Fernando Siñeriz

Synthesis and biological activities of a library of glycosaminoglycans mimetic oligosaccharides

Biologically active oligosaccharides related to glycosaminoglycans are accumulating increased attention because of their therapeutic potential and for their value in mechanistic studies. Heparan mimetics (HMs) are a family of dextran based polymer known to mimic the properties of glycosaminoglycans, and particularly those of heparan sulfates, as to interact with heparin binding proteins. HMs have shown to stimulate tissue repair in various animal models. Here, we use different methods to depolymerize HMs in order to produce a library of related oligosaccharides and study their biological activities. Since HMs were resistant to endoglycanases activities, depolymerization was achieved by chemical approaches. In vitro biological studies showed that HM oligosaccharides can differentially potentiate FGF-2 mitogenic and antithrombotic activities. In vivo, a selected oligosaccharide (H-dp12) showed to be able to regenerate tissue almost as well as the related polymeric product. The very low anticoagulant activity and high biological activity of low mass oligosaccharides give to these products a new therapeutic potential.

Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies.

Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR(4131)] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR(4131)] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC.

Synthesis of a trisulfated heparan sulfate disaccharide analog and its use as a template for preliminary molecular imprinting studies.

A heparan sulfate disaccharide analog was synthesized by a multistep route. This synthesis was designed in such a way that one intermediate could be selectively deprotected to provide versatility during both synthesis and homologation of heparan sulfate related polysaccharides. Non-covalent imprinted polymers were prepared by using the synthesized disaccharide as a template and a primary amine functionalized acrylate as the key functional monomer suitable for specific sulfated sugar recognition. The binding of related sugars to the imprinted and non-imprinted polymers and the binding of template to the chemically modified polymers have been also investigated.

Heparin affin regulatory peptide modulates the endogenous anticoagulant activity of heparin and heparan sulphate mimetics.

Pleiotrophin, also known as heparin affin regulatory peptide (HARP), is a growth factor expressed in various tissues and cell lines. In this work, HARP was tested for its capacity to modulate the anticoagulant activity of heparin and heparan sulphate mimetics (OTR4120). We used both in vitro and in vivo assays. HARP was found to be differently effective for neutralization of the anticoagulant activity of the mimetic heparan sulphate (OTR4120) and heparin in purified system and human plasma. HARP was shown to compete with both antithrombin and thrombin for binding to heparin and to OTR4120, respectively. In the presence of OTR4120, the Vmax was constant and the calculated maximum velocity was 1.56 U/min; the thrombin Km value (0.011 nM) was affected by HARP concentrations. The Km (HARP) value was 0.085 nM, which is consistent with high affinity of HARP to OTR4120. Under the same conditions, initial velocity patterns for antithrombin–heparin were determined in the presence or in the absence of HARP. The antithrombin value Km (0.022 nM) was affected by HARP (0.077 nM). HARP exhibits efficacy equivalent to or greater than protamine. Interestingly, intraperitoneally administered HARP decreased the anticoagulant activity of heparin and of OTR4120 in mice. Taken together, these data provide the first evidence for a physiological role of HARP in the modulation of anticoagulant activity of heparin and heparin‐like material.

Poly(ethylene glycol acrylate)-functionalized hydrogels for heparan sulfate oligosaccharide recognition

Heparan sulfates are complex polysaccharides belonging to the family of glycosaminoglycans that participate to the regulation of cell behavior and tissue homeostasis. The biological activities conferred to heparan sulfates are largely dependent on the content and positioning of the sulfate groups along their saccharidic units. At present, identification of particular sulfation patterns in biologically relevant heparan sulfate sequences remains challenging. Although several approaches for structure analysis exist, the complexity of heparan sulfates makes new and original approaches still required. Here, we used molecular imprinting technologies to prepare a library of polyethylene glycol acrylate functionalized hydrogels with the aim to investigate their applicability as specific recognizing systems for fondaparinux, a synthetic pentasaccharide analog to the antithrombin binding site of heparin. Adequate choice of the hydrogel composition and controlling rebinding conditions were important determinants for improving the sulfated oligosaccharide recognition specificity and selectivity. Our results suggest that molecular imprinting approaches could be a possibility for the specific recognition of biologically active sequences in heparan sulfates.

Randomized controlled trial demonstrates the benefit of RGTA® based matrix therapy to treat tendinopathies in racing horses

A randomized controlled trial was performed on racing horses, to evaluate the efficacy of a new class of therapeutic agents in regenerative medicine—ReGeneraTing Agents® (RGTA®), to treat tendinopathies. Preliminary uncontrolled studies on tendon healing in racing horses with RGTA® (OTR4131)—Equitend® showed encouraging results, justifying performing a randomized, controlled, multicenter study with a two-year racing performance follow up. The objective of this study was to evaluate the effect of Equitend® versus placebo on acute superficial digital flexor tendonitis in racing French Standardbred Trotters (ST). Twenty-two ST were randomly and blindly assigned to receive with a ratio of 2 to 1, a single Equitend® (n = 14) or placebo (n = 8) intralesional injection under ultrasonographic guidance. Horses were evaluated over 4 months, by clinical and ultrasonographic evaluations (day 0, months 1, 2, 4), and their racing performances followed up over the 2 years after treatment. During the first month of treatment, a significant decrease in the cross-sectional area (CSA) was found in the Equitend® group (p = 0.04). After 4 months, the number of Equitend® treated horses with an improved CSA was significantly higher than the placebo-treated horses (p = 0.03571). The Equitend® group returned to their pre-injury performance level, racing in, and winning, significantly more races than the placebo group (p = 0.01399 and 0.0421, respectively). Furthermore, recurrence was significantly higher in the placebo group than in the Equitend® group (71.4% vs 16.6%, p = 0.02442). In conclusion, we measured a significant, short-term, reduction effect on CSA and demonstrated a long-term beneficial effect of intralesional injection of Equitend® for the treatment of superficial digital flexor tendonitis on racing ST, racing 2. 3 times more often than placebo, with 3.3 times fewer recurrences maintaining pre-injury performance level. This study may open the way for the development of a human treatment of tendonitis.