|Program and Abstracts
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Hypoxia Preconditioned Plasma (HPP) and its Role in promoting Angiogenesis-supported Wound Regeneration
P.Niclas Broer, MD, Ektoras Hadjipanayi, MD PhD, Anna T. Bauer, MD, Philipp Moog, MS, Sarah von Isenburg, MD, Milomir Ninkovic, MD PhD, Hans-Günther Machens, MD PhD, Arndt F. Schilling, MD PhD.
Klinikum rechts der Isar , Technische Universität München, München, Germany.
PURPOSE: Hypoxic preconditioning of cells has been shown to be a promising strategy for generating complex angiogenic factor protein mixtures, which can be delivered to ischemic tissues in order to support regeneration. Peripheral blood cells (PBCs) are an ideal autologous cell type for hypoxic stress stimulation, given that their ease of harvest and ample availability circumvents lengthy cell population expansion cycles. Hypoxia preconditioned plasma (HPP), i.e. plasma derived following extracorporeal conditioning of anticoagulated blood under physiological temperature (37°C) and physiological hypoxia (1-5% O₂), represents a novel form of conditioned culture medium. Here, we tested the angiogenic potential of HPP factor-loaded gels in in vitro Matrigel assays, and assessed its regenerative effect in two select patients.
METHODS: Peripheral blood (5 ml) was collected into EDTA-Vacutainer tubes from 19 healthy subjects (9 males; age = 38.3 ± 6 yrs; 10 females; age = 30.7 ± 2.2 yrs). Samples were cultured in a normoxic or hypoxic incubator (3% O₂) for 7 days. Plasma media were analyzed with a
Proteome Assay, and ELISA for vascular endothelial growth factor (VEGF), angiogenin (ANG), and thrombospondin-1 (TSP-1). Hypoxia-conditioned plasma media were co-incubated with type I collagen gel carriers to enable factor loading. The angiogenic potential of gel releasates was investigated by assessing tubule formation in calcein-stained human umbilical vein endothelial cells, seeded on factor-reduced Matrigel. The extent of capillary-like network formation was examined by counting the number of tubules and nodes, and quantification of tubule length. The ability of releasates to induce directional endothelial cell invasion through Matrigel-coated PET membranes was carried out by exclusively visualizing invasive cells labeled with DilC12 Fluorescent Dye. Gel releasates were also tested in an aortic ring model to assess their ability to induce microvessel sprouting.
RESULTS: It was found that, relative to the normoxic baseline, hypoxia
significantly affected the expression of a range of angiogenesis-related
factors including VEGF, ANG and TSP-1. HPP obtained after 5 days culture had a five-fold higher concentration of the angiogenesis-stimulator VEGF, and a two-fold lower concentration of the angionesis-inhibitor TSP-1, compared to fresh plasma (p<0.05). While both factors eventually underwent down-regulation over time under hypoxia, there was significant variation in their temporal expression profile. Releasates of HPP factor-loaded gels increased endothelial cell tubule formation and directional migration relative to control medium (Fig.1), while microvessel sprouting was found to be significantly improved compared to recombinant VEGF (p<0.05). Two patients with non-healing wounds treated with application of 5% HPP factor-containing emulsion showed complete wound closure within 20 and 50 days, respectively.
CONCLUSION: Our findings indicate that HPP provides a potent autologous, angiogenic medium. It can be readily generated, and its
factor composition is determined by the physiological and patient-specific
cellular responses that mediate effective wound healing. Observations made in preliminary clinical applications merit further investigation in a controlled clinical trial.
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