Paracrine release from nonviral engineered adipose-derived stem cells promotes endothelial cell survival and migration in vitro

Abstract

Stem cells hold great potential for therapeutic angiogenesis due to their ability to directly contribute to new vessel formation or secrete paracrine signals. Adipose-derived stem cells (ADSCs) are a particularly attractive autologous cell source for therapeutic angiogenesis due to their ease of isolation and relative abundance. Gene therapy may be used to further enhance the therapeutic efficacy of ADSCs by overexpressing desired therapeutic factors. Here, we developed vascular endothelial growth factor (VEGF)-overexpressing ADSCs utilizing poly(β-amino esters) (PBAEs), a hydrolytically biodegradable polymer, and examined the effects of paracrine release from nonviral modified ADSCs on the angiogenic potential of human umbilical vein endothelial cells (HUVECs) in vitro. PBAE polymeric vectors delivered DNA into ADSCs with high efficiency and low cytotoxicity, leading to an over 3-fold increase in VEGF production by ADSCs compared with Lipofectamine 2000. Paracrine release from PBAE/VEGF-transfected ADSCs enhanced HUVEC viability and decreased HUVEC apoptosis under hypoxia. Further, paracrine release from PBAE/VEGF-transfected ADSCs significantly enhanced HUVEC migration and tube formation, two critical cellular processes for effective angiogenesis. Our results demonstrate that genetically engineered ADSCs using biodegradable polymeric nanoparticles may provide a promising autologous cell source for therapeutic angiogenesis in treating cardiovascular diseases.

FIG. 1.

Efficiency of gene delivery to human adipose-derived stem cells (ADSCs) utilizing poly(β-amino ester) (PBAE) nanoparticles. Under leading transfection conditions, PBAE (a) delivered enhanced green fluorescence protein (EGFP) DNA into ADSCs with 4 times the efficiency of Lipofectamine 2000 (Lipo) (b). Fluorescence microscopy showed markedly increased GFP signals in ADSCs transfected using PBAE nanoparticles (d) compared with cells transfected using Lipo (e). No GFP expression was observed in non-transfected cells (c, f). Scale bar: 500 μm. Color images available online at www.liebertpub.com/scd

FIG. 2.

Effects of nanoparticle dose on transfection efficiency and cell viability. (a) PBAEs led to significantly increased transfection efficiency in ADSCs using doses as low as 2 μg/well compared with Lipo (^#p<0.001 compared with Lipo). (b) Cells transfected using PBAEs at all doses demonstrated high cell viability, which were about 1-fold higher compared with the cells transfected using Lipo (^#p<0.01 vs. Lipo; *p<0.05 vs. cells only).

FIG. 3.

Vascular endothelial growth factor (VEGF) release from ADSCs after transfection using polymeric nanoparticles. (a) VEGF release from PBAE-modified ADSCs increased with DNA dose and reached a maximum at DNA dose of 6 μg, which was significantly higher than groups transfected using Lipo (^#p<0.05) and cells alone (*p<0.05). (b) Accumulated VEGF release showed continuously enhanced VEGF release from cells transfected using PBAE/VEGF nanoparticles compared with Lipo control.

FIG. 4.

Effects of hypoxia on VEGF production and cell viability of nonviral engineered ADSCs. (a) Hypoxia (1% O₂ for 48 h) enhanced VEGF release from nonviral engineered ADSCs in a dose-dependent manner. (b) Hypoxia treatment led to an increase in ADSC viability in most PBAE-transfected groups, except in the groups transfected with 6 μg PBAE/DNA or Lipo.

FIG. 5.

Effects of paracrine signals from nonviral engineered ADSCs on endothelial cell (EC) viability and apoptosis. (a) Paracrine release from ADSCs significantly improved human umbilical vein endothelial cell (HUVEC) viability under hypoxia [*p<0.05 vs. endothelial basal medium (EBM)], with no significant difference in cell viability due to VEGF overexpression. (b) Paracrine release from ADSCs markedly decreased HUVEC apoptosis under hypoxia, and PBAE/VEGF-transfected group led to the greatest decrease in cell apoptosis [*p<0.05 vs. EBM; ^#p<0.05 vs. Lipo VEGF/ADSC–conditioned medium (CM)]. (c–f) Representative images of TUNEL-stained HUVECs undergoing apoptosis. Scale bars=200 μm. Color images available online at www.liebertpub.com/scd

FIG. 6.

Paracrine release from PBAE/VEGF-engineered ADSCs enhanced EC migration. (a–c) Representative images of migrated HUVECs in response to CM from ADSCs or EBM (negative control). (d) Percentage of migrated HUVECs toward CM (*p<0.05 vs. EBM only; ^#p<0.05 vs. ADSC-CM only). Color images available online at www.liebertpub.com/scd

FIG. 7.

Paracrine release from PBAE/VEGF-engineered human ADSCs enhanced EC tube formation. (a–d) CM from PBAE/VEGF-transfected ADSCs led to formation of interconnected tubular structures by HUVECs (scale bars=500 μm). (e) The number of branch points formed by HUVECs in response to CM from all groups (*p<0.05 vs. EBM; ^#p<0.05 compared with Lipo).