Use of Oral Anticoagulation and Diabetes Do Not Inhibit the Angiogenic Potential of Hypoxia Preconditioned Blood-Derived Secretomes

Abstract

Patients suffering from tissue ischemia, who would greatly benefit from angiogenesis-promoting therapies such as hypoxia preconditioned blood-derived secretomes commonly receive oral anticoagulation (OA) and/or have diabetes mellitus (DM). In this study, we investigated the effect of OA administration on the in vitro angiogenic potential of hypoxia preconditioned plasma (HPP) and serum (HPS), prepared from nondiabetic/diabetic subjects who did not receive OA (n = 5) or were treated with acetylsalicylic acid (ASA, n = 8), ASA + clopidogrel (n = 10), or nonvitamin K antagonist oral anticoagulants (n = 7) for longer than six months. The effect of DM was differentially assessed by comparing HPP/HPS obtained from nondiabetic (n = 8) and diabetic (n = 16) subjects who had not received OA in the past six months. The concentration of key proangiogenic (vascular endothelial growth factor or VEGF) and antiangiogenic (thrombospondin-1 or TSP-1 and platelet factor-4 or PF-4) protein factors in HPP/HPS was analyzed via ELISA, while their ability to induce microvessel formations was examined in endothelial cell cultures. We found that OA use significantly reduced VEGF levels in HPP, but not HPS, compared to non-OA controls. While HPP and HPS TSP-1 levels remained largely unchanged as a result of OA usage, HPS PF-4 levels were significantly reduced in samples obtained from OA-treated subjects. Neither OA administration nor DM appeared to significantly reduce the ability of HPP or HPS to induce microvessel formations in vitro. These findings indicate that OA administration does not limit the angiogenic potential of hypoxia preconditioned blood-derived secretomes, and therefore, it does not prohibit the application of these therapies for supporting tissue vascularization and wound healing in healthy or diabetic subjects.

Effect of Hypoxia Preconditioned Secretomes on Lymphangiogenic and Angiogenic Sprouting: An in Vitro Analysis

“…Hypoxia preconditioned blood-derived secretomes represent a new generation of autologous growth factor preparations [23][24][25][26][27][28]30] that can be produced through extracorporeal conditioning of peripheral blood cells (PBCs) under wound-simulating conditions, namely physiological temperature and hypoxia [25,26]. We had previously demonstrated that hypoxia preconditioned plasma (HPP) and serum (HPS) supply angiogenesis-specific signaling, similar to that naturally In this experiment we included adipose-derived cell suspension (ADCS), a secretome with lymphangiogenic properties [34], and fetal calf serum (FCS), a powerful lymphangiogenic stimulator [10], as positive controls.…”

“…Incubation was carried out for 4 days (blood incubation time), without any prior centrifugation. Pericellular (local) hypoxia (~1% O 2 ) was generated in situ through cell-mediated O 2 consumption, by controlling the blood volume per unit area (BVUA > 1 mL/cm 2 ) in the blood-containing syringe, and consequently, the PBC seeding density [25,29,30]. After 4 days, the blood was passively separated into three layers, from bottom to top: red blood cell (RBC) component, buffy coat/clot, and HPP/HPS, so that the top layer comprising hypoxia preconditioned plasma or serum could be filtered (0.2-µm pore filter, Sterifix ® , Braun AG, Melsungen, Germany) into a new syringe, removing cells/cellular debris.…”

“…These angiogenic compositions can be obtained through the method of Extracorporeal Wound Simulation (EWS), using peripheral (venous) blood [23][24][25][26][27][28][29]. The ability to condition PBCs under the same environmental conditions encountered within a wound, i.e., physiological temperature and hypoxia, offers an easy way to optimize the angiogenic potential of Hypoxia Preconditioned Plasma (HPP) and Hypoxia Preconditioned Serum (HPS), which can be differentially prepared by adjusting blood coagulation prior to hypoxic conditioning [23][24][25][26][27][28][29][30]. More specifically, we have shown that the angiogenic potential of blood-derived secretomes is defined by the complex stoichiometry of their component pro-and anti-angiogenic factor proteins, rather than the concentration of one or more individual growth factors [28,29].…”

“…These secretomes have also been extensively examined for their angiogenic activity, showing that they are able to induce microvessel formation and sprouting in vitro, as well as promote wound healing in vivo [23,27,28]. The angiogenic potency of hypoxia preconditioned secretomes is further highlighted by the fact that they maintain their pro-angiogenic activity in vitro, even when they are prepared from peripheral blood that is obtained from patients who receive oral anticoagulation due to underlying vascular pathology or who suffer from diabetes mellitus [30].…”

Special Issue: Hypoxia-Inducible Factors: Regulation and Therapeutic Potential

“…In this Special Issue, we invited research and review papers in various areas of oxygen biology research that focused on the fundamental understanding of HIF signaling pathways and related gene expression profiling, as well as pharmacogenomic biomarkers, molecular targets driving the regulation of human physiology and pathophysiology, and validation in animal models. As a result, we published six original papers and three review articles in this Special Issue [ 1 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ].…”