Supplementary Material
Supplementary Figure Ledgend
SUPPLEMENTAL FIGURE 1. Time before TEM as a function of subendothelial stiffness.
The average time for all MNCs was 18 ± 1.7 minutes. Substrate stiffness did not significantly
influence the time MNCs required prior to TEM (p > 0.05).
SUPPLEMENTAL FIGURE 2. MNC transmigration is effected by VCAM-1 in a
subendothelial stiffness dependent manner. Blocking VCAM-1 significantly reduced TEM on
all substrates. However the degree of reduction was not the same for all subendothelial
stiffnesses. There is about a 50% reduction in TEM on stiff substrates (5 kPa to glass), a 80%
reduction for the physiological case (3 kPa), and 96% for ECs on soft substrates (1 kPa).
*p<0.05, ** p<0.001 compared to 1 kPa with the same treatment.
Video 1. MNC transmigration through HAEC monolayer on a glass substrate. The
migration and transmigration of 57 initial MNCs over an activated HAEC monolayer cultured on
glass can be observed in this 14 sec. video. White or gray MNCs are on top of the endothelium
while dark gray MNCs have transmigrated through the endothelium. The video consists of 721
frames taken over a 1 hr. timelapse using phase contrast microscopy. Scale bar represents 10 µm.
Video 2. MNC migration over an activated and VCAM-1 blocked HAEC monolayer on a
280 kPa gel. Video detailing the migration and transmigration of 39 initial MNCs over an
activated HAEC monolayer that was cultured on 280 kPa polyacrylamide gel and blocked for
VCAM-1 using anti-human VCAM-1 antibodies. The video duration is 14 sec. and consists of
721 frames taken over a 1 hr. timelapse using phase contrast microscopy. Scale bar represents 10
Supplementary Figures
Time untill TEM
Time (minutes)
1 kPa
3 kPa
5 kPa
13 kPa
280 kPa
Sub-endothelial Stiffness
SUPPLEMENTAL FIGURE 1. Hayenga and Aranda-Espinoza.
VCAM-1 Blocked
% Transmigrated
1 kPa
3 kPa
5 kPa
13 kPa
280 kPa
Sub-endothelial Substrate Stiffness
SUPPLEMENTAL FIGURE 2. Hayenga and Aranda-Espinoza.
Online Supplementary Discussion
Others have shown activation of all PMNs on cytokine-activated HUVEC endothelium
after only 30 minutes8 and the percent of TEM is greater for PMNs than MNCs.2 Therefore, what
is contributing to less MNCs being activated and transmigrating compared to PMNs? On the
endothelium selectins (e.g. P-selectin and E-selectin) and adhesion molecules (e.g. ICAM-1 and
VCAM-1) are presented to both PMNs and MNCs, therefore the difference in activation may be
due differences in molecular at the surface to leukocytes and not ECs. Generally PMNs are
considered the first responders in the acute inflammatory response whereas MNCs play a larger
role in chronic inflammation. Furthermore, perhaps subpopulations of the MNCs (e.g.
lymphocytes) are less prone to transmigrate. Further experiments are needed to elucidate the
higher activation threshold for MNCs and hence less TEM compared to PMNs.
The morphology of monocyte migration on the apical surface of the endothelium is also
different than neutrophil migration. After the initial capture monocytes flatten (turn gray in phase
contrast microscopy) and then migrate until reaching the desired TEM location, at which point
they transmigrate (and appear dark gray in phase contrast microscopy) (see Fig. 1B, 4B and 5A).
On the contrary neutrophils do not flatten but remain white until TEM.8 Adhesion and migration
of both monocytes and neutrophils is mediated by the β1/β2 integrin families and members of
CAMs on endothelial cells. However, the specificity and distribution of these receptor-ligand
interactions differ between neutrophils and monocytes.4, 9 Monocytes can also use integrins α4β1
(VLA-4), in addition to αLβ2 (LFA-1) to adhere to the activated endothelium.5,
monocytes primary use LFA-1 to migrate,1 whereas neutrophils are dependent on αMβ2 (Mac1).3, 7 These differences may contribute to the flattening prior to transmigration that is observed
in migrating monocytes but not neutrophils.
Online Supplemental References:
Auffray, C., Fogg, D., Garfa, M., Elain, G., Join-Lambert, O., Kayal, S., Sarnacki, S., Cumano, A.,
Lauvau, G., and Geissmann, F. Monitoring of Blood Vessels and Tissues by a Population of
Monocytes with Patrolling Behavior. Science. 317: 666-670, 2007.
Hakkert, B. C., Kuijpers, T. W., Leeuwenberg, J. F., van Mourik, J. A., and Roos, D. Neutrophil and
Monocyte Adherence to and Migration across Monolayers of Cytokine-Activated Endothelial
Cells: The Contribution of Cd18, Elam-1, and Vla-4. Blood. 78: 2721-2726, 1991.
Henderson, R. B., Lim, L. H., Tessier, P. A., Gavins, F. N., Mathies, M., Perretti, M., and Hogg, N.
The Use of Lymphocyte Function-Associated Antigen (Lfa)-1-Deficient Mice to Determine the
Role of Lfa-1, Mac-1, and Alpha4 Integrin in the Inflammatory Response of Neutrophils. The
Journal of experimental medicine. 194: 219-226, 2001.
Ley, K., Laudanna, C., Cybulsky, M. I., and Nourshargh, S. Getting to the Site of Inflammation:
The Leukocyte Adhesion Cascade Updated. Nature reviews. Immunology. 7: 678-689, 2007.
Luscinskas, F. W., Kansas, G. S., Ding, H., Pizcueta, P., Schleiffenbaum, B. E., Tedder, T. F., and
Gimbrone, M. A., Jr. Monocyte Rolling, Arrest and Spreading on Il-4-Activated Vascular
Endothelium under Flow Is Mediated Via Sequential Action of L-Selectin, Beta 1-Integrins, and
Beta 2-Integrins. The Journal of cell biology. 125: 1417-1427, 1994.
Meerschaert, J., and Furie, M. B. The Adhesion Molecules Used by Monocytes for Migration
across Endothelium Include Cd11a/Cd18, Cd11b/Cd18, and Vla-4 on Monocytes and Icam-1,
Vcam-1, and Other Ligands on Endothelium. J Immunol. 154: 4099-4112, 1995.
Phillipson, M., Heit, B., Colarusso, P., Liu, L., Ballantyne, C. M., and Kubes, P. Intraluminal
Crawling of Neutrophils to Emigration Sites: A Molecularly Distinct Process from Adhesion in the
Recruitment Cascade. The Journal of experimental medicine. 203: 2569-2575, 2006.
Stroka, K. M., and Aranda-Espinoza, H. Endothelial Cell Substrate Stiffness Influences Neutrophil
Transmigration Via Myosin Light Chain Kinase-Dependent Cell Contraction. Blood. 118: 16321640, 2011.
Xie, X., Raud, J., Hedqvist, P., and Lindbom, L. In Vivo Rolling and Endothelial Selectin Binding of
Mononuclear Leukocytes Is Distinct from That of Polymorphonuclear Cells. European journal of
immunology. 27: 2935-2941, 1997.

Supplementary Figures - Springer Static Content Server