Relationship between serum levels of sSema4D,CXCL12 and left ventricular diastolic function in young and middle-aged patients with essential hypertension / 国际检验医学杂志

Objective To explore the relationship between serum soluble semaphorin 4D(sSema4D),CXC chemokine ligand 12(CXCL12)levels and left ventricular diastolic function in young and middle-aged patients with essential hypertension.Methods A total of 148 young and middle-aged patients with essential hyperten-sion admitted to a hospital from November 2020 to November 2022 were selected as the study subjects,and were grouped into left ventricular diastolic dysfunction group(n=41)and normal left ventricular diastolic function group(n=107)according to their left ventricular diastolic function.The serum levels of sSema4D and CXCL12 were detected by enzyme-linked immunosorbent assay.Pearson correlation analysis was applied to analyze the correlation between the serum levels of sSema4D and CXCL12 and the left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),left ventricular septal thickness(IVST),left ventricular end-diastolic posterior wall thickness(LVPWT),left ventricular ejection fraction(LVEF),E peak/A peak(E/A)and maximum velocity of tricuspid regurgitation(TRVmax).The predictive value of ser-um sSema4D and CXCL12 levels in left ventricular diastolic dysfunction in young and middle-aged patients with essential hypertension was analyzed by receiver operating characteristic(ROC)curve.Results There were significant differences in diastolic blood pressure and gender between the left ventricular diastolic dys-function group and the left ventricular diastolic function normal group(P＜0.05).Compared with the normal left ventricular diastolic function group,serum levels of sSema4D,CXCL12 in the left ventricular diastolic dys-function group were obviously increased,and the difference was statistically significant(P＜0.05).Compared with normal left ventricular diastolic function group,IVST and LVPWT in the left ventricular diastolic dys-function group were significantly increased,and E/A was significantly decreased,with statistical significance(P＜0.05).Pearson correlation analysis showed that serum sSema4D and CXCL12 levels were positively cor-related with LVEDD,IVST and LVPWT(P＜0.05),and negatively correlated with E/A(P＜0.05).ROC curve analysis showed that the area under the curve(AUC)of serum sSema4D and CXCL12 combined in pre-dicting left ventricular diastolic dysfunction in young and middle-aged patients with essential hypertension was 0.894(95％CI:0.833-0.939),which was significantly greater than that of sSema4D alone in predicting left ventricular diastolic dysfunction in young and middle-aged patients with essential hypertension(Z=3.142,P=0.002)and CXCL12 alone predicted the AUC of left ventricular diastolic dysfunction in young and middle-aged patients with essential hypertension(Z=3.268,P=0.001).Conclusion Serum sSema4D and CXCL12 levels are associated with left ventricular diastolic function in young and middle-aged patients with essential hypertension.

Study on Correlation Between Semaphorin 3E and 1-month Poor Prognosis After Interventional Embolization in Patients With Intracranial Aneurysm / 中国微创外科杂志

Objective To investigate the serum levels of semaphorin 3E(Sema3E)in patients with intracranial aneurysms,revealing the correlation between Sema3E and 1-month poor prognosis after interventional embolization.Methods This study was a prospective single-center cohort study,recruiting 102 consecutive patients with intracranial aneurysms who underwent interventional surgery from June 2020 to January 2022 in our hospital.Among them,11 patients were excluded.Clinical and radiological profiles were collected.Peripheral blood was collected after admission,and serum Sema3E levels were determined by enzyme-linked immunosorbent assay.All the aneurysms were treated with endovascular coil embolization or stent-assisted coil embolization.The primary outcome was evaluated with the Glasgow Outcome Scale(GOS)1 month after interventional therapy.The favorable outcome was defined as a GOS score of 4-5,and a poor outcome was defined as a GOS score of 1-3(severe disability,vegetative state,or death).Univariate and multivariate logistic regression analyses were used to identify potential prognostic factors after interventional therapy.Results The average age of 91 patients with intracranial aneurysm was 59.9±11.0 years old,including 70 cases(76.9%)with favorable prognosis and 21 cases(23.1%)with poor prognosis.The mean preoperative Glasgow Coma Scale(GCS)score of the poor prognosis group(9.4±4.5)was significantly lower than that of the favorable prognosis group(13.3±2.5;P<0.001).In the poor prognosis group,the Hunt-Hess grade(3.6±0.6 vs.2.0±1.3,P<0.001)and the serum Sema3E levels[(6.21±1.58)μg/L vs.(4.38±1.77)μg/L,P<0.001]were significantly higher than those in the favorable prognosis group.Logistic regression analysis showed the Hunt-Hess grade(OR =7.150,P =0.003),stent-assisted coil embolization(OR =15.777,P =0.010),and the serum Sema3E level(OR =1.756,P =0.027)were independent prognostic factors for intracranial aneurysms after interventional therapy.Conclusions The serum Sema3E level is closely correlated with the severity of intracranial aneurysms.The serum Sema3E level is a prognostic factor for interventional treatment,which can be used as a biomarker for predicting poor outcomes.

Reduced expression of semaphorin 3A in osteoclasts causes lymphatic expansion in a Gorham-Stout disease (GSD) mouse model / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Gorham-Stout disease (GSD) is a sporadic chronic disease characterized by progressive bone dissolution, absorption, and disappearance along with lymphatic vessel infiltration in bone-marrow cavities. Although the osteolytic mechanism of GSD has been widely studied, the cause of lymphatic hyperplasia in GSD is rarely investigated. In this study, by comparing the RNA expression profile of osteoclasts (OCs) with that of OC precursors (OCPs) by RNA sequencing, we identified a new factor, semaphorin 3A (Sema3A), which is an osteoprotective factor involved in the lymphatic expansion of GSD. Compared to OCPs, OCs enhanced the growth, migration, and tube formation of lymphatic endothelial cells (LECs), in which the expression of Sema3A is low compared to that in OCPs. In the presence of recombinant Sema3A, the growth, migration, and tube formation of LECs were inhibited, further confirming the inhibitory effect of Sema3A on LECs in vitro. Using an LEC-induced GSD mouse model, the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo. We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss, whereas the injection of lentivirus expressing Sema3A short hairpin RNA (shRNA) into the tibiae caused GSD-like phenotypes. Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment, compared with the control. Based on the above results, we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.

Expression and clinical significance of NDRG3 and SEMA3A in elderly patients with acute ischemic stroke / 中华老年心脑血管病杂志

Objective To analyze the expression levels and clinical significance of N-myc down-stream-regulated gene 3(NDRG3)and semaphoring 3A(SEMA3A)in elderly patients with acute ischemic stroke(AIS).Methods A total of 100 elderly AIS patients admitted to Department of Geriatrics of Liaocheng People's Hospital from September 2020 to September 2022 were included as the study group.According to their NIHSS score at admission,they were divided into mild(34 cases),moderate(31 cases)and severe(35 cases)subgroups.All patients were followed up for 3 months after discharge.And they were assigned into good prognosis group(69 cases)and poor prognosis group(31 cases)based on the modified Rankin scale score.Another 100 healthy individ-uals who underwent physical examination in our hospital during the same period were recruited as the control group.Western blotting was used to detect the protein expression of NDRG3 and SEMA3A in peripheral blood mononuclear cells(PBMC).ELISA was applied to measure the con-tents of VEGF,TGF-1,TNF-α,and IL-17 in peripheral blood samples.Spearman rank correlation analysis was performed to analyze the correlation of NDRG3 and SEMA3A levels with NIHSS score,and ROC curve was plotted to analyze the values of NDRG3 and SEMA3A in predicting poor prognosis in elderly AIS patients.Results The expression levels of NDRG3 and SEMA3A in PBMC were obviously higher in the study group than the control group(1.11±0.16 vs 0.76± 0.13,0.78±0.13 vs 0.42±0.09,P＜0.01).The levels in the mild,moderate and severe subgroups were significantly higher than that of the control group(P＜0.01).The poor prognosis group had statistically higher expression levels of NDRG3 and SEMA3A than the good prognosis group(P＜0.01).Spearman rank correlation analysis showed that the NIHSS score was positively corre-lated with the expression levels of NDRG3 and SEMA3A in elderly AIS patients(r=0.597,P＜0.01;r=0.618,P＜0.01),while the NDRG3 level was positively correlated with that of SEMA3 A(r=0.477,P＜0.01).ROC curve analysis indicated that the AUC value of combined NDRG3 and SEMA3A levels was superior to that of NDRG3 and SEMA3A alone in predicting of poor progno-sis(0.962 vs 0.861,0.880,P＜0.01).Conclusion The levels of NDRG3 and SEMA3A proteins are up-regulated in elderly AIS patients,and are closely associated with the severity and prognosis of the disease.

Expression of Semaphorin 3A after spinal cord injury / 中国骨伤

OBJECTIVE@#To investigate expression of Semaphorin 3A in rats after spinal cord injury and explore possible mechanism of inhibiting of axonal regeneration after SCI.@*METHODS@#Forty healthy female SD rats, 8 weeks old, weighing (210.00±9.88) g, were randomly divided into control group(20 rats in group A) and model group(20 rats in group B). In control group, removal of T@*RESULTS@#After a simple spinal cord transection injury, hemorrhagic necrosis, localized edema, neurodegeneration, necrosis, and cyst formation occurred in the injured area, and glial scar formation occurred in glial cells. Semaphorin 3A expression levels in control group was low in the gray matter area. There was no expression of Semaphorin 3A in the injured area of spinal cord injury in model group 3 days after operation. On the 14th day, the expression of Semaphorin 3A in the injured area of spinal cord injury increased significantly and was at a high level. On the 28th day, the expression of Semaphorin 3A was moderate. On the 42th day, the positive expression of Semaphorin 3A returned to normal level.@*CONCLUSION@#The increased expression of Semaphorin 3A after spinal cord injury may be one of the mechanisms that inhibit axonal regeneration.

Association of Semaphorin 3A with thrombocytopenia in systemic lupus erythematosus / 北京大学学报(医学版)

OBJECTIVE@#To measure the level of serum Semaphorin 3A (Sema3A) and to analyze the relationship between serum Sema3A and systemic lupus erythematosus (SLE) with thrombocytopenia.@*METHODS@#The concentration of serum Sema3A was detected by enzyme-linked immuno sorbent assay (ELISA) in 170 SLE patients, 50 Sjögren's syndrome (SS) patients, 19 hypersplenism (HS) patients and 150 healthy controls (HC). Based on the presence of thrombocytopenia and whether the thrombocytopenia was in remission, the SLE patients were divided into three groups: SLE with thrombocytopenia (41 cases), SLE with thrombocytopenia remission (28 cases), and SLE without thrombocytopenia (101 cases). According to whether there was thrombocytopenia, the SS patients were divided into SS with thrombocytopenia (18 cases) and SS without thrombocytopenia (32 cases). The 28 SLE patients who underwent bone marrow aspiration biopsy were divided into two groups from the aspect of whether the bone marrow hyperplasia was normal (19 cases) or low (9 cases), as well as from the aspect of whether the maturity disturbance of megakaryocyte was positive (8 cases) or negative (20 cases). The serum Sema3A levels in SLE, SS, HS with HC were compared, meanwhile, the correlation between serum Sema3A level and platelet (PLT) in the patients with different diseases analyzed.@*RESULTS@#(1) Serum Sema3A levels in SLE were significantly lower than in HC [(3.84±2.76) μg/L vs. (6.96±2.62) μg/L, P < 0.001], serum Sema3A levels in SS were also obviously lower than in HC [(4.35±3.57) μg/L vs. (6.96±2.62) μg/L, P < 0.001], and in HS it was lower than HC at a certain extant [(5.67±2.26) μg/L vs. (6.96±2.62) μg/L, P=0.041]. (2) Serum Sema3A levels in SLE were slightly lower than in SS, but there was no significant difference [(3.84±2.76) μg/L vs. (4.35±3.57) μg/L, P=0.282]. However, when compared with HS, serum Sema3A levels in SLE were significantly lower [(3.84±2.76) μg/L vs. (5.67±2.26) μg/L, P=0.006]. (3) Serum Sema3A concentration in SLE with thrombocytopenia was significantly lower than in SLE with thrombocytopenia remission [(1.28±1.06) μg/L vs. (3.83±2.65) μg/L, P < 0.001], and in SLE patients without thrombocytopenia [(1.28±1.06) μg/L vs. (4.87±2.60) μg/L, P < 0.001]. There was no significant difference between SLE with thrombocytopenia remission and SLE without thrombocytopenia [(3.83±2.65) μg/L vs. (4.87±2.600 μg/L, P=0.123]. Serum Sema3A concentration in SLE with thrombocytopenia was slightly lower than in SS with thrombocytopenia, but there was no significant difference [(1.28±1.06) μg/L vs. (1.68±1.11) μg/L, P=0.189]. (4) Strong positive correlations were found between serum Sema3A and PLT in SLE (r=0.600, P < 0.001). Positive correlations were also found between serum Sema3A and PLT in SS (r=0.573, P < 0.001). However, there was no such correlation showed in HS patients (P=0.393). (5) There was no significant difference of serum Sema3A concentration in SLE whether the bone marrow hyperplasia was normal or low. And the same situation appeared in the patients whether the maturity disturbance of megakaryocyte was positive or negative (P>0.05).@*CONCLUSION@#Serum Sema3A was significantly reduced in SLE patients, and it was highly correlated with the blood damage. Similar conclusions could be drawn in patients with SS. The serum level of Sema3A was generally decreasing in desmosis which merged thrombocytopenia, and was obviously positive correlated with platelet counts.

Inhibitory effect of recombinant human semaphorin 3A on angiogenesis of gastric cancer and the associated mechanisms / 解剖学报

Objective To investigate the expression of semaphorin 3A (Sema3A) and its receptor neuropilin-1 (NRP-1) in gastric cancer and its correlation with microvessel density (MVD), and then to explore the effect of recombinant human Sema3A on angiogenesis of gastric cancer and the associated mechanisms. Methods Forty cases of gastric cancer tissues and its corresponding adjacent normal tissues were used to detecte the expression of Sema3A, NRP-1 and MVD in tissues by immunohistochemistry method . The expression level of Sema3A in serum of gastric cancer patient group and normal control group were measured by Enzyme-linked immuno-sorbent assay (ELISA). Western blotting was used to detect the expression of Sema3A and NRP-1 in five gastric cancer cell lines (MGC-803,HGC-27,MKN-28,SGC-7901,MKN-45) and human gastric mucosal epithelial cell (GES-1). Transwell chamber was used to construct non-contact in vitro co-culture system, in which the effects of different concentrations of recombinant human Sema3A on angiogenesis in gastric cancer were analyzed by tube formation assay preliminarily. The expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and NRP-1 in co-culture system were detected by Western blotting. Results The expression levels of Sema3A in gastric cancer tissues, cell lines and patient serum were significantly lower than that in the control group(P<0. 05), while the expression of NRP-1 in gastric cancer tissues and MKN-28 cells was significantly increased, and both of them were associated with TNM staging of gastric cancer (P < 0. 05) . In vitro co-culture system, The tube forming abilities of human umbilical vein endothelial cell (HUVEC) were decreased in recombinant human Sema3A treated group, and this phenomenon was concentration dependent. The expression of VEGFR2 protein was down-regulated by recombinant human Sema3A. Conclusion The expression of Sema3A was decreased in gastric cancer tissues, cell lines and patient serum, and negatively correlated with microvessel density. The recombinant human Sema3A could inhibit the angiogenesis of gastric cancer in vitro, which may be related to down-regulation of VEGFR2 protein expression.

Lung cancer cell-derived Sema4D mediates osteolytic bone destruction in lung cancer by inhibiting osteoblast differentiation / 肿瘤

Objective: To determine the role of semaphorin 4D (Sema4D) in osteolytic bone destruction of bone metastasis in lung cancer, and to explore its mechanism. Methods: Immunohistochemistry was used to detect the expression of Sema4D in bone metastases of lung cancer and normal bone tissues. The expression levels of Sema4D protein and mRNA in four kinds of lung cancer cell lines were detected by Western blotting and real-time fluorescent quantitative PCR, respectively. The conditioned medium of lung cancer cells was collected, and ELISA was used to detect the secretion level of Sema4D in lung cancer conditioned medium. The conditioned medium from lung cancer cells with Sema4D high expression (PC9 cells) or low expression (A549 cells) was collected and co-cultured with osteoblastic precursor MC3T3-E1 cells, then the osteoblast differentiation ability was detected by alkaline phosphatase staining, the mineralization ability of osteoblasts was detected by alizarin red staining, the expression levels of osteoblast differentiation genes alkaline phosphatase, liver/bone/kidney (ALPL), Oxterix and collagen type alpha 1 (Col1α1) were detected by real-time fluorescent quantitative PCR. Sema4D shRNA was transfected into lung cancer PC9 cells, then the expression and secretion levels of Sema4D in lung cancer cells were detected by Western blotting, real-time fluorescent quantitative PCR and ELISA, respectively. The conditioned medium of lung cancer PC9 cells transfected with Sema4D shRNA was collected and co-cultured with preosteoblasts, and the effect of the conditioned medium from lung cancer cells with Sema4D interference on the osteoblast differentiation was determined by alkaline phosphatase staining, alizarin red staining and real-time fluorescent quantitative PCR, respectively. Results: Compared with the normal bone tissues, Sema4D was highly expressed in osteolytic bone metastases of lung cancer. Sema4D was expressed and secreted in different lung cancer cell lines in different degrees. The expression and secretion levels of Sema4D were the highest in lung cancer PC9 cells, but lowest in lung cancer A549 cells (P < 0.05). The conditioned medium of lung cancer cells significantly inhibited the osteogenic differentiation of osteoblasts induced by osteogenic inducer, which was characterized by the reduction of relative staining area of alkaline phosphatase staining and alizarin red staining (both P < 0.05), and the decreased expression levels of osteogenic differentiation genes ALPL, Oxterix and Col1α1 (all P < 0.05). The conditioned medium of PC9 cells with Sema4D high expression exhibited more aggressive inhibitory effect on osteoblast differentiation than the conditioned medium of A549 cells with Sema4D low expression (P < 0.05). shRNA transfection significantly reduced the expression and secretion of Sema4D in lung cancer cells (both P < 0.05), and significantly attenuated the inhibitory effect of lung cancer conditioned medium on osteoblast differentiation (all P < 0.05). Conclusion: Lung cancer-derived Sema4D plays an important role in osteolytic bone destruction by inhibiting osteoblast differentiation, suggesting that it is expected to become a new therapeutic target for bone metastasis of lung cancer.

SEMA3B but Not CUL1 as Marker for Pre-Eclampsia Progression

@#Background: An imbalance between pro- and anti-angiogenic factors contributes to impaired trophoblast invasion during pregnancy, leading to failure of uterine spiral artery remodeling, blood vessel ischemia, and pre-eclampsia (PE). Anti-angiogenic semaphorin 3B (SEMA3B) and pro-angiogenic cullin 1 (CUL1) are expressed in both the placenta and maternal blood. The present study investigated correlations between serum and placental SEMA3B as well as CUL1 levels in late-onset PE. Methods: This cross-sectional study included 50 patients with late-onset (≥ 32 weeks gestation) PE. Maternal serum was obtained before delivery, and placentas were obtained immediately after delivery. SEMA3B and CUL1 levels were evaluated by ELISA. Results were statistically analysed by Spearman correlation test, with a P < 0.05 considered statistically significant. Results: While elevated serum SEMA3B levels significantly correlated with increased placental SEMA3B levels in late-onset PE (R = 0.620, P = 0.000), alteration of serum CUL1 levels did not correlate with alteration of placental CUL1. Conclusion: Alteration of circulating maternal SEMA3B, but not CUL1, levels can potentially be used to monitor PE progression during pregnancy.

Semaphorin 3A-stimulated bone marrow mesenchymal stem cells sheets promotes osteogenesis of type 2 diabetic rat / 中华口腔医学杂志

Objective@#To evaluate the effect of semaphorin 3A (Sema3A) pre-treated bone marrow mesenchymal stem cells (BMSC) sheets on new bone formation in type 2 diabetes mellitus rats.@*Methods@#Type 2 diabetes mellitus (T2DM) were induced by injection of streptozotocin, and the BMSC were isolated, controlled, identified and induced into cell sheets. Fifteen T2DM rats were randomly divided into control, sheets and Sema3A-sheets group and the calvarial critical size defect (CSD) model of rats were established. The defect zone of rats from control group were implanted with bone powder. The defect zone of rats from sheets group were implanted with bone powder and BMSC sheets. The defect zone of rats from Sema3A-sheets group were implanted with bone powder and BMSC sheets pretreated with 1.0 mg/L Sema3A. After 8 weeks, the bone samples were harvested and analyzed by micro-CT scanning, HE staining for the evaluation of new bone formation, and the immunohistochemical analysis for the expression of osteogenesis-related proteins including type Ⅰ collagen (COL- Ⅰ ), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN).@*Results@#The BMSC were isolated and cultured, and oil red O and Alizarin red S staining proved the multi-potential differentiation. Eight weeks after the establishment of calvarial CSD model, Sema3A-sheet group showed the most abundant new bone formation (0.516±0.070), with increased bone volume fraction, namely bone volume/tissue volume (BV/TV) compared with sheets group (0.319±0.050) and control group (0.224±0.037) (P<0.05), and the sheets group showed increased BV/TV compared with control group (P<0.05). While trabecular thickness (Tb.Th) control group showed no difference in three groups (P>0.05). HE staining also confirmed that Sema3A-sheets group showed the most new bone formation. Sheet group (0.174±0.051) compared showed difference with control group (0.099±0.033) (P< 0.05), and Sema3A-sheet group (0.421±0.069) showed increased bone formation compared with sheet group and control group (P<0.05). Immunohistochemistry showed that BMSC sheet increased the expression of osteogenesis-related proteins including COL-Ⅰ, BMP-2 and OCN, while Sema3A pretreatment showed more obvious increase of the expression of COL-Ⅰ and OCN.@*Conclusions@#The combined implantation of bone powder and Sema3A stimulated BMSC sheets significantly increased bone regeneration in vivo. Therefore, Sema3A pre-treated BMSC sheets transplantation provides a new strategy for restoring bone defect in T2DM.

Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Glioblastoma (GBM) is classified as one of the most aggressive and lethal brain tumor. Great strides have been made in understanding the genomic and molecular underpinnings of GBM, which translated into development of new therapeutic approaches to combat such deadly disease. However, there are only few therapeutic agents that can effectively inhibit GBM invasion in a clinical framework. In an effort to address such challenges, we have generated anti-SEMA3A monoclonal antibody as a potential therapeutic antibody against GBM progression. MATERIALS AND METHODS: We employed public glioma datasets, Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas, to analyze SEMA3A mRNA expression in human GBM specimens. We also evaluated for protein expression level of SEMA3A via tissue microarray (TMA) analysis. Cell migration and proliferation kinetics were assessed in various GBM patient-derived cells (PDCs) and U87-MG cell-line for SEMA3A antibody efficacy. GBM patient-derived xenograft (PDX) models were generated to evaluate tumor inhibitory effect of anti-SEMA3A antibody in vivo. RESULTS: By combining bioinformatics and TMA analysis, we discovered that SEMA3A is highly expressed in human GBM specimens compared to non-neoplastic tissues. We developed three different anti-SEMA3A antibodies, in fully human IgG form, through screening phage-displayed synthetic antibody library using a classical panning method. Neutralization of SEMA3A significantly reduced migration and proliferation capabilities of PDCs and U87-MG cell line in vitro. In PDX models, treatment with anti-SEMA3A antibody exhibited notable tumor inhibitory effect through down-regulation of cellular proliferative kinetics and tumor-associated macrophages recruitment. CONCLUSION: In present study, we demonstrated tumor inhibitory effect of SEMA3A antibody in GBM progression and present its potential relevance as a therapeutic agent in a clinical framework.

The expression of Semaphorin 5A in patients with rheumatoid arthritis and its effect on osteoclastogenesis / 中华内科杂志

Objective To investigate the clinical significance of soluble Semaphorin 5A(Sema 5A) in patients of rheumatoid arthritis(RA) and the effect of Sema 5A on osteoclastogenesis in RAW264.7 cells.Methods (1)Soluble Sema 5A was detected in the serum of 62 RA patients,30 osteoarthritis(OA) patients and 48 healthy controls(HC) by enzyme-linked immunosorbent assay(ELISA).The relationships between serum Sema 5A and disease activity,radiographic severity and laboratory parameters were investigated in RA patients.(2)RAW264.7 cells were treated with different concentrations of Sema 5A(0,0.5,1,2.5,5 μg/ml).After 7 days,tartrate-resistant acid phosphate(TRAP) staining was performed.The mRNA levels of TRAP,cathepsin-K and matrix metallopeptidase 9(MMP-9) were tested using real-time polymerase chain reaction (RT-PCR).(3)Bone resorption area of dentine slides cultured with RAW264.7 cells was calculated after Sema 5A (5μg/ml) treatment.Results (1)The serum Sema 5A in RA patients[(5.24±0.59)μg/L] was significantly higher than those in healthy controls[(2.93±0.34)μg/L,P＜0.01] and OA patients[(2.68±0.47)μg/L,P＜0.05].The Sema 5A level in RA patients was positively correlated with disease activity score with 28 joint using C-reactive protein(DAS28-CRP),clinical disease activity index (CDAI),C-reactive protein(CRP) and sharp scores(P＜0.05 or P＜0.01).In addition,the serum Sema 5A in RA patients with positive anti-cyclic citrullinated peptide(CCP) antibody was significantly greater than that of anti-CCP antibody-negative patients (P＜0.05).(2)After RAW264.7 cells were treated with Sema 5A,the number of TRAP positive osteoclasts increased according to the increase of Sema 5A concentration with maximal effect at 5 μg/ml.Meanwhile,Sema 5A promoted mRNA expression of TRAP,Cathepsin-K and MMP-9.(3)Bone resorption area increased when RAW264.7 cells were treated with Sema 5A(5 μg/ml).Conclusions Serum Sema 5A is elevated in RA patients and correlated with disease activity and radiographic severity.Sema 5A promotes osteoclastogenesis of RAW264.7 cells.

Importance of the putative furin recognition site 742RNRR745 for antiangiogenic Sema3C activity in vitro

Angiogenesis is one of the key processes in the growth and development of tumors. Class-3 semaphorins (Sema3) are characterized as axon guidance factors involved in tumor angiogenesis by interacting with the vascular endothelial growth factor signaling pathway. Sema3 proteins convey their regulatory signals by binding to neuropilins and plexins receptors, which are located on the effector cell. These processes are regulated by furin endoproteinases that cleave RXRR motifs within the Sema, plexin-semaphorins-integrin, and C-terminal basic domains of Sema3 protein. Several studies have shown that the furin-mediated processing of the basic domain of Sema3F and Sema3A is critical for association with receptors. It is unclear, however, if this mechanism can also be applied to other Sema3 proteins, including the main subject of this study, Sema3C. To address this question, we generated a variant of the full-length human Sema3C carrying point mutation R745A at the basic domain at the hypothetical furin recognition site 742RNRR745, which would disable the processing of Sema3C at this specific location. The effects produced by this mutation were tested in an in vitro angiogenesis assay together with the wild-type Sema3C, Sema3A, and Sema3F proteins. Our results showed that the inhibitory effect of Sema3C on microcapillary formation by human umbilical vein endothelial cells could be abrogated upon mutation at the Sema3C basic domain within putative furin cleavage site 742RNRR745, indicating that this site was essential for the Sema3 biological activity.

Urinary semaphorin 3A as an early biomarker to predict contrast-induced acute kidney injury in patients undergoing percutaneous coronary intervention

Contrast-induced acute kidney injury (CI-AKI) is a serious complication of diagnostic coronary angiograph and percutaneous coronary intervention (PCI). However, the exact pathophysiological mechanisms underlying CI-AKI development are largely unknown. The present study examined whether urinary semaphorin 3A levels predict the development of CI-AKI in patients undergoing PCI. This study enrolled 168 patients with stable angina undergoing elective PCI. Serial urine samples, obtained at baseline and 2, 6, 12, 24, 36, and 48 h post-PCI were analyzed by semaphorin 3A and neutrophil gelatinase-associated lipocalin (NGAL) ELISA kit. AKI was defined as an increase in serum creatinine beyond 50% according to the RIFLE classification system. Receiver operator characteristic (ROC) curve analyses identified optimal semaphorin 3A and NGAL values for diagnosing CI-AKI. CI-AKI occurred in 20 of 168 patients. There were no significant differences in the baseline clinical characteristics and angiographic findings between non-AKI patients group and AKI patients group. Both urinary semaphorin 3A and NGAL levels significantly increased at 2 and 6 h post-PCI. ROC analysis showed that the cut-off value of 389.5 pg/mg semaphorin 3A at 2 h post-PCI corresponds to 94% sensitivity and 75% specificity and the cut-off value of 94.4 ng/mg NGAL at 2 h post-PCI corresponds to 74% sensitivity and 82% specificity. Logistic regression showed that semaphorin 3A levels at 2 and 6 h post-PCI were the significant predictors of AKI in our cohort. Urinary semaphorin 3A may be a promising early biomarker for predicting CI-AKI in patients undergoing PCI.

Influences of semaphorin 3A over-expression on H2O2-induced injury in human umbilical vein endothelial cells / 中国病理生理杂志

AIM:To explore the influences of semaphorin 3A (Sema 3A) on hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs).METHODS:Sema 3A over-expression vectors were constructed and transfected into the HUVECs by Lipofectamine 2000, and the over-expression effect was verified by qPCR and Western blot.The HUVECs in different groups were treated with or without 200 μmol/L H2O2 for 4 h.The levels of inflammatory cytokines were measured by qPCR.The levels of lactic dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by corresponding colorimetry.The cell viability was measured by MTT assay.The cell apoptosis was analyzed by flow cytometry.The levels of apoptosis-related proteins cleaved caspase-3 and Bcl-2 were determined by Western blot.RESULTS:H2O2 induced inflammatory cytokine secretion, increased the levels of LDH and MDA, decreased SOD activity and cell viability, and increased cell apoptosis in the HUVECs.Over-expression of Sema 3A enhanced the above processes.No injury effect of Sema 3A over-expression on HUVECs without H2O2 treatment was observed, indicating that the injury effects of Sema 3A on HUVECs depended on H2O2.CONCLUSION:Sema 3A markedly enhances H2O2-induced injury in the HUVECs, which depends on H2O2.Sema 3A may promote oxidative stress-caused endothelial cell injury.

Polymorphism in the Promoter Region of SEMA5A Is Associated with Sociality Traits in Korean Subjects with Autism Spectrum Disorders

In this study, we evaluated the association between autism spectrum disorders (ASDs) and 10 single-nucleotide polymorphisms (SNPs) in the 5' region of the semaphorin 5A gene (SEMA5A) for 250 Korean trios including children with ASDs. Family-based association testing and haplotype analysis revealed a statistically significant association between rs194085 and multiple sociality traits with Korean ASDs in the dominant model (p < 0.001, corrected p=0.035). This indicates that genetic variations in the 5' region of SEMA5A play a role in the genetic predisposition to sociality traits in Korean ASDs.

Effect of semaphorin 4D on the proliferation, migration and angiogenic of human pancreatic carcinoma cells / 新乡医学院学报

Objective To investigate the effects of semaphorin 4D(Sema4D) on the proliferation,migration and angiogenic of human pancreatic carcinoma cells.Methods Sema4D-siRNA was designed and synthesized and transfected into human pancreatic carcinoma cells.After 48 hours of transient infection,the changes of expression of Sema4D mRNA before and after transfection were detected by reverse transcription-polymeruse chain reaction method.And after 72 hours of transient infection,the changes of expression of Sema4D protein before and after transfection were detected by Western blot method.The changes of growth of the transfected cells were observed by methyl thiazolyl terazolium assay.Using transwell migration test and scratch repair test to detect the changes of migration ability of human pancreatic carcinoma cells after transfection.Using tubule formation assay to observe the effect of supernatant of pancreatic carcinoma cell cultures on angiogenesis after transfection.Results Compared with the negative control group and blank control group,the expression of Sema4D mRNA and Sema4D protein and the growth rate of pancreatic carcinoma cells decreased significantly (P ＜ 0.05).In transwell migration test and scratch repair test,it was observed that Pancreatic cancer cells penetrating cell number and scratch repair rate were significantly lower than that in negative control group and blank control group (P ＜ 0.05).Tubule formation assay showed that there were significant differences in angiogenesis numbers among siRNA transfection group(0.5 ± 0.02),negative control group(1.45 ± 0.60) and blank control group (1.37 ± 0.52) (P ＜ 0.05).Conclusion Sema4D-siRNA can induce RNA interference in pancreatic carcinoma cells and down-regulate the expression of Sema4D gene,which can inhibit the proliferation of pancreatic carcinoma cells,significantly reduce the migration ability of pancreatic carcinoma ceils and inhibit angiogenesis.

Regulation of semaphorin 4D expression and cell proliferation of ovarian cancer by ERalpha and ERbeta

Ovarian cancer is one of the most common malignancies in women. Semaphorin 4D (sema 4D) is involved in the progress of multiple cancers. In the presence of estrogen-like ligands, estrogen receptors (ERα and ERβ) participate in the progress of breast and ovarian cancers by transcriptional regulation. The aim of the study was to investigate the role of sema 4D and elucidate the regulatory pattern of ERα and ERβ on sema 4D expression in ovarian cancers. Sema 4D levels were up-regulated in ovarian cancer SKOV-3 cells. Patients with malignant ovarian cancers had significantly higher sema 4D levels than controls, suggesting an oncogene role of sema 4D in ovarian cancer. ERα expressions were up-regulated in SKOV-3 cells compared with normal ovarian IOSE80 epithelial cells. Conversely, down-regulation of ERβ was observed in SKOV-3 cells. Forced over-expression of ERα and ERβ in SKOV-3 cells was manipulated to establish ERα+ and ERβ+ SKOV-3 cell lines. Incubation of ERα+ SKOV-3 cells with ERs agonist 17β-estradiol (E2) significantly enhanced sema 4D expression and rate of cell proliferation. Incubated with E2, ERβ+ SKOV-3 cells showed lower sema 4D expression and cell proliferation. Blocking ERα and ERβ activities with ICI182-780 inhibitor, sema 4D expressions and cell proliferation of ERα+ and ERβ+ SKOV-3 cells were recovered to control levels. Taken together, the data showed that sema 4D expression was positively correlated with the progress of ovarian cancer. ERα positively regulated sema 4D expression and accelerated cell proliferation. ERβ negatively regulated sema 4D expression and inhibited cell multiplication.

Effects of short hairpin RNA-mediated semaphorin 5A gene silencing on proliferation, metastasis and invasion ;of malignant melanoma cell line A375 / 中华皮肤科杂志

Objective To study the effects of semaphorin 5A (SEMA5A) gene silencing by lentivirus?mediated short hairpin RNA(shRNA)on biological activity of malignant melanoma cell line A375. Methods Two pairs of interference sequences for SEMA5A gene(shRNA1 and A375?shRNA2)and a pair of control interference sequences were designed to build lentiviral vectors, which were then transfected into HEK293T cells to gain lentivirus. A375 cells were divided into three groups:experimental group(A375?shRNA1 and A375?shRNA2 cells)transfected with the lentivirus containing shRNA1 or shRNA2, negative control group (A375?con cells) transfected with that containing the control shRNA, and blank control group(A375 cells)receiving no transfection. The A375 cells with stable knockdown of SEMA5A gene expression were screened by puromycin. Subsequently, reverse transcription?PCR and Western?blot analysis were performed to detect mRNA and protein expressions of Semaphorin 5A in these cells, and methyl thiazolyl tetrazolium(MTT)assay was applied to evaluate the growth of cells. The scratch assay and invasion assay were conducted to estimate migration and invasion ability of cells. Results The lentivirus containing the SEMA5A?targeting shRNAs or control shRNA was successfully transfected into A375 cells, and stably transfected cells were gained after puromycin selection. The expressions of semaphorin 5A mRNA and protein in the A375?shRNA2 cells were significantly reduced compared with those in the A375?con and A375 cells(all P 0.05). The scratch assay showed that there was no obvious cell migration into the scratch in the experiment group, whereas the scratch was almost covered by cells in the negative control group and blank control group. The invasion assay showed that the number of A375?shRNA2 cells passing through the Transwell chamber was significantly smaller than that of A375 and A375?con cells(both P 0.05). Conclusion The silencing of SEMA5A gene by lentivirus?mediated shRNA could effectively down?regulate the expression of semaphorin 5A, and inhibit the growth, invasion and migration of A375 cells.

Hypoxia inhibits the expression of semaphorin 3A in breast cancer cells and regulates the differentiation of preosteoblast cells / 肿瘤

Objective: To compare the effect of MCF-7 conditioned medium under normoxia or hypoxia condition on the differentiation of osteoblastic precursor MC3T3-E1 cells after exposing breast cancer MCF-7 cells to hypoxia, and to investigate the role of semaphorin 3A (Sema3A) on osteogenic differentiation regulated by hypoxia in breast cancer with bone metastasis. Methods: The conditioned medium was collected from breast cancer MCF-7 cells cultured under the normoxia or hypoxia condition for 48 h, and used to incubate osteoblastic precursor MC3T3-E1 cells. The content and activity of alkaline phosphatase (ALP) in MC3T3-E1 cells were evaluated by BCIP/NBT ALP color development kit and ALP activity assay kit, respectively. The mineralization of MC3T3-E1 cells was detected by alizarin red S (ARS) staining. The expressions of Sema3A mRNA and protein in MCF-7 cells under normoxia or hypoxia condition for 0, 12, 24 and 48 h were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The recombinant human Sema3A (rhSema3A) was added into MC3T3-E1 cells incubated with MCF-7 hypoxia conditioned medium, then the mRNA expression levels of osteogenic differentiation-related proteins ALP, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and Osterix (Osx) in MC3T3-E1 cells were measured by real-time fluorescent quantitative PCR. Results: Compared with normoxia cultured MCF-7 conditioned medium (NorCM) group, the activity of ALP in MC3T3-E1 cells was significantly decreased in hypoxia cultured MCF-7 conditioned medium (HyCM) group (P<0.05), and the area of mineralization nodes stained with ARS was also markedly reduced in HyCM group (P<0.05). The mRNA and protein expressions of Sema3A in MCF-7 cells after hypoxia culture for 24 and 48 h were significantly lower than those after normoxia culture for the same time (all P<0.05). After rhSema3A was used to treat MC3T3-E1 cells which were incubated with HyCM, the mRNA expression levels of ALP, RUNX2, OCN and Osx were significantly increased as compared with the rhSema3A untreated group (all P<0.05). Conclusion: Hypoxia down-regulates the expression of Sema3A in MCF-7 cells in vitro, and inhibits the differentiation of osteoblast, which indicates that Sema3A is probably an important factor for the inhibition of osteoblastic differentiation by breast cancer conditioned medium under hypoxic environment.