S1 File - Molecular and Cellular Effects of In Vitro Shockwave Treatment on Lymphatic Endothelial Cells

Supplementary Information. S1 Figure, Influences of IVSWT on MG63 proliferation. Shockwave treatment with different energy flux densities had no visible effects on MG63 proliferation. S2 Figure, IVSWT-induced changes of EC adhesion and LEC permeability. (A) The ability of LECs to reattach to a fibronectin-coated surface after shockwave treatment was significantly decreased when cells were stimulated on Cytodex-1 microcarrier beads. (B) HUVEC adhesion was not influenced by IVSWT. (C) Quantification of LEC monolayer permeability demonstrates an increase in permeability right after treatment which decays after 4 hours. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5. S3 Figure, IVSWT-mediated influences on in vitro vasculogenesis. (A) Stimulation of HUVEC/ASC co-cultures on day 0 with following incubation for 7 days showed no visible changes in the amount of junctions, tubules, total and mean length of the developed networks. (B) Network stimulation on day 2 after seeding with fixation on day 4 revealed no effect of IVSWT on vasculogenesis. (C) Choosing an EC∶ASC ratio of 1∶0.5 instead of 1∶1 revealed a converse effect to the 1∶1 ratio results (Shown in figure 2). The amount of junctions, tubules and the total length of tubules decreased whereas the mean tubule length increased. Scale bar = 200 µm. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5. S4 Figure, Flow cytometry analyses of LEC and HUVEC marker expression after IVSWT. (A) Cell surface expression of CD31, VE-Cadherin, VEGFR2, VEGFR3 and LYVE-1 on LECs did not change upon IVSWT whereas a significant upregulation of podoplanin was observed. (B) The expression of CD31, VE-Cadherin, CD146, VEGFR2 and Tie-2 on HUVECs did not change significantly after IVSWT. (C) The upregulation of podoplanin on LECs is energy flux density dependent. 0.03 and 0.09 mJ/mm2 suppressed, whereas 0.07 and 0.09 mJ/mm2 increased podoplanin expression. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5. S5 Figure, Gene ontology (GO) Analysis of genes differentially expressed in the sorted LEC subpopulations. (A) Functional annotation cluster of all annotated genes that are higher expressed in podoplaninhigh cells (A1), or higher expressed in podplaninlow cells (A2). (B) Functional annotation chart, including tissue expression, of all annotated genes that are higher expressed in podoplaninhigh cells (B1), or higher expressed in podplaninlow LECs (B2). All analyses were performed using DAVID (Database for Annotation, Visualization and Integrated Discovery) at http://david.abcc.ncifcrf.gov/home.jsp. S6 Figure, Correlation analysis of putative mRNA∼ microRNA networks in IVSWT treated LECs. (A) Network visualization of mRNA transcripts down-regulated in podoplaninhigh LECs (yellow boxes) and targeting microRNAs (blue boxes) with negatively correlating expression (i.e. up-regulated in podoplaninhigh LECs). (B) Analogous network visualization for mRNA transcripts up-regulated in podoplaninhigh LECs and targeting microRNAs with negatively correlating expression. S1 Table, Predicted microRNA binding sites and correlation analysis in down-regulated genes (Podoplaninhigh vs Podoplaninlow). 5 genes showed predicted binding sites and correlation with miRNAs. S2 Table, Predicted microRNA binding sites and correlation analysis in up-regulated genes (Podoplaninhigh vs Podoplaninlow). 9 genes showed predicted binding sites and correlation with miRNAs. (DOC)