Platelet rich plasma extract promotes angiogenesis through the angiopoietin1-Tie2 pathway

Highlights

• Platelet rich plasma enhances angiogenesis through the Ang1-Tie2 pathway in vitro.

• Platelet rich plasma enhances mouse neonatal retinal angiogenesis.

• Platelet rich fibrin recapitulates angiogenesis inside the matrix in vivo.

Abstract

Development and regeneration of tissues and organs require precise coordination among endothelial, epithelial and mesenchymal morphogenesis. Angiogenesis plays key roles in normal development, wound healing, recovery from ischemic disease, and organ regeneration. It has been recognized that the combination of various angiogenic factors in an appropriate physiological ratio is critical for long-term functional blood vessel formation. Here we show that mouse soluble platelet-rich-plasma (PRP) extract, which includes abundant angiopoetin-1 (Ang1) and other angiogenic factors, stimulates endothelial cell growth, migration and differentiation in cultured human dermal microvascular endothelial cells in vitro and neonatal mouse retinal angiogenesis in vivo. Mouse platelet rich fibrin (PRF) matrix, the three-dimensional fibrin matrix that releases angiogenic factors with similar concentrations and proportions to the PRP extract, also recapitulates robust angiogenesis inside the matrix when implanted subcutaneously on the living mouse. Inhibition of Ang1-Tie2 signaling suppresses PRP extract-induced angiogenesis in vitro and angiogenic ability of the PRF matrix in vivo. Since human PRP extract and PRF matrix can be prepared from autologous peripheral blood, our findings may lead to the development of novel therapeutic interventions for various angiogenesis-related diseases as well as to the improvement of strategies for tissue engineering and organ regeneration.

Introduction

Angiogenesis is indispensable for organ development and homeostasis (Carmeliet and Tessier-Lavigne, 2005, Chung and Ferrara, 2011, Heinke et al., 2012). In adults, appropriate angiogenesis plays important roles in wound healing, recovery from ischemic diseases and tissue regeneration (Banai et al., 1994, Barrientos et al., 2008, Navaratna et al., 2009, Takeshita et al., 1994, Werner and Grose, 2003). Deregulation of angiogenesis contributes to the pathogenesis of many diseases, including cancer, retinopathy, chronic lung disease and arthritis (Carmeliet and Jain, 2011, Chung and Ferrara, 2011, Koch and Claesson-Welsh, 2012, Voelkel et al., 2007). Importantly, blood vessels, generated through the process of angiogenesis, not only deliver oxygen and nutrients but also provide instructive regulatory signals to surrounding cells in the local tissues and thereby play key roles in organ morphogenesis and regeneration (Butler et al., 2010, Crivellato, 2011, Crivellato et al., 2007, Ding et al., 2010, Ding et al., 2011, Matsumoto et al., 2001, Sakaguchi et al., 2008). Thus, in order to regenerate damaged organs or engineer organs, we need to modulate or recapitulate local angiogenesis. Currently, most angiogenic therapies rely on the use of a single angiogenic factor (e.g., vascular endothelial growth factor (VEGF)) (Navaratna et al., 2009, Wang et al., 2013). However, it is becoming clear that angiogenesis operates through the cooperation of a variety of angiogenic factors and their receptors (Carmeliet and Jain, 2011, Chung and Ferrara, 2011, Herbert and Stainier, 2011). Therefore, the use of a single angiogenic factor does not ensure stable angiogenesis and the combination of various angiogenic factors in the appropriate physiological ratio is rather preferred to generate long-term functional blood vessels in tissue (Emanueli and Madeddu, 2005, Yancopoulos et al., 2000, Zhou et al., 2007).

In addition to coagulation factors, platelets also store and release many bioactive angiogenic factors including platelet-derived epidermal growth factor (PD-EGF), platelet-derived growth factor (PDGF), VEGF, basic fibroblast growth factor (bFGF), angiopoietins (Angs) and transforming growth factor beta (TGF-β) (Italiano et al., 2008, Klement et al., 2009, Pintucci et al., 2002), making platelets important players in angiogenesis (Kisucka et al., 2006, Patzelt and Langer, 2012, Pinedo et al., 1998). They attract endothelial cells, macrophages, mesenchymal stem cells and osteoblasts to enhance angiogenesis, tissue regeneration and wound healing (Borzini and Mazzucco, 2005, Coppinger et al., 2004, Demidova-Rice et al., 2012, Jurk and Kehrel, 2005, Nachman and Rafii, 2008, Weyrich et al., 2009). Consequently, platelet rich plasma (PRP), a platelet extract that contains combinations of various angiogenic factors, has been extensively used in the orthopedic field for the last two decades (Everts et al., 2006, Sanchez-Gonzalez et al., 2012). Recently, platelet rich fibrin (PRF) matrix, which stably releases platelet-derived growth factors, has been used as a three-dimensional (3D) scaffold to accelerate wound healing and tissue engineering in the orthopedic and periodontal fields (Dohan Ehrenfest et al., 2009, Roy et al., 2011). In this report, we found that mouse PRP extract includes abundant Ang1 and other angiogenic factors. Ang1, a secreted 70 kDa glycoprotein of the angiopoietin family, is a ligand for the Tie2 receptor and Ang1-Tie2 signaling plays a pivotal role in angiogenesis during development and regeneration (Jeansson et al., 2011, Suri et al., 1996, Suri et al., 1998). Given its role in stabilizing blood vessel integrity and inducing distinctive vascular remodeling through highly organized angiogenesis (Suri et al., 1998), Ang1-Tie2 signaling has been targeted as a promising candidate for therapeutic angiogenesis and vascular protection (Koh, 2013). Since platelets store and release Ang1 and thus potentially maintain vascular integrity (Huang et al., 2000, Li et al., 2001), we explored whether Ang1-Tie2 signaling mediates the angiogenic effects of PRP and PRF in vitro and in vivo.

Here we characterized the angiogenic ability of PRP extract from mouse whole blood on cultured microvascular endothelial cells in vitro and on mouse neonatal retinal angiogenesis in vivo. We also engineered 3D PRF matrix, which releases angiogenic factors with similar proportion to the PRP extract, and found that robust angiogenesis is induced inside the matrix through Ang1-Tie2 signaling when implanted subcutaneously on the living mouse. Since human PRP extract and PRF matrix are (1) Food and Drug Administration (FDA)-approved and have been extensively used in the orthopedic field in humans (Everts et al., 2006, Foster et al., 2009, Kurita et al., 2011, Sanchez-Gonzalez et al., 2012), (2) cost effective, (3) made from autologous patients' peripheral blood and hence elicit little immunological rejection and minimize unnecessary pathogen transfer, and (4) abundant in cell adhesion molecules and angiogenic factors in a physiological ratio (Everts et al., 2006, Sanchez-Gonzalez et al., 2012), our findings with mouse PRP extract and PRF matrix will lead to the development of new therapeutic strategies for angiogenesis-related diseases and significant advances in clinical practice in the field of regenerative medicine.