David A Pye

Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms.

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.

Monomer Complexes of Basic Fibroblast Growth Factor and Heparan Sulfate Oligosaccharides Are the Minimal Functional Unit for Cell Activation

The interaction of basic fibroblast growth factor (bFGF) with heparan sulfate (HS)/heparin has been shown to strongly enhance the activity of the growth factor although the mechanism of activation is unclear. We have addressed the issue of the minimal stoichiometry of an active HS oligosaccharide·bFGF complex by chemically cross-linking the two components to form novel covalent conjugates. The cross-linking procedure produced both monomeric and dimeric bFGF·oligosaccharide complexes, which were purified to homogeneity. Dimer conjugates were shown to have been formed as a result of disulfide bridging of monomer conjugates. These monomer conjugates were subsequently found to be biologically active in a mitogenesis assay. We therefore conclude that a monomeric bFGF·oligosaccharide complex is the minimal functional unit required for mitogenic stimulation.

Structural Studies of Heparan Sulfate Hexasaccharides : New Insights into Iduronate Conformational Behavior

There is a growing opinion that the conformational dynamics within HS chains is critical to their observed biological activities. Investigations into HS conformational dynamics are problematic, given the structural complexity and heterogeneity of HS chains. However, this goal will be more obtainable once we understand the important roles HS sequence/sulfation patterns play in determining the conformational dynamics of iduronate units. This is the first study to compare isomers of N-sulfated oligosaccharides, with respect to the conformational versatility of their internal iduronates. Characterization by NMR spectroscopy of two HS oligosaccharides derived from porcine mucosal HS enabled the measurement of iduronate coupling constants, while under the influence of different flanking saccharide sequences. By fitting our coupling constant data to a new set of theoretical coupling constants, calculated using explicit water molecular dynamic simulations, we are able to offer new insights into the role sequence/sulfation patterns play in influencing iduronate conformational behavior. Fitting of experimental data, using our new theoretically derived coupling constants, suggests that replacement of the N-sulfate group to the reducing side of IdoUA by an N-acetyl group has little effect on the balance of IdoUA conformational equilibrium. Fitting of coupling constants for sequences GlcNS-IdoUA(2S)-GlcNS and GlcNS(6S)-IdoUA(2S)-GlcNS suggests that the flanking 6-O-sulfate group alters the balance of the IdoUA(2S) equilibrium more toward the (2)S0 conformation. There is also the suggestion that a cooperative effect may exist for N- and 6-O sulfation. These observations could be the key to understanding the important regulatory function attributed to 6-O-sulfation within HS chains.

Quantitation of endothelial cell specific protein E-9 employing a single monoclonal antibody in an indirect sandwich ELISA

An indirect enzyme-linked immunosorbent assay is described for the quantitation of protein E-9 which is specifically expressed on human vascular endothelial cells. The assay capitalizes on the dimeric structure of the E-9 protein by utilizing a single monoclonal antibody as both the capture and detection reagent. Detection is achieved by conjugating the Mab with biotin and is followed by the addition of streptavidin peroxidase to provide high sensitivity. Bound activity is measured by enhanced chemiluminescence utilizing standard Amerlite chemistry. The optimised assay is reproducible and is highly sensitive. Using this assay it was possible to detect the presence of E-9 protein in tissue culture media of endothelial cells and in serum samples--in one case even at 1/100 dilution. In vitro, X irradiation resulted in a greater than two-fold increase (P < or = 0.005) in the level of E-9 protein in culture supernatants of human umbilical vein endothelial cells (HUVEC). There are potential applications for measurements of E-9 protein in body fluids and tissue extracts from patients with a vast variety of diseases characterised by vascular endothelial damage and/or activation.

Fractionated high dose rate brachytherapy moulds – a precise treatment for carcinoma of the pinna

BACKGROUND AND PURPOSE The aim of this paper is to describe a fractionated high dose rate brachytherapy procedure for the treatment of small superficial cancers of the pinna and to report the outcome in a small series of patients. MATERIALS AND METHODS Thirteen patients with superficial cancers of the pinna, not invading cartilage, have been treated and in the majority of cases the tumour thickness was determined by a transdermal ultrasound measurement. For the single-plane moulds the prescribed surface dose was 45 Gy in eight fractions over 5 days and the moulds were constructed such that the full thickness of the disease, as determined by the ultrasound measurement, would lie within the 80% isodose surface. One case was treated with a sandwich mould and in this case the dose was reduced to 42.5 Gy. The treatment machine was a high dose rate microselectron, which contains a single stepping iridium source. RESULTS The radiation reactions were of moderate severity, but were limited to the high dose volume. In all cases there was complete tumour resolution and rapid healing occurred leaving a barely perceptible scar. There were no recurrences over a minimum follow-up time of 18 months and there were no late radiation complications in this period. CONCLUSIONS The treatment of superficial carcinoma of the pinna by means of HDR moulds is a safe and reliable technique. In this small series of patients there was total tumour control with excellent cosmesis.

Non-Invasive Pulsed Ultrasound Quantification of the Resolution of Basal Cell Carcinomas After Photodynamic Therapy

Abstract.The probability of local control of basal cell carcinomas (BCC) treated by photodynamic therapy (PDT) depends strongly on lesion thickness, thicker lesions often requiring two treatments. We examine the utility of 20 MHz pulsed ultrasound (US) for the non-invasive measurement of thickness and rate of regression after PDT treatment. PDT was by topically applied 20% aminolaevulinic acid, followed at 6 h by a standard 100 J/cm2 of 630 nm light. Patients (n=60) were selected as being difficult to treat with existing modalities for reasons of likely poor quality of healing or of cosmesis in this very largely elderly population. Ultrasound ‘A’ scans were made immediately before treatment, and at first and subsequent follow-ups. Parameters measured non-invasively for BCC, adjacent normal skin, and for fibroses after previous conventional therapies, were (a) thickness of skin or lesion, (b) linear density of ultrasound echoes and (c) linear density of high-amplitude echoes. Prior to treatment, median skin thickness (to the dermal/subcutaneous boundary) was 2.6 mm (range 1.2–5.7), fibroses 2.5 mm (1.4–5.6) and BCC 1.5 mm (0.5–4.4). Median linear density of echoes for normal skin, fibroses and BCC plus underlying tissue were 5.6, 5.5 and 4.5, respectively, the BCC values being significantly lower (p=0.002). The corresponding medians for high-amplitude echoes were 1.9, 1.9 and 1.1 (skin or fibrosis versus BCC, p=0.001). Patients whose BCCs appeared clinically to be controlled at up to 220 days after a single treatment, all had values of ultrasound parameters corresponding to skin/fibrosis and were significantly different from measurements on the same site prior to treatment. Patients whose tumours appeared to be reverting to the original BCC ultrasound pattern were subsequently found to be recurring as judged clinically. Non-invasive pulsed ultrasound indicates that rates of resolution vary widely between BCC of similar initial thickness and that the probability of clearance of BCC by PDT is determined largely by the deepest, sometimes small, regions within a lesion, with the overall area being relatively unimportant.

Molecular attributes of bovine aortic endothelial cell heparan sulfate

Heparan sulfate (HS) secreted into the medium of bovine aortic endothelial cell (BAEC) cultures was subjected to chemical and enzymatic degradation followed by analysis using gel-filtration and ion-exchange chromatography. Treatment with HNO2 showed that 41% of the disaccharides were N-sulfated. Degradation by Heparin lyases I (Hep I) showed that 8-9% of the disaccharides contained IdoA(2S) residues. Heparin lyase III (Hep III) degradation produced mainly disaccharides with 67% of the molecules glycosidic linkages susceptible to cleavage. Further degradation of Hep III-resistant fragments with Hep I showed that IdoA(2S) residues were predominantly positioned centrally within the repeating GlcNSO3(+/- 6S)alpha 1-4IdoA containing domains. Digestion with a mixture of Heparin lyases I, II and III degraded the molecule almost entirely to disaccharides, with small amounts of tetrasaccharides containing resistant linkages, suggesting the presence of 3-O sulfated GlcNSO3. Further analysis of the disaccharide products by ion-exchange chromatography and comparison with the data from single enzymatic digestion, allowed an estimate of the disaccharide composition to be made. The results suggest an ordered arrangement of structural domains; however, variations in the structure of these domains results in a heterogeneous population of HS chains. It is suggested that biosynthetic differences in HS structure may act as a regulator of bFGF induced cellular responses.

Irradiation of bovine aortic endothelial cells enhances the synthesis and secretion of sulphated glycosaminoglycans

The effect of X-irradiation on the synthesis of heparan sulphate (HS) and chondroitin/dermatan sulphate (CS/DS) by bovine aortic endothelial cells (BAEC), was studied by measuring the incorporation of [35S]sulphate and [3H]glucosamine into newly synthesized glycosaminoglycan (GAG) chains. Medium extracts from irradiated cultures (5Gy) were found to contain approx. 130% more HS and 200% more CS/DS than unirradiated controls. Smaller increases were observed in cellular extracts, irradiated cultures (5Gy) containing approx. 60% more HS and 100% more CS/DS than unirradiated controls. Structural studies showed no significant changes occurred upon irradiation in either the amounts or distribution of N- and O-sulphate groups in the HS molecule. Values for N-sulphation of 41.1% control and 41.5% irradiated (5Gy) were obtained, the corresponding values for O-sulphation being 19.9% control and 20.2% irradiated. Isotope incorporation data indicated that sulphation of CS/DS may decrease after irradiation, however, analysis of chondroitin ABC lyase derived disaccharides showed no changes in the proportion of non-sulphated and O-sulphated disaccharides. The present study indicates that X-irradiation stimulates the synthesis and secretion of HS and CS/DS proteoglycans (PGs) by BAEC. This could be relevant to many features which are found to be indicative of radiation-induced damage.