Human anti-fibronectin antibodies in systemic lupus erythematosus: occurrence and antigenic specificity.

Sera from patients with connective tissue diseases exhibit autoantibodies to a spectrum of extracellular matrix proteins. Antibodies binding to solid phase bovine fibronectin (Fn) were investigated by ELISA in patients with systemic lupus erythematosus (SLE). Sera showing binding of 2 s.d. above the mean of the normal human control were considered positive and 43/150 SLE sera (28.7%) demonstrated such binding. These antibodies were mainly IgG and IgA as determined by isotype-specific ELISA. Specificity studies on selected positive sera revealed that binding was inhibited by preincubation with soluble Fn, but not with thyroglobulin or type 1 collagen. The binding was demonstrated not to be related to interactions with rheumatoid factor, complement components or immune complexes. Additional studies to determine which Fn fragment is bound by naturally occurring anti-Fn antibodies demonstrated that the binding was predominantly to the 30-kD collagen binding domain (CBD) of Fn molecule. Inhibition studies using 120-kD, 40-kD and 30-kD Fn fragments confirmed that this binding site was the 30-kD CBD.     

神经干细胞克隆在不同细胞外基质上的生长特性

目的：观察不同细胞外基质对培养神经干细胞生长特性的影响。方法：经体外扩增和传代,神经干细胞克隆被接种于多聚赖氨酸、多聚鸟氨酸和鼠尾胶原形成的细胞外基质上,观察其增殖和迁移特性。结果：未经包被的塑料培养板作为基质时,体积较大的神经干细胞克隆才能贴壁,贴壁后的克隆仍呈球形,在其底部有细胞呈放射状迁出,数量逐渐增多,以梭形为主,细胞排列稀疏;多聚鸟氨酸和多聚赖氨酸作为细胞外基质时,迁移细胞排列密集,形态多样;鼠尾胶原作为细胞外基质时,促贴壁迁移的作用最差。当多聚赖氨酸和鼠尾胶原形成界限时可明显观察到鼠尾胶原对细胞迁移的抑制作用。结论：在各种基质上,克隆体积均可继续增长,但在鼠尾胶原上增长较慢。     

The role of chemokines and extracellular matrix components in the migration of T lymphocytes into three-dimensional substrata

The role of chemokines and their interactions with extracellular matrix components (ECM) or the capacity of T cells to migrate into and accumulate within three-dimensional (3D) collagen type 1 substrata was studied. We examined the influence of chemokines and fibronectin on the infiltration properties of non-infiltrative (do not migrate into 3D substrata) and spontaneously infiltrative (migrate into 3D substrata) T-cell lines. Infiltrative and non-infiltrative T-acute lymphocytic leukaemic cell lines exhibited no consistent differences with respect to the expression of various chemokine receptors or beta(1)-integrins. Chemokines presented inside the collagen increased the depth of migration of infiltrative T-cell lines, but did not render non-infiltrative T-cell lines infiltrative, although they augmented the attachment of non-infiltrative T-cell lines to the upper surface of the collagen. The presence of fibronectin inside the collagen did not render non-infiltrative T-cell lines infiltrative, but markedly augmented the migration of 'infiltrative' T-cell lines into collagen. Both infiltrative and non-infiltrative T-cell lines showed migratory responses to chemokines in Boyden assays (migration detected on 2D substrata). These results indicate that the process of T-cell infiltration/migration into 3D substrata depends on a tissue penetration mechanism distinguishable from migration on 2D substrata and that the basic capacity of T cells to infiltrate is independent of chemokines and ECM components applied as attractants.     

Modulation of human mesangial cell behaviour by extracellular matrix components--the possible role of interstitial type III collagen.

We have investigated the effects of various extracellular matrix (ECM) components on the behaviour of human mesangial cells (HMC) in a gel culture system using a modified MTT assay method. When cultured on a reconstituted basement membrane, Matrigel (M gel), HMC aggregated and formed isolated colonies initially, then extended an array of cell processes to form a dendritic network structure and proliferated very slowly as the culture period progressed. On type I collagen gel (CI gel), however, HMC developed elongated bipolar shapes, migrated into the gel, and showed rapid cell growth. Next, separate ECM components, such as type III and IV collagens, laminin, heparin and heparan sulphate, were incorporated into CI gel and HMC proliferation was assessed. Although attachment of HMC to each gel did not differ significantly, HMC proliferation was inhibited markedly on gels containing type III collagen, heparin and heparan sulphate; type IV collagen suppressed HMC proliferation slightly; and laminin had no significant effect. These data suggest that interstitial type I and III collagens, which are often observed in diseased glomeruli, as well as the basement membrane components, may play important roles in the regulation of HMC proliferation under pathophysiological conditions in vivo. We conclude that HMC behaviour is affected by the surrounding ECM constituents, which appear to function as a refined modulator.     

Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components

The aim of the present study was to explore whether nitric oxide (NO) interferes with the attachment of human melanocytes to the extracellular matrix (ECM) components. Consequently, the effects have been investigated of the NO-releasing compounds 3-morpholino-sydnonimine (SIN-1) and S-nitroso-glutathione (GSNO) on the in vitro adhesion of human melanocytic cells to fibronectin. The NO donors induced a concentration-dependent reduction in the adhesion of both ⁵¹CrO₄²⁻-labelled melanocytes and melanoma cells to fibronectin. Pigmented M14 melanoma cells were more susceptible to the effect of SIN-1 (half-maximal inhibiting effect at about 0·5 mm) than normal human melanocytes and also than the non-pigmented melanoma cells Mel57 (half-maximal inhibiting effects between 0·9 and 2 mm). This effect of SIN-1 also appeared to be related to the melanin content of normal melanocytes, whereas GSNO was significantly less active. Both flow cytometric analysis and immunocytochemical staining showed expression of neuronal NO synthase in all cell lines. The results of this study suggest that aberrant in vivo production of NO during infection and inflammation may contribute to loss of melanocytes in, for example, vitiligo, by reducing de novo attachment of melanocytes to the ECM. These findings could also be important for understanding the process of metastasis. © 1997 John Wiley & Sons, Ltd.     

Expression of adhesion molecules and extracellular matrix proteins in glioblastomas: relation to angiogenesis and spread

We studied the immunohistochemical expression of inducible adhesion molecules, integrins and extracellular matrix proteins in 10 cases of glioblastoma multiforme in order to investigate their angiogenesis, local invasiveness, poor metastasizing properties and their lack of tumour infiltrating leukocytes. In glioblastomas endothelial proliferations represent the majority of vascular structures; they were positive for endothelial markers (vWF, CD31, VE-cadherin) and negative for macrophage markers (CD68, PAM-1). Immunohistologically, they were subtyped into: 1 solid-glomeruloid ICAM-1, α₂β₁, α₃β₁, α₅β₁ negative; 2 channelled-branching ICAM-1 negative and α₂β₁, α₃β₁, α₅β₁ positive; 3 channelled-telangiectatic ICAM-1, α₂β₁, α₃β₁, α₅β₁ positive. In channelled proliferations, the expression and distribution of tenascin and merosin in the basal membrane was similar to that of normal brain vessels. The expression of all these molecules might indicate different steps of maturation of endothelial proliferations. The majority of endothelial proliferations may be immunohistologically considered as incomplete vascular structures; this might account for the low metastasizing tendency and low recruitment of leukocytes by these tumours. Neoplastic astrocytes were GFAP-1, ICAM-1, VCAM-1, α₂β₁, α₃β₁ and α₅β₁ immunoreactive and α₆β₄ negative; this allows them to interact with extracellular matrix proteins and might, in part, explain the tendency of glioblastomas to infiltrate locally.     

细胞外基质成分及其构筑对平滑肌细胞黏着斑激酶表达的影响

目的:探讨细胞外基质纤维连接蛋白(FN)、层黏连蛋白(LN)及三维基膜Matrigel胶对平滑肌细胞黏着斑激酶(FAK)表达的影响。方法:免疫荧光组织化学技术对生长在FN、LN和Matrigel胶里的平滑肌细胞FAK的表达进行检测。结果:生长存FN、LN和Matrigel胶里的平滑肌细胞FAK的表达依次为最高、次之和最低,各组之间有统计学意义;平滑肌细胞FAK磷酸化水平和FAK的表达呈正相关,生长在FN上的平滑肌细胞FAK(py397)的表达水平最高,生长在Matrigel胶里的平滑肌表达最低。结论:细胞外基质成分及其构筑对平滑肌细胞FAK的表达具有重要调节作用。     

细胞外基质对培养神经元细胞存活和轴突生长的影响

目的：探讨不同的细胞外基质对体外培养胚胎大鼠脊髓运动神经元和背根神经节感觉神经元生长的影响。方法：取大鼠胚胎脊髓腹侧组织和背根神经节体外分离培养，选取不同生长底物包括多聚赖氮酸(PLL)、Ⅰ型胶原(CoⅠ)、层粘连蛋白(LN)、PLL联合LN进行包被培养板，观察运动神经元和感觉神经元的体外生长状况。结果：PLL和LN联合包被时，神经元存活率高，细胞分散良好。CoⅠ包被时细胞聚集成团．突起粗长。结论：不同的细胞外基质影响神经元的生长方式，PLL联合LN包被是体外研究单一神经元胞体和突起的较好方法。     

The extracellular matrix in pigmented skin lesions: an immunohistochemical study

In recent years the interaction between tumour cells and the surrounding extracellular matrix in the process of tumour development, invasion and metastasis has been a focus of interest. We studied frozen sections of nine naevocellular naevi (junctional, compound and intradermal), 40 dysplastic naevi, six pagetoid in situ melanomas and 12 superficial spreading melanomas in order to determine the expression of: the basement membrane proteins collagen type IV and laminin, the interstitial collagen types I, III and VI, and fibronectin and tenascin. An indirect immunoperoxidase technique was used. In the various stages of melanocytic tumour progression we observed: 1 loss of type IV collagen and laminin within dermal melanocytic cell nests; 2 de novo expression of basement membrane type IV collagen and increased expression of the interstitial collagen types I, III and VI, as well as tenascin and fibronectin in the dermal stroma surrounding dysplastic naevus cells and melanoma cells; 3 presence of extracellular matrix components in close association with intra-epidermally located invading atypical melanocytes. These data demonstrate the complex alterations of the composition of the extracellular matrix from bland naevi through lesions with progressive atypia to invasive melanoma. The changes described result in a molecular environment which melanocytes with an altered adhesion molecule profile are able to invade.     

IL-4-induced B cell migration involves transient interactions between {beta}1 integrins and extracellular matrix components

IL-4 has previously been shown to stimulate motile responsesin murine B lymphocytes. This was studied as acquisition ofmotile morphology and migration through filters in microchemotaxischambers. In this paper, we investigated IL-4-stimulated migrationof B cells into gels of native collagen fibers, which may bea more physiologically relevant assay. When IL-4 was presentin the gel and/or in the medium above, B cells were able toinvade the collagen gel. Migration was dependent on the doseof IL-4 and was optimal after 45 h of incubation. It appearedthat IL-4 acted by inducing both chemokinesis and chemotaxis.Fibronectin (FN) was found to be an important factor for B celllocomotion, since low concentrations of FCS or FN in the gelmatrix greatly improved migration. B cell locomotion was inhibitedby antibodies specific for ß1, 4 and 5 integrins,indicating the presence of integrin-extracellular matrix (ECM)interactions in lymphocyte motility responses. Migration wasnot associated with an up-regulatlon of ß1, 4 or 5integrins. The adhesion between substrate and cells is likelyto be of low affinity, since IL-4-stimulated, as well as non-stimulatedB cells, did not adhere to ECM-coated culture wells. Our datasuggest that transient interactions between integrins and theECM matrix may favour B cell migration.     

Immunohistochemical characterization of extracellular matrix components of granulosa cell tumor of ovary

In order to clarify the characteristics of granulosa cell tumors of the ovary, extracellular matrix components were investigated by immunohistochemical techniques. Twenty-three granulosa cell tumors (GCT; eight juvenile and 15 adult type) were studied in comparison with non-neoplastic granulosa cells of human ovaries. In all 23 cases of GCT, chondroitin 6-sulfate proteoglycan revealed with antibody 3B3 was characteristically observed in the extracellular matrix in the solid nest, as well as in microfollicles. In the juvenile cases, the extracellular matrix also contained large proteoglycan (PG) revealed with antibody 2B1. Macrofollicles as well as micro-follicles contained PG chondroitin 6-sulfate side chains with a significant amount of chondroitin 4-sulfate. By biochemical analysis using high pressure liquid chromatography, it was also found that disaccharide composition of glycosaminog-lycan fractions extracted from granulosa cell tumor tissues consisted mainly of 2-acetamide-2-deoxyl-3-0-(β-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (δ Di-6S). The characteristic feature of granulosa cell tumors is the accumulation of chondroitin sulfate PG, especially chondroitin 6-sulfate PG, which may be synthesized by the tumor cells themselves. Immunohistochemical characterization of the extracellular matrix components (collagen, laminin, heparan sulfate PG, chondroitin 4-sulfate PG) was also studied in relation to chondroitin 6-sulfate PG localization.     

细胞外基质对气道平滑肌细胞增殖活性的促进作用及其机制

目的 观察纤维粘连蛋白（FN），层粘连蛋白（LN），及胶原蛋白I（ColI)等主要细胞外基质成分，对气道平滑肌细胞（ASMCs)增殖活性的影响，并探讨其作用机制。方法 将原代培养的大鼠ASMCs，接种于不同细胞外基质蛋白包被的培养板，观察各时间点细胞的贴壁数量。在培养的ASMCs中分别加入两种浓度（20和60mg/L)的不同细胞外基质蛋白溶液，即FN组，LN组和Col I组，采用^3H-TdR掺入法，检测作用8，12和24h后各组ASMCs增殖活性的变化。间接免疫荧光染色法检测RAS蛋白在各组ASMCs中的表达。结果 与无包被组相比较，FN，ColI包被组及LN包被组ASMCs的贴壁数量明显高（P＜0．01或P＜0．05）。与正常对照组相比较，FN和Col I各浓度组ASMCs的增殖活性增高显著（P＜0．01），且与浓度和作用时间成正比；而LN组ASMCs的活性无明显差异。同时，FN和Col I组ASMCs的RAS染色呈强阳性；对照组及LN组ASMCs的染色则为弱阳性。结论 ASMCs对FN，LN和Col I均有良好的粘附性，但只有FN的ColI 能明显提高ASMCs的增殖活性，其作用是通过RAS信号转导途径而实现的。表明细胞外基质是哮喘气道重塑的重要刺激因子之一。     

Somatostatin receptor (SSTR) expression and function in normal and leukaemic T-cells. Evidence for selective effects on adhesion to extracellular matrix components via SSTR2 and/or 3

We have examined normal T-cells and T-cell lines with respect to expression of various somatostatin receptor subtypes (SSTR1--5) using RT-PCR and PCR. To evaluate the function of these receptors we have further studied the effects of subtype specific signalling on T-cell adhesion using somatostatin analogs specific for various receptors as probes. Human T-lymphocytes showed SSTR expression related to activation and stage of differentiation. Normal T-cells (peripheral blood, T-cell clone) and T-leukaemia cell lines expressed SSTR2, SSTR3 and SSTR4. Normal T-cells expressed SSTR1 and SSTR5 while T-leukaemia lines did not. SSTR5 was selectively expressed in activated normal T-cells. T-lymphocytes produced no somatostatin themselves. Somatostatin and somatostatin analogs specific for SSTR2 and/or SSTR3 enhanced adhesion of T-cells to fibronectin (FN), and to a certain extent, also to collagen type IV (CIV) and laminin (LAM). T-lymphocytes express multiple SSTR and somatostatin may therefore regulate lymphocyte functions via distinct receptor subtypes as shown here for adhesion to extracellular matrix components (ECM) via SSTR2 and SSTR3. SSTR expression also distinguishes normal and leukaemic T-cells. Our findings suggest that SSTR subtypes may be useful targets for therapy during inflammatory diseases and malignancies affecting lymphocytes.     

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus

A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue-level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue-level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra- and/or peri-cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies.     

Morphological and biological changes of a hepatocellular carcinoma cell line cultured in a three-dimensional matrix of collagen

A human hepatocellular carcinoma (HCC) cell line, KIM-1, was cultured in three different concentrations (0.1, 0.2 and 0.3%) of type-l collagen gel matrix and its morphologic features, growth kinetics and alpha-fetoprotein and albumin productions were compared with each other or with those of cells growing on a plastic dish. KIM-1 cells in any concentration of collagen gel matrix formed various sized threedimensional colonies with compact to trabecular cell arrangement. Larger colonies with a more definitive trabecular cell arrangement, resembling the in vivo structure of HCC, tended to form in the collagen gel matrix of a low concentration. The prolongation of doubling time was identified as the collagen concentration in the gel became higher. The cells on a plastic dish proliferated in a monolayered sheet with a shorter doubling time than others. Ultrastructurally, the cells in collagen gel matrix have more distinct cell membranes, junctional complexes and bile canaliculus-like structures, and less cytoskeletons than those on plastic dishes, similar to those in vivo. The productions of alphafetoprotein per 10⁴ cells and albumin per 1O⁵ cells were much higher in the collagen gel matrix culture than on a plastic dish in a stationary phase. These data suggest that collagen gel matrix culture is suitable to monitor the morphologic features and protein production of the tumor cells in similar conditions to those in vivo, and that the three-dimensional presence of an extracellular matrix is important in cellular proliferation and differentiation.     

TAB2 c.1398dup variant leads to haploinsufficiency and impairs extracellular matrix homeostasis

Transforming growth factor β‐activated kinase 1 (TAK1) mediates multiple biological processes through the nuclear factor κ‐light‐chain‐enhancer of activated B cells (NF‐κB) and the mitogen‐activated protein kinase (MAPK) signaling pathways. TAK1 activation is tightly regulated by its binding partners (TABs). In particular, binding with TAB2 is crucial for cardiovascular development and extracellular matrix (ECM) homeostasis. In our previous work, we reported a novel multisystem disorder associated with the heterozygous TAB2 c.1398dup variant. Here, we dissect the functional effects of this variant in order to understand its molecular pathogenesis. We demonstrate that TAB2 c.1398dup considerably undergoes to nonsense‐mediated messenger RNA decay and encodes a truncated protein that loses its ability to bind TAK1. We also show an alteration of the TAK1 autophosphorylation status and of selected downstream signaling pathways in patients’ fibroblasts. Immunofluorescence analyses and ECM‐related polymerase chain reaction‐array panels highlight that patient fibroblasts display ECM disorganization and altered expression of selected ECM components and collagen‐related pathways. In conclusion, we deeply dissect the molecular pathogenesis of the TAB2 c.1398dup variant and show that the resulting phenotype is well explained by TAB2 loss‐of‐function. Our data also offer initial insights on the ECM homeostasis impairment as a molecular mechanism probably underlying a multisystem disorder linked to TAB2.     

Trypanosoma cruziand myoid cells from seminiferous tubules: interaction and relation with fibrous components of extracellular matrix in experimental Chagas' disease

The main transmission route of Trypanosoma cruzi is by triatomine bugs. However, T. cruzi is also transmitted through blood transfusion, organ transplantation, ingestion of contaminated food or fluids, or is congenital. Sexual transmission, although suggested since the discovery of Chagas' disease, has remained unproven. Sexual transmission would require T. cruzi to be located at the testes and ovaries. Here we investigated whether T. cruzi is present in the gonads of mice infected with 10⁴ T. cruzi trypomastigotes from the CL strain. Fourteen days after experimental infection, histopathological examination showed alterations in the extracellular matrix of the lamina propria of the seminiferous tubules. Furthermore, amastigotes were present in seminiferous tubules, within myoid cells, and in the adjacencies of the basal compartment. These results indicate that T. cruzi is able to reach seminiferous tubule lumen, thus suggesting that Chagas' disease could potentially be transmitted through sexual intercourse. Complementary studies are required to demonstrate that Chagas' disease can be transmitted by coitus.