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

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.


Introduction
Hutchinson-Gillford progeria syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including skin, musculoskeletal and cardiovascular systems [1,2]. HGPS patients suffer from early severe cardiovascular diseases which are characterized by progressive atherosclerosis [3,4], adventitial fibrosis and left ventricular hypertrophy [5]. They typically die from myocardial infarction or ischemic attack at the average age of 13 [6]. HGPS belongs to laminiopathies associated with point mutations in nuclear A/C lamin gene (LMNA), which results in the production of a truncated lamin A protein known as progerin. Lack of normal lamin A and accumulation of progerin result in abnormal nuclear envelope shape and chromatin architectures [7,8], and causes disruption of cell division [9,10]. A recent study showed that cytosolic Ca 2+ may cause a conformational change in progerin, affecting its posttranslational processing, which may be crucial for disease pathogenesis [11]. However, the mechanisms of premature atherosclerosis and death in HGPS patients remain obscure.
Studies have showed that HGPS cells display an increase in mechanosensitivity [9,10], which may be associated with a decrease in viability and an increase in apoptosis under repetitive mechanical strain [9]. Vascular cells, including vascular smooth muscle cells and endothelial cells, are the primary targets of progerin accumulation [10]. These cells are constantly exposed to fluid shear stress and mechanical strain in the vessel wall [9].
Moreover, it has been suggested that an increase in mechanical sensitivity of vascular cells from HGPS patients may impair cell cycle activation, which contributes to necrotic and apoptotic vascular cell death, leading to severe vascular diseases including atherosclerosis [9]. Although an increase in mechanosensitivity of HGPS vascular cells appears to play a critical role in vascular cell death and cardiovascular diseases, there are still no reports on ''mechanosensors'' that serve to monitor mechanical stimuli in HGPS vascular cells.
Our recent studies showed that human induced pluripotent stem cell (iPSC) could be derived from normal subjects as well as from patients with HGPS [20][21][22]. Moreover, iPSC-derived vascular cells from HGPS patients are a good model for studying the correlation between premature senescence phenotypes and vascular ageing [21]. The aim of this present study was to explore the possible role of TRP channels in mechanosensation in iPSCderived endothelial cells (iPSC-ECs) from HGPS patients.

Hypotonicity-induced [Ca 2+ ] i rise in iPSC-ECs from HGPS patients and normal individuals
We used a single wavelength dye Fluo-4 to measure [Ca 2+ ] i change. Figure 1A and 1B show the effect of hypotonicity on [Ca 2+ ] i in the iPSC-ECs derived from normal individuals (IMR90-iPSC-ECs) and HGPS patients (HGPS-iPSC-ECs), respectively. Upon perfusion with hypotonic solution (210 mOsm), [Ca 2+ ] i increased rapidly and peaked within 5467 sec in IMR90-iPSC-ECs and 6764 sec in HGPS-iPSC-ECs. Subsequently, elevated [Ca 2+ ] i gradually reverted back to its resting level in IMR90-iPSC-ECs with F1/F0 value of 1.060.1 (n = 6). In contrast, [Ca 2+ ] i elevation in HGPS-iPSC-ECs sustained with the F1/F0 value maintained at ,3.0 over 10 min (n = 7) ( Figure 1A and B). In the absence of extracellular Ca 2+ , hypotonicity failed to induce [Ca 2+ ] i rise in both cell types ( Figure 1C), suggesting that the hypotonicityinduced [Ca 2+ ] i rise was mostly due to Ca 2+ influx but not intracellular Ca 2+ store release. Basal [Ca 2+ ] i level was measured  2+ ] i response to ATP was slightly higher in HGPS-iPSC-ECs than that in IMR90-iPSC-ECs ( Figure 1E and F). However, because it is well documented that ATP-induced [Ca 2+ ] i transients in endothelial cells were mostly due to Ca 2+ release from IP 3 -sensitive intracellular Ca 2+ stores [23] but unrelated to mechanosensitive Ca 2+ influx, no further studies were carried out.
The specificity of anti-TRPV2 antibody was verified. The TRPV2 antibody could recognize expected TRPV2 bands in TRPV2overexpressing HEK293 cells ( Figure 3C), similar to previous reports by others [24]. No bands were observed in non-transfected HEK293 cells ( Figure 3C).

The effects of TRPV2 inhibitors on hypotonic-induced caspase 8 activation
The effect of hypotonicity on cellular apoptosis was determined by measuring caspase 8 activity, which has been reported as a hallmark of apoptotic cell death [27]. Hypotonicity treatment for 10 min significantly increased caspase 8 activity in HGPS-iPSC-ECs but not in IMR90-iPSC-ECs ( Figure 6). The hypotonicityinduced caspase 8 activation in the HGPS-iPSC-ECs was abolished by 100 mM tranilast treatment ( Figure 6).

Discussion
Major findings of the present study are as follows: 1) iPSC-ECs from HGPS patients expressed much higher levels of TRPV2 mRNAs and proteins compared to iPSC-ECs from normal individuals. 2) Hypotonicity induced a transient [Ca 2+ ] i rise in iPSC-ECs from normal individuals, but evoked a sustained [Ca 2+ ] i elevation in iPSC-ECs from HGPS patients. 3) The sustained phase of hypotonicity-induced [Ca 2+ ] i rise was completely abrogated by tranilast or RuR in iPSC-ECs from HGPS patients. Furthermore, the transient phase of hypotonicity-induced [Ca 2+ ] i rise was partially inhibited by RuR or tranilast. 4) Hypotonicity treatment for 10 min caused substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in iPSC-ECs from normal individuals. Tranilast could inhibit the hypotonicityinduced increase in caspase 8 activity. Taken together, our data strongly suggested that TRPV2 was a major mechanosensitive channel that contributed to hypotonicity-induced [Ca 2+ ] i rise in iPSC-ECs. More importantly, this channel was exclusively responsible for sustained Ca 2+ influx under hypotonicity and contributed to apoptotic cell death in HGPS-iPSC-ECs.
It has been well documented that most TRP channels are Ca 2+permeable and many of them, including TRPC1, TRPC5, TRPV2, TRPV4, TRPM3, TRRM7 and TRPP2, can be activated by hypotonicity-induced cell swelling [28]. Thus, we compared the expression profile of TRP channel mRNAs in iPSC-ECs from HGPS patients with that from normal individuals. iPSC-ECs from both subjects expressed multiple hypotonicityactivated TRP channels, including TRPC1, TRPV2, TRPV4, TRPM7 and TRPP2. However, while expression levels of other   channel in other cell types, including murine aortic smooth muscle [25] and mouse odontoblasts [26].
Our findings may have significant pathological significance. HGPS cells displayed an increase in mechanosensitivity [9,10]. Such increase in mechanosensitivity has been suggested to be a causative factor for necrotic and apoptotic vascular cell death, contributing to severe vascular diseases such as atherosclerosis [9]. However, the underlying mechanism of how an increase in mechanosensitivity could lead to cell death was not known. This present study provided a mechanistic explanation. From our results, it is conceivable that mechanical strain may stimulate TRPV2, which was excessively expressed in HGPS vascular cells and caused a sustained Ca 2+ entry, leading to Ca 2+ overload and subsequent vascular cell death. Indeed, our data clearly showed that TRPV2 mediated the hypotonicity-induced activation of caspase 8, an index for apoptosis, in iPSC-ECs from HGPS patients. These data were in agreement with the generally accepted notion that Ca 2+ overload could result in cell death [17,18]. Interestingly, TRPV2-mediated Ca 2+ overloading was previously reported to contribute to muscular dystrophy and/or cardiomyopathy in humans and animal models [29].
In conclusion, we found that an upregulation in TRPV2 expression is responsible for the sustained [Ca 2+ ] i elevation under mechanical stress in iPSC-ECs from HGPS patients. This could be an underlying reason for mechanical stress-induced vascular cell death in HGPS patients.  [20]. The cells were maintained on Matrigel TM (BD Biosciences, MA)-coated dishes with mTeSR TM medium (Stem Cell Technologies, BC, Canada) [20]. Differentiation of endothelial-like cells from human iPSCs was performed as described [30]. Briefly, embryoid bodies (EBs) were generated by digesting the human iPSCs with 1 mg/ml dispase (Gibco, Gaithersburg, MD) and the cell clusters were then re-suspended in differentiation medium which consisted of knockout-DMEM with 20% fetal calf serum (Hyclone, Logan, UT), 2 mM L-glutamine, 0.1 mM non-essential amino acids and 0.1 mM b-mercaptoethanol (Invitrogen, Carlsbad, CA) in noncoated dishes for 9 days. The EBs were then plated on gelatincoated dish for 7 days. Then the central portions of the attached EBs were manually dissected and isolated from the surrounding outgrowth cells for further expansion using endothelial growth medium-2 (EGM-2, Lonza, Walkersville, MD) for 14 days for endothelial-like cell differentiation. CD45 2 -CD31 + cells were then isolated by MoFlow XPD cell sorter (Beckman-Coulter, Fullerton, CA) and were ultimately designated as human iPSC-ECs. For characterization, human iPSC-ECs were treated with 4.8 mg/ml of DiI-labeled acetylated low-density lipoprotein (DiI-AcLDL, Molecular Probes, Eugene, OR) for 5 hours at 37uC. Cells were washed by phosphate buffered saline followed by fixation with 2% paraformaldehyde, and immunostained with 10 mg/ml of Ulexeuropaeus Lectin-FITC (Sigma Aldrich, St Louis, MO, USA) for 1 hr at room temperature [31]. Total RNA isolation and RT-PCR Cells were incubated with 1 ml of Trizol reagent (Invitrogen, USA), and total RNA was extracted via following the manufacturer's instructions. In brief, lyates were mixed with chloroform and centrifuged at 12,000 g for 15 min at 4uC. RNA was precipitated with isopropanol, and washed with 75% ethanol, followed by centrifugation at 7,500 g for 5 min at 4uC. The RNA pellet was air-dried, resuspended in RNase-free water. UV absorbance of the RNA samples was measured using a NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scientific Inc, USA). The ratio of the absorbance at 260 and 280 nm was found to be 2.0, indicated its adequate purity. For cDNA synthesis, 1.5 mg of total RNA was mixed with reverse-transcription master mix using a high-capacity cDNA reverse transcription kits (AppliedBiosystems, USA). Reverse transcription reactions were performed in a thermal cycler (Bio-Rad Laboratories Inc, USA). Primer sequences are shown in table 1. cDNA samples (0.4 ml) and primers (0.5 ml) were added with 24.1 ml of AmpliTaqGoldH DNA Polymerase (Applied Biosystems, USA). 35 cycles (94uC for 1 min, 58uC for 1 min, 72uC for 1 min) were performed with a PCR machine (PTC-200, MJ Research, USA). The amplified PCR products were run on a 1.5% agarose gels containing ethidium bromide.

Immunoblots
Cells were rinsed with ice-cold PBS, harvested in lysis buffer containing protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Germany), and centrifuged at 14,000xg for 20 minutes at 4uC. Equal amount of proteins were mixed with laemmi buffer, incubated at 70uC for 3 min. Protein samples were separated by using 12% SDS-polyacrylamide mini-gels. The gels were transferred to polyvinylidene-difluoride membrane using Trans-blot SD semi-dry electrophoretic transfer cell (Bio-Rad Laboratories, USA) and blocked with 5% skimmed milk in TBS buffer at room temperature for 2 hr. The membranes were then incubated with the primary antibodies against TRPV2 (VRL-1) (Novus Biologicals, CO, USA) or b-actin (Santa Cruz Biotechnology Inc., USA) overnight at 4uC. Immunoreactivity was detected with the secondary anti-rabbit or anti-mouse antibody conjugated to HRP (Santa Cruz Biotechnology Inc., USA). The blots were developed using a chemiluminescence's reagent (Bio-Rad Laboratories, USA), with films being exposed and analyzed using Image J (National Institutes of Health, USA).

[Ca 2+ ] i measurement
The cells were loaded with 5 mM Fluo-4/AM or 10 mM Fura-2/AM in the presence of 0.02% pluronic F-12 for 1 hr, and maintained in normal physiological saline solution (NPSS