Monomeric C-reactive protein--a key molecule driving development of Alzheimer's disease associated with brain ischaemia?

Alzheimer's disease (AD) increases dramatically in patients with ischaemic stroke. Monomeric C-reactive protein (mCRP) appears in the ECM of ischaemic tissue after stroke, associating with microvasculature, neurons and AD-plaques, Aß, also, being able to dissociate native-CRP into inflammatory, mCRP in vivo. Here, mCRP injected into the hippocampal region of mice was retained within the retrosplenial tract of the dorsal 3rd ventrical and surrounding major vessels. Mice developed behavioural/cognitive deficits within 1 month, concomitant with mCRP staining within abnormal looking neurons expressing p-tau and in beta-amyloid 1-42-plaque positive regions. mCRP co-localised with CD105 in microvessels suggesting angiogenesis. Phospho-arrays/Western blotting identified signalling activation in endothelial cells and neurons through p-IRS-1, p-Tau and p-ERK1/2-which was blocked following pre-incubation with mCRP-antibody. mCRP increased vascular monolayer permeability and gap junctions, increased NCAM expression and produced haemorrhagic angiogenesis in mouse matrigel implants. mCRP induced tau244-372 aggregation and assembly in vitro. IHC study of human AD/stroke patients revealed co-localization of mCRP with Aß plaques, tau-like fibrils and IRS-1/P-Tau positive neurons and high mCRP-levels spreading from infarcted core regions matched reduced expression of Aß/Tau. mCRP may be responsible for promoting dementia after ischaemia and mCRP clearance could inform therapeutic avenues to reduce the risk of future dementia.

Anti-angiogenic activity of sesterterpenes; natural product inhibitors of FGF-2-induced angiogenesis.

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. We have investigated the anti-angiogenic potential of two naturally occurring sesterterpenes, leucosesterterpenone (compound 1) and leucosterlactone (compound 2) isolated from the Himalayan plant Leucosceptrum canum and identified as having biological activity in preliminary screening. Compound 1 inhibited fibroblast growth factor-2-induced proliferation, migration in a wounding assay, chemotaxis and tube formation with small vessel (human dermal) and large vessel (bovine aortic) endothelial cells while compound 2 was largely inactive. Both compounds were also active in an in vivo angiogenic model using the chick chorioallantoic membrane. Neither compounds showed inhibitory activity in the absence of fibroblast growth factor-2. We were able to demonstrate in a binding assay that compounds 1 and 2 bound to the fibroblast growth factor-2 receptor-1 with IC(50) values of 1.4 +/- 0.956 and 132.47 +/- 7.90 muM, respectively, with a concomitant down regulation of phosphorylated ERK1/2 but did not bind to receptor-2. Compound 1 was less hydrophobic than compound 2 and this may contribute to its increased activity. Compound 1 is a new addition to the small number of inhibitors of fibroblast growth factor-2-induced angiogenesis. The compound was a specific inhibitor in that it had no effect on vascular endothelial growth factor or epithelial growth factor-induced angiogenesis. Since angiogenesis is essential for tumour development we conclude that these compounds may have potential as anti-tumour agents.

Application of mathematical morphology to the quantification of in vitro endothelial cell organization into tubular-like structures.

Experiments of in vitro angiogenesis are important tools for studying both the mechanisms of formation of new blood vessels and the potential development of therapeutic strategies to modulate neovascularisation (e.g., screening of new pharmacological molecules). One of the most frequently used angiogenesis assays is the culture of endothelial cells on a reconstituted basement membrane named Matrigel, since the cells constitute a capillary-like network which can be quantified by image analysis. In this paper, a global, robust and fully automated methodology is proposed to segment and quantify in vitro endothelial cell networks from greyscale images using mathematical morphology operators. After extracting the established cell network by means of a top-hat transformation and separating the tubular structures and the cell aggregates by size and shape an interpolation algorithm yields a reconstituted closed network. Using these image data results different kinds of quantitative parameters are calculated: size/shape, morphological distribution, spatial organisation, etc. In this paper, we have established an automatic quantitative analysis to evaluate a modulator effect of a sulphated exopolysaccharide on FGF-2-induced in vitro angiogenesis, according to different parameters. Experimental results allow us to draw a discussion of the pertinence of the alternative morphological parameters to evaluate the characteristics and behaviour of cell cultures.

Modulation of vascular human endothelial and rat smooth muscle cell growth by a fucosylated chondroitin sulfate from echinoderm.

Fucosylated chondroitin sulfate is a glycosaminoglycan extracted from the sea cucumber Ludwigothurea grisea. This polysaccharide has the same structure as a mammalian chondroitin sulfate but some of the glucuronic acid residues display sulfated fucose branches. Anticoagulant and antithrombotic properties of fucosylated chondroitin sulfate have already been described. In order to further investigate its potential therapeutic use as an antithrombotic agent, we studied its effect on vascular smooth muscle cell (SMC) proliferation and endothelial cell proliferation, migration and Tissue Factor Pathway Inhibitor (TFPI) release. The experiments were performed on SMC from rat thoracic aorta and on human umbilical vein endothelial cell (HUVEC) in culture with or without added fibroblast growth factors (FGF-1 and FGF-2). Our results showed that: (i) fucosylated chondroitin sulfate had a strong inhibitory effect on SMC proliferation (IC50 =10 +/- 5 microg/ml) and (ii) no effect on HUVEC proliferation and migration assays, in the absence of exogenous FGF, while heparin had inhibitory effects; (iii) fucosylated chondroitin sulfate (10 microg/ml) enhanced FGF-1 and FGF-2 induced HUVEC proliferation by 45% (145.4 +/- 7.2%) and 27% (126.9 +/- 4.2%), respectively; (iv) on FGF-induced HUVEC migration, fucosylated chondroitin sulfate (10 microg/ml) had a strong enhancing effect with FGF-1, +122% (222.2 +/- 15.8%), three times higher than that of heparin, and a lower enhancing effect with FGF-2, +43% (142.7 +/- 4.6%), whereas heparin had no effect; (v) fucosylated chondroitin sulfate stimulated TFPI release, mainly on the free form. +98% (198.2 +/- 25%). In addition, the structural features of the polysaccharide associated with its biological activity were resolved using chemically modified fucosylated chondroitin sulfates. Sulfated fucose branches groups are essential to the potentiating effect of the polysaccharide on HUVEC proliferation and migration. Surprisingly, removal of fucose branches from the fucosylated chondroitin sulfate did not abolish TFPI release. Finally, partial reduction of the glucuronic acid carboxyl groups limited the potentiating effect on HUVEC proliferation and migration but did not affect TFPI release. In conclusion, this fucosylated chondroitin sulfate from invertebrate origin reveals useful properties for an antithrombotic agent: inhibition of SMC proliferation, enhancement of endothelium wound repair and TFPI release. These properties on vascular cells, associated with a low bleeding tendency and an antithrombotic activity, strongly suggest its potential use as a new therapeutic agent in arterial thrombosis and restenosis, with a more favorable effect than heparin.

Fucoidan, as heparin, induces tissue factor pathway inhibitor release from cultured human endothelial cells.

Fucoidan, a sulfated polysaccharide extracted from brown seaweeds, has antithrombotic properties, the mechanism of which is not yet completely understood. Tissue factor pathway inhibitor (TFPI), which regulates the tissue factor-dependent pathway of blood coagulation, is released from the endothelium by heparin, a mechanism contributing to its antithrombotic activity. In this study, we demonstrated that fucoidan, as heparin, induces TFPI release from cultured human umbilical vein endothelial cells (HUVEC). The TFPI accumulation in the HUVEC supernatants depends on the incubation time and polysaccharide concentration. After 30 to 60 minutes of incubation, TFPI concentration (total antigen level) was twice higher in the presence of both polysaccharides than in their absence. After one hour of incubation, in the presence of increasing concentrations of each polysaccharide, an optimal stimulation was observed for 0.5 microg/ml of fucoidan and 5 microg/ml of heparin, as evidenced by a raise of the basal TFPI level: a 2-fold increase for the total antigen and a 3-fold increase for the free antigen. These data suggest that TFPI released from vascular endothelial cells may contribute to the antithrombotic effect of fucoidan.

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin.

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.