Hypoxic culture and in vivo inflammatory environments affect the assumption of pericyte characteristics by human adipose and bone marrow progenitor cells

Abstract

Previous studies have shown that exposure to a hypoxic in vitro environment increases the secretion of pro-angiogenic growth factors by human adipose-derived stromal cells (hASCs) [Cao Y, et al., Biochem Biophys Res Commun 332: 370-379, 2005; Kokai LE, et al., Plast Reconstr Surg 116: 1453-1460, 2005; Park BS, et al., Biomed Res (Tokyo) 31: 27-34, 2010; Rasmussen JG, et al., Cytotherapy 13: 318-328, 2010; Rehman J, et al., Circulation 109: 1292-1298, 2004]. Previously, it has been demonstrated that hASCs can differentiate into pericytes and promote microvascular stability and maintenance during angiogenesis in vivo (Amos PJ, et al., Stem Cells 26: 2682-2690, 2008; Traktuev DO, et al., Circ Res 102: 77-85, 2008). In this study, we tested the hypotheses that angiogenic induction can be increased and pericyte differentiation decreased by pretreatment of hASCs with hypoxic culture and that hASCs are similar to human bone marrow-derived stromal cells (hBMSCs) in these regards. Our data confirms previous studies showing that hASCs: 1) secrete pro-angiogenic proteins, which are upregulated following culture in hypoxia, and 2) migrate up gradients of PDGF-BB in vitro, while showing for the first time that a rat mesenteric model of angiogenesis induced by 48/80 increases the propensity of both hASCs and hBMSCs to assume perivascular phenotypes following injection. Moreover, culture of both cell types in hypoxia before injection results in a biphasic vascular length density response in this model of inflammation-induced angiogenesis. The effects of hypoxia and inflammation on the phenotype of adult progenitor cells impacts both the therapeutic and the basic science applications of the cell types, as hypoxia and inflammation are common features of natural and pathological vascular compartments in vivo.

Fig. 1.

Secretion of growth factors and matrix enzymes by progenitor cells after 48 h (0-h time point) of normal culture conditions (NCC) or hypoxic culture conditions (HCC) and 48 h (48-h time point) of subsequent NCC culture. A: hepatocyte growth factor (HGF) secretion was highest for NCC human adipose-derived stromal cell (hASCs) at the 0-h time point but highest for HCC hASCs at the 48-h time point. B: all three cell types secrete matrix metalloproteinase-2 (MMP-2). C: among HCC hASCs, normally cultured NCC hASCs, and human bone marrow-derived stromal cells (hBMSCs), hBMSCs secrete the most vascular endothelial growth factor (VEGF) per cell. HCC hASCs secrete a statistically similar amount of HGF as NCC hASCs and a similar amount of VEGF as hBMSCs. D: hASCs produce more tissue inhibitor of metalloproteinases-1 (TIMP-1) than hBMSCs, regardless of culture condition. Values are means ± SD; *P < 0.001; †P < 0.01; #P < 0.05; XP < 0.001 vs. 0-h time point; OP < 0.05 vs. 0-h time point.

Fig. 2.

A: in scratch test migration assays, NCC hASCs consistently showed higher baseline migration and higher migration in the presence of platelet-derived growth factor BB (PDGF-BB) than HCC hASCs. hBMSCs did not exhibit an increase in migration in the scratch test assay in the presence of PDGF-BB (shaded bars) compared with untreated controls (solid bars). Values are means ± SD; *P < 0.001 vs. untreated control same cell group/time point; #P < 0.05 vs. untreated control same cell group/time point; †P < 0.001 vs. hBMSC at the same time point; ‡P < 0.05 vs. hBMSC at the same time point; §P < 0.01 vs. NCC hASC at the same time point. B: NCC ASCs were able to migrate significantly more toward a gradient of PDGF-BB, but not VEGF, than untreated negative controls in Boyden chamber migration assays. BMSCs were able to migrate more in the presence of both VEGF and PDGF-BB than negative controls, but baseline hASC migration was higher than that of BMSCs. Values are means ± SD; *P < 0.001 vs. untreated control same cell group/time point; #P < 0.01 vs. untreated control same cell group/time point; †P < 0.001 vs. hBMSC at the same time point; ‡P < 0.05 vs. hBMSC at the same time point; @P < 0.01 vs. hASCs treated with VEGF.

Fig. 3.

1,1′-Dioctadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate (DiI)-labeled hASCs (red) exhibit perivascular positioning with respect to blood vessels (blue) in inflamed and angiogenic rat mesenteric windows as well as pericyte markers like neuron glial antigen-2 (NG2) and smooth muscle α-actin (SMA) (A and C, respectively). SMA and NG2 staining of inflamed rat mesenteries injected with vehicle PBS instead of cells showed positive staining of native pericytes (B) and smooth muscle cells (D). Nerves in the vascular bed also stained positively for NG2 (A).

Fig. 4.

For all injected cell groups, the number of cells associating closely with blood vessels was significantly lower by day 60 in unstimulated mesenteries compared with inflamed mesenteries. hBMSCs showed steady increase in perivascular association in these tissues, and HCC hASCs showed a biphasic response in stimulated tissues. Values are means ± SE, *P < 0.001; †P < 0.01; #P < 0.05; XP < 0.001 vs. unstimulated; +P < 0.01 vs. unstimulated; OP < 0.05 vs. unstimulated.

Fig. 5.

The number of cells exhibiting pericyte-like phenotypes (A: concurrent PDGFβR expression and perivascular positioning; B: concurrent NG2 expression and perivascular positioning; C: concurrent SMA expression and perivascular positioning) in each group was low at all time points in unstimulated mesenteries, but all groups showed increases in pericyte-like phenotype by day 60 when injected into inflamed mesenteries. hBMSCs showed an increase in pericyte-like phenotype slightly earlier than either hASC group in the Compound 48/80-treated tissues. Values are means ± SE, *P < 0.001; †P < 0.01; #P < 0.05; XP < 0.001 vs. unstimulated; +P < 0.01 vs. unstimulated; OP < 0.05 vs. unstimulated.

Fig. 6.

A: hBMSCs and NCC hASCs showed a general increase in vascular length density over time in inflamed mesenteries, but HCC hASCs produced a biphasic length density profile over time. B: angiogenic efficiency (length density per cell) was found to increase over 60 days for NCC hASCs, but hBMSCs seemed to peak in angiogenic efficiency at day 30. HCC hASCs showed an increase in angiogenic efficiency from day 10 to day 60 in unstimulated mesenteries but no change in angiogenic efficiency when injected into stimulated mesenteries. Values are means ± SE, *P < 0.001; †P < 0.01; #P < 0.05; XP < 0.001 vs. unstimulated; +P < 0.01 vs. unstimulated; OP < 0.05 vs. unstimulated.