Aging and diabetes impair the neovascular potential of adipose-derived stromal cells

Abstract

Background: Aging and diabetes are major risk factors for poor wound healing and tissue regeneration that reflect an impaired ability to respond to ischemic insults. The authors explored the intrinsic neovascular potential of adipose-derived stromal cells in the setting of advanced age and in type 1 and type 2 diabetes.

Methods: Adipose-derived stromal cells isolated from young, aged, streptozotocin-induced, and db/db diabetic mice were exposed to normoxia and hypoxia in vitro. Vascular endothelial growth factor (VEGF) expression, proliferation, and tubulization were measured. Conditioned media harvested from adipose-derived stromal cell cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation (n = 3 and n = 3).

Results: Young adipose-derived stromal cells demonstrated significantly higher levels of VEGF production, proliferation, and tubulogenesis than those derived from aged, streptozotocin-induced, and db/db mice in both normoxia and hypoxia. Although aged and diabetic adipose-derived stromal cells retained the ability to up-regulate VEGF secretion, proliferation, and tubulogenesis in response to hypoxia, the response was blunted compared with young controls. Conditioned media derived from these cells cultured in normoxia in vitro also had a significantly greater ability to increase human umbilical vein endothelial cell proliferation compared with media harvested from aged, streptozotocin-induced, and db/db adipose-derived stromal cells. This effect was magnified in conditioned media harvested from hypoxic adipose-derived stromal cell cultures.

Conclusions: This study demonstrates that aging and type 1 and type 2 diabetes impair intrinsic adipose-derived stromal cell function; however, these cells may still be a suitable source of angiogenic cells that can potentially improve neovascularization of ischemic tissues.

Fig. 1

Adipose-derived stromal cells from passages 3 to 5 expressed identical cell surface antigens when analyzed by fluorescence-activated cell sorting. Adipose-derived stromal cells were positive for the cell surface markers Sca-1, CD90, and CD44 and negative for all lineage markers (CD4, CD8, CD11b, B220, GR-1, and TER-119), CD45, c-kit/CD117, CD31, and Flk-1. These data are congruent with the marker profile of murine-derived stromal cells described in previous studies. Data are expressed as a histogram plot, with black representing isotype control and red representing experimental.

Fig. 2

Adipose-derived stromal cell supernatants were analyzed for VEGF production by enzyme-linked immunosorbent assay after a 24-hour exposure to either normoxia (21% oxygen; red bars) or hypoxia (1% oxygen; blue bars). Significantly higher levels of VEGF secretion were seen in adipose-derived stromal cells derived from young mice than were seen in adipose-derived stromal cells derived from aged, streptozotocin-induced, and db/db mice under normoxic (**p < 0.05) and hypoxic conditions (***p < 0.05). However, aged, streptozotocin-induced, and db/db adipose-derived stromal cells retained the ability to up-regulate VEGF secretion in response to hypoxia but not to the extent seen in young adipose-derived stromal cells (*p < 0.05) (n = 3 and N = 3).

Fig. 3

Adipose-derived stromal cell proliferation rates were measured by bromodeoxyuridine incorporation after a 24-hour exposure to either normoxia (21% oxygen, red bars) orhypoxia (1%oxygen, blue bars). Adipose-derived stromal cells derived from young mice demonstrated significantly increased bromodeoxyuridine incorporation than that seen in adipose-derived stromal cells derived from aged, streptozotocin-induced, and db/db mice under normoxic (**p < 0.05) and hypoxic conditions (***p < 0.05). However, aged, streptozotocin-induced, and db/db adipose-derived stromal cells retained the ability to up-regulate the rate of proliferation in response to hypoxia but not to the extent seen in young adipose-derived stromal cells (*p < 0.05) (n = 3 and N = 3).

Fig. 4

Proliferation rates of human umbilical vein endothelial cells were measured by bromodeoxyuridine incorporation after 24 hours of incubation in conditioned media harvested from adipose-derived stromal cells that were exposed to normoxia (21% oxygen, red bars) or hypoxia (1% oxygen, blue bars) for 72 hours. Normoxic conditioned media harvested from young adipose-derived stromal cells had a significantly greater ability to increase proliferation of human umbilical vein endothelial cells than conditioned media harvested from aged, streptozotocin-induced, or db/db adipose-derived stromal cells (**p < 0.05). Conditioned media from hypoxic cultures also generated a significant increase in human umbilical vein endothelial cell proliferation that was even more pronounced than normoxia-exposed conditioned media (***p < 0.05) (n = 3 and N = 3).

Fig. 5

Representative images of tubulization of adipose-derived stromal cells harvested from young (first row), aged (second row), streptozotocin-induced type I diabetic (third row), and db/db type II diabetic mice (last row) in normoxic (left panels) and hypoxic (right panels) conditions.

Fig. 6

Quantification of adipose-derived stromal cell tubulization after seeding on Matrigel for 6 hours in either normoxia (21% oxygen) or hypoxia (1% oxygen). Adipose-derived stromal cells derived from young mice demonstrated a significantly superior ability to undergo tubulogenesis on Matrigel than that seen in aged, streptozotocin-induced, or db/db adipose-derived stromal cells under normoxic conditions (red bars; **p < 0.05). Hypoxia exposure increased tubule formation in all groups (blue bars); however, the response in young adipose-derived stromal cells far exceeded the degree of tubulization seen in other groups (***p< 0.05). Of note, the diabetic adipose-derived stromal cells demonstrated the most severe impairment in hypoxia-induced tubulogenesis (n = 3 and N = 3).