The Extracellular Matrix and Cell Migration

As we know all too well, pancreatic cancer has a very poor prognosis largely due to its early tendency to invade, locally and distantly. Recently, scientists in the field have increasingly focused on the desmoplastic reaction, which is characteristic of most pancreatic cancers. This reaction is associated with proliferation of fibroblastic cells, sometimes outnumbering local tumor cells, and consists of abundant extracellular matrix (ECM) proteins. Importantly, the processes of invasion and metastasis take place within this tumor microenvironment. Stroma and tumor cells exchange signals to modify the local ECM, which subsequently stimulates cell migration and promotes proliferation and survival. Even though recognition of the significance of these microenvironment interactions exists, knowledge on the mechanisms ofthe interplay among pancreatic cells, myofibroblasts, and the ECM is lacking. Therefore, this ‘Pancreatology and the Web’ focuses on websites that provide information on the ECM and cell migration.     

Urokinase Plasminogen Activator Receptor and Soluble Matrix Metalloproteinase-9 in Acute Myeloid Leukemia Patients: A Possible Relation to Disease Invasion

Extracellular proteolytic enzymes of the urokinase-type plasminogen activator (uPA) and metalloproteinase (MMP) family play a crucial role in the matrix degradation and tissue remodeling process characteristic of malignant disorders. The receptor for urokinase plasminogen activator (uPAR) serves to localize and intensify the action of UPA and is expressed on the surface of malignant cells. Although the biological significance of MMP-9 and soluble urokinase receptor in growth and progression of lymphoid neoplasm is understood, its clinical significance in acute myeloid leukemia (AML) has not been fully elucidated. In this study, we determined the levels of soluble urokinase-type plasminogen activator receptor (suPAR), cellular uPAR and sMMP-9 in 43 newly diagnosed AML patients at diagnosis, before chemotherapy, and also studied 10 normal subjects served as a control group. After chemotherapy suPAR and MMP-9 were determined at remission and relapse. The levels of suPAR, cellular PAR were significantly higher (P=0.001, 0.001) and MMP-9 was significantly lower (P=0.001) in AML patients at diagnosis as compared to controls. suPAR and MMP-9 levels were significantly lower in AML patients who achieved complete remission (CR) as compared to those who did not (P=0.001 for both). Levels of suPAR and MMP-9 were significantly correlated to peripheral blood blast cells (r=0.88, P=0.001; r=0.65, P=0.001, respectively) and blast cell distribution ratio (BCDR, r=0.84, P=0.001; r=65, P=0.001, respectively). suPAR, cellular PAR and MMP-9 were significantly higher in patients with extramedullary infiltration as compared with those without (P=0.001, 0.001, <0.05). The suPAR, cellular uPAR, and MMP-9 levels were uneven in AML FAB subtypes being highest in M5(P<0.05 for all). MMP-9 and suPAR levels were correlated with the disease status. In AML survivors, MMP-9, cellular uPAR and suPAR were significantly lower as compared to non-survivors (P=0.001 for all).

In conclusion, MMP-9 and suPAR levels might be used as a marker for disease activity and may contribute to blast cell dissemination. MMP-9 and suPAR may be target molecules in the strategy of treatment of AML.     

Stimulation of Extracellular Matrix Synthesis in the Developing Cornea by Glycosaminoglycans

Previously, it was demonstrated that the embryonic corneal epithelium produces the chondroitin sulfate and heparan-sulfate-like compounds and the collagen of the primary corneal stroma. Synthesis of all of these extracellular materials is greatly enhanced in vitro when isolated epithelium is grown on collagenous substrata instead of Millipore filters. We report here that chondroitin sulfate, heparin, and heparan sulfate added to the culture medium at a concentration of 200 mug/ml enhance the synthesis by the epithelium of chondroitin sulfate and heparan-sulfate-like compounds 2-fold, whether or not collagenous substrata are employed. Collagen synthesis is unaffected by adding glycosaminoglycan to the medium. Chondroitin sulfate proteoglycan (chondromucoprotein) has the same stimulatory effect as chondroitin sulfate, but dermatan sulfate and hyaluronate have no measurable effect on glycosaminoglycan production by epithelial cells. Keratan sulfate however, seems to depress glycosaminoglycan synthesis. Thus, in this system, only sulfated polyanions like those produced by the corneal epithelium have a stimulatory effect on glycosaminoglycan synthesis. The results are discussed in terms of how the tissues of the cornea (epithelium, endothelium, keratocytes) may interact by changing the composition of the stromal extracellular matrix.     

TG、VLDL、0X-VLDL对L-02和HLF细胞增殖及合成细胞外基质的影响

以MTT法、透明质酸(HA)放免测定法及~3H-脯氮酸掺人法,观察4种不同浓度的甘油三酯(TG)、极低密度脂蛋白(VLDL)、氧化极低密度脂蛋白(OX-VLDL)对无血清培养正常成人肝细胞(L-02细胞)、人胚肺成纤维细胞(HLF细胞)增殖及合成细胞外基质(ECM)的影响。结果发现.TG、VLDL、OX.VLDL可影响HLF细胞和/或L.02细胞的增殖,促进其胶原蛋白和HA的合成,其中以OX-VLDL的作用最为明显。提示肝内脂质(特别是OX-VLDL)过多可通过加强脂质过氧化反应促进肝纤维化的发生和发展。     

The Extracellular Matrix and the Cytoskeleton in Heart Hypertrophy and Failure

Cell characteristics and phenotype depend on the nature of the extracellular matrix, the type and organization of integrins and cytoskeleton. The interactions between these components are poorly known at the myocyte level and during cardiac remodeling associated with cardiac hypertrophy and heart failure. We analyze here the nature and organization of extracellular matrix (ECM) proteins, cytoskeleton and integrins and their regulation by growth factors, such as angiotensin II, in normal myocyte growth and in pathological growth (hypertrophy) of the myocardium and heart failure.     

Impact of the Protein Content of Red Cell Concentrates on the Optimum Use of Blood

It was previously shown, in theory and in practice, that a blood component program utilizing 80--85% of all donations as red cell concentrates with a hematocrit of 70% contributes substantially to the coverage of national albumin demands without increasing the need for that protein. The current trend towards the production of red cell concentrates with hematocrits around 85% by the removal of more plasma prompted a computer simulation study of the effects of this modification on the protein and monetary economy of a component program. The results suggest that a hematocrit around 70% constitutes an optimum in these respects. In a general way, they point to the desirability of examining the repercussions of isolated modifications of the processing of blood donations on the system as a whole prior to their large-scale application.     

Histological and Immunohistochemical Studies on the Preparation of Leukocyte-Poor Red Cell Concentrates by Filtration: The Filtration Process on Cellulose Acetate Fibers

A histological and immunohistochemical investigation of slices from the Cellselect leukocyte filter showed that the capture of lymphocytes within these filters depended on trapping of the cells in the cellulose acetate fiber network more than on adherence. Probably, additional mechanisms played a role in granulocyte removal. Microaggregates of granulocytes and platelets were found at the top of the Cellselect leukocyte filter. Therefore, it is likely that granulocyte and platelet removal, at least in part, is due to the formation of cell clusters on the surface of the fibers. Although a part of the original, nonfiltered, leukocytes already showed morphologic characteristics of cell necrosis, more necrotic leukocytes were detected within the filter.     

Differentiation Between Intracellular and Cell Surface Glycosyl Transferases: Galactosyl Transferase Activity in Intact Cells and in Cell Homogenate

Intact BHK (baby hamster kidney) cells catalyze the hydrolysis of UDP-galactose to free galactose. The generation of galactose from UDP-galactose and its intracellular utilization impede the detection of possible galactosyl transferases on the cell surface of intact cells. Several independent procedures have been used to distinguish between intracellular and cell surface glycosyl transferases. With these procedures, no evidence was obtained for the presence of detectable amounts of galactosyl transferase activity on the surface of BHK cells. The data suggest that galactosyl transferases do not play a general role in the phenomena of cell adhesion and contact inhibition.     

Serological and Immunogenic Activity of Soluble Mouse Transplantation Antigens Controlled by the H-2 Locus

The solubilization and partial purification of mouse transplantation antigens were monitored by (1) an in vitro assay for alloantigenic specificities and (2) an in vivo assay for transplantation antigens controlled by the H-2 histocompatibility locus. Antigens from A/J mice were solubilized by papain and fractionated on a Sephadex G-150 column. The eluate showed a 280 nm absorbance peak (F(1)) in the excluded volume and two peaks (F(2) and F(3)) in the included volume. H-2 specificities 1, 3, 4, 5, 11, 23, and 28 were confined to a single peak in the F(2) fraction.Fractions F(1), F(2), and F(3), were tested for their ability to accelerate skin graft rejection in noncoisogenic strains which differ at both H-2 and non-H-2 loci, and coisogenic strains which differ only at the H-2 locus. All fractions produced significant acceleration of graft rejection in the noncoisogenic strains, but only fraction F(2) produced significant acceleration in the coisogenic strains. These findings indicate that H-2 transplantation antigens detected by our in vivo assay, and H-2 alloantigenic specificities detected by our in vitro assay are solubilized by papain and are eluted in the same peak during Sephadex fractionation.     

阿托伐他汀对心肌肥厚大鼠细胞外基质重塑的抑制作用

目的探讨阿托伐他汀对去甲肾上腺素诱导的心肌肥厚大鼠细胞外基质重塑的影响及其可能的机制.方法雄性SD大鼠随机分为三组(1)对照组,(2)去甲肾上腺素组[1.06 mg/(kg·d)×15 d],(3) 去甲肾上腺素+阿托伐他汀组[50 mg/(kg·d)×15 d].去甲肾上腺素ip,2次/d,15 d,建立心肌肥厚模型.应用超声心动图及病理学方法评价整体心肌肥厚及组织胶原表达.用逆转录-聚合酶链反应法(RT-PCR)及免疫组化检测细胞外基质调节因子-基质金属蛋白酶(MMP-9)及其生理性抑制剂(TIMP-1)和转化生长因子β1(TGF-β1)mRNA和蛋白表达.结果去甲肾上腺素组大鼠发生左心室肥厚及纤维化,胶原含量及MMP-9、TIMP-1和TGF-β-1蛋白、mRNA表达显著高于健康对照组(P<0.01).阿托伐他汀能减少心肌中总体胶原及Ⅰ、Ⅲ型胶原的合成及MMP-9、TGF-β-1表达(P<0.01).结论 MMP-9、TIMP-1和TGF-β-1与心肌肥厚大鼠的细胞外基质重塑有关.阿托伐他汀能有效防治心肌纤维化及细胞外基质重塑,这一效应与其降低心肌中高表达的MMP-9和TGF-β-1有关.     

苯那普利对SHR大鼠左心室细胞外基质的影响

目的：探讨苯那普利（一种血管紧张素转换酶抑制剂）是否具有将自发性高血压大鼠（ＳＨＲ）左心室肥厚（ＬＶＨ）时增生的胶原和其它细胞外基质成份恢复到正常水平的作用。方法：６周龄的性ＳＨＲ给予口服苯那普利（１０ｍｇ．ｋｇ＾－１．ｄ＾－１）１６周,性别、年龄配对的ＳＨＲ和Ｗｉｓｔａｒ－Ｋｙｏｔｏ（ＷＫＹ）大鼠作为对照。测量血压、体重、左心室肥厚的指标,采用免疫组化方法作纤维粘连蛋白（ＦＮ）、层粘连蛋白（ＬＮ     

苯那普利对SHR大鼠左心室细胞外基质的影响

目的:探讨苯那普利(一种血管紧张素转换酶抑制剂)是否具有将自发性高血压大鼠(SHR)左心室肥厚(LVH)时增生的胶原和其它细胞外基质成份恢复到正常水平的作用.方法:6周龄的雄性SHR给予口服苯那普利(10 mg.kg-1.d-1)16周,性别、年龄配对的SHR和Wistar-Kyoto(WKY)大鼠作为对照.测量血压、体重、左心室肥厚的指标,采用免疫组化方法作纤维粘连蛋白(FN)、层粘连蛋白(LN)和IV型胶原染色,计算机图像半定量分析. 结果:(1)苯那普利治疗组SHR的SBP和LVM/BW比显著低于对照组SHR;(2)治疗组I V型胶原、FN和LN含量也显著低于未治疗组.结论:苯那普利可减轻SHR大鼠左心室肥厚的发展,伴有心肌细胞外基质的减少.     

Ethanol Feeding Selectively Impairs the Spreading of Rat Perivenous Hepatocytes on Extracellular Matrix Substrates

BACKGROUND: Hepatocytes require attachment and subsequent spreading on an extracellular matrix for their proper growth, function and survival. Our previous studies have shown that ethanol feeding selectively impairs perivenule hepatocyte attachment to various extracellular matrices. This study was undertaken to determine whether zonal differences in hepatocyte spreading in response to ethanol feeding occurs and to ascertain the influence of ethanol consumption on the zonal expression of the beta1 subunit of integrins, which are the major surface receptors responsible for matrix binding and subsequent interactions. METHODS: Hepatocytes from the perivenous and periportal regions of the liver were isolated by digitonin/collagenase perfusion from rats that were pair-fed for 2 to 3 weeks with a liquid diet containing either ethanol or isocaloric carbohydrate. The ability of perivenous and periportal hepatocytes to spread on plates coated with either type IV collagen, laminin, fibronectin or polylysine was determined. In addition, the isolated cells were used for the analysis of total cellular and surface beta1 integrin expression. RESULTS: With all of the matrix substrates tested, the spreading of perivenous hepatocytes isolated from the ethanol-fed animals was markedly impaired, while the spreading of periportal hepatocytes was essentially unaffected by ethanol feeding. Both the total cellular as well as the surface expression of the beta1 integrin subunit in perivenous cells from the ethanol-fed rats were significantly higher than from the perivenous control cells, whereas the total and surface expression of the beta1 integrin in periportal cells isolated from ethanol-fed and control rats were not significantly different. CONCLUSIONS: The results indicated that in addition to impairing hepatocyte attachment, ethanol feeding also impairs another critical step of the adhesion process, that of hepatocyte spreading on extracellular matrix substrates. This defect occurred preferentially in perivenous cells and not periportal cells and was associated with an increase in beta1 integrin expression, suggesting that a compensatory mechanism occurs as an attempt by the perivenous cells to overcome impaired cell-matrix interactions caused by ethanol. Overall, these alterations in extracellular matrix-hepatocyte interactions could lead to alterations of hepatocyte structure and function and potentially play a role in alcoholic liver injury.     

Relative Efficiency of Leucocyte Removal Procedures for the Production of Leucocyte-Poor Red Cell Concentrates Assessed by Flow Cytometry

Flow cytometry was used to: (1) determine residual leucocyte numbers in red cell suspensions following the range of leucocyte depletion procedures used in our organisation, and (2) to characterise phenotypically the leucocytes using direct immuofluorescence with monoclonal antibodies to cell surface receptors. Under the conditions used, a lower limit of detection of 2.5 leucocytes per μl (equivalent to 3.43 log₁₀ or 99.96% removal) could be achieved. Filtration through polyester filters was found to remove up to >99.96% of the initial leucocytes; however, a significant differential efficacy was observed between filters from different manufacturers even when filters with similar costs were compared. The order of filter brands with respect to leucocyte removal found was Pall BPF4 = Erypur Optima G-O > Sepacell R500 > Pall RC50. Pheno-typing revealed that increasing filtration efficacy was associated with a preferential removal of lymphocytes; conversely, a second filtration over one brand of filter allowed proportionately more lymphocytes to pass through compared with the first filtration. A saline wash following filtration removed a further 0.5% of the initial leucocyte content, and was associated with a preferential loss of granulocytes. Freeze-thawing the red cell suspension removed fewer leucocytes (96.3%) than did filtration (98.74% to>l99.6%) or filtration followed by washing (99.22%), and also led to preferential loss of granulocytes. Flow cytometry provides a reliable tool for the quality control of leuco-depleted red cells, and allows a qualitative assessment of the residual leucocytes. This information is of value in choosing procedures aimed at decreasing the risk of alloimmunisation and post-transfusion reactions.     

Activity of Platelet-Derived Growth Factor (PDGF) in Platelet Concentrates and Cryopreserved Platelets Determined by PDGF Bioassay

The bioassay of platelet-derived growth factor (PDGF) in platelets was established using the rat fibroblast cell line 3Y1. 3Y1 cells (5 X 10(3)/well) were cultured in 24-well microculture plates with RPMI 1640 medium using 1% of PDGF-free serum and 5% of platelet extracts for 4 days. Cell proliferation was measured by the increase of DNA content. This assay made it possible to measure the biological PDGF activity. PDGF activity of platelet concentrates kept the same level as that of fresh platelet samples during preservation for up to 120 h at 22 degrees C; cryopreserved platelets also retained PDGF activity well.     

The Role of Hyaluronan and the Extracellular Matrix in Islet Inflammation and Immune Regulation

Type 1 diabetes (T1D) is a disease that in most individuals results from autoimmune attack of a single tissue type, the pancreatic islet. A fundamental, unanswered question in T1D pathogenesis is how the islet tissue environment influences immune regulation. This crosstalk is likely to be communicated through the extracellular matrix (ECM). Here, we review what is known about the ECM in insulitis and examine how the tissue environment is synchronized with immune regulation. In particular, we focus on the role of hyaluronan (HA) and its interactions with Foxp3+ regulatory T-cells (Treg). We propose that HA is a "keystone molecule" in the inflammatory milieu and that HA, together with its associated binding proteins and receptors, is an appropriate point of entry for investigations into how ECM influences immune regulation in the islet.     

Extracellular Matrix and Integrin Interactions in the Skeletal Responses to Mechanical Loading and Unloading

The adaptation of the skeleton to changes in mechanical loading depends on the ability of bone cells first to detect then to convert diverse mechanical forces into chemical signals that regulate cell behavior, a process known as mechanotransduction. A network of interactions between the extracellular matrix, integrin receptors that span the cell membrane, and intracellular cytoskeletal and signaling elements participates in mechanotransduction along with other proteins, most notably ion channels. Integrins are a family of heterodimeric proteins with binding specificity for extracellular matrix proteins, and the ability to influence cell behavior via various key effector functions in addition to mechanotransduction, including adhesion, motility, differentiation, proliferation, survival, and gene expression. The vast majority of what we know now about the relevance of extracellular matrix–integrin interactions for mechanical loading is based on evidence gathered from cells in culture, although the increasing use of animal models confirm the relevance of this network both in health and disease. Dissecting the integrin-extracellular matrix pathway provides mechanistic insight into skeletal changes during loading, distraction osteogenesis, and musculoskeletal disuse, and yields new therapeutic strategies both to inhibit bone resorption and improve the integration and survival of bone and dental implants.     

Investigations on Enzyme Absorption on the Red Cell Surface

Abstract. The absorption of blood group A-decomposing enzyme from Clostridium tertium on the red cell surface was examined. The enzyme was thoroughly absorbed on the surface of red cells of blood group A. Cells which were treated for a few minutes and separated from the enzyme solution lost their A-activity more rapidly than those which were maintained in enzyme solution. Enzyme absorption occurs with biochemical and biophysical specificity; i.e. A-enzyme is absorbed solely on A-cells and it competes with anti-A agglutinin from Euphadra periomphala for their reactive sites. N-acetyl-D-galactosamine and D-galactosamine are considered to release cell-fixed A-enzyme by an elution process.