Graciela C. Calabrese

Low-molecular-weight heparin inhibition in classical complement activaton pathway during pregnancy

INTRODUCTION Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications. MATERIALS AND METHODS The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH(50) assay). RESULTS The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03+/-1.20 microg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 microg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG. CONCLUSIONS We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.

Dermatan sulfate/chitosan polyelectrolyte complex with potential application in the treatment and diagnosis of vascular disease

Cardiovascular disease is the largest single cause of morbid-mortality in the world. However, there is still no pharmaceutical treatment that directly targets the blood vessel wall instead of just controlling the risk factors. Here, we produced polyelectrolyte complexes (PECs) by a simple and reproducible polyelectrolyte complexation method between low molecular mass dermatan sulfate (polyanionic polysaccharide) and chitosan (polycationic polysaccharide), and evaluated the cellular uptake by vascular endothelial cells. The composition and the composition homogeneity of PECs were confirmed by (13)C-CP-MAS spectroscopy and by polyacrylamide gel electrophoresis, respectively. The hydrodynamic radius, determined by dynamic light scattering, was 729±11nm. PECs were not cytotoxic for a murine heart endothelium-derived cell line. Fluorescent confocal microscopy showed the specific uptake of fluorescently-labeled PECs by endothelial cells when they were cultured alone or in the presence of macrophages. Overall, these findings confirmed the potential of these PECs for targeting different agents to the vessel wall in the prevention, diagnosis, and therapy of vascular disease.

Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model.

Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high‐cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal‐to‐media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo‐intima rearrangement were observed in high‐cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high‐cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high‐cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high‐cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6‐sulphate to 4‐sulphate disaccharides was increased in high‐cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.

Low molecular mass dermatan sulfate modulates endothelial cells proliferation and migration.

Low molecular mass dermatan sulfate, obtained by depolymerization, induced the entrance in S phase of mitosis, enhanced the activity of matrix metalloproteinase-2, and could modulate cell migration of endothelial cells, through mechanisms independent of TNF-α autocrine regulation. LMMDS located at the injured sites could influence early stages of angiogenesis.

Antithrombin and first complement protein recognize the same active heparin fraction

Antithrombin (AT) high affinity of unfractionated heparin (UFH) resides in a specific pentasaccharide sequence. Heparin also regulates complement activity on the classical and the alternative pathways. Most experimental pieces of evidence accumulated show that these important activities reside in different segments of the heparin molecule. We demonstrated in previous papers that a low ionic strength and the presence of calcium ions are essential to detect specific interactions between glycosaminoglycans and proteins. Then these very strict conditions were used, and we demonstrated that the first protein complex of the human complement cascade recognizes in the UFH a fraction with very high anticoagulant activity. After isolation from the precipitate of the interaction, this fraction of heparin also contained the pentasaccharide sequence responsible for the great affinity with AT: in fact, it was strongly bound to a resin of AT agarose, and to detach it, an ionic strength of 0.6 M sodium chloride was required. In this way, the heparin regions responsible for the anticoagulant activity and also for the effects over the complement system were identified on the same short segment of the heparin molecule, which includes the active fraction of the glycosaminoglycan. The differences with early results could be explained by our experimental conditions of low ionic strength and the presence of calcium ions used for the interaction of the protein and the glycosaminoglycan.

Prostanoid production in endothelial and Kupffer liver cells from monocrotaline intoxicated rats

A single dose of monocrotaline, a pyrrolizidine alkaloid, was injected into rats in order to produce 25 (Group I) and 45 (Group II) days later a progressive and so called delayed liver injury. The present study investigated the prostanoid production of Kupffer cells and endothelial cells separated from Monocrotaline and saline (Group III) injected rat livers. Kupffer cells: formation of 6 keto Prostaglandin F1 a, the major prostacyclin metabolite, gradually decreased in Groups I vs II (P50.01) and in both Groups I and II vs Controls (P50.01). In addition Prostaglandin F2 a showed a significant increase in Groups I and II when compared to Group III, (P50.001), and Thromboxane B2 was present in both Groups of Monocrotaline treated animals, while it was not detectable in the control Group III. Endothelial cells:6ketoProstaglandin F1 a decreased in Groups I vs II. This differences was significant when compared, and compared to controls (Group III, P50.001). Prostaglandin E2 was detected only in Groups I and II. Prostaglandin F2 a and Thromboxane B2 could not be detected in any Group. Ultramicroscopy showed morphological cell damage in nonparenchymal cells in Monocrotaline intoxication in Group II, rats sacrified 45 days after the injection, while it shows normal features in those treated animals sacrified 25 days after the injection, as well as in control group. Conclusion: Asingle Monocrotaline injection produces, 25 and 45 days later, severe and progressive alterations in the prostanoid production in Kupffer and Endothelial cells, while ultramicroscopic alterations was only observed 45 days after the injection of Monocrotaline. A decreased production of vasodilators and the presence of vasoconstrictor prostanoids that can participate in the production of the circulatory derangements enhancing liver injury and portal hypertension were also observed.

VLDL Induced Modulation of Nitric Oxide Signalling and Cell Redox Homeostasis in HUVEC

High levels of circulating lipoprotein constitute a risk factor for cardiovascular diseases, and in this context, the specific role of the very-low-density lipoproteins (VLDL) is poorly understood. The response of human umbilical vein endothelial cells (HUVEC) to VLDL exposure was studied, especially focusing on the pathways involved in alteration of redox homeostasis and nitric oxide (NO) bioavailability. The results obtained by the analysis of the expression level of genes implicated in the NO metabolism and oxidative stress response indicated a strong activation of inducible NO synthase (iNOS) upon 24 h exposure to VLDL, particularly if these have been preventively oxidised. Simultaneously, both mRNA and protein expression of endothelial NO synthase (eNOS) were decreased and its phosphorylation pattern, at the key residues Tyr495 and Ser1177, strongly suggested the occurrence of the eNOS uncoupling. The results are consistent with the observed increased production of nitrites and nitrates (NOx), reactive oxygen species (ROS), 3-nitrotyrosine (3-NT), and, at mitochondrial level, a deficit in mitochondrial O2 consumption. Altogether, these data suggest that the VLDL, particularly if oxidised, when allowed to persist in contact with endothelial cells, strongly alter NO bioavailability, affecting redox homeostasis and mitochondrial function.

Learning from Synthetic Models of Extracellular Matrix; Differential Binding of Wild Type and Amyloidogenic Human Apolipoprotein A-I to Hydrogels Formed from Molecules Having Charges Similar to Those Found in Natural GAGs

Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases. The composition of these molecules is highly diverse and a key issue seems to be the equilibrium between physiological and pathological events. In order to have a model in which the composition of the matrix could be finely controlled, we designed and synthesized crosslinked hydrophilic polymers, the so-called hydrogels varying the amounts of negative charges and hydroxyl groups that are prevalent in GAGs. We checked and compared by fluorescence techniques the binding of human apolipoprotein A-I and a natural mutant involved in amyloidosis to the hydrogel scaffolds. Our results indicate that both proteins are highly retained as long as the negative charge increases, and in addition it was shown that the mutant is more retained than the Wt, indicating that the retention of specific proteins in the ECM could be part of the pathogenicity. These results show the importance of the use of these polymers as a model to get deep insight into the studies of proteins within macromolecules.