Blood-derived factor preparations are being clinically employed as tools for promoting tissue repair and regeneration. Here we set out to characterize the in vitro angiogenic potential of two types of frequently used autologous blood-derived secretomes: platelet-rich plasma (PRP) and hypoxia preconditioned plasma (HPP)/serum (HPS). The concentration of key pro-angiogenic (VEGF) and anti-angiogenic (TSP-1, PF-4) protein factors in these secretomes was analyzed via ELISA, while their ability to induce microvessel formation and sprouting was examined in endothelial cell and aortic ring cultures, respectively. We found higher concentrations of VEGF in PRP and HPP/HPS compared to normal plasma and serum. This correlated with improved induction of microvessel formation by PRP and HPP/HPS. HPP had a significantly lower TSP-1 and PF-4 concentration than PRP and HPS. PRP and HPP/HPS appeared to induce similar levels of microvessel sprouting; however, the length of these sprouts was greater in HPP/HPS than in PRP cultures. A bell-shaped angiogenic response profile was observed with increasing HPP/HPS dilutions, with peak values significantly exceeding the PRP response. Our findings demonstrate that optimization of peripheral blood cell-derived angiogenic factor signalling through hypoxic preconditioning offers an improved alternative to simple platelet concentration and release of growth factors pre-stored in platelets.

Blood-derived growth factor preparations have long been employed to improve perfusion and aid tissue repair. Among these, platelet-rich plasma (PRP)-based therapies have seen the widest application, albeit with mixed clinical results to date. Hypoxia-preconditioned blood products present an alternative to PRP, by comprising the complete wound healing factor-cascade, i.e., hypoxia-induced peripheral blood cell signaling, in addition to platelet-derived factors. This study set out to characterize the preparation of hypoxia preconditioned serum (HPS), and assess the utility of HPS–fibrin hydrogels as vehicles for controlled factor delivery. Our findings demonstrate the positive influence of hypoxic incubation on HPS angiogenic potential, and the individual variability of HPS angiogenic factor concentration. HPS–fibrin hydrogels can rapidly retain HPS factor proteins and gradually release them over time, while both functions appear to depend on the fibrin matrix mass. This offers a means of controlling factor retention/release, through adjustment of HPS fibrinogen concentration, thus allowing modulation of cellular angiogenic responses in a growth factor dose-dependent manner. This study provides the first evidence that HPS–fibrin hydrogels could constitute a new generation of autologous/bioactive injectable compositions that provide biochemical and biomaterial signals analogous to those mediating physiological wound healing. This therefore establishes a rational foundation for their application towards biomimetic tissue regeneration.

Hypoxia Preconditioned Plasma (HPP) and Serum (HPS) are two blood-derived autologous growth factor compositions that are being clinically employed as tools for promoting tissue regeneration, and have been extensively examined for their angiogenic activity. As yet, their ability to stimulate/support lymphangiogenesis remains unknown, although this is an important but often-neglected process in wound healing and tissue repair. Here we set out to characterize the potential of hypoxia preconditioned secretomes as promoters of angiogenic and lymphangiogenic sprouting in vitro. We first analysed HPP/HPS in terms of pro- (VEGF-C) and anti- (TSP-1, PF-4) angiogenic/lymphangiogenic growth factor concentration, before testing their ability to stimulate microvessel sprouting in the mouse aortic ring assay and lymphatic sprouting in the thoracic duct ring assay. The origin of lymphatic structures was validated with lymph-specific immunohistochemical staining (Anti-LYVE-1) and lymphatic vessel-associated protein (polydom) quantification in culture supernatants. HPP/HPS induced greater angiogenic and lymphatic sprouting compared to non-hypoxia preconditioned samples (normal plasma/serum), a response that was compatible with their higher VEGF-C concentration. These findings demonstrate that hypoxia preconditioned blood-derived secretomes have the ability to not only support sprouting angiogenesis, but also lymphangiogenesis, which underlines their multimodal regenerative potential.

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.

The ability to use the body’s resources to promote wound repair is increasingly becoming an interesting area of regenerative medicine research. Here, we tested the effect of topical application of blood-derived hypoxia preconditioned serum (HPS) on wound healing in a murine wound model. Alginate hydrogels loaded with two different HPS concentrations (10 and 40%) were applied topically on full-thickness wounds created on the back of immunocompromised mice. We achieved a significant dose-dependent wound area reduction after 5 days in HPS-treated groups compared with no treatment (NT). On average, both HPS-10% and HPS-40% -treated wounds healed 1.4 days faster than NT. Healed tissue samples were investigated on post-operative day 15 (POD 15) by immunohistology and showed an increase in lymphatic vessels (LYVE-1) up to 45% with HPS-40% application, while at this stage, vascularization (CD31) was comparable in the HPS-treated and NT groups. Furthermore, the expression of proliferation marker Ki67 was greater on POD 15 in the NT-group compared to HPS-treated groups, in accordance with the earlier completion of wound healing observed in the latter. Collagen deposition was similar in all groups, indicating lack of scar tissue hypertrophy as a result of HPS-hydrogel treatment. These findings show that topical HPS application is safe and can accelerate dermal wound healing in mice.

Strategies for therapeutic lymphangiogenesis are gradually directed toward the use of growth factor preparations. In particular, blood-derived growth factor products, including Hypoxia Preconditioned Serum (HPS) and Platelet-rich Plasma (PRP), are both clinically employed for accelerating tissue repair and have received considerable attention in the field of regenerative medicine research. In this study, a comparative analysis of HPS and PRP was conducted to explore their lymphangiogenic potential. We found higher pro-lymphangiogenic growth factor concentrations of VEGF-C, PDGF-BB, and bFGF in HPS in comparison to normal serum (NS) and PRP. The proliferation and migration of lymphatic endothelial cells (LECs) were promoted considerably with both HPS and PRP, but the strongest effect was achieved with HPS-40% dilution. Tube formation of LECs showed the highest number of tubes, branching points, greater tube length, and cell-covered area with HPS-10%. Finally, the effects were double-validated using an ex vivo lymphatic ring assay, in which the highest number of sprouts and the greatest sprout length were achieved with HPS-10%. Our findings demonstrate the superior lymphangiogenic potential of a new generation blood-derived secretome obtained by hypoxic preconditioning of peripheral blood cells—a method that offers a novel alternative to PRP.