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Molecular and cellular effects of in vitro shockwave treatment on lymphatic endothelial cells.

Rohringer S, Holnthoner W, Hackl M, Weihs AM, Rünzler D, Skalicky S, Karbiener M, Scheideler M, Pröll J, Gabriel C, Schweighofer B, Gröger M, Spittler A, Grillari J, Redl H - PLoS ONE (2014)

Bottom Line: We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs.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.Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria.

ABSTRACT
Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro 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 podoplaninhigh and podoplaninlow 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 in vivo.

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Related in: MedlinePlus

Regulation of miRNA transcription in sorted subpopulations.(A) Heatmap visualization of log2-transformed fold changes between podoplaninhigh and podoplaninlow 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 podoplaninhigh and podoplaninlow 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).
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pone-0114806-g005: Regulation of miRNA transcription in sorted subpopulations.(A) Heatmap visualization of log2-transformed fold changes between podoplaninhigh and podoplaninlow 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 podoplaninhigh and podoplaninlow 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).

Mentions: Moreover, we also analyzed the miRNome from the podoplaninhigh and podoplaninlow populations. Hybridization shows that after imposing a fold change cut-off >1.5 fold, the majority (41 miRNAs) were upregulated in podoplaninhigh LECs, compared to 9 down-regulated miRNAs (Fig. 5A). To test the validity of these results, the expression of the top-regulated miRNAs (miR-16, miR-17, miR-181d, miR-23b, miR-92a) as well as two other miRNAs (miR-29b, miR-31) were confirmed by qPCR analysis, resulting in a significant correlation as indicated by a pearson correlation coefficient of 0.86 (Fig. 5B).


Molecular and cellular effects of in vitro shockwave treatment on lymphatic endothelial cells.

Rohringer S, Holnthoner W, Hackl M, Weihs AM, Rünzler D, Skalicky S, Karbiener M, Scheideler M, Pröll J, Gabriel C, Schweighofer B, Gröger M, Spittler A, Grillari J, Redl H - PLoS ONE (2014)

Regulation of miRNA transcription in sorted subpopulations.(A) Heatmap visualization of log2-transformed fold changes between podoplaninhigh and podoplaninlow 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 podoplaninhigh and podoplaninlow 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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263473&req=5

pone-0114806-g005: Regulation of miRNA transcription in sorted subpopulations.(A) Heatmap visualization of log2-transformed fold changes between podoplaninhigh and podoplaninlow 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 podoplaninhigh and podoplaninlow 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).
Mentions: Moreover, we also analyzed the miRNome from the podoplaninhigh and podoplaninlow populations. Hybridization shows that after imposing a fold change cut-off >1.5 fold, the majority (41 miRNAs) were upregulated in podoplaninhigh LECs, compared to 9 down-regulated miRNAs (Fig. 5A). To test the validity of these results, the expression of the top-regulated miRNAs (miR-16, miR-17, miR-181d, miR-23b, miR-92a) as well as two other miRNAs (miR-29b, miR-31) were confirmed by qPCR analysis, resulting in a significant correlation as indicated by a pearson correlation coefficient of 0.86 (Fig. 5B).

Bottom Line: We analyzed migration, proliferation, vascular tube forming capability and marker expression changes of LECs after IVSWT compared with HUVECs.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.Our findings help to understand the cellular and molecular mechanisms underlying shockwave-induced lymphangiogenesis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, Vienna, Austria; Austrian Cluster for Tissue Regeneration, Vienna, Austria.

ABSTRACT
Extracorporeal shockwave treatment was shown to improve orthopaedic diseases and wound healing and to stimulate lymphangiogenesis in vivo. The aim of this study was to investigate in vitro 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 podoplaninhigh and podoplaninlow 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 in vivo.

Show MeSH
Related in: MedlinePlus