Employing antagonistic C-X-C motif chemokine receptor 4 antagonistic peptide functionalized NaGdF4 nanodots for magnetic resonance imaging-guided biotherapy of breast cancer.

C-X-C motif chemokine receptor 4 (CXCR4) is a promising therapeutic target of breast cancer because it is overexpressed on cell surface of all molecular subtypes of breast cancer including triplenegative breast cancer (TNBC). Herein, CXCR4 antagonistic peptide-NaGdF4 nanodot conjugates (termed as anti-CXCR4-NaGdF4 NDs) have been constructed for magnetic resonance imaging (MRI)-guided biotherapy of TNBC through conjugation of the C-X-C Motif Chemokine 12 (CXCL12)-derived cyclic peptide with tryptone coated NaGdF4 nanodots (5 ± 0.5 nm in diameter, termed as Try-NaGdF4 NDs). The as-prepared anti-CXCR4-NaGdF4 NDs exhibits high longitudinal relaxivity (r1) value (21.87 mM-1S-1), reasonable biocompatibility and good tumor accumulation ability. The features of anti-CXCR4-NaGdF4 NDs improve the tumor-MRI sensitivity and facilitate tumor biotherapy after injection in mouse-bearing MDA-MB-231 tumor model in vivo. MRI-guided biotherapy using anti-CXCR4-NaGdF4 NDs enables to suppress 46% tumor growth. In addition, about 47% injection dose of anti-CXCR4-NaGdF4 NDs is found in the mouse urine at 24 h post-injection. These findings demonstrate that anti-CXCR4-NaGdF4 NDs enable to be used as renal clearable nanomedicine for biotherapy and MRI of breast cancer.

Safety and effectiveness of mogamulizumab in relapsed or refractory CC chemokine receptor 4-positive peripheral T-cell lymphoma and relapsed or refractory cutaneous T-cell lymphoma: A post-marketing surveillance in Japan.

Mogamulizumab is a humanized antibody targeting CC chemokine receptor 4 (CCR4). This post-marketing surveillance was conducted in Japan as a regulatory requirement from 2014 to 2020 to ensure the safety and effectiveness of mogamulizumab in patients with relapsed or refractory (r/r) CCR4-positive peripheral T-cell lymphoma (PTCL) or r/r cutaneous T-cell lymphoma (CTCL). Safety and effectiveness data were collected for up to 31 weeks after treatment initiation. A total of 142 patients were registered; safety was evaluated in 136 patients. The median number of doses was 8.0 (range, 1-18). The main reasons for treatment termination were insufficient response (22.1%) and adverse events (13.2%). The frequency of any grade adverse drug reaction was 57.4%, including skin disorders (26.5%), infections and immune system disorders (16.2%), and infusion-related reactions (13.2%). Graft-versus-host disease, grade 2, developed in one of two patients who underwent allogeneic-hematopoietic stem cell transplantation after receiving mogamulizumab. Effectiveness was evaluated in 131 patients (103 with PTCL; 28 with CTCL). The best overall response rate was 45.8% (PTCL, 47.6%; CTCL, 39.3%). At week 31, the survival rate was 69.0% (95% confidence interval, 59.8%-76.5%) [PTCL, 64.4% (54.0%-73.0%); CTCL, 90.5% (67.0%-97.5%)]. Safety and effectiveness were comparable between patients <70 and ≥ 70 years old and between those with relapsed and refractory disease. The safety and effectiveness of mogamulizumab for PTCL and CTCL in the real world were comparable with the data reported in previous clinical trials. Clinical Trial Registration.

CXCR4-mediated neutrophil dynamics in periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a common oral disease closely related to immune response and this study is aimed to identify the key immune-related pathogenic genes and analyze the infiltration and function of immune cells in the disease using bioinformatics methods. METHODS: Transcriptome datasets and single-cell RNA sequencing (scRNA-seq) datasets were downloaded from the GEO database. We utilized weighted correlation network analysis and least absolute selection and shrinkage operator, protein-protein interaction network construction to screen out key pathogenic genes as well as conducted the cell-type identification by estimating relative subsets of RNA transcripts algorithm to analyze and characterize immune cell types in periodontal tissues. In addition to bioinformatics validations, clinical and cell samples were collected and mouse periodontitis models were constructed to validate the important role of key genes in periodontitis. RESULTS: Bioinformatics analysis pointed out the positive correlation between CXCR4 expression and periodontitis, and revealed the increased infiltration of neutrophils in periodontal inflammatory. Similar results were obtained from clinical samples and animal models. In addition, the clustering and functional enrichment results based on CXCR4 expression levels included activation of immune response and cell migration, implying the possible function of CXCR4 on regulating neutrophil dynamics, which might contribute to periodontitis. Subsequent validation experiments confirmed that the increased expression of CXCR4 in neutrophils under periodontitis, where cell migration-related pathways also were activated. CONCLUSION: CXCR4 could be the key pathogenic gene of periodontitis and CXCR4/CXCL12 signal axial might contribute to the development of periodontitis by mediating neutrophil dynamics, suggesting that CXCR4 could be a potential target to help identify novel strategies for the clinical diagnosis and treatment of periodontitis.

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease.

BACKGROUND: Moyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. METHODS: Our study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. RESULTS: The results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. CONCLUSION: These findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

Autologous transplantation of green tea epigallocatechin-3-gallate pretreated adipose-derived stem cells increases cardiac regenerative capability through C-X-C motif chemokine receptor 4 expression in the treatment of rats with diabetic cardiomyopathy.

Cardiomyopathy is one of complications related to diabetes. Stem cell transplantation shows potential in diabetic cardiomyopathy treatment. Epigallocatechin-3-gallate (EGCG) is one of the major components found in green tea. Although stem cell transplantation and green tea EGCG supplementation show therapeutic effects on cardiomyopathy, the detailed cellular mechanisms in stem cell transplantation coupled with EGCG treatment remain unclear. This study investigates whether adipose-derived stem cells (ADSC) pretreated with EGCG show better protective effect on diabetic cardiomyopathy than ADSC without EGCG pretreatment. A cell model indicated that ADSC pretreated with EGCG increased cell functions including colony formation, migration and survival markers. All of these functions are blocked by small interfering C-X-C motif chemokine receptor 4 (siCXCR4) administration. These findings suggest that ADSC pretreatment with EGCG increases cell functions through CXCR4 expression. A diabetic animal model was designed to verify the above findings, including Sham, DM (diabetes mellitus), DM+ADSC (DM rats receiving autologous transplantation of ADSC) and DM+E-ADSC (DM rats receiving EGCG pretreated ADSC). Compared to the Sham, we found that all of pathophysiological signalings were activated in the DM group, including functional changes (decrease in ejection fraction and fractional shortening), structural changes (disarray and fibrosis) and molecular changes (increases in apoptotic, fibrotic, hypertrophic markers and decreases in survival and longevity markers). E-ADSC (DM+E-ADSC) transplantation shows significant improvement in the above pathophysiological signalings greater than ADSC (DM+ADSC). Therefore, ADSC pretreated with EGCG may contribute to clinical applications for diabetic patients with cardiomyopathy.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Corrigendum: Identification of ACKR4 as an immune checkpoint in pulmonary arterial hypertension.

[This corrects the article DOI: 10.3389/fimmu.2023.1153573.].

Effects of electroacupuncture on repairing neurological damage following ischemic stroke based on miR-381-mediated SDF-1/CXCR4 signaling pathway. / 基于miR-381调控SDF-1/CXCR4信号通路探讨电针对缺血性脑卒中后神经损伤修复的影响.

OBJECTIVES: To investigate the effects of electroacupuncture (EA) on the miR-381, leucine-rich repeat C4 protein (LRRC4), and downstream stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling pathway in rat model of ischemic stroke, and to explore the mechanism by which EA improves neurological damage following ischemic stroke. METHODS: Among 50 SPF male SD rats, 10 rats were randomly selected into a sham surgery group, and the remaining rats were used to establish the middle cerebral artery occlusion (MCAO) model. The 30 successfully modeled rats were randomly divided into a model group, an EA group, and an agonist group, with 10 rats in each group. The rats in the EA group received EA at "Baihui" (GV 20) and "Dazhui" (GV 14), with disperse-dense wave, a frequency of 2 Hz/10 Hz, and a current intensity of 1 mA, 30 min per session, once daily for a total of 14 days. The rats in the agonist group received miR-381 agonist injections into the lateral ventricle, with 10 µL per injection, every 7 days for a total of 2 injections. After intervention, ZeaLonga neurobehavioral deficit score was observed in each group. HE staining was performed to observe the morphological changes in the ischemic brain tissue of rats in each group. ELISA was used to measure the levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and nerve growth factor (NGF) in serum. Western blot was employed to detect the protein expression of LRRC4, SDF-1, CXCR4, and extracellular regulated protein kinase 1 (ERK1) in the ischemic brain tissue. Real-time PCR was utilized to assess the expression of miR-381 and LRRC4, SDF-1, CXCR4, ERK1 mRNA in the ischemic brain tissue. RESULTS: After intervention, the brain tissue showed disordered cell arrangement, reduced quantity, and significant interstitial edema, with numerous vacuoles in the model group. The pathological changes mentioned above were alleviated in the brain tissue of rats in the EA group and the agonist group. Compared with the sham surgery group, the rats in the model group exhibited increased ZeaLonga neurobehavioral deficit scores, elevated levels of serum TNF-α and IL-6 (P<0.01), and decreased serum NGF level (P<0.01)；the protein expression of SDF-1, CXCR4 and ERK1 in ischemic brain tissue was reduced (P<0.01), while LRRC4 protein expression was increased (P<0.01)；the expression of miR-381, as well as SDF-1, CXCR4 and ERK1 mRNA in ischemic brain tissue was decreased (P<0.01), while LRRC4 mRNA expression was increased (P<0.01). Compared with the model group, the rats in the EA group and the agonist group showed decreased ZeaLonga neurobehavioral deficit scores and reduced levels of serum TNF-α and IL-6 (P<0.05, P<0.01), and increased serum NGF levels (P<0.05, P<0.01); the protein expression of SDF-1, CXCR4 and ERK1 in ischemic brain tissue was increased (P<0.01), while LRRC4 protein expression was decreased (P<0.01)；the expression of miR-381, as well as SDF-1, CXCR4 and ERK1 mRNA in ischemic brain tissue was increased (P<0.05, P<0.01), while LRRC4 mRNA expression was decreased (P<0.01). CONCLUSIONS: EA at "Baihui" (GV 20) and "Dazhui" (GV 14) may promote the repair of neurological damage following ischemic stroke by up-regulating miR-381 to selectively inhibit LRRC4 expression, thereby activating the SDF-1/CXCR4 signaling pathway.

Esketamine inhibits the c-Jun N-terminal kinase pathway in the spinal dorsal horn to relieve bone cancer pain in rats.

Cancer-induced bone pain (CIBP) is one of the most common and feared symptoms in patients with advanced tumors. The X-C motif chemokine ligand 12 (CXCL12) and the CXCR4 receptor have been associated with glial cell activation in bone cancer pain. Moreover, mitogen-activated protein kinases (MAPKs), as downstream CXCL12/CXCR4 signals, and c-Jun, as activator protein AP-1 components, contribute to the development of various types of pain. However, the specific CIBP mechanisms remain unknown. Esketamine is a non-selective N-methyl-d-aspartic acid receptor (NMDA) inhibitor commonly used as an analgesic in the clinic, but its analgesic mechanism in bone cancer pain remains unclear. We used a tumor cell implantation (TCI) model and explored that CXCL12/CXCR4, p-MAPKs, and p-c-Jun were stably up-regulated in the spinal cord. Immunofluorescence images showed activated microglia in the spinal cord on day 14 after TCI and co-expression of CXCL12/CXCR4, p-MAPKs (p-JNK, p-ERK, p-p38 MAPK), and p-c-Jun in microglia. Intrathecal injection of the CXCR4 inhibitor AMD3100 reduced JNK and c-Jun phosphorylations, and intrathecal injection of the JNK inhibitor SP600125 and esketamine also alleviated TCI-induced pain and reduced the expression of p-JNK and p-c-Jun in microglia. Overall, our data suggest that the CXCL12/CXCR4-JNK-c-Jun signaling pathway of microglia in the spinal cord mediates neuronal sensitization and pain hypersensitivity in cancer-induced bone pain and that esketamine exerts its analgesic effect by inhibiting the JNK-c-Jun pathway.

A genetic variant of CXCR4 predicts pegylated interferon-alpha treatment response in HBeAg-positive chronic hepatitis B patients.

Chemokine receptor 4 (CXCR4) plays a vital role in immunoregulation during hepatitis B virus (HBV) infection. This study aimed to screen single-nucleotide polymorphisms (SNPs) of CXCR4 for predicting pegylated interferon-alpha (PegIFNα) therapy response in chronic hepatitis B (CHB) patients. This retrospective cohort study enrolled a total of 945 CHB patients in two cohorts (Cohort 1, n = 238; Cohort 2, n = 707), and all the patients were hepatitis B e antigen (HBeAg)-positive and treated with PegIFNα for 48 weeks and followed up for 24 weeks. Twenty-two tag SNPs were selected in CXCR4 and its flanking region. A polygenic score (PGS) was utilized to evaluate the cumulative effect of multiple SNPs. The relationships between CXCR4 SNPs and PGS and PegIFNα treatment response were explored in the two cohorts. Among the 22 candidate SNPs of CXCR4, rs28367495 (T > C) was significantly linked to PegIFNα treatment response in both cohorts. In patients with more number of rs28367495 C allele, a higher rate of combined response (CR, defined as HBeAg seroconversion and HBV DNA level < 3.3 log10 IU/mL; P = 1.51 × 10-4), a lower mean hepatitis B surface antigen (HBsAg) level (P = 4.76 × 10-4), and a higher mean HBsAg decline (P = 3.88 × 10-4) at Week 72 were achieved. Moreover, a PGS integrating CXCR4_rs28367495 and five previously reported SNPs was strongly correlated with CR (P = 1.26 × 10-13), HBsAg level (P = 4.90 × 10-4), and HBsAg decline (P = 0.005) in all the patients of the two cohorts. CXCR4_rs28367495 is a promising indicator for predicting the responsiveness to PegIFNα treatment for HBeAg-positive CHB patients. The new PGS may further improve the prediction performance.

Stromal cell-derived factor-1 downregulation contributes to neuroprotection mediated by CXC chemokine receptor 4 interactions after intracerebral hemorrhage in rats.

AIM: Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have a substantial role in neuronal formation, differentiation, remodeling, and maturation and participate in multiple physiological and pathological events. In this study, we investigated the role of SDF-1/CXCR4 in neural functional injury and neuroprotection after intracerebral hemorrhage (ICH). METHODS: Western blot, immunofluorescence and immunoprecipitation were used to detect SDF-1/CXCR4 expression and combination respectively after ICH. TUNEL staining, Lactate dehydrogenase assay, Reactive oxygen species assay, and Enzyme-linked immunosorbent assay to study neuronal damage; Brain water content to assay brain edema, Neurological scores to assess short-term neurological deficits. Pharmacological inhibition and genetic intervention of SDF-1/CXCR4 signaling were also used in this study. RESULTS: ICH induced upregulation of SDF-1/CXCR4 and increased their complex formation, whereas AMD3100 significantly reduced it. The levels of TNF-α and IL-1ß were significantly reduced after AMD3100 treatment. Additionally, AMD3100 treatment can alleviate neurobehavioral dysfunction of ICH rats. Conversely, simultaneous SDF-1/CXCR4 overexpression induced the opposite effect. Moreover, immunoprecipitation confirmed that SDF-1/CXCR4 combined to initiate neurodamage effects. CONCLUSION: This study indicated that inhibition of SDF-1/CXCR4 complex formation can rescue the inflammatory response and alleviate neurobehavioral dysfunction after ICH. SDF-1/CXCR4 may have applications as a therapeutic target after ICH.

CXCR4-directed PET/CT with [68 Ga]Ga-pentixafor in solid tumors-a comprehensive analysis of imaging findings and comparison with histopathology.

BACKGROUND: C-X-C motif chemokine receptor 4 (CXCR4) is overexpressed in various solid cancers and can be targeted by CXCR4-directed molecular imaging. We aimed to characterize the in-vivo CXCR4 expression in patients affected with solid tumors, along with a comparison to ex-vivo findings. METHODS: A total 142 patients with 23 different histologically proven solid tumors were imaged with CXCR4-directed PET/CT using [68 Ga]Ga-pentixafor (total number of scans, 152). A semi-quantitative analysis of the CXCR4-positive tumor burden including maximum standardized uptake values (SUVmax) and target-to-background ratios (TBR) using blood pool was conducted. In addition, we performed histopathological staining to determine the immuno-reactive score (IRS) from patients' tumor tissue and investigated possible correlations with SUVmax (by providing Spearman's rho ρ). Based on imaging, we also assessed the eligibility for CXCR4-targeted radioligand therapy or non-radioactive CXCR4 inhibitory treatment (defined as more than five CXCR4-avid target lesions [TL] with SUVmax above 10). RESULTS: One hundred three of 152 (67.8%) scans showed discernible uptake above blood pool (TBR > 1) in 462 lesions (52 primary tumors and 410 metastases). Median TBR was 4.4 (1.05-24.98), thereby indicating high image contrast. The highest SUVmax was observed in ovarian cancer, followed by small cell lung cancer, desmoplastic small round cell tumor, and adrenocortical carcinoma. When comparing radiotracer accumulation between primary tumors and metastases for the entire cohort, comparable SUVmax was recorded (P > 0.999), except for pulmonal findings (P = 0.013), indicative for uniform CXCR4 expression among TL. For higher IRS, a weak, but statistically significant correlation with increased SUVmax was observed (ρ = 0.328; P = 0.018). In 42/103 (40.8%) scans, more than five TL were recorded, with 12/42 (28.6%) exhibiting SUVmax above 10, suggesting eligibility for CXCR4-targeted treatment in this subcohort. CONCLUSIONS: In a whole-body tumor read-out, a substantial portion of prevalent solid tumors demonstrated increased and uniform [68 Ga]Ga-pentixafor uptake, along with high image contrast. We also observed a respective link between in- and ex-vivo CXCR4 expression, suggesting high specificity of the PET agent. Last, a fraction of patients with [68 Ga]Ga-pentixafor-positive tumor burden were rendered potentially suitable for CXCR4-directed therapy.

Clinical Value of 68Ga-Pentixafor PET/CT in Subtype Diagnosis of Primary Aldosteronism Patients with Adrenal Micronodules.

Our objective was to investigate the clinical value of 68Ga-pentixafor PET/CT in subtype diagnosis of primary aldosteronism (PA) patients with adrenal micronodules less than 1 cm in diameter and compare it with the routine clinical methods. Methods: We used prospective enrollment of PA patients with adrenal micronodules identified by adrenal CT scans to undergo 68Ga-pentixafor PET/CT. Patients were divided into surgically eligible and ineligible groups based on surgical pathology and postoperative follow-up or adrenal venous sampling (AVS) results. Patient management was discussed by a multidisciplinary team. The semiquantitative parameters of PET/CT included SUVmax for adrenal lesion and SUV ratios for lesion to liver and lesion to normal adrenal gland. Results: In total, 123 PA patients with adrenal micronodules were examined using 68Ga-pentixafor PET/CT, and 104 patients who underwent surgery or successful AVS were included in the analysis (48 ± 10 y old). The sensitivity, specificity, and accuracy of visual analysis using 68Ga-pentixafor PET/CT to identify surgically eligible patients were 90.2%, 72.7%, and 86.5%, respectively, which were significantly higher than those of adrenal CT (73.1%, 53.8%, and 68.3%, respectively) and yielded consistent results in different CT morphologic or age subgroups. In 36 patients who had both AVS and 68Ga-pentixafor PET/CT, the tests showed a 66.7% concordance rate. However, PET/CT was significantly more concordant with surgical outcomes than was AVS in 17 patients who underwent adrenalectomy (82.4% vs. 68.86%). Among the 183 adrenal micronodules included in the study, the semiquantitative diagnostic thresholds for 92 lesions eligible for surgical treatment were an SUVmax of at least 4.55, an SUV ratio of at least 2.17 for lesion to liver, and an SUV ratio of at least 1.90 for lesion to normal adrenal gland. All patients benefited from surgical removal of 68Ga-pentixafor-avid microlesions. Conclusion: In PA patients with adrenal micronodules, 68Ga-pentixafor PET/CT demonstrated promising diagnostic accuracy in classification and appeared to perform better than adrenal CT. Furthermore, there was also a suggestion of some potential in predicting postoperative efficacy compared with AVS, although these observations require further investigation and verification in larger cohorts.

[Electroacupuncture combined with bone marrow mesenchymal stem cell transplantation promotes repair of thin endometrium by regulating SDF-1/CXCR4 signaling].

OBJECTIVE: It is to explore, based on stromal cell derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signal axis, whether the electroacupuncture (EA) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation can promote thin endometrium regeneration and improve endometrial receptivity, so as to further study its mechanisms underlying improvement of promoting BMSCs homing to repair thin endometrium. METHODS: Thirty matured female SD rats were randomly divided into normal control , model , BMSCs transplantation (BMSCs), BMSCs+AMD3100 (a specific antagonist of CXCR4, BMSCs+AMD3100), BMSCs+EA, and BMSCs+EA+AMD3100 groups, with 5 rats in each group. The thin endometrial model was established by intrauterine injection of 95% ethanol during the period of estrus. Rats of the model group received intravenous injection of PBS solution (tail vein) on day 1, 3 and 7 of modeling and intraperitoneal injection of normal saline once daily for 3 estrous cycles. Rats of the BMSCs group received intravenous injection of BMSCs suspension on day 1,3 and 7 of modeling, and those of the BMSCs+EA group received BMSCs transplantation and EA stimulation. EA (2 Hz/15 Hz, 1 mA) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao"(SP9), "Zigong" (EX-CA1) for 15 min, once daily for 3 estrous cycles. Rats of the BMSCs+AMD3100 group received intravenous injection of BMSCs suspension (1×106/mL) and intraperitoneal injection of AMD3100 (5 mg/kg), and those of the BMSCs+EA+AMD3100 group received administration of BMSCs, AMD3100 and EA, with both groups being once daily for 3 estrous cycles. H.E. staining was used to observe histopathological changes of endometrium tissues, and immunohistochemistry was used to detect the expressions of cytokeratin (CK19) and vimentin in endometrium (for evaluating the damage and repair of endometrium). The expression levels of homeobox A10 (HOXA10), leukemia inhibitory factor (LIF), SDF-1 and CXCR4 proteins were detected by Western blot, and those of SDF-1 and CXCR4 mRNAs in the endometrium detected by real-time PCR. RESULTS: In comparison with the normal control group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression leve-ls of HOXA10, LIF and CXCR4 proteins and CXCR4 mRNA were significantly down-regulated (P<0.01), and the expression levels of SDF-1 protein and mRNA significantly up-regulated (P<0.05) in the model group. Compared with the model group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, and the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs group, and the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA, and SDF-1 protein and mRNA in the BMSCs+EA group were significantly up-regulated (P<0.05, P<0.01). Compared to the BMSCs group, the number of endometrial glands, and the expression levels of LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA group were up-regulated (P<0.01, P<0.05)； the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+AMD3100 group were down-regulated (P<0.01). Compared to the BMSCs+EA group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA+AMD3100 group were down-regulated (P<0.01). Results of H.E. staining showed thin endometrium with absence of epithelial cells, and sparse glands and blood vessels, with smaller glandular cavity in the model group, which was relative milder in BMSCs and BMSCs+EA groups. CONCLUSION: EA can promote the transfer of transplanted BMSCs to the damaged site through SDF-1/CXCR4 signaling related stem cell homing, thereby promoting thin endometrial regeneration, repairing endometrial injury, and improving endometrial tolerance in rats with thin endometrium.

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration.

BACKGROUND: G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer. METHODS: We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, ß-arrestin recruitment, ligand binding, and transwell migration assays. RESULTS: We observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and ß-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists. CONCLUSIONS: The presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions. Video Abstract.

Inhibition of Cxcr4 Disrupts Mouse Embryonic Palatal Mesenchymal Cell Migration and Induces Cleft Palate Occurrence.

Many processes take place during embryogenesis, and the development of the palate mainly involves proliferation, migration, osteogenesis, and epithelial-mesenchymal transition. Abnormalities in any of these processes can be the cause of cleft palate (CP). There have been few reports on whether C-X-C motif chemokine receptor 4 (CXCR4), which is involved in embryonic development, participates in these processes. In our study, the knockdown of Cxcr4 inhibited the migration of mouse embryonic palatal mesenchymal (MEPM) cells similarly to the use of its inhibitor plerixafor, and the inhibition of cell migration in the Cxcr4 knockdown group was partially reversed by supplementation with C-X-C motif chemokine ligand 12 (CXCL12). In combination with low-dose retinoic acid (RA), plerixafor increased the incidence of cleft palates in mice by decreasing the expression of Cxcr4 and its downstream migration-regulating gene Rac family small GTPase 1 (RAC1) mediating actin cytoskeleton to affect lamellipodia formation and focal complex assembly and ras homolog family member A (RHOA) regulating the actin cytoskeleton to affect stress fiber formation and focal complex maturation into focal adhesions. Our results indicate that the disruption of cell migration and impaired normal palatal development by inhibition of Cxcr4 expression might be mediated through Rac1 with RhoA. The combination of retinoic acid and plerixafor might increase the incidence of cleft palate, which also provided a rationale to guide the use of the drug during conception.

Development and Validation of CXCR4 Nomogram-Based Immune Infiltration/Tumor Inflammation in Primary Glioblastoma.

Glioblastoma (GBM) is a highly malignant and aggressive tumor with poor prognosis. Therefore, the discovery of new prognostic molecular markers is of great significance for clinical prognosis. The CXC chemokine receptor (CXCR) members play a key regulatory role in many cancers. In this study, we explore the clinical value and application of the CXCR members in primary glioblastoma. Two GBM datasets from The Cancer Genome Atlas (TCGA) and The China Glioma Genome Atlas (CGGA) databases were used to explore the relationship between differential expression of CXCRs and GBM subtypes as well as immune infiltration. C-X-C motif chemokine receptor 4 (CXCR4) was screened as an independent prognostic factor, and a nomogram and risk prediction model were developed and tested in the CGGA database using the TCGA database. Receiver operating curve (ROC) and decision curve analysis (DCA) found good accuracy and net benefit of the models. The correlation of CXCR4 with immune infiltration and tumor was analyzed using CancerSEA and TIMER. In in vitro experiments, we found that CXCR4 was significantly overexpressed in glioblastoma and was closely related to the inflammatory response of U251/U87 cells. CXCR4 is an excellent independent prognostic factor for glioblastoma and positively correlates with tumor inflammation.

Identification of ACKR4 as an immune checkpoint in pulmonary arterial hypertension.

Objective: Inflammation is recognized as a contributor in the development of pulmonary arterial hypertension (PAH), and the recruitment and functional capacity of immune cells are well-orchestrated by chemokines and their receptors. This study is aimed at identification of critical chemokines in the progression of PAH via transcriptomic analysis. Methods: Differentially expressed genes (DEGs) from lungs of PAH patients were achieved compared to controls based on Gene Expression Omnibus (GEO) database. Gene set enrichment analysis (GSEA) was applied for functional annotation and pathway enrichement. The abundance of immune cells was estimated by the xCell algorithm. Weighted correlation network analysis (WGCNA) was used to construct a gene expression network, based on which a diagnostic model was generated to determine its accuracy to distinguish PAH from control subjects. Target genes were then validated in lung of hypoxia-induce pulmonary hypertension (PH) mouse model. Results: ACKR4 (atypical chemokine receptor 4) was downregulated in PAH lung tissues in multiple datasets. PAH relevant biological functions and pathways were enriched in patients with low-ACKR4 level according to GSEA enrichment analysis. Immuno-infiltration analysis revealed a negative correlation of activated dendritic cells, Th1 and macrophage infiltration with ACKR4 expression. Three gene modules were associated with PAH via WGCNA analysis, and a model for PAH diagnosis was generated using CXCL12, COL18A1 and TSHZ2, all of which correlated with ACKR4. The ACKR4 expression was also downregulated in lung tissues of our experimental PH mice compared to that of controls. Conclusions: The reduction of ACKR4 in lung tissues of human PAH based on transcriptomic data is consistent with the alteration observed in our rodent PH. The correlation with immune cell infiltration and functional annotation indicated that ACKR4 might serve as a protective immune checkpoint for PAH.

CXCR4, regulated by HIF1A, promotes endometrial breakdown via CD45+ leukocyte recruitment in a mouse model of menstruation.

Menstruation is a specific physiological phenomenon in female humans that is regulated by complex molecular mechanisms. However, the molecular network involved in menstruation remains incompletely understood. Previous studies have suggested that C-X-C chemokine receptor 4 (CXCR4) is involved; however, how CXCR4 participates in endometrial breakdown remains unclear, as do its regulatory mechanisms. This study aimed to clarify the role of CXCR4 in endometrial breakdown and its regulation by hypoxia-inducible factor-1 alpha (HIF1A). We first confirmed that CXCR4 and HIF1A protein levels were significantly increased during the menstrual phase compared with the late secretory phase using immunohistochemistry. In our mouse model of menstruation, real-time PCR, western blotting, and immunohistochemistry showed that CXCR4 mRNA and protein expression levels gradually increased from 0 to 24 h after progesterone withdrawal during endometrial breakdown. HIF1A mRNA and HIF1A nuclear protein levels significantly increased and peaked at 12 h after progesterone withdrawal. Endometrial breakdown was significantly suppressed by the CXCR4 inhibitor AMD3100 and the HIF1A inhibitor 2-methoxyestradiol in our mouse model, and HIF1A inhibition also suppressed CXCR4 mRNA and protein expression. In vitro studies using human decidual stromal cells showed that CXCR4 and HIF1A mRNA expression levels were increased by progesterone withdrawal and that HIF1A knockdown significantly suppressed the elevation in CXCR4 mRNA expression. CD45+ leukocyte recruitment during endometrial breakdown was suppressed by both AMD3100 and 2-methoxyestradiol in our mouse model. Taken together, our preliminary findings suggest that endometrial CXCR4 expression is regulated by HIF1A during menstruation and may promote endometrial breakdown, potentially via leukocyte recruitment.

Efficacy of Rectal Systemic Administration of Mesenchymal Stem Cells to Injury Sites via the CXCL12/CXCR4 Axis to Promote Regeneration in a Rabbit Skeletal Muscle Injury Model.

Mesenchymal stem cells (MSCs) have been transplanted directly into lesions or injected intravenously. The administration of MSCs using these delivery methods requires specialized knowledge, techniques, and facilities. Here, we describe intrarectal systemic administration of MSCs, a simple, non-invasive route for homing to the injury sites to promote the regeneration of skeletal muscle injuries. Using a cardiotoxin (CTX)-induced rabbit skeletal muscle injury model, homing to the site of muscle injury was confirmed by intrarectal administration of MSCs; the time required for homing after intrarectal administration was approximately 5 days. In addition, the C-X-C chemokine ligand 12 (CXCL12)/C-X-C chemokine receptor-4 (CXCR4) axis was found to be involved in the homing process. Histopathological examinations showed that skeletal muscle regeneration was promoted in the MSCs-administered group compared to the CTX-only group. Myosin heavy polypeptide 3 (Myh3) expression, an indicator of early muscle regeneration, was detected earlier in the intrarectal MSCs group compared to the CTX-only group. These findings indicate that intrarectal administration of MSCs is effective in homing to the injured area, where they promote injury repair. Since intrarectal administration is a simple and non-invasive delivery route, these findings may be valuable in future research on stem cell therapy.