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STUDY DESIGN: Experimental human study. PURPOSE: To determine whether angiopoietin-like protein 2 (ANGPTL2) is highly expressed in the hyperplastic facet joint (FJ) synovium and whether it activates interleukin-6 (IL-6) secretion in FJ synoviocytes. OVERVIEW OF LITERATURE: Mechanical stress-induced synovitis is partially, but significantly, responsible for degenerative and subsequently osteoarthritic changes in the FJ tissues in patients with lumbar spinal stenosis (LSS). However, the underlying molecular mechanism remains unclear. IL-6 is highly expressed in degenerative FJ synovial tissue and is responsible for local chronic inflammation. ANGPTL2, an inflammatory and mechanically induced mediator, promotes the expression of IL-6 in many cells. METHODS: FJ tissues were harvested from five patients who had undergone lumbar surgery. Immunohistochemistry for ANGPTL2, IL-6, and cell markers was performed in the FJ tissue samples. After cultured synoviocytes from the FJ tissues were subjected to mechanical stress, ANGPTL2 expression and secretion were measured quantitatively using real-time quantitative reverse-transcription–polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Following ANGPTL2 administration in the FJ synoviocytes, anti-nuclear factor-κB (NF-κB) activation was investigated using immunocytochemistry, and IL-6 expression and secretion were assayed quantitatively with or without NF-κB inhibitor. Moreover, we assessed whether ANGPTL2-induced IL-6 modulates leucocyte recruitment in the degenerative process by focusing on the monocyte chemoattractant protein-1 (MCP-1) expression. RESULTS: ANGPTL2 and IL-6 were highly expressed in the hyperplastic FJ synovium samples. ANGPTL2 was co-expressed in both, fibroblast-like and macrophage-like synoviocytes. Further, the expression and secretion of ANGPTL2 in the FJ synoviocytes increased in response to stimulation by mechanical stretching. ANGPTL2 protein promoted the nuclear translocation of NF-κB and induced IL-6 expression and secretion in the FJ synoviocytes. This effect was reversed following treatment with NF-κB inhibitor. Furthermore, ANGPTL2-induced IL-6 upregulated the MCP-1 expression in the FJ synoviocytes. CONCLUSIONS: Mechanical stress-induced ANGPTL2 promotes chronic inflammation in the FJ synovium by activating IL-6 secretion, leading to FJ degeneration and subsequent LSS.
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Humans , Chemokine CCL2 , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Inflammation , Interleukin-6 , Spinal Stenosis , Stress, Mechanical , Synovial Membrane , Synovitis , Zygapophyseal JointABSTRACT
Objective To investigate the influence factors of serum Angptl 2 levels is to explore the relationship between serum Angptl 2 and carotid atherosclerosis (CAS) in patients with type 2 diabetes mellitus (T2DM). Methods 114 cases of T2DM in clinic were selected.According to the results of color doppler ultrasonography,the patients were divided into T2DM with CAS group(T2DM + CAS,n=58)and simple T2DM group(n= 56).56 healthy subjects were selected as normal control (NC)group during the same period.ELISA method was used to detect the level of serum Angptl 2. Results Serum Angptl 2 levels were significantly higher in T2DM + CAS group and T2DM group than in NC group [(69.83 ± 12.07) vs (42.93 ± 10.44)vs (24.26 ± 8.78)ng/ml,P< 0.01].Correlation analysis showed that serum Angptl 2 was positively correlated with age,BMI,SBP,DBP,TC,TG,LDL-C,FPG and HbA1c in T2DM patients (r=0.574,0.325,0.528,0.308,0.396,0.387,0.295,0.536 and 0.601,respectively,P<0.01),and negatively correlated with HDL-C and FIns in T2DM patients (r= -0.248,-0.352,P<0.01).Similarly,multiple regression analysis showed that FIns,and HbA1c were the independent factors of serum Angptl 2 (β= -2.186,2.680,P<0.01). Conclusion Serum Angptl 2 level is closely related to obesity,blood pressure,blood glucose,blood lipid and insulin.Serum Angptl 2 may play an important role in the development of T2DM and carotid atherosclerosis.
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Objective To detect the serum levels of angiopoietin-like protein 2 (ANGPTL 2) and vascular endothelial growth factor (VEGF) in patients with rheumatoid arthritis (RA),and to analyze their value in the differential diagnosis of RA in order to clarify whether they are synergetic in the pathogenesis of RA.Methods Seventy-seven patients with RA and 25 subjects for heath check-up in the same period were selected.According to the relevant clinical information,the RA patients were divided into three subgroups:high disease activity group [(DAS28)＞5.1],moderate disease activity group (3.2＜DAS28 ≤5.1),low disease activity group (DAS28 ≤3.2).Enzyme-linked immunosorbent assay (ELISA) was used to masure the levls of ANGPTL2 and VEGF of those groups.The data were analyzed by t test,Mann-Whitney U test,Kruskal-Wallis H test and Speraman correlation analysis.Results ① The serum levels of ANGPTL2 and VEGF in RA patients were significantly higher than those in control group [(5.3±1.1) ng/ml vs (3.3±0.9) ng/ml,Z=-6.644;(106±30) pg/ml vs (93±12) pg/ml,Z=-4.4115,P＜0.05].② Serum level of ANGPTL2 in RA patients with high disease activity was higher than low disease group and healthy control group {[5.7(1.3)] ng/ml vs [4.5(0.3)]ng/ml,H=4.257,P＜0.05;[5.7 (1.3)] ng/ml vs [3.3 (0.9)] ng/ml,H=7.639,P＜0.05}.Serum level of VEGF in RA patients with high disease activity was higher than low disease group and healthy control group {[116(37)]pg/ml vs [96(8)] pg/ml,H=3.579,P＜0.05;[116(37)] pg/ml vs [92.90(12.24)] pg/ml,H=5.698,P＜0.05].③ There was a positive correlation between the serum level of ANGPTL2 and DAS28 score,erythrocyte sedimentation rate,swollen joint count and tender joint count (rDAS28=0.703,rESR=0.311,rSJC=0.503,rTJC=0.670,P＜0.05),so was VEGF (rDAS28=0.553,rESR=0.260,rSJC=0.399,rTJC=0.538,P＜0.05).④ In RA patients,there was a positive correlation between ANGPTL2 and VEGF (r=0.853,P＜0.05).Conclusion ANGPTL2 might coordinate with VEGF and be involved in the immune process of RA,and it might be regarded as one of the reference indexes of RA activity,so that it may contribute to access and monitor the condition of RA.
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Objective To explore Angptl2(angiopoietin-like protein 2)expression in tumor tis-sue and serum in patients with breast cancer(BC). Methods A total of 78 BC patients were enrolled in this study. Immunohistochemistry was used to detect Angptl2 expression in tumor and adjacent normal tis-sues. Preoperative and postoperative serum Angptl2 levels were determined via ELISA. Correlation among Angptl2 expression,clinicopathological factors and progression-free survival(PFS)were further evalua-ted. The receiver operating characteristics(ROC)curve was constructed to describe diagnostic specificity and sensitivity. Results Angptl2 was positively expressed in 64(82. 1% )cases. The rates of high and moderate expression of Angpt l2 in tumor and adjacent tissues were 53. 8%(42 / 78)and 7. 7%(6 / 78), respectively(P ﹤ 0. 001). Moreover,the elevated Angptl2 protein was significantly associated with lymph metastasis and adverse PFS. In addition,serum Angptl2 level in BC patients was significantly higher than that in benign controls[(218. 72 ± 88. 02)vs(80. 88 ± 30. 59)ng/ ml,P ﹤ 0. 01]. The area under the curve(AUC)was 0. 909,which indicated a good function for diagnosis. Postoperative serum Angptl2 level was decreased to(142. 43 ± 60. 29)ng/ ml,but still higher than that in controls(P ﹤ 0. 01). Conclusion The expression of Angptl2 may increase in tumor tissue and serum of BC and it may be a potential tumor marker for diagnosis and prognosis.
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Objective To explore the expressions and clinical value of angiopoietin like protein 1 (Ang-1)and angiopoietin like protein 2 (Ang-2)in patients with gastric cancer.Methods 65 patients with gastric cancer and 50 healthy subjects for con-trol group were collected from June in 2012 to December in 2014.The level of Ang-1 and Ang-2 were determined by enzyme-linked immunosorbent assay (ELISA).The results were compared.Results Compared with control group (19.8±2.1 μg/L),the level of Ang-1 in patients with gastric cancer (19.8±2.1 μg/L)was no statistics difference (P >0.05),but Ang-2 (2.3±0.8 μg/L)was significantly higher than those of healthy subjects (0.8±0.2 μg/L,t=2.50,P 0.05).The level of Ang-2 in TNM stage Ⅲ~Ⅳ (2.6±0.5 μg/L)was obviously higher than that in stage.Ⅰ~Ⅱ (1.6±0.4 μg/L).Ang-2 levels were signif-icantly higher in patients with lymph node metastasis (2.7±0.5 μg/L)or postoperative recurrence after one year (2.0±0.6μg/L)than those without lymph node metastasis (1.6 ± 0.5 μg/L)or postoperative recurrence (1.2 ± 0.5 μg/L,P <0.05).The level of Ang-2 in gastric cancer was significantly positive correlated with tumor stage and lymph node metastasis (r=0.31 and 0.33 respectively,P <0.01).Conclusion Ang-2 level was significantly increased in gastric cancer.Its level was significantly correlated with tumor stage,lymph node metastasis and prognosis,which has important application value for the cancer progression,clinical observation and prognosis evaluation of gastric cancer.
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[Summary] This paper was to investigate whether serum angiopoietin-like protein 2 (Angptl 2) is associated with macrovascular complications in type 2 diabetes mellitus.The results showed that there were statistically significant differences in serum Angptl 2 levels among control group and groups of type 2 diabetic patients with or without carotid atherosclerosis [0.98 (0.82-1.22),3.70 (2.69-4.85),1.17 (0.76-2.47) ng/ml].Logistic regression showed that Angptl 2 was an independent risk factor of macrovascular complications in the patients with type 2 diabetes.
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Objective To study the assosiation of angiopoietin-like protein 2 (ANGPTL2) to lower extremity arterial disease in type 2 diabetes mellitus.Methods A total of 360 type 2 diabetic patients were divided into three groups:without (group A),with mild to moderate (group B),and severe (group C) lower extremity arterial disease according to the ankle brachial index.And,120 healthy subjects serveed as control group.The levels of ANGPTL2,high sensitivity C-reactive protein (hsCRP),free fatty acid,biochemical index,and fasting insulin were detected.And,waist-to-hip ratio (WHR),body mass index (BMI),insulin resistance index were calculated.Results The level of ANGPTL2 was getting higher and higher as lower extremity arterial disease progressing.The level of ANGPTL2 in group B was higher than that in group A [1.27 (1.09,1.51) vs 0.88 (0.66,1.07) μg/L,P＜0.05],and the level of ANGPTL2 in group C was higher than that in group B [1.70 (1.45,1.91) vs 1.27 (1.09,1.51)μg/L,P＜0.05].Pearson correlation analysis showed that the ANGPTL2 level in serum positively correlated with hsCRP,BMI,WHR,cholesterol,low-density lipoprotein-cholesterol,fasting insulin,and insulin resistance index.Logistic regression analysis showed that the levels of ANGPTL2,hsCRP,and glycosylated hemoglobin were significantly associated with lower extremity arterial disease in type 2 diabetes mellitus.Conclusion It is possible that ANGPTL2 is one of the risk factors of lower extremity arterial disease in type 2 diabetes mellitus,and which is likely to be of great significance in the early prediction,disease assessment,and prognosis evaluation for lower extremity arterial disease in type 2 diabetes mellitus.
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Objective To observe the effect of irbesartan on the expression of angiopoietinlike protein 2 (ANGPTL2) in the diabetic rats kidney and explore the underlying mechanism.Methods A total of sixty male SD rats were divided into normal control group (NC group,n=15) and experimental group (n=45) randomly.The experimental group was fed with high sugar-fat diet and given a low dose streptozocin (STZ 30 mg/kg)to establish type 2 diabetic model.Rats successfully induced diabetes were randomly divided into 2 groups:diabetes group (DM) and irbesartan group (DI).Weight,blood pressure,blood glucose,serum creatinine (Scr),blood urea nitrogen(BUN),24 hour urinary albumin(UAL) and renal histomorphology were observed after drug intervention at the 4th,8th and 12thweeks.The expression of ANGPTL2 in renal tissue were detected by immunohistochemistry,real-time PCR and Western blotting.Results The levels of Scr,BUN,TG,TC and UAL in group DM were higher than in group NC at the 4th,8th and 12th week (all P ＜ 0.05).Compared with that in group DM,above indexes were lower in group DI at the 4th,8th and 12th week (all P ＜ 0.05).The pathological changes of the kidney in group DM were more serious than that in group DI.The expression of ANGPTL2 in group DM was much higher than that in group NC at the 4th,8th and 12th week (all P ＜0.05),and irbesartan treatment inhibited the up-regulation of ANGPTL2 in group DI(all P ＜ 0.05).Conclusion The expression of ANGPTL2 increases in T2DM rats kidney tissue with time and irbesartan can inhibit the up-regulation of ANGPTL2 in T2DM rats.
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Background The special pathological change of diabetic retinopathy(DR)is microvascular disorder.Angiopoietin-like protein 2(Ang-2)is a new protein associated with genesis of blood vessels.Quercetin has multiple pharmacological action,including improving the microcircularion and the permeability of blood capillary.However,the action mechanism of Ang-2 on DR was unclear.Objective The present study was to investigate the effects of quercetin on Ang-2 and its receptor Tie2 expression in retina with diabetes mellitus.Methods Sixty clean male Wistar rats were randomized into 7 groups and 10 rats for each group,and 10 rats served as blank control group.Streptozotocin of 35 mg/kg was intraperitoneally injected in 60 rats to establish the diabetic models.Quercetins encapsulated by liposome with the doses of 50,150 and 250 mg/(kg · d)(3-5 ml)were used to gavage in different groups of models for 12 weeks,and normal saline solution and calcium dobesilate were used at the same fashion as the negative control group and positive control group,respectively.Twelve weeks later,the animals were sacrificed and retinas were isolated.Expressions of Ang-2 protein and Tie2 mRNA in retinas were detected by ELISA and RT-PCR,respectively.The usage and rearing of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Sciences and Technology Commission.Results ELISA showed that the A450 of Ang-2 in 150 and 250 mg/(kg · d)quercetin groups was 0.796±0.057 and 0.842±0.043 respectively and was lower than that negative group(1.012±0.046),showing statistically significant differences(q =2.95,2.698,P＜0.05).RT-PCR assay showed that expression of Tie2 mRNA(Tie2 mRNA/GAPDH mRNA)in retinas was 0.712±0.092 and 0.821±0.087,presenting statistically significant differences in comparison with negative group(1.182±0.098)(q =3.497,2.852,P＜0.05).The expression levels of Ang-2 and Tie2 mRNA in retina were lowest in 150 mg/(kg · d)quercetin group.Conclusions Quercetin can improve the retinal microcirculation by downregulating the expressions of Ang-2 and its receptor in early period of diabetic rats.
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Kidney samples were obtained from db/db mice and their db/m littermates at the age of 4, 8, 12, 16, 20 and 24 weeks. The expression of ANGPTL2 was assessed by RT-PCR and immunohistochemistry, and then correlated with biochemical and histological indices of kidney injury. No significant difference of ANGPTL2 expression was found in the kidney of db/db mice and the control db/m mice at the age of 4 weeks, a time when all biochemical and histological indices indicated that the db/db mice were in pre-diabetic condition. However, with the development of obesity, hyperglycemia and proteinuria, the expression of ANGPTL2 in the kidney of db/db mice increased, which indicated that the increased expression of ANGPTL2 associated with diabetic nephropathy. ANGPTL2 protein was found distributed mainly in the glomerulus. It is along the capillary loop, located just outside the basement membrane, and in the same location as podocytes, which suggested that podocytes are the main origin of ANGPTL2 in the kidney. Besides, increased expression of ANGPTL2 was found in older db/m mice though it has not reached to a significant level, which may suggest that ANGPTL2 might have some thing to do with the kidney injury caused by ageing.
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Objective: ANGPTL2 is a newly found angiogenesis-associated protein.In the previous studies,we found that the renal expression of ANGPTL2 is involved in the development of diabetic nephropathy,but failed to reveal the exact distribution and synthesis of renal ANGPTL2.This study aimed to analyze the expression of the ANGPTL2 gene in the glomerulus and tubules in the kidney of db/db mice with diabetic nephropathy and to determine the cells responsible for the synthesis of renal ANGPTL2.Methods: Glomerulus and tubules were microdissected from the renal tissue of diabetic db/db mice and the expression of ANGPTL2 mRNA was analyzed by RT-PCR.The distribution and synthesis of ANGPTL2 in the kidney of the db/db mice were determined by immunohistochemistry,dual-labeling immunofluorescence and immunoelectron microscopy.Results: Significantly increased expression of ANGPTL2 mRNA was found in the glomeruli but not in the tubules of the diabetic db/db mice,as compared with the controls.Immunohistochemistry revealed that the ANGPTL2 protein was distributed in a podocyte-like pattern;dual-labeling immunofluorescence analysis showed colocalization of ANGPTL2 with WT1(Wilms' tumor 1,a marker of podocyte) staining,suggesting that renal ANGPTL2 was expressed specifically by podocytes,which was confirmed by immunoelectron microscopy.Conclusion: ANGPTL2 is specifically expressed by podocytes in diabetic nephropathy mice.
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Objective: To construct an angiopoietin-like protein 2(ANGPTL2) expression vector and obtain ANGPTL2 over-expression endothelial cell strains.Methods: Plasmid phrGFP-C was used to amplify hrGFP protein coding sequence by polymerase chain reaction.The amplified sequence was cloned into the A multiple cloning sites of pIRES to construct plasmid pIRES-hrGFP.Complementary oligonucleotides containing the recognition sequence of BamH I,Sal I,Xba I and SSe8387 I were synthesized,annealed and cloned into a BamH I site on the backbone of plasmid pIRES-hrGFP to obtain vector pIRES-hrGFP-MS.ANGPTL2 cDNA was cloned by RT-PCR while human renal RNA was used as the templet and then inserted into the B multiple cloning sites of the vector pIRES-hrGFP-MS.The newly constructed ANGPTL2 expression vector pIRES-hrGFP-MS-ANGPTL2 was linearized by Xba I and introduced into human umbilical vein endothelial cells.ANGPTL2 over-expressed endothelial clones were screened out by G418 selection and identified by the expression levels of both hrGFP and ANGPTL2 genes in these cell clones.Results: The ANGPTL2 expression vector pIRES-hrGFP-MS-ANGPTL2 was constructed successfully and two ANGPTL2 over-expression endothelial strains were obtained.The cells displayed a significantly extended appearance quite different from that of the control cells.Conclusion: The successful construction of the ANGPTL2 expression vector pIRES-hrGFP-MS-ANGPTL2 and the obtainment of two ANGPTL2 over-expression HUVEC strains have paved the way for further investigation into the function of ANGPTL2 and its possible role in diabetic nephropathy.
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Objective:To prepare human angiopoietin like protein 2(ANGPTL2) monoclonal antibody.Methods:The purified recombinant human ANGPTL2 was used to immunize BALB/c mice.Then,the mouse spleen cells were isolated and fused with mouse myeloma cells.After selecting with HAT medium and analyzing with ELISA assay,the hybridoma cell clones stably secreting human ANGPTL2 antibody were screened out.The monoclonal antibody against humain ANGPTL2 was purified by ammonium sulfate precipitation method from the supernatant liquid of hybridoma cell culture.Western blotting,Cell immunostaining,and immunohistochemisty staining were used to characterize the antibody.Results:A strain of hybridoma cell clones stably secreting human ANGPTL2 antibody was screened out.The ANGPTL2 monoclonal antibody prepared was proven useful. Conclusion:A monoclonal antibody against human ANGPTL2 was successfully prepared,which provide a basis for basic study of ANGPLTL2.