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.

Akt-centered amplification loop plays a critical role in vascular endothelial growth factor/stromal cell-derived factor 1-αcross-talk and cardioprotection

Background Vascular endothelial growth factor （VEGF） is one of major mediators of angiogenesis and survival factor in some tissue, however, its direct effects on cardiomyocytes remain poorly understood. Methods Rat neonatal ventricular myocytes were cultured in vitro. Akt phosphorylation was measured by Western blotting; the expression of stromal cell-derived factor α（SDF-1α）/CXCR4 axis was evaluated by real-time PCR and Western blotting. LY294002 and AMD3100 were used to interfere with the signaling of VEGF and SDF-1α/CXCR4 axis. Cardiac myocytes viability and injury were evaluated by trypan blue staining and lactate dehydrogenase （LDH） release. Results Treatment of neonatal rat ventricular myocytes with VEGF induced phosphorylation of Akt in a dose and FIk-1 dependent manner. VEGF attenuated H202 induced cardiac myocyte death. The phosphoinositol-3-kinase （PI3K） inhibitor, LY294002 and FIk-1 antibody abolished the beneficial effects of VEGF on H202 induced cell death. In the mean time SDF-1α-CXCR4 axis was up-regulated by VEGF through PI3K-Akt signaling and contributed to the protective effects of VEGF on H202 induced cell death. Interestingly, SDF-1α also promoted production of VEGF in cultured cardiac myocytes and LY294002 reversed the up-reguLation of VEGF induced by SDF-1α. Conclusion VEGF has direct protective effects on cardiomyocytes; a crosstalk between VEGF and SDF-1α through PI3K-Akt serves a survival role in cardiomvocvtes in vitro.

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.

Expression of stromal cell-derived factor-1 in diabetic retinopathy

Background Neovascularization can cause vision loss in proliferative diabetic retinopathy （PDR） and may be affected by many factors. Stromal cell-derived factor-1 （SDF-1） is a potent stimulator of angiogenesis. The study was aimed to investigate the expression of SDF-1 and its correlation with vascular endothelial growth factor （VEGF） in the eyes with diabetic retinopathy. Methods The levels of SDF-1 and VEGF were measured by enzyme-linked immunosorbent assay in the vitreous of 41 eyes of 41 patients with PDR and 12 eyes of 12 patients with idiopathic macular hole （IMH）. Vitreous fluid samples and fibrovascular preretinal membranes were obtained at vitrectomy. SDF-1 and VEGF were localized using immunohistochemistry. Results The vitreous concentration of VEGF was significantly higher in eyes with PDR （（2143.7~1685.21） pg/ml） than in eyes with IMH （（142.42~72.83） pg/ml, P 〈0.001）. The vitreous level of SDF-1 was also significantly higher in eyes with PDR （（306.37~134.25） pg/ml） than in eyes with IMH （（86.91~55.05） pg/ml, P 〈0.001）. The concentrations of both VEGF and SDF-1 were higher in eyes with active PDR than in eyes with inactive PDR. Panretinal photocoagulation （PRP） could decrease the SDF-1 levels in the vitreous of PDR patients. The vitreous concentration of SDF-1 correlated with that of VEGF in eyes with PDR （r=0.61, P 〈0.001）. The costaining of SDF-1 and VEGF was confined to the vascular components in preretinal membranes. Conclusions SDF-1 protein is highly expressed in both the vitreous and preretinal membranes of PDR patients; SDF-1 may be correlated with VEGF in angiogenesis in PDR.

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.

Tropism mechanism of stem cells targeting injured brain tissues by stromal cell-derived factor-1

Objective: To explore the role and function of stromal cell-derived factor- 1 (SDF- 1) in stem cells migrating into injured brain area.Methods: Rat-derived nerve stem cells (NSCs) were isolated and cultured routinely. Transwell system was used to observe the migration ability of NSCs into injured nerve cells. Immunocytochemistry was used to explore the expression of chemotactic factor receptor-4 (CXCR-4) in NSCs. In vivo, we applied immunofluorescence technique to observe the migration of NSCs into injured brain area. Immunofluorescence technique and Western blotting were used to test expression level of SDF- 1. After AMD3100 (a special chemical blocker) blocking CXCR-4, the migration ability of NSCs was tested in vivo and in vitro, respectively.Results: NSCs displayed specific tropism for injured nerve cells or traumatic brain area in vivo and in vitro. The expression level of SDF-1 in traumatic brain area increased remarkably and the expression level of CXCR-4 in the NSCs increased simultaneously. After AMD3100 blocking the expression of CXCR-4, the migration ability of NSCs decreased significantly both in vivo and in vitro.Conclusions: SDF-1 may play a key role in stem cells migrating into injured brain area through specially combining with CXCR-4.

CXCR4基因转染的猪BMSCs体外诱导分化为心肌样细胞的实验研究

目的：探讨基质细胞衍生因子-1（SDF-1）受体CXCR4基因转染的猪骨髓间充质干细胞（BMSCs）体外诱导分化为心肌样细胞的可行性。方法：按照Nance方法培养BMSCs,转染带荧光的CXCR4基因,流式细胞仪检测转染后BMSCs的细胞周期,用5-氮胞苷（5-aza）诱导,并行结蛋白、肌球蛋白重链、心肌特异性肌钙蛋白I免疫组化鉴定。对照组为未转染CXCR4基因的BMSCs。结果：体外培养的原代BMSCs 10~14 d达到融合,CXCR4基因转染后的BMSCs能成功表达CXCR4,转染前后细胞周期无明显改变。与对照组相同,经5-aza刺激后,部分BMSCs成梭形,结蛋白、肌球蛋白重链、心肌特异性肌钙蛋白I免疫组化染色均为阳性。结论：CXCR4基因转染的猪BMSCs在体外条件下生长稳定,传代后仍保持未分化状态,有分化成心肌样细胞的潜能。

基质细胞衍生因子1α培养心肌细胞和成纤维细胞的增殖与迁移

背景:在心脏缺损边缘造成新创面能够促进缺损自愈,基质细胞衍生因子1α能够促进血管生成以及心脏功能。目的:探讨物理损伤及基质细胞衍生因子1α对心肌细胞增殖的影响及心肌细胞对心脏成纤维细胞迁移的趋化作用。方法:原代分离培养大鼠心肌细胞和心脏成纤维细胞,采用刮伤法建立物理损伤模型,并应用10-160μg/L的基质细胞衍生因子1α进行干预;CCK-8法检测心肌细胞的增殖能力;Transwell法检测心肌细胞趋化下心脏成纤维细胞的迁移能力。结果与结论:不同浓度的基质细胞衍生因子1α均可提高物理损伤下心肌细胞的增殖能力,尤以80μg/L基质细胞衍生因子1α作用下心肌细胞增殖最明显。同时,物理损伤联合基质细胞衍生因子1α培养的心肌细胞可显著增加心脏成纤维细胞的迁移能力,并且随着趋化培养时间的延长,迁移能力增加更明显。可见基质细胞衍生因子1α能提高物理损伤心肌细胞的增殖能力,物理损伤联合基质细胞衍生因子1α培养的心肌细胞对心脏成纤维细胞的迁移具有促进作用。

The impact of hypoxic-ischemic brain injury on stem cell mobilization,migration,adhesion,and proliferation

Neonatal hypoxic-ischemic encephalopathy continues to be a significant cause of death or neurodevelopmental delays despite standard use of therapeutic hypothermia.The use of stem cell transplantation has recently emerged as a promising supplemental therapy to further improve the outcomes of infants with hypoxic-ischemic encephalopathy.After the injury,the brain releases several chemical mediators,many of which communicate directly with stem cells to encourage mobilization,migration,cell adhesion and differentiation.This manuscript reviews the biomarkers that are released from the injured brain and their interactions with stem cells,providing insight regarding how their upregulation could improve stem cell therapy by maximizing cell delivery to the injured tissue.

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.