European Journal of Pharmaceutics and Biopharmaceutics
Research paperPharmacologically active microcarriers for endothelial progenitor cell support and survival☆,☆☆
Graphical abstract
Pharmacologically active microcarriers (FN-PAMs), made with poly (D, L-lactic-coglycolic acid) releasing VEGF-A (FN-VEGF-PAMs) were assessed as support and survival for early endothelial progenitor cells (eEPCs). Data indicate that FN-VEGF-PAMs increased eEPCs ability to adhere to microcarriers which also supported their survival. VEGF-A released by FN-VEGF-PAMs stimulated in vitro HUVEC migration and proliferation.
Introduction
Ischemic tissue disease remains one of the primary causes of morbidity and mortality. Conventional therapies are not yet sufficient to promote adequate recovery of the blood flow in ischemic areas [1]. Initially, growth factor-based approaches without additional cell application were widely used to enhance neovascularization, but rapid protein degradation in vivo hinders sustainable success [2]. More recently, cell-based therapies have attracted great interest, since improved neovascularization in both experimental hind limb ischemia models and clinical studies has demonstrated [3], [4]. Early endothelial progenitor cells (eEPCs) are able to promote vasculogenesis: they can be isolated from blood, bone marrow, and blood vessels [5], [6], expanded ex vivo, and then transplanted into damaged tissue. However, this technique has some drawbacks that limit the real efficacy of the treatment, such as the death of a large percentage of transplanted cells within a few hours from their injection and their clearing through both lymphatic and blood vessels [7]. Vasculogenic progenitor cell therapy for ischemic diseases still requires further optimization to justify its clinical application, and the outcome of patients treated with cell therapy is still poor. Optimization protocols for enhanced cell therapy have been proposed [8], [9]. The disadvantages associated with cell injection could be overcome by using pharmacologically active scaffolds that allow cell delivery and enhance the survival of stem cells.
Among the various polymers that are commonly used for the production of scaffolds suitable for regenerative medicine, biodegradable and biocompatible microparticles made of poly(d,l-lactic-coglycolic acid) (PLGA) [10] commercially available product with current human application have been used [11], [12]. These carriers with a biomimetic surface may be loaded with growth factors stimulating either transported stem cells or resident mature cells [10]. These molecules may improve survival and differentiation of the cells and may also affect the immediate environment, thus allowing better graft integration. Cell carriers or microspheres delivering growth factors have been used in animal models of neurodegenerative diseases [13], [14], [15], [16]. Pharmacologically active microcarriers (PAMs) transporting stem or progenitor cells with a biomimetic surface and delivering growth factors have also been effectively used for tissue repair in Parkinson’s disease [17], [18] and to promote cartilage formation [19]. However, no information is available on the use of PAMs for the support eEPCs.
The aim of the present study was to investigate whether eEPCs could be cultured onto PAMs with a fibronectin biomimetic surface (FN-PAMs) through evaluation of their adhesion, survival, and differentiation. We also compared eEPC adhesion and phenotype when cultured with FN-PAMs loaded with VEGF-A (FN-VEGF-PAMs). The effect of FN-PAMs delivering VEGF-A on human mature endothelial cell activation was also investigated.
Our data demonstrate that FN-PAMs were a good support for eEPCs. Moreover, the release of VEGF-A by FN-PAMs supported the eEPC phenotype, increased their survival and stimulated mature endothelial cell migration and proliferation.
Section snippets
Preparation of polymeric biodegradable microspheres of PLGA
Poly(lactic-co-glycolic acid) (PLGA)-microspheres of an average diameter ranging from around 30 to 60 μm were prepared using a previously described emulsion solvent extraction–evaporation process [18], [20]. The total protein loading was 0.6% w/w of the amount of polymer, i.e. 0.1% VEGF165 (VEGF-A) (Peprotech, France) and 0.5% HSA. First, NaCl and glycofurol, a water-miscible protein non-solvent, were used to precipitate the proteins separately, as previously described [18]. For VEGF-A, an NaCl
eEPC adhesion to empty PAMs
Early EPCs at increasing concentrations, ranging from 5 × 104 to 18 × 105, were seeded in the presence of 0.5 mg empty FN-PAMs, in complete medium. The attached cells were observed under the microscope after 6, 12, 24, 48 h and 5 days. Results showed that the optimal EPC concentration (1.25 × 105/0.5 mg PAM) attached to FN-PAMs within a few hours (Fig. 2A) and increased in a time-dependent manner, with maximal effect after 48 h (Fig. 2E). It is possible, indeed, to observe many cells attached to the
Discussion
The formation of a functional vessel network via angiogenesis represents a crucial and critical step in the wound healing process and in the repair of ischemic tissue. Effective angiogenesis occurs as a result of the proliferation of pre-existing mature endothelial cells in response to stimuli released in the wound site, but may also involve the direct or indirect action of EPCs. Direct incorporation of EPCs into neo-vessels has been demonstrated together with the production of paracrine
Acknowledgements
Supported by Compagnia di San Paolo (Turin, Italy) and INRC (National Institute of Cardiovascular Research, Italy), Institut National de la Santé et de la Recherche Médicale and Angers-Loire Métropole.
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The aim of the present study was to investigate whether human early endothelial progenitor cells (eEPCs) could be efficiently cultured in pharmacologically active microcarriers (PAMs) coated with fibronectin (FN-PAMs), with or without controlled delivery of VEGF-A. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-PAMs releasing VEGF-A increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation.
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These in vitro data strongly sustain the use of FN-PAMs for supporting eEPCs growth, and their combined effect on progenitor and mature endothelial cells seems to be of potential interest for in vivo applications.