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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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IVSWT-mediated HUVEC vasculogenesis.(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.
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pone-0114806-g002: IVSWT-mediated HUVEC vasculogenesis.(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.

Mentions: We have shown before that endothelial cells form vascular networks in fibrin gels in the presence of ASCs after one week [25]. In the present study, this model was used to determine the ability of IVSWT to induce vascular tube formation in vitro. Various shockwave treatment setups were tested (S3A–S3C Figures in S1 File). Finally, a three times treatment turned out to be necessary for visualizing the influence of shockwaves. Fig. 2A illustrates the time schedule for shockwave treatment of the gels. The gels were stimulated right after preparation, and on the second and fifth day. Applying these parameters IVSWT enhanced HUVEC-mediated vasculogenesis in vitro (Fig. 2B). The total number of junctions, tubules and the total tubule length were significantly higher in the stimulated group compared to non-treated controls whereas the mean tubule length decreased (Fig. 2C). In contrast, using LECs instead of HUVECs, we did not find significant differences between shockwave-treated and control groups (Fig. 2B).


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)

IVSWT-mediated HUVEC vasculogenesis.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114806-g002: IVSWT-mediated HUVEC vasculogenesis.(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.
Mentions: We have shown before that endothelial cells form vascular networks in fibrin gels in the presence of ASCs after one week [25]. In the present study, this model was used to determine the ability of IVSWT to induce vascular tube formation in vitro. Various shockwave treatment setups were tested (S3A–S3C Figures in S1 File). Finally, a three times treatment turned out to be necessary for visualizing the influence of shockwaves. Fig. 2A illustrates the time schedule for shockwave treatment of the gels. The gels were stimulated right after preparation, and on the second and fifth day. Applying these parameters IVSWT enhanced HUVEC-mediated vasculogenesis in vitro (Fig. 2B). The total number of junctions, tubules and the total tubule length were significantly higher in the stimulated group compared to non-treated controls whereas the mean tubule length decreased (Fig. 2C). In contrast, using LECs instead of HUVECs, we did not find significant differences between shockwave-treated and control groups (Fig. 2B).

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