An upregulation in the expression of vanilloid transient potential channels 2 enhances hypotonicity-induced cytosolic Ca²⁺ rise in human induced pluripotent stem cell model of Hutchinson-Gillford Progeria

Abstract

Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca²⁺ ([Ca²⁺](i)) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca²⁺](i) rise in iPSC-ECs from normal individuals but a sustained [Ca²⁺](i) elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca²⁺](i) rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca²⁺](i) rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca²⁺](i) elevation in HGPS-iPSC-ECs under hypotonicity, consequently resulting in apoptotic cell death. This mechanism may contribute to the pathogenesis of vascular diseases in HGPS patients.

Figure 1. Effect of hypotonicity and ATP on [Ca²⁺]_i in IMR90-iPSC-ECs and HGPS-iPSC-ECs.

(A and B), Representative traces (A) and data summary (B) showing the effect of hypotonicity (210 mOsm) on [Ca²⁺]_i (fluorescence ratio F1/F0) in IMR90-iPSC-ECs and HGPS-iPSC-ECs bathed in isotonic solution. n  =  6-7 experiments. (C), Representative traces showing the effect of hypotonic solution (210 mOsm) on [Ca²⁺]_i in cells bathed in Ca²⁺-free isotonic saline. n  =  8 experiments. D. Basal [Ca²⁺]_i level in IMR90-iPSC-ECs and HGPS-iPSC-ECs as determined by Fura-2 dye. n  =  8. ^** p<0.01 unpaired t-test compared with the sustained [Ca²⁺]_i level in IMR90-iPSC-EC group in B or compared with basal [Ca²⁺]_i level in D. (E and F), Representative traces showing the effect of ATP (1 µM) on [Ca²⁺]_i in cells bathed in normal physiological saline. Representative from 3 experiments.

Figure 2. Expression of TRP channel transcripts in IMR90-iPSC-ECs and HGPS-iPSC-ECs.

Shown were the expressional levels of transcripts for TRPV (A), TRPC (B), TRPM (C) and TRPP (D) in IMR90-iPSC-ECs (I) and HGPS-iPSC-ECs (H). n  =  4 independent experiments. ^* p<0.05 unpaired t-test compared with IMR90-iPSC-ECs.

Figure 3. Expression of TRPV2 proteins in IMR90-iPSC-ECs and HGPS-iPSC-ECs.

(A and B), representative images (A) and data summary (B) of TRPV2 protein expression in IMR90- and HGPS-iPSC-ECs. n  =  5 experiments. ^* p<0.05 unpaired t-test compared with IMR90-iPSC-EC. (C), Representative immunoblot images showing that the TRPV2-antibody recognized the targeted bands in TRPV2-overexpressing HEK293 cells (+) but not in non-transfected HEK293 cells (-). n  =  3 experiments.

Figure 4. Effect of ruthenium red (RuR) on hypotonicity-induced [Ca²⁺]_i rise.

(A-B), Representative traces showing the effect of RuR (20 µM) on hypotonicity-induced [Ca²⁺]_i rise in IMR90-iPSC-ECs (A) and HGPS-iPSC-ECs (B). (C and D), Summarized data showing the effect of RuR on transient phase (C) and sustained phase (D) of hypotonicity-induced [Ca²⁺]_i rise. n  =  6–7 independent experiments, 5–10 cells per experiment. ^** p<0.01 or ^## p<0.01 unpaired t-test compared with corresponding vehicle control.

Figure 5. Effect of tranilast on hypotonicity-induced [Ca²⁺]_i rise.

(A-B), Representative traces showing the effect of tranilast (100 µM) on hypotonicity-induced [Ca²⁺]_i rise in IMR90-iPSC-ECs (A) and HGPS-iPSC-ECs (B). (C and D), Summarized data showing the effect of tranilast on transient phase (C) and sustained phase (D) of hypotonicity-induced [Ca²⁺]_i rise. n  =  6–7 independent experiments, 5–10 cells per experiment. ^** p<0.01 unpaired t-test compared with vehicle control.

Figure 6. Effect of tranilast on hypotonicity-stimulated caspase-8 activity.

Data summary showing the inhibitory effect of tranilast on hypotonicity-induced activation of caspase-8 in HGPS-iPSC-ECs. n  =  6 independent experiments for each group. ^** p<0.01 unpaired t-test compared with isotonicity. ^## p<0.01 unpaired t-test compared with hypotonicity without tranilast.