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

Abstract

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.

Hypoxia Preconditioned Serum (HPS)-Hydrogel Can Accelerate Dermal Wound Healing in Mice—An In Vivo Pilot Study

“…We have shown that the localized delivery of peripheral blood-derived hypoxia-induced growth factor-mixtures can serve as a tool for the natural promotion of spatio-temporally controlled regeneration [ 15 , 16 , 17 , 18 ]. In accordance to the principle that the host response for optimal wound healing depends on multiple regulatory pathways [ 19 , 20 ], these paracrine proteins are used to stimulate a range of targeted cellular responses including wound angiogenesis, lymphangiogenesis, matrix deposition and re-epithelialization through fibroblast migration and proliferation [ 16 , 18 , 20 , 21 , 22 , 23 , 24 , 25 ]. Previous work by our group has already shown that collagen scaffolds containing hypoxia-induced growth factor proteins, produced by dermal fibroblasts, can promote vascularization and improve deep scaffold oxygenation when implanted in vivo in a rabbit model [ 26 , 27 ].…”

“…Based on these fundamental concepts, we had previously developed a novel approach for providing a biomimetic cocktail of blood-derived growth factor proteins within a locally-deployable carrier [ 16 , 17 , 18 ]. For this purpose, the primary stimulus of the wound healing angiogenic response, i.e., hypoxia, is used to stimulate PBCs to produce ‘new’ angiogenic and lymphangiogenic protein factors [ 16 , 18 , 23 , 24 , 25 ]. To obtain these complex growth factor mixtures we utilize a method of hypoxia-adjusted in vitro preconditioning, by cultivating PBCs ex vivo within a self-regulated low-oxygen microenvironment [ 16 , 17 , 21 ].…”

“…In HUVEC (human umbilical vein endothelial cell)-sprouting assays and aortic ring assays, HPS stimulated a greater invasion of endothelial cells, more vascular sprouts (sprouting angiogenesis) and greater sprout length compared to the recombinant VEGF- and PRP-treated groups [ 18 , 23 ]. Regarding lymphangiogenesis, HPS has been tested in a ductus thoracicus sprouting assay and exhibited superior formation of new lymphatic vessels after 4 days compared to other lymphatic stimulants such as FCS 20% [ 25 ]. Importantly, we have shown that there was no loss of pro-angiogenic activity in vitro when blood was derived from patients who receive oral anticoagulation due to underlying vascular pathology or those who suffer from diabetes mellitus type 1 and type 2 [ 22 ].…”

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 ].…”

“…The term “secretome” is a general term that includes not only soluble proteins released from cells via the endoplasmic reticulum (ER)-Golgi pathway, but also extracellular matrix proteins and cleaved fragments of membrane proteins. Moog et al have shown that the properties of the secretome are affected by hypoxia in a series of studies [ 16 , 17 , 18 ].…”