Supplementary Information
Surface density of vascular endothelial growth factor
modulates endothelial proliferation and differentiation
Richard J. Galas Jr.a and Julie C. Liua,b,*
a
School of Chemical Engineering, Purdue University, West
Lafayette, IN 47907-2100
b
Weldon School of Biomedical Engineering, Purdue University,
West Lafayette, IN 47907-2100
*Corresponding Author: Email: julieliu@purdue.edu, Phone: 765494-1935, Fax: 765-494-0805
Supplementary Figure 1: Absorbance spectra of BSA, sulfo-SANPAH (SS), and BSA
conjugated to SS (BSA-SS) were used to confirm (A) the NHS ester reaction conditions,
(B) exposure duration for loss of azide peak, and (C) conjugation to TCPS. (A) BSA was
reacted with 50 molar excess of sulfo-SANPAH for 5 h and dialyzed against water. The
resulting product (BSA-SS) had the azide peak found in pure SS and also had a slight
increase in absorbance around 300 nm due to BSA. (B) Exposure of BSA-SS in water
to ultraviolet light attenuated the azide absorbance peak in a time-dependent manner.
Five minutes of exposure was used for all subsequent experiments. (C) BSA-SS was
dried on a surface and exposed to UV light. The disappearance of the azide peak
confirmed the success of the photocrosslinking step for conjugating BSA to a surface.
Supplementary Figure 2: Most adsorbed VEGF can be removed in acidic conditions. A
solution of VEGF was dried onto TCPS surfaces overnight in a 40°C oven and was
either stored dry for two days or incubated in acidic solution for two days. (A) Surfaces
were evaluated with an ELISA to determine the amount of correctly folded VEGF
proteins remaining on the surface. (B) To confirm that VEGF was removed and not just
denatured by acidic conditions, a BCA assay was used to assess total protein content
after treatment. (*) indicates that after treatment, surfaces were significantly different
(p<0.05) as determined by a t-test. Error bars indicate one standard deviation.
Supplementary Figure 3: Surface-bound VEGF did not affect the expression of
endothelial proteins or ac-LDL uptake. Expression of endothelial proteins (A) VECadherin, (B) VEGF R2, (C) vWF, (D) the venous marker EphB4, and (E) the arterial
marker Eprhin-B1, as well as internalization of (F) DiI-ac-LDL were quantified using
populations (n=3) of at least 100 cells. MSCs (stripes) were differentiated on TCPS
(white), TCPS with surface-bound VEGF (grey, defined as s-X where X denotes the
surface density in ng VEGF/cm2), or TCPS with 50 or 100 ng/mL soluble VEGF (black,
labeled 50 VEGF and 100 VEGF, respectively). Letters indicate statistically similar
groups as determined from Tukey’s post hoc analysis (p>0.05) of a two-way ANOVA. (*)
represent groups statistically different from proliferating MSCs (stripes) as determined
from Dunnett’s post hoc analysis (p<0.05) of a one-way ANOVA. Error bars indicate one
standard deviation. The data for the control, or comparison, groups (MSCs, None, 50
VEGF, and 100 VEGF) were previously published by Galas and Liu (2013).
References
Galas RJ, Liu JC. 2013. Vascular endothelial growth factor does not accelerate
endothelial differentiation of human mesenchymal stem cells. J Cell Physiol, in press,
doi: 10.1002/jcp.24421.