Stromal Cell-derived Factor-1 Treatment Research Articles

The Effect of Dosing of Stromal Cell-Derived Factor 1 on Anal Sphincter Regeneration.

Aim: The aim of this study is to investigate if a high dose of the stromal cell-derived factor-1 (SDF-1) plasmid improves outcome in a minipig model of chronic anal sphincter injury. Methods: Twenty-two female minipigs underwent excision of the posterior hemicircumference of the anal sphincter complex and were allowed to recover for 6 weeks. They were randomly allocated (n = 6) to receive either 5% dextrose (sham) or 2, 4, or 8 mg of SDF-1 plasmid at the defect site. Two control pigs received no surgery/treatment. Outcome measures included anal manometry at preinjury/pretreatment and 2, 4, and 8 weeks after treatment, recording the mean of eight pressure channels and the posterior channel alone, histopathology using Masson's trichrome, and immunohistochemistry using PGP9.5 for staining of neural structures, and CY3 staining for blood vessels. Data are expressed as mean ± standard error. Manometry analysis used two-way analysis of variance (ANOVA) followed by the Holm-Sidak test. Quantification of muscle/fibrosis was analyzed with a Kruskal-Wallis one-way ANOVA on ranks. Results: Posterior anal pressures were significantly decreased in sham treated animals compared with controls (p = 0.04). In contrast, mean anal pressures at the four time points were not significantly different between groups (p > 0.05). The defect area of the sham treated group showed irregular muscle bundles, while all three SDF-1 treatment groups show organized muscle bundles, with the most organization in the higher dose groups. Quantification of Masson-stained slides showed no statistically significant differences between groups, but did show increased muscle volume in the area of defect in the treatment groups compared with sham. PGP9.5 and CY3 staining showed increased fluorescence in the higher dose groups compared with sham treatment. Conclusion: A single higher dose of the plasmid encoding SDF-1 may increase muscle volume in the area of a chronic defect. Impact statement Fecal or bowel incontinence as a result of a torn anal sphincter complex remains undetected for many years. The resulting defect does not respond well to surgical repair. Regenerating the anal sphincter complex with functional muscle has been a long-term goal. Stem cells home to a site of a chronic injury and cause regeneration when a cell signaling mechanism is available. Stromal cell-derived factor-1 is one such cytokine that has been well researched by us and others to have this effect. It is easy to use clinically and has been used in other applications in humans and considered safe.

Exogenous stromal cell-derived factor-1 (SDF-1) suppresses the NLRP3 inflammasome and inhibits pyroptosis in synoviocytes from osteoarthritic joints via activation of the AMPK signaling pathway

ObjectiveNLRP3 inflammasome may play a key role in OA pathogenesis. Stromal cell-derived factor-1 (SDF-1) is a homeostatic CXC chemokine. Since the role of SDF-1 in OA has not been explored, this study aimed to examine the effect of SDF-1 on NLRP3 inflammasome and pyroptosis in synoviocytes from OA joints.Materials and methodsHuman synovium was obtained from OA patients for isolation of primary synoviocytes and a murine model of collagenase-induced OA was established for testing intra-articular injections of SDF-1. Immunoblotting assays were used to examine the effects and underlying mechanism of action of SDF-1 on NLRP3 inflammasome and synoviocyte pyroptosis in synoviocytes. Inhibitors of AMPK and PI3K–mTOR were utilized to investigate the key signaling pathways involved in SDF-1-mediated OA inflammasome formation and pyroptosis.ResultsSynoviocytes from OA joints exhibited significantly higher expression of NLRP3 inflammasome and biomarkers of synoviocyte pyroptosis relative to healthy individuals. This was confirmed in the collagenase-induced OA model, where OA synoviocytes had a significantly lower SDF-1 expression than healthy ones. SDF-1 treatment in synoviocytes of OA patients and collagenase-induced OA led to significant downregulation in the expression of NLRP3 inflammasome and synoviocyte pyroptosis biomarkers. Inhibition of the AMPK signaling pathway significantly suppressed the inhibitory effect of SDF-1 on NLRP3 inflammasome expression of OA synoviocytes. However, blocking the SDF-1-activated PI3K–mTOR signaling pathway could still suppress the expression of NLRP3 inflammasome and synoviocyte pyroptosis biomarkers.ConclusionsSDF-1 ameliorates NLRP3 inflammasome and pyroptosis in OA synoviocytes through activation of the AMPK signaling pathway. Therefore, SDF-1 may be a novel therapeutic target for OA.

Macrophages contribute to liver repair after monocrotaline-induced liver injury via SDF-1/CXCR4.

Monocrotaline (MCT) administration induces liver injury in rodents that mimics the pathology of human sinusoidal obstruction syndrome. MCT-induced SOS models are used to investigate the mechanism of injury and optimize treatment strategies. However, the processes underlying liver repair are largely unknown. Specifically, the role of macrophages, the key drivers of liver repair, has not been elucidated. The current study aimed to examine the role of macrophages in the repair of MCT-induced liver injury in male C57/BL6 mice. Maximal liver injury occurred at 48 h post-MCT treatment, followed by repair at 120 h post-treatment. Immunofluorescence analysis revealed that CD68+ macrophages were recruited to the injured regions after MCT treatment. This was associated with the decreased expression of genes related to a pro-inflammatory macrophage phenotype and the increased expression of those associated with a reparative macrophage phenotype during the repair phase. The results also revealed that stromal cell-derived factor-1 (SDF-1) and its receptor C-X-C chemokine receptor-4 (CXCR4) were upregulated, and CD68+ macrophages were co-localized with CXCR4 expression. Treatment of mice with AMD3100, a CXCR4 antagonist, delayed liver repair and increased the expression of genes related to a pro-inflammatory macrophage phenotype. In contrast, SDF-1 treatment stimulated liver repair and increased the expression of genes related to a reparative macrophage phenotype. The results suggested that macrophages accumulate in the liver and repair damaged tissue after MCT treatment, and that the SDF-1-CXCR4 axis is involved in this process.

Role of the stromal cell derived factor-1 in the biological functions of endothelial progenitor cells and its underlying mechanisms.

Stromal cell derived factor-1 (SDF-1) is a chemokine that plays a critical role in the homing of stem and progenitor cells, including endothelial progenitor cells (EPCs). However, little research has been undertaken to evaluate the roles of SDF-1 in the biological functions of EPCs and related signaling pathways. The present study aimed to investigate the biological functions of EPCs in response to SDF-1, as well as the underlying mechanisms. The effects of SDF-1 treatment on EPC proliferation, migration and tube formation were assessed by performing MTS, Transwell and in vitro tube formation assays, respectively. The phosphorylation status of Akt and ERK was evaluated by western blotting. The present results indicated that SDF-1 treatment enhanced EPC proliferation, migration and tube formation compared with the control group. Furthermore, SDF-1-induced EPC proliferation was significantly reduced following treatment with a C-X-C Motif Chemokine Receptor 4 antagonist (AMD3100), a PI3K inhibitor (LY294002) and the mitogen-activated protein kinase kinase inhibitor (MEK; PD98059). SDF-1-induced migration and angiogenesis were significantly suppressed by the PI3K inhibitor, but not the MEK inhibitor. Moreover, SDF-1 significantly increased the protein expression levels of phosphorylated (p)-Akt and p-ERK; however, SDF-1-induced effects on protein expression were suppressed by AMD3100, LY294002 and PD98059. Thus, SDF-1-induced EPC proliferation was mediated by activation of the Akt and ERK signaling pathways, whereas SDF-1-mediated EPC migration and tube formation only involved activation of the Akt signaling pathway.

Effect of genipin crosslinked chitosan scaffolds containing SDF-1 on wound healing in a rat model.

Prolonged healing is a severe problem for elderly and diabetic patients. Impaired angiogenesis, stem cell differentiation, and migration have been shown to delay wound healing. The chemokine stromal cell-derived factor-1 (SDF-1) plays an essential role in recruiting cells to wound sites and is suggested to be a candidate for tissue engineering. In this study, chitosan (CHI) scaffolds were crosslinked with nontoxic genipin (Gp) and further heparinized for SDF-1 immobilization. Then, the structures were evaluated for their physicochemical properties (porosity, swelling ratio, and water vapor transmission rate (WVTR)). The interaction between SDF-1 and heparin could sustain SDF-1 release, which has been shown to enhance human umbilical vein endothelial cell (HUVEC) 2D/3D migration. The investigation of the wound-healing activity of the SDF-1-loaded CHI scaffolds revealed a better wound recovery rate in vivo in healthy and streptozotocin-induced diabetic Sprague-Dawley (SD) rats. The histological analysis illustrated that the local of SDF-1 treatment scaffold at the wound site enhanced neovascularization. The wounds treated with SDF-1 scaffolds also exhibited higher vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) expression in Western blot assays. Based on the wound-healing activity and beneficial characteristics, the SDF-1-loaded CHI scaffold demonstrates potential as a material for treating skin wounds.

Effect of integrin α5β1 inhibition on SDF-l/CXCR4-mediated choroidal neovascularization.

To investigate the roles of integrins in choroidal neovascularization (CNV) and their associations with the stromal cell-derived factor-1 (SDF-1)/CXCR4 axis. CNV lesions were induced in mice using laser photocoagulation. After CNV induction, all animals were randomly assigned to: control, SDF-1, SDF-1+age-related macular degeneration (AMD) 3100 (CXCR4 inhibitor), and SDF-1+ATN161 (integrin α5β1 inhibitor) groups; their effects on CNV progression were observed using hematoxylin eosin (HE) staining, fundus fluorescein angiography (FFA) grading and optical coherence tomography (OCT), and their effects on CXCR4/integrin α5 expression were evaluated using Western blot and double immunofluorescence staining. Hypoxia-exposed endothelial cells (ECs) were used to simulate CNV in vitro, they were treated with SDF-1, combined with CXCR4 siRNA/AMD3100 or ATN161, and expression of integrin α5, cell migration and tube formation were analyzed. Integrin subunit α5 increased at 3rd and 7th day and decreased at 14th day in CNV mice, with no significant change of β1-integrin. CXCR4 expression in CNV mice had persistent increase within 14d after induction. SDF-1 treatment significantly promoted the CNV progression during 3-14d. The mean CNV length in AMD3100 and ATN161 group at day 7 was 270.13 and 264.23 µm in HE images, significantly lower than the mean length in SDF-1 (345.70 µm) group. AMD3100 and ATN161 also significantly reduced thickness and leakage of CNV induced by SDF-1. Mean integrin α5 positive area in SDF-1 group reached 2.31×104 µm2, significantly higher than control (1.25×104 µm2), which decreased to 1.78×104 µm2 after AMD3100 treatment. About 61.36% of ECs in CNV lesions expressed α5 in SDF-1 group, which significantly decreased to 43.12% after AMD3100 treatment. In vitro, integrin α5 peaked by 6 folds after 6h of hypoxia exposure and CXCR4 gradually increased by up to 2.3 folds after 24h of hypoxia. Approximately 25.12% of ECs expressed integrin α5 after SDF-1 stimulation, which decreased to 7.2%-9.5% after si-CXCR4 or AMD3100 treatment. ATN161 exerted an inhibitory effect comparable to that of si-CXCR4 on EC migration and tube formation in the presence of SDF-1. SDF-1/CXCR4 signaling induces integrin α5β1 expression in ECs to promote CNV.

Retraction Note to "SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma".

Stromal cell-derived factor 1 (SDF-1)/CXCR4 ligand-receptor axis is widely recommended as an attractive target for cancer therapy. Meanwhile, epithelial-mesenchymal transition (EMT) process is linked to disease pathophysiology. As one of inhibitors of apoptosis proteins, survivin is implicated in the onset and development of cancer. In the present study, we tried to determine the cause-effect associations between SDF-1/CXCR4 axis and survivin expression in glioblastoma U-251 cell line. Survivin activation and inhibition were induced with exogenous SDF-1 and survivin small interfering RNA (survivin siRNA), respectively. Western blot was used to detect relevant proteins in SDF-1/CXCR4 axis. Western blot analysis revealed that survivin expression in U-251 increased in a dose- and time-dependent manner in response to SDF-1 treatment. However, the interference with MEK/ERK and PI3K/AKT pathway prohibited SDF-1-induced survivin up-regulation. Importantly, survivin knockdown abrogated cell cycle progression and the expression of snail and N-cadherin, compared with non-transfectants. In conclusion, the present study shows that SDF-1 up-regulates survivin via MEK/ERK and PI3K/AKT pathway, leading to cell cycle progression and EMT occurrence dependent on survivin. The blockade of survivin will allow for the treatment of glioblastoma.

Retraction Note to: Nuclear Protein C23 on the Cell Surface Plays an Important Role in Activation of CXCR4 Signaling in Glioblastoma.

The chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 (SDF-1) plays an important role in tumor progression and are associated with angiogenesis. Meanwhile, the implications of C23 in multiple signaling pathways have been also investigated. However, the effects of C23 on CXCR4 pathway in glioblastoma are not fully characterized. In the present study, C23 and CXCR4 of U87 cell line were inhibited by anti-C23 and anti-CXCR4 antibodies, respectively; and then C23 and CXCR4 siRNAs were used to knock down endogenous C23 and CXCR4, respectively. In addition, MTT assay was also introduced. Our data showed that either anti-C23 or anti-CXCR4 antibodies efficaciously repressed the phosphorylation levels of ERK (p < 0.000) and AKT (p < 0.000) compared with SDF-1 alone and control. As expected, either C23 or CXCR4 siRNAs indeed resulted in C23 and CXCR4 knockdown and further suppressed the expression of p-ERK and p-AKT. Most importantly, immunoprecipitation revealed C23 interacted with CXCR4 once U87 was exposed to SDF-1 treatment. In addition, MTT assay identified that C23 or CXCR4 siRNAs could obviously decreased cell proliferation capacity (p = 0.002). In conclusion, our results suggest that C23 plays a crucial role in activation of SDF-1-induced ERK and PI3K/AKT pathways via interacting with CXCR4. Furthermore, C23 could be recommended as an important element in glioblastoma development and a new target for glioblastoma treatment.

Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia.

Endothelial progenitor cells (EPCs) respond to stromal cell-derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus-induced EPCs dysfunction remains unknown. To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3β phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3β/Fyn pathway via increased activity of Nrf2.

Effect of stromal cell-derived factor-1 on myocardial apoptosis and cardiac function recovery in rats with acute myocardial infarction

The aim of the study was to investigate the effect of stromal cell-derived factor-1 (SDF-1) on myocardial apoptosis and cardiac function recovery in rats with acute myocardial infarction (AMI) and the mechanism of the Toll-like receptor (TLR)-4/nuclear factor-κB (NF-κB) signaling pathway. A total of 64 healthy male F344 rats were randomly divided into the sham operation, model, SDF-1 intervention and SDF-1 antibody groups, with 16 rats in each group. The method of Olivette was used to establish the AMI model by ligation of the left anterior descending artery. Day 1 after establishing the animal model, the rats in the SDF-1 intervention group were injected with 10 µl recombinant SDF-1 (400 ng/ml) in five regions including the myocardial infarction area and the four surrounding areas. The rats in the model group were injected with 10 µl normal saline including the myocardial infarction area and the four surrounding areas, and those in the SDF-1 antibody group were injected with 1 ml SDF-1 antibody (2 µg/ml). Four rats were sacrificed after 1, 3, 7 and 14 days after the intervention, and the analysis was carried out. TUNEL in situ labeled apoptotic cells were used for cell counting, and immunohistochemical staining was performed to measure vascular density. The animal echocardiographic measurement was for the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular fractional shortening (FS) and ejection fraction (EF) values. The results showed that the number of apoptotic cells in the SDF-1 treatment group was significantly lower than those in the other groups at each time-point. The vessel densities in the 3–14 days were significantly greater than those in other groups. At each time-point, the LVEDd and LVESd values were smaller compared with the model group, but greater than the sham operation group and decreased over time. FS and EF values were higher than those in the model group at each time-point, but less than those of the sham operation group and increased over time. The expression levels of TLR-4 and NF-κB at each time-point were significantly higher than those of the remaining groups (p<0.05). In conclusion, SDF-1 is capable of decreasing the apoptosis of cardiac muscle cells in AMI, promoting angiogenesis and improving cardiac function, which may be associated with the activation of the TLR-4/NF-κB signaling pathway.

Abstract 279: Stromal Cell-derived Factor-1 Prevents From Diabetic Cardiomyopathy via Cxcr7/ampk-mediated Nrf2 Activation in Type 2 Diabetic Mice

We have demonstrated that stromal cell-derived factor 1(SDF-1) protects against palmitate-induced cardiac cell death via CXCR7-mediated activation of AMPK signaling (Diabetes 62:2545-2558, 2013). Whether SDF-1 prevents diabetic cardiomyopathy (DCM) and what the underlying mechanism has not been addressed. Here we evaluated the prevention of SDF-1 from DCM in a high fat diet plus streptozotocin (HFD/STZ)-induced type 2 diabetic model in C57BL/6J mice. After 1 month on HFD, the HFD-fed mice were injected with one low dose STZ (100mg/kg body weight, ip). Five days after STZ injection, mice with blood glucose levels ≥250 mg/dl were defined as diabetic. In parallel, the age-matched normal diet-fed mice injected with a same volume of vehicle were used as control. After onset of diabetes, the mice were maintained on HFD or normal diet for another 4 months with or without SDF-1 treatment. Then cardiac function was assayed again, and the mice were sacrificed and cardiac tissue collected for cardiomyopathic index assay. We found that 1 month HFD feeding induced a significant insulin resistance without effect on cardiac function, but continued HFD feeding after STZ injection significantly increased insulin resistance and blood glucose, as well as blood insulin, triglyceride and cholesterol levels. Furthermore, HFD/STZT significantly impaired cardiac function, which were accompanied by increased cardiac inflammation, oxidative stress and fibrotic remodeling. Treatment with SDF-1 dose-dependently prevented diabetes-induced cardiac dysfunction, inflammation and remodeling, but without significant effects on the above mentioned other pathophysiological changes. Mechanistic study demonstrated that diabetes significantly inhibited the function of AMPK and Nrf2, and the expression of CXCR7, but not CXCR4; while treatment with SDF-1 significantly preserved AMPK and Nrf2 function and CXCR7 expression. Knockout CXCR4 did not affect SDF-1 preservation of AMPK and Nrf2 function, but SiRNA knockdown of AMPK resulted in blockade of SDF-1 preservation of Nrf2 function. These results indicate that SDF-1 prevents from DCM via CXCR7/AMPK-mediated Nrf2 activation in type 2 diabetic mice.

Spinal Cord Injury Repair by Intrathecal Infusion of Stromal Cell-Derived Factor-1/CXC Chemokine Receptor 4 in a Rat Model

Background: Stromal cell-derived factor-1 (SDF-1)/CXC Chemokine receptor 4 (CXCR4) is an important cytokine, with multiple functions, which plays a crucial role in the recruitment of multiple stem cell types in the defect sites of central nervous system (CNS). Various strategies have been managed to improve functional recovery after spinal cord injury (SCI). One of these strategies is the use of factors to limit damage and increase recovery. Objectives: In this study we investigated the effect of SDF-1 in spinal cord injury repair in a rat model. Materials andMethods: Adult male Wistar rats were randomly divided to four groups (n = 5) as follows: Sham, SCI, SDF-1 and Vehicle. Spinal cord injury model was created by contusion of T8-T9 by clips and SDF-1 infusion pump implanted in the neck region. One week after injury, 5-Bromo-20-Deoxyuridine (BrdU) was injected to trace the proliferative cells. Basso-Beattie-Bresnahan (BBB) test was performed to evaluate locomotor activity following SCI. Immunohistochemistry test was performed to determine proliferating cells, and real time polymerase chain reaction (PCR) was performed to detect the CXCR4 cells in tissue. Results: Significant improvements in locomotor function were detected in the SDF-1 group compared with the SCI and vehicle groups (P < 0.05). The results showed that SDF-1 treatment increased proliferative cells at the spinal cord injury site. Real time PCR revealed that these proliferative cells are CXCR4 positive that intake Bromodeoxyuridine (Brdu). Conclusions: These results showed that the administration of SDF-1a increases the number of proliferating cells in the injured area in the spinal cord and improves functional recovery.

Protein phosphatase 2A plays an important role in migration of bone marrow stroma cells.

Administration of bone marrow stroma cells (BMSCs) has the potential to ameliorate degenerative disorders and to repair injured sites. The homing of transplanted BMSCs to damaged tissues is a critical property of engraftment. Therefore, it is important to understand signal molecules controlling migration of BMSCs. Here, we demonstrate that serine-threonine protein phosphatase 2A (PP2A) is responsive to migration of BMSCs. Pharmacological Inhibition of PP2A, using okadaic acid (OA), leads to attenuated cell migration in rat primary BMSCs both in the absence or presence of stromal cell-derived factor-1 (SDF-1). Consistent with the above findings, knockdown of the main catalytic subunit PP2Acα using small interfering RNA also attenuates chemotaxis of BMSCs. On the other hand, cell viability of BMSCs remains unchanged with OA treatment or knockdown of PP2Acα subunit. Moreover, we observed an upregulation of PP2A-B55β in transcription level after SDF-1 treatment, indicating their potential role as the functioning regulatory subunit of PP2A phosphatase in BMSCs migration model. Collectively, these data provide first insight into the modulation of BMSCs migration by PP2A phosphatase activity and lay a foundation for exploring PP2A signaling as a modulating target for BMSCs transplantation.

RGS1 and RGS13 Regulate SDF-1-Mediated Responses and Homing in Human Cord Blood CD34+ Hematopoietic Stem/Progenitor Cells

The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis is crucial to migration and homing of hematopoietic stem/progenitor cells (HSC). In a previous study, we reported the expression profile of human cord blood CD34+ cells in response to a short-term exposure of SDF-1 (Leung et al, Blood, 2011). We further identified that expression levels of several R4 Regulator of G-protein signaling (RGS) family proteins were upregulated upon SDF-1 treatment. RGS family proteins are known to negatively control G-protein-coupled receptor signal transduction through their GTPase-accelerating activity. In several types of hematopoietic cells (e.g., B lymphocytes), responses to SDF-1 are subjected to regulation by RGS proteins. However, their expression patterns and functional roles in HSC are poorly characterized. We first assessed the basal expressions of 10 R4 RGS proteins by quantitative RT-PCR. Our results demonstrated that cord blood CD34+ cells expressed relatively high mRNA level of RGS1, RGS2 and RGS3, and moderate level of RGS5, RGS13, RGS16 and RGS18 (n = 3). Expression of RGS8 was barely detectable, while RGS4 and RGS21 expression was not detectable. A short-term exposure (1-24 hours) of CD34+ cells to SDF-1 (100 ng/mL, n = 4) significantly increased expressions of RGS1 (1.5 fold, P = .002), RGS13 (1.8 fold, P = .044), and to a lesser extent RGS2 and RGS3 (both 1.3 fold; P < .032). Expressions of RGS5, RGS16 and RGS18 were not affected by SDF-1 stimulation. Preincubation of CD34+ cells with the CXCR4 antagonist AMD3100 resulted in 82.2% and 56.2% inhibition of SDF-1-induced RGS1 and RGS13 expressions respectively. To investigate the functional roles of RGS1 and RGS13 in SDF-1-mediated responses, we introduced GFP-tagged cDNA clones into cord blood CD34+ cells by lentiviral transduction. Using the spleen focus-forming virus (SFFV) promoter, we achieved 55.2% transduction efficiency in control GFP vector-transduced cells, 39.4% in RGS1-transduced cells and 51.7% in RGS13-transduced cells. Quantitative RT-PCR further confirmed RGS1 (76 fold) and RGS13 (3,641 fold) overexpression in transduced CD34 cells. We showed that SDF-1-directed migration was inhibited by 18.5% in RGS1-transduced cells, but not in RGS13-transduced cells. In RGS1-transduced cells, significantly less GFP+ cells was found in the migrating fraction (51% in non-migrating vs 28% in migrating fraction). We further demonstrated that actin polymerization, an early response to SDF-1 stimulus, was reduced by 9.5% in RGS13-transduced cells but not in RGS1-transduced cells. In the NOD/SCID mouse xenotransplantation model, preliminary results showed that bone marrow homing of CD34+ cells was impaired in RGS1-overexpressing (54.4% reduction) or RGS13-overexpressing (28.1% reduction) cells, when compared to control GFP-transduced cells. Splenic homing of CD34+ cells was not affected by either RGS1 or RGS13. Taken together, we provided the first expression profile of R4 RGS proteins in cord blood CD34+ cells, and their expressional changes in response to SDF-1 stimulation. We also provided evidence that RGS1 and RGS13 regulate specific SDF-1-mediated responses and, importantly, both negatively affect homing of CD34+ cells. Strategies to inhibit RGS1 or RGS13 signaling could be a potential method for enhancing HSC homing and their long-term engraftment for immunologic reconstitution. DisclosuresNo relevant conflicts of interest to declare.

Stromal cell-derived factor 1 promoted migration of adipose-derived stem cells to the wounded area in traumatic rats

BackgroundAdipose-derived stem cells (ADSCs) were effective in treating wound. Stromal cell-derived factor-1 (SDF-1), a chemokine usually called CXCL12, is well known for its chemotaxis in induction of cell migration. However, little is known about the SDF-1responsible for the complex migration of ADSCs from residence to injured sites. ObjectiveHerein, we firstly showed SDF-1 is a major regulator involved in migration of ADSCs during wound repair in vivo. MethodsTrauma in rats was induced by surgical operation. The levels of SDF-1 in wounded tissue were assayed by ELISA. ADSCs were labeled with Green Fluorescent Protein (GFP), and then were transferred to injured rats by intracarotid injection. The plasma levels of ADSCs during wound healing were detected by flow cytometry, and ADSCs in injured tissue were evaluated by bioluminescence imaging in vivo and laser confocal microscopy (LCM), respectively. ResultsADSCs were successfully labeled with GFP. SDF-1 level reached to the peak value on 24 h after injury and then decreased continuously. Additionally, levels of plasma ADSCs in SDF-1 treated rats reached to the peak value (12%) at d21 after medicine delivery, while those of normal and injured rats showed the peak values of 6.28% and 9.84% at d7 and d21, respectively. Finally, the results of LCM indicated treatment of ectogenic SDF-1 obviously enhanced GFP–ADSCs distribution in wounded tissues. ConclusionOur results indicated that SDF-1 treatment obviously promoted the migration and directed distribution of ADSCs in traumatic tissue.

Abstract 315: Stromal Cell-derived Factor 1 Protects From Diabetic Cardiomyopathy

We have demonstrated that stromal cell-derived factor 1(SDF-1) protects against palmitate-induced cardiac apoptosis, which is mediated by NOX-activated nitrosative damage and endoplasmic reticulum stress, via CXCR7, to activate AMPK/p38 MAPK-mediated IL-6 generation (Diabetes 62:2545-2558, 2013). Whether SDF-1 prevents diabetic cardiomyopathy has not been addressed. Here we evaluated the preventive effects of SDF-1 from diabetic cardiomyopathy in a high fat diet plus streptozotocin (HFD/STZ)-induced type 2 diabetic model in C57BL/6J mice. After 1 month on HFD, cardiac function was assayed by echocardiography, and then HFD-fed mice were injected with one low dose STZ (100mg/kg body weight, ip). Five days after STZ injection, mice with blood glucose levels ≥250 mg/dl were defined as diabetic. In parallel, the age-matched normal diet-fed mice injected with a same volume of citrate buffer (pH4.5) were used as control. After onset of diabetes, the mice were maintained on HFD or normal diet for another 4 months with or without SDF-1 treatment. Then cardiac function was assayed again, and the mice were sacrificed and cardiac tissue collected for cardiomyopathic index assay. We found that 1 month HFD feeding induced a significant insulin resistance without effect on cardiac function, but continued HFD feeding after STZ injection significantly impaired cardiac function, which were accompanied by increased insulin resistance and blood glucose, as well as blood insulin, triglyceride and cholesterol levels. Treatment with SDF-1 dose-dependently prevented diabetes-induced cardiac dysfunction but without significant effects on the above mentioned other pathophysiological parameters. These results indicate that SDF-1 possibly prevents diabetic cardiomyopathy via a direct cardiomyocyte action, which needs to be further defined in future study.

Effect of dual treatment with SDF-1 and BMP-2 on ectopic and orthotopic bone formation.

PurposesThe potent stem cell homing factor stromal cell-derived factor-1 (SDF-1) actively recruits mesenchymal stem cells from circulation and from local bone marrow. It is well established that bone morphogenetic protein-2 (BMP-2) induces ectopic and orthotopic bone formation. However, the exact synergistic effects of BMP-2 and SDF-1 in ectopic and orthotopic bone regeneration models have not been fully investigated. The purpose of this study was to evaluate the potential effects of simultaneous SDF-1 and BMP-2 treatment on bone formation.Materials and MethodsVarious doses of SDF-1 were loaded onto collagen sponges with or without BMP-2.These sponges were implanted into subcutaneous pockets and critical-size calvarial defects in C57BL/6 mice. The specimens were harvested 4 weeks post-surgery and the degree of bone formation in specimens was evaluated by histomorphometric and radiographic density analyses. Osteogenic potential and migration capacity of mesenchymal cells and capillary tube formation of endothelial cells following dual treatment with SDF-1 and BMP-2 were evaluated with in vitro assays.ResultsSDF-1-only-treated implants did not yield significant in vivo bone formation and SDF-1 treatment did not enhance BMP-2-induced ectopic and orthotopic bone regeneration. In vitro experiments showed that concomitant use of BMP-2 and SDF-1 had no additive effect on osteoblastic differentiation, cell migration or angiogenesis compared to BMP-2 or SDF-1 treatment alone.ConclusionsThese findings imply that sequence-controlled application of SDF-1 and BMP-2 must be further investigated for the enhancement of robust osteogenesis in bone defects.

A critical role of Src family kinase in SDF-1/CXCR4-mediated bone-marrow progenitor cell recruitment to the ischemic heart

The G protein-coupled receptor CXCR4 and its ligand stromal-cell derived factor 1 (SDF-1) play a crucial role in directing progenitor cell (PC) homing to ischemic tissue. The Src family protein kinases (SFK) can be activated by, and serve as effectors of, G proteins. In this study we sought to determine whether SFK play a role in SDF-1/CXCR4-mediated PC homing. First, we investigated whether SDF-1/CXCR4 signaling activates SFK. Bone-marrow mononuclear cells (BM MNCs) were isolated from WT and BM-specific CXCR4-KO mice and treated with SDF-1 and/or CXCR4 antagonist AMD3100. SDF-1 treatment rapidly induced phosphorylation (activation) of hematopoietic Src (i.e., Lyn, Fgr, and Hck) in WT cells but not in AMD3100-treated cells or CXCR4-KO cells. Then, we investigated whether SFK are involved in SDF-1/CXCR4-mediated PC chemotaxis. In a combined chemotaxis and endothelial-progenitor-cell (EPC) colony assay, Src inhibitor SU6656 dose-dependently inhibited the SDF-1-induced migration of colony-forming EPCs. Next, we investigated whether SFK play a role in SDF-1/CXCR4-mediated BM PC homing to the ischemic heart. BM MNCs from CXCR4BAC:eGFP reporter mice were i.v. injected into WT and SDF-1BAC:SDF1-RFP transgenic mice following surgically-induced myocardial infarction (MI). eGFP+ MNCs and eGFP+c-kit+ PCs that were recruited in the infarct border zone in SDF-1BAC:SDF1-RFP recipients were significantly more than that in WT recipients. Treatments of mice with SU6656 significantly reduced eGFP+ and eGFP+c-kit+ cell recruitment in both WT and SDF-1BAC:RFP recipients and abrogated the difference between the two groups. Remarkably, PCs isolated from BM-specific C-terminal Src kinase (CSK)-KO (Src activated) mice were recruited more efficiently than PCs from WT PCs in the WT recipients. In conclusion, SFK are activated by SDF-1/CXCR4 signaling and play an essential role in SDF-1/CXCR4-mediated BM PC chemotactic response and ischemic cardiac recruitment.

Stromal cell-derived factor 1 (SDF-1) accelerated skin wound healing by promoting the migration and proliferation of epidermal stem cells.

Epidermal stem cells could contribute to skin repair through the migration of cells from the neighboring uninjured epidermis, infundibulum, hair follicle, or sebaceous gland. However, little is known about the factors responsible for the complex biological processes in wound healing. Herein, we will show that the attracting chemokine, SDF-1/CXCR4, is a major regulator involved in the migration of epidermal stem cells during wound repair. We found that the SDF-1 levels were markedly increased at the wound margins following injury and CXCR4 expressed in epidermal stem cells and proliferating epithelial cells. Blocking the SDF-1/CXCR4 axis resulted in a significant reduction in epidermal stem cell migration toward SDF-1 in vitro and delayed wound healing in vivo, while an SDF-1 treatment enhanced epidermal stem cell migration and proliferation and accelerated wound healing. These results provide direct evidence that SDF-1 promotes epidermal stem cell migration, accelerates skin regeneration, and makes the development of new regenerative therapeutic strategies for wound healing possible.

SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma.

Stromal cell-derived factor 1 (SDF-1)/CXCR4 ligand-receptor axis is widely recommended as an attractive target for cancer therapy. Meanwhile, epithelial-mesenchymal transition (EMT) process is linked to disease pathophysiology. As one of inhibitors of apoptosis proteins, survivin is implicated in the onset and development of cancer. In the present study, we tried to determine the cause-effect associations between SDF-1/CXCR4 axis and survivin expression in glioblastoma U-251 cell line. Survivin activation and inhibition were induced with exogenous SDF-1 and survivin small interfering RNA (survivin siRNA), respectively. Western blot was used to detect relevant proteins in SDF-1/CXCR4 axis. Western blot analysis revealed that survivin expression in U-251 increased in a dose- and time-dependent manner in response to SDF-1 treatment. However, the interference with MEK/ERK and PI3K/AKT pathway prohibited SDF-1-induced survivin up-regulation. Importantly, survivin knockdown abrogated cell cycle progression and the expression of snail and N-cadherin, compared with non-transfectants. In conclusion, the present study shows that SDF-1 up-regulates survivin via MEK/ERK and PI3K/AKT pathway, leading to cell cycle progression and EMT occurrence dependent on survivin. The blockade of survivin will allow for the treatment of glioblastoma.