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(Circulation Research. 2008;102:347.)
© 2008 American Heart Association, Inc.

Cellular Biology

Role of TRPM2 Channel in Mediating H₂O₂-Induced Ca²⁺ Entry and Endothelial Hyperpermeability

Claudie M. Hecquet, Gias U. Ahmmed, Stephen M. Vogel, Asrar B. Malik

From the Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago.

Correspondence to Asrar B. Malik, Department of Pharmacology, University of Illinois at Chicago, College of Medicine, 835 South Wolcott Ave (M/C 868), Chicago, IL 60612. E-mail abmalik{at}uic.edu

	Abstract

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Oxidative stress through the production of oxygen metabolites such as hydrogen peroxide (H₂O₂) increases vascular endothelial permeability. H₂O₂ stimulates ADP-ribose formation, which in turn opens transient receptor potential melastatin (TRPM)2 channels. Here, in endothelial cells, we demonstrate transcript and protein expression of TRPM2, a Ca²⁺-permeable, nonselective cation channel. We further show the importance of TRPM2 expression in signaling of increased endothelial permeability by oxidative stress. Exposure of endothelial cell monolayers to sublytic concentrations of H₂O₂ induced a cationic current measured by patch-clamp recording and Ca²⁺ entry detected by intracellular fura-2 fluorescence. H₂O₂ in a concentration-dependent manner also decreased trans-monolayer transendothelial electrical resistance for 3 hours (with maximal effect seen at 300 µmol/L H₂O₂), indicating opening of interendothelial junctions. The cationic current, Ca²⁺ entry, and transendothelial electrical resistance decrease elicited by H₂O₂ were inhibited by siRNA depleting TRPM2 or antibody blocking of TRPM2. H₂O₂ responses were attenuated by overexpression of the dominant-negative splice variant of TRPM2 or inhibition of ADP-ribose formation. Overexpression of the full-length TRPM2 enhanced H₂O₂-mediated Ca²⁺ entry, cationic current, and the transendothelial electrical resistance decrease. Thus, TRPM2 mediates H₂O₂-induced increase in endothelial permeability through the activation of Ca²⁺ entry via TRPM2. TRPM2 represents a novel therapeutic target directed against oxidant-induced endothelial barrier disruption.

Key Words: transient receptor potential channels • Ca²⁺ influx • endothelial vascular barrier • permeability • lung injury

	Introduction

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Reactive oxygen species (ROS) are important mediators of vascular barrier dysfunction in settings such as acute respiratory distress syndrome, ischemia/reperfusion, and hyperoxia.^1–4 Evidence suggests that oxidants increase Ca²⁺ permeability of endothelial cell membrane.^5–7 The resulting elevation of intracellular Ca²⁺ could contribute to barrier disruption because Ca²⁺ entry into endothelial cells is recognized to promote interendothelial gap formation.^5,8–10 The molecular mechanisms of oxidant-induced change in endothelial Ca²⁺ permeability remains an important area of inquiry.

Transient receptor potential melastatin (TRPM)2 is an oxidant-activated channel belonging to the TRP family of cation channels. TRPM2,¹¹ first named TRPC7 and later LTRPC-2,¹² is a nonselective cation channel widely expressed in mammalian tissues, including the brain, peripheral blood cells such as neutrophils,^11,13 bone marrow, spleen, heart, and liver,¹⁴ and lungs.¹⁵ TRPM2 opening after exposure to oxidants is induced by the binding of the intracellular second messenger adenosine diphosphoribose (ADP-ribose) or related molecules to the Nudix box sequence motif (NUDT9-H)^14,16,17 in its carboxyl-terminal domain.¹⁴ Because the Nudix box has significant homology with a pyrophosphatase, NUDT9 ADP-ribose hydrolase,^17–21 TRPM2 was dubbed a "chanzyme." Besides free ADP-ribose primarily generated from poly(ADP-ribose) polymerase (PARP) activity, NAD (possibly through conversion to ADP-ribose)^17–19,22 and cyclic adenosine diphosphoribose (cADP-ribose)²² may also activate TRPM2.

Hydrogen peroxide (H₂O₂) produced in the cytosol during oxidative stress¹⁵ stimulates ADP-ribose formation in the nucleus and mitochondria.²³ TRPM2 channels may thus participate in signaling oxidative stress–induced Ca²⁺ entry, thereby eliciting Ca²⁺-dependent cellular processes.^15,18,23 Although most investigators can demonstrate an indirect action of H₂O₂ in stimulating ADP-ribose formation in nuclei and mitochondria,²³ direct agonist action of H₂O₂ on TRPM2 is also proposed for myeloid cells.^24,25

In addition to full-length functional TRPM2 (TRPM2-L), several TRPM2 isoforms have been identified in human hematopoietic cells, including a short splice variant (TRPM2-S).²⁴ TRPM2-S (short) lacks the entire carboxyl terminus of the long variant, 4 of 6 carboxyl-terminal transmembrane domains, including the putative Ca²⁺-permeable pore, and functions in a dominant-negative fashion to inhibit TRPM2-L activity.^24,25 TRPM2-S directly interacts with TRPM2-L to suppress H₂O₂-induced Ca²⁺ influx through TRPM2-L in transfected 293T cells.²⁴ Thus, TRPM2-S is an important isoform of TRPM2 that may modulate channel activity and cell death induced by oxidative stress activation of TRPM2-L.²⁴

Here, we demonstrate that H₂O₂ at noncytolytic concentrations elicits marked Ca²⁺ influx via TRPM2 channels, which thereby signals increased endothelial permeability. Inhibition of endogenous TRPM2 expression and function in endothelial cells by RNA silencing, a specific TRPM2-blocking antibody, overexpression of TRPM2-S isoform, or inhibition of ADP-ribose generation significantly decreased H₂O₂-induced increase in [Ca²⁺]_i and the resulting increase in endothelial permeability. These data demonstrate a critical role of TRPM2 in the mechanism of endothelial barrier disruption following oxidative stress.

	Materials and Methods

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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

Endothelial Cell Culture and Transfection
Human pulmonary artery endothelial (HPAE) cells (Clonetics, La Jolla, Calif) were cultured in gelatin-coated flasks using endothelial basal medium 2 (EBM2) supplemented with bullet kit additives plus 10% fetal bovine serum, and used in experiments between passages 3 and 6.

Human full-length TRPM2, tagged on its carboxyl terminus with the blue fluorescent protein (BFP)–TRPM2-L, was subcloned into pQBI50 (QbioGene, Carlsbad, Calif). Short splice variant, tagged on its carboxyl terminus with the green fluorescent protein (GFP)–TRPM2-S, was inserted into pTracer-CMV (Invitrogen). HPAE cells grown to 60% to 80% confluence were transfected for 4 hours with 1 µg/mL each BFP–TRPM2-L or GFP–TRPM2-S cDNA or with vector alone (control cells) using LipofectAMINE Plus.²⁶ Cells transfected by TRPM2-L were susceptible to apoptosis; therefore, we added caspase 9 inhibitor (Ac-LEHD-CHO, 20 µmol/L) to the medium. After 48 hours, transfected or control cells forming confluent monolayers were used for experiments. Successful transfection of cells with TRPM2-S or TRPM2-L was verified by detection of GFP (excitation, 478 nm; emission, 535 nm) or BFP (excitation, 380 nm; emission, 435 nm) with laser-scanning confocal microscope (Zeiss LSM 510). Transfection efficiency was 80% to 90%.

Small Interfering RNA Transfection
HPAE cells were transiently transfected with 50 nmol/L each 2 predesigned small interfering (si)RNAs using TransIT-TKO transfection reagent according to the instructions of the manufacturer. siRNAs were targeted to exon 1 or 4 of the TRPM2 mRNA sequence. We cotransfected both siRNAs for maximal knockdown of TRPM2 expression. As a control, we used commercially available nonspecific siRNA. Experiments and RNA extractions were performed at 48 hours after transfections. Transfection efficiency was >75%.

Whole-Cell Patch-Clamp Recording in Endothelial Cells, [Ca²⁺]_i Measurements, Transendothelial Electrical Resistance Measurement, and Western Blotting Analyses
Methods are detailed in the online data supplement.

Detection of TRPM2 by RT-PCR
Two-step RT-PCR was performed using the Eppendorf Mastercycler gradient system (Eppendorf, New York) and real-time PCR using the ABI Prism 7700 Sequence Detection System (Perkin-Elmer, Applied Biosystems). PCR was performed for 36 cycles (denaturation at 94°C for 15 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute for 36 cycles). The primers used to amplify the TRPM2-L transcript targeted its carboxyl-terminal region. The forward sequence used was 5'-TCGGACCCAACCACACGCTGTA-3', and the reverse sequence was 5'-CGTCATTCTGGTCCTGGAAGTG-3'. Primers targeting both transcripts (TRPM2-L and -S) were: forward, 5'-GAAGAGCATTTTCCGCAGA-3'; reverse, 5'-ATGAGCTCGCCTTCCTTGT-3'. We used coamplified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. The amplified products were separated on 1.4% agarose gels.

	Results

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H₂O₂ and ADP-Ribose Analog 3-Deaza-cADP-Ribose Activate TRPM2 Ionic Current in Endothelial Cells
To record the ionic current induced by oxidants in single HPAE cells, we voltage clamped the cells at –50 mV after achieving a whole-cell configuration. Addition of H₂O₂ (300 µmol/L, Figure 1A) rapidly elicited an inward current above background. Isosmotic replacement of all external Na⁺ by the impermeant organic cation N-methyl-D-glucamine (NMDG⁺) abolished the H₂O₂-induced inward current. Repletion of external Na⁺ restored the current (Figure 1A). TRPM2 knockdown using siRNA abrogated the H₂O₂ effects (Figure 1B), indicating that expression of TRPM2 channels is required. Overexpression of TRPM2-L, which we verified as described (see Materials and Methods), augmented the H₂O₂-induced current (Figure 1C versus Figure 1A). The current–voltage relationship for cationic current was linear over a wide potential range (–80 to +80 mV) and passed through the origin. This current–voltage characteristic is typical of TRPM2 channels studied in expression systems.²⁴ Transduction of TRPM2-L, which caused overexpression of the functional channel (see below), increased the H₂O₂-induced current at all clamp potentials; the current–voltage curve was steeper in slope but unaltered in linearity or reversal potential. By contrast, TRPM2 silencing, which we also verified (see below), markedly reduced the slope. As a functional assay for TRPM2 channels, we tested a nonhydrolysable cADP-ribose analog (3-deaza-cADP-ribose, 10 nmol/L). Control experiments showed that saline vehicle elicited no current (Figure 1E). Internal application of the analogue via the patch pipette induced an inward current (Figure 1F), implying the presence of TRPM2 channel activity in HPAE cells. To further test the involvement of TRPM2 in the H₂O₂-induced inward current, we pretreated cultures with a cell-permeable inhibitor of PARP {100 µmol/L 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) for 45 minutes} that prevents the generation of ADP-ribose²⁷ without directly blocking TRPM2 channel.¹⁶ H₂O₂ was inactive in the presence of inhibitor (Figure 1G), indicating that endogenous TRPM2 activators ADP-ribose and cADP-ribose mediate effects of H₂O₂ in HPAE cells. The results, summarized in Figure 1H, show TRPM2 expression and cADP-ribose generation to be essential for elicitation of inward cationic current by H₂O₂.

View larger version (23K): [in this window] [in a new window]   Figure 1. H2O2- and 3-deaza-cADP-ribose–induced currents in HPAE cells. Subconfluent cultures of HPAE cells were prepared for whole-cell patch-clamp recording as described (Materials and Methods) and voltage-clamped to a standard holding potential (–50 mV). A through C, H2O2 (300 µmol/L) addition to the bath (arrow) induced an inward current in control cells, which was reversibly blocked by isosmotic substitution of Na+ for NMDG+, an impermeant organic cation (A). TRPM2 silencing suppressed the current (B) in normal medium, and TRPM2-L overexpression enhanced it (C). D, Current–voltage relationship for membrane current induced by H2O2 in untransfected cells, TRPM2 siRNA–transfected cells, and in TRPM2-L–overexpressing cells. E, Addition of control buffer to bath fluid had no effect above background. F, 3-Deaza-cADP-ribose (10 nmol/L) added to the pipette solution induced an inward current on establishing the whole-cell configuration. G, Pretreatment for 45 minutes with DPQ (D; 100 µmol/L), an inhibitor of PARP, prevented the H2O2-induced (300 µmol/L) current. H, Bar graph quantifying peak currents (in pA) obtained in A through F. Results are given as means±SEM (n=3 to 4 HPAE cells). *Significant difference in current amplitude from untreated control.

Action of H₂O₂ on Ca²⁺ Entry
Because the electrophysiological observations suggest that H₂O₂ can stimulate Ca²⁺ entry by activating the Ca²⁺-permeable channel TRPM2-L, we next measured intracellular Ca²⁺ responses to H₂O₂ in HPAE cells. We used a "Ca²⁺ add-back" protocol designed to rule out indirect effects of H₂O₂ on Ca²⁺ entry via Ca²⁺-store depletion (Figure 2). In the absence of extracellular Ca²⁺, H₂O₂ application (300 µmol/L) did not produce a Ca²⁺ transient, although in the same experiment, Ca²⁺ repletion (in the continued presence of H₂O₂) elicited a brisk Ca²⁺ response. Control experiments showed that Ca²⁺ repletion per se (no H₂O₂ added) was completely ineffective in evoking Ca²⁺ transients (Figure 2). The amplitude of Ca²⁺-repletion transients depended on the concentration of H₂O₂ (EC₅₀, 58.6 µmol/L; Figure 2, inset). These results indicate that H₂O₂ increases intracellular Ca²⁺ by stimulating Ca²⁺ entry without provoking Ca²⁺-store depletion. Interestingly, at a concentration of 500 µmol/L, H₂O₂ elicited a Ca²⁺ transient in the absence of extracellular Ca²⁺, suggesting that H₂O₂, at the highest level used, releases sequestered Ca²⁺ (Figure 2). TRPM2 siRNA or TRPM2-blocking antibodies did not block this effect (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 2. Concentration-dependent effects of H2O2 on Ca2+ entry. HPAE cells in culture were loaded with fura-2, washed, and transferred to Ca2+-free medium. H2O2 (0 to 500 µmol/L) was added at the arrow and CaCl2 (2.0 mmol/L) was repleted at the fifth minute; the resulting Ca2+-repletion transient reflects Ca2+ entry. The Ca2+ ionophore ionomycin (ion) was added at the end of the experimental recordings for calibration purposes. Each tracing is the average response of 60 to 99 cells in HPAE monolayers. The abscissa indicates time in seconds; the ordinate, relative [Ca2+]i level. Experiments were repeated 3 to 5 times with similar results. The inset displays the dose–response curve of best fit for the calcium-repletion transient (EC50, 58.6 µmol/L). The data points are mean values (n=3 per point), and the bars indicate ±SEM. At >300 µmol/L, H2O2 mobilized stored intracellular Ca2+.

TRPM2 Expression in Endothelial Cells
RT-PCR and Western Blot analysis gave evidence of TRPM2 channel expression in HPAE cells (Figure 3A and 3B). TRPM2 transcript was detected in untreated wild-type HPAE cells and to an even greater extent in TRPM2-L transfected cells (Figure 3A). The channel was not detected in TRPM2 siRNA–treated cells. Western blot analysis showed TRPM2-L protein expression as a prominent band at 171 kDa (Figure 3B). The level of TRPM2 detected in untreated HPAE cells was significantly reduced in TRPM2 siRNA–transfected cells; treatment with control siRNA was ineffective. Cells overexpressing TRPM2-L had a significantly intensified band at 171 kDa by densitometric analysis, confirming specificity of the antibody used.

View larger version (23K): [in this window] [in a new window]   Figure 3. TRPM2-activated Ca2+ entry in endothelial cells. HPAE cells were grown to confluence in cell culture and prepared for RT-PCR analysis (A), Western blotting (B), or intracellular Ca2+ measurements using fura-2 (C through G). A, TRPM2 expression in HPAE cells detected by RT-PCR. Untreated HPAE cells showed constitutive transcript expression (lanes 2 and 3). TRPM2 silencing suppressed transcript expression (lanes 4 and 5), whereas TRPM2-L transduction caused overexpression (lanes 6 and 7). B, Representative Western blots for TRPM2-L and β-actin (molecular masses of 171 and 45 kDa, respectively). Protein was quantified by densitometry. TRPM2 densities have been normalized to β-actin values and plotted as the percentage of the untreated control (mean±SEM for 3 runs). *Significance from control. C through G, Ca2+ mobilization assays using a Ca2+add-back protocol. Insets display tracings in Ca2+-containing medium for comparison. Addition (buffer or H2O2) was made shortly after 0 minutes. C and D, Untransfected cells received control buffer (arrow) (C) or 300 µmol/L H2O2 (arrow) (D). Note the marked intracellular Ca2+ response to H2O2 application. Ionomycin (ion) addition is shown at the end of tracings. E and F, Inhibition of Ca2+ entry in cells receiving 300 µmol/L H2O2 after siRNA transfection to inhibit expression of TRPM2 (E) or after pretreatment with TRPM2 blocking antibody (5 µg/mL for 16 hours) (F). Preincubation of cells with control IgG did not modify Ca2+ entry induced by H2O2 (see the legend of Figure 7), confirming the role of TRPM2 channels in H2O2-evoked Ca2+ entry. The experiments were repeated 3 to 5 times with similar results. The abscissa indicates time in seconds; the ordinate, relative [Ca2+]i level. G, Mean ratiometric values (±SEM) for steady-state [Ca2+]i (n=3 to 4) obtained in C through F. Note that inhibition of TRPM2 activity prevents H2O2-mediated Ca2+entry.

TRPM2 Regulates H₂O₂-Induced Ca²⁺ Entry in Endothelial Cells
Monolayers of HPAE cells adhering to glass coverslips had submicromolar levels of intracellular Ca²⁺ in Ca²⁺-containing medium (Figure 3C, inset). Addition of H₂O₂ (300 µmol/L) produced Ca²⁺ transients (Figure 3D, inset). TRPM2 knockdown (siRNA-transfected HPAE cells) nearly abolished the H₂O₂ effect (Figure 3E, inset). Anti-TRPM2 blocking antibody (Bethyl Laboratories, Montgomery, Tex) also prevented the H₂O₂-induced Ca²⁺ transients (Figure 3F, inset). We added back Ca²⁺ after first depleting extracellular Ca²⁺ for less than 5 minutes to rule out indirect effects of H₂O₂ via Ca²⁺-store depletion. This alone without H₂O₂ had no effect on intracellular Ca²⁺ levels (Figure 3C). In Ca²⁺-free medium, H₂O₂ addition of released no intracellular Ca²⁺, whereas Ca²⁺ repletion in the continued presence of H₂O₂ elicited a Ca²⁺ transient (Figure 3D), which represented the Ca²⁺ entry stimulated by H₂O₂. A similar Ca²⁺-repletion–dependent transient did not occur on TRPM2 depletion with siRNA transfection (Figure 3E) or anti-TRPM2 blocking antibody (5 µg/mL for 8 to 16 hours; Figure 3F). Transfection of HPAE cells with TRPC4 siRNA (as negative control) did not affect the ability of H₂O₂ to induce Ca²⁺ entry (Figure 3G). Figure 3G quantifies all data obtained for Ca²⁺-containing media and for the Ca²⁺-repletion protocol.

TRPM2 Depletion Reduces H₂O₂-Mediated Endothelial Hyperpermeability
Increased intracellular Ca²⁺ causes opening of interendothelial junctions, which is detectable as reduction in transendothelial electrical resistance (TER).^5,8–10 Because H₂O₂ increased intracellular Ca²⁺, we tested H₂O₂ for its ability to decrease TER. HPAE cells were grown to confluence on gold microelectrodes and treated with H₂O₂ (300 to 600 µmol/L), and changes in TER were followed for 4 hours. Recordings of TER in Figure 4A show that H₂O₂ decreases TER. The effect was transitory; at <500 µmol/L, H₂O₂-mediated response recovered to basal levels within 2 hours. Results of quantification of the peak TER response at each H₂O₂ concentration are displayed with curve of best fit (EC₅₀, 254 µmol/L; Figure 4A).

View larger version (31K): [in this window] [in a new window]   Figure 4. H2O2-induced increase in endothelial barrier permeability depends on TRPM2-L expression. A, Concentration-dependent action of H2O2 on endothelial barrier function. HPAE cells were grown to confluence on gold microelectrodes, the cells were treated with H2O2 (concentration indicated), and TER was followed for 4 hours. Each tracing is the average response of 4 wells. Experime- nts were repeated 3 times with similar results. The abscissa indicates time in hours; the ordinate, normalized resistance (relative to basal value). The inset shows the corresponding dose–response curve (n=12; bars, ±SEM). H2O2 (0 to 600 µmol/L) caused a rapid, dose-dependent decrease in TER with an EC50 of 254 µmol/L. At <500 µmol/L, H2O2 effects were transitory. B, Real-time RT-PCR showing specificity of the siRNA used toward TRPM2 but not TRPM7 or GAPDH in confluent HPAE cultures transfected by TRPM2-L. C, Influence of TRPM2 siRNA in TRPM2-L–transduced cells. In these experiments, HPAE cells were transfected with a fluorescent form of TRPM2 (BFP–TRPM2-L). Confocal image of control cells expressing the construct is shown (left). Cotransfection with TRPM2-silencing RNA (siRNA) eliminated expression of the fusion protein (center). Cotransfection with scrambled siRNA (negative control) was ineffective (right). D, TER decrease on H2O2 exposure (300 µmol/L). Left, Note that TRPM2 silencing inhibits H2O2 responses relative to the untreated group (no transfection) or the negative control group (scrambled siRNA; n=4 per group). The abscissa indicates time in hours; the ordinate, normalized resistance. Right, Mean value (±SEM) of peak TER responses to H2O2 (n=12). Experiments were repeated 3 times with similar results. H2O2-induced TER decrease was significantly attenuated in cells transfected by TRPM2-specific siRNAs compared with untreated control or negative control group transfected with a scrambled siRNA.

We next compared TER responses to H₂O₂ with or without TRPM2 silencing. To show the effect of siRNAs on TRPM2 protein expression, we transduced HPAE cultures with BFP-tagged TRPM2-L, a fluorescent fusion protein forming functional channels. RT-PCR analysis showed the specificity of siRNAs. TRPM2 siRNA transfection reduced TRPM2 transcript expression by 75% without affecting expression of TRPM7 or GAPDH (Figure 4B). Control siRNA had no effect on TRPM2 expression (Figure 4B). We determined the percentage of fluorescent cells by confocal imaging. Without siRNA, 85% to 90% of cells were fluorescent (Figure 4C, left). TRPM2 siRNAs greatly reduced this percentage and the fluorescent intensity (Figure 4C, center). Nonspecific siRNA was ineffective (Figure 4C, right). HPAE cells were plated to confluence on gold electrodes without or with siRNA, and TER was measured. TRPM2 silencing reduced the TER response to H₂O₂ (300 µmol/L) by 42% relative to control (no siRNA transfection) or negative control (nonspecific siRNA transfection; Figure 4D).

Transduction of TRPM2-S Inhibits H₂O₂-Induced Ca²⁺ Entry and Endothelial Hyperpermeability
The short splice variant of the functional channel, TRPM2-S, acts in a dominant-negative fashion to inhibit TRPM2-L activity.^24,25 We overexpressed TRPM2-S in HPAE cells to determine its effects on H₂O₂-mediated responses. To quantify the expression level of TRPM2-S, we transfected HPAE cells with the fluorescent fusion protein GFP–TRPM2-S. We divided the cells for use in parallel determinations of intracellular Ca²⁺ and TER changes. Confocal imaging showed that 80% of cells expressed GFP–TRPM2-S (Figure 5A). RT-PCR analysis demonstrated that construct overexpression did not alter expression of endogenous TRPM2-L isoform (Figure 5B). Using the Ca²⁺ add-back protocol, we observed that overexpression of GFP–TRPM2-S inhibited H₂O₂-mediated (300 µmol/L) Ca²⁺entry by 83±13% (Figure 5C). Overexpression of GFP–TRPM2-S also attenuated the peak TER response to H₂O₂ (300 µmol/L) by 31±10% and markedly reduced its duration (Figure 5D, n=4).

View larger version (20K): [in this window] [in a new window]   Figure 5. Transduction of TRPM2-S ("short" dominant-negative splice variant of TRPM2) inhibits H2O2-induced Ca2+ entry and increase in endothelial permeability. The short splice variant of the channel, TRPM2-S, lacking the putative Ca2+-permeable pore, acts in a dominant-negative fashion to inhibit TRPM2-L activity. HPAE cells were transfected by a fluorescently tagged TRPM2-S isoform (GFP–TRPM2-S). A, Confocal images of HPAE monolayer expressing GFP–TRPM2-S. B, RT-PCR analysis showing that construct expression does not alter endogenous level of TRPM2-L isoform (ie, the channel-forming isoform). C, Left, Ca2+ entry assay as described in Materials and Methods (see also the legend of Figure 2). Right, Mean ratiometric values (±SEM) for steady-state [Ca2+]i (n=6). Note that the overexpression of GFP–TRPM2-S prevents H2O2-mediated Ca2+entry. D, Changes in TER after H2O2 (300 µmol/L) addition (time 0) with or without transfection of the TRPM2-S isoform. Left, Original TER recordings (each trace is the average of 4 responses). Right, Summary of TER data. Note that the overexpression of GFP–TRPM2-S attenuates H2O2-induced (300 µmol/L) resistance changes. *Significance at P<0.05.

Overexpression of TRPM2-L Augments H₂O₂-Induced Ca²⁺ Entry and Endothelial Hyperpermeability
If the balance between short and long TRPM2 isoforms determines channel activity, overexpression of TRPM2-L should enhance H₂O₂ effects; thus, we transfected HPAE cells with the fluorescent BFP–TRPM2-L isoform (see Figure 4) and monitored H₂O₂ responses (Figure 6). H₂O₂ addition increased Ca²⁺ entry above the vector control by 29±14% (Figure 6A; n=4) and also increased the TER response above the control by 151±7% (Figure 6B; n=4).

View larger version (18K): [in this window] [in a new window]   Figure 6. Overexpression of TRPM2-L in endothelial cells augments H2O2-induced Ca2+ entry and endothelial permeability. HPAE cells were transiently transfected with the BFP–TRPM2-L isoform. A, Left, Ca2+mobilization assay in control and TRPM2-L–overexpressing HPAE cells; H2O2 (300 µmol/L) was added at the arrow. Right, Mean ratiometric values (±SEM) for steady-state [Ca2+]i (n=4). B, Left, Changes in TER on H2O2 (300 µmol/L) addition. Right, Summary of TER changes in control (empty transfection) vs TRPM2-L–transfected cells (n=12). Results show that overexpression of TRPM2-L in HPAE cells augmented H2O2-induced (300 µmol/L) Ca2+ entry and increased barrier permeability in a sustained fashion.

TRPM2 Blocking Antibody Prevents H₂O₂-Induced Endothelial Barrier Disruption
We next tested the blocking effect of a TRPM2 antibody (see Materials and Methods) on the TER response to H₂O₂. Confluent HPAE monolayers were treated overnight with either blocking antibody (5 µg/mL) or isotype-matched control antibody (5 µg/mL). Treatment with the specific antibody markedly reduced Ca²⁺ entry evoked by 300 µmol/L H₂O₂ compared with control (Figure 7A; n=3 for each group). TRPM2 antibody also diminished the TER response to H₂O₂ by 44±9% (Figure 7B; n=3); control antibody had no effect.

View larger version (28K): [in this window] [in a new window]   Figure 7. Anti-TRPM2 blocking antibody protects endothelial cells from H2O2-induced barrier dysfunction. Confluent HPAE monolayers were treated overnight with a specific TRPM2 antibody or control IgG (5 µg/mL). Intracellular Ca2+ (A) or TER changes (B) were measured in response to H2O2 challenge (300 µmol/L). A, TRPM2 antibody markedly reduced H2O2-induced Ca2+ entry compared with control groups (no treatment or isotype matched antibody) (n=3 for each group). B, Left, Original TER recordings. Right, Mean drop in TER (relative to basal). Anti-TRPM2 antibody inhibited the TER decrease caused by H2O2 (n=3).

Inhibition of PARP Suppresses H₂O₂-Mediated Ca²⁺ Entry and Permeability Increase
TRPM2-L is activated by the binding of ADP-ribose to its binding cleft in TRPM2 carboxyl terminus.¹³ H₂O₂ stimulates PARP to generate ADP-ribose, whereas inhibitors of PARP prevent agonist formation.¹⁶ Therefore, we tested 2 PARP inhibitors, DPQ and 3-aminobenzamide (3-AB), for their ability to prevent H₂O₂ responses.^16,28 HPAE cells were treated with 3-AB (1 mmol/L) or DPQ (100 µmol/L) for 45 minutes, and intracellular Ca²⁺ was measured in response to H₂O₂ (300 µmol/L) using the Ca²⁺ add-back protocol. Both 3-AB and DPQ significantly reduced Ca²⁺-repletion transients (60±11% and 62±9%, respectively) compared with untreated cells (Figure 8A). We obtained similar results in Ca²⁺-containing media on stimulating cells with H₂O₂ (300 µmol/L) (see Figure 8A, inset). In parallel experiments (Figure 8B), both PARP inhibitors also significantly reduced TER responses to H₂O₂ by 43±12% (3-AB) and 40±15% (DPQ).

View larger version (24K): [in this window] [in a new window]   Figure 8. Inhibition of ADP-ribose polymerase suppresses H2O2-mediated Ca2+ entry and permeability increase. HPAE cells were pretreated with inhibitors of ADP-ribose polymerase (1 mmol/L 3-AB or 100 µmol/L DPQ) for 45 minutes. A and B, In response to H2O2 challenge (300 µmol/L), we measured intracellular Ca2+ in complete (A, inset) vs. Ca2+-free Hanks’ balanced salt solution (A) and also TER changes in separate monolayers (B). A, Left, Original recordings of intracellular Ca2+ transients without or with pretreatment by specified inhibitors. CaCl2 (2.0 mmol/L) and Ca2+ ionophore ionomycin were added as indicated. Right, Summary plot of the data. Note that either polymerase inhibitor significantly reduced Ca2+ rise induced by H2O2 (n=3). B, Left, Recordings of TER in the absence or presence of polymerase inhibitors. Right, ADP-ribose polymerase inhibitors suppressed the increase in barrier permeability caused by H2O2 (n=12). These observations are consistent with known properties of TRPM2 channel that is selectively activated by H2O2 by the intracellular ligand ADP-ribose.

	Discussion

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The present study demonstrates the expression and function of TRPM2-L channels in endothelial cells. Western blot analysis indicated the presence of the channel protein. Bath-applied H₂O₂ or internally applied ADP-ribose (the endogenous activator of TRPM2 channels¹³) elicited inward currents consistent with opening of nonselective cation channels. The ability to abolish H₂O₂-induced current by blocking cellular generation of ADP-ribose suggests that TRPM2 channel is responsible for the observed electrophysiological effects of H₂O₂. TRPM2-silencing studies confirmed that TRPM2 is requisite for H₂O₂-induced currents. The linearity of the current–voltage curve for TRPM2 channels and reversal potential obtained agrees with findings in microglia, which also express functional TRPM2 channels.²⁰ Under physiologic ionic conditions (140 mmol/L Na⁺, 2 mmol/L Ca²⁺), Na⁺ was the main carrier of inward current via endothelial TRPM2 channels, because NMDG substitution for Na⁺ abolished the current. Ca²⁺ entry via the channel was also detected using the fura-2 method. Because oxidant-activated TRPM2 channels are Ca²⁺-permeable, we hypothesized their involvement in the oxidant-induced rise in intracellular Ca²⁺ and the resulting increase in paracellular permeability.

We observed that pathophysiologically relevant H₂O₂ concentrations of 100 to 300 µmol/L²⁹ did not release Ca²⁺ from intracellular stores but did significantly stimulate extracellular Ca²⁺ entry, indicating that H₂O₂ (<300 µmol/L) does not activate store-operated channels in HPAE cells.^30,31 We determined whether oxidants (300 µmol/L) activated Ca²⁺ entry via TRPM2 channels by suppressing TRPM2 expression or activity by various means, eg, siRNAs, TRPM2-blocking antibody, overexpression of TRPM2-S, and PARP inhibitors that prevent generation of ADP-ribose.^16,24,25,28 All of these interventions abolished H₂O₂-induced Ca²⁺ entry, indicating the crucial role of activation of TRPM2 channels in mediating Ca²⁺ entry caused by H₂O₂ in endothelial cells.

We also determined the role of TRPM2 in mediating the H₂O₂-induced decrease in TER resulting from opening of interendothelial junctions. Suppression of TRPM2 activity caused only a 50% reduction in the TER response of H₂O₂, suggesting that TRPM2 channels mediate approximately half of the permeability-increasing effect of H₂O₂. The residual effect of H₂O₂, which appears to be independent of Ca²⁺ entry, remains to be elucidated.

We showed that TRPM2-S overexpression suppressed Ca²⁺ entry and TER responses to H₂O₂ in endothelial cells. Importantly, the cells normally expressed both isoforms because the relative expression levels observed by real-time time RT-PCR were greater using the primer targeting both isoforms, as opposed to the primer specifically targeting the long form. TRPM2-S in human hematopoietic cells is generated by alternative splicing of the full-length protein.²⁴ Coimmunoprecipitation and functional studies in HEK293T cells have also demonstrated direct interaction between short and long TRPM2 isoforms, resulting in suppression of H₂O₂-induced current and Ca²⁺ entry via TRPM2-L.²⁴ Thus, endothelial responses to H₂O₂ may well depend on the relative abundance of the 2 isoforms.

Our electrophysiological observations suggest that a small population of TRPM2 channels can account for the whole-cell current induced by H₂O₂. Given an estimated single-channel conductance of 67 pS,^20,32 and our observed whole-cell current of 125 pA (at –50 mV), we calculated 40 functional channels per cell. However, this apparent value presumably underestimates the actual expression of TRPM2 in the endothelial cell membrane because of the presence of the inhibitory short isoform TRPM2-S.

Lung endothelial injury, particularly in the setting of sepsis, is the result of oxidant generation by endothelial cells themselves and neutrophils and other inflammatory cells adherent to vessels.^33,34 The generally held belief is that the resulting oxidants, including H₂O₂, directly damage endothelium.^1–4 Our observations demonstrate a novel mechanism of H₂O₂-mediated disruption of endothelial barrier function that, in part, is attributable to a rise in intracellular Ca²⁺ mediated by Ca²⁺ entry through oxidant-sensitive TRPM2 channels. In this regard, increased microvessel endothelial permeability secondary to ischemia/reperfusion and neutrophil adhesion and activation may depend on H₂O₂-mediated activation of TRPM2 and influx of Ca²⁺. Therefore, inhibition of TRPM2 may provide a useful therapeutic strategy for the treatment of endothelial barrier dysfunction and vascular inflammation.

	Acknowledgments

 
We thank Dr Barbara Miller for providing the BFP–TRPM2-L and GFP–TRPM2-S constructs used in our experiments.

Sources of Funding

This work was supported in part by NIH training grants T32 HL007829 (to C.M.H.), P01 HL060678, and R01 HL045638.

Disclosures

None.

	Footnotes

 
Original received July 18, 2007; revision received October 31, 2007; accepted November 19, 2007.

	References

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