CO-EXPRESSION OF MACROPHAGE COLONY-STIMULATING FACTOR WITH ITS RECEPTOR IN HUMAN HEPATOMA CELLS AND ITS POTENTIAL ROLES

Objective: To investigate the potential role of macrophage colony-stimulating factor (M-CSF) and macrophage colony-stimulating factor receptor (M-CSF-R) on the growth of human hepatoma cells. Methods: Specimens of different origin, including tissues of human hepatocellular carcinoma (HCC), human fetal liver (FL) and normal liver (NL), the hepatoma cell lines, as well as the peripheral blood mononuclear cells (PBMC) from patients with HCC or liver metastatic tumor (LMT), were used to detect the expression levels of M-CSF and M-CSF-R by ABC immunohistochemistry staining and reverse transcription polymerase chain reaction methods the expression levels of M-CSF and M-CSF-R. Influence of monoclonal antibody against M-CSF (B5) or M-CSF-R (RE2) on proliferation ability of hepatoma cell linesin vitro was also studied. Results: The results showed that hepatoma tissues produced elevated levels of both M-CSF and M-CSF-R compared with those of fetal liver (P<0.001). The M-CSF/M-CSF-R expression levels of PBMC from hepatoma patients were higher than those of LMT patients (P<0.01,P<0.05) and the normal people (P<0.001). The hepatoma cell lines showed strong positive for M-CSF and M-CSF-R production. Both B5 and RE2 displayed a dose-dependent inhibitory effect on the growth and proliferation of hepatoma cells. Conclusion: The study indicates a co-expression model for M-CSF-R in hepatoma cells, suggesting an involvement of M-CSF/M-CSF-R in growth signaling of those malignant cells. The M-CSF/M-CSF-R seems to function through an autonomy mechanism in human hepatoma.

DETERMINATION OF SERUM SOLUBLE MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR LEVELS IN PATIENTS WITH HEMATOLOGICAL DISEASES

Objective: To investigate the serum levels of soluble macrophage colony-stimulating factor receptor (M-CSFsR) in normal subjects and patients with hematological diseases and its clinical implications in hematological diseases. Methods: The concentration of M-CSFsR was determined by ELISA. The serum M-CSFsR was identified and characterized by immunoprecipitation and Western blotting. Results: The mean serum level of M-CSFsR of 123 normal individuals was 0.48 ng/ml ± 0.41 ng/ml. Immunoprecipitation and Western blotting assay revealed a ～ 90kD band of serum M-CSFsR. The mean serum M-CSFsR level of 60 patients with acute lymphoblastic leukemia (ALL), 36 patients with acute myeloblastic leukemia (AML), 13 patients with myelodysplastic syndrome (MDS) and 42 patients with aplastic anemia (AA) .were 0.22 ng/ml±0.23 ng/ml, 0.17 ng/ml±0.16 ng/ml, 0.19 ng/ml±0.16 ng/ml and 0.23 ng/ml±0.21 ng/ml, respectively, which were significantly lower than that of normal subjects (P=0.002 ,P<0.0001,P<0.0001 andP<0.0001). The mean serum M-CSFsR level of 51 idiopathic thrombocytopenic purpura (ITP) patients was significantly higher than that of normal subjects (2.05 ng/ml±2.75 ng/ml,P<0.0001). Conclusion: The serum M-CSFsR levels of patients with ALL, AML, MDS and AA were significantly lower, while the level of patients with ITP was significantly higher than that of normal individuals. Patients with severe ITP (platelet count<30×l09/L) had the highest M-CSFsR level. It suggested that the abnormal levels of serum M-CSFsR may associate with some hematological diseases and may contribute to the pathological process.

Granulocyte-macrophage colony-stimulating factor protects mice against hepatocellular carcinoma by ameliorating intestinal dysbiosis and attenuating inflammation

BACKGROUND Hepatocellular carcinoma(HCC)is the third leading cause of cancer mortality worldwide.The gut microbiota can help maintain healthy metabolism and immunity.Granulocyte-macrophage colony-stimulating factor(GM-CSF)is a critical factor in promoting health and homeostasis;it promotes intestinal immunity,stimulates bone marrow precursors to generate macrophage colonies,and enhances the antibacterial and antitumor activity of circulating monocytes.As such,GM-CSF may protect against HCC development by regulating immunity as well as intestinal microecology.AIM To investigate the impact of GM-CSF on the gut microbiome and metabolic characteristics of HCC.METHODS Thirty-six male BALB/c nude mice were divided into three groups:Control(n=10),HCC(n=13),and HCC+GM-CSF(GM-CSF overexpression,n=13).We utilized HCC cells to establish orthotopic transplantation tumor models of HCC with normal and over-expressing GM-CSF.Liver injury,immune inflammatory function and intestinal barrier function were evaluated.The fecal microbiome and metabolome were studied using 16S rRNA absolute quantification sequencing and gas chromatography-mass spectrometry.RESULTS GM-CSF overexpression significantly affected the gut microbiome of mice with HCC and resulted in a high abundance of organisms of the genera Roseburia,Blautia and Butyricimonass,along with a significant reduction in Prevotella,Parabacteroides,Anaerotruncus,Streptococcus,Clostridium,and Mucispirillum.Likewise,GM-CSF overexpression resulted in a substantial increase in fecal biotin and oleic acid levels,along with a prominent decrease in the fecal succinic acid,adenosine,fumaric acid,lipoic acid,and maleic acid levels.Correlation analysis revealed that the intestinal microbiota and fecal metabolites induced by GM-CSF were primarily involved in pathways related to reducing the inflammatory response,biotin metabolism,and intestinal barrier dysfunction.CONCLUSION GM-CSF can protect against HCC development by regulating immunity and modulating the abundance of specific intestinal microorganisms and their metabolites.This study provides new insights into the therapeutic approaches for HCC.

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR WITH GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR GENE

Objective: To construct the eukaryotic expression vector that express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene for making highly express in mammalian cells. Methods: Extract totally RNA from the induced human fetal lung (HFL) cell line. HGM-CSF cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR), and then directionally subcloned into the HindIII and EcoRI site on the pcDNA3.1 plasmid, which was controlled by the CMV promoter, to form the recombinant expressing vector pcDNA3.1-GM-CSF. Results: The PCR amplification was identified and the sequence was analyzed, the results showed that hGM-CSF was properly inserted into the vector and the sequence was correct.

Granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells for the treatment of ischemic stroke

Adult, male, Sprague-Dawley rats were injected with granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells （GM-CSF-BMSCs） into the ischemic boundary zone at 24 hours after onset of middle cerebral artery occlusion. Results showed reduced infarct volume, decreased number of apoptotic cells, improved neurological functions, increased angiogenic factor expression, and increased vascular density in the ischemic boundary zone in rats that underwent GM-CSF-BMSCs transplantation compared with the BMSCs group. Experimental findings suggested that GM-CSF-BMSCs could serve as a potential therapeutic strategy for ischemic stroke and are superior to BMSCs alone.

Antiviral Activity of Silkworm Expressed Recombinant Human Macrophage Colony-Stimulating Factor in Vitro and Its Mechanism

Cells, pretreated with the recombinant human macrophage colony-stimulating factor (rhM-CSF) expressed in silkworm larvae, were inoculated with several viruses to observe the effect of rhM-CSF on viral replication. The results showed that in cultures of fibroblast derived from human fetal skin-muslce tissues infected with the viruses (including VSV, rhinovirus, influenza virus type A3, HSV-1, HSV-2, adenovirus type 6 and type 11), rhM-CSF inhibited the virus-induced cytopathy, which defered or relieved the cytophathy and that in the cells derived from breast feeding rabbit kidney infected with HSV-1, rhM-CSF reduced titer of the virus in a rhM-CSF dose-dependent pattern,in which rhM-CSF with the dose of 1×106 U/L made the viral titer drop dwon over 200 times. This antiviral activity of rhM-CSF could be partially neutralized by anti-IFN-βMcAb, but not by antiTNF-α, anti-IFN-α, or anti-IFA-γ McAb, indicating the mechanism of the antiviral activity of MCSF is related to the induction of IFN-β.

Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma

AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor(HSVGM-CSF) in pancreatic carcinoma.METHODS: Tumor blocks were homogenized in a sterile grinder in saline.The homogenate was injected into the right armpit of each mouse.After vaccination,the mice were randomly assigned into four groups: a control group,a high dose HSVGM-CSFgroup [1 × 107plaque forming units(pfu)/tumor],a medium dose HSVGM-CSF group(5 × 106pfu/tumor) and a low dose HSVGM-CSF group(5 × 105pfu/tumor).After initiation of drug administration,body weights and tumor diameters were measured every 3 d.Fifteen days later,after decapitation of the animal by cervical dislocation,each tumor was isolated,weighed and stored in 10% formaldehyde solution.The drug effectiveness was evaluated according to the weight,volume and relative volume change of each tumor.Furthermore,GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1,2,3 and 4 d after injection of HSVGM-CSF.RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group,and dosedependent effects were observed: the relative tumor proliferation rates of the low dose,medium dose and high dose groups on 15 d after injection were 45.5%,55.2% and 65.5%,respectively.The inhibition rates of the tumor weights of the low,middle,and high dose groups were 41.4%,46.7% and 50.5%,respectively.Furthermore,the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF.The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.CONCLUSION: Our study provides the first evidence that HSVGM-CSFcould inhibit the growth of pancreatic cancer.The enhanced GM-CSF expression might be responsible for the phenomenon.

Impaired granulocyte-macrophage colony-stimulating factor bioactivity accelerates surgical recurrence in ileal crohn's disease

AIM To examine the relationship between elevated granulocyte-macrophage colony-stimulating factor(GMCSF) auto-antibodies(Ab) level and time to surgical recurrence after initial surgery for Crohn's disease(CD). METHODS We reviewed 412 charts from a clinical database at tertiary academic hospital. Patients included in the study had ileal or ileocolonic CD and surgical resection of small bowel or ileocecal region for management of disease. Serum samples were analyzed for serological assays including GM-CSF cytokine, GM-CSF Ab, ASCA Ig G and Ig A, and genetic markers including SNPs rs2066843, rs2066844, rs2066845, rs2076756 and rs2066847 in NOD2, rs2241880 in ATG16 L1, and rs13361189 in IRGM. Cox proportional-hazards models were used to assess the predictors of surgical recurrence.RESULTS Ninety six percent of patients underwent initial ileocecal resection(ICR) or ileal resection(IR) and subsequently 40% of patients required a second ICR/IR for CD. GMCSF Ab level was elevated at a median of 3.81 mcg/mL. Factors predicting faster time to a second surgery included elevated GM-CSF Ab [hazard ratio(HR) 3.52, 95%CI: 1.45-8.53, P = 0.005] and elevated GM-CSF cytokine(HR = 2.48, 95%CI: 1.31-4.70, P = 0.005). Factors predicting longer duration between first and second surgery included use of Immunomodulators(HR = 0.49, 95%CI: 0.31-0.77, P = 0.002), the interaction effect of low GM-CSF Ab levels and smoking(HR = 0.60, 95%CI: 0.45-0.81, P = 0.001) and the interaction effect of low GM-CSF cytokine levels and ATG16 L1(HR = 0.65, 95%CI: 0.49-0.88, P = 0.006).CONCLUSION GM-CSF bioavailability plays a critical role in maintaining intestinal homeostasis. Decreased bioavailability coupled with the genetic risk markers and/or smoking results in aggressive CD behavior.

Granulocyte-macrophage colony-stimulating factor （GM-CSF） and T-cell responses： what we do and don＇t know

Granulocyte 巨噬细胞刺激殖民地的因素(GM-CSF ) 是一个重要造血的生长因素和有免疫力的调节的人。GM-CSF 也在各种各样的传播白血球的功能的活动有深刻效果。它被许多房间类型在收到有免疫力的刺激之上包括 T 房间，巨噬细胞， endothelial 房间和成纤维细胞生产。尽管 GM-CSF 局部地被生产，它能以一种 paracrine 方式行动到在主人防卫提高他们的功能的成员传播 neutrophils，单核白血球和淋巴细胞。最近的集中的调查为它增加树枝状的房间(DC ) 成熟和功能以及巨噬细胞活动的能力作为一个有免疫力的助手在 GM-CSF 的应用程序上被集中。在经历化疗的癌症病人对待嗜中性白血球减少症临床上被使用，在在治疗期间的爱滋病病人，并且在在骨髓移植以后的病人。有趣地， GM-CSF-deficient 老鼠的造血的系统看起来正常；最重要的变化在一些特定的 T 房间回答。尽管 GM-CSF 的分子的克隆用 T 房间的 cDNA 图书馆被执行， T 房间在激活以后生产 GM-CSF，是众所周知的，在 T 房间功能上有在由 T 房间和它的效果的生产的这 cytokine 的系统的调查的缺乏。在这篇文章，我们将在 T 房间主要集中于 GM-CSF 的免疫生物学。

Macrophage colony-stimulating factor expressed in non-cancer tissues provides predictive powers for recurrence in hepatocellular carcinoma

AIM To investigate the role of macrophage colony-stimulating factor(M-CSF) in patients with hepatocellular carcinoma(HCC) after surgery. METHODS Expression of M-CSF, distribution of M2 macrophages(Mφs), and angiogenesis were assessed in the liver, including tumors and peritumoral liver tissues. The prognostic power of these factors was assessed. Mouse isolated hepatic Mφs or monocytes were cultured with media containing M-CSF. The concentration of vascular endothelial growth factor(VEGF) in media was assessed. Furthermore, the role of the M-CSF-matured hepatic Mφs on proliferation of the vascular endothelial cell(VEC) was investigated. RESULTS A strong correlation between the expressions of M-CSF and CD163 was observed in the peritumoral area. Also, groups with high density of M-CSF, CD163 or CD31 showed a significantly shorter time to recurrence(TTR) than low density groups. Multivariate analysis revealedthe expression of M-CSF or hepatic M2Mφs in the peritumoral area as the most crucial factor responsible for shorter TTR. Moreover, the expression of M-CSF and hepatic M2Mφs in the peritumoral area had better predictable power of overall survival. Values of VEGF in culture media were significantly greater in the hepatic Mφs compared with the monocytes. Proliferation of the VEC was greatest in the cells co-cultured with hepatic Mφs when M-CSF was present in media.CONCLUSION M-CSF increases hepatocarcinogenesis, most likely by enhancing an angiogenic factor derived from hepatic Mφ and could be a useful target for therapy against HCC.

The roles of macrophage migration inhibitory factor in retinal diseases

Macrophage migration inhibitory factor(MIF),a multifunctional cytokine,is secreted by various cells and participates in inflammatory reactions,including innate and adaptive immunity.There are some evidences that MIF is involved in many vitreoretinal diseases.For example,MIF can exacerbate many types of uveitis;measurements of MIF levels can be used to monitor the effectiveness of uveitis treatment.MIF also alleviates trauma-induced and glaucoma-induced optic nerve damage.Furthermore,MIF is critical for retinal/choroidal neovascularization,especially complex neovascularization.MIF exacerbates retinal degeneration;thus,anti-MIF therapy may help to mitigate retinal degeneration.MIF protects uveal melanoma from attacks by natural killer cells.The mechanism underlying the effects of MIF in these diseases has been demonstrated:it binds to cluster of differentiation 74,inhibits the c-Jun N-terminal kinase pathway,and triggers mitogen-activated protein kinases,extracellular signal-regulated kinase-1/2,and the phosphoinositide-3-kinase/Akt pathway.MIF also upregulates Toll-like receptor 4 and activates the nuclear factor kappa-B signaling pathway.This review focuses on the structure and function of MIF and its receptors,including the effects of MIF on uveal inflammation,retinal degeneration,optic neuropathy,retinal/choroidal neovascularization,and uveal melanoma.

Granulocyte-macrophage colony-stimulating factor and interleukin 4 induce the malignant transformation of the bone marrow- derived human adult mesenchymal stem cells

Background The purpose of the study was to examine the effects of granulocyte-macrophage colony-stimulating factor （GM-CSF） and interleukin 4 （IL-4） on the bone-marrow-derived human adult mesenchymal stem cells （hMSCs）. Methods The hMSCs were isolated and cultured with GM-CSF and IL-4 for a period of one month. A single colony of transformed cells was then isoloated and their phenotype was characterized by morphology, surface marker expression, and in vivo tumorigenesis.Results After one month culture, the transformed mesenchymal cells exhibited the morphology and phenotype similar to those of tumor cells, and also caused multiple fast growing lung deposits when it was injected into immunodeficient mice.Conclusion Cytokines-driven malignant transformation of hMSCs may be a useful model for studying signaling pathways initiating malignant transformation of hMSC.

Clinical Study of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor on Chemotherapy-Induced Leukopenia.

We have studied the efficacy and safery of recombinant human granulocyte-macrophate colony-stimulating factor

Enhancing effects of silkworm expressed recombinant human macrophage colony-stimulating factor on hematopoietic recovery of irradiation-injured mice

Objective To study the hematopoietic enhancing effects of recombinant human macrophage colony stimulating factor (rhM CSF) expressed in silkworm. Method Balb/c mice were irradiated with sublethal dose of 60 Coγ rays and then administered intraperitoneally with silkworm expressed rhM CSF (1000 U per individual for 7 days) in treatment group or with normal saline (for 7 days as well) in control group. The hematopoietic recovery of irradiation mice was observed by comparing peripheral white blood cell (WBC) counts, differential counts of WBC and bone marrow hematopoietic progenitor cell colony forming assay in soft agar at different time after irradiation. Results The total WBC counts (×10 9 /L) of treatment group at day 15 and 20 after irradiation (3.42±1.20, 5.56±2.50, respectively) were significantly higher than those of control group (2.03±0.90, 3.72± 2.30; both P<0.05). On days 10, 15, 20, 25 and 30 after irradiation, the monocyte counts (×10 9 /L) of treatment group (0.08±0.06, 0.16±0.10, 0.48± 0.35, 0.47±0.21 and 0.33±0.17, respectively) were all significantly higher than those of control group (0.025±0.016, 0.05±0.04, 0.23±0.16, 0.33± 0.19 and 0.17±0.13; all P<0.05). On days 15, 20 and 25, the granulocyte count (X10 9 /L) of treatment group (1.03±0.61, 2.18±1.19 and 3.28±1.09) were also higher than those of control group (0.62± 0.37, 1.40±0.99 and 2.20±0.74; all P<0.05). On day 9 after irradiation, the bone marrow CFU GM yield of control group (19±11/10 6 cells) was significantly lower than that of treatment group (78±30/10 6 cells, P< 0.05). Conclusion rhM CSF expressed in silkworm could accelerate hematopoietic recovery in irradiated mice.

Mudskipper interleukin-34 modulates the functions of monocytes/macrophages via the colony-stimulating factor-1 receptor 1

Interleukin-34(IL-34)is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colonystimulating factor-1 receptor(CSF-1R).However,information on the function of IL-34 in fish remains limited.In the present study,we identified an IL-34 homolog from mudskippers(Boleophthalmus pectinirostris).In silico analysis showed that the mudskipper IL-34(BpIL-34)was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper(Epinephelus coioides)homolog.BpIL-34 transcripts were constitutively expressed in various tissues,with the highest level of expression found in the brain.Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues.The recombinant mature BpIL-34 peptide(rBpIL-34)was purified and used to produce anti-rBpIL-34 IgG.Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages(MOs/MФs)was N-glycosylated.In vitro,rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs,as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factorα(BpTNF-α)and BpIL-1βin these cells.Furthermore,the knockdown of mudskipper CSF-1R1(BpCSF-1R1),but not mudskipper BpCSF-1R2,significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФfunction.In conclusion,our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.

Internalization and half-life of membrane-bound macrophage colony-stimulating factor

The binding of recombinant human macrophage colony-stimulating factor (M-CSF) soluble receptor (rh-M-CSF-sR) to membrane-bound macrophage colony stimulating factor (m-M-CSF) and the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes mediated by m-M-CSF were studied with a model of J6-1 cell line. The results indicated that m-M-CSF bound rh-M-CSF-sR with high affinity (Kd= 1.78×10 12 mol/L) and mediated a temperature- and energy-dependent internalization of rh-M-CSF-sR, and that internalized rh-M-CSF-sR could return to the cell surface in an m-M-CSF-bound state, suggesting that m-M-CSF may have a capability to mediate the internalization and recycling of rh-M-CSF-sR/m-M-CSF complexes. In addition, the half-lives of cell-associated M-CSF and its receptor of stimulated normal human cord blood mononuclear cells (CBMCs) and 4 leukemic cell lines were measured by indirect immunoflu-orescence and flow cytometry. The results showed that the half-lives of the various kinds of M-CSF isoforms and

Receptor role of membrane bound macrophage colony-stimulating factor

According to the definition of cytokine, the direction of signaling should be from cytokine to receptor. The counter receptor was presented. Membrane bound macrophage colony-stimulating factor (m-M-CSF) and its receptor (M-CSF-R) were shown in human leukemic cell line J6-1 as autojuxtacrine mechanism. Soluble M-CSF receptor (sM-CSF-R), which was isolated from J6-1 cells membrane, was added into J6-1 cell culture. It was observed inhibition of J6-1 cell proliferation, decreasing of mitosis index and ratio of multinuclear cells, enlargement of cell diameter and volume, down regulation of numerous surface antigens. Dramatic change of intracellular pH was shown between several min to 20 min after treatment of sM-CSF-R. It suggested that some information was transmitted via m-M-CSF from sM-CSF-R. This counter signaling was not influenced by saccharification of m-M-CSF.

Serum profiles of circulating granulocyte-macrophage colony-stimulating factor in acute myocardial infarction and relation with post-infarction left ventricular function

Accumulating evidence indicates that inflammation plays an important role in cardiac repairing and remodeling after acute myocardial infarction （AMI）, process of which is mediated by a cytokine reaction cascade. 1 Granulocytemacrophage colony-stimulating factor （GM-CSF） is a cytokine, which belongs to the family of haemopoietic cell colony-stimulating factor and regulates the proliferation and differentiation of myeloid progenitor cells. In addition to its growthpromoting effects, this pro-inflammation cytokine stimulates the function of mature neutrophils, monocytes and eosinophils, including regulation of leukocyte adhesion, augmentation of surface antigen expression, superoxide anion generation, enhancement or induction of other cytokine production.

Th17/Treg balance and macrophage polarization ratio in lower extremity arteriosclerosis obliterans

Objective:To explore the balance of peripheral blood T helper 17 cells/regulatory T cell(Th17/Treg)ratio and the polarization ratio of M1 and M2 macrophages in lower extremity arteriosclerosis obliterans(ASO).Methods:A rat model of lower extremity ASO was established,and blood samples from patients with lower extremity ASO before and after surgery were obtained.ELISA was used to detect interleukin 6(IL-6),IL-10,and IL-17.Real-time RCR and Western blot analyses were used to detect Foxp3,IL-6,IL-10,and IL-17 expression.Moreover,flow cytometry was applied to detect the Th17/Treg ratio and M1/M2 ratio.Results:Compared with the control group,the iliac artery wall of ASO rats showed significant hyperplasia,and the concentrations of cholesterol and triglyceride were significantly increased(P<0.01),indicating the successful establishment of ASO.Moreover,the levels of IL-6 and IL-17 in ASO rats were pronouncedly increased(P<0.05),while the IL-10 level was significantly decreased(P<0.05).In addition to increased IL-6 and IL-17 levels,the mRNA and protein levels of Foxp3 and IL-10 in ASO rats were significantly decreased compared with the control group.The Th17/Treg and M1/M2 ratios in the ASO group were markedly increased(P<0.05).These alternations were also observed in ASO patients.After endovascular surgery(such as percutaneous transluminal angioplasty and arterial stenting),all these changes were significantly improved(P<0.05).Conclusions:The Th17/Treg and M1/M2 ratios were significantly increased in ASO,and surgery can effectively improve the balance of Th17/Treg,and reduce the ratio of M1/M2,and the expression of inflammatory factors.

Single-Nucleus RNA Sequencing Reveals Cardiac Macrophage Landscape in Hypoplastic Left Heart Syndrome

Background:Hypoplastic left heart syndrome(HLHS)is one of the most challenging congenital heart diseases in clinical treatment.In cardiac tissues,resident macrophages fulfill critical functions in maintaining a stable cardiac state and have strong regenerative capacity and organ specificity.However,the molecular mechanisms of macro-phages in HLHS remained unclear.Methods:Single-nucleus RNA sequencing(snRNA-seq)data of HLHS and healthy control(donors)samples obtained from the Gene Expression Omnibus(GEO)database were normalized and clustered using the Seurat package.The“FindMarkers”function was used to screen differentially expressed genes(DEGs)between the HLHS and donor groups and to analyze the functional enrichment of the set of genes of interest.Finally,cell-cell communication,pseudotime,and single-cell regulatory network inference and cluster-ing(SCENIC)analyses were used to study the mechanisms of macrophages in HLHS.Results:Based on the snRNA-seq data of HLHS and donors,we identified a total of 9 cell clusters,among which the proportion of macrophages was significantly less in the HLHS group than in the control group.Subdivision of macrophage subpopulations(Macrophages 1,2,and 3)showed that Macrophages 1 was mainly involved in nervous system development,angiogenesis,and apoptotic processes.In addition,analysis of communication between Macro-phages 1 and cardiomyocytes revealed that ligand-acceptor pairs such as GAS6/AXL,IL6,IGF1,THY1,and L1CAM were present only in the donor group.Finally,pesudotime and SCENIC analyses demonstrated that FOXO3 and ELF2 played a critical role for Macrophages 1 to maintain cardiac function in patients with HLHS.Conclusion:Our study improved the current understanding of the molecular mechanisms of macrophage devel-opment in HLHS,showing that manipulating the regulatory role of macrophages in the heart can be a novel treat-ment for HLHS.