Thrombospondin as a mediator of cancer cell adhesion in metastasis

Summary Thrombospondin (TSP) is a 450 kDa adhesive glycoprotein. It is present in high concentrations in the platelet -granule and can readily be secreted following platelet activation where local concentrations can be increased by 3–4 orders of magnitude. TSP is also synthesized by a variety of other cells and is incorporated into their extracellular matrix. TSP is a homotrimer with a number of functional domains, at least four of which might serve as receptor recognizing regions. The amino-terminal heparin binding domain interacts with heparin, other glycosaminoglycans and glycolipids and likely recognizes specific cell surface proteoglycans. The central disulfide cross-linked region, 210 kDa non-reduced and 70 kDa reduced, contains a peptide motif CSVTCG which is apparently responsible for binding to glycoprotein IV (CD36) with high affinity. Immediately adjacent to the calcium binding region of TSP, which undergoes considerable molecular relaxation in the absence of calcium, is an RGDA sequence. TSP has been demonstrated to bind to integrins of the _v3 and _IIb3 class. The carboxy-terminal region of TSP also contains at least one binding epitope for a cell receptor. There are 2 well characterized genes for TSP and truncated forms of TSP have been detected which have inhibitory effects on angiogenesis. Finally, TSP can interact with fibrinogen and fibronectin, perhaps on cellular surfaces, which might serve as secondary receptor-like mechanisms for TSP binding and subsequent mediation of cell adhesion.     

Pancreatic tumor growth is regulated by the balance between positive and negative modulators of angiogenesis

There is increasing evidence for the implication of tumor-derived angiogenic and anti-angiogenic factors in controlling tumor growth in vivo. In this study, we documented the production of inhibitors of angiogenesis by pancreatic cancer cells and examined how changes in the balance between pro- and anti-angiogenic factors regulate tumor growth in vivo. The human pancreatic cancer cell line Hs-776T (HS-W) produces slow-growing tumors in SCID mice. Cells of a variant form (HS-R) of Hs-776T produced faster-growing tumors compared to HS-W. Characterization of HS-W and HS-R cells in vitro showed similar proliferation rates and production of the angiogenic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Analyzes of anti-angiogenic factors showed comparable levels of angiostatin and thrombospondin 1 and 2, but endostatin was only detected in conditioned media of HS-W cells and was absent in HS-R. Cell proliferation was similar in both tumor types in vivo, whereas HS-W tumors demonstrated increased apoptosis with a high percentage of apoptotic endothelial cells (EC). Subsequently, VEGF was over-expressed in Hs-776T cells (HS-VF), resulting in rapidly growing tumors and lowering tumor and EC apoptosis. Collectively, our study confirms that tumor growth is dependent on its ability to increase the angiogenic stimulus or to reduce the amounts of endogenous anti-angiogenic factors.     

Evaluation of a chromogenic commercial assay using VWF-73 peptide for ADAMTS13 activity measurement

Introduction

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy (TMA), related to a severe functional deficiency of ADAMTS13 activity (< 10% of normal). ADAMTS13 activity is thus crucial to confirm the clinical suspicion of TTP, to distinguish it from other TMAs, and to perform the follow-up of TTP patients.

Material and methods

We compared the performance of the commercial chromogenic assay Technozym® ADAMTS13 Activity ELISA (chromogenic VWF73 substrate, Chr-VWF73, Technoclone, Vienna, Austria), to that of our in-house FRETS-VWF73 used as reference method. A large group of 247 subjects (30 healthy volunteers and 217 patients with miscellaneaous TMAs) was studied.

Results

The lower limit of detection of the Chr-VWF73 was 3%, which is well adapted to the clinically relevant threshold for TTP diagnosis (10%). Our results showed a reasonable agreement between FRETS-VWF73 and Chr-VWF73 assays to distinguish samples with an ADAMTS13 activity < 10% from those with an ADAMTS13 activity > 10%. However, Chr-VWF73 assay provided false negative results in ~ 12% of acute TTP patients. Inversely, the Chr-VWF73 assay globally underestimated ADAMTS13 activity in detectable values ranging from 11 to 100% (with a great variability compared to FRETS-VWF73), which may be a concern for the follow-up of TTP patients in remission.

Conclusion

In-house assays developed and performed by expert laboratories remain the reference methods that should be used without limitation to control values provided by commercial assays when needed. Also, the development of an international reference preparation will be crucial to improve standardization.     

多配体蛋白聚糖-4和新型金属蛋白酶-1在组织愈合过程中的作用

多配体蛋白聚糖-4是一种新发现的多肽,广泛存在于多种组织中,调控着多种生物学效应。含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS)-1是一种新型金属蛋白酶,广泛存在于哺乳动物和无脊椎动物体内,在保持凝血系统的稳态、器官生成、炎症和生育等方面起着重要的作用。二者均参与组织的创伤愈合过程,且ADAMTS-1特异性地作用于多配体蛋白聚糖-4,使其胞外区脱落。本文就多配体蛋白聚糖、ADAMTS-1、多配体蛋白聚糖-4与ADAMTS-1、多配体蛋白聚糖-4与牙周组织等研究进展作一综述,为探究牙周组织的损伤修复机制提供理论依据。     

Thrombospondin I activates the macrophage Toll-like receptor 4 pathway

Previously, we demonstrated that macrophages from thrombospondin 1 （TSP1）-deficient mice have a reduced inflammatory phenotype, suggesting that TSP1 plays a role in macrophage activation. In this study, we determined how TSP1 regulates macrophage function. We found that recombinant or purified piatelet human TSP1 treatment stimulated tumor-necrosis factor （TNF）-α expression in bone marrow-derived macrophages in a time- and dose-dependent manner. Toll-like receptor 4 （TLR4） expression （at the mRNA and protein levels） and nuclear factor-kappaB （NF-KB） activity were also stimulated by TSP1 treatment. The TSPl-mediated increase in TNF-a production was abolished in TLR4-deficient macrophages, suggesting that TSP1 activates macrophages through a TLR4-dependent pathway. TSP1 also stimulated TLR4 activation in macrophages in vivo. Furthermore, TSPl-mediated macrophage activation was attenuated by using a peptide or an antibody to block the association between TSP1 and CD36. Taken together, these data suggest that the stimulation of the macrophage TLR4 pathway by TSP1 is partially mediated by the interaction of TSP1 with its receptor, CD36.     

Simvastatin Increases ADAMTS13 Expression in Podocytes

Introduction

ADAMTS13 is a specific von Willebrand factor–cleaving protease. Severe deficiency of ADAMTS13 is the main cause of thrombotic thrombocytopenic purpura. ADAMTS13 is mainly synthesized and released from hepatic stellate cells and endothelial cells, but is also expressed in other cells, including kidney podocytes. Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, has a beneficial effect on atherosclerosis and also has anti-inflammatory and antithrombotic properties. A recent study indicates that ADAMTS13 reduces inflammatory plaque formation during early atherosclerosis in mice. In our study, we investigated the effects of simvastatin on inflammatory cytokines–induced ADAMTS13 expression in podocytes.

Materials and Methods

A conditionally immortalized mouse podocyte cell line was utilized to study the expression of ADAMTS13 in podocytes. The influence of TNF-α, IL-4, IL-6 and simvastatin on ADAMTS13 was investigated. ADAMTS13 mRNA levels in podocytes were measured by using real-time PCR and protein levels were detected by Western blotting.

Results

Simvastatin significantly up-regulated the expression levels of ADAMTS13 mRNA and protein in podocytes. IL-6 decreased ADAMTS13 expression, and TNF-α had no significant effects on ADAMTS13 expression in podocytes. IL-4 reduced ADAMTS13 mRNA expression but not its protein level. Simvastatin was able also reversed the inhibitory effect of IL-6.

Conclusions

We demonstrate that simvastatin increases the expression of ADAMTS13 in a dose-dependent manner in podocytes, which likely contributes to the antithrombotic property of statin. Different inflammatory cytokines have different effects on the levels of ADAMTS13 mRNA expression and protein within podocytes.