Angiogenic Events Positively Modulated by Complex Magnetic Fields in an In Vitro Endothelial Cell Model

Figures

• 

  Figure 1. Next Generation CMFs instrument. The device is composed of the machine (A) and the CMFs emitting plaque (B). During the stimulation, the emitting plaque is located in the incubator with the multiwell plates on it (C).

• 

  Figure 2. Study design of the selected experimental conditions.

• 

  Figure 3. MTT test performed on EA.hy926 cell line 24 h (A) and 48 h (B) after stimulation with RTP of CMFs. UT1 = cells with no RTP stimulation; 1 cycle = cells stimulated with single RTP cycle; 2 cycles = cells stimulated with two consecutive RTP cycles; 3 cycles = cells stimulated with three consecutive RTP cycles; T0 + T4 = cells stimulated with two RTP cycles with 4 h of interval from one to another; T0 + T8 = cells stimulated with two RTP cycles with 8 h of interval from one to another; T0 + T4 + T8 = cells stimulated with three RTP cycles with 4 h of interval from one to another; UT2 = cells with no RTP stimulation but remain in culture one day more compared to UT1; T0 + T24 = cells stimulated with two RTP cycles with 24 h of interval from one to another; 2T0 + 2T24 = cells stimulated with two consecutive RTP cycles with 24 h of interval. Metabolic activity of cells in the first treatment block has been normalized to UT1; metabolic activity of cells in the second treatment block has been normalized to UT2. Data shown represent the mean ± SD of three independent experiments. **** p < 0.0001; *** p < 0.001.

• 

  Figure 4. LDH release from EA.hy926 cells 14 h (A) and 24 h (B) after 2 cycles, T0 + T8 and T0 + T24 experimental conditions. Data shown represent the mean ± SD of three independent experiments.

• 

  Figure 5. (A) Cell morphology observed by light phase contrast microscopy 24 h after 2 cycles, T0 + T8 and T0 + T24 RTP stimulations. Magnification, 10×. (B,C) Number of live cells/mL established by Trypan blue exclusion test 24 h (B) and 48 h (C) after 2 cycles, T0 + T8 and T0 + T24 RTP stimulations. Data shown represent the mean ± SD of three independent experiments. *** p < 0.001; ** p < 0.01; * p < 0.05. The number of cells in the first treatments block has been normalized to UT1; the number of cells in the second treatments block has been normalized to UT2.

• 

  Figure 6. (A,B) Relative gene expression of eNOS in EA.hy926 cells 6 h (A) and 14 h (B) after stimulation with CMFs. The first treatments block data are expressed as relative to UT1 (calibrator sample, defined as 1); the second treatment block data are expressed as relative to UT2 (calibrator sample, defined as 1). Y-axis, fold change. Values represent means ± SD of three independent experiments. *** p < 0.001; * p < 0.05. (C,D) p-eNOS (ser1177), total eNOS immunoreactive bands and the p-eNOS (ser1177)/eNOS ratio relative protein expression levels in EA.hy926 cells 14 h (C) and 24 h (D) after stimulation with CMFs. (E,F) p-eNOS (thr495), total eNOS immunoreactive bands, and p-eNOS (thr495)/eNOS ratio relative protein expression levels in EA.hy926 cells 14 h (E) and 24 h (F) after stimulation with CMFs. The first treatments block data are expressed as relative to UT1; the second treatments block data are expressed as relative to UT2. Tubulin is used as a loading control. The bar graph displays densitometric values (reported as Integrated Optical Intensity) obtained from the immunoreactive bands analysis and are expressed as ±SD normalized on loading control. **** p < 0.0001; *** p < 0.001;…

• 

  Figure 7. Intracellular NO levels 14 h (A) and 24 h (B) after CMF stimulation. The first treatments block data are expressed as relative to UT1; the second treatment block data are expressed as relative to UT2. Values represent means ± SD of three independent experiments. **** p < 0.0001; ** p < 0.01; * p < 0.05.

• 

  Figure 8. Relative gene expression of MMP-2 in EA.hy926 cells 6 h (A) and 14 h (B) after stimulation with CMFs. The first treatments block data are expressed as relative to UT1 (calibrator sample, defined as 1); the second treatment block data are expressed as relative to UT2 (calibrator sample, defined as 1). Values represent means ± SD of three independent experiments. Y-axis, fold change. ** p < 0.01.

• 

  Figure 9. (A,B) MMP-2 immunoreactive bands and their relative protein expression levels in EA.hy926 cells 14 h (A) and 24 h (B) after stimulation with CMFs. (C,D) MMP-9 immunoreactive bands and their relative protein expression levels in EA.hy926 cells 14 h (C) and 24 h (D) after stimulation with CMFs. (E,F) MMP-1 immunoreactive bands and their relative protein expression levels in EA.hy926 cells 14 h (E) and 24 h (F) after stimulation with CMFs. The first treatments block data are expressed as relative to UT1; the second treatment block data are expressed as relative to UT2. Tubulin is used as a loading control. The bar graph displays densitometric values (reported as Integrated Optical Intensity) obtained from the immunoreactive bands analysis and are expressed as ± SD normalized on loading control. **** p < 0.0001; *** p < 0.001; ** p < 0.01.

• 

  Figure 10. Representative images of scratch wound-healing assay performed on EA.hy926 cells at T0 (starting point, when cell monolayer was wounded) and 14, 24, and 48 h after 2 cycles, T0 + T8 and T0 + T24 RTP stimulations.

• 

  Figure 11. Bar graph displays the cut width reduction % obtained from scratch wound-healing assay, quantified by using ImageJ software. The cut reduction 14, 24 and 48 h after the stimulation is normalized on T0. **** p < 0.0001; ** p < 0.01; * p < 0.05.

• 

  Figure 12. (A,B) Integrin β1 immunoreactive bands and their relative protein expression levels in EA.hy926 cells 14 h (A) and 24 h (B) after stimulation with CMFs. (C,D) p-Erk/Erk immunoreactive bands and p-Erk/Erk ratio relative protein expression levels in EA.hy926 cells 14 h (C) and 24 h (D) after stimulation with CMFs. (E,F) Cdk2 immunoreactive bands and their relative protein expression levels in EA.hy926 cells 14 h (E) and 24 h (F) after stimulation with CMFs. The first treatments block data are expressed as relative to UT1; the second treatment block data are expressed as relative to UT2. Tubulin is used as a loading control. The bar graph displays densitometric values (reported as Integrated Optical Intensity) obtained from the immunoreactive bands analysis and are expressed as ± SD normalized on loading control. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.