Abstract
IntroductionAdipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization.MethodsThe stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay.ResultsCD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells.Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs. Quantitative analysis of angiogenesis at 14 days after implantation demonstrated that neovascularization of the implants containing SVF CD14+ cells or PB monocytes previously co-cultured with MSCs was 3.5 or 2 times higher than that observed in the implants with SVF-derived MSCs. Moreover, immunofluorescence of Matrigel sections revealed that SVF CD14+ cells differentiated into endothelial cells and contributed to vascular endothelium.ConclusionsThe results from this study suggest that adipose tissue-resident monocytes should contribute to tissue vascularization. Because SVF CD14+ cells were more efficient in inducing angiogenesis than SVF-derived MSCs, and differentiated into vascular endothelial cells, they may constitute a new cell source for cell-based therapeutic angiogenesis.
Highlights
Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth
Hematopoietic cell population from the stromal vascular fraction (SVF) contains CD14+ cells expressing CD34 and displaying an endothelium-like phenotype Human adipose tissue SVF reportedly contains a variable proportion of CD34+ cells [1,19,20]
Most CD34+ cells expressed CD90 and CD13, which was indicative of their mesodermal origin, and a lower proportion were positive for CD31 (Table 2)
Summary
Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization. It has become evident that in addition to endothelial cell sprouting from preexisting blood vessels, endothelial progenitor cells and/or myeloid cells contribute to the angiogenic process [4,5]. Adipogenesis and angiogenesis are two closely related processes during embryogenesis and in adult life [2,7,8], and accumulating evidence demonstrates a functional link among endothelial cells, adipocytes, and monocytes. Conditioned media from mature adipocytes induce overexpression of the intracellular adhesion molecule and platelet/endothelial cell adhesion molecule (PECAM)-1 in adipose endothelial cells, which in turn increases blood monocyte adhesion and migration to/through endothelial cells [11]. Leptin, which is elevated in the plasma of obese subjects [12], increases blood monocyte adhesion and transmigration in a concentration-dependent manner [11] and induces angiogenesis [13]
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