含表皮生长因子受体的外泌体诱导肿瘤特异性调节T细胞

目的探讨树突状细胞(DC)能否捕获含表皮生长因子受体(EGFR)外泌体并转化为成熟DC,诱导生成肿瘤特异性调节T细胞,及后者对肿瘤特异性CD8+T细胞的作用。方法提纯NSCLC肿瘤标本中的外泌体并验证其有EGFR成分,通过外泌体诱导DC转化为免疫耐受型,观察后者诱导生成的调节T细胞对肿瘤特异性CD8+T细胞的影响。结果 NSCLC样本中的EGFR阳性率为80%,而对照肺组织仅为2%。与外泌体共培养7 d后,DC呈现IDO表达高于对照组(80.8%±3.2%vs 65.6%±6.4%,P<0.05)。IDO+DC诱导生成调节T细胞亦明显超过对照组(24.1%±5.2%vs 4.2%±2.3%,P<0.01),进而明显抑制肿瘤特异性CD8+T细胞增殖(5.4%±0.2%vs 86.7%±9.3%,P<0.01)。结论肿瘤细胞的EGFR能够通过外泌体形式排出细胞外,EGFR+外泌体能够诱导免疫耐受IDO+DC产生,进而诱导调节T细胞生成,后者对肿瘤特异性CD8+T细胞有强大抑制作用。     

海洋硫酸多糖DPS抑制血管平滑肌细胞释放血管紧张素Ⅱ和内皮素-1作用机理的探讨

采用碱性成纤维细胞生长因子 (b FGF)诱导的大鼠血管平滑肌细胞 (VSMC)增殖模型 ,对海洋硫酸多糖 DPS抑制 VSMC释放血管紧张素 (Ang )和内皮素 - 1(ET- 1)的机理进行了初步探讨。取生长稳定的 5～ 8代 VSMC,以密度为每孔 5× 10 4 接种在 2 4孔培养板内 ,在含 10 % M199的培养液中 37℃孵育。待细胞贴壁生长稳定后 ,将细胞分为正常对照组 ,b FGF组、DPS组、DPS与 L - NAME联合用药 (DPS L - NAME)组。DPS组和 DPS L- NAME组分别在加入含有 DPS(1mg· L-1)的 M199培养液或含有 DPS(1mg· L-1)和一氧化氮合酶抑制剂 (L - NAME,0 .1mg· L-1)的培养液中预孵 2 4 h后 ,正常对照组除外 ,各组均加入含终浓度为 50 mg· L-1的 b FGF继续培养 2 4 h。用均相竞争放射免疫分析法测定 VSMC上清液 Ang 和 ET- 1的含量。结果表明 ,0 .1mg·m L-1的 L- NAME能完全阻断 DPS抑制 VSMC释放 Ang 的作用 ,但对 ET- 1的释放未见明显影响。提示一氧化氮可能介导 DPS抑制 VSMC释放 Ang 的作用 ,对抑制 ET- 1的释放则无介导作用。     

Leptin stimulates endothelin-1 expression via extracellular signal-regulated kinase by epidermal growth factor receptor transactivation in rat aortic smooth muscle cells

Obesity is a major risk factor for the development of hypertension. Recent studies have suggested that leptin, a 167-amino acid peptide hormone produced by white adipose tissue, is related to the pathogenesis of obesity-related hypertension. However, the signaling mechanisms underlying the effects of leptin remain to be extensively examined. In this study, we found that leptin induced extracellular signal-regulated kinase phosphorylation and endothelin-1 expression in rat aortic smooth muscle cells. Both PD98059 and U0126, inhibitors of the upstream activator of mitogen-activated protein kinase kinase, inhibited augmentation of endothelin-1 expression stimulated with leptin. Leptin induced significant tyrosine phosphorylation of epidermal growth factor receptor, which was significantly attenuated by two inhibitors, an epidermal growth factor receptor tyrosine kinase inhibitor, AG1478, and a broad-spectrum matrix metalloproteinase inhibitor, GM6001. This indicates that the pathway of epidermal growth factor receptor transactivation induced by leptin is dependent on proteolytically released epidermal growth factor receptor ligands. Pretreatment of cells with AG1478 significantly reduced the degree of phosphorylation of extracellular signal-regulated kinase and endothelin-1 expression. Our results reveal that epidermal growth factor receptor transactivation is involved in the leptin signaling pathway in vascular smooth muscle cells, which may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects.     

Resveratrol inhibits angiotensin II-induced endothelin-1 gene expression and subsequent proliferation in rat aortic smooth muscle cells

Resveratrol is a phytoestrogen naturally found in grapes and is the major constituent of wine thought to have a cardioprotective effect. The aims of this study were to examine whether resveratrol alters angiotenisn II-induced cell proliferation and endothelin-1 gene expression and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with resveratrol then stimulated with angiotensin II, after which [3H]thymidine incorporation and endothelin-1 gene expression were examined. The intracellular mechanism of resveratrol in cellular proliferation and endothelin-1 gene expression was elucidated by examining the phosphorylation level of angiotensin II-induced extracellular signal-regulated kinase (ERK). The inhibitory effects of resveratrol (1-100 microM) on angiotensin II-induced DNA synthesis and endothelin-1 gene expression were demonstrated with Northern blot and promoter activity assays. Measurements of 2'7'-dichlorofluorescin diacetate, a redox-senstive fluorescent dye, showed a resveratrol-mediated inhibition of intracellular reactive oxygen species generated by the effects of angiotensin II. The inductive properties of angiotensin II and H2O2 on ERK phosphorylation and activator protein-1-mediated reporter activity were found reversed with resveratrol and antioxidants such as N-acetyl-cysteine. In summary, we speculate that resveratrol inhibits angiotensin II-induced cell proliferation and endothelin-1 gene expression, and does so in a manner which involves the disruption of the ERK pathway via attenuation of reactive oxygen species generation. Thus, this study provides important insight into the molecular pathways that may contribute to the proposed beneficial effects of resveratrol on the cardiovascular system.     

Nitric oxide stimulates elastin expression in chick aortic smooth muscle cells

Nitric oxide (NO), an endothelium-dependent relaxing factor, regulates relaxation, proliferation, and migration of smooth muscle cells (SMCs) and most likely attenuates developing vascular disease such as atherosclerosis. We investigated whether or not NO is associated with regulation of aortic elasticity. S-Nitrosoglutathione (GSNO), a NO donor, stimulated tropoelastin synthesis in cultured SMCs during both the quiescent and proliferating phases. The stimulation of tropoelastin synthesis was dose-dependent within 1-100 nM. Maximum stimulation was detected by treatment with 100 nM GSNO for 24 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), an exogenous cyclic GMP analog, also upregulated tropoelastin synthesis. Tropoelastin and lysyl oxidase mRNA expression, as assessed by Northern blot analysis, was also stimulated by GSNO. Administration of KT5823, a cyclic GMP-dependent protein kinase inhibitor, inhibited the GSNO-induced tropoelastin synthesis. These results indicate that the stimulatory effects of GSNO are due to cyclic GMP dependent protein kinase (PKG) activation by NO. In conclusion, NO seems to enhance aortic elasticity via tropoelastin and lysyl oxidase upregulation.     

Transforming growth factor-beta(1) induces apoptosis via connective tissue growth factor in human aortic smooth muscle cells

We examined the possible involvement of connective tissue growth factor (CTGF) in the apoptosis induced by transforming growth factor-beta(1) (TGF-beta(1)) in human aortic vascular smooth muscle cells (HASC). In quiescent HASC, TGF-beta(1) induced the mRNA and protein of CTGF. A CTGF antisense oligonucleotide inhibited this induction. TGF-beta(1) significantly reduced cell viability and induced DNA fragmentation, and the CTGF antisense oligonucleotide reversed these effects. Moreover, TGF-beta(1) activated caspase 3 in HASC, and the CTGF antisense oligonucleotide reduced this activation. These findings show that CTGF plays a key role in the TGF-beta(1)-induced apoptosis in HASC.     

Ca2+ entry channels in rat thoracic aortic smooth muscle cells activated by endothelin-1.

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellular Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patch-clamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (< or = 0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (> or = 1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.     

Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway

Aim:

To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).

Methods:

Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.

Results:

Thrombin (0.3–3.0 U/mL) caused a concentration- and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist {"type":"entrez-protein","attrs":{"text":"SCH79797","term_id":"1052762130","term_text":"SCH79797"}}SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH₂ (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH₂ (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH₂ (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3–30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3–3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1–10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.

Conclusion:

Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.     

Connective tissue growth factor induces apoptosis via caspase 3 in cultured human aortic smooth muscle cells

Connective tissue growth factor (CTGF) stimulates proliferation of fibroblasts and endothelial cells, but nothing is known about its role in smooth muscle cells. In this study, the effects of recombinant human CTGF (r-hCTGF, 0.5-10 microgram/ml) on cultured human aortic vascular smooth muscle cells were investigated. r-hCTGF significantly reduced cell viability, increased apoptosis, and augmented caspase 3 activity. Moreover, r-hCTGF-induced apoptosis was significantly inhibited by an antibody to CTGF and a caspase-3 inhibitor, Z-Asp(Ome)-Glu-(Ome)Val-Asp(Ome)-FMK. These results suggest that r-hCTGF activates caspase 3 and induces apoptosis.     

Regulation of endothelin-1 production in cultured rat vascular smooth muscle cells

Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-beta1 (TGF-beta), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-beta and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET- 1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET- 1, whereas approximately 90% of that from ECs was ET-1. By TGF-beta stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.     

曲匹地尔阻抑内皮素—1诱导的培养大鼠主动脉平滑肌细胞增生

AIM: To study the effect of trapidil (Tra) on endothelin-1-induced proliferation of cultured rat aortic vascular smooth muscle cells (VSMC). METHODS: The [3H]TdR incorporation into DNA assay, the number of VSMC, and cell cycle distribution were measured. RESULTS: Pretreated with endothelin-1 100 nmol.L-1, cell number, [3H]TdR uptake, and cell mitogenic activity increased 134% +/- 23%, 210% +/- 70%, and 86% +/- 18%, respectively. This proliferation was inhibited by Tra 5, 50, 500 mumol.L-1. The inhibitory rates were 12%-48%, 35%-54% and 15%-47%, respectively. Tra did not influence the proliferation of VSMC without endothelin-1 pretreatment. CONCLUSION: Tra antagonized the proliferation of VSMC induced by endothelin-1.