Regulation of miRNA transcription in sorted subpopulations.

<p>(A) Heatmap visualization of log₂-transformed fold changes between podoplanin^high and podoplanin^low LECs. The top 50 regulated miRNAs according to log₂-fold change were chosen for visualization. Average log₂ fold changes from n = 2 biological replicates per sample are shown. (B) Specific miRNAs were selected for confirmation of fold-changes by quantitative PCR (indicated by boxes in the heatmap). Average log₂-transformed fold changes between podoplanin^high and podoplanin^low LECs derived from microarray and RT-qPCR (reference gene  = 5S rRNA) are shown (n = 2 per sample) and linear correlation was estimated using Pearson correlation (PCC  = 0.8646).</p

Molecular and Cellular Effects of <i>In Vitro</i> Shockwave Treatment on Lymphatic Endothelial Cells

<div><p>Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis <i>in vivo</i>. The aim of this study was to investigate <i>in vitro</i> shockwave treatment (IVSWT) effects on lymphatic endothelial cell (LEC) behavior and lymphangiogenesis. We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs. Finally, transcriptome- and miRNA analyses were conducted to gain deeper insight into the IVSWT-induced molecular mechanisms in LECs. The results indicate that IVSWT-mediated proliferation changes of LECs are highly energy flux density-dependent and LEC 2D as well as 3D migration was enhanced through IVSWT. IVSWT suppressed HUVEC 3D migration but enhanced vasculogenesis. Furthermore, we identified podoplanin^high and podoplanin^low cell subpopulations, whose ratios changed upon IVSWT treatment. Transcriptome- and miRNA analyses on these populations showed differences in genes specific for signaling and vascular tissue. Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis <i>in vivo</i>.</p></div

Flow cytometry analyses of IVSW-treated LECs.

<p>(A) Podoplanin expression on LECs was significantly enhanced by IVSWT. (B) LEC populations differ in FSC values and podoplanin expression. (C) Shockwave stimulation mediates a morphology change of LECs by increasing the amount of podoplanin^high LECs. The amount of podoplanin^low LECs did not change significantly. (D)Neither the podoplanin expression on podoplanin^high, nor the expression on podoplanin^low LECs changed upon IVSWT. All analyses were performed with n = 15. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5.</p

Gene expression profile of sorted subpopulations.

<p>(A) Transcripts with more than two-fold stronger expression in one of the respective populations (B) Heatmap visualization of 3 replicates showing log₂-transformed gene expression of podoplanin high and podoplanin low LECs. Affymetrix.CEL files were mas5 normalized. Log2-transformed expression values were normalized centred to the median of the 25 plotted transcripts.</p

LEC and HUVEC proliferation and migration changes upon IVSWT.

<p>(A) LEC proliferation was enhanced by stimulation with 0.07 and 0.09 mJ/mm², but decreased by 0.03 and 0.19 mJ/mm². (B) HUVEC proliferation was unaffected by IVSWT. (C) The reduction of a scratched, cell-free area within a time frame of 6 h was significantly enhanced by IVSWT in LECs. (D) No changes of migration upon IVSWT were observed in HUVECs. (E) IVSWT mediated a significant migration of LECs away from Cytodex-3 microcarrier beads embedded in fibrin gels. (F) HUVEC 3D migration was reduced in a 3D migration model. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5.</p

IVSWT-mediated HUVEC vasculogenesis.

<p>(A) Overview of the treatment setup for stimulation of EC/ASC co-cultures in fibrin. (B) Fluorescent images of non-treated versus treated LEC and HUVEC networks on day 7. (C) Quantifications of HUVEC networks. IVSWT increased the number of junctions, tubules and the total tubule length. The mean tubule length was decreased. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5.</p

Gene expression profile of sorted subpopulations.

<p>(A) Transcripts with more than two-fold stronger expression in one of the respective populations (B) Heatmap visualization of 3 replicates showing log₂-transformed gene expression of podoplanin high and podoplanin low LECs. Affymetrix.CEL files were mas5 normalized. Log2-transformed expression values were normalized centred to the median of the 25 plotted transcripts.</p