Research ArticleAPPLIED SCIENCES AND ENGINEERING

Conjugated polymers optically regulate the fate of endothelial colony-forming cells

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Science Advances  27 Sep 2019:
Vol. 5, no. 9, eaav4620
DOI: 10.1126/sciadv.aav4620
  • Fig. 1 Polymer devices for optical stimulation of ECFC cultures.

    (A) P3HT polymer optical absorption spectrum. Insets show the chemical structure of the conjugated polymer and a sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. (B) ECFC viability at fixed time points after plating (24, 48, and 72 hours). Cell cultures were kept in dark conditions at controlled temperature (37°C) and fixed CO2 levels (5%). No statistically significant difference was observed between the glass and polymer substrates at any fixed time point (unpaired Student’s t test). (C) Experimental setup and optical excitation protocol for evaluation of polymer-mediated cell photoexcitation effects on cell fate. Polymer and control samples are positioned within a sterilized, home-designed petri holder. Light scattering effects are completely screened. The geometry and the photoexcitation protocol have been implemented to minimize overheating effects and to keep the overall extracellular bath temperature fairly unaltered. Thirty-millisecond-long green light pulses are followed by 70 ms in dark condition.

  • Fig. 2 Polymer-mediated optical activation of TRPV1 stimulates proliferation in ECFCs.

    (A) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT thin films, together with corresponding control samples kept in dark conditions. Cell proliferation was measured after 36 hours of culture in the presence of EBM-2 supplemented with 2% fetal calf serum. (B) Relative variation of the proliferation rate of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence (CTRL) and presence of 10 μM capsazepine (CPZ), 10 μM ruthenium red (RR), 20 μM RN-1734 (RN-1734), and 30 μM BAPTA-AM (BAPTA). The results are represented as the means ± standard error of the mean (SEM) of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way analysis of variance (ANOVA) coupled with Tukey (A) or Dunnett’s (B) post hoc test. *P < 0.05.

  • Fig. 3 Light-induced photoexcitation stimulates tubulogenesis in ECFC cultures.

    (A to D) Representative images of in vitro tubular networks of ECFCs subjected to long-term optical excitation seeded on both bare glass and P3HT, as well as on corresponding control samples in dark conditions. Cultures were observed up to 24 hours, but their appearance did not substantially change after pictures were taken after 8-hour culture. Scale bars, 250 μm. (E) Sketch representing the main features typical of the capillary-like network that were considered for the topologic analysis. Number of master segments (F), master junctions (G), and meshes (H) analyzed in the different conditions. The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Tukey post hoc test. **P < 0.01 and ***P < 0.001.

  • Fig. 4 Light-induced TRPV1 activation promotes in vitro tubulogenesis in ECFC cultures.

    (A to D) Representative optical images of in vitro tubular network of ECFCs subjected to long-term optical excitation seeded either on bare glass or on P3HT thin films and treated respectively with CPZ (A), RR (B), RN-1734 (C), and BAPTA-AM (D). Scale bars, 250 μm. (E to G) Relative variation of number of master segments (E), master junctions (F), and meshes (G) of ECFCs subjected to long-term optical excitation seeded on P3HT in the absence [control (CTRL)] and presence of 10 μM CPZ, 10 μM RR, 20 μM RN-1734 (RN-1734), and 30 μM BAPTA-AM (BAPTA). The results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with one-way ANOVA coupled with Dunnett’s post hoc test. *P < 0.05 and **P < 0.01.

  • Fig. 5 Phototransduction mechanisms.

    (A) An electrically insulating, thermally conducting material (photoresist) is successfully used as an ECFC seeding substrate. (B) Photoresist long-term photoexcitation does not lead to sizable enhancement in tubulogenesis parameters. (C) Evaluation of intracellular ROS production following long-term photoexcitation protocol of ECFC cultures on polymer and glass substrates (glass dark, n = 629; glass light, n = 656; P3HT dark, n = 686; and P3HT light, n = 583). For each panel, the results are represented as the means ± SEM of three different experiments conducted on cells harvested from three different donors. The significance of differences was evaluated with unpaired Student’s t test (A and B) or one-way ANOVA coupled with Tukey post hoc test (C). ***P < 0.001.

  • Fig. 6 Light-induced photostimulation promotes p65 NF-κB nuclear translocation and induces the expression of proangiogenic genes in ECFCs.

    ECFCs seeded on P3HT samples and glass controls are subjected to long-term photostimulation protocol. Corresponding control samples are kept in dark conditions. After photostimulation, p65 NF-κB nuclear translocation (A and B) and mRNA levels of tubulogenic/angiogenic genes that have been shown to be activated downstream of NF-κB (C) are evaluated. (A) Representative images of immunofluorescence staining showing p65 NF-κB (green) nuclear translocation. Cell nuclei are detected by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. (B) Quantitative evaluation of p65 NF-κB nuclear translocation, as evidenced by colocalization experiments. Results are expressed as means ± SEM of the relative percentage of p65 nuclei–positively stained cells to the total number of cells (glass dark, n = 151; glass light, n = 125; P3HT dark, n = 147; and P3HT light, n = 159). Ten fields per condition are analyzed. Data are obtained from two different experiments conducted on cells harvested from two different donors. (C) mRNA levels of intercellular adhesion molecule 1 (ICAM1), selectin E (SELE), and matrix metalloproteinases (MMP1, MMP2, and MMP9) are quantified by real-time polymerase chain reaction (PCR). Data are expressed as means ± SEM of percentage variation with respect to cells grown in the dark (n = 6). The significance of differences was evaluated with unpaired Student’s t test (C) or one-way ANOVA coupled with Tukey post hoc test (B). *P < 0.05 and **P < 0.01.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaav4620/DC1

    Fig. S1. Local and global evaluation of the extracellular bath temperature.

    Fig. S2. TRPV1 is endogenously expressed in ECFCs, and it is efficiently activated by polymer photostimulation.

    Fig. S3. Current clamp measurements in HEK-293 cells.

    Fig. S4. Pharmacological study on ECFCs seeded on polymer substrates in the dark—Evaluation of effect on tubulogenesis.

    Fig. S5. Polymer photostability.

    Fig. S6. p65 NF-κB nuclear translocation is unaltered in ECFCs seeded on glass subjected to light-induced photostimulation.

    Fig. S7. mRNA levels of proangiogenic genes downstream of NF-κB signaling.

    Table S1. List of oligonucleotide primers used for real-time PCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Local and global evaluation of the extracellular bath temperature.
    • Fig. S2. TRPV1 is endogenously expressed in ECFCs, and it is efficiently activated by polymer photostimulation.
    • Fig. S3. Current clamp measurements in HEK-293 cells.
    • Fig. S4. Pharmacological study on ECFCs seeded on polymer substrates in the dark—Evaluation of effect on tubulogenesis.
    • Fig. S5. Polymer photostability.
    • Fig. S6. p65 NF-κB nuclear translocation is unaltered in ECFCs seeded on glass subjected to light-induced photostimulation.
    • Fig. S7. mRNA levels of proangiogenic genes downstream of NF-κB signaling.
    • Table S1. List of oligonucleotide primers used for real-time PCR.

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