Adipose-derived stromal vascular fraction cells isolated from old animals exhibit reduced capacity to support the formation of microvascular networks

Abstract

Adipose-derived regenerative and stem cells, defined collectively as the stromal vascular fraction (SVF), support the formation of neovascular networks at the site of implantation. The effect of advancing age on SVF cell population effectiveness towards stimulated neovascularization was evaluated.

Methods: SVF was enzymatically isolated from adipose of young (ySVF, 4 months) or old (oSVF, 24 months) Fisher-344 rats, combined with type I collagen and polymerized. Encapsulated SVF was implanted subcutaneously into young Rag1 mice for two or four weeks. Angiogenic function of age-dependent SVF was also extensively evaluated in vitro using standard assays.

Results: In vitro studies indicated no difference in angiogenic function between ySVF and oSVF (viability, proliferation, migration, and tube-formation). At two weeks post-implantation, there was no age-related difference in percent apoptosis in explanted constructs. By four weeks post-implantation, oSVF implants displayed 36% less total vessels/mm(2), 43% less perfused vessels/mm(2), and exhibited greater percent apoptosis compared to ySVF (n ≥ 12). Blocking thrombospondin-1 (Thbs-1), a protein found to be highly expressed in oSVF but not ySVF, increased the percent of perfused vascular volume and vessel diameters in oSVF constructs after two weeks compared to oSVF implants treated with control antibody.

Conclusions: Advancing donor age reduces the potential of adipose-derived SVF to derive a mature microcirculation, but does not hinder initial angiogenesis. However, modulation of Thbs-1 may improve this outcome. This data suggests that greater pruning, dysfunctional structural adaptation and/or poor maturation with initiation of blood flow may occur in oSVF.

Keywords: Aging; Angiogenesis; Apoptosis; Inosculation; Ischemia; Maturation; Thrombospondin-1; Vascular remodeling.

Figure 1

Reduced neovascularization potential of SVF from old donors. Collagen constructs containing freshly isolated SVF from young or old donors (young n=13, old n=11) were implanted in Rag1 mice for four weeks. Prior to explant, perfused vessels were stained with dextran-rhodamine and post-explant all vessels were stained with GS-1-fluorescein. Constructs were imaged by confocal microscopy (A). SVF constructs from old donors showed a reduction in total and perfused vessel density (B, C). A reduction in the percent of perfused vessel volume (volume perfused vessels/volume total vessels X100) was observed in networks formed by old SVF (D). Analysis of the size distribution of vessels showed a shift towards smaller diameters in old SVF constructs at four weeks (young n =7, old n=8) (E). Scale bars are 100 µm, *P<0.05.

Figure 2

Donor age effects expression of angiogenesis-related factors in SVF. Live/Dead assay (young n=9, old n=15) showed no age related difference in viability of freshly isolated SVF (A). Flow cytometry of freshly isolated SVF (n=3 per group) revealed age-dependent expression of the cell surface receptors VEGFR2, CD47, and CXCR2, but not CD11b, CD31, CD34, cKit, or PDGFR (B). Western blot analysis showed increased expression of the anti-angiogenic matricellular protein Thrombospondin-1 in old SVF (n=7 both groups). Representative blot and quantification are shown (E). *P<0.05.

Figure 3

Time-dependent in vivo apoptosis in SVF constructs. TUNEL staining of SVF constructs revealed a decrease in apoptosis of young SVF from two to four weeks while levels of apoptosis remained elevated in old SVF (Two week young n=2, old n=5; Four week n=10 both groups) (A). A corresponding reduction in cell density was noted in old SVF at four weeks (Two week young n=2, old n=5; Four week n=10 per group) (B). *P<0.05 notes differences between age groups, #P<0.05 notes differences between time points within an age group.

Figure 4

Perfused volume of vessels formed by old SVF is improved by Thrompospondin-1 antibody incubation. Old SVF was treated with control IgG or Thrombospondin-1 blocking antibody, suspended in collagen constructs, and implanted in Rag1 mice for two weeks. Perfused vessels were labeled with dextran-rhodamine and all vessels were labeled with GS-1-fluorescin (old+IgG ab n=4, old+ α-Thbs-1 n=3). Representative images are shown (A). No differences were seen between groups in total or perfused vessel density (B,C). Treatment with α-Thbs-1 significantly increased the percent of vessel volume that was perfused (vol. perfused vessels/vol. total vessels ×100) (D). Analysis of the size distribution of vessels showed a shift towards larger diameter in α-Thbs-1 treated SVF constructs (E). Scale bars are 100 µm, *P<0.05.