SDF-1α gene-activated collagen scaffold enhances provasculogenic response in a coculture of human endothelial cells with human adipose-derived stromal cells

Abstract

Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1α GAS) could be used to enhance functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1α GAS with a gene-free scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the VE-cadherin. On the 14th day, the coculture on the SDF-1α GAS showed enhanced maturation than its gene-free equivalent through the elevation of provasculogenic genes (SDF-1α-7.4-fold, CXCR4-1.5-fold, eNOS-1.5-fold). Furthermore, we also found that the coculture on SDF-1α GAS secretes bioactive factors that significantly (p < 0.01) enhanced human Schwann cells' clustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1α GAS could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between endothelial cells and ADSCs.

Fig. 1

Effect of PEI–pSDF-1α transfection on HUVECs and its impact on ADSCs. A PEI–pSDF-1α transfected HUVECs exhibited significantly enhanced survivability response compared to those transfected nontherapeutic PEI–pLuc polyplex. B Transfection with PEI–pSDF-1α induced transient production of the target protein over a period of 2 weeks. C Phase-contrast microscopy images of morphological changes in HUVECs and its coculture with ADSCs at day 14. i Untransfected HUVECs maintained its cobblestone-like morphology. ii Transfected HUVECs appeared more polarized. iii Addition of ADSCs to untransfected HUVECs resulted in the formation of interconnected cellular network within the monolayer assembly. iv Elongated, pseudo three-dimensional dense cellular clusters were formed in transfected coculture group. Data are plotted as mean ± standard deviation (n = 6)

Fig. 2

Temporal regulation of soluble VE-cadherin from endothelialized gene-free scaffold and SDF-1α GAS. SDF-1α GAS strongly affects the vascular growth of endothelial cells by suppressing the release of soluble VE-cadherin. Coculturing with ADSCs offers further control on the release of soluble VE-cadherin from endothelial cells. HUVECs on SDF-1α GAS demonstrated significant reduction in the levels of soluble VE-cadherin at days 7 (p < 0.05) and 10 (p < 0.0005) relative to HUVECs on gene-free scaffold. Coculture on SDF-1α GAS strongly attenuated the release of VE-cadherin at all time points. Data are presented as mean ± standard deviation. One-way ANOVA was used to deduce statistical significance. *, **, ***, and **** indicate statistical significance of p < 0.05, p < 0.01, p < 0.005, and p < 0.0005, respectively

Fig. 3

Gene expression analysis of HUVECs and its coculture on gene-free scaffolds and SDF-1α GAS. SDF-1α GAS significantly increased the expression of mRNAs for SDF-1α and its cognate receptor CXCR4 in HUVECs. Coculture with ADSCs significantly elevated the expression of downstream effector genes of SDF-1α—CXCR4 and eNOS than the HUVECs on SDF-1α GAS. Data are plotted as mean ± standard deviation (n = 3). *, **, and *** indicate statistical significance of p < 0.05, p < 0.01, and p < 0.005, respectively

Fig. 4

Visualization of endothelial anastomosis and SDF-1α expression. A All the cultures showed strong immunoreactivity to CD31 and exhibited endothelial morphogenesis representative of capillary-like network. B HUVECs on the gene-free scaffold expressed the lowest amount of SDF-1α proteins. C Magnified images of the endothelial network showing the differences in spatial distribution of SDF-1α between the groups. D Quantitative representation of SDF-1α protein expression showing an increasing trend toward the coculture group. Data are presented as mean ± standard deviation. * indicates statistical significance of p < 0.05

Fig. 5

Cellular organization of human Schwann cells in response to CM derived from coculture of endothelial cells and ADSCs. A A scratched monolayer of Schwann cells. B Schwann cells invading the wounded zone 24 h post exposure to CM. C Schwann cells undergoing morphological changes 48 h post exposure to CM. D At 48 h, Schwann cells exposed to CM from the coculture on SDF-1α GAS organized into significantly (p < 0.01) larger interconnected clusters than the Schwann cells exposed to CM from the coculture on gene-free scaffold. Data are presented as mean ± standard deviation