Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Abstract

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF's blood vessel forming potential prior to clinical implantation.

Keywords: Adipose; Angiogenesis; Stromal vascular fraction; Vasculogenesis.

Figures

Figure 1.

×4 phase contrast images of SVF grown on 3 mg/mL collagen I (A), 1% gelatin (B), Matrigel (C), 5 mg/mL fibrin (D), or 2 μg laminin 332 (E) after 112 h of incubation. Monolayers of cells are formed on collagen I (A), gelatin (B) and laminin 332 (E) with cell clustering demonstrated on Matrigel (C) and fibrin (D). SVF forms microvascular networks on Matrigel (C).

Figure 2.

×4 phase contrast images of SVF grown on Matrigel at 18 h (A, B); 36 h (C, D); 60 h (E, F); and 112 h (G, H). Images B, D, F, and H are ×3 optical zooms of boxed sections of images A, C, E, and G, respectively. White arrows demonstrate initial cell clustering occurring at 18 h. Tip cell formation occurs at 36 h as marked by the red arrow. Stalk cell formation and elongation occurs at 60 h as marked by the blue arrow. Branching (green arrow) and inosculation (yellow arrow) can be seen at 118 h in vitro.

Figure 3.

×10 fluorescent images of SVF grown on 1% gelatin (A) or Matrigel (B) for 112 h. Blue: DAPI, green: Griffonia simplicifolia 1 endothelial specific lectin-FITC, and red: α smooth muscle actin-rhodamine.

Figure 4.

Schematic of SVF microvascular assembly on Matrigel in vitro. Digested SVF cells (A) self-assemble into a cluster of cells by 18 h (B). By 36 h, endothelial cells sprout out of the cluster with dendritic-like extensions (tip cells: orange ●) (C). DAPT, an inhibitor of the γ-secretase that cleaves notch to its soluble ligand, blocks tip cell signaling to stalk cells, inhibiting stalk cell formation. Stalk cell formation occurs when notch signaling leads to a higher expression of VEGR-1 on the adjacent cell. ZM 306416 blocks VEGFR-1 and thus, stalk cell phenotype. In the absence of inhibitors, stalk cells (black ) migrate away from clusters behind tip cells by 60 h (D). Microvascular networks continue to grow after 112 h and demonstrate increased complexity (E). Networks from adjacent clusters inosculate with one another (yellow *) and recruit perivascular support cells to endothelial tubes (purple ▲) (F). PDGFR-B is found on perivascular support cells and activated by endothelial PDGF-β, which signals pericyte recruitment and tube stabilization. Imatinib mesylate inhibits PDGFR-B.

Figure 5.

Number of SVF cell clusters (grouping of two or more cells over 25 μm in total size) at 18, 36, 60, and 112 h of incubation in the presence of inhibitors (A). Cluster size (micrometer) at 18, 36, 60, and 112 h of incubation in the presence of inhibitors (B). Percentage of clusters containing tip cells at 18, 36, 60, and 112 h of incubation in the presence of inhibitors (C). Percentage of clusters containing stalk cells at 18, 36, 60, and 112 h of incubation in the presence of inhibitors (D). Angiogenic inhibitors (in DMSO) were added at days 0, 2, 4, and 6. p < 0.05 *, p < 0.005 **, p < 0.0005 ***.