Angiogenic Properties of Concentrated Growth Factors (CGFs): The Role of Soluble Factors and Cellular Components

Blood-derived concentrated growth factors (CGFs) represent a novel autologous biomaterial with promising applications in regenerative medicine. Angiogenesis is a key factor in tissue regeneration, but the role played by CGFs in vessel formation is not clear. The purpose of this study was to characterize the angiogenic properties of CGFs by evaluating the effects of its soluble factors and cellular components on the neovascularization in an in vitro model of angiogenesis. CGF clots were cultured for 14 days in cell culture medium; after that, CGF-conditioned medium (CGF-CM) was collected, and soluble factors and cellular components were separated and characterized. CGF-soluble factors, such as growth factors (VEGF and TGF-β1) and matrix metalloproteinases (MMP-2 and -9), were assessed by ELISA. Angiogenic properties of CGF-soluble factors were analyzed by stimulating human cultured endothelial cells with increasing concentrations (1%, 5%, 10%, or 20%) of CGF-CM, and their effect on cell migration and tubule-like formation was assessed by wound healing and Matrigel assay, respectively. The expression of endothelial angiogenic mediators was determined using qRT-PCR and ELISA assays. CGF-derived cells were characterized by immunostaining, qRT-PCR and Matrigel assay. We found that CGF-CM, consisting of essential pro-angiogenic factors, such as VEGF, TGF-β1, MMP-9, and MMP-2, promoted endothelial cell migration; tubule structure formation; and endothelial expression of multiple angiogenic mediators, including growth factors, chemokines, and metalloproteinases. Moreover, we discovered that CGF-derived cells exhibited features such as endothelial progenitor cells, since they expressed the CD34 stem cell marker and endothelial markers and participated in the neo-angiogenic process. In conclusion, our results suggest that CGFs are able to promote endothelial angiogenesis through their soluble and cellular components and that CGFs can be used as a biomaterial for therapeutic vasculogenesis in the field of tissue regeneration.

Angiogenic properties of human endothelial colony-forming cells in Granulomatosis with Polyangiitis

BACKGROUND Endothelial progenitor cells are essential for vascular homeostasis. Considering the recurrent nature of granulomatosis with polyangiitis (GPA) the aim of the study was to evaluate the angiogenic capacity of endothelial colony-forming cells (ECFC), which have the capacity for neovasculogenesis in vitro, of the patients with GPA, before and after plasma stimulation. METHODS Thirteen GPA PR3-positive patients and 15 healthy controls were included. ECFC were isolated from periferic blood and characterized by flow cytometry (FACS). Capillary tube formation (Matrigel assay) and scratching assay were measured during 24 hours. The migration assay was also performed after overnight incubation with healthy control plasma and active GPA patient plasma. Three patients with active disease where submitted to recollection of ECFC after treatment for longitudinal evaluation. RESULTS The culture was successful in 62% of GPA patients and 57% of controls. In the matrigel assay the ECFC from patients and controls showed similar capillary structures formation, however ECFC from inactive GPA alone showed early loss of angiogenic capacity between 15 and 24 hours. In scratching assay, there was an impairment in the proliferative capacity of the ECFC between GPA patients and controls without significant difference (12th hour, p = 0.05). When incubated with control plasma, ECFC of remission GPA patients showed a significant lower migration capacity after the 4th hour (p = 0.0001). In longitudinal analysis, ECFC isolated after treatment showed significantly lower migration rates. CONCLUSIONS PR3-positive remission GPA ECFC demonstrated early loss of tube formation and less proliferative capacity compared to those of healthy controls, suggesting impairment of endothelial function.

Abstract 293: Embryological Origin of Human Smooth Muscle Cells Influences Their Ability to Support Vasculogenesis.

Lineage-tracking studies in avian and mouse embryos have revealed that SMCs in different regions of the vasculature are derived from different embryological origins. We have developed an in vitro protocol to differentiate human embryonic stem cells (hESCs) into SMCs through different embryonic lineages, namely neuroectoderm, lateral plate mesoderm (LM) and paraxial mesoderm. As mural cells are thought to augment the formation of functional blood vessels during revascularisation, this study aims to determine whether embryologically-distinct SMCs differ in their ability to support vasculogenesis. We assessed the hypothesis that LM-SMCs are superior in supporting endothelial network formation in vitro. Using an mStrawberry-expressing hESC line, we derived the three origin-specific SMCs and co-cultured them with GFP-expressing HUVECs in a 3D in vitro matrigel assay. Endothelial network formation was assessed using real-time confocal imaging. Quantitative analysis revealed that LM-SMCs alone had a supportive effect on network formation and survival, with an increase in HUVEC network area after 4 days (3.23-fold increase ± 1.04-fold vs HUVECs alone; p<0.05; n=3) and 8 days (6.38-fold increase ± 0.39-fold vs HUVECs alone; p<0.001; n=3). In addition, LM-SMCs appear to facilitate more complex endothelial networks, with narrower cords and more branch points, compared with the other SMC types and HUVECs alone. To identify whether the LM-SMC-specific influence on network formation and survival was a paracrine effect, the three SMC types were tested in a paracrine 3D in vitro assay, where they shared media with HUVECs seeded in an adjacent well. Preliminary results reveal that the supportive effect of LM-SMCs is, at least partly, paracrine, with an increase in HUVEC network area after 4 days (7.08-fold increase vs HUVECs alone; n=1) and 8 days (21.39-fold increase vs HUVECs alone; n=1). Currently, we are exploring possible soluble factors that may be responsible and investigating this effect in an in vivo matrigel assay. In conclusion, the embryological origin of SMCs influences their functional ability to support vasculogenesis. This research will provide insight into which SMC type will be most effective in future revascularisation therapy.

Adrenomedullin augments the angiogenic potential of late outgrowth endothelial progenitor cells

The therapeutic utility of endothelial progenitor cells (EPCs) in cardiovascular disease is potentially hampered by their low numbers in the circulation, impaired functional activity, and inhibitory factors in the recipient. These obstacles can possibly be circumvented by the use of proangiogenic cytokines and peptides. We sought to examine the effect of the endogenous vasoactive peptide adrenomedullin (AM) on the angiogenic potential of late outgrowth EPCs and their release of proangiogenic and proinflammatory cytokines/chemokines. Human peripheral blood mononuclear cells were cultured until the appearance of typical late outgrowth EPC colonies. The effect of AM on EPC proliferation was assessed using a colorimetric MTS proliferation assay while differentiation and formation of tubular structures in an EPC/fibroblast coculture or matrigel assay was used to assess the angiogenic potential of the cells. Finally, the release and mRNA transcripts of cytokines/chemokines were quantified in stimulated vs. nonstimulated EPCs using real-time PCR and a bead-based multiplex assay. The cultured EPCs possessed an endothelial phenotype and expressed the AM receptor (calcitonin receptor-like receptor/receptor activity modifying protein-2). AM stimulation induced proliferation of EPCs compared with controls ( P < 0.05). Furthermore, AM produced a 36% and 80% increase in the formation of tubular networks in the EPC/fibroblast coculture and matrigel assay, respectively ( P < 0.05). These effects seemed to be mediated through the phosphatidylinositol 3-kinase/Akt signaling pathway. AM did not seem to significantly influence the release or production of IL-6, IL-8, VEGF, stromal cell-derived factor 1, or the expression of CXCR-4 or VEGF receptor 2. In conclusion, adrenomedullin augmented the growth and angiogenic properties of late outgrowth EPCs, but did not influence their paracrine properties.