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

Abstract

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.

Keywords: angiogenesis; biomaterials; concentrated growth factors (CGFs); endothelial cells; endothelial markers; endothelial progenitor cells (EPCs); matrix metalloproteinases; pro-angiogenic factors; tissue regeneration; vasculogenesis.

Figure 1

Experimental design.

Figure 2

CGFs promote endothelial cell migration and tube formation. A scratch wound was performed on endothelial monolayers of HMEC-1 that were stimulated with CGF-CM (1%, 5%, 10% or 20%) for 16 h (A). Cell migration was quantified and monitored under phase-contrast microscopy (C). HMEC-1 cells were plated onto a three-dimensional collagen gel (Matrigel) surface and then stimulated with CGF-CM (1%, 5%, 10%, or 20%) or VEGF (10 ng/mL) for 16 h (B,D). Tube formation was monitored under phase-contrast microscopy, photographed, and analyzed. Images are representative of cell migration (A) and capillary-like tube formation (×100 magnification) (B). Data are representative of three independent experiments, expressed as means ± SD, and presented as percentage of wound closure (C) and branch points per field (D). Each experiment consisted of four replicates for each condition. * p < 0.05 and ** p < 0.01 vs. control (CTR).

Figure 3

CGFs induce the expression and release of VEGF and matrix metalloproteinases in endothelial cells. HUVECs were incubated with CGF-CM (10%) or culture medium (CTR) for 4 h and mRNA levels of VEGF. (A) MMP-9 and MMP-2 (C) were analyzed by qRT-PCR and expressed as fold over unstimulated control (CTR) (mean ± SD). HUVECs were incubated with CGF-CM (10%) or culture medium (CTR) for 4 h, and then culture media were removed and replaced with fresh media for 48 h. Media were collected, and VEGF (B), MMP-9, and MMP-2 (D) concentrations were analyzed by ELISA. Data are representative of three independent experiments, expressed as means ± SD. Each experiment consisted of four replicates for each condition. * p < 0.05 and ** p < 0.01 vs. control (CTR).

Figure 4

CGFs enhance the expression of pro-angiogenic factors in endothelial cells. HUVECs were incubated with CGF-CM (10%) or culture medium (CTR) for 4 hm and mRNA levels of Ang-1, Ang-2, PDGF-B, FGF-2, TGF-β1, and BMP-2 were analyzed by qRT-PCR and expressed as fold over unstimulated control (CTR) (mean ± SD). Each experiment consisted of four replicates for each condition. * p < 0.05 vs. CTR.

Figure 5

CGF-derived cell characterization and expression of pro-angiogenic factors. Cells were released by CGF clot and cultured in M199 medium (A). CGF cell morphological characterization was determined by hematoxylin–eosin staining (B). RNA was extracted from CGF-derived cells, and levels of pro-angiogenic factor expression were analyzed by qRT-PCR. GAPDH was considered a housekeeping gene. The expression level of genes is referred to with BMP-2 (=1) as the lesser expressed gene (C). Data are representative of three independent experiments.

Figure 6

CGF-derived cells express hematopoietic stem cells and endothelial markers and participate in endothelial angiogenesis. Immunostaining analysis was performed for CD34, VE-cadherin, VEGFR-2 and eNOS (A), and negative controls for anti-mouse (NC1), and anti-rabbit (NC2) IgG antibodies were reported. RNA was extracted from CGF-derived cells, and the expression of hematopoietic stem cell (CD133 and CD34) and endothelial cell (CD31, VE-cadherin, VEGFR-2 and eNOS) markers was analyzed by qRT-PCR. GAPDH was considered a housekeeping gene. The expression level of genes is referred with CD133 (=1) as the lesser expressed gene (B). CGF-derived cells were loaded with DilC18 and plated with endothelial cells onto a Matrigel surface for 16 h. Tube formation was monitored and photographed. Images are representative of capillary-like tube formation under light microscopy on the left and fluorescence microscopy on the right (×100 magnification) (C). Green and yellow arrows indicate HUVEC and CGF-derived cells, respectively (C). Each experiment consisted of four replicates for each condition.

Figure 7

CXCL-12 and CXCR-4 expression in CGF-derived cells and CGF-CM-treated endothelial cells. mRNA was extracted from CGF-derived cells and CGF-CM (10%)-treated HUVECs for 4 h, and the expression levels of CXCL-12 and CXCR-4 were analyzed by qRT-PCR and expressed as fold over unstimulated HUVEC control (mean ± SD). Each experiment consisted of four replicates for each condition. * p < 0.05 and ** p < 0.01 vs. HUVEC control.