C-X-C Chemokine Receptor Type 4 mRNA Expression Research Articles

Different Modes of Mechanism of Gamma-Mangostin and Alpha-Mangostin to Inhibit Cell Migration of Triple-Negative Breast Cancer Cells Concerning CXCR4 Downregulation and ROS Generation.

Two mangostin compounds, gamma-mangostin and alpha-mangostin, show anticancer properties through the inhibition of cell proliferation and cell migration. Metastatic triple-negative breast cancer (TNBC) cells, including MDA-MB-231, highly express C-X-C chemokine receptor type 4 (CXCR4) to maintain reactive oxygen species (ROS) and cell migration. This study was performed to analyze and compare different modes of action of γ-mangostin and α-mangostin as antimigratory effects targeted on CXCR4 in MDA-MB-231 as a model of TNBC cell. This study investigated the effect of γ-mangostin and α-mangostin using a series of assays, including Cell Counting Kit-8 (CCK-8) assay for cytotoxicity, wound healing assay for migration study, quantitative real-time polymerase chain reaction (qRT-PCR) for gene expression analysis, and flow cytometry for ROS measurement, along with in silico study to observe the binding between the compound and CXCR4. The findings revealed half maximal inhibitory concentration (IC50) values of 25 and 20 μM for γ-mangostin and α-mangostin in MDA-MB 231 cells, respectively. Moreover, a concentration of 10 μM was used for the migration assay. Both γ-mangostin and α-mangostin significantly suppressed cell migration within 24 hours. The present gene expression studies revealed the downregulation of key migration-associated genes, namely Farp, CXCR4, and LPHN2, upon γ-mangostin treatment but not α-mangostin. Additionally, both γ-mangostin and α-mangostin increased cellular ROS generation, highlighting the same effect of γ-mangostin and α-mangostin ROS elevation to inhibit cancer cell migration. Molecular docking simulations further suggested a potential interaction between γ-mangostin and α-mangostin with CXCR4 in high affinity. These findings suggest that both γ-mangostin and α-mangostin inhibit breast cancer cell migration and induce cellular ROS levels in MDA-MB-231 cells; notably, γ-mangostin suppresses CXCR4 mRNA expression that might correlate to its activity to inhibit MDA-MB-231 cell migration.

Suppressing the activity of CXCR4 down-regulates the expression of renal fibrosis related genes in primary glomerular cells.

BackgroundC-X-C chemokine receptor type 4 (CXCR4) has a certain effect on renal fibrosis, and there are few specific studies in cells. We want to investigate the impact of suppressing CXCR4 activity on the expression of renal fibrosis-related genes in primary glomerular endothelial cells, mesangial cells, and podocytes.MethodsImmunofluorescence assays were used to determine the purity of isolated glomerular endothelial cells, mesangial cells, and podocytes. CXCR4 knockdown cell lines were established by transfecting the short hairpin (sh)RNA against CXCR4. T140 and AMD3100 were used to inhibit the activity of CXCR4. LY294002 was used to inhibit the activity of phosphoinositide 3-kinase (PI3K). The mRNA expression of CXCR4 was determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The protein expression of CXCR4, collagen IV, matrix metallopeptidase (MMP)-9, PI3K, Rac1, and vascular cell adhesion protein 1 (VCAM-1) was evaluated by Western blot analysis.ResultsHigh purity was observed on isolated primary glomerular endothelial cells and podocytes. However, the purity of isolated mesangial cells was relatively low. The mRNA expression of CXCR4 was significantly suppressed by the transfection of shRNA. Compared to control cells, the expression of CXCR4, collagen IV, MMP-9, PI3K, Rac1, and VCAM-1 were dramatically downregulated in cell lines transfected with shRNA against CXCR4. Furthermore, cell lines treated with T140, AMD3100, or LY294002 also showed downregulated expression of these proteins compared to untreated cells. No significant differences were observed in the protein expression of these proteins between control cells and cells transfected with the shRNA negative control (NC).ConclusionsSuppressing the activity of CXCR4 downregulated the expression of renal fibrosis-related genes in primary glomerular cells, even under a non-inflammatory state.

Clinical Significance of Chemokine Receptor CXCR4 and CCR7 mRNA Expression in Patients With Colorectal Cancer.

The chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 7 (CCR7) play an important role in the invasion and metastasis of cancer. This study investigated the relationship between relative expression of CXCR4 and CCR7 mRNA, clinicopathological factors, and outcomes in patients with colorectal cancer (CRC). We studied 202 patients who underwent surgery for CRC. The expression levels of CXCR4 and CCR7 mRNA in cancerous tissue were measured using quantitative real-time reverse-transcriptase polymerase chain reaction. High CCR7 mRNA expression levels in CRC tissues were positively associated with tumour size and were more frequently associated with cancer of the rectum than of the colon. Moreover, outcomes were significantly poorer in patients with high CCR7 mRNA expression than in those with low expression. On multivariate Cox regression analysis, a higher CCR7 mRNA expression level was a significant independent predictor of poorer overall survival in patients with CRC. Overexpression of CCR7 mRNA may be a useful independent prognostic factor in patients with CRC.

Hypoxic-inflammatory responses under acute hypoxia: In Vitro experiments and prospective observational expedition trial.

Induction of hypoxia-inducible-factor-1α (HIF-1α) pathway and HIF-target genes allow adaptation to hypoxia and are associated with reduced incidence of acute mountain sickness (AMS). Little is known about HIF-pathways in conjunction with inflammation or exercise stimuli under acute hypobaric hypoxia in non-acclimatized individuals. We therefore tested the hypotheses that (1) both hypoxic and inflammatory stimuli induce hypoxic-inflammatory signaling pathways in vitro, (2) similar results are seen in vivo under hypobaric hypoxia, and (3) induction of HIF-dependent genes is associated with AMS in 11 volunteers. In vitro, peripheral blood mononuclear cells (PBMCs) were incubated under hypoxic (10%/5% O2) or inflammatory (CD3/CD28) conditions. In vivo, Interleukin 1β (IL-1β), C-X-C Chemokine receptor type 4 (CXCR-4), and C-C Chemokine receptor type 2 (CCR-2) mRNA expression, cytokines and receptors were analyzed under normoxia (520 m above sea level (a.s.l.)), hypobaric hypoxia (3883 m a.s.l.) before/after exercise, and after 24 h under hypobaric hypoxia. In vitro, isolated hypoxic (p = 0.004) or inflammatory (p = 0.006) stimuli induced IL-1β mRNA expression. CCR-2 mRNA expression increased under hypoxia (p = 0.005); CXCR-4 mRNA expression remained unchanged. In vivo, cytokines, receptors, and IL-1β, CCR-2 and CXCR-4 mRNA expression increased under hypobaric hypoxia after 24 h (all p ≤ 0.05). Of note, proinflammatory IL-1β and CXCR-4 mRNA expression changes were associated with symptoms of AMS. Thus, hypoxic-inflammatory pathways are differentially regulated, as combined hypoxic and exercise stimulus was stronger in vivo than isolated hypoxic or inflammatory stimulation in vitro.

Stromal cell-derived factor-1 and CXC chemokine receptor type-4 are associated with cardiovascular disease in patients under hemodialysis

Background:Cardiovascular disease (CVD) is the most common cause of death among patients with end-stage renal disease especially whom under hemodialysis (HD). Stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor type-4 (CXCR4) could contribute to CVD. The main aim of this study was to evaluate the association between SDF-1 and CXCR4 with CVD and its related risk factors in patients under HD.Methods:Sixty patients under HD and 29 healthy subjects were recruited in the study. The serum levels and relative messenger RNA (mRNA) expressions of SDF-1 and CXCR4 were measured using enzyme-linked immunosorbent assay and real-time polymerase chain reaction in patients and controls, respectively. CVD history of the patients was obtained.Results:Twenty patients (33.3%) had a history of CVD. The mean levels of serum and relative mRNA expressions of SDF-1 and CXCR4 were higher in patients than controls and also in patients with a history of CVD than patients without it. The serum levels and relative expressions of SDF-1 and CXCR4 were positively correlated with blood urea nitrogen, parathyroid hormone, and high-sensitivity C-reactive protein and inversely correlated with hemoglobin. The history of CVD was the independent predictor of serum levels of SDF-1 and CXCR4 and also relative mRNA expression of CXCR4.Conclusions:The higher levels of serum and relative mRNA expressions of SDF-1 and CXCR4 were associated with CVD in patients under HD. Furthermore, SDF-1 and CXCR4 were associated with several traditional and uremia-related CVD risk factors in such patients.

Correlation of CXCR4/CXCR7 signaling pathway with pre-eclampsia.

The possible role of C-X-C chemokine receptor type 4 (CXCR4)/CXCR7 signaling pathway in pre-eclampsia (PE) was investigated to study the expression of CXCR4/CXCR7 in serum of PE patients and its correlation and relationship with PE. Twenty patients with mild PE and 40 cases with severe PE enrolled into the PE group and 60 cases of the normal pregnancy group during the same period were selected as the objects of study. The changes in expression of serum CXCR4 messenger ribonucleic acid (mRNA) and CXCR7 mRNA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and CXCR4/CXCR7 protein concentration in serum was determined by enzyme-linked immunosorbent assay (ELISA). The pregnancy termination time of gravid was earlier in the severe PE group than that in the normal pregnancy group (P<0.05); the mean arterial pressure (MAP), systolic pressure, diastolic pressure were higher in patients with mild and severe PE than those in the normal pregnancy group (P<0.05). The results of RT-qPCR showed that the mRNA expression of serum CXCR4 and CXCR7 in PE patients were distinctly higher than those in the normal pregnancy group (P<0.05). The expression level of CXCR4 mRNA was positively correlated with that of CXCR7 mRNA (r=0.567, P=0.02). The results of ELISA displayed that the content of CXCR4/CXCR7 in serum of patients with PE was remarkably higher than that in the normal pregnancy group (P<0.05); the expression of serum CXCR4/CXCR7 in patients with severe PE was higher than in those with mild PE (P<0.05). The expression level of serum CXCR4 protein was positively correlated with that of CXCR7 protein (r=0.563, P=0.01). The expression level of CXCR4/CXCR7 may be closely related to the formation of PE.

Stromal cell‑derived factor‑1 induces matrix metalloproteinase expression in human endplate chondrocytes, cartilage endplate degradation in explant culture, and the amelioration of nucleus pulposus degeneration in vivo.

Intervertebral disc (IVD) degeneration is a strong etiological factor in chronic lower back pain. Stem cell migration toward the site of IVD degeneration for regeneration is restricted by avascularity and distance. Our previous study indicated that the expression of stromal cell‑derived factor‑1(SDF‑1) and its receptor, C-X-C chemokine receptor type 4 (CXCR4) was upregulated in degenerated cartilage endplate (CEP) and nucleus pulposus (NP). In the present study, SDF‑1 increased CXCR4 mRNA and protein expression in human endplate chondrocytes in a dose‑dependent manner. The results of reverse transcription-quantitative polymerase chain reaction, western blotting and zymography indicated that SDF‑1 increased matrix metalloproteinase(MMP)‑1, ‑3 and ‑13 mRNA and protein expression in human endplate chondrocytes in a dose‑dependent manner. The results of zymography suggested that SDF‑1 also increased MMP‑2 and ‑9 protein expression in a dose‑dependent manner. The CXCR4‑specific chemical inhibitor AMD3100 significantly decreased the levels of MMP‑1, ‑2, ‑3, ‑9 and ‑13 expression. In a human cartilage explant culture model, SDF‑1 accelerated the degradation of extracellular matrix (ECM), and AMD3100 decreased cartilage cleavage. However, in a rat tail disc degeneration model, the injection of SDF‑1 into the NP resulted in the retention of dense areas of proteoglycan matrix and enhanced NP regeneration. These results suggest that SDF‑1, as an inflammatory cytokine, induces MMP expression in human endplate chondrocytes and that ECM remodeling in the CEP may be a favorable factor of endogenous stem cell homing into the NP for regeneration invivo.

The prenylflavonoid Icaritin enhances osteoblast proliferation and function by signal transducer and activator of transcription factor 3 (STAT-3) regulation of C-X-C chemokine receptor type 4 (CXCR4) expression

In this study, we examined the effects of a natural prenylflavonoid Icaritin (ICT), on human osteoblast proliferation and osteogenic function. We observed that ICT dose-dependently enhanced osteoblast proliferation by ~15% over a 7day period. This increase in cell proliferation was associated with corresponding increases in osteoblast functions as measured by ALP secretion, intracellular calcium ions influx and calcium deposition. These anabolic effects were associated with a 4-fold increase in CXCR4 mRNA and protein expression. Silencing of CXCR4 protein expression using small interfering RNA reversed ICT-induced increase in cell proliferation, ALP activity and calcium deposition. Interestingly, we observed that ICT dose-dependently increased STAT-3 phosphorylation; and this resulted in increased binding of phosphorylated STAT-3 to the promoter region of the CXCR4 gene, to increase CXCR4 protein expression. Furthermore, we found that inhibition of STAT-3 phosphorylation resulted in a decrease in CXCR4 protein expression; whilst increasing phosphorylation of STAT-3 using a constitutive active STAT-3 vector significantly increased CXCR4 levels. Moreover, the chemical inhibition of STAT-3 phosphorylation annulled our previously observed ICT-induced increases of osteoblast proliferation and function. Finally, in a rat model of estrogen-deficient osteoporosis, ICT restored both osteoblasts numbers and CXCR4 expression. Taken together, both cellular and animal models support the novel findings that ICT; through the phosphorylation of STAT-3, up-regulated CXCR4, to increase osteoblast proliferation and function.

Chronic Prostatitis Affects Male Reproductive Health and Is Associated with Systemic and Local Epigenetic Inactivation of C-X-C Motif Chemokine 12 Receptor C-X-C Chemokine Receptor Type 4

Background/Aims/Objectives: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has detrimental effects on the quality of life including the aspect of sexual dysfunction. The aim of the study was to identify if there was an adverse effect on the male genital compartment and if there are systemic or compartment-specific local signals for epigenetic dysregulation of inflammatory factors in CP/CPPS patients. Methods: One hundred five NIH IIIb CP/CPPS patients and 41 healthy men were recruited and underwent investigations of urines, semen and blood. Promoter methylation and expression of the chemokine C-X-C motif chemokine 12 and its receptor C-X-C chemokine receptor type 4 (CXCR4) (involved in the recruitment of mast cells) were analyzed in prostate epithelial cell lines and in healthy volunteers' and patients' blood, ejaculate cell pellets, and separated ejaculate fractions (sperm and seminal somatic cells). Results: Independently from age, CP/CPPS NIH IIIb was associated with significant impairment of sperm motility, morphology and semen pH (p < 0.001). Patients older than 33 years showed significantly increased seminal interleukin-8 and serum prostate specific antigen values. In patients, the CXCR4 mRNA-expression was significantly decreased in whole blood and ejaculate cell pellets due to promoter hypermethylation. Analyses on separated fractions of sperm and seminal somatic cells revealed that sperm DNA was unaffected, whereas somatic cell DNA was differentially methylated. Conclusions: NIH IIIb CP/CPPS has negative effects on surrogate parameters of male fertility and is associated significantly with systemic and local epigenetic inactivation of CXCR4.

Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide.

The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line.

Combining cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) has demonstrated marked efficacy in the treatment of elderly and relapsed/refractory patients with acute myeloid leukemia (AML); however, the role of G-CSF remains poorly understood. The present study aimed to investigate the ability of G-CSF to overcome stromal-mediated drug resistance and the underlying molecular mechanism. Two types of co-culture models were established in the HS-5 human bone marrow/stromal and HL-60 human promyelocytic leukemia cell lines, in order to imitate the interactions between stromal and leukemia cells in vitro, which is mediated by the stromal cell-derived factor (SDF)-1α signaling axis. In the present study, HL-60 cells were attracted and adhered to HS-5 cells using migration assay and flow cytometry, respectively; however, these interactions were inhibited by treatment with G-CSF and/or the C-X-C chemokine receptor type 4 (CXCR4) antagonist, AMD3100. Co-culture with HS-5 cells, including direct and indirect contact, protected HL-60 cells against spontaneous apoptosis or drug-induced apoptosis; however, these protective effects were disrupted by treatment with G-CSF and/or AMD3100. Notably, G-CSF and/or AMD3100 did not alter cell viability or apoptosis when HL-60 cells were cultured with medium alone. In addition, G-CSF significantly reduced the expression levels of surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA, and significantly upregulated the expression of microRNA (miR)-146a. Conversely, AMD3100 significantly reduced surface CXCR4 expression levels, but not the total CXCR4, CXCR4 mRNA or miR-146a expression levels. The results of the present study suggested that interfering with the CXCR4/SDF-1α signaling axis via G-CSF inhibited the migration and adhesion of HL-60 cells to HS-5 cells and eliminated HS5 cell-mediated protective effects. Furthermore, G-CSF administration reduced CXCR4 expression levels by upregulating the expression of miR-146a, whereas AMD3100 appeared to be predominantly dependent on receptor internalization. Therefore, a G-CSF/miR-146a/CXCR4 pathway may explain how G-CSF inhibits CXCR4/SDF-1α signaling and overcomes stromal cell-mediated drug resistance in acute myeloid leukemia.

Aberrant CXCR4 and β-catenin expression in osteosarcoma correlates with patient survival.

To determine the clinical significance of C-X-C chemokine receptor type 4 (CXCR4) and β-catenin in osteosarcoma, their protein expression levels were assessed in 96 osteosarcoma and 20 osteochondroma cases using immunohistochemistry. Additionally, CXCR4 and β-catenin mRNA expression levels were measured in 16 fresh osteosarcoma and 16 adjacent healthy tissue samples using fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In the osteosarcoma samples, the positive CXCR4 protein expression rate was significantly higher than the rate in the osteochondroma samples (68.75 vs. 20.00%; P<0.01). Furthermore, β-catenin protein expression was detected in 61.46% of osteosarcoma cases and 25.00% of osteochondroma cases. Similarly, the RT-qPCR data identified increased CXCR4 and β-catenin mRNA expression levels in the osteosarcoma compared with adjacent control tissues. It was determined that CXCR4 (P<0.01) and β-catenin (P<0.05) expression were significantly associated with the clinical Enneking stage, metastasis and survival of osteosarcoma. Furthermore, multivariate analysis identified CXCR4 and β-catenin protein expression levels, as well as clinical stage and metastasis, as significant risk factors for survival in patients with osteosarcoma (P<0.05). In conclusion, the present study determined that CXCR4 and β-catenin are abnormally expressed in osteosarcoma tissues, and, therefore, may be important during osteosarcoma progression.

Recruitment of mesenchymal stem cells by stromal cell-derived factor 1α in pulp cells from deciduous teeth.

Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.

A meta-analysis for CXCR4 as a prognostic marker and potential drug target in non-small cell lung cancer.

BackgroundRecent reports have shown that C-X-C chemokine receptor type 4 (CXCR4) is a candidate oncogene in several types of human tumors, including non-small cell lung cancer (NSCLC). However, the correlation between CXCR4 expression and clinicopathological characteristics of NSCLC remains controversial and has not been emphasized. The aim of this study is to quantitatively evaluate the association of CXCR4 expression with the incidence of NSCLC and clinicopathological characteristics by performing a meta-analysis.MethodsA detailed literature search was carried out for related research publications. Only articles in which CXCR4 expression was detected by immunohistochemical staining were included. Odds ratio (OR) and hazard ratio (HR) with 95% confidence intervals (CIs) were calculated and summarized.ResultsFinal analysis of 1,872 NSCLC patients from 19 eligible studies was performed. We observed that CXCR4 expression was significantly higher in NSCLC than in normal lung tissue, based on the pooled OR from ten studies, including 678 NSCLCs and 189 normal lung tissues (OR =16.66, 95% CI =6.94–40.02, P<0.00001). CXCR4 expression was also significantly associated with clinical stages, metastatic status, and overall survival (OS) in NSCLC patients. In addition, CXCR4 mRNA high expression was found to correlate with worse OS of all NSCLC patients followed for 20 years, HR =1.24, P=0.0047.ConclusionThe present meta-analysis indicated that CXCR4 protein expression is associated with an increased risk and worse survival in NSCLC patients. The aberrant CXCR4 protein and mRNA expression play an important role in the carcinogenesis and metastasis of NSCLC.

Activation of the CXCL12/CXCR4 signaling axis may drive vascularization of the ovine placenta

Early pregnancy, when most embryonic losses occur, is a critical period in which vital placental vascularization is established. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, and factors that regulate VEGF function, expression, or both may ultimately affect vascularization. Activation of the C-X-C chemokine receptor type 4 (CXCR4) by its cognate ligand, C-X-C chemokine ligand 12 (CXCL12), increases VEGF synthesis and secretion, which in turn stimulates CXCL12 and CXCR4 production and this synergistic regulation may influence placental vascularization. We hypothesized that expression of CXCL12, CXCR4, select angiogenic factors, and their receptors would increase in placental tissues during early pregnancy and that treatment of ovine trophectoderm cells with CXCL12 would increase production of angiogenic factors. To test this hypothesis, maternal caruncle (CAR) and fetal extraembryonic membrane (FM) tissues were collected on days 18, 20, 22, 25, 26, and 30 of pregnancy and on day 10 of the estrous cycle (control, NP) to determine relative mRNA or protein expression of CXCL12 and CXCR4 and selected angiogenic factors. In CAR, expression of mRNA for CXCR4 increased on day 18, 20, 22, and 25 and CXCL12 increased on day 18 and 20 compared with NP ewes. CXCL12 protein followed a similar pattern in CAR tissue, with greater levels on day 20 than in NP tissue. Greater levels of fibroblast growth factor 2 (FGF2) mRNA was observed in CAR on day 20 of gestation than on day 30. In FM, CXCL12, CXCR4, angiopoietin 1, VEGF, and VEGF receptor 1 were enhanced with advancing pregnancy, whereas FGF2 and kinase insert domain receptor (or VEGF receptor 2) peaked on day 25. An increase in protein levels occurred on day 25 compared with day 20 in FM for CXCL12 and CXCR4, as well as a similar tendency for FGF2 protein. Both CXCL12 and CXCR4 are specifically localized to trophoblast cells and to the uterine luminal and glandular epithelium. Treatment of ovine trophectoderm cells with CXCL12 increased mRNA expression for VEGF and FGF2. The relationship between VEGF, FGF2, and the CXCL12/CXCR4 signaling underscores the potential role for this chemokine axis in driving placentation.

Human Breast Adipose-Derived Stem Cells Transfected with the Stromal Cell-Derived Factor-1 Receptor CXCR4 Exhibit Enhanced Viability in Human Autologous Free Fat Grafts

Background: The main complication of autologous free fat tissue transplantation is fat resorption and calcification due to the ischemic necrosis of fat. The promotion of transplant neovascularization soon after autologous free fat grafts may reduce these outcomes. In adulthood, stromal cell-derived factor-1 (SDF-1) and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4) are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. We hypothesized that CXCR4 may improve the long-term survival of free fat tissue transplants by recruiting endothelial progenitor cells (EPCs) and may therefore improve graft revascularization. In this study, we aimed to determine the effect of human breast adipose-derived stem cells (HBASCs) transfected with the CXCR4 gene on the survival rate of human autologous free fat transplants in nude mice. Methods: Human breast adipose-derived stem cells (HBASCs) were expanded ex vivo for 3 passages, labeled with green fluorescent protein (GFP) and transfected with CXCR4 or left untransfected. Autologous fat tissues were mixed with the GFP-labeled, CXCR4-transfected HBASCs (group A), GFP-labeled HBASCs (group B), the known vascularization-promoting agent VEGF (group C), or medium (group D) and then injected subcutaneously into 32 nude mice at 4 spots in a random fashion. Six months later, the transplanted tissue volume and histology were evaluated, and neo-vascularization was quantified by counting the capillaries. CXCR4 and SDF-1α mRNA expression in the transplants was determined using real-time quantitative PCR analysis (qPCR). Results: The data revealed that the control (group D) transplant volume survival was 28.3 ± 4.5%. Mixing CXCR4-transfected (group A) and untransfected (group B) HBASCs significantly increased transplant volume survival (79.5 ± 8.3% and 67.2 ± 5.9%, respectively), whereas VEGF-transfected HBASCs (group C) were less effective (41.2 ± 5.1%). Histological analysis revealed that both types of HBASCs-treated transplants consisted predominantly of adipose tissue, unlike the control transplants, and also presented significantly less fat necrosis and fibrosis. The CXCR4-transfected HBASCs-treated transplants had a significantly higher capillary density than did the other transplants and showed GFP and CD31 double-positive cells (i.e., ASCs-derived endothelial cells). The mRNA expression of CXCR4 and SDF-1α was much higher in the CXCR4-transfected HBASCs transplants than in the other three transplants. Conclusions: Our data demonstrated that HBASCs can enhance the survival and quality of transplanted free fat tissues. Moreover, CXCR4 transfection of these HBASCs could augment this effect. Stimulation of angiogenesis and decreased fat cell apoptosis due to the recruitment of endothelial progenitor cells (EPCs) and an increase in graft revascularization are potential mechanisms underlying the improved long-term survival of free fat transplants following CXCR4-transfected HBASCs treatment.

Sprouting of Nervous Fibers and Upregulation of C‐X‐C Chemokine Receptor Type 4 Expression in Hippocampal Formation of Rats with Enhanced Spatial Learning and Memory

Hippocampal mossy fiber sprouting following training in the Morris water maze (MWM) is associated with spatial learning, memory and neural plasticity. The C-X-C chemokine receptor type 4 (CXCR4) is the main receptor for stromal cell-derived factor-1 (SDF-1), which is a chemokine that can regulate axonal elongation. This study aimed to investigate the relationship between the morphological plasticity of hippocampal formation and CXCR4 expression. A model of spatial learning and memory was established in rats by training using the MWM. Mossy fiber sprouting in the striatum oriens of the CA3 area of the hippocampus was found in trained rats by Neo-Timm's method. As shown by immunohistochemistry, the CXCR4 immunopositive neurons were distributed in all layers and areas of hippocampal formation. There were no differences among groups regarding the distribution or shape of the immunopositive neurons. However, the immunoreactive staining intensity was increased in trained rats as compared with the control rats. Both CXCR4 gene transcription and translation were significantly upregulated in the trained group as compared with the control group (P < 0.01). Morphological plasticity in the form of axonal sprouting in the hippocampal formation can be induced by enhanced spatial learning and memory activity, and CXCR4 mRNA and protein expression is upregulated, indicating a positive correlation between CXCR4 expression and axonal sprouting.