Fibronectin in synovial fluid and tissue in rheumatoid arthritis

Fibronectin is a glycoprotein found in body fluids, loose connective tissue matrix and in basement membranes. Fibronectin in rheumatoid arthritis synovial fluid was immunologically indistinguishable from the plasma form, as shown by double-diffusion analysis. Fibronectin isolated from rheumatoid synovial fluid by affinity chromatography on gelatin--Sepharose had a polypeptide pattern similar to that of plasma fibronectin in SDS--polyacrylamide gel electrophoresis. In fifty-one patients with rheumatoid arthritis and related diseases fibronectin concentrations is synovial fluid were 445 +/- 103 micrograms/ml (mean +/- SD) and within normal range, 335 +/- 52 micrograms/ml, in plasma. Immunofluorescence staining showed a prominent increase of fibronectin in the proliferating synovial connective tissue in rheumatoid arthritis as compared to normal synovial membrane. The results suggest an increased local production of fibronectin in rheumatoid synovial tissue.     

Microstructural Characteristics of Extracellular Matrix Produced by Stromal Fibroblasts

The overall objective of this investigation was to characterize the extracellular matrix deposited by the stromal fibroblasts as a function of time in culture and matrix microstructure. Stromal fibroblasts were seeded onto collagen matrices and cultured for up to 5 weeks. The collagen matrices contained collagen fibrils with an average diameter of 215 ± 20 nm. When cultured on a collagen film, an average fibril diameter of 62 ± 39 nm was observed for single layer films with only slight variations with time in culture, and after 1 week of culture between two film layers 67 ± 47 nm fibrils were observed after 1 week. When the film surface was molded into 1 and 2 μm microgrooves, the initial average fibril diameter of the extracellular matrix was 73 ± 21 and 73 ± 31 nm respectively. When cultured on a collagen sponge, an average fibril diameter of 107 ± 20 nm was initially observed and decreased to 47.5 ± 17 nm after 1 week in culture. For cells cultured on a collagen sponge, Western blotting showed an increase in myofibroblast phenotype expression with time in culture. Shifts in phenotype were less distinct for cells cultured on collagen films. The microstructure, rather than geometry, of the matrix substrate appeared to influence the newly synthesized extracellular matrix and cell phenotype.     

转染结缔组织生长因子基因的大鼠肾系膜细胞纤连蛋白和Ⅳ型胶原表达的改变

目的观察大鼠肾系膜细胞（MsC）转染结缔组织生长因子（CTGF）基因转染阳性细胞克隆中纤连蛋白（FN）和Ⅳ型胶原（ColⅣ）表达的改变,探讨CTGF在肾小球硬化发生中的作用。方法经脂质体介导将含有CTGF的重组表达质粒转染至大鼠MsC,用G418筛选及Western blot和半定量逆转录聚合酶链反应作鉴定;检测阳性克隆FN、ColⅣ蛋白及其mRNA表达的改变。结果成功建立高表达CTGF的阳性MsC克隆（MCT-1,2）,并证实其与对照组相比,阳性MsC克隆FN、ColⅣ的表达均有上调,其FN蛋白及其mRNA表达分别增高2．9倍和3．2倍,ColⅣ蛋白和mRNA表达分别增高为2．4倍和3．8倍。结论CTGF可促进MsC的FN、ColⅣ的表达。表明其在肾小球硬化发生中起促进作用。     

Minute changes in composition of polymer substrates produce amplified differences in cell adhesion and motility via optimal ligand conditioning

We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.     

Infrared LED light therapy influences the expression of fibronectin and tenascin in skin wounds of malnourished rats—A preliminary study

The aim of this investigation was to evaluate the effect of infrared (λ 846 ± 20 nm) LED irradiation on the expression profile of the extracellular matrix protein components, tenascin and fibronectin on skin wounds induced in well nourished and malnourished rats. Eighteen albino rats (21 days old) were randomly divided into a well-nourished group (standard diet) and a malnourished group (regional basic diet). After receiving the diet for 70 days, skin wounds were created and the animals were subdivided into three groups: well-nourished control (n = 6), malnourished control (n = 6), and malnourished + LED irradiated (λ 846 ± 20 nm, 100 mW, 4 J/cm²) (n = 6). The animals were sacrificed 3 and 7 days after injury and histological sections were immunostained for both proteins. They were examined for the presence, intensity, distribution and pattern of immunolabeling. At 3 days, the distribution of tenascin was shown to be greater in the wound bed of malnourished animals compared to the well-nourished group. The intensity and distribution of tenascin was shown to be lower in the malnourished LED irradiated group compared to the malnourished control. There was a significant difference regarding the presence of fibronectin in the malnourished and well-nourished groups after 7 days (p = 0.03). The intensity of fibronectin was slight (100%) in the irradiated group and moderate to intense in the malnourished control group. The results of the present study indicate that infrared LED irradiation modulates positively the expression of tenascin and particularly fibronectin.     

细胞因子调节对纤连蛋白在孕早期小鼠子宫内膜表达的研究

目的:通过研究细胞因子对FN表达的调节,探讨FN在围着床期子宫内膜表达的调节机理。方法:给孕早期小鼠注射不同的细胞因子,收集子宫内膜。分别用Immuno-blot法及RT-PCR法测定子宫内膜FN蛋白和nRNA的水平。结果:LIF低、高剂量组的FNmRNA和蛋白水平都比对照组高,统计分析差异有显著性（P<0.05,P<0.01）,IL-1和IL-1ra各组FN水平与对照组比无显著差异。结论:LIF在转录和转录后水平上调早期子宫内膜FN的表达,LIF参与着床与调节粘附分子的表达有关。IL-1系统则不影响子宫内膜FN的合成。     

右归丸对膝骨关节炎模型鼠软骨组织显微结构及热休克蛋白90α蛋白表达的影响

目的:观察右归丸对膝骨关节炎(knee osteoarthritis,KOA)模型大鼠软骨组织显微结构和热休克蛋白90α(heat shock protein 90α,Hsp90α)蛋白表达的影响,进一步揭示右归丸防治KOA的机制。方法:将大鼠随机分为假手术对照组、KOA模型组、硫酸氨基葡萄糖组和右归丸(高、中、低剂量)组,每组10只。采用改良Hulth法制备大鼠KOA模型,分别予相应药物灌胃8周。HE染色法观察软骨组织的形态学变化,并进行Makin评分;应用免疫组化法检测各组大鼠软骨组织Hsp90α、纤连蛋白(fibronectin,Fn)和Ⅱ型胶原(collagen typeⅡ,COL-Ⅱ)的表达;RT-qPCR法检测各组大鼠软骨组织Hsp90α、白细胞介素1β(interleukin-1β,IL-1β)、基质金属蛋白酶3(matrix metalloproteinase-3,MMP-3)和MMP-13的mRNA表达水平,应用Western blot法检测各组大鼠软骨组织Hsp90α和COL-Ⅱ的表达。结果:与假手术组比较,KOA模型组大鼠软骨组织Makin评分明显升高,软骨组织H...     

Interaction of transforming growth factor β1 with human glomerular epithelial cells in culture: opposite effects on synthesis of matrix proteins and on urokinase plasminogen activator

The effect of transforming growth factor- (TGF-) was analyzed on the synthesis of fibronectin, collagen type IV, and urokinase plasminogen activator in human glomerular epithelial cells in culture. An increase in the abundance of specific mRNA was found for collagen type IV and fibronectin. Fibronectin protein synthesis was also increased in TGF- treated cells; most of the de novo synthesized fibronectin was found as an unsoluble protein associated with extracellular matrix. In the same cells the amount of plasminogen activator mRNA was found leading also to a decreased surface expression of urokinase plasminogen activator. The data support the concept that by upregulating matrix protein synthesis and downregulating the plasminogen activator system, TGF- favors the development of sclerosis.Abbreviations FN Fibronectin - GEC Glomerular epithelial cells - TGF- Transforming growth factor - uPA Urokinase-type plasminogen activator     

Chondrocyte phenotypes on different extracellular matrix monolayers

Chondrocytes undergo a process of dedifferentiation in monolayer culture that is characterized by a transition to a fibroblast-like phenotype. This behavioral change poses a challenge for tissue-engineered cartilage constructs, as approaches using autologous cells require expansion in vitro. Because chondrocytes express a variety of integrin receptors specific to different adhesive proteins, we hypothesized that chondrocytes expanded on various underlying protein monolayers would have different phenotypic responses. Bovine articular chondrocytes were cultured for up to 2 weeks on tissue culture plastic, fibronectin, collagen type I or collagen type II substrate in the presence or absence of ascorbate. Contrary to our hypothesis, the extracellular matrix protein substrates used in this study did not significantly alter the changes in chondrocyte morphology, gene expression, matrix formation, or cytoskeletal organization. Cells on all substrates assembled equivalent matrices, which may have subsequently regulated cell behavior. In cultures with ascorbate, populations of round and spread cells emerged after 1 week, with round cells expressing collagen type II and the differentiated phenotype and spread cells dedifferentiating. In cultures without ascorbate, chondrocytes rapidly adhered and spread onto organized fibronectin matrices via the ₅β₁ integrin, which has been associated with survival and proliferation of chondrocytes in vitro. These findings indicate that expanding chondrocytes on protein monolayers may not be an effective solution to preventing dedifferentiation and improving autologous chondrocyte transplantation.     

The control of endothelial cell adhesion and migration by shear stress and matrix-substrate anchorage

Endothelial cells constitute the natural inner lining of blood vessels and possess anti-thrombogenic properties. This characteristic is frequently used by seeding endothelial cells on vascular prostheses. As the type of anchorage of adhesion ligands to materials surfaces is known to determine the mechanical balance of adherent cells, we investigated herein the behaviour of endothelial cells under physiological shear stress conditions. The adhesion ligand fibronectin was anchored to polymer surfaces of four physicochemical characteristics exhibiting covalent and non-covalent attachment as well as high and low hydrophobicity. The in situ analysis combined with cell tracking of shear stress-induced effects on cultured isolated cells and monolayers under venous (0.5 dyn/cm²) and arterial (12 dyn/cm²) shear stress over a time period of 24 h revealed distinct differences in their morphological and migratory features. Most pronounced, unidirectional and bimodal migration patterns of endothelial cells in or against flow direction were found in dependence on the type of substrate-matrix anchorage. Combined by an immunofluorescent analysis of the actin cytoskeleton, cell-cell junctions, cell-matrix adhesions, and matrix reorganization these results revealed a distinct balance of laminar shear stress, cell-cell contacts and substrate-matrix anchorage in affecting endothelial cell fate under flow conditions. This analysis underlines the importance of materials surface parameters as well as primary and secondary adhesion ligand anchorage in the context of artificial blood vessels for future therapeutic devices.