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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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Flow cytometry analyses of IVSW-treated LECs.(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 podoplaninhigh LECs. The amount of podoplaninlow LECs did not change significantly. (D)Neither the podoplanin expression on podoplaninhigh, nor the expression on podoplaninlow LECs changed upon IVSWT. All analyses were performed with n = 15. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5.
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pone-0114806-g003: Flow cytometry analyses of IVSW-treated LECs.(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 podoplaninhigh LECs. The amount of podoplaninlow LECs did not change significantly. (D)Neither the podoplanin expression on podoplaninhigh, nor the expression on podoplaninlow LECs changed upon IVSWT. All analyses were performed with n = 15. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5.

Mentions: To identify possible target molecules involved in IVSWT influences on LEC behavior, flow cytometry analyses were performed. Lymphatic endothelial markers VEGFR3 and LYVE-1 as well as pan-endothelial markers such as CD31, VEGFR2 and CD144 showed no regulation upon IVSWT in LEC and HUVEC (S4A,B Figure in S1 File). In contrast, the expression of podoplanin was significantly increased after IVSWT in LECs (Fig. 3A). This effect was highly energy flux density dependent (S4C Figure in S1 File). Whereas low (0.03 mJ/mm2) and high (0.19 mJ/mm2) energy flux densities downregulated podoplanin expression, increases were observed at average energy flux densities (0.07 and 0.09 mJ/mm2), the same energy flux densities that also increased proliferation. Interestingly, we continuously identified two different subpopulations of LECs differing in their forward scatter (FSC) values (Fig. 3B, left panel). By gating these populations we found that the smaller cells expressed more podoplanin (mean geometric mean 4.04) than the larger population (mean geometric mean 1.92, Fig. 3B, right panel). The populations are further termed podoplaninhigh and podoplaninlow LECs throughout the manuscript. IVSWT mediated a significant increase in the relative amount of podoplaninhigh LECs. The podoplaninlow LEC population concomitantly decreased, albeit not to a significant extent (Fig. 3C). However, IVSWT did not alter the podoplanin expressionin the respective populations (Fig. 3D). Thus, IVSWT did not upregulate podoplanin per se, but mediated an increase in the podoplaninhigh LEC population.


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)

Flow cytometry analyses of IVSW-treated LECs.(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 podoplaninhigh LECs. The amount of podoplaninlow LECs did not change significantly. (D)Neither the podoplanin expression on podoplaninhigh, nor the expression on podoplaninlow LECs changed upon IVSWT. All analyses were performed with n = 15. 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-g003: Flow cytometry analyses of IVSW-treated LECs.(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 podoplaninhigh LECs. The amount of podoplaninlow LECs did not change significantly. (D)Neither the podoplanin expression on podoplaninhigh, nor the expression on podoplaninlow LECs changed upon IVSWT. All analyses were performed with n = 15. P-values: *** ≤0.01, ** ≤0.1, * ≤0.5.
Mentions: To identify possible target molecules involved in IVSWT influences on LEC behavior, flow cytometry analyses were performed. Lymphatic endothelial markers VEGFR3 and LYVE-1 as well as pan-endothelial markers such as CD31, VEGFR2 and CD144 showed no regulation upon IVSWT in LEC and HUVEC (S4A,B Figure in S1 File). In contrast, the expression of podoplanin was significantly increased after IVSWT in LECs (Fig. 3A). This effect was highly energy flux density dependent (S4C Figure in S1 File). Whereas low (0.03 mJ/mm2) and high (0.19 mJ/mm2) energy flux densities downregulated podoplanin expression, increases were observed at average energy flux densities (0.07 and 0.09 mJ/mm2), the same energy flux densities that also increased proliferation. Interestingly, we continuously identified two different subpopulations of LECs differing in their forward scatter (FSC) values (Fig. 3B, left panel). By gating these populations we found that the smaller cells expressed more podoplanin (mean geometric mean 4.04) than the larger population (mean geometric mean 1.92, Fig. 3B, right panel). The populations are further termed podoplaninhigh and podoplaninlow LECs throughout the manuscript. IVSWT mediated a significant increase in the relative amount of podoplaninhigh LECs. The podoplaninlow LEC population concomitantly decreased, albeit not to a significant extent (Fig. 3C). However, IVSWT did not alter the podoplanin expressionin the respective populations (Fig. 3D). Thus, IVSWT did not upregulate podoplanin per se, but mediated an increase in the podoplaninhigh LEC population.

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