AB loss was estimated by the periodontal ligament area in the furcation. In the periodontitis study, the P group showed higher numbers of Ly6G+ cells and CD115+ cells in blood and periodontal tissue than the C group. The P + AMD group showed a greater number of Ly6G+ cells and CD115+ cells in blood, but not in periodontal tissue compared to the P group. There was no difference in AB loss between the P and P + AMD groups. In the air pouch study, the LPS group had higher levels of Ly6G+ CD11b+ cells and CD115+ cells in both blood and exudates than the C group. The number of these cells in the LPS+AMD group was higher in blood than in the LPS group, but not in the exudates. The CXCR4 antagonist further increased neutrophil and monocyte/macrophage populations in the blood, but did not alter the levels in the periodontal tissue and exudates in mice with periodontitis and LPS-injected air pouches. These results suggest that during inflammatory conditions such as periodontitis, CXCR4 is involved in the distribution of neutrophils and monocytes/macrophages in the blood, but not in inflamed peripheral tissues.

Prec-O1-04 - CXCR4/CXCL12 axis as a potential therapeutic target in mycosis fungoides: an in-vitro study

The C-X-C chemokine receptor 4 (CXCR4) is overexpressed in the tumor cells of many solid and hematologic neoplasms. Its ligand, C-X-C chemokine ligand 12 (CXCL12), is secreted by cells in the tumor microenvironment, mostly by stromal fibroblasts. The engagement of CXCL12 to CXCR4 results in activation of several intracellular signal transduction pathways, including, the proapoptotic p38, the prosurvival AKT, and the chemotaxis mediator ERK. The balance between activation of AKT and activation of p38 will determine the fate of cell viability. Plerixafor, AMD3100 (AMD), is a CXCR4 antagonist that reversibly blocks the binding of CXCR4 to CXCL12. AMD is already in clinical use as bone marrow stem cell or progenitor cell mobilizers. AMD and other CXCR4 inhibitors are investigated either alone or in combination with other systemic treatments in numerous clinical trials in cancer. CXCR4 and CXCL12 are known to be overexpressed in MF skin biopsies, but the exact differential contribution of the lymphoma vs the reactive T-cells is not clear. We previously have shown that primary MF-fibroblast culture (MF-Fs) established from MF biopsies, secrete high CXCL12 which promotes the migration of MyLa (MF cell line), protect them from chemotherapy and both effects were suppressed by AMD. We studied the role of CXCR4 as essential key protein in the survival of MF, and the effect and mechanism of CXCR4 antagonist on MyLa cell viability. We found that MyLa cells express higher CXCR4 vs CD3+CD4+ PBMCs from healthy donor (FACS). Single cell analysis of skin MF biopsy by flow mass cytometry (CyTOF) revealed sub-population of T- cells expressing the highest intensity of CXCR4, CD30, PD-1, and PDL-1 compared to all other immune cells, and therefore are suspected as the lymphoma cells overexpressing CXCR4. AMD was toxic to MyLa cells with higher toxicity to MyLa co-cultured with MF-Fs than MyLa alone (MTT viability assay, trypan blue staining), and promotes their death through apoptosis (FACS). AMD-induced death was selective to MyLa cells compared to healthy PBMCs, when both were co-cultured with MF-Fs. The phosphorylation of p38 and AKT in MyLa co-cultured with MF-Fs were reduced upon AMD treatment (Western blot). Spheroids of MyLa cells with MF-Fs were established to show the toxic effect of AMD in 3D co-culture model. The intracellular signaling pathways mediated by AMD was studied by phospho-proteomic analysis of MyLa cells co-cultured with MF-Fs. In sum, we showed the overexpression of CXCR4 in subpopulation of T cells suspected as the lymphoma cells in skin biopsy, and the pivotal role of overexpressed CXCR4 in the survival of MF cell line. The apoptotic effect mediated by AMD indicates that disrupting the interaction between stroma derived CXCL12 and MF derived CXCR4 might be a new therapeutic option in MF.

CXCR4-Receptor-Targeted Liposomes for the Treatment of Peritoneal Fibrosis.

Peritoneal fibrosis (PF) is a common complication of long-term peritoneal dialysis (PD). It is considered as the main reason for dialysis inadequacy and PD withdrawal. Transforming growth factor beta (TGF-β) regulates the expression of stromal cell-derived factor 1 (SDF-1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) on human peritoneal mesothelial cells (HPMCs), resulting in an increased migratory potential of HPMCs and extracellular matrix (ECM) deposition in the scar tissue and eventually fibrosis. Because SDF-1α/CXCR4 activation has a vital role in the pathogenesis of PF, codelivery of a CXCR4-receptor targeting agent with an antifibrotic agent in a single nanocarrier can be a promising strategy for treating PF. Here, for the first time, AMD3100 (AMD), a CXCR4-receptor antagonist, was coformulated with sulfotanshinone IIA sodium (STS IIA) into a liposome (STS-AMD-Lips) to develop a CXCR4 receptor targeting form of combination therapy for PF. CXCR4 targeting increased the ability of liposomes to target fibrotic peritoneal mesothelial cells overexpressing CXCR4 and facilitated the ability of STS IIA treatment at the fibrotic site. The liposome had an average diameter of 103 nm with encapsulated efficiencies of above 50%. The in vivo studies confirmed the reversal of PD solution-induced epithelial-to-mesenchymal transition by STS-AMD-Lips in HPMCs. The in vivo studies also revealed the precise biodistribution of the liposomes to peritoneum. Significant reduction of the morphological lesions and decreased level of ECM proteins were observed in rats treated with STS-AMD-Lips, proving that the liposomal nanocarrier has excellent ability to reverse PF. It has been concluded that the STS-AMD-Lips exhibit specific peritoneal targeting ability and could be used to improve STS-AMD combination delivery for the treatment of PF.

CXCR4-targeted liposomal mediated co-delivery of pirfenidone and AMD3100 for the treatment of TGFβ-induced HSC-T6 cells activation.

Background: Liver fibrosis is a chronic liver disease associated with an excessive accumulation of extracellualr matrix (ECM) proteins which ultimately lead to cirrohosis and hepatocellular carcinoma.Purpose: Liver fibrosis therapies that use combination approaches with the ability to affect multiple disease pathways have proven higher efficacies. This study aimed at optimizing and characterizing the co-encapsulation of pirfenidone (PF) and AMD3100 (AMD) into CXCR4-targeted combination liposomes (CTC liposome) for CXCR4 targeting, and the inhibition of major molecular culprits ie α-SMA, CXCR4, TGFβ, and P-p38 involved in liver fibrosis in-vitro.Methods: The CTC liposomes were prepared using the thin-film hydration method. The concentration of encapsulated AMD and PF was measured by HPLC and UV spectrophotometry, respectively. Tramsmission electron microscopy (TEM) was used to determine the liposomal morphology. The CXCR4 targeting ability was determined by CXCR4 redistribution assay. Confocal microscopy and flowcytometry were used to determine the CXCR4 mediated cell uptake. The apoptosis inducing and protein downreguating ability of CTC liposomes were determined by apoptosis assay and western blot analysis, respectively. In-vivo biodistribution and Hoechst staining were used to confirm the feasibility of CTC liposome for the in-vivo applications and drug targeted accumulation, respectively.Results: The TEM studies revealed that CTC liposomes were spherical in shape. The cumulative release of AMD and PF from CTC liposome was 67% and 84%, respectively, at 48 h. Compared to the free drug counterparts, encapsulated drugs displayed higher cell viability. The CXCR4 redistribution assay confirmed the CXCR4 targeting and antagonistic ability of CTC liposomes. The CTC liposomes were internalized more effectively via caveolae-mediated endocytic pathways. CTC liposomes displayed aggressive apoptosis (87.3%) in TGFβ-induced activated HSC-T6 cells suggesting a propensity to fibrosis regression. Also, CTC liposomes significantly reduced α-SMA (65%), CXCR4 (77%), TGFβ (89%), and P-p38 (66%) expressions, better than free drugs. CTC@IR780 liposomes (CTC liposomes incorporating IR780 dye) were more accumulated in fibrotic livers compared to free IR780, as judged by in-vivo imaging, biodistribution analysis, and Hoechst staining. These findings suggest that this simple and stable CTC liposomal system holds a great promise for the treatment and prevention of liver fibrosis.

Role of CXCR4 in dorsal root ganglia in incisional pain in rats

Objective To evaluate the role of C-X-C chemokine receptor type 4 (CXCR4) in the dorsal root ganglia (DRG) in incisional pain in rats. Methods Thirty-two male Sprague-Dawley rats, aged 7-10 weeks, weighing 250-300 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table method: sham operation group (group S), CXCR4 antagonist AMD3100 plus sham operation group (group A+ S), incisional pain group (group I) and CXCR4 antagonist AMD3100 plus incisional pain group (group A+ I). Rats were anesthetized with sevoflurane.AMD3100 20 μg (in 10 μl of normal saline) was intrathecally injected, and no incision was made 30 min later in group A+ S.A 1-cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the left hindpaw in group I. AMD3100 20 μg (in 10 μl of normal saline) was intrathecally injected, and 30 min later the model of incisional pain was established in group A+ I.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before surgery and 2, 4, 8, 16 and 24 h after surgery.The rats were sacrificed after the last measurement of pain threshold and the DRGs of the lumbar segment (L4-6) were removed for detecting the expression of CXCR4, phosphorylated extracellular regulated protein kinase (p-ERK) and total ERK (t-ERK) by Western blot. Results Compared with group S, MWT was significantly decreased and TWL was shortened at T1-5 in group I and group A+ I, and the expression of CXCR4 and p-ERK in DRGs was significantly up-regulated (P 0.05). Compared with group I, MWT was significantly increased and TWL was prolonged at T1-5, the expression of CXCR4 and p-ERK in DRGs was down-regulated (P 0.05). Conclusion CXCR4 in DRGs is involved in incisional pain, and the mechanism may be related to activating ERK1/2 signaling pathway in rats. Key words: Pain, postoperative; Dorsal root ganglia; Receptors, CXCR4

Mechanism of tissue-engineered bone recruiting endogenous mesenchymal stem cells towards bone regeneration

Objective To investigate the mechanism of implanted tissue-engineered bone (TEB) recruiting endogenous mesenchymal stem cells (BMSCs) towards bone regeneration after traumatic bone defect. Methods In vivo experiments: 2 mm of diaphysis and periosteum were removed from the middle of the femoral shaft in 8 week old FVB/N mice to form a large segment of bone defect. Demineralized bone matrix (DBM) and TEB were implanted into the defect area and fixated. All mice were randomly divided into DBM group (n=18) and TEB group (n=18). The results were observed 24 hours after implantation: (1) flow cytometry was used to evaluate the number of mobilized host BMSCs into the blood; (2) non-invasive bioluminescent imaging was used to observe the ability of two groups in recruiting mouse bone marrow derived mesenchymal stem cells (mBMSCs) in peripheral blood to the defect area; (3) ELISA was used to evaluate the stromal cell-derived factor 1 (SDF-1) content in peripheral blood of two groups. In vitro experiments: (1) transwell assay was conducted to evaluate the ability of SDF-1 (100 ng/ml) in promoting the migration of human bone marrow derived mesenchymal stem cells (hBMSCs). SDF-1/C-X-C motif chemokine receptor-4 (CXCR4) pathway was blocked by the selective CXCR4 antagonist Plerixafor (AMD3100). The experimental groups were divided into control group, SDF-1 group, and SDF-1+ AMD3100 group. (2) The co-culture system of human umbilical vein endothelial cells (hUVECs) and hBMSCs was established, and cells were stimulated by SDF-1. The experimental groups were divided into hBMSCs group, hBMSCs+ hUVECs group, and hBMSCs+ hUVECs (AMD3100 pretreatment) group. Transwell assays were used to compare the migration of hBMSCs in each group. ELISA was used to detect the concentration of hepatocyte growth factor (HGF) in the co-culture supernatant. (3) In vitro cultured hUVECs were stimulated by SDF-1 and SDF-1/CXCR4 pathway was antagonized by AMD3100. The experimental groups were divided into control group, SDF-1 group, and SDF-1+ AMD3100 group. Quantitative real-time polymerase chain reaction (qRT PCR) was used to evaluate the expression of HGF in each group. Results In vivo experiments: 24 h after transplantation, the number of BMSCs and SDF-1 concentration in the TEB group were significantly higher than those in the DBM group (P 0.05); (3) qRT-PCR showed that the expression of HGF was significantly increased in the SDF-1 group compared with the control group (P<0.05). After antagonizing SDF-1/CXCR4, HGF expression in the SDF-1+ AMD3100 group was significantly lower than that in the SDF-1 group. Conclusions TEB transplantation in traumatic bone defect can significantly increase the concentration of chemokine SDF-1 in vivo and effectively promote the mobilization of endogenous MSCs and recruitment of circulating MSCs. SDF-1 not only directly promotes the migration of hBMSCs through SDF-1/CXCR4 pathway, but also up-regulates the expression and secretion of HGF in vascular cells to further amplify the chemotactic effect of SDF-1 on hBMSCs. Key words: Tissue engineering; Mesenchymal stem cells; Stromal cell-derived factor-1

Role of chemokine receptor CXCR4 in sorafenib resistance of renal cell carcinoma

Objective To investigate the role and possible mechanism of Chemokine receptor CXCR4 in the drug resistance of sorafenib in renal cell carcinoma. Methods 786-O cells were inoculated into the anterior sciatic region of nude mice subcutaneously, 5×106 cells per point. The mice were given normal saline and sorafenib intragastric (80 mg/kg, 1 time/day) when the transplanted tumor volume reached about 100 mm3. The tumor volume in the saline group was more than 1 500 mm3 at the 5th week, and the tumor was taken as the control tissue. Sorafenib group tumors started to grow accelerately at week 8, and the tumor volume was more than 1 500 mm3 at week 13. The 13th week tumors were used as resistant tissue. The expression of CXCR4 in control tissues and drug resistant tissues was detected by real-time quantitative PCR, western blotting and immunohistochemistry. The pcDNA3.1-CXCR4 plasmid was constructed and transfected into 786-O cells. The expression of CXCR4 was detected by real-time quantitative PCR and western blotting. The drug reactivity of the cells was measured by CCK-8 and monoclonal assay to compare the drug resistance of the control group, CXCR4 overexpression group and CXCR4 overexpression + CXCR4 inhibitor AMD3100 group. The phosphorylation of PKB, ERK and STAT3 in the control group, the sorafenib alone group, the overexpressing CXCR4+ sorafenib group and the overexpressing CXCR4+ sorafenib+ AMD3100 group were determined by Western blotting. Results Compared with the control tissues, the mRNA levels of CXCR4 in the drug-resistant tissues increased (3.22±0.23) times, and the levels of protein expression increased (2.33±0.47) according to western blotting, the differences were statistically significant (P<0.01). After overexpression of CXCR4, the mRNA expression of CXCR4 increased (78.3±5.3) times, and the protein expression level increased (2.80±0.95) times, and the differences were statistically significant (P<0.01), indicating that the expression model was established successfully. The drug response curves of the control group, CXCR4 overexpression group and CXCR4 overexpression+ AMD3100 group on sorafenib were measured by cck8 method, and the IC50 was (7.5±0.8) μmol/L, (10.3±0.7) μmol/L, (5.7±0.6) μmol/L, the differences were statistically significant (P<0.05); The numbers of clones formed in the above three groups were 26±5, 56±12 and 42±9, respectively. The differences were statistically significant (P<0.05). Sorafenib could reduce the phosphorylation of PKB, ERK and STAT3, and overexpression of CXCR4 could reverse the inhibition of phosphatidylation of PKB, ERK and STAT3 by sorafenib. After inhibition of chemokine receptor CXCR4 activity by AMD3100, PKB, ERK, STAT3 phosphorylation was re-suppressed. Conclusions CXCR4 can promote renal cell carcinoma sorafenib resistance. The expression of CXCR4 increased in secondary resistant tumor tissue increased; CXCR4 may promote drug resistance by activating the cell viable pathway. The inhibition of CXCR4 signaling pathway is expected to improve the therapeutic effect of sorafenib in renal cell carcinoma. Key words: Renal cell carcinoma; Chemokine receptor CXCR4; Sorafenib; Drug resistant

Role of SDF-1/CXCR4 axis in the invasion and migration of human choriocarcinoma cells and its significance in preeclampsia

Objective To investigate the effects of stromal cell-derived factor 1 (SDF-1) and an CXC chemokine receptor 4 (CXCR4) antagonist (AMD3100) on the invasion and migration capabilities of the huaman choriocarcinoma cell line JAR for further elucidating the role of SDF-1/CXCR4 axis in the pathogenesis of preeclampsia. Methods JAR cells were divided into four groups: SDF-1 group (treated with 50 ng/ml of SDF-1), SDF-1+ AM3100 mixed group (first treated with 100 ng/ml of AMD3100 for 2 hours and then treated with 50 ng/ml of SDF-1), AMD3100 group (treated with 100 ng/ml of AMD3100) and blank control group (without any treatment). RT-PCR was performed to detect the expression of CXCR4 at mRNA level in JAR cells. Western blot assay was used to measure the expression of CXCR4 and p-AKT at protein level. MTT assay was used to analyze the effects of different concentrations of SDF-1 (10, 30, 50 and 100 ng/ml) on the proliferation of JAR cells at different time points (0, 24, 48, 72 h). Transwell invasion assay and wound-healing assay were used to test the changes in invasion and migration capabilities of JAR cells after different treatments. Results (1) Results of the RT-PCR showed that the expression of CXCR4 at mRNA level in JAR cells was increased in the SDF-1 group (1.839±0.083) as compared with that in the blank control group (1.372±0.086), AMD3100 group (0.694±0.045) or SDF-1+ AM3100 mixed group (0.703±0.093). Moreover, the differences between the SDF-1 group and the other three groups were statistically significant (F=30.67, P<0.05). Compared with the blank control group, the expression of CXCR4 at mRNA level in JAR cells was decreased in the AMD3100 group (P<0.01). (2) Results of the Western blot assay showed that the expression of CXCR4 and p-AKT at protein level in JAR cells were enhanced in the SDF-1 group as compared with that in the blank control group, AMD3100 group or SDF-1+ AM3100 mixed group. Compared with the blank control group, the expression of CXCR4 and p-AKT at protein level in JAR cells were inhibited in the AMD3100 group. (3) Results of the MTT assay showed that SDF-1, especially at the concentration of 50 ng/ml, could enhance the proliferation of JAR cells (P<0.05) and its best effect on proliferation was seen at 48 h. (4) Results of the Transwell invasion assay showed that the number of transmembrane cells in the SDF-1 group (70.49±2.42) was more than that in the blank control group (54.36±2.26), AMD3100 group (21.68±8.31), or SDF-1+ AMD3100 mixed group (28.18±4.61). The differences between the SDF-1 group and the other three groups were statistically significant (F=116.26, P<0.01). Compared with the blank control group, the number of transmembrane cells was reduced in the AMD3100 group (P<0.05). (5) Results of the wound-healing assay showed that the relative migration distance was increased in the SDF-1 group (1.162±0.034) as compared with that in the blank control group (0.823±0.101), AMD3100 group (0.160±0.047), or SDF-1+ AMD3100 mixed group (0.183±0.064). The differences between the SDF-1 group and the other three groups were statistically significant (F=30.500, P<0.05). Compared with the blank control group, the relative migration distance was decreased in the AMD3100 group (P<0.01). Conclusion The invasion and migration of huaman choriocarcinoma JAR cells can be enhanced by SDF-1, but inhibited by AMD3100. This study indicates that the blocked biological axis of SDF-1/CXCR4 may play an important role in the pathogenesis of preeclampsia through inducing abnormal activation of PI3K/AKT pathway, which results in inhibited invasion and migration of trophoblast cells and placenta abnormality. Key words: SDF-1/CXCR4 axis; Invasion; Migration; PI3K/AKT pathway; Trophoblast cell; Preeclampsia

The effects of stromal cel1-derived factor 1 on the migration of neural stem cells and the recovery of lower limb function after spinal cord injury

Objective To assess the effect of stromal cel1-derived factor 1 (SDF-1) on the migration of neural stem cells and recovery of lower limb function after spinal cord injury (SCI). Methods A modified version of Allen's method was used to establish a SCI model in each of 96 adult male Sprague-Dawley rats. They were then randomly divided into group A which received an injection of phosphate buffer solution, group B which received an injection of neural stem cells, group C which received a combination of SDF-1 and neural stem cells, and group D which received AMD3100 and neural stem cells 7 days after the modeling. The functioning of the hind limbs of all of the rats was assessed on the 7th, 14th, 21st and 28th day after the modeling. The rats were then sacrificed and frozen sections of their spinal cords were stained with hematoxylin-eosin (HE) and marked with CM-Dil under fluorescent light. Results An accumulation of fluorescing cells were observed in spine cords from both group B and C, with the counts of group B [(23.6±3.7), (18.9±5.6)and(15.2±4.3)respectively] at the day 14, 21 and 28 after modeling significantly smaller than those of group C [(27.4±4.7), (20.4±5.2) and (18.3±3.9) respectively]. During the same period of time, the average BBB scores of groups B and C were significantly better than those of groups A and D. Moreover, the average score of group C was significantly higher than group B's at all time points. Conclusions Transplanted neural stem cells can migrate to the injured site, surviving and differentiating to promote the recovery of limb function in rats with spinal cord injury. SDF-1 can promote that migration and proliferation. Moreover, CXCR4 receptor's antagonist AMD3100 can significantly hinder neural stem cells' migration to the injured site on the spinal cord. Key words: Neural stem cells; Spinal cord injury; Stromal cel1 derived factor

The mobilization of stem cells promote the recovery of the ischemia brain injury after cardiopulmonary resuscitation

Objective To explore the therapeutic potential and mechanism of stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) and AMD3100 to repair global cerebral ischemia injuries in a rat model of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). Methods Cardiac arrest was induced by asphyxia. Fifty-six SD rats were randomly assigned into four groups: G-CSF group,G-CSF + AMD3100 group,CPR control group and sham operated group. The animals were sacrificed at 3d and 6d after CPR respectively. The neurological status and morphological changes of damaged cerebrum,the apoptosis of nerve cells and vascular endothelial growth factor (VEGF) expressed in brain tissue and capillary density in hippocampus and temporal lobe cortex were measured and analyzed by means of neurological deficit score (NDS), adhesive tape removal test (TRT), ELISA,MRI and immunofluorescence. Results NDS in G-CSF + AMD3100 group (61.4 ±10.7) was significantly higher than that in CPR control group (49.9 ±10.4) at 3 d after CPR (P <0.05). And less time consumption for TRT found in G-CSF + AMD3100 group (85.5 ±28.9) s rather than was in CPR control group (148.1 ± 23.8) s and G-CSF group (118.5 ±30.4) s (P <0.05). The severity of cerebral injury assessed by MRI was significantly milder at both 3 d and 6 d in the two stem cell mobilization groups. The apoptosis rate of nerve cells in G-CSF + AMD3100 group (0.23 ± 0.06) was significantly lower than that in G-CSF group (0.34 ±0.08) at 3 d after CPR,and that in both stem cell mobilization groups was lower than that in CPR control group (0.44 ±0.09) (P <0.05). At 3 d and 6 d after CPR,the levels of VEGF in brain tissue were (106.2 ±23.3) pg/ mL and (79.9 ±18.4) pg/ mL in G-CSF + AMD3100 group,and were (50.6 ± 13.7) pg/ mL and (73.9 ±16.6) pg/ mL in G-CSF group,which were both significantly higher than that in CPR control group (23.1 ±10.2) pg/ mL and (36.2 ±12.8) pg/ mL (P <0.05). At 3 d after CPR,the cerebral capillary density (351.8 ± 67.9) branches in every high power field (A/ HPF) was significantly higher in G-CSF + AMD3100 group than that (301.4 ±77.3) A/ HPF in G-CSF group and (250.4 ±48.0) A/ HPF in CPR control group (P <0.05). The cerebral capillary density in G-CSF group elevated to (348.4 ±76.7) A/ HPF at 6 d after CPR which was significantly higher than that at 3 d (P <0.05), and there was no difference between that at 3 d and 6 d in G-CSF + AMD3100 group. Conclusions The mobilization stem cells improve the impaired neurological function. The increased expression of VEGF in brain tissue,the neo-vascularization promoted by the mobilized stem cells and the inhibition of nerve cell apoptosis may be associated with the protective effects of the stem cell mobilization. Key words: Cardiac arrest; Cardiopulmonary resuscitation; Stem cell mobilization; Rats; Granulocyte colony-stimulating factor; Neurological deficit score

Role of chemokine receptor CXCR4 in spinal dorsal horn in development of morphine tolerance in rats with bone cancer pain

Objective To evaluate the role of chemokine receptor CXCR4 in spinal dorsal horn in the development of morphine tolerance in rats with bone cancer pain (BCP).Methods Forty female Sprague-Dawley rats,weighing 180-220 g,were equally randomized into 5 groups using a random number table:sham operation group (group S),BCP group (group B),BCP + AMD3100 (specific CXCR4 antagonist) group (group BA),BCP + morphine group (group BM),BCP + morphine + AMD3100 group (group BMA).BCP was induced by injecting Walker 256 mammary gland cancer cell suspension (4 × 105 cells/ml) 5 μl into the bone marrow of the right tibia of rats anesthetized with chloral hydrate.On 6 days after injection of mammary gland cancer cells,AMD3100 2 mg/kg was intraperitoneally injected twice a day for 9 days in BA group,and morphine 10 mg/kg was subcutaneously injected twice a day for 9 days in BM group.AMD3100 was intraperitoneally injected and morphine was subcutaneously injected as previously described at the corresponding time point in BMA group.Before injection of mammary gland cancer cells (T0) and on 4,6,8,10,12 and 14 days after injection of mammary gland cancer cells (T1-6),paw withdrawal threshold to von Frey hair mechanical stimulation (PWMT) was measured.The rats were then sacrificed and L3-5 segments of the spinal cord were removed for determination of c-fos expression in spinal dorsal horn using immunofluorescence.Results Compared with S group,PMWT was significantly decreased at T2-6 in B and BA groups and at T4-6 in BM group,and c-fos expression was up-regulated at T6 in BM group (P ＜0.01).PMWT was significantly higher at T3-5 in BM group and at T3-6 in BMA group than in group B (P ＜ 0.01).Compared with BM group,PMWT was significantly increased at T5,6 and c-fos expression was down-regulated at T6 in BMA group (P ＜ 0.01).Conclusion Chemokine receptor CXCR4 in spinal dorsal horn is involved in the development of morphine tolerance in rats with BCP and the mechanism may be related to activation of c-fos. Key words: Receptors,CXCR4; Spinal cord; Bone neoplasms; Morphine; Drug tolerance

Role of CXCR4-FAK signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells towards damaged tissues resulting from spinal cord ischemia-reperfusion injury in rats

Objective To investigate the role of CXC chemokine receptor 4-focal adhesion kinase (CXCR4-FAK) signaling pathway in migration and adhesion of hypoxia-preconditioned bone marrow mesenchymal stem cells (BMSCs) towards damaged tissues resulting from spinal cord ischemia-reperfusion (I/R) injury in rats.Methods Part I Rat BMSCs transfected with recombinant adenovirus-mediated green fluorescent protein gene 3 were seeded in 24-well plates and randomly divided into 5 groups (n =18 wells each):control group (group C),normoxia-incubated group (group N),HP group (group H),HP + CXCR4 antagonist AMD3100 group (group HA) and HP + FAK inhibitor FAK-related nonkinase group (group HF).In group C,BMSCs were incubated in DMEM culture medium.In group N,BMSCs were exposed to 21％ O2-74％ N2-5％ CO2 for 36 h.In group H,BMSCs were exposed to 0.5％O2-94.5％ N2-5.0％CO2 for 24 h followed by 12 h exposure to normoxia.In groups HA and HF,5 μg/ml AMD3100 and 10 μg/ml FAK-related nonkinase were added to the culture medium before HP,respectively.The expression of stromal derived factor-1α (SDF-1α),CXCR4 and phosphorylated FAK (p-FAK) in BMSCs was determined by Western blot.The migratory capability and adhesive ability of BMSCs were measured by Transwell invasive assay and fibronectin adhesive assay,respectively.Part Ⅱ Two hundred and sixteen male Sprague-Dawley rats weighing 300-350 g were used and 210 out of the 216 rats underwent spinal cord ischemia by occlusion of the thoracic aorta combined with controlled hypotension.Thirty-six rats were chosen and sacrificed before spinal cord I/R and at 12 h and 1,3,5 and 7 days of reperfusion (T0-5) and the lumbar segment of spinal cord was removed for detection of the content of SDF-1α.The left 180 rats with spinal cord I/R were randomly divided into 5 groups (n =36 each).IT DMEM medium 300 μl was injected in group C and BMSC suspension 300 μl (1 × 106/ml) was injected in groups N,H,HA and HF immediately after onset of reperfusion.Neurological function was scored at T0-5.The animals were then sacrificed and the lumbar segment of spinal cord was removed for detection of the degree of BMSC aggregation.Results There was no significant difference in the expression of SDF-1α,CXCR4 and p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation between groups C and N (P ＞ 0.05).Compared with group N,the expression of SDF-1α,CXCR4 and p-FAK was significantly up-regulated,and migratory capability and adhesive ability of BMSCs,neurological function scores and the degree of BMSC aggregation were increased in group H,while no significant change was found in the expression of p-FAK,migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation in HA and HF groups (P ＞ 0.05).Compared with group H,the expression of p-FAK was down-regulated and the migratory capability and adhesive ability of BMSCs,neurological function scores and degree of BMSC aggregation were decreased in groups HA and HF (P ＜0.05).The content of SDF-1α was significantly higher at T2,3 than at T0.Conclusion HP can promote migration and adhesion of BMSCs towards damaged tissues resulting from spinal cord I/R injury through CXCR4-FAK signaling pathway in rats; thus CXCR4-FAK signal pathway provides the protective effect on spinal cord. Key words: Receptors, CXCR4; Focal adhesion protein-tyrosine kinases; Mesenchymal stem cell transplantation ; Cell Hypoxia ; Ischemic preconditioning ; Cell movement; Cell adhesion; Spinal cord ; Reperfusion injury

Distribution of CXCL12 and CXC chemokine receptor-4 in glioblastoma microenviroment and the role of them in invasion and migration

Objective To detect the expression of CXCL12/CXC chemokine receptor-4 (CXCR4) in glioblastoma microenviroment and explore the role of CXCL12/CXCR4 in invasion and migration.Methods The surgically resected specimens were collected from the core of mass,junctional zones between tumor and peritumoral areas and peritumoral edematous areas in 45 patients with glioblastoma.Immunohistochemistry was employed to detect the expression of CXCL12,CXCR4 and matrix metalloproteinase (MMP)-9 in tissue specimens.Human glioma U251 cells were treated with CXCL12 and AMD3100 (CXCR4 inhibitor).The expression of MMP-9 in treated and control cells was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.Results In the core of mass,junctional zones and peritumoral edematous areas,the scores of CXCL12 were 4.2 ± 1.3,2.8 ±0.8 and 1.6 ± 0.5,those of CXCR4 were 4.6 ± 0.9,3.0 ± 0.7 and 1.8 ± 0.8,and those of MMP-9 were 5.2 ±0.8,3.8 ±0.8 and 2.6 ±0.5,respectively.The expression of CXCL12,CXCR4 and MMP-9 showed significant difference in above different areas (P ＜ 0.05).The expression of CXCL12/CXCR4 and MMP-9 was positively correlated (CXCL12 and MMP-9 r =0.769,P ＜ 0.05,CXCR4 and MMP-9 r =0.948,P ＜0.05).The expression values of MMP-9 mRNA were 0.968 ±0.065,0.187 ±0.028 and 0.294 ±0.042 in CXCL12 treated,AMD3100 treated and control groups,and the expression values of MMP-9 protein were 0.605 ±0.011,0.358 ±0.009 and 0.449 ±0.026,respectively.There was significant difference among those groups (P ＜ 0.05).Conclusion CXCL12/CXCR4 may promote the invasion and migration of glioma cells via the upregulated expression of MMP-9.CXCR4 could be a potential target against the invasion and migration of glioblastoma. Key words: Chemokine ; Glioblastoma ; Microenviroment ; Invasion ; Migration

Relationship between chemokine axis CXCL12-CXCR4 and epithelial ovarian cancer

To explore the relationship between chemokine axis CXCL12-CXCR4 and the pathogenesis and severity of epithelial ovarian cancer. SKOV3 transfected with plasmid, SKOV3 transfected with vector and SKOV3 were cultured in vitro. Methyl thiazolyl tetrazolium (MTT) was used to analyze the effects of different concentrations of CXCL12 on the proliferation, migration and invasion of three cell lines and examine the inhibition of neutralizing CXCR4 antibody or antagonist AMD3100. And the load and weight of acquired tumor were determined at different concentrations of CXCL12. CXCL12 could promote the proliferation, migration and invasion of SKOV3/CXCR4 cells in a dose-dependent fashion (P < 0.05). The effect on CXCL12 tumorigenesis could be inhibited by neutralizing CXCR4 antibody or antagonist AMD3100 (P < 0.05). Significant differences existed in the mean survival time, load and weight of metastatic tumors among the three nude mice. A close correlation exists between chemokine axis CXCL12-CXCR4 and the pathogenesis, metastasis of epithelial ovarian cancer. The above axis may be an important pathogenic factor of epithelial ovarian cancer. And the antibody of CXCL12-CXCR4 is probably effective in its management.

Effects of stromal cell-derived factor-1 on the proliferation and apoptosis of articular chondrocytes in rabbits

Objective To observe the effect of stromal cell-derived factor-1 (SDF-1) on the proliferation of articular chondrocytes in rabbits and discuss the role of SDF-1 in chondrocyte apoptosis.Methods The chondrocytes were isolated and cutured,then divided into 4 groups:control group,50 μg/L SDF-1 group,50 μg/L SDF-1 + 5 mg/L CXC chemokine receptor-4 specific antagonist (AMD3100) group,and 5 mg/L AMD3100 group.The MTT assay and methods for cell cycle were used to observe the proliferation of chondrocytes.In addition,each group was exposured to the condition of 250 μmol/L hydrogen peroxide.Then cell viability was detected by using trypan blue exclusion assay,apoptosis rate of chondrocytes was detected by using flow cytometry,and the dynamic changes of Caspase-3 were measured in each group.Furthermore,the apoptosis of chondrocytes was evaluated by TdT-mediated dUTP nick end labeling (TUNEL) assay.Results The absorbance value of SDF-1 set (0.504 ±0.024) was higher than other groups,and the S +G2/M cell ratio in SDF-1 group was higher than other groups with the differnce bing statistically significant (all P ＜0.05).In terms of apoptosis,the viable rate in SDF-1 group was higher than other groups (P ＜ 0.01),while the Annexin V-FITC (+) proportion of cartilage cells in the SDF-1 group (4.00 ± 0.92) ％ was lower than other groups (P ＜ 0.05).Caspase-3 activity in SDF-1 group [(40.03 ± 2.56) nmolpNA/(h·mg) protein] was lower than other groups (P ＜0.01),and in addition,the number of apoptotic cells in SDF-1 group was less than other groups (P ＜ 0.05),but there was no significant difference among control group,SDF-1 + AMD3100 group and AMD3100 group in all experiments.Conclusion SDF-1 could contribute to the proliferation of rabbit chondrocytes in vitro,and in 50 μg/L group the Proliferation activity is reasonable,meanwhile SDF-1 can protect the chondrocytes from apoptosis induced by hydrogen peroxide. Key words: Stromal cell-derived factor-1 ; Apoptosis; Proliferation; Chondrocyte; Rabbit

Study on effects of G-CSF and AMD3100 on proliferation, migration and adhesion of rat mesenchymal stem cells

Objective To study the effects ofgranulocyte colony-stimulating factor(G-CSF) and AMD3100 on the proliferation,migration and adhesion of mesenchymal stem cells (MSCs).Methods The proliferation,migration and adhesion of MSCs were detected by MTT chromometry,transwell and adhesion test.Results When the concentration of G-CSF was 200 μg/L and AMD3100 was 0.5 mg/L,the proliferation and migration of MSCs were the strongest.When the concentration of G-CSF was 200 μg/L and AMD3100 was 0 mg/L,the adhesion of MSCs was the strongest.Conclusion The proliferation,migration,adhesion of MSCs are promoted by G-CSF,and inhibited by AMD3100. Key words: Granulocyte colony-stimulating factor; AMD3100; Mesenchymal stem cells; Proliferation; Migration; Adhesion

Effects of hypoxic preconditioning on the migration of bone marrow derived mesenchymal stem cells

To explore the effects and probable mechanism of CoCl2-induced hypoxic preconditioning on the migration of bone marrow derived mesenchymal stem cells (BMSC). BMSC were cultured by whole bone marrow adherence and identified by surface markers (CD29, CD90 and CD45) with flow cytometry (FCM). The methods of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and FCM were applied to establish the model of CoCl2-induced hypoxic preconditioning. The migratory capacity of BMSC with hypoxic preconditioning was analyzed by the assays of scratch wound healing and transwell migration. The protein and mRNA expressions of HIF-1α and CXCR4 of BMSC were detected by Western blot and real-time polymerase chain reaction (PCR). After silencing HIF-1α by siRNA technique and blocking CXCR4 by its antagonist AMD3100, the changes of migratory capacity of BMSC were also tested. Cultured BMSC were uniformly positive for CD29 and CD90 and negative for CD 45. According to the results of MTT and FCM, 200 µmol/L CoCl2 and 24 h culture time was the ideal hypoxic preconditioning model of BMSC. The migratory capacity of BMSC in hypoxic preconditioning group was higher than the one in control group (scratch wound healing assay: (0.396 ± 0.018) mm vs (0.200 ± 0.011) mm, transwell migration assay: 21.0 ± 4.5 vs 8.5 ± 1.7, both P < 0.05). The protein and mRNA levels of HIF-1α and CXCR4 of BMSC in hypoxic preconditioning group were significantly higher than in control group. After silencing HIF-1α or blocking CXCR4 by AMD3100, the migratory capacity of BMSC in hypoxic preconditioning group decreased and had no difference with the control group. Hypoxic preconditioning may enhance the migratory capacity of BMSC in vitro. And it is partially attributable to the up-regulation of HIF-1α/CXCR4 axis after preconditioning.

Role of stromal cell derived factor/CXC chemokine receptor axis in regulation of expression of CD133 in gastric cancer cells via PI3K/Akt pathway in vitro

Objective To observe the effects of stromal cell derived factor/CXC chemokine recep- tor （SDF-1α/CXCR4） axis on the expression of CD133 via PI3K/Akt signaling pathway in gastric cancer （GC） in vitro. Methods The morphological expression of CXCR4, Akt, p-Akt and CD133 in KATO-1α cells were detected by using immunocytochemistry. SDF-1α, AMD3100 and LY294002 were respectively used to stimulate/inhibit KATO-m ceils. The semi-quantitative enzyme-linked polymerization assay for CXCR4 and CD133 mRNA expression was applied. The expression levels of CXCR4, Akt, p-Akt and CD133 proteins were detected by using Western blotting. Results Positive staining of CXCR4, Akt, p-Akt and CD133 was observed in the cultured cells. The expression levels of CXCR4 protein （0. 980 ±0. 083）, p-Akt protein （0. 770 ± 0. 071 ）, and CD133 protein （0. 890 ±0. 078 ） were significantly higher （ P 〈 0. 05） in SDF-1α group than in control group （0. 750 ±0. 042, 0. 680 ±0. 038, 0. 720 ±0. 034 respectively）. The expression levels of CD133 mRNA （0. 890 ±0. 061 ） were significantly higher （P 〈 0. 05 ） in SDF-1α group. In AMD3100 group, the expression levels of CXCR4 protein （0. 430 ±0. 055）, p-Akt protein （0. 350 ±0. 050） and CD133 protein （0. 110 ±0. 060） were decreased significantly as compared with those in control group （0. 750 ±0. 042, 0. 680 .SO. 038, and 0. 720 ±0. 034 respectively, P 〈0. 05）. In LY294002 group, the expression levels of p-Akt protein （ 0. 100 ± 0. 033 ） and CD133 protein （0. 440 ±0. 055 ） were decreased significantly as compared with those in control group （0. 680 ±0. 038 and 0. 720 ± 0. 034 respectively, P 〈0. 05） ; In comparison with that in control group （0. 540 ±0. 036）, CD133 mRNA （0. 310 ±0. 021 ） was decreased significantly （P 〈0. 05）. Conclusion The axis of SDF-la/CXCR4 stimulated or inhibited can up-regulate or down-regulate the CD133 expression via PI3K/Akt signaling pathway in GC in vitro. Key words: Gastric cancer; Stromal cell derived factor/CXC chemokine receptor; CD133; PI3K/Akt pathway

Inhibitory effect of CXCR4 inhibitor combined with anti-vascular endothelial growth factor antibody on experimental choroidal neovascularization

Objective To observe the effects of CXCR4 inhibitor (AMD3100) combined with antivascular endothelial growth factor (VEGF) antibody on experimental choroidal neovascularization.Methods Choroidal neovascularization (CNV) was induced in 48 Brown-Norway (BN) rats by Krypton red laser photocoagulation,and those rats were randomly divided into AF564 group (group A),AMD3100 group (group B),combined treatment group (group C) and PBS group (group D),12 rats in each group.Left eyes were the experimental eyes.The rats of group A - D received intravitreal injection of 5μl of AF564,AMD3100,AF564/AMD3100 and PBS after laser photocoagulation respectively.Fourteen days after photocoagulation,fundus fluorescein angiography (FFA),pathological section analysis and choroidal vascular wholemount were used to observe the degree of fluorescein leakage,the relative thickness and areas of CNV.Results Fourteen days after photocoagulation,the scores of fluorescein leakage in group A - D were 2.16±0.91,2.16±0.91,1.92±1.03,1.39±0.93 respectively.Fluorescein leakage in group A - C was obviously reduced compared to group D (F =12.91,P＜0.001 ),while fluorescein leakage in group C was reduced compared to group A and B (F=9.21,P＜0.05 ).The CNV relative thicknesses in group A-D were 1.82±0.11,1.90±0.22,1.12±0.12,2.82±0.29 respectively.Group A -C had thinner CNV compared to group D (F=5.92,P＜0.001),while group C had thinner CNV compared to group A and B (F=5.16,P＜0.05).The CNV areas in group A - D were (8204± 122),(9332±211),(6533± 101),and (13644±255) μm2 respectively.Group A - C had smaller CNV area compared to group D (F=147.50,P＜0.001),while group C had smaller CNV area compared to group A and B (F=112.60,P＜0.05).Conclusion Combined treatment with CXCR4 inhibitor and anti-VEGF antibody can inhibit laser-induced CNV significantly. Key words: Choroidal neovascularization/therapy; Receptors, CXCR4/ drug effects; Vascular endothelial growth factors; Chemokine CXCL12 ; Disease models, animal

CXCR4 blockade augments bone marrow progenitor cell recruitment to the neovasculature and reduces mortality after myocardial infarction.

We hypothesized that a small molecule CXCR4 antagonist, AMD3100 (AMD), could augment the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs), thereby enhancing neovascularization and functional recovery after myocardial infarction. Single-dose AMD injection administered after the onset of myocardial infarction increased circulating EPC counts and myocardial vascularity, reduced fibrosis, and improved cardiac function and survival. In mice transplanted with traceable BM cells, AMD increased BM-derived cell incorporation in the ischemic border zone. In contrast, continuous infusion of AMD, although increasing EPCs in the circulation, worsened outcome by blocking EPC incorporation. In addition to its effects as a CXCR4 antagonist, AMD also up-regulated VEGF and matrix metalloproteinase 9 (MMP-9) expression, and the benefits of AMD were not observed in the absence of MMP-9 expression in the BM. These findings suggest that AMD3100 preserves cardiac function after myocardial infarction by enhancing BM-EPC-mediated neovascularization, and that these benefits require MMP-9 expression in the BM, but not in the ischemic region. Our results indicate that AMD3100 could be a potentially useful therapy for the treatment of myocardial infarction.