Quantitation of microparticles released from coated-platelets

Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated-platelets, a sub-population of cells known formerly as COAT-platelets (collagen and thrombin). Coated-platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated-platelets also release microparticles. Platelets were prelabeled with 2.5 microm Bodipy-maleimide and then stimulated with convulxin plus thrombin. Microparticles, 0.3-0.5 microm in diameter, were observed by fluorescence confocal microscopy. Confocal microscopy was also used to demonstrate that microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9, and PS, but negative for fibrinogen and thrombospondin. Furthermore, microparticles released from Bodipy-labeled platelets were observed by flow cytometry, and activation with convulxin plus thrombin produced 15 +/- 5 microparticles per coated-platelet. In contrast, platelets stimulated with thrombin or convulxin alone produced few microparticles. Phenylarsine oxide and diamide, both of which potentiate the mitochondrial permeability transition pore and coated-platelet production, significantly increased the number of microparticles released per coated-platelet.     

Antisense-mediated reduction in thrombospondin-1 expression reduces cell motility in malignant glioma cells.

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein implicated in cancer cell adhesion, migration, invasion, inhibition of angiogenesis and activation of latent transforming growth factor-beta. The involvement of TSP-1 in the motility of malignant glioma cells was investigated by transfection of TSP-1 complementary deoxyribonucleic acid (cDNA) sense and antisense expression vectors into the glioblastoma cell line T98G-G7 that secretes high amounts of TSP-1. TSP-1 production in the 3 antisense cDNA-transfected clones was significantly reduced to 51%, 43% and 47% compared to the host T98G-G7 cells. Motility of the 3 clones was evaluated by invasion assay and compared to the motility of host T98G-G7 cells and 2 sense-transfected clones. Migration of cells was significantly reduced in the 3 antisense-transfected clones with reduced TSP-1 production to 56%, 61% and 43% compared to the host T98G-G7 cells. The host T98G-G7 and another TSP-1-secreting A172 and YMG5 glioblastoma cells were also treated with a synthetic peptide, WSHWSPWSSCSVTCG, which includes 3 consecutive sequences of the adhesion sites in the TSP-1 molecule and with a control peptide. The synthetic peptide significantly inhibited the migration of T98G-G7 and A172 cells in a dose-related manner. Maximum inhibition of migration was achieved by 100 microg/ml of the peptide and the reduction of cell motility compared to untreated cells was 34.6 % and 53.9 %, respectively. On the other hand, the inhibition of migration by the peptide was minimal in YMG5 cells, which secretes a smaller amount of TSP-1 than T98G-G7 and A172 cells. These results suggest that TSP-1 secreted by malignant glioma cells is involved in the motility of glioma cells.     

瘢痕组织中微血管数和TSP-1mRNA表达

目的研究不同瘢痕组织和正常皮肤中血小板反应蛋白-1(TSP-1)mRNA的表达和微血管(MV)计数及其意义.方法应用原位分子杂交技术研究TSP-1 mRNA的表达水平,应用免疫组化的方法研究MV的计数.结果TSP-1 mRNA在增生性瘢痕和瘢痕疙瘩中表达的阳性率低于在正常皮肤和外科瘢痕组织中的表达(P＜0.05);TSP-1 mRNA表达阴性病例组的MVC显著高于TSP-1 mRNA阳性病例组的MCV(P＜0.05);增生性瘢痕和瘢痕疙瘩组织中微血管数多于正常皮肤和外科瘢痕组织的微血管.结论血管内皮细胞的过度增殖分化而抑制不足可能是瘢痕过度增生的重要因素.抑血管生成疗法可能是防治病理性瘢痕的新途径.     

Redistribution of Platelet Membrane Glycoprotein IV and Release of Intracellular α-granule Thrombospondin in Patients with Chronic Myelogenous Leukemia

Plateletmembraneglycopr0tein(GP)IV,als0knownasGPIIIbandCD36,hasbeenproposedasthemembranerecept0rthrombospondin(TSP).TSPreleasedfromplateleta-granuleshasbeenshownt0playaroleinitsbindingt0GPlVonplateletmem-braneandtobecriticalfortheconsolidationofaggregatingplateletinthromb0sisandhem0stasis[lJ.Duringchronicmyelogenousleukemia(CML),plateletmernbraneGPsundergochangesintheirdistributions,sug-gestingqualitativeand/orquantitativeab-normalitiesandtherebyaffectingfunction0fplateletadhesionand…     

Thrombospondin-4, tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14: Novel extracellular matrix modulating factors in cardiac remodelling

Abstract

Cardiac remodelling is defined as changes in the size, shape, and function of the heart, which are most commonly caused by hypertension-induced left ventricular hypertrophy and myocardial infarction. Both neurohumoral and inflammatory factors have critical roles in the regulation of cardiac remodelling. A characteristic feature of cardiac remodelling is modification of the extracellular matrix (ECM), often manifested by fibrosis, a process that has vital consequences for the structure and function of the myocardium. In addition to established modulators of the ECM, the matricellular protein thrombospondin-4 (TSP-4) as well as the tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 has been recently shown to modulate cardiac ECM. TSP-4 null mice develop pronounced cardiac hypertrophy and fibrosis with defects in collagen maturation in response to pressure overload. TWEAK and Fn14 belong to the tumour necrosis factor superfamily of proinflammatory cytokines. Recently it was shown that elevated levels of circulating TWEAK via Fn14 critically affect the cardiac ECM, characterized by increasing fibrosis and cardiomyocyte hypertrophy in mice.

Here we review the literature concerning the role of matricellular proteins and inflammation in cardiac ECM remodelling, with a special focus on TSP-4, TWEAK, and its receptor Fn14.     

糖尿病视网膜病变患者血清VEGF、ES、TSP的含量分析

目的探讨糖尿病视网膜病变(diabetic retinopathy,DR)患者血清中血管内皮生长因子(vascular endothelial growth fac-tor,VEGF)、内皮抑素(endostatin,ES)及血小板反应蛋白(thrombospondin,TSP)的含量,及其与眼底病变程度的相关性。方法采用ELISA法检测51例(51眼)DR患者(DR组)和10例(20眼)无DR表现的糖尿病患者(对照组)血清中VEGF、ES及TSP的含量,分析其与眼底病变程度的关系。结果 VEGF含量在对照组、轻度NPDR、中度NPDR组、高度NPDR组、PDR组中分别为(143.7450±23.6566)ng.L-1、(163.2792±32.5337)ng.L-1、(224.8375±84.5714)ng.L-1、(297.3173±81.4124)ng.L-1、(503.0682±77.4703)ng.L-1,各组之间差异有显著统计学意义(P<0.01),ES含量在对照组与非增生性DR组之间差异有显著统计学意义(P<0.01),TSP含量在不同程度DR组之间差异无统计学意义(P>0.05)。VEGF与眼底病变程度具有显著相关性(r=0.84,P<0.05);对照组与非增生性DR患者血清中ES与眼底病变程度具有显著相关性(r=0.82,P<0.05),而增生性DR患者无相关性(r=0.16,P>0.05);TSP与眼底病变程度无相关性(r=-0.06,P>0.05)。结论血管生成因子和血管抑制因子作用的失衡可能是DR新生血管生成的主要原因。     

Prosaposin inhibits tumor metastasis via paracrine and endocrine stimulation of stromal p53 and Tsp-1

Metastatic tumors can prepare a distant site for colonization via the secretion of factors that act in a systemic manner. We hypothesized that non- or weakly metastatic human tumor cells may act in an opposite fashion by creating a microenvironment in distant tissues that is refractory to colonization. By comparing cell lines with different metastatic potential, we have identified a tumor-secreted inhibitor of metastasis, prosaposin (Psap), which functions in a paracrine and endocrine fashion by stimulating the expression of thrombospondin-1 (Tsp-1) in fibroblasts present in both primary tumors and distant organs, doing so in a p53-dependent manner. Introduction of Psap in highly metastatic cells significantly reduced the occurrence of metastases, whereas inhibition of Psap production by tumor cells was associated with increased metastatic frequency. In human prostate cancer, decreased Psap expression was significantly associated with metastatic tumors. Our findings suggest that prosaposin, or other agents that stimulate p53 activity in the tumor stroma, may be an effective therapy by inhibition of the metastatic process.