Transient receptor potential canonical channels are required for in vitro endothelial tube formation

Abstract

In endothelial cells Ca(2+) entry is an essential component of the Ca(2+) signal that takes place during processes such as cell proliferation or angiogenesis. Ca(2+) influx occurs via the store-operated Ca(2+) entry pathway, involving stromal interaction molecule-1 (STIM1) and Orai1, but also through channels gated by second messengers like the transient receptor potential canonical (TRPC) channels. The human umbilical vein-derived endothelial cell line EA.hy926 expressed STIM1 and Orai1 as well as several TRPC channels. By invalidating each of these molecules, we showed that TRPC3, TRPC4, and TRPC5 are essential for the formation of tubular structures observed after EA.hy926 cells were plated on Matrigel. On the contrary, the silencing of STIM1 or Orai1 did not prevent tubulogenesis. Soon after being plated on Matrigel, the cells displayed spontaneous Ca(2+) oscillations that were strongly reduced by treatment with siRNA against TRPC3, TRPC4, or TRPC5, but not siRNA against STIM1 or Orai1. Furthermore, we showed that cell proliferation was reduced upon siRNA treatment against TRPC3, TRPC5, and Orai1 channels, whereas the knockdown of STIM1 had no effect. On primary human umbilical vein endothelial cells, TRPC1, TRPC4, and STIM1 are involved in tube formation, whereas Orai1 has no effect. These data showed that TRPC channels are essential for in vitro tubulogenesis, both on endothelial cell line and on primary endothelial cells.

FIGURE 1.

Ca²⁺ dependence of the in vitro tube formation process on EA.hy926 cells. The EA.hy926 were seeded on growth factor-reduced Matrigel and visualized 10–14 h later, in control conditions, or in the presence of 30 μm SK&F96365 (a Ca²⁺ entry blocker), or 50 μm BAPTA/AM (an intracellular Ca²⁺ chelator). Bar graph shows the percent of the surface covered by tubes in control condition and upon SK&F96365 or BAPTA/AM treatment. Data are mean ± S.E. (error bars) of three independent experiments. Scale bars, 200 μm. ***, p < 0.001.

FIGURE 2.

Effects of protein silencing on tube formation on EA.hy926 cells. A, 48 h after transfection with siRNA (against STIM1, Orai1, TRPC3, TRPC4, and TRPC5), the EA.hy926 cells were seeded on growth factor-reduced Matrigel and visualized 10–14 h later. B, tube formation was quantified in control condition and upon siRNA treatment. Data are mean ± S.E. (error bars) of three to five independent experiments. *, p < 0.05; ***, p < 0.001. C, cells were seeded on growth factor-reduced Matrigel in control conditions and in the presence of 10 μm TRPC3 inhibitor Pyr3 or 2 μm phospholipase C inhibitor U73122. Scale bars, 200 μm.

FIGURE 3.

EA.hy926 cell proliferation assay. A, quantification of BrdU incorporation in control conditions and in the presence of 30 μm SK&F96365 and 50 μm BAPTA/AM. B, quantification of BrdU incorporation in EA.hy926 cells transfected with control siRNA and siRNA against STIM1, Orai1, and STIM1/Orai1. C, quantification of BrdU incorporation in EA.hy926 cells transfected with control siRNA and siRNA against TRPC3, TRPC4, and TRPC5. Data are mean ± S.E. (error bars) of minimum three independent experiments in duplicate. ns, not significant; ***, p < 0.001.

FIGURE 4.

Spontaneous Ca²⁺ oscillations on EA.hy926 cells during tube formation. A–D, cytosolic Ca²⁺ measured at 37 °C on EA.hy926 cells loaded with 2 μm Fura-2 (supplemental Movies 1 and 2). Intracellular Ca²⁺ variations were measured after the first 1–2 h of tube formation on Matrigel, after siControl treatment (A), siSTIM1 (B), siTRPC3 (C) or siTRPC4 (D). Acquisition was done for 10 min, and each panel displays the responses of a representative coverslip. E, number of Ca²⁺ oscillations during 10 min in cells treated with siControl, siSTIM1, siOrai1, siTRPC3, siTRPC4, and siTRPC5. F, percentage of cells showing at least one Ca²⁺ transient during the 10-min recording time (n ranges from 64 to 168 cells). Error bars, S.E. ns, not significant; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Histamine-induced Ca²⁺ response on EA.hy926 cells after tube formation. Cytosolic Ca²⁺ responses were measured at 37 °C once the tubular structures were formed (14 h after cell adhesion on Matrigel). Before stimulation, no Ca²⁺ oscillations were detected. A–D, Ca²⁺ responses elicited by histamine after siControl treatment (A), siSTIM1 (B), siOrai1 (C), or siTRPC3 (D). E, number of Ca²⁺ oscillations during 10 min in cells treated with siControl, siSTIM1, siOrai1, siTRPC3, siTRPC4, and siTRPC5. F, amplitude of Ca²⁺ oscillations for each conditions shown in E (n ranges between 25 and 91 cells). Error bars, S.E. ns, not significant; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Histamine-induced Ca²⁺ entry on EA.hy926 cells grown on glass. Cytosolic Ca²⁺ concentration was assessed on EA.hy926 cells loaded with 2 μm Fura-2 and stimulated with 1 μm histamine in 2 mm Ca²⁺-containing medium. A, EA.hy926 cells were transiently transfected with control siRNA (scramble, siControl), or siRNA against STIM1 or Orai1. Each trace is the mean of a representative coverslip. B, quantification of the effects of siSTIM1 and siOrai1 on the difference between the ratio at 10 min and the basal ratio (n ranges from 81 to 201 cells). C, representative traces of 1 μm histamine stimulation of cells transiently transfected with siControl or siTRPC3. Each trace represents the mean of a representative coverslip. D, quantification of the effects of siTRPC3, siTRPC4, and siTRPC5 on the difference between the ratio after 10 min of stimulation and the basal ratio (n ranges from 67 to 85 cells). E, EA.hy926 cells were transiently transfected with the ER-targeted cameleon probe D1_ER. Representative recording of a cell stimulated sequentially with 1 μm histamine in Ca²⁺-containing medium and in Ca²⁺-free medium is shown. F, quantification of ER Ca²⁺ release in EA.hy926 cells stimulated by 1 μm histamine in Ca²⁺-containing medium (CA), Ca²⁺-free medium (CF), and by 1 μm thapsigargin (Tg) (in CA). Error bars, S.E. ***, p < 0.001.

FIGURE 7.

Expression of TRPC isoforms on HUVEC and effects of protein silencing on tube formation. A, mRNA amplification of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 isoforms by real-time PCR in HUVECs. B, mRNA levels assessed by quantitative RT-PCR 48 h after transfection (mean ± S.E. (error bars), 2–4 independent experiments). C, 48 h after transfection with siRNA (against TRPC1, TRPC4, STIM1, and Orai1), the HUVECs were seeded on growth factor-reduced Matrigel and visualized 10–14 h later. D, quantification of tube formation in control condition and upon siRNA treatment. Data are mean ± S.E. of three to five independent experiments. Scale bar, 200 μm. ns, not significant; ***, p < 0.001.

FIGURE 8.

Spontaneous Ca²⁺ oscillations during the tube formation on HUVECs. A and B, cytosolic Ca²⁺ measured at 37 °C on HUVECs loaded with 2 μm Fura-2. Intracellular Ca²⁺ variations were measured after the first 1–2 h of tube formation on Matrigel, after siControl treatment (A) and siTRPC4 (B). Acquisition was done for 10 min, and each panel displays the responses of a representative coverslip. C, percentage of cells showing at least one Ca²⁺ transient during the 10-min recording time (n ranges from 39 to 135 cells). D, number of Ca²⁺ oscillations during 10 min in cells treated with siControl, siTRPC1, siTRPC4, siSTIM1, and siOrai1. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.