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

BACKGROUND Intervertebral disc(IVD) degeneration is a condition characterized by a reduction in the water and extracellular matrix content of the nucleus pulposus(NP) and is considered as one of the dominating contributing factors to low back pain. Recent evidence suggests that stromal cell-derived factor 1α(SDF-1α) and its receptor CX-C chemokine receptor type 4(CXCR4) direct the migration of stem cells associated with injury repair in different musculoskeletal tissues.AIM To investigate the effects of SDF-1α on recruitment and chondrogenic differentiation of nucleus pulposus-derived stem cells(NPSCs).METHODS We performed real-time RT-PCR and enzyme-linked immunosorbent assay to examine the expression of SDF-1α in nucleus pulposus cells after treatment with pro-inflammatory cytokines in vitro. An animal model of IVD degeneration was established using annular fibrosus puncture in rat coccygeal discs. Tissue samples were collected from normal control and degeneration groups.Differences in the expression of SDF-1α between the normal and degenerative IVDs were analyzed by immunohistochemistry. The migration capacity of NPSCs induced by SDF-1α was evaluated using wound healing and transwell migration assays. To determine the effect of SDF-1α on chondrogenic differentiation of NPSCs, we conducted cell micromass culture and examined the expression levels of Sox-9, aggrecan, and collagen II. Moreover, the roles of SDF-1/CXCR4 axis in the migration and chondrogenesis differentiation of NPSCs were analyzed by immunofluorescence, immunoblotting, and real-time RT-PCR.RESULTS SDF-1α was significantly upregulated in the native IVD cells cultured in vitro with pro-inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, mimicking the degenerative settings. Immunohistochemical staining showed that the level of SDF-1α was also significantly higher in the degenerative group than in the normal group. SDF-1α enhanced the migration capacity of NPSCs in a dose-dependent manner. In addition, SDF-1α induced chondrogenic differentiation of NPSCs, as evidenced by the increased expression of chondrogenic markers using histological and immunoblotting analyses. Realtime RT-PCR, immunoblotting, and immunofluorescence showed that SDF-1αnot only increased CXCR4 expression but also stimulated translocation of CXCR4 from the cytoplasm to membrane, accompanied by cytoskeletal rearrangement.Furthermore, blocking CXCR4 with AMD3100 effectively suppressed the SDF-1α-induced migration and differentiation capacities of NPSCs.CONCLUSION These findings demonstrate that SDF-1α has the potential to enhance recruitment and chondrogenic differentiation of NPSCs via SDF-1/CXCR4 chemotaxis signals that contribute to IVD regeneration.

Upregulation of stromal cell-derived factor-1 alpha/CXCR4 axis-induced migration of human neural progenitors by tumor necrosis factor-alpha and interleukin-8

BACKGROUND： Studies of several animal models of central nervous system diseases have shown that neural progenitor cells （NPCs） can migrate to injured tissues. Stromal cell-derived factor 1 alpha （SDF-la）, and its primary physiological receptor CXCR4, have been shown to contribute to this process. OBJECTIVE： To investigate migration efficacy of human NPCs toward a SDF-1α gradient, and the regulatory roles of tumor necrosis factor-α （TNF-α） and interleukin-8 （IL-8） in SDF-1α/CXCR4 axis-induced migration of NPCs. DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, cellular and molecular biology study was performed at the Laboratory of Department of Cell Biology, Medical College of Soochow University between October 2005 and November 2007. MATERIALS： SDF-1α and mouse anti-human CXCR4 fusion antibody were purchased from R＆D Systems, USA. TNF-αwas purchased from Biomyx Technology, USA and IL-8 was kindly provided by the Biotechnology Research Institute of Soochow University. METHODS： NPCs isolated from forebrain tissue of 9 to 10-week-old human fetuses were cultured in vitro. The cells were incubated with 0, 20, and 40 ng/mL TNF-α, or 0, 20, and 40 ng/mL IL-8, for 48 hours prior to migration assay. For antibody-blocking experiments, cells were further pretreated with 0, 20, and 40 μg/mL mouse anti-human CXCR4 fusion antibody for 2 hours. Subsequently, the transwell assay and CXCR4 blockade experiments were performed to evaluate migration of human NPCs toward a SDF-1α gradient. Serum-free culture medium without SDF-1α served as the negative control. MAIN OUTCOME MEASURES： The transwell assay was performed to evaluate migration of human NPCs toward a SDF-1α gradient, which was blocked by fusion antibody against CXCR4. In addition, CXCR4 expression in human NPCs stimulated by TNF-α and IL-8 was measured by flow cytometry. RESULTS： Results from the transwell assay demonstrated that SDF-1α was a strong chemoattractant for human NPCs （P 〈 0.01）, and 20 ng/mL produced the highest levels of migration. Anti-human CXCR4 fusion antibody significantly blocked the chemotactic effect （P 〈 0.05）. Flow cytometry results showed that treatment with TNF-α and IL-8 resulted in increased CXCR4 expression and greater chemotaxis efficiency of NPCs towards SDF-1α（P 〈 0.01）. CONCLUSION： These results demonstrated that SDF-la significantly attracted NPCs in vitro, and neutralizing anti-CXCR4 antibody could block part of this chemotactic function. TNF-α and IL-8 increased chemotaxis efficiency of NPCs towards the SDF-1αgradient by upregulating CXCR4 expression in NPCs.

Leukocyte cell-derived chemotaxin-2 and fibroblast growth factor 21 in alcohol-induced liver cirrhosis

BACKGROUND The importance of early diagnosis of alcoholic liver disease underscores the need to seek better and especially non-invasive diagnostic procedures.Leukocyte cellderived chemotaxin-2(LECT2)has been widely studied to determine its usefulness in monitoring the course of non-alcoholic fatty liver disease but not for alcoholic liver cirrhosis(ALC).AIM To determine the concentration of LECT2 in the blood serum of patients in relation to progressive stages of ALC,its relation to fibroblast growth factor 1(FGF-1)and FGF-21,and to examine the possible wider use of LECT2 in diagnosing ALC.METHODS A retrospective case-control study was conducted with 69 ALC cases and 17 controls with no ALC.Subjects were recruited from the region of Lublin(eastern Poland).Liver cirrhosis was diagnosed based on clinical features,history of heavy alcohol consumption,laboratory tests,and abdominal ultrasonography.The degree of ALC was evaluated according to Pugh-Child criteria(the Pugh-Child score).Blood was drawn and,after centrifugation,serum was collected for analysis.LECT2,FGF-1,and FGF-21 were determined using enzyme-linked immunosorbent assay kits.RESULTS The LECT2 Levels in the control group were 18.99±5.36 ng/mL.In the study groups,they declined with the progression of cirrhosis to 11.06±6.47 ng/mL in one group and to 8.06±5.74 ng/mL in the other(P<0.0001).Multiple comparison tests confirmed the statistically significant differences in LECT2 Levels between the control group and both test groups(P=0.006 and P<0.0001).FGF-21 Levels were 44.27±64.19 pg/mL in the first test group,45.4±51.69 pg/mL in the second(P=0.008),and 13.52±7.51 pg/mL in the control group.The difference between the control group and the second test group was statistically significant(P=0.007).CONCLUSION We suggest that LECT2 may be a non-invasive diagnostic factor for alcoholinduced liver cirrhosis.The usefulness of LECT2 for non-invasive monitoring of alcohol-induced liver cirrhosis was indirectly confirmed by the multiple regression model developed on the basis of our statistical analysis.

血清生长停滞特异基因6和基质细胞衍生因子1水平与糖尿病视网膜病变的相关性

目的探讨生长停滞特异基因6(Gas6)、基质细胞衍生因子1(SDF-1)在糖尿病视网膜病变(DR)发病机制中的作用。方法收集我院就诊的2型糖尿病患者120例,并根据糖尿病分期分为糖尿病无视网膜病变组(NDR)、单纯型DR组(BDR)及增生型DR组(PDR)3组,每组40例;正常组我院体检的健康成年人40例;测定血中白细胞和中性粒细胞的数量,总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDLC)、低密度脂蛋白(LDL-C)的含量,酶联免疫吸附试验(ELISA)测定受试者血清中Gas6、SDF-1α和SDF-1β的水平。结果 PDR组血清中TC水平明显高于其余3组(P=0.023、0.041、0.046);PDR组白细胞计数显著降低,与其余3组比较差异有统计学意义(P=0.016、0.021,0.019),BDR组的中性粒细胞计数高于PDR组(P=0.028),正常组和NDR组HDL-C水平高于PDR组(P=0.031),4组的血清TG水平差异无统计学意义(P=0.42);正常组血清Gas6水平明显高于3组糖尿病患者,差异有统计学意义(P=0.028);NDR组、BDR组和PDR组患者血清SDF-1α水平高于正常组(P=0.02),且BDR组和PDR组高于NDR组(P=0.04);BDR组和PDR组血清SDF-1β水平明显高于正常组和NDR组(P=0.014);DR患者血清Gas6与TC、TG水平均呈弱正相关(P<0.05);DR患者血清SDF-1β水平与白细胞的数量呈弱正相关(P<0.05)。结论 Gas6可能是通过改变患者的脂质代谢状况而参与DR的发病过程;DR病变后,由于视网膜部位的缺血、缺氧诱导了SDF-1α、SDF-1β的高表达,参与视网膜血管的新生;Gas6、SDF-1与DR的发病机制有关。

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.

Apelin and vascular endothelial growth factor are associated with mobilization of endothelial progenitor cells after acute myocardial infarction

This study was designed to determine the levels of early endothelial progenitor cells （EPCs）, apelin, vascu- lar endothelial growth factor （VEGF） and stromal cell-derived growth factor-1 （SDF-1） after acute myocardial infarction （AMI）, and to investigate the relationships between these cytokines and early EPCs. Early EPCs, de- fined as CD133＋, KDR＋, and CD34~ cells, were quantified by flow cytometry. The levels of early EPCs and those cytokines in AMI patients were significantly different from those with coronary artery disease or controls （P 〈 0.05）. Plasma apelin levels were inversely correlated with Gensini score and early EPCs （both P 〈 0.01）. Early EPCs, VEGF and SDF-1 showed different patterns of changes in AMI patients during the first 24 h. The trend in the change of early EPCs was proportionally correlated with that of VEGF （P 〈 0.05）. AMI patients exhibited in- creased early EPCs with remarkably decreased apelin levels and enhanced VEGF levels.

强制性运动疗法对脑缺血后神经修复的作用及分子机制

目前强制性运动疗法广泛地应用于康复领域,并被证实能够有效促进脑缺血后肢体运动功能的恢复。功能磁共振成像和经颅磁刺激等神经影像技术证明,强制性运动疗法能够促进脑缺血后功能重组,影响神经再生。强制性运动疗法通过上调间质细胞源性因子-1(SDF-1)来增强内源性神经干细胞的激活,而SDF-1又通过抑制Rho激酶的活性来促进神经元轴突延伸。抑制Rho激酶活性还会消除勿动蛋白(Nogo)受体介导的抑制作用从而促进轴突再生,进而明显改善神经功能缺失。因此,SDF-1、Nogo及Rho激酶可能是强制性运动疗法促使脑缺血后神经再生的重要分子。

低氧诱导趋化因子受体表达对视网膜前体细胞迁移能力的影响

背景体外研究表明，趋化性细胞因子受体4（CXCR4）及其配体基质细胞衍生因子-1（SDF．1）在诱导视网膜前体细胞（RPCs）定向迁移的过程中可能起重要作用。RPCs表达CXCR4升高能增强干细胞的趋化活性，从而提高移植细胞的定向迁移能力。目的探讨RPCs在低氧条件下CXCR4受体的表达。方法分离孕龄17d的NIH小鼠的胚胎视网膜细胞并制备成含5×10^6～10×10^6个／L细胞的悬液，将细胞接种到25cm2培养瓶中，用全神经球贴壁培养法进行培养。RPCs在正常O2（体积分数16％O2）和低O2（体积分数10％O2）环境中培养12h和24h后，用逆转录聚合酶链反应（RT—PCR）法检测CXCR4和缺氧诱导因子-1（HIF-1）mRNA的表达；流式细胞仪（FACS）检测RPCs中CXCR4阳性细胞的百分比；Boyden小室实验观察30μg／L的SDF-1对RPCs的趋化效应。结果10％O2培养12h和24h后，RPCs中CXCR4mRNA的表达量（CXCR4mRNA／B—actinmRNA）分别为0．28±0．07和0．48±0．17，比正常氧培养组的0．16±0．02升高了1．75倍和3．00倍，10％O2培养12h和24h后RPCs中HIF-1mRNA表达量（HIF—1mRNA／B—actinmRNA）分别为0．18±0．07和0．38±0．13，比正常氧培养组的0．06±0．01升高了3．00倍和6．30倍，差异有统计学意义（P〈0．01）。Boyden小室实验表明，10％O2培养12h和24h后SDF-1对RPCs的趋化效应由正常氧的13．00％分别上升到36．00％和46．00％。FACS检测表明，10％O2诱导12h和24h后，RPCs中CXCR4阳性细胞率由正常氧浓度的9．01％分别上升到26．90％和46．10％，差异均有统计学意义（P〈0．01）。结论RPCs在低氧条件下CXCR4受体表达增加，同时对SDF-1的趋化能力增强。HIF-1的表达增加是CXCR4表达增高的可能机制。

Effects of Gas1 on gliomas:a review on current preclinical studies

Glioblastoma multiforme(GBM)is the most common and lethal brain tumor.Its prognosis remains very poor,despite the use of combined treatments such as surgical resection,radiation and chemotherapy.The major limitations for the treatment of GBM are its high invasiveness,tumor recurrence and resistance to treatments.Therefore,gene therapy appears as a relevant strategy for its treatment.Thus,we have investigated the use of growth-arrest-specific 1(Gas1)for the treatment of GBM.Gas1 is a tumor suppressor protein that inhibits glioma growth by inducing arrest and apoptosis of tumor cells.Moreover,we have shown that a soluble form of Gas1 acting in both autocrine and paracrine manners is also effective inhibiting tumor growth in animal models,indicating its potential as an adjuvant for the treatment of GBM.

The molecular basis of hypertrophic scars

Hypertrophic scars(HTS)are caused by dermal injuries such as trauma and burns to the deep dermis,which are red,raised,itchy and painful.They can cause cosmetic disfigurement or contractures if craniofacial areas or mobile region of the skin are affected.Abnormal wound healing with more extracellular matrix deposition than degradation will result in HTS formation.This review will introduce the physiology of wound healing,dermal HTS formation,treatment and difference with keloids in the skin,and it also review the current advance of molecular basis of HTS including the involvement of cytokines,growth factors,and macrophages via chemokine pathway,to bring insights for future prevention and treatment of HTS.

Effect of Fufang Xueshuantong Capsule(复方血栓通胶囊) on A Rat Model of Retinal Vein Occlusion

Objective：To establish a retinal vein occlusion（RVO） animal model and observe the therapeutic effect of a Chinese herbal composition（Fufang Xueshuantong Capsule,复方血栓通胶囊,FXC） in ischemic retinal disease.Methods：Fifteen adult male Sprague-Dawley rats underwent laser photothrombosis to induce RVO on their right eyes and were subsequently randomized to receive FXC（the intervention group,n=7） or placebo treatment（the control group,n=8）.Fundus fluorescein angiography was performed after 2,4 and 8 weeks of treatment.Real-time reverse transcription-PCR was used to quantify the mRNA expression of vascular endothelial growth factor（VEGF） and stromal cell-derived factor-1（SDF-1）.The main outcomes were the mRNA copies of VEGF and SDF-1 and the counts of RVO signs.Results：Laser photothrombosis procedure induced typical lesions of RVO,including hemorrhage,leakage,retinal detachment,capillary non-perfusion,filling defect of retinal vessels,and lateral circulation/dilation of small vessels.The retinal lesions were associated with an increased expression of VEGF（P0.05）.No significant change of SDF-1 expression was noticed.Compared with the control group,the intervention group had numerically fewer RVO lesions at week 2（1.71±0.76 vs.3.50±1.51,t=-2.82,P0.05）.The benefit of intervention remained at weeks 4 and 8.Conclusions：A rat model of laser photothrombosis-induced RVO was established and an increase in the VEGF expression was observed in the retinal lesion.The FXC had therapeutic benefit in improving retinal lesions in the rat model of RVO.

Acupuncture Alters Pro-inflammatory Cytokines in the Plasma of Maternally Separated Rat Pups

Objective: To investigate the potential alleviating effects of acupuncture on maternal separation(MS)-induced changes in plasma pro-inflammatory cytokine levels of rat pups. Methods: On postnatal day 15, rat pups were randomly assigned to 4 groups(n=6 per group) using a random number table: normal, MS, MS with acupuncture stimulation at Shenmen(HT 7) acupoint(MS+HT 7), and MS with acupuncture stimulation at Chengshan(BL 57) acupoint(MS+BL 57) groups. Rat pups in the normal group were housed with their mothers under standard conditions; those in the MS, MS+HT 7 and MS+BL 57 groups were maternally separated and individually maintained. Acupuncture stimulation was performed at HT 7 or BL 57 acupoints once a day for 7 consecutive days. A tail suspension test was performed to measure immobility time of rats and the plasma was collected on postnatal day 21, then levels of corticosterone(CORT), interleukin(IL)-1β, IL-6 and glial cell-derived neurotrophic factor(GDNF) in plasma were measured. Results: Compared with the normal group, the immobility time and the plasma levels of CORT, IL-1β, IL-6 and GDNF in the MS group were significantly increased(P<0.05 or P<0.01). Compared with the MS group, the immobility time and the plasma levels of CORT, IL-1β, IL-6 and GDNF were significantly reduced in the MS+HT 7 group(P<0.05 or P<0.01). Moreover, the immobility time and plasma levels of IL-1β and IL-6 in the MS+HT 7 group were significantly lower than those in the MS+BL 57 group(P<0.05). Conclusion: Acupuncture stimulation at HT 7 can alleviate the behavioral impairment and changes of the cytokines by MS, indicating that acupuncture can help to relieve MS-induced depression.