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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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LEC and HUVEC proliferation and migration changes upon IVSWT.(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.
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pone-0114806-g001: LEC and HUVEC proliferation and migration changes upon IVSWT.(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.

Mentions: Recent in vivo studies revealed ESWT-mediated upregulation of VEGF and its receptor VEGFR2 [30], [31] as well as VEGF-C and VEGFR3 [14], [15]. To reproduce these findings in vitro, LECs and HUVECs were stimulated with different energy flux densities at constant pulse number and frequency to ascertain possible energy-dependent proliferation changes. As it is shown in Fig. 1A, proliferation of LECs was enhanced when cells were stimulated with 0.07 and 0.09 mJ/mm2. In contrast, 0.03 and 0.19 mJ/mm2 suppressed proliferation compared to a non-stimulated control group. However, HUVEC proliferation was not altered by IVSWT using the same parameters used to determine LEC proliferation changes (Fig. 1B). We further examined IVSWT effects on the non-endothelial cell line MG63 and found no changes in proliferation after treatment (S1 Figure in S1 File). To measure the influence of shockwaves on cell migration artificially created scratches in cell monolayers were stimulated with 0.07 mJ/mm2 since different preliminary experiments identified this energy flux density level as the one inducing the highest responses of LECs (data not shown). The change of migration of LECs after 6 h in a 2D set-up was significantly higher with IVSWT compared to a non-stimulated control (Fig. 1C) whereas HUVEC migration remained unchanged after stimulation (Fig. 1D). Moreover, LEC and HUVEC migration responses to IVSWT were further determined by using a more physiological 3D migration model. Fig. 1E demonstrates a significant increase of LEC migration in a 3D setup upon IVSWT. HUVEC migration was significantly decreased after treatment (Fig. 1F). LEC adhesion assays revealed a significant reduction when cells were stimulated on Cytodex-1 microcarrier beads, however no effects were observed when cells were stimulated on other extracellular matrices (S2A Figure in S1 File). In addition, HUVEC adhesion did not change at all (S2B Figure in S1 File). Moreover, the permeability of LEC monolayers was higher 30 minutes after IVSWT compared to non-treated cells, but no differences were observable after 20 hours (S2C Figure in S1 File).


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)

LEC and HUVEC proliferation and migration changes upon IVSWT.(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.
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Related In: Results  -  Collection

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pone-0114806-g001: LEC and HUVEC proliferation and migration changes upon IVSWT.(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.
Mentions: Recent in vivo studies revealed ESWT-mediated upregulation of VEGF and its receptor VEGFR2 [30], [31] as well as VEGF-C and VEGFR3 [14], [15]. To reproduce these findings in vitro, LECs and HUVECs were stimulated with different energy flux densities at constant pulse number and frequency to ascertain possible energy-dependent proliferation changes. As it is shown in Fig. 1A, proliferation of LECs was enhanced when cells were stimulated with 0.07 and 0.09 mJ/mm2. In contrast, 0.03 and 0.19 mJ/mm2 suppressed proliferation compared to a non-stimulated control group. However, HUVEC proliferation was not altered by IVSWT using the same parameters used to determine LEC proliferation changes (Fig. 1B). We further examined IVSWT effects on the non-endothelial cell line MG63 and found no changes in proliferation after treatment (S1 Figure in S1 File). To measure the influence of shockwaves on cell migration artificially created scratches in cell monolayers were stimulated with 0.07 mJ/mm2 since different preliminary experiments identified this energy flux density level as the one inducing the highest responses of LECs (data not shown). The change of migration of LECs after 6 h in a 2D set-up was significantly higher with IVSWT compared to a non-stimulated control (Fig. 1C) whereas HUVEC migration remained unchanged after stimulation (Fig. 1D). Moreover, LEC and HUVEC migration responses to IVSWT were further determined by using a more physiological 3D migration model. Fig. 1E demonstrates a significant increase of LEC migration in a 3D setup upon IVSWT. HUVEC migration was significantly decreased after treatment (Fig. 1F). LEC adhesion assays revealed a significant reduction when cells were stimulated on Cytodex-1 microcarrier beads, however no effects were observed when cells were stimulated on other extracellular matrices (S2A Figure in S1 File). In addition, HUVEC adhesion did not change at all (S2B Figure in S1 File). Moreover, the permeability of LEC monolayers was higher 30 minutes after IVSWT compared to non-treated cells, but no differences were observable after 20 hours (S2C Figure in S1 File).

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