The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

Evidence for Cell Surface Extracellular Matrix Binding Proteins in Hydra vulgaris

The present study was designed to identify and functionally characterize potential cell surface extracellular matrix binding proteins in Hydra vulgaris. Using [³H]-laminin as a probe, radioreceptor analysis of a dissociated mixed hydra cell preparation indicated that the average number of laminin binding sites per cell was about 10,000 with a dissociation constant of 1.49 nM. These binding sites could be displaced with unlabelled laminin in a dose-dependent manner and with high concentrations (500 nM) of unlabelled fibronectin. No displacement with type-IV collagen and type-I collagen was observed. Immunoscreerting studies with a battery of antibodies raised to mammalian extracellular matrix (ECM) binding proteins indicated potential cell surface binding sites for the anti-β₁ integrin monoclonal antibody, mAb JG22. Cell adhesion studies indicated that mAb JG22 blocked binding of hydra cells to laminin, but did not affect their binding to fibronectin, type-IV collagen, or type-I collagen. Light and electron microscopic immunocytochemical studies indicated that mAb JG22 localized to the basal plasma membrane of ectodermal and endodermal epithelial cells. Immunoprecipitation studies identified two major bands with masses of about 196 kDa and 150 kDa under reducing conditions, and two bands with masses of >200 kDa under non-reducing conditions. Functional studies indicated that mAb JG22 could reversibly block morphogenesis of hydra cell aggregates, and could block in vivo interstitial cell migration in hydra grafts. These observations indicate that hydra has cell surface binding sites for ECM components which are functionally important during development of this simple Cnidarian     

Role of matrix metalloproteinases in epithelial migration

In response to injury, epithelial cells migrate across the denuded tissue to rapidly close the wound and restore barrier, thereby preventing the entry of pathogens and leakage of fluids. Efficient, proper migration requires a range of processes, acting both inside and out of the cell. Among the extracellular responses is the expression of various matrix metalloproteinases (MMPs). Though long thought to ease cell migration simply by breaking down matrix barriers, findings from various models demonstrate that MMPs facilitate (and sometimes repress) cell movement by other means, such as affecting the state of cell–matrix interactions or proliferation. In this Prospect, we review some key data indicting how specific MMPs function via their activity as proteinases to control closure of epithelial wounds. J. Cell. Biochem. 108: 1233–1243, 2009. © 2009 Wiley‐Liss, Inc.     

Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.     

Depressed Immune Surveillance Against Cancer: Role of Deficient T Cell: Extracellular Matrix Interactions

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

炎性体与器官移植

炎性体是指存在于细胞质内能够激活半胱天冬酶-1（caspase-1）的大分子复合物,活化的caspase-1通过酶切白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）前体分子而生成具有生物学活性的IL-1β和IL-18.近来研究发现,炎性体分子NLRP1、NLRP2、NLRP5、CARD8、CASP5的基因型与移植的临床结果相关,心脏移植排斥反应时ASC和IL-1β的表达升高,缺血再灌注损伤中NLRP3炎性体激活增加IL-1β分泌,表明炎性体的激活与移植排斥反应和缺血再灌注损伤密切相关,但诱导炎性体活化的配体和参与的炎性体分子有待进一步研究.     

Emerging Role of Syndecans in Extracellular Matrix Remodeling in Cancer

The extracellular matrix (ECM) offers a structural basis for regulating cell functions while also acting as a collection point for bioactive molecules and connective tissue cells. To perform pathological functions under a pathological condition, the involved cells need to regulate the ECM to support their altered functions. This is particularly common in the development of cancer. The ECM has been recognized as a key driver of cancer development and progression, and ECM remodeling occurs at all stages of cancer progression. Thus, cancer cells need to change the ECM to support relevant cell surface adhesion receptor–mediated cell functions. In this context, it is interesting to examine how cancer cells regulate ECM remodeling, which is critical to tumor malignancy and metastatic progression. Here, we review how the cell surface adhesion receptor, syndecan, regulates ECM remodeling as cancer progresses, and explore how this can help us better understand ECM remodeling under these pathological conditions     

半椎蛋白的生物学作用及其与人类疾病的关系

半椎蛋白作为一种细胞外基质蛋白质,在细胞的生长、分化、迁移以及细胞与基底膜的稳定接触中发挥非常重要的作用。线虫、小鼠等多种动物模型的研究发现:该蛋白质参与稳定细胞有丝分裂时出现的分裂沟、促进胞浆的正常分裂,并且是一种细胞黏附体系B-LINK的重要组成部分;调控新生小鼠心室心肌成纤维细胞对TGF-β信号的反应。多项研究表明,该蛋白质参与到多种人类疾病的发生发展之中,包括舍格伦综合症、年龄相关性黄斑变性、心血管疾病、肾疾病和胰岛瘤。本文主要就该胞外基质蛋白质的生物学作用,以及与人类疾病关系的相关研究进展进行综述。     

肿瘤细胞粘附、迁移与转移的相关性

肿瘤细胞的粘附、迁移能力与癌转移密切相关. 细胞粘附分子选择素、整合素、免疫球蛋白超家族及钙粘素介导同型或异型细胞间以及细胞与基质间的粘附,其在肿瘤细胞表面表达数量或分布方式的改变直接或间接影响着转移潜能,是肿瘤细胞从原发瘤脱落以及着床的关键性环节.肿瘤细胞的迁移能力被认为是癌转移的限速环节.一般情况下,肿瘤细胞在体内或体外的迁移能力与其转移潜能呈正相关性,肿瘤细胞通过对迁移刺激物的趋化性及趋触性应答而完成向远离器官的转移,其具体分子机制目前还不清楚.     

Diabetic Nephropathy and Extracellular Matrix

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typicalmorphological changes are the result of changes in the extracellular matrix (ECM). Thus,basement membranes are thickened and the glomerular mesangial matrix and thetubulointerstitial space are expanded, due to increased amounts of ECM. One important ECMcomponent, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs inbasement membranes are decreased but increased in the mesangium and the tubulointerstitialspace. The amounts and structures of heparan sulfate chains are changed, and such changesaffect levels of growth factors regulating cell proliferation and ECM synthesis, with cellattachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfatestructures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classesalso modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serineproteases, such as plasminogen activator, as well as their corresponding inhibitors. Thelevels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN.Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesisand turnover in DN. Whether ECM components can be used as markers for early kidney changesis an important research topic, whereas at present, the clinical use remains to beestablished.     

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient''s blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis¹. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.     

Glomerular Extracellular Matrix Components and Integrins

It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits α3 and α6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys.

The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative α chains of about 395 (m) and of 375 kDa (cry), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins α1β1, α2β, α3Aβ1, α5β1, αvβ3, αvβ5, and small amounts of α6Aβ1 and α6Bβ1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins α2β1, αAβ1, α6Aβ4, αBβ4 and the integrin subunit av were found to be expressed.

We show that during nephrogenesis, the laminin α1 chain disappears and is replaced by another a chain, possibly one of the two as yet unidentified α chains mentioned above. The laminin β1 chain is replaced by the β2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin α3 or α6 subunits could be demonstrated during nephrogenesis.

Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular stucture of the glomerular basement membrane. Furthermore our results indicate that laminin αxβ2γ1 and αβ2γ1 isoforms are important in the glomerulus of adult kidney and that the integrin α3Aβ1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.     

Functional Interactions of Fibronectin and TNFα: A Paradigm of Physiological Linkage Between Cytokines and Extracellular Matrix Moieties

The ECM is composed of various cell-adhesive glycoproteins, such as, fibronectin (FN), laminin (LN), and different types of glycosaminoglycans and proteoglycans. These building blocks of the ECM are linked together to form a dense and complex tissue that fills the interstitial spaces and comprises the boundaries between cells and tissues. The ECM is the major milieu in which immune cells function during inflammatory processes (Shimizu and Shaw, 1991; Yamada, 1991). Recognition of ECM-glycoproteins by immune cells is mediated by very late activation (VLA) receptors, also referred to as integrins of the β₁-subfamily (Hynes, 1992). A prerequisite of lymphocyte-ECM interactions is activation of the cells by mitogens, or via their CD3-T cell receptor complex, either of these types of activation modulates the affinity of otherwise inactive β₁-integrins (Shimizu, et al., 1990).     

靶向细胞外基质磷酸糖蛋白的microRNA的筛选及鉴定

目的:寻找靶向细胞外基质磷酸糖蛋白(MEPE)基因的微小RNA(miRNA),并检测其对人HeLa细胞内源性Mepe基因表达的影响。方法:通过NCBI检索人源Mepe的3’UTR,利用miRNA预测工具TargetScan预测可能靶向Mepe的所有miRNA,通过双萤光素酶报告基因系统检测miRNA与Mepe3’UTR的结合情况,从而初步筛选出可能靶向Mepe的miRNA;同时,用Western印迹检测miRNA经转染后对Mepe基因表达的影响。结果:利用TargetScan预测出36条可能靶向Mepe的miRNA,根据分值及匹配情况从中挑选出6条进行验证;与转染空载体pGL3-cm的相对荧光素值相比,转染miR-376a的相对荧光素值降低较为明显,而当Mepe3’UTR与miR-376a结合位点突变后,miR-376a不能抑制萤光素酶的活性;Western印迹结果显示miR-376a能明显抑制MEPE的表达。结论:miRNA-376a可能是靶向Mepe基因的miRNA,为进一步研究MEPE的功能奠定了基础。     

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).