Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow

Homing and engraftment of hematopoietic stem/progenitor cells (HSPCs) in bone marrow is the major determining factor in success of hematopoietic stem cell transplantation. This is a complex, multistep process orchestrated by the coordinated interplay between adhesion molecules, cytokines, growth factors, and regulatory cofactors, many of which remain to be defined. Recent studies have highlighted the pivotal role of unique stromal-derived factor-1 (SDF-1)/CXCR4 signaling in the regulation of HSPC homing and subsequent engraftment. In addition, studies suggest that SDF-1/CXCR4 signaling acts as an essential survival-promoting factor of transplanted HSPCs as well as maintenance of quiescent HSCs in bone marrow niche. These pleiotropic effects exerted by SDF-1/CXCR4 axis make this unique signaling initiator very promising, not only for optimal hematopoietic reconstitution but also for the development of innovative approaches to achieve restoration, regeneration, or repair of other damaged tissues potentially amendable to reversal by stem cell transplantation. This goal can only be achieved when the role of SDF-1/CXCR4 axis in hematopoietic transplantation is clearly defined. Hence, this review presents current knowledge of the mechanisms through which SDF-1/CXCR4 signaling promotes restoration of hematopoiesis by regulating the homing and engraftment of HSPCs.     

基质细胞衍生因子1及其受体CXCR4在内源性神经干细胞迁移中的作用

背景：神经干细胞具有体外、体外迁移的特性,但关于其迁移的具体机制还不十分清楚。 目的：探讨基质细胞衍生因子1及其受体CXCR4在神经干细胞体内迁移中的作用。 方法：制备脑皮质微量注射脂多糖脑损伤大鼠模型,于建模后3,5,7 d灌注取脑,采用冰冻切片免疫组织化学方法,动态监测基质细胞衍生因子1和nestin表达的时相变化。 结果与结论：免疫组织化学染色观察显示：基质细胞衍生因子1随时间推移表达的量逐渐增多。Nestin阳性细胞有明显向外延伸和迁移的迹象,尖端指向脂多糖注射区。结果可见基质细胞衍生因子1趋化体内表达其特异性受体的内源性神经干细胞向脂多糖注射区迁移。     

The notch receptor and its ligands are selectively expressed during hematopoietic development in the mouse.

Members of the Notch family of transmembrane receptors are found on primitive hematopoietic precursors, and Notch ligand expression has been demonstrated on the surface of stromal cells, suggesting a role for Notch signaling in mammalian blood cell development. The current report examines the expression of Notch receptors and their ligands in murine hematopoietic tissues to determine: A) which blood cell lineages in the adult are influenced by Notch activity, and B) whether fetal hematopoiesis in the embryo involves the Notch pathway. In the adult mouse, a combination of flow cytometry, immunohistochemistry and Northern analysis was used to examine Notch receptor or ligand expression in bone marrow and spleen. In the embryo, Northern analysis and in situ hybridization were used to characterize Notch receptor and ligand expression in fetal liver on embryonic day 12 (E12) through E17, an active period encompassing both erythropoiesis and granulopoeisis. Flow cytometry demonstrated the presence of Notch1 and Notch2 receptors on bone marrow-derived myeloid cells but not on erythroid cells positive for the marker, Ter-119. In situ hybridization of E12 through E17 fetal liver demonstrated widespread expression of Jagged1 and Delta1 in a pattern similar to but less abundant than that of the erythropoietin receptor. Taken together with earlier functional results, the current expression data suggest a role for Notch activity in establishing definitive hematopoiesis in fetal liver, as well as a selective use of Notch signaling in adult erythropoiesis and granulopoiesis. Notch receptors in the adult are most likely utilized by early erythroid precursors and intermediate-stage granulocytes, but not by terminally differentiating cells of either subset.     

Expression of stromal cell-derived factor-1 and of its receptor CXCR4 in liver regeneration from oval cells in rat

Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.     

Stromal cell-derived factor-1 from biliary epithelial cells recruits CXCR4-positive cells: implications for inflammatory liver diseases

Although stromal cell-derived factor-1 (SDF-1) plays an important role in hematopoiesis in the fetal liver, the role after birth remains to be clarified. We investigated the role of SDF-1 and its receptor, CXCR4, in 75 patients; this included controls and patients with viral hepatitis, liver cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Interestingly, SDF-1 appeared up-regulated in biliary epithelial cells (BEC) of inflammatory liver disease. Furthermore, in inflammatory liver diseases, SDF-1 was expressed by BEC of interlobular and septal bile ducts and by proliferated bile ductules. The message expression of SDF-1 in BEC was confirmed at a single-cell level by RT-PCR and laser capture microdissection. The plasma levels of SDF-1 were significantly higher in patients with liver diseases than in normal controls. Flow cytometric analysis of the surface expression of CXCR4 showed that most liver-infiltrating lymphocytes express CXCR4 and the intensity was up-regulated more significantly in liver-infiltrating lymphocytes than in peripheral blood lymphocytes. These results suggest that increased SDF-1 production by BEC may play an important role in the recruitment of CXCR4-positive inflammatory cells into the diseased livers. These data are significant because modulation of the SDF-1/CXCR4 interaction has therapeutic implications for inflammatory liver diseases.     

The chemokine receptor CXCR4 is expressed within the mast cell lineage and its ligand stromal cell-derived factor-1alpha acts as a mast cell chemotaxin

In this study we provide evidence that the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) acts as a mast cell chemoattractant through interactions with its receptor CXCR4 expressed on mast cell progenitors in the blood as well as on in vitro-developed and leukemic mast cells. We found expression of CXCR4 on cord blood-derived mast cells (CBMC) and on the human mast cell line HMC-1, analyzed by RNAse protection assay and flow cytometry. SDF-1alpha induced intracellular calcium mobilization in HMC-1 cells and was chemotactic for both HMC-1 cells and CBMC. The activity of SDF-1alpha was completely blocked by treating the cells with pertussis toxin, indicating the involvement of Gi-proteins in the signaling. By applying a transwell assay we could show that SDF-1alpha induces migration of a cell population in peripheral blood that is enriched for cells with the capacity to differentiate into mast cells. These findings thus suggest a mechanism by which human mast cell progenitors may be recruited from circulation into the tissue.     

Possible regulation of migration of intrahepatic cholangiocarcinoma cells by interaction of CXCR4 expressed in carcinoma cells with tumor necrosis factor-alpha and stromal-derived factor-1 released in stroma

Intrahepatic cholangiocarcinoma (ICC) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. We examined roles of CXCR4 and its ligand, stromal cell-derived factor (SDF)-1, in migration of ICC with respect to tumor-stromal interaction by using two ICC cell lines, a fibroblast cell line (WI-38), and 28 human ICC tissues. The two ICC cell lines expressed CXCR4 mRNA and protein, and WI-38 fibroblasts expressed SDF-1 mRNA and protein. Migration of cultured ICC cells in Matrigel was induced by co-culture with WI-38 fibroblasts and by incubation with SDF-1. Anti-SDF-1 antibody suppressed migration, demonstrating that SDF-1 released from WI-38 fibroblasts was responsible for this migration. Tumor necrosis factor (TNF)-alpha pretreatment of ICC cells up-regulated CXCR4 mRNA and protein expression in a concentration-dependent manner. Administration of SDF-1 and TNF-alpha increased synergistically ICC cell migration, which was suppressed by the CXCR4 antagonist AMD3100. In ICC tissue, TNF-alpha was mainly expressed in infiltrated macrophages, CXCR4 in ICC cells, and SDF-1 in stromal fibroblasts. In conclusion, the interaction of SDF-1 released from fibroblasts and CXCR4 expressed on ICC cells may be actively involved in ICC migration, and TNF-alpha may enhance ICC cell migration by increasing CXCR4 expression. CXCR4 could be a therapeutic target to prevent ICC invasion.     

基质细胞衍生因子1对转染CXCR4基因支气管上皮细胞的增殖和 定向趋化作用

背景：早期的诱导替代物表面的气管黏膜上皮覆盖并恢复其功能,是气管替代物研究中的关键问题。基质细胞衍生因子1/CXCR4通路在加快组织修复中发挥重要作用。 目的：观察基质细胞衍生因子1对转染CXCR4基因支气管上皮细胞的增殖和定向趋化作用。 方法：构建CXCR4慢病毒表达载体并转染人支气管上皮细胞。实验分为3组,空白组：未感染任何病毒的细胞组;对照组：感染未转染CXCR4的慢病毒细胞组;实验组：转染CXCR4慢病毒表达载体的细胞组。将消化好的病毒浸染的人支气管上皮细胞和普通人支气管上皮细胞悬液以不同浓度基质细胞衍生因子1(1 μmol/L,100 nmol/L, 10 nmol/L,1 nmol/L,0.1 nmol/L,0 nmol/L)处理。 结果与结论：实验成功构建了CXCR4慢病毒表达载体。正常人支气管上皮细胞的CXCR4蛋白基础表达较低。转染CXCR4的人支气管上皮细胞中CXCR4 mRNA和蛋白都有较高水平的表达,且随着基质细胞衍生因子1浓度增加,其吸光度逐渐增加,并呈浓度依赖性(P < 0.05)。提示基质细胞衍生因子1增强了转染其受体CXCR4基因支气管上皮细胞的增殖作用和定向迁移能力。     

Vascular endothelial growth factor, its receptor Flk-1, and hypoxia inducible factor-1alpha are involved in malignant transformation in dysplastic nodules of the liver

Dysplastic nodules (DNs) are regarded as a premalignant lesion of hepatocellular carcinoma (HCC). Sinusoidal capillarization and unpaired arteries are reported in HCC and also to a lesser degree in DN. However, the mechanism and significance of these vascular alterations remain unclear. In this study, these vascular changes were examined with respect to vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1), hypoxia inducible factor-1alpha (HIF-1alpha), and CD34, by using 20 surgically resected cases of DNs and 36 cases of conventional HCC. The expression of these molecules was examined immunohistochemically. Although sinusoidal capillarization characterized by CD34 expression was found diffusely in HCC, such changes were found mainly in the areas around the portal tracts and also in other areas in DNs (focal in 6 cases, zonal in 7 cases, and extensive distribution in 7 cases). These capillarized areas were frequently associated with unpaired arteries, and the infiltration of neoplastic hepatocytes into the portal tracts and loss of reticulin fibers in DNs, particularly those with a zonal and extensive distribution. VEGF was diffusely expressed in neoplastic hepatocytes of DNs and HCC. Interestingly, Flk-1 and HIF-1alpha were mostly expressed in endothelial cells and neoplastic hepatocytes in the capillarized areas around portal tracts in DNs, respectively. In conclusion, the capillarized areas with increased numbers of unpaired arteries in DNs may represent an early malignant transformation. Increased expression of Flk-1 and HIF-1alpha associated with VEGF may be involved in sinusoidal capillarization and the increased numbers of unpaired arteries in these areas.     

SDF-1/CXCR4轴活化诱导内皮祖细胞增殖、迁移及管型形成

目的观察趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成的作用。方法用免疫细胞化学检测内皮祖细胞SDF-1和CXCR4表达;用MTT法、Millicell趋化法及Matrigel体外三维成型法分别检测不同浓度的趋化因子SDF-1促内皮祖细胞增殖、迁移和管型形成。并应用CXCR4受体抑制剂AMD3100观察上述指标的变化。结果免疫细胞化学显示内皮祖细胞表达SDF-1和CXCR4蛋白。SDF-1可促进内皮祖细胞的增殖、迁移和体外小管样结构的形成。AMD3100可抑制SDF-1的诱导作用。结论SDF-1/CXCR4轴在内皮祖细胞参与血管新生中可能发挥重要作用。     

Vascular endothelial growth factor and its receptor Flk-1 are expressed in the hippocampus following entorhinal deafferentation

Vascular endothelial growth factor (VEGF) has been thought of as a mitogen that promotes proliferation of endothelial cells and as a neurotrophic factor that stimulates neurogenesis and axonal growth in both peripheral and central nervous systems. To investigate the potential involvement of VEGF in the lesion-induced reorganization in the brain, the expression changes of VEGF and its receptor Flk-1 were analyzed in the mouse hippocampus after transections of the entorhinal afferents. In situ hybridization and immunohistochemistry showed the time-dependent expression upregulation of VEGF mRNA and protein in the entorhinally denervated hippocampal stratum lacunosum-moleculare and dentate outer molecular layer, which initiated by 3 days postlesion, reached its maximum at 7-15 days postlesion, still persisted by 30 days postlesion for protein, and recovered to the normal levels at 30 days postlesion for mRNA and at 60 days postlesion for protein. Double labeling of VEGF and glial fibrillary acidic protein revealed that VEGF-expressing cells in the denervated areas were reactive astrocytes. Semi-quantitative RT-PCR analysis showed that VEGF receptor Flk-1 mRNA was also time-dependently upregulated in the deafferented hippocampus with its maximal elevation at 7-15 days postlesion while the Flt-1 mRNA levels remained unchanged at any time point we examined. Immunohistochemistry analysis also displayed the upregulation of Flk-1 protein in the denervated stratum lacunosum-moleculare and outer molecular layer with a time course similar to that of VEGF mRNA upregulation. Flk-1 receptors were found to be expressed not only by reactive astrocytes but also by neurites, which most likely belong to sprouting axons by 7 days postlesion and regrowing dendrites by 15-30 days postlesion. From these data we suggest that the spatiotemporal upregulation of VEGF and Flk-1 in the hippocampus is induced by entorhinal deafferentation and that VEGF may be involved in the structural reorganization in the deafferented hippocampus via directly or indirectly promoting neurite growth.     

Circulating bone marrow-derived osteoblast progenitor cells are recruited to the bone-forming site by the CXCR4/stromal cell-derived factor-1 pathway

Previous studies demonstrated the existence of osteoblastic cells in circulating blood. Recently, we reported that osteoblast progenitor cells (OPCs) in circulation originated from bone marrow and contributed to the formation of ectopic bone induced by implantation of a bone morphogenetic protein (BMP)-2-containing collagen pellet in mouse muscular tissue. However, the character of circulating bone marrow-derived osteoblast progenitor cells (MOPCs) and the precise mechanisms involving the circulating MOPCs in the osteogenic processes, such as signals that recruit the circulating MOPCs to the osseous tissues, have been obscure. In this report, we demonstrated for the first time that the MOPCs were mobilized from intact bones to transiently occupy approximately 80% of the mononuclear cell population in the circulating blood by BMP-2-pellet implantation. The mobilized MOPCs in the circulation did not express the hematopoietic marker CD45 on their surface, but they expressed CD44 and CXCR4, receptors of osteopontin and stromal cell-derived factor-1 (SDF-1), respectively. The MOPCs isolated from the mouse peripheral blood showed the ability to be osteoblasts in vitro and in vivo. Furthermore, the MOPCs in the circulation efficiently migrated to the region of bone formation by chemoattraction of SDF-1 expressed in vascular endothelial cells and the de novo osteoblasts of the region. These data may provide a novel insight into the mechanism of bone formation involving MOPCs in circulating blood, as well as perspective on the use of circulating MOPCs to accelerate bone regeneration in the future.     

Expression of CXCR4, the receptor for stromal cell-derived factor-1 on fetal and adult human lympho-hematopoietic progenitors.

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine produced by stromal cells that acts as a chemoattractant for human CD34+ progenitor cells. We investigated the expression of CXCR4, the receptor for SDF-1, on CD34+ cells from different hematopoietic sites and developmental stages. CXCR4 was detected by flow cytometry on 37 % of fetal bone marrow (BM) [gestation weeks (gw) 14-23] and 40% of adult BM CD34+ cells. Interestingly, in fetal liver CD34+ cells, CXCR4 was expressed at lower levels at later stages (9%, gw 20-23) compared to early stages of development (39%, gw 7.5-18), suggesting a development-related change in the migratory capacity of progenitors. CXCR4 was detected at similar levels on both phenotypically primitive and committed progenitors from fetal and adult sites. However, B cell lineage progenitor and precursor cells expressed CXCR4 at the highest density (80% of BM CD34+/CD10+ pro-B cells are CXCR4+). CXCR4 was also expressed in the fetal thymus in early T cell precursors and found to be down-regulated during T cell maturation. Finally, we found that stem cell factor, alone or in combination with other cytokines, can up-modulate CXCR4 expression on CD34+ cells by three- to fourfold. In conclusion, our results suggest that CXCR4 may play an important role in the local and systemic trafficking of human CD34+ cells as well as in human B lymphopoiesis and that its expression can be modulated by cytokines.     

Enhanced functional response to CXCL12/SDF-1 through retroviral overexpression of CXCR4 on M07e cells: implications for hematopoietic stem cell transplantation

The chemokine CXCL12 (stromal cell derived factor-1/SDF-1) stimulates hematopoietic stem and progenitor cells (HSCs/HPCs) through the corresponding chemokine receptor CXCR4. CXCL12 is thought to be important for both proper HSC homing, retention, and engraftment into the bone marrow (BM) and mobilization out of the BM. Previous studies suggest that breaking the CXCL12-CXCR4 interaction mobilizes HPCs, blocking CXCR4 inhibits HSC homing, and overexpression increases HSC/HPC repopulation. The efficiency of mobilization and engraftment therefore appears to be dependent on the response of HSCs/HPCs to CXCL12, which is in turn dependent upon levels of CXCR4 expressed on HSCs/HPCs. However, expression of CXCR4 on the surface of HSCs/HPCs appears to be variable. To study the function of CXCR4 on HSCs/HPCs, we used the MSCV-based bicistronic (EGFP) retroviral vector MIEG3 to overexpress CXCR4 on M07e cells, an established model of human HPC. CXCR4 overexpression resulted in significant increases in CXCL12-induced chemotaxis and cell survival. Most importantly, cells overexpressing CXCR4 responded to CXCL12 at levels typically too low induce a response. These data suggest that an increased transplant efficiency resulting from CXCR4 overexpression is likely a function of increased HSC/HPC homing and increased HSC/HPC survival in the recipient's BM. These experiments also validate the ability of the MIEG3-CXCR4 retroviral construct to overexpress CXCR4 efficiently and the use of MIEG3-CXCR4 M07e cells for further study. Finally, this information may have future potential therapeutic implications for improvements in transplant efficiency.     

The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4

In the bone marrow, progenitor (pro-) and precursor (pre-) B cells depend on close contact with stromal cells for growth and maturation. Stromal cell-derived factor 1 (SDF-1), also known as pre-B cell growth-stimulating factor, is produced by bone marrow stromal cells and was reported to act together with interleukin-7 as co-mitogen for pre-B cells. SDF-1 was recently shown to be a chemokine which is chemotactic for different types of leukocytes and acts via the chemokine receptor CXCR4. Using sorted B220⁺ bone marrow cells and several B cell lines characteristic for different stages of B lymphopoiesis, we now show that SDF-1 is a potent attractant for pro- and pre-B cells, but is inactive on B cells at later stages of development. In early B cell precursors, SDF-1 induced intracellular Ca²⁺ mobilization and in vitro migration with a potency and efficacy similar to that observed for chemokines acting on blood leukocytes. These responses were mediated via CXCR4 as they could be inhibited by an anti-receptor antibody. SDF-1 is the first chemokine shown to act on early-stage B cell precursors. Mice lacking SDF-1 die perinatally and show a severe deficiency in B lymphopoiesis. We propose that SDF-1 released from the stromal cells exerts its critical hematopoietic function by selectively attracting and confining early B cell precursors within the bone marrow microenvironment that provides the necessary factors for growth and differentiation.     

C1qR(P), a myeloid cell receptor in blood, is predominantly expressed on endothelial cells in human tissue.

C1qR(P) is a type I cell surface glycoprotein that has been shown to enhance ingestion of suboptimally opsonized targets by phagocytes in vitro. In this study, we developed and characterized polyclonal antibodies to study the tissue distribution of this receptor targeted to either the N- or C-terminal portion of the molecule. C1qR(P) was detected in vascular endothelial cells and in a subset of pyramidal neurons in the brain, as well as neutrophils, but it was absent in most tissue macrophages. Analysis of in vitro differentiation of blood monocytes to dendritic cells demonstrated a down-regulation of the receptor as monocytes differentiate to dendritic cells, providing a possible explanation for the lack of reactivity of these cells in tissue. The predominant presence of C1qR(P) in endothelial cells, while compatible with a phagocytic role in host defense and/or clearance of cellular material, suggests other possible novel roles for this receptor.     

Urotensin-II and its receptor (UT-R) are expressed in rat brain endothelial cells, and urotensin-II via UT-R stimulates angiogenesis in vivo and in vitro

Urotensin-II (UII), along its receptor UT-R, is widely expressed in the cardiovascular system, where it exerts regulatory actions under both physiological and pathological conditions. Real-time PCR and immunocytochemistry demonstrated the expression of UII and UT-R as mRNA and protein in rat neuromicrovascular endothelial cells (NECs). UII did not affect the proliferation rate of cultured NECs, but exerted a strong angiogenic action in both an in vitro assay on Matrigel and an in vivo assay on chorioallantoic membrane. The angiogenic effect of UII was similar to that of FGF-2, and was abolished by the UT-R antagonist Palosuran. Collectively, our findings allow us to include UII in the group of cytokines (e.g. endothelin-1 and adrenomedullin), which are expressed in endothelial cells and exert a pro-angiogenic effect acting in an autocrine-paracrine manner.