Release Of Heparin-binding Growth Factor Research Articles

The mechanism of action of a novel neuroprotective low molecular weight dextran sulphate: New platform therapy for neurodegenerative diseases like Amyotrophic Lateral Sclerosis.

Background: Acute and chronic neurodegenerative diseases represent an immense socioeconomic burden that drives the need for new disease modifying drugs. Common pathogenic mechanisms in these diseases are evident, suggesting that a platform neuroprotective therapy may offer effective treatments. Here we present evidence for the mode of pharmacological action of a novel neuroprotective low molecular weight dextran sulphate drug called ILB®. The working hypothesis was that ILB® acts via the activation of heparin-binding growth factors (HBGF). Methods: Pre-clinical and clinical (healthy people and patients with ALS) in vitro and in vivo studies evaluated the mode of action of ILB®. In vitro binding studies, functional assays and gene expression analyses were followed by the assessment of the drug effects in an animal model of severe traumatic brain injury (sTBI) using gene expression studies followed by functional analysis. Clinical data, to assess the hypothesized mode of action, are also presented from early phase clinical trials. Results: ILB® lengthened APTT time, acted as a competitive inhibitor for HGF-Glypican-3 binding, effected pulse release of heparin-binding growth factors (HBGF) into the circulation and modulated growth factor signaling pathways. Gene expression analysis demonstrated substantial similarities in the functional dysregulation induced by sTBI and various human neurodegenerative conditions and supported a cascading effect of ILB® on growth factor activation, followed by gene expression changes with profound beneficial effect on molecular and cellular functions affected by these diseases. The transcriptional signature of ILB® relevant to cell survival, inflammation, glutamate signaling, metabolism and synaptogenesis, are consistent with the activation of neuroprotective growth factors as was the ability of ILB® to elevate circulating levels of HGF in animal models and humans. Conclusion: ILB® releases, redistributes and modulates the bioactivity of HBGF that target disease compromised nervous tissues to initiate a cascade of transcriptional, metabolic and immunological effects that control glutamate toxicity, normalize tissue bioenergetics, and resolve inflammation to improve tissue function. This unique mechanism of action mobilizes and modulates naturally occurring tissue repair mechanisms to restore cellular homeostasis and function. The identified pharmacological impact of ILB® supports the potential to treat various acute and chronic neurodegenerative disease, including sTBI and ALS.

Heparin-conjugated alginate multilayered microspheres for controlled release of bFGF

In order to effectively immobilize and control release of basic fibroblast growth factor (bFGF) from alginate microspheres, heparin-conjugated alginate (H-Alg) was first synthesized by covalent binding. Then multilayered H-Alg microspheres (multilayered microspheres) were fabricated via an electrostatic droplet generation technique followed by a layer-by-layer (LbL) self-assembly technique. Several techniques such as Fourier transform infrared spectroscopy (FTIR), 1H-NMR, zeta potential analysis and scanning electron microscopy (SEM) were used to characterize the properties of H-Alg (FTIR and 1H-NMR) and multilayered microspheres (FTIR, zeta potential analysis and SEM). bFGF binding efficiency, release profiles of bFGF from multilayered microspheres and the biological activity of released bFGF were well investigated. It was found that the bFGF binding efficiency of H-Alg microspheres was increased up to five times higher than that of the alginate microspheres. Additionally, the release profiles of bFGF from multilayered microspheres were sustained for two weeks with relieved initial burst release, and the release rate to bFGF could be regulated by controlling the number of deposited layers. Importantly, the released bFGF still retained its biological activity as assessed by the in vitro proliferation of NIH-3T3 mouse fibroblasts. In conclusion, this study presented an easy yet effective method for the controlled, sustained release of heparin-binding growth factors, using polyelectrolyte multilayer-coated heparin-conjugated alginate microspheres.

Assessment of in vitro impact of low molecular weight heparin on expression of heparanase and heparin binding growth factors in the endometrium of women with impaired reproduction

Heparin has a beneficial effect in the treatment of recurrent miscarriages and positively affects implantation rates in the IVF procedure in women with reproductive disorders not associated with thrombophilia. Several studies have indicated that heparin, by blocking the enzymatic activity of heparanase, may affect the structure and function of the extracellular matrix (ECM) and related growth factors. Disturbances in the remodeling (ECM) are believed to be the potential cause of implantation disorders and recurrent miscarriages. The aim of the study was the evaluation, on an in vitro model, of the effect of low molecular weight heparin (LMWH) on the expression of heparanase (HPSE) and, important for successful implantation and invasion of trophoblast, heparan sulfate (HS)--binding growth factors, i.e., heparin-binding epidermal growth factor-like (HB-EGF), vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF2) in the endometrium, during the implantation window in women with recurrent miscarriage. Biopsy samples, obtained from 10 patients with two or more unexplained miscarriages, were used to construct a co-culture of glandular epithelial cells and stroma. Endometrium in vitro model was supplemented with steroid hormones and enoxaparin 5 ug/ml. Using the qPCR, we assessed, relative levels of the HPSE, HB-EGF, VEGF-A and FGF2 transcripts in glandular epithelium and stroma in cell culture. Using ELISA, we measured con- centrations of the mentioned above factors in culture medium. A statistically significant increase in the relative level of HPSE, HB-EGF VEGF-A, FGF2 transcripts in the cells of the glandular epithelium and stroma (p<0.001), as well as their increased concentration in the medium of cultures treated with steroid hormones (p<0.001) were observed. However we found no effect of LMWH supple- mentation on the level of the investigated factors. Our results show that the importance of the HPSE hydrolytic activity in the endometrium, during the implantation window, may have a secondary function, and/or that beneficial effects of LMWH in women with impaired reproduction have no significant, direct connection with the, catalyzed by HPSE, reconstruction of the ECM and with release of heparin-binding growth factors.

Minimizing the passive release of heparin-binding growth factors from an affinity-based delivery system

We consider a mathematical model that describes the leakage of heparin-binding growth factors from an affinity-based delivery system. In the delivery system, heparin binds to a peptide which has been covalently cross-linked to a fibrin matrix. Growth factor in turn binds to the heparin, and growth factor release is governed by both binding and diffusion mechanisms, the purpose of the binding being to slow growth factor release. The governing mathematical model, which in its original formulation consists of six partial differential equations, is reduced to a system of just two equations. It is usually desirable that there be no passive release of growth factor from a device, with all of the growth factor being held in place via binding until such time as it is actively released by invading cells. However, there will inevitably be some passive release, and so it is of interest to identify conditions that will make this release as slow as possible. In this paper, we identify a parameter regime that ensures that at least a fraction of the growth factor will release slowly. It is found that slow release is assured if the matrix is prepared with the concentration of cross-linked peptide greatly exceeding the dissociation constant of heparin from the peptide, and with the concentration of heparin greatly exceeding the dissociation constant of the growth factor from heparin. Also, for the first time, in vitro experimental release data are directly compared with the theoretical release profiles generated by the model. We propose that the two stage release behaviour frequently seen in experiments is due to an initial rapid out-diffusion of free growth factor over a diffusion time scale (typically days), followed by a much slower release of the bound fraction over a time scale depending on both diffusion and binding parameters (frequently months).

Sulfated glyco-block copolymers with specific receptor and growth factor binding to support cell adhesion and proliferation

We report on the successful preparation of thin glyco-block copolymer films with a combined thermoresponsive and heparin-like functionality. The copolymers were synthesized from poly( N-isopropylacrylamide) and glucose units and were covalently fixed onto glass supports by means of low pressure plasma cross-linking. The thin films retain the thermoresponsive characteristics of poly( N-isopropylacrylamide) with a transition temperature around 33 °C. Additionally, it could be shown that sulfation of the glucose moieties introduces a heparin-like functionality to the films. An increase in binding of basic fibroblast growth factor (bFGF) as well as specific adhesion of endothelial cells and hematopoietic progenitor cells could be demonstrated. The functional coupling of bFGF to the glyco-block copolymer surfaces was further proven by the dose-dependent response of endothelial cell proliferation. The results show that the newly synthesized glyco-block copolymers allow for the preparation of biomimetic surfaces with dual functionalities of thermoresponsive and heparin-like characteristics for the application in cell culture experiments with specific binding and release of heparin-binding growth factors and cell adhesion receptors.

Transforming growth factor β‐3 bound with sulfate polysaccharide in synthetic extracellular matrix enhanced the biological activities for neocartilage formation in vivo

The thermoreversible hydrogel of poly(N-isopropylacrylamide-co-acrylic acid) [p(NiPAAm-co-AAc)] was utilized in an injectable protein (growth factor) delivery system for use in tissue regeneration protocols. The transforming growth factor beta (TGF beta) proteins, which have been shown to bind to heparin, have been well established to induce chondrogenic differentiation in chondrocytes. In this study, we assessed the effects of heparin on the TGF beta-3 activities in rabbit chondrocytes embedded in thermoreversible hydrogel. The hydrogels embedded with chondrocytes and heparin-bound TGF beta-3 were injected subcutaneously into nude mice, which were monitored for up to 8 weeks after injection. P(NiPAAm-co-AAc) hydrogels with TGF beta-3 were compared with the hydrogels with heparin bound to TGF beta-3 for the assessment of the effects of heparin binding on chondrogenic differentiation. To monitor the release of growth factor via in vivo analysis, we assessed the Cy5.5-NHS-conjugated growth factors via the bioimaging method. In terms of the release of growth factors, the release of heparin-binding growth factor was slower than that of the growth factor itself. Additionally, the proliferation rate and cartilage-specific ECM production were both found to be significantly higher in the presence of heparin binding than was observed with the controls. The quantity of cartilage-associated ECM proteins was assessed via immunohistochemical staining (collagen type II) and safranin-O staining. These data indicate the potential use of heparin-binding TGF beta-3 for the reconstruction of neocartilage formation.

Controlled release of heparin-binding growth factors using heparin-containing particulate systems for tissue regeneration

The strategy of growth factor delivery to specific sites for therapeutic applications has been considered an essential process in biomedical fields despite some obstacles, such as a non-controlled release with initial burst. This article focuses on particulate systems using heparin for the controlled delivery of heparin-binding growth factors (HBGFs), an emerging area in the tissue engineering field. Since heparin has been widely utilized for growth factor delivery due to its electrostatic nature and specific affinity with HBGFs, heparin-containing polymeric particulates can be utilized as functional carriers to deliver growth factors in a controlled manner. In particular, examples of the HBGF delivery systems containing heparin, perspectives and potential applications are described and discussed.

Decidual Heparanase Activity Is Increased During Pregnancy in the Baboon (Papio anubis) and in In Vitro Decidualization of Human Stromal Cells1

Implantation is a complex process involving interactions between the embryo and the uterus. Adhesion, remodeling of the maternal vasculature, and decidualization are crucial events necessary for successful implantation to occur. Heparanase (HPSE), an endo-beta-D-glucuronidase, cleaves heparan sulfate at specific sites, leading to release of growth factors that may be involved in decidualization and remodeling of the maternal vasculature. HPSE also can function as a cell adhesion molecule. The aim of this study was to determine the expression of HPSE in the uteri of nonpregnant and pregnant baboons as well as in human stromal fibroblasts decidualized in vitro. We examined the localization and expression of HPSE using immunohistochemistry, Western blotting, RT-PCR, and activity assays. In nonpregnant baboon uteri, HPSE expression was localized to the apical surface of the glandular epithelia and in glandular secretions. However, in pregnant baboon uteri, HPSE was localized primarily in decidua. Uteri obtained at midpregnancy had higher heparanase activity compared with the nonpregnant uteri. A slight increase in HPSE expression was observed in human stromal fibroblasts decidualized in vitro. HPSE and HPSE2 mRNA transcripts were present in both decidualized tissue and cells. Increases in heparanase activity in the decidua from pregnant baboon uteri compared with tissue from nonpregnant animals and in human stromal fibroblasts decidualized in vitro suggest that HPSE plays a role in extracellular matrix remodeling and in increasing heparin-binding growth factor release during embryo implantation.

Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor

Synthetic hydrogel mimics of the extracellular matrix (ECM) were created by crosslinking a thiol-modified analog of heparin with thiol-modified hyaluronan (HA) or chondroitin sulfate (CS) with poly(ethylene glycol) diacrylate (PEGDA). The covalently bound heparin provided a crosslinkable analog of a heparan sulfate proteoglycan, thus providing a multivalent biomaterial capable of controlled release of basic fibroblast growth factor (bFGF). Hydrogels contained >97% water and formed rapidly in <10 min. With as little as 1% (w/w) covalently bound heparin (relative to total glycosaminoglycan content), the rate of release of bFGF in vitro was substantially reduced. Total bFGF released increased with lower percentages of heparin; essentially quantitative release of bFGF was observed from heparin-free hydrogels. Moreover, the hydrogel-released bFGF retained 55% of its biological activity for up to 28 days as determined by a cell proliferation assay. Finally, when these hydrogels were implanted into subcutaneous pockets in Balb/c mice, neovascularization increased dramatically with HA and CS hydrogels that contained both bFGF and crosslinked heparin. In contrast, hydrogels lacking bFGF or crosslinked heparin showed little increase in neovascularization. Thus, covalently linked, heparin-containing glycosaminoglycan hydrogels that can be injected and crosslinked in situ constitute highly promising new materials for controlled release of heparin-binding growth factors in vivo.

Development of fibrin derivatives for controlled release of heparin-binding growth factors

The goal of this work was to develop a growth factor delivery system for use in wound healing that would provide localized release of heparin-binding growth factors in a biomimetic manner, such that release occurs primarily in response to cell-associated enzymatic activity during healing. A key element of the drug delivery system was a bi-domain peptide with an N-terminal transglutaminase substrate and a C-terminal heparin-binding domain, based on antithrombin III. The bi-domain peptide was covalently cross-linked to fibrin matrices during coagulation by the transglutaminase activity of factor XIIIa and served to immobilize heparin electrostatically to the matrix, which in turn immobilized the heparin-binding growth factor and slowed its passive release from the matrix. Basic fibroblast growth factor (bFGF) was considered as an example of a heparin-binding growth factor, and cell culture experimentation was performed in the context of peripheral nerve regeneration. A mathematical model was developed to determine the conditions where passive release of bFGF would be slow, such that active release could dominate. These conditions were tested in an assay of neurite extension from dorsal root ganglia to determine the ability of the delivery system to release bioactive growth factor in response to cell-mediated processes. The results demonstrated that bFGF, immobilized within fibrin containing a 500-fold molar excess of immobilized heparin relative to bFGF, enhanced neurite extension by up to about 100% relative to unmodified fibrin. A variety of control experiments demonstrate that all components of the release system are necessary and that the bi-domain peptide must be covalently bound to the fibrin matrix. The results thus suggest that these matrices could serve as therapeutic materials to enhance peripheral nerve regeneration through nerve guide tubes and may have more general usefulness in tissue engineering.