Ligand Stromal Cell-derived Factor-1 Research Articles

CXCR4 as a prognostic marker in Egyptian chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) has variable clinical presentations, and molecular and biological prognostic markers. The C-X-C chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of lymphocytes and monocytes. The aim was to study lymphocyte expression of CXCR4 and its prognostic value in CLL. A case control study was carried out on 30 newly diagnosed CLL cases and 30 healthy controls. Fludarabine, cyclophosphamide, and rituximab (FCR) was the standard treatment. Flowcytometric measurement of CXCR4 expression on lymphocytes was done. CXCR4 was significantly higher in patients than controls (81.67 ± 17.95 vs. 11.78 ± 2.78; P< 0.001). CXCR4 was significantly higher (P<0.001) in high risk CLL (93.63 ± 6.78) vs. intermediate risk (82.50 ± 7.13) and low risk (75.84 ± 12.23). CXCR4 was significantly higher (P<0.001) in non-responders (91.63 ± 6.98) vs. partial responders (83.11 ± 5.55) and complete responders (70.11 ± 4.44). CXCR4 was significantly lower in survivors vs. non-survivors (80.89 ± 5.09 vs. 85.43 ± 5.51; P< 0.001. CXCR4 had significant positive correlation with WBCs (r=0.45, P=0.01) and lymphocytes (r=0.40, P=0.01) measured at diagnosis. In conclusions, expression of CXCR4 in newly diagnosed CLL is significantly high. CXCR4 increased expression is associated with poor prognosis and resistance to the therapy.so it can be used as prognostic tool.

Immune Modulation of Platelet-Derived Mitochondria on Memory CD4+ T Cells in Humans.

CD4+ T cells are one of the key immune cells contributing to the immunopathogenesis of type 1 diabetes (T1D). Previous studies have reported that platelet-derived mitochondria suppress the proliferation of peripheral blood mononuclear cells (PBMC). To further characterize the immune modulation of platelet-derived mitochondria, the purified CD4+ T cells were treated, respectively, with platelet-derived mitochondria. The data demonstrated that MitoTracker Deep Red-labeled platelet-derived mitochondria could directly target CD4+ T cells through C-X-C motif chemokine receptor 4 (CXCR4) and its ligand stromal cell-derived factor-1 (SDF-1), regulating the anti-CD3/CD28 bead-activated CD4+ T cells. The result was an up-regulation of Naïve and central memory (TCM) CD4+ T cells, the down-regulation of effector memory (TEM) CD4+ T cells, and modulations of cytokine productions and gene expressions. Thus, platelet-derived mitochondria have a translational potential as novel immune modulators to treat T1D and other autoimmune diseases.

Abstract 4: Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4

Abstract Introduction: CXCR4 is a member of chemokine receptor and G protein coupled receptor (GPCR) families. Its interaction with the chemotactic ligand stromal cell-derived factor 1 (SDF-1 or CXCL12) plays important roles in physiological and pathological processes. Recent studies have showed that the blockade of CXCL12-CXCR4 interaction by small molecules can be used in several clinical applications, including sensitizing cancer cells to chemotherapy, mobilizing hematopoietic stem cells (HSCs) to the blood for HSC collection, and inhibiting the tumor metastasis. To date, AMD3100 (Plerixafor) is the only clinically approved drug used in combination with G-CSF for mobilizing HSCs. Here, we report the discovery and development of a new class of highly potent small molecule CXCR4 inhibitors which can strongly antagonize CXCL12-CXCR4 interaction and mobilizing HSCs. Methods: Based on the crystal structures of CXCR4 showing a major and minor subpockets, we applied a fragment-based combinatorial design (FBCD) strategy to design a new class of hybrid molecules to recognize these two subpockets. A series of designed compounds were chemically synthesized and tested in a panel of biological assays including competitive CXCR4 binding, chemotaxis, calcium mobilization and CXCR4 internalization to characterize their biological activities. Furthermore, site-directed mutagenesis coupled with molecular docking simulation was carried out to determine the binding modes of the most potent compounds. Results: More than 30 compounds were synthesized and characterized. Among them, compound HFX51116 showed the most potent CXCR4 binding activity (IC50 = 12.2 nM), which was more potent than AMD3100 tested in parallel (IC50 = 325.3 nM). Furthermore, HFX51116 displayed remarkable inhibition of calcium mobilization (IC50 = 1.478 nM) and chemotaxis activity (IC50 = 252.7 nM), both of which are better than those of AMD3100. In addition, mutational studies and molecular docking simulation suggested that HFX51116 recognizes the minor subpocket mainly and major subpocket partially. Conclusions: Our findings have demonstrated that HFX51116 is a promising novel lead compound for developing a new class of small molecule therapeutics targeting CXCR4. Citation Format: Xiong Fang, Qian Meng, Xiao Fang, Siyu Zhu, Yazi Huang, Jing An, Yan Xu, Ziwei Huang. Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4.

Atypical Chemokine Receptor 3 Generates Guidance Cues for CXCL12-Mediated Endothelial Cell Migration.

Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the ACKR3 zebrafish ortholog ackr3b in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that ackr3b is a CXCL12-binding ortholog of human ACKR3. ackr3b is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of ackr3b becomes restricted to the basal part of the somites. After ackr3b knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker hlx1. In addition, ackr3b morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for ackr3b in endothelial cell guidance, CXCL12 gradient generated by ACKR3 expression in CHO cell transfectants guides human endothelial cell migration in an in vitro cell co-culture chemotaxis assay. Our results demonstrate that ackr3b plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12.

Differential interactions of missing in metastasis and insulin receptor tyrosine kinase substrate with RAB proteins in the endocytosis of CXCR4

Missing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAi-mediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins.

Role of CXCL12 and CXCR4 in the pathogenesis of hematological malignancies

The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1/CXCL12) are important players in the cross-talk among lymphoma, myeloma and leukemia cells and their microenvironments. In hematological malignancies and solid tumors, the overexpression of CXCR4 on the cell surface has been shown to be responsible for disease progression, increasing tumor cell survival and chemoresistance and metastasis to organs with high CXCL12 levels (e.g., lymph nodes and bone marrow (BM)). Furthermore, the overexpression of CXCR4 has been found to have prognostic significance for disease progression in many type of tumors including lymphoma, leukemia, glioma, and prostate, breast, colorectal, renal, and hepatocellular carcinomas. In leukemia, CXCR4 expression granted leukemic blasts a higher capacity to seed into BM niches, thereby protecting leukemic cells from chemotherapy-induced apoptosis, and was correlated with shorter disease-free survival. In contrast, neutralizing the interaction of CXCL12/CXCR4 with a variety of antagonists induced apoptosis and differentiation and increased the chemosensitivity of lymphoma, myeloma, and leukemia cells. The role of CXCL12 and CXCR4 in the pathogenesis of hematological malignancies and the clinical therapeutic potential of CXCR4 antagonists in these diseases is discussed.

Angiogenic T cell expansion correlates with severity of peripheral vascular damage in systemic sclerosis.

The mechanisms underlying endothelial cell injury and defective vascular repair in systemic sclerosis (SSc) remain unclear. Since the recently discovered angiogenic T cells (Tang) may have an important role in the repair of damaged endothelium, this study aimed to analyze the Tang population in relation to disease-related peripheral vascular features in SSc patients. Tang (CD3+CD31+CXCR4+) were quantified by flow cytometry in peripheral blood samples from 39 SSc patients and 18 healthy controls (HC). Circulating levels of the CXCR4 ligand stromal cell-derived factor (SDF)-1α and proangiogenic factors were assessed in paired serum samples by immunoassay. Serial skin sections from SSc patients and HC were subjected to CD3/CD31 and CD3/CXCR4 double immunofluorescence. Circulating Tang were significantly increased in SSc patients with digital ulcers (DU) compared either with SSc patients without DU or with HC. Tang levels were significantly higher in SSc patients with late nailfold videocapillaroscopy (NVC) pattern than in those with early/active NVC patterns and in HC. No difference in circulating Tang was found when comparing either SSc patients without DU or patients with early/active NVC patterns and HC. In SSc peripheral blood, Tang percentage was inversely correlated to levels of SDF-1α and CD34+CD133+VEGFR-2+ endothelial progenitor cells (EPC), and positively correlated to levels of vascular endothelial growth factor and matrix metalloproteinase-9. Tang were frequently detected in SSc dermal perivascular inflammatory infiltrates. In summary, our findings demonstrate for the first time that Tang cells are selectively expanded in the circulation of SSc patients displaying severe peripheral vascular complications like DU. In SSc, Tang may represent a potentially useful biomarker reflecting peripheral vascular damage severity. Tang expansion may be an ineffective attempt to compensate the need for increased angiogenesis and EPC function. Further studies are required to clarify the function of Tang cells and investigate the mechanisms responsible for their change in SSc.

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.

The Pivotal Role of CXCR7 in Stabilization of the Pulmonary Epithelial Barrier in Acute Pulmonary Inflammation.

Acute pulmonary inflammation is still a frightening complication in intensive care units and has a high mortality. Specific treatment is not available, and many details of the pathomechanism remain unclear. The recently discovered chemokine receptor CXCR7 and its ligand stromal cell-derived factor (SDF)-1 are known to be involved in inflammation. We chose to investigate the detailed role of CXCR7 in a murine model of LPS inhalation. Inflammation increased pulmonary expression of CXCR7, and the receptor was predominantly expressed on pulmonary epithelium and on polymorphonuclear neutrophil (PMNs) after transepithelial migration into the alveolar space. Specific inhibition of CXCR7 reduced transepithelial PMN migration by affecting the expression of adhesion molecules. CXCR7 antagonism reduced the most potent PMN chemoattractants CXCL1 and CXCL2/3. After inhibiting CXCR7, NF-κB phosphorylation was reduced in lungs of mice, tight junction formation increased, and protein concentration in the bronchoalveolar lavage diminished, showing the impact of CXCR7 on stabilizing microvascular permeability. In vitro studies with human cells confirmed the pivotal role of CXCR7 in pulmonary epithelium. Immunofluorescence of human lungs confirmed our in vivo data and showed an increase of the expression of CXCR7 in pulmonary epithelium. Highlighting the clinical potential of CXCR7 antagonism, nebulization of the agent before and after the inflammation showed impressive anti-inflammatory effects. Additional CXCR7 inhibition potentiated the effect of SDF-1 antagonism, most probably by downregulating SDF-1 and the second receptor of the chemokine (CXCR4) expression. In conclusion, our data identified the pivotal role of the receptor CXCR7 in pulmonary inflammation with a predominant effect on the pulmonary epithelium and PMNs.

Ibrutinib inhibits CD20 upregulation on CLL B cells mediated by the CXCR4/SDF-1 axis

AbstractAgents targeting B-cell receptor (BCR) signaling-associated kinases such as Bruton tyrosine kinase (BTK) or phosphatidylinositol 3-kinase can induce mobilization of neoplastic B cells from the lymphoid tissues into the blood, which makes them potentially ideal to combine with anti-CD20 monoclonal antibodies (such as rituximab, obinutuzumab, or ofatumumab) for treatment of B-cell lymphomas and chronic lymphocytic leukemia (CLL). Here we show that interactions between leukemia cells and stromal cells (HS-5) upregulate CD20 on CLL cells and that administering ibrutinib downmodulates CD20 (MS4A1) expression in vivo. We observed that CLL cells that have recently exited the lymph node microenvironment and moved into the peripheral blood (CXCR4dimCD5bright subpopulation) have higher cell surface levels of CD20 than the cells circulating in the bloodstream for a longer time (CXCR4brightCD5dim cells). We found that CD20 is directly upregulated by CXCR4 ligand stromal cell-derived factor 1 (SDF-1α, CXCL12) produced by stromal cells, and BTK-inhibitor ibrutinib and CXCR4-inhibitor plerixafor block SDF-1α–mediated CD20 upregulation. Ibrutinib also downmodulated Mcl1 levels in CLL cells in vivo and in coculture with stromal cells. Overall, our study provides a first detailed mechanistic explanation of CD20 expression regulation in the context of chemokine signaling and microenvironmental interactions, which may have important implications for microenvironment-targeting therapies.

Caveolin‐1 Deficiency May Predispose African Americans to Systemic Sclerosis–Related Interstitial Lung Disease

Interstitial lung disease (ILD) is the leading cause of death in patients with systemic sclerosis (SSc; scleroderma). Although SSc-related ILD is more common and severe in African Americans than in Caucasians, little is known about factors underlying this significant health disparity. The aim of this study was to examine the role that low expression of caveolin-1 might play in susceptibility to ILD among African Americans. Assays of monocyte migration toward stromal cell-derived factor 1 (SDF-1) were performed using monocytes from Caucasian and African American healthy donors and patients with SSc. For fibrocyte differentiation studies, total peripheral blood mononuclear cells were incubated on fibronectin-coated plates. Protein expression was evaluated by immunohistochemistry and Western blotting. Monocytes from healthy African American donors and those from patients with SSc had low caveolin-1 levels, enhanced migration toward the CXCR4 ligand SDF-1, and enhanced differentiation to fibrocytes. Enhanced migration and differentiation of monocytes from African Americans and patients with SSc appeared to be attributable to the lack of caveolin-1, because restoring caveolin-1 function using a caveolin-1 scaffolding domain peptide inhibited these processes. Although they differed from monocytes from Caucasians, monocytes from both African Americans and patients with SSc were not identical, because SSc monocytes showed major increases from baseline in ERK, JNK, p38, and Smad2/3 activation, while monocytes from African Americans showed only limited ERK activation and no activation of JNK, p38, or Smad2/3. In contrast, SDF-1 exposure caused no additional ERK activation in SSc monocytes but did cause significant additional activation in monocytes from African Americans. African Americans may be predisposed to SSc-related ILD due to low baseline caveolin-1 levels in their monocytes, potentially affecting signaling, migration, and fibrocyte differentiation. The monocytes of African Americans may lack caveolin-1 due to high levels of transforming growth factor β in their blood.

Complexity of metastasis-associated SDF-1 ligand signaling in breast cancer stem cells.

Tumor metastasis is the main cause of cancer’s lethality. Much research has been dedicated to understand the signaling networks underlying tumor metastasis, with the ultimate goal of identifying signaling nodes or pathways whose inhibition will prevent metastasis. Although previous studies have identified the interaction of the chemokine ligand stromal cell-derived factor-1 (SDF-1) and the G protein-coupled receptor chemokine (C-X-C motif) receptor 4 (CXCR4) as essential for breast cancer metastasis (1), the signaling pathways downstream of SDF-1/CXCR4 have not been well established. In PNAS, Yi et al. use quantitative, liquid chromatography tandem mass spectrometry (LC-MS/MS) to greatly expand our knowledge of the protein phosphorylation networks downstream of SDF-1/CXCR4 in breast cancer stem cells (2).

Hematopoietic MicroRNA-126 Protects against Renal Ischemia/Reperfusion Injury by Promoting Vascular Integrity

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.

The Disparate Twins: A Comparative Study of CXCR4 and CXCR7 in SDF-1α–Induced Gene Expression, Invasion and Chemosensitivity of Colon Cancer

In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor.

Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats

BackgroundTumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel–forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)–1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts.MethodsWe used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea.ResultsRats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide.ConclusionsInhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

Inhibition of the CXCL12/CXCR4-Axis as Preventive Therapy for Radiation-Induced Pulmonary Fibrosis

BackgroundA devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process.Methodology/Principal FindingsThe radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process.Conclusions/SignificanceCXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.

Expression of stromal cell-derived factor-1 receptors CXCR4 and CXCR7 on circulating platelets of patients with acute coronary syndrome and association with left ventricular functional recovery

Surface expression of stromal cell-derived factor-1 (SDF-1) on platelets is enhanced during ischaemic events and might play an important role in peripheral homing and myocardial repair. As SDF-1 effects are mediated through CXCR4/CXCR7, we investigated platelet expression of SDF-1/CXCR4/CXCR7 in patients with coronary artery disease (CAD). Expression of SDF-1, CXCR4, and CXCR7 in platelets was investigated by western blot analysis, immunofluorescence confocal microscopy, and flow cytometry among healthy subjects and patients with acute coronary syndrome (ACS) and stable CAD. In a cohort study, platelet surface expression of CXCR4, CXCR7, and SDF-1 was measured in 215 patients with symptomatic CAD (stable CAD = 112, ACS = 103) at the time of percutaneous coronary intervention. Course of left ventricular ejection fraction (LVEF) was followed up during intrahospital stay and at 3 months. Both CXCR4 and CXCR7 are surface expressed on human platelets and to a higher degree in CAD patients when compared with healthy controls. Platelet surface expression of CXCR7 but not CXCR4 was enhanced in patients with ACS when compared with patients with stable CAD (mean fluorescence intensity 17.8 vs. 15.3, P = 0.004 and 29.0 vs. 26.3, P = 0.122, respectively). CXCR4 and CXCR7 significantly correlated with their ligand SDF-1 on platelets (ρ = 0.273, P < 0.001 and ρ = 0.454, P < 0.001, respectively). Additionally, high CXCR7 expression above the median correlated with the absolute improvement of LVEF% after 5 days and 3 months (46.2, 49.8, 53.7; P = 0.003). These findings indicate that platelet surface expression of CXCR4 and CXCR7 might differentially contribute to SDF-1-mediated effects on regenerative mechanisms following ACS. Studies are warranted to further evaluate the regulatory mechanisms of CXCR4/-7 expression and its prognostic impact on CAD.

Inhibition of CXCL12 Signaling Attenuates the Postischemic Immune Response and Improves Functional Recovery after Stroke

After stroke, brain inflammation in the ischemic hemisphere hampers brain tissue reorganization and functional recovery. Housing rats in an enriched environment (EE) dramatically improves recovery of lost neurologic functions after experimental stroke. We show here that rats housed in EE after stroke induced by permanent occlusion of the middle cerebral artery (pMCAO), showed attenuated levels of proinflammatory cytokines in the ischemic core and the surrounding peri-infarct area, including a significant reduction in the stroke-induced chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor-1 (CXCL12). To mimic beneficial effects of EE, we studied the impact of inhibiting CXCL12 action on functional recovery after transient MCAO (tMCAO). Rats treated with the specific CXCL12 receptor antagonist 1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclo-tetradecan (AMD3100) showed improved recovery compared with saline-treated rats after tMCAO, without a concomitant reduction in infarct size. This was accompanied by a reduction of infiltrating immune cells in the ischemic hemisphere, particularly cluster of differentiation 3-positive (CD3(+)) and CD3(+)/CD4(+) T cells. Spleen atrophy and delayed death of splenocytes, induced by tMCAO, was prevented by AMD3100 treatment. We conclude that immoderate excessive activation of the CXCL12 pathway after stroke contributes to depression of neurologic function after stroke and that CXCR4 antagonism is beneficial for the recovery after stroke.

Anti-Notch treatment prevents multiple myeloma cells localization to the bone marrow via the chemokine system CXCR4/SDF-1

Multiple myeloma (MM) is a deadly hematopoietic malignancy characterized by proliferation of malignant plasma cells in the bone marrow (BM) and bone disease. Interactions between myeloma and BM cells facilitate tumor progression and resistance to therapies. CXCR4 and its ligand Stromal cell-derived factor-1 (SDF-1) have a primary role in this process and are associated with poor prognosis. The Notch pathway is active in myeloma cells, resulting in increased proliferation, resistance to apoptosis and osteolytic activity. We hypothesized that the CXCR4/SDF-1 axis mediates the effects of Notch signals in myeloma cells. Here we show that Notch positively controls CXCR4/SDF-1 expression and functions in myeloma cell lines, and that forced CXCR4 activation partially rescues tumor cells from the outcomes of Notch inhibition. Additionally, we provide evidences that Notch blocking in vivo significantly reduces BM infiltration by human myeloma cells in mouse xenografts. This is the first evidence that a Notch-targeted approach effectively prevents MM cell migration, proliferation and resistance to apoptosis by reducing CXCR4 and SDF-1 levels.

Development of a Unique Small Molecule Modulator of CXCR4

BackgroundMetastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites.Methodology/Principal FindingsWe describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles.Conclusions/SignificanceWe developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.