Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate.

The structural properties of fibroblast heparan sulfate (HS) that are necessary for it to bind strongly to basic fibroblast growth factor (bFGF) have been investigated using bFGF affinity chromatography. Specific enzymic and chemical scission of HS, together with chemical N-desulfation, revealed that N-sulfate groups and iduronate-2-sulfates (IdoA(2-OSO3)) were essential for the interaction. bFGF-affinity chromatography of sulfated oligosaccharides released from HS by treatment with heparitinase led to the identification of an oligosaccharide component (oligo-H), seven disaccharides in length, with a similar affinity for bFGF as the parent molecule. Heparinase treatment of this fraction abolished the high affinity binding to bFGF. Analysis of oligo-H indicated that 74% of the disaccharide units had the structure IdoA(2-OSO3)alpha 1,4GlcNSO3; the remainder comprised N-acetylated and N-sulfated units, the majority of which were devoid of O-sulfate groups. Oligo-H was fully degraded to disaccharides by treatment with nitrous acid. These results indicate that the sequence of oligo-H is as shown below. delta GlcA beta 1,4GlcNSO3 alpha 1,4[IdoA(2-OSO3)alpha 1,4GlcNSO3]5 alpha 1, 4IdoA alpha 1,4GlcNAc Sulfated oligosaccharides of similar size but with a lower affinity for bFGF had a reduced concentration of IdoA(2-OSO3) but significant quantities of GlcNSO3(6-OSO3) and GlcNAc(6-OSO3). The data indicate a primary role for contiguous sequences of IdoA(2-OSO3)alpha 1,4GlcNSO3 in mediating the high affinity binding between fibroblast HS and bFGF.     

The basic fibroblast growth factor-saporin mitotoxin acts through the basic fibroblast growth factor receptor.

We have confirmed the hypothesis that a mitotoxin resulting from the conjugation of basic fibroblast growth factor and saporin exerts its cytotoxic effect through specific interaction with the basic fibroblast growth factor (FGF) receptor. Accordingly, the mitotoxin stimulates tyrosine phosphorylation of the 90 kD substrate that characterizes the initial cellular response to basic FGF. Cross-linking experiments show that radio-labeled basic fibroblast growth factor-saporin (FGF-SAP) binds to the receptor. Suramin, an inhibitor of growth factor receptor binding, inhibits the cytotoxicity of basic FGF-SAP. In a study of 4 different cell types, there is a decrease in the ED50 of the mitotoxin as the receptor number per cell increases. We have verified the cytotoxicity of the mitotoxin in 3 different assay systems. As expected, it is effective in the inhibition of protein synthesis and DNA synthesis, as well as of cell count. Binding of basic FGF-SAP which will result in cytotoxicity occurs very rapidly; 5 minutes of incubation of 10 nM basic FGF-SAP with cells results in 80% inhibition of cell count. The in vitro data indicate that the basic FGF-SAP is a receptor specific and potent suicide antagonist of basic FGF. Its potential as an anti-FGF for therapeutic and research uses in vivo is discussed.     

Colony-forming fibroblast precursors of the circulating blood

Colony-forming fibroblast precursors were detected in circulating blood of adult guinea pigs by CFUf in vitro colony assay. SFU amount to 0.9 +/- 0.2 (M +/- m) per 10(5) explanted leucocytes ranging from 0.04 X 10(-5) to 3.0 X 10(-5) for individual donors. The presence of collagen type I and lack of factor VIII antigen and of FC-receptors proved that CFU-derived colonies in the blood cultures were composed of fibroblasts.     

Thrombin inactivates acidic fibroblast growth factor but not basic fibroblast growth factor

Incubation of bovine brain derived acidic fibroblast growth factor (aFGF) with bovine or human thrombin, 0.5 NIH unit/mL, for 24 h at 37 degrees C results in cleavage of the mitogen, generating a 14-kilodalton fragment which has significantly reduced affinity for immobilized heparin as compared to aFGF, and is at least 50-fold less potent at stimulating mitogenesis. In addition, an 18 amino acid peptide, aFGF(123-140), is generated, identifying one of the thrombin cleavage sites as the Arg-122/Thr-123 bond. The peptide, aFGF(123-140), is neither mitogenic itself nor an inhibitor of the mitogenic activity of aFGF. The cleavage of aFGF by thrombin is inhibited by heparin (50 micrograms/mL) and is completely blocked by the irreversible thrombin inhibitors D-Phe-Pro-Arg chloromethyl ketone and hirudin. Incubation of aFGF with 50 units/mL thrombin at 37 degrees C results in rapid cleavage of the mitogen into several fragments. In contrast, incubation of bovine brain derived basic fibroblast growth factor with 1 unit/mL thrombin for 24 h, or 50 units/mL thrombin for 6 h, does not result in significant cleavage of mitogen. The results show that the C-terminal region of aFGF is of functional importance in both mitogenesis and heparin binding. Most importantly, a novel role for anionic heparin-binding growth factors and their fragments is indicated in physiologic and pathologic situations associated with thrombin generation.     

Single-domain antibodies that compete with the natural ligand fibroblast growth factor block the internalization of the fibroblast growth factor receptor 1

Single-domain antibodies in VHH format specific for fibroblast growth factor receptor 1 (FGFR1) were isolated from a phage-display llama naïve library. In particular, phage elution in the presence of the natural receptor ligand fibroblast growth factor (FGF) allowed for the identification of recombinant antibodies that compete with FGF for the same region on the receptor surface. These antibodies posses a relatively low affinity for FGFR1 and were never identified when unspecific elution conditions favoring highly affine binders were applied to panning procedures. Two populations of competitive antibodies were identified that labeled specifically the receptor-expressing cells in immunofluorescence and recognize distinct epitopes. Antibodies from both populations effectively prevented FGF-dependent internalization and nuclear accumulation of the receptor in cultured cells. This achievement indicates that these antibodies have a capacity to modulate the receptor physiology and, therefore, constitute powerful reagents for basic research and a potential lead for therapeutic applications.     

成纤维细胞和巨噬细胞的染色

动物组织学实验中,学生在观察疏松结缔组织时,往往不容易区别细胞成分中的成纤维细胞和巨噬细胞,我们在制做教学标本中,按传统的方法先注射染料后取材铺片,往往因注射染料易引起皮下感染并发生粘连影响取材,而且做出的标本也不理想,经反复实验,改用猫的皮下组织,免去注射?..     

Conversion of a paracrine fibroblast growth factor into an endocrine fibroblast growth factor

FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.     

Aggregation-induced alterations in fibroblast morphology

Summary The different stages during aggregation of diploid human skin fibroblasts have been examined by transmission and scanning electron microscopy. As a result of aggregation, fibroblasts form a complex tissue configuration. Numerous intercellular junctions can be observed, while the cells remain polygonal and do not develop an organised intracellular cytoskeleton. Cell division occurs only rarely. After aggregation, signs of progressive auto-digestion develop.Adhesion to a substrate results in outgrowth of the cells and monolayer formation, even when extensive cell damage had occurred. The morphology of fibroblasts in aggregates and in the monolayers, from which they were derived, is compared and the contribution of the aggregate system to the study of fibroblast behavior is discussed.J.J. Cassiman is Aangesteld Navorser van The National Foundation for Scientific Research, Belgium     

Prostacyclin analogs inhibit fibroblast migration

The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PGI(2)) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI(2) on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PGI(2) analog carbaprostacyclin (10(-6) M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 microg/ml) 58.0 +/- 13.2% (P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 +/- 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI(2) analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both 10(-6) M), significantly inhibited fibroblast migration to fibronectin. In summary, PGI(2) appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.     

Reaction of the skin fibroblast cytoskeleton to micromanipulation interventions

Micromanipulation is a strong mechanical intervention into cellular integrity and induces large changes in the fine structure of the treated cells. Human diploid skin fibroblasts (KF1 and KF2 cell lines) were chosen as an experimental model. Special hatching needles were used for defined micromanipulation interventions (deformation of plasma membrane). Changes in cytoskeletal structures were visualized by using fluorescent and confocal microscopy. The actin cytoskeleton showed a more sensitive response to micromanipulation than microtubules. Characteristic changes in microfilaments, i.e., thickenings and knot formation, were visible in treated cells fixed immediately after micromanipulation and were the result of hatching-needle pressure on the plasma membrane as well as a reaction of actin filaments localized near the plasma membrane deformation. These direct changes and also other specific alterations in the actin filament network were detectable 14 to 16 h after treatment, but they were not observed when longer reparation intervals were used.     

Acidic fibroblast growth factor in the developing rat embryo

Compared to basic fibroblast growth factor (bFGF), a widely distributed, broad spectrum mitogen and mesoderm inducer, acidic fibroblast growth factor (aFGF) is reported to have an essentially neural distribution and to be undetectable in the early embryo. In the present investigation, we used immunoblotting and immunochemistry to assess the cellular and tissue distributions of aFGF and bFGF in 11-20-d rat embryos. Immunoblotting of crude and heparin-bound embryo extracts revealed faint bands at the expected 17-18-kD and predominant bands at an apparent molecular mass of 26 to 28-kD (despite reducing conditions) using multiple specific antibodies for aFGF and bFGF. Pretreatment with 8 M urea yielded 18-20-kD aFGF and bFGF and some 24-26-kD bFGF. Immunoreactivity for both aFGF and bFGF was positive and similar in the cytoplasm, nuclei, and extracellular matrix of cells of neuroectodermal and mesodermal origin, while it was negative in endoderm-derived cells. The distribution of immunoreactive aFGF and bFGF also showed changes during development that were associated with the process of cellular and tissue differentiation. For example, intensity and extent of immunoreactivity for both peptides progressively increased in the middle layer of the spinal cord with increasing differentiation of the neural cells. The immunostaining patterns were very similar for aFGF and bFGF for each organ and at each stage. In conclusion, high molecular mass forms of immunoreactive aFGF and bFGF are present in the rat embryo. Acidic FGF and bFGF are both widely distributed in tissues of neuroectodermal and mesodermal origin, and their distribution was very similar.     

Investigation of the fibroblast chalone (author's transl)]

By ultrafiltration of a cytoplasm preparation from diploid human fibroblasts (Flow 2000) we obtained a fraction which inhibited the proliferation of the same cells. We succeeded in demonstrating fibroblast chalone, because the activity was endogenous, reversible and affected murine fibroblasts, but not human cervical carcinoma cells (HeLa S3) or neuronal tumor cells of rats (B104). The chalone activity was found in the range of molecular weights between 10 000 and 100 000. The active part was of peptide nature, for proteolytic treatment destroyed the chalone activity.     

Acridine orange staining of the mammalian fibroblast cell coat

Acridine orange selectively binds to glycos- and galactosaminoglycan (GAG) compounds in the presence of Na+ in low concentrations. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface GAG components. The method consists of a glutaraldehyde prefixation, an acridine orange block-staining for 48 h and an OsO4 postfixation for some hours. The specificity of the staining was verified with the help of fluorimetric--in vitro--measurements and enzymatic digestions, respectively.     

Proteome annotations and identifications of the human pulmonary fibroblast

We hereby report on a three year project initiative undertaken by our research team encompassing large-scale protein expression profiling and annotations of human primary lung fibroblast cells. An overview is given of proteomic studies of the fibroblast target cell involved in several diseases such as asthma, idiopatic pulmonary disease, and COPD. It has been the objective within our research team to map and identify the protein expressions occurring in both activated-, as well as resting cell states. The JGGL database www.2DDB.org has been built around these data, allowing advanced hypothesis building using the interactive query bioinformatic tools developed. Gene ontology has been applied to these annotations, classifying and correlating protein expressions to function. The localization as well as the biological processes involved for the annotations are being presented including an annotation-, and sequence-identification strategy, resulting in close to 2000 protein identities. Both gel based, high resolution 2D-gels, and liquid-phase separation (three-dimensional HPLC), as well as the combination of gel- and LC-based approaches (1D-gels and nano-capillary LC, reversed-phase) were utilized. Protein sequencing and structure identities were acquired by a combination of MALDI-, and electrospray-mass spectrometry techniques. Phenotypical and morphological characterizations were also made for this human disease target cell in both stimulated- and resting-cell states. The use of functional assays that demonstrate the key regulating role of growth factors and cytokine stimuli such as PDGF, TGF-beta, and EGF and the effect of ECM molecules such as Biglycan, are also presented and discussed.     

Acridine orange staining of the mammalian fibroblast cell coat

Summary Acridine orange selectively binds to glycos- and galactosaminoglycan (GAG) compunds in the presence of Na⁺ in low concentrations. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface GAG components. The method consists of a glutaraldehyde prefixation, an acridine orange block-staining for 48 h and an OsO₄ postfixation for some hours. The specificity of the staining was verified with the help of fluorimetric-in vitro-measurements and enzymatic digestions, respectively.