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CELLULAR AND MOLECULAR

Dominant Role for Calpain in Thromboxane-Induced Neuromicrovascular Endothelial Cytotoxicity

Departments of Pediatrics and Pharmacology, Centre de Recherche de l'Hôpital Ste-Justine, Université de Montréal, Montréal, Québec, Canada (C.Q., F.S., M.H.B., D.C., I.L., S.B., F.G., M.S., A.K., P.H., A.P., S.C.); Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada (D.C., S.B., M.S., A.K., S.C.); Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada (C.Q.); Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec, Canada (F.G.); and Institut National de la Santé et Recherche Médicale, Unite 598, Paris, France (F.S.)

Received August 9, 2005; accepted October 6, 2005.

	Abstract

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Thromboxane A₂ (TXA₂) is an important lipid mediator generated during oxidative stress and implicated in ischemic neural injury. This autacoid was recently shown to partake in this injury process by directly inducing endothelial cytotoxicity. We explored the mechanisms for this TXA₂-evoked neural microvascular endothelial cell death. Stable TXA₂ mimetics 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1,4,5(Z),6,(1E,3S)]]-9,11-dedioxy-9,11-methanolpoxy (U-46619) [as well as [1S-[1,2(Z),3(1E,3S^*),4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; I-BOP] induced a retinal microvascular degeneration in rat pups in vivo and in porcine retinal explants ex vivo and death of porcine brain endothelial cells (in culture). TXA₂ dependence of these effects was corroborated by antagonism using the selective TXA₂ receptor blocker (-)-6,8-difluoro-9-p-methyl-sulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid (L670596). In all cases, neurovascular endothelial cell death was prevented by pan-calpain and specific m-calpain inhibitors but not by caspase-3 or pan-caspase inhibitors. Correspondingly, TXA₂ (mimetics) augmented generation of known active m-calpain (but not µ-calpain) form and increased the activity of m-calpain (cleavage of fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; and of -spectrin into specific fragments) but not of pan-caspase or specific caspase-3 (respectively, using sulforhodamine-Val-Arg-Asp-fluoromethyl ketone and detecting its active 17- and 12-kDa fragments). Interestingly, these effects were phospholipase C (PLC)-dependent [associated with increase in inositol triphosphate and inhibited by PLC blocker 1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122 [GenBank] )] and required calcium but were not associated with increased intracellular calcium. U-46619-induced calpain activation resulted in translocation of Bax to the mitochondria, loss of polarization of the latter (using potentiometric probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; JC-1) and in turn release of cytochrome c into the cytosol and depletion of cellular ATP; these effects were all blocked by calpain inhibitors. Overall, this work identifies (specifically) m-calpain as a dominant protease in TXA₂-induced neurovascular endothelial cell death.

Cytotoxic effects of TXA₂ have largely been attributed to its hemodynamic actions elicited through vasoconstriction and platelet aggregation (FitzGerald et al., 1987). But more recently, direct toxicity in response to activation of the TXA₂ receptor has been uncovered in thymocytes (Ushikubi et al., 1993), throphoblasts (Yusuf et al., 2001), renal tubule epithelial cells (Jariyawat et al., 1997), ventricular myocytes (Shizukuda and Buttrick, 2002) as well as in (rat, porcine, and human) neural microvascular endothelial cells (Lahaie et al., 1998; Beauchamp et al., 2001). In contrast, stimulation of receptors for related prostanoids prostaglandin E₂ and prostaglandin F₂ did not lead to toxicity. Of relevance, the major peroxidation products the isoprostanes exert neurovascular endothelial cytotoxicity via TXA₂/TP pathway (Beauchamp et al., 2001; Brault et al., 2003). Furthermore, an important role for TXA₂ has been demonstrated in retinal vaso-obliteration associated with ischemic retinopathies before platelet aggregation (which itself generates TXA₂) (Beauchamp et al., 2002). This TXA₂/TP-induced neurovascular endothelial cell death is delayed by 12 to 18 h, albeit it does not exhibit classic features of apoptosis such as chromatin condensation, terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity, and frequently observed caspase dependence; accordingly, a role for other cysteine proteases may be inferred.

Caspases, calcium-dependent calpains, and cathepsins compose the three major groups of cysteine proteases. Conversely, ubiquitous calpain isoforms Calp I (µ-calpain) and Calp II (m-calpain) are abundantly expressed in the central nervous system (Ray et al., 2003), and calpain activity is increased in the process of cell death applied to neurodegeneration and ischemic central nervous system events (Saito et al., 1993; Majno and Joris, 1995). Interestingly, calpain activity has mostly been associated with necrosis, whereas caspase activity is largely associated with classic apoptosis (Wang, 2000). However, whether calpains participate in TXA₂-induced endothelial cytotoxicity and the mechanisms involved in this process have yet to be described.

Therefore, we investigated the mechanisms of TXA₂-induced neurovascular endothelial cell death, with particular emphasis on the role of calpains; for this purpose, established stable mimics of this prostanoid known to activate its TP receptor were used (U-46619 and at times I-BOP). Our findings reveal that TXA₂ (mimics) elicited a neuroretinal microvascular degeneration in vivo and in tissue explants (ex vivo) and in primary neural endothelial cells (cultures) via a mechanism dependent upon calpain activity and that degeneration was prevented specifically by m-calpain inhibitor and mediated in turn by increased mitochondrial Bax/Bcl-2 ratio associated with loss of mitochondrial membrane polarity and consequent ATP depletion. In contrast, pan-caspase and more specifically caspase-3 activity and role were not involved. Thus, we hereby disclose an important mode of action of TXA₂ in eliciting neurovascular endothelial cytotoxicity.

	Materials and Methods

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Chemicals and Materials. L670596 was a gift of Merck Frosst (Pointe-Claire, QC, Canada). The following materials were purchased: ceramide, dimethyl sulfoxide, cycloheximide, staurosporine, Nonidet P-40, and 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich, St. Louis, MO); U-46619 and I-BOP (Cayman Chemical, Ann Arbor, MI); JC-1, valinomycin, Hoescht 33342, and propidium iodine (Invitrogen, Burlington, ON, Canada); calpain inhibitors IV and V, U73122 [GenBank] (Calbiochem, San Diego, CA); and Z-DEVD-fmk caspase-3 inhibitor (R&D Systems, Minneapolis, MN). Other materials were purchased from Fisher Scientific Co. (Montreal, QC, Canada).

TXA₂-elicited effects were tested using stable analogs of the prostanoids, namely, U-46619 and I-BOP, both of which are known to stimulate specifically the TP receptor (Beauchamp et al., 2001). TXA₂-induced actions were further corroborated using the selective TP antagonist L670596 (Beauchamp et al., 2001).

Animals. Newborn Sprague-Dawley rats (Charles River Canada, Montreal, PQ, Canada) and 1- to 3-day-old Yorkshire piglets (Fermes Ménard, L'Ange-Gardien, QC, Canada) were used according to a protocol of the Hôpital Sainte-Justine Animal Care Committee and in accordance with regulations of the Canadian Council of Animal Care.

Intravitreous Injections. Retinovascular degeneration was studied in rat pups as reported previously (Beauchamp et al., 2001). Rat pups (postnatal day 7) were injected intravitreously [1 µl (capillary injector)] with vehicle or U-46619 in absence or presence of TXA₂ receptor blocker L670596 [estimated final concentration 1 µM (30-µl ocular volume) as described previously; Lahaie et al., 1998; Beauchamp et al., 2001], pan-calpain inhibitor (Calp V; final concentration, 1 µM), or caspase-3 inhibitor Z-DEVD-fmk (final concentration, 50 µM). Some preparations were treated with the pan-caspase inhibitor Z-VAD-fmk (50 µM). Rats were euthanized on postnatal day 10, and retinas were isolated for endothelial cell staining with the TRITC-conjugated lectin Griffonia simplicifolia (Sigma-Aldrich). Retinas were visualized using a fluorescent Nikon Eclipse E800 microscope and photographed with a Nikon digital camera DMX1200. Vascular density was determined using a computer software (Image-Pro Plus 4.1; Media Cybernetics, Inc., Silver Spring, MD) as reported previously (Lahaie et al., 1998; Beauchamp et al., 2001).

Retinal Explants. To ascertain that the vasculotoxic effects of TXA₂ are hemodynamic-independent and can be reproduced in different species, the effects of the TXA₂ analog U-46619 were tested on retinal explants of 1- to 3-day-old pigs. Dissected retinas were cut into 5-mm² fragments, placed on a Track-Etch membrane (What-man, Maidstone, UK), and left to float on the surface of DMEM (2% fetal bovine serum) culture medium at 37°C with 5% CO₂ in six-well plates (three explants per membrane). Retinal explants were treated with vehicle or U-46619 (1 µM) in the absence or presence of L670596 (1 µM), m-calpain inhibitor Calp IV (Angliker et al., 1992) (k₂ = 28,900 M^-1 s^-1 (1 µM), pan-calpain inhibitor Calp V (Esser et al., 1994) (1 µM), or Z-DEVD-fmk (50 µM). Some preparations were treated with the pan-caspase inhibitor Z-VAD-fmk (50 µM). µ-Calpain inhibitors are not yet available. After 3 days of incubation, the medium was removed, and the explants were fixed at room temperature with formalin and permeabilized with ice-cold 100% methanol. Tissues were washed three times with 1% Triton X-100 in PBS and stained with 1:100 TRITC-lectin G. simplicifolia (in 1% Triton X) overnight. Explants were flat mounted and visualized with a fluorescent microscope, and microvasculature was quantified as described above for the intravitreal injection experiments.

Cells. Neural microvessels (25 µm) were isolated from piglet brains as detailed previously (Lahaie et al., 1998; Beauchamp et al., 2001, 2002). Microvessels were suspended in selective endothelial growth media (Cambrex Bio Science Walkersville, Walkersville, MD), and endothelial cells were grown to confluence as reported previously (Lahaie et al., 1998; Beauchamp et al., 2001). Endothelial cells were identified morphologically and by their positive reactivity to FVIII and negative reactivity to smooth muscle-specific actin and glial fibrillary acidic protein. Only low passage (less than seven) cell cultures were used.

View larger version (58K): [in this window] [in a new window]   Fig. 1. TXA2-mediated retinal microvascular vaso-obliteration. Rat pups on postnatal day 7 were injected intravitreously (1 µl) with vehicle or U-46619 in the absence or presence of TXA2 receptor antagonist L670596 [estimated final concentration 1 µM (30-µl ocular volume) as described previously; Lahaie et al., 1998; Beauchamp et al., 2001, 2002], pancalpain inhibitor Calp V (final concentration, 1 µM), or Z-DEVD-fmk caspase-3 inhibitor (final concentration, 50 µM). Retinas were removed on postnatal day 10, and endothelium was stained with TRITC-conjugated lectin G. simplicifolia to determine vessel density. A, representative retinal flat mounts depicting vascular network. Note decreased vessel density after U-46619, which is noticeably prevented by L670596 and Calp V. B, compiled vessel density of retinas treated as described above. Values are mean ± S.E.M. of three to four experiments. *, P < 0.05 compared with all other values without asterisks.

Caspase and Calpain Activity. Pan-caspase activity was determined using the fluorogenic substrate SR-VAD-fmk (BIOMOL Research Laboratories, Plymouth Meeting, PA) which fluoresces (red) upon binding to the active enzyme (Grabarek et al., 2002). Essentially, microvascular endothelial cells were seeded on coverslips and treated as described above; staurosporine (1 µM) was used as positive control activator of caspase. At the end of drug exposure period, SR-VAD-fmk was added to the cell media for 1 h at 37°C. Cells were counterstained with Hoescht dye 33342. Cells were photographed under fluorescent microscopy and quantified with the Image-Pro software described above. In addition, caspase-3 activity was determined by immunoreactivity of the 12- and 17-kDa caspase fragments; antibody to 12 and 17 kDa does not recognize full caspase-3 protein.

Calpain activity was measured using the fluorogenic synthetic substrate Suc-LLVY-AMC (calpain activity kit; Calbiochem). Hydrolysis of the substrate yields the fluorescent product AMC (Debiasi et al., 1999). In brief, microvascular cells (24-well plates) incubated for a given number of hours with U-46619 were added with Suc-LLVY-AMC for 15 min at 37°C, as described by the manufacturer. The formation of AMC was read with a fluorescence plate reader at the following wavelength settings: excitation 360-380 nm and emission 440-460 nm. Enzyme activity was measured as nanomoles of free AMC released per minute per amount of total cell lysate protein. Calpain activity was also determined by specific cleavage of -spectrin to yield a 145- to 150-kDa immunoreactive fragment.

Inositol Phosphate Measurements. Endothelial cells grown in 12-well plates were labeled with 1 to 2 µCi/ml [³H]myo-inositol (17 Ci/mmol; GE Healthcare, Little Chalfont, Buckinghamshire, UK) overnight. The cells were preincubated in DMEM containing 10 mM LiCl with or without the phospholipase C (PLC) inhibitor U73122 [GenBank] (0.1 µM) or L670596 (1 µM) and treated with U-46619 (0.1 µM) for 30 min at 37°C. The reaction was terminated by addition of 0.5 volume of NaOH (100 mM), followed by acidification with 2 mM formic acid. Total inositol phosphates were separated by using Dowex AG1X8 (formate form) and 1.2 M ammonium formate in 0.1 N formic acid as the eluant. Radioactivity of phosphoinositides was determined in liquid scintillation cocktail.

Intracellular Ca²⁺ Measurements. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using the fluorescent Ca²⁺ indicator fura 2-acetoxymethyl ester as described previously. Cells were resuspended in Hanks' balanced salt solution with Ca²⁺ (2.5 mM) and 1% fetal bovine serum, and they were then stimulated with U-46619 (0.1 µM), EGTA (5 mM), or bradykinin (1 µM). [Ca²⁺]_i was determined in 2 ml of fura 2-acetoxymethyl ester-loaded cell suspension and measured using a spectrofluorometer (model LS 50; PerkinElmer Life and Analytical Sciences, Beaconsfield, UK); excitation wavelengths were 340 and 380 nm, and emission was at 510 nm. Calibration of the fluorescent signal was determined on 2 ml of cell suspension by sequential addition of 0.2% Triton X-100 to obtain the maximal fluorescence ratio (R_max) and of 5 mM EGTA to obtain the minimal fluorescence ratio (R_min). Autofluorescence was determined by measuring fluorescence from nonloaded cells and subtracting it from the fluorescence produced by fura 2-loaded cells to calculate the fluorescence ratio R corresponding to the values produced at 340 and 380 nm. The [Ca²⁺]_i was calculated from the equation [Ca²⁺]_i = K_d [(R - R_min)/(R_max - R)](S_f2/S_b2), where K_d (224 nM) is the effective dissociation constant of the fura 2-Ca²⁺ complex, and S_f2/S_b2 is the ratio of fluorescence intensity at 380-nm wavelength in the presence of EGTA to that in the presence of Triton X-100 (Grynkiewicz et al., 1985).

View larger version (84K): [in this window] [in a new window]   Fig. 2. Microvascular degeneration induced on retinal explants by U-46619. Porcine retinas were incubated for 3 days with vehicle or U-46619 (0.1 µM) without or with L670596 (1 µM), Calp V (1 µM), Calp IV (1 µM), or Z-DEVD-fmk (50 µM) as described under Materials and Methods. Vascular network was revealed with TRITC-conjugated lectin G. simplicifolia. A, representative flat mounts of incubated retinas treated with different compounds. Note marked decrease in vessel density induced by U-46619 alone. B, histographic representation of compiled vessel density data of tissues treated as described above. Values are mean ± S.E.M. of three to four experiments. *, P < 0.05 compared with all other values without asterisks.

Mitochondrial and Cytosol Fraction Isolation. Cell fractionation was performed as described in detail previously (Gobeil et al., 2003). Stimulated cells were rinsed with PBS and concentrated by centrifugation (500g). Cells were suspended in lysis buffer, pH 7.4 (10 mM Tris-HCl, 10 mM NaCl, 3 MgCl₂, and 30 mM sucrose) and homogenized with a glass homogenizer (300 strokes). Lysate was centrifuged at 700g for 10 min at 4°C to remove nuclei and debris, and the supernatant was recentrifuged at 10,000g for 15 min. The mitochondrial-containing pellet was resuspended in phosphate buffer and used for corresponding experiments (see below), whereas the supernatant was centrifuged again at 120,000g for 1 h to obtain the S100 cytosolic fraction.

Mitochondrial Membrane Potential. Mitochondrial membrane depolarization was determined using the potentiometric probe JC-1 (Smiley et al., 1991). JC-1 selectively enters the polarized mitochondria and is driven by the mitochondrial membrane electrochemical gradient. In polarized membranes, it forms red fluorescent aggregates and when depolarized, JC-1 stays dispersed as a monomer and fluoresces in green. Microvascular endothelial cells were split in six-well plates and treated as described above for viability assays. After the 24-h incubation period, cells were trypsinized, centrifuged, and resuspended in 500 µl of PBS. JC-1 (1 mM) was added for 15 min at 37°C; valinomycin (100 nM) was used as a positive control of mitochondrial depolarization. Monomers and aggregates of JC-1 were detected in the FL1 and FL2 channels, respectively, with a FACScalibur (BD Biosciences, San Jose, CA). For microscopic visualization, cells were photographed as described above.

ATP Assay. ATP content was determined using a commercial kit (Calbiochem). Microvascular endothelial cells were split in 12-wells plates, starved, and treated. Cells were suspended in boiling 100 mM Tris-HCl, pH 7.75, 4 mM EDTA buffer for 10 min to inactivate released ATPases. The suspension was centrifuged and cooled. Fifty milliliters of cell suspension was mixed with 50 ml of HEPES buffer and 25 ml luciferin-luciferase solution. ATP was determined by measuring the light generated, using the following formula described in the manufacturer's manual: D-luciferin + ATP-Mg²⁺ + O₂ luciferase oxyluciferin + AMP + CO₂ + Ppi-Mg²⁺ + light.

Statistical Analysis. Data were analyzed by one- or two-way analysis of variance factoring for treatment and/or time or concentration. Comparison among means was analyzed by Tukey-Kramer method. Statistical significance was set at P < 0.05. Values are presented as mean ± S.E.M.

View larger version (45K): [in this window] [in a new window]   Fig. 3. A, time and concentration dependence of effects of TXA2 analogs U-46619 and I-BOP on porcine neurovascular endothelial cell viability determined by MTT assay (see Materials and Methods). B, LDH activity in endothelial cell media at given times after treatment with U-46619 (0.1 µM) with or without L670596 (1 µM). C, cell viability determined by MTT assay after 24-h treatment with vehicle or U-46619 (0.1 µM) with or without (1 µM) L670596, Calp IV, Calp V, calphostin-C, U73122 [GenBank] , or Z-DEVD-fmk (50 µM). D, PI (red) incorporation after 24-h treatment of cells with indicated treatments at concentrations described in C. Cells were counterstained with Hoescht 33342 (blue); inhibitors alone did not affect PI incorporation. Representative photomicrographs are shown on the left, and quantification in histogram format is presented on the right. Values are mean ± S.E.M. of three to six experiments. *, P < 0.05 compared with other values without asterisks.

	Results

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Cytotoxic Effects of TXA₂ and Role of Calpain Specifically Apply to the Neural Microvascular Endothelium. To ascertain that the cytotoxic effects of TXA₂ and thus far presumed (pharmacological) role of calpain specifically apply to the neural microvascular endothelium, the latter was studied on primary neurovascular endothelial cell cultures. TXA₂ analogs U-46619 as well as I-BOP induced a concentration- and time-dependent decrease in cell viability, determined by MTT assay and LDH release (Fig. 3, A and B) (and confirmed by direct cell counting); accordingly, cellular PI incorporation was increased by U-46619 (Fig. 3D). This U-46619-triggered cytotoxicity was largely prevented by the selective TXA₂ receptor blocker L670596, Calp IV, and Calp V as well as by the PLC and PKC inhibitors U73122 [GenBank] and calphostin-C, respectively (Fig. 3, B-D), whereas Z-DEVD-fmk [or Z-VAD-fmk; data not shown)] was ineffective, consistent with in vivo and ex vivo observations (Figs. 1 and 2).

TXA₂-Mediated Changes in Caspase and Calpain Activities. Effects of TXA₂ (mimetic) on caspase and calpain activities were specifically studied. U-46619 (at 6 and 24 h) failed to activate caspase-3 in contrast to staurosporine as determined by increased immunoreactivity of the 17- and 12-kDa fragments (Fig. 4C). In addition, pan-caspase activity determined using the enzyme-binding fluorescent substrate SR-VAD-fmk did not reveal any activity upon treatment [24 h as well as 12 h (data not shown)] with U-46619 (Fig. 4, A and B); the positive control staurosporine (Gao et al., 2000) readily evoked caspase activation, which was inhibited by Z-DEVD-fmk.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Caspase activity in porcine neurovascular endothelial cells treated with U-46619. A, representative micrographs of pan-caspase activity determined using the fluorogenic substrate SR-VAD-fmk; cells were counterstained with Hoescht 33342. Cells were treated with U-46619 (0.1 µM) for 12 h (data not shown) or 24 h, or with staurosporine (1 µM) for 1 h without or with caspase-3 inhibitor Z-DEVD-fmk (50 µM). Green fluorescence was not detected after treatment with U-46619 but was with staurosporine (positive control), which was prevented by Z-DEVD-fmk. B, histographic representation of pan-caspase activity determined using SR-VAD-fmk. Values are mean ± S.E.M. of three experiments. * P < 0.01 compared with other values without asterisks. C, activated caspase-3 fragments determined by Western blot (representative of three experiments). U-46619 (0.1 µM) did not increase immunoreactivity to the caspase-3 fragments; the positive control staurosporine (1 µM; 1 h) induced an increase in caspase-3 fragment immunoreactivity. Values are mean ± S.E.M. of three experiments. *, P < 0.05 compared with other values without asterisks.

In contrast, U-46619 evoked a significant increase in the formation of the 58-kDa m-calpain active fragment within 8 h (Fig. 5A) (Weber et al., 2004; Park and Ferreira, 2005), whereas µ-calpain was unaltered. Generation of m-calpain active fragment was inhibited by treatment with PKC and PLC inhibitors calphostin-C and U73122 [GenBank] , respectively; correspondingly, U-46619 evoked an increase in inositol phosphate generation, which was blocked by U73122 [GenBank] (Fig. 5B). Calpain activity (at 6 h) was also found to increase dose dependently in response to U-46619 as shown using the fluorogenic synthetic substrate Suc-LLVY-AMC (Fig. 5, C and D). This effect was again blocked by U73122 [GenBank] and calphostin-C and as expected by L670596 and Calp IV. This increase in calpain activity was further confirmed by induced hydrolysis of cytoskeletal -spectrin, which generates a Calp IV-sensitive 145- to 150-kDa-specific fragment of spectrin detectable by Western blot (Fig. 5E).

View larger version (43K): [in this window] [in a new window]   Fig. 5. Calpain activation and activity. A, calpain activation detected by Western blot of 58-kDa fragment resulting from the U-46619 (0.1 µM)-induced hydrolysis of the 80-kDa latent m-calpain at 8 h in the absence or presence of the PKC inhibitor calphostin (1 µM), PLC inhibitor U73122 [GenBank] (1 µM), and the TXA2 receptor antagonist L670596 (1 µM). Representative blots are found on left, whereas compiled data are presented in histogram format on the right. B, inositol phosphate generation in response to U-46619 (0.1 µM) in the absence or presence of L670596 (1 µM) or U73122 [GenBank] (1 µM). C, calpain activity, determined using the fluorogenic substrate Suc-LLVY-AMC (see Materials and Methods) in response to U-46619 (at 6 h). D, calpain activity measured as in B in the absence or presence of calphostin-C (1 µM), U73122 [GenBank] (1 µM), L670596 (1 µM), or Calp IV (1 µM). E, representative (of three experiments) calpain activity detected by Western blot of a 145- to 150-kDa-specific fragment of -spectrin resulting from hydrolysis; 250-kDa band represents the full -spectrin. -Actin was used as loading control. Endothelial cells were treated for 6 or 18 h with U-46619 (1 µM) without or with Calp IV (1 µM). U-46619 induced a robust increase in immunoreactivity of the 145- to 150-kDa fragment of -spectrin. F, net peak calcium transients in neurovascular endothelial cells in response to 1 µM U-46619 and bradykinin with or without 1 mM EGTA. One notes absent response to U-46619. Values are mean ± S.E.M. of three to four experiments. * P < 0.05 compared with other values without asterisks.

TXA₂-Induced Mitochondrial Dysfunction. Cytotoxicity secondary to calpain activation is reported to occur in a number of instances via sequential activation of Bax, leading to mitochondrial dysfunction (Chen et al., 2002; Liu et al., 2004). Therefore, we determined the immunoreactivity of the procell death protein Bax relative to that of the anticell death protein Bcl-2 in cytosol and mitochondria of endothelial cells treated with the TXA₂ mimetic U-46619. Bax expression in mitochondria increased, whereas that in cytosol decreased over time after stimulation with U-46619, consistent with a translocation of Bax to the mitochondria (Fig. 6A). This led to a corresponding rise in Bax/Bcl-2 ratio in the mitochondria, which was virtually abrogated by L670596, Calp IV, and Calp V (Fig. 6A). Therefore, U-46619 caused (at 18 and 24 h) mitochondrial membrane depolarization [appearance of green and loss of red fluorescence (colors are superimposed in Fig. 6B)], associated with release of cytochrome c in the cytosol (Fig. 6C) and ATP depletion (Fig. 6D). U-46619-induced changes in mitochondrial function were markedly attenuated by the mitochondrial permeability transition pore blocker bongkrekic acid (Halestrap and Brennerb, 2003) as well as by L670596, Calp IV, and Calp V (Fig. 6B). Valinomycin served as a positive control.

View larger version (44K): [in this window] [in a new window]   Fig. 6. TXA2-mediated mitochondrial dysfunction of porcine neurovascular endothelial cells. A, U-46619 (0.1 µM)-induced translocation of Bax to the mitochondria. Drug concentrations were as described in Fig. 3. Values are mean ± S.E.M. of three to four experiments. B, mitochondrial depolarization evoked by U-46619 (0.1 µM) in the presence or absence of L670596 (1 µM), Calp V (1 µM), or the adenine nucleotide translocator of the mitochondrial permeability transition pore bongkrekic acid (50 µM). Mitochondrial membrane depolarization was determined using the potentiometric probe JC-1 (see Materials and Methods). Some preparations were treated with valinomycin (100 nM), a known inducer of mitochondrial depolarization. Photomicrographs are merged images (orange-yellow) of polarized (red) and depolarized (green) mitochondria. Representative micrographs are shown on the left, and compiled data in histogram format are presented on the right; inhibitors alone did not affect mitochondrial depolarization. Values are mean ± S.E.M. of three experiments. *, P < 0.05 compared with other values without asterisks. C, effects of U-46619 (0.1 µM) on cytochrome c (Cyt C) release in the cytosol. The 15-kDa cytochrome c was detected in the cytosol by immunoblotting (representative of three experiments); -actin (bottom band) was used as control. Cells were treated as indicated; concentration of inhibitors is presented in B. Note increase in cytosolic cytochrome c upon treatment with U-46619 alone and prevented by inhibitors. D, ATP cell content as a function of time after treatment with U-46619 (0.1 µM) with or without L670596 (1 µM) or Calp V (1 µM). Values are mean ± S.E.M. of three experiments. *, P < 0.05 compared with all other corresponding values.

	Discussion

Top Abstract Materials and Methods Results Discussion References

A dominant feature in this study is the role of calpain without that of caspase in TXA₂-induced neurovascular endothelial cell death. The TXA₂ mimetic U-46619 elicited early m-calpain activation (within 6 h) detected by distinct techniques (Fig. 5, A and C-E), whereas pan-caspase and caspase-3 activation were not detected at early and later times (6 and 24 h) (Fig. 4, A-C). More importantly, calpain and specifically m-calpain inhibitors, but not caspase-3 or pan-caspase inhibitors (Beauchamp et al., 2001), prevented (to a similar extent) microvascular degeneration in vivo and ex vivo as well as endothelial cell death in vitro (Figs. 1, 2, 3). Findings point to a major role for m-calpain in TXA₂-evoked neural microvascular endothelial cell death. Cell death often but not exclusively involves an interaction between calpains and caspases (Neumar et al., 2003; Rami, 2003). For example, the degradation of the endogenous calpain inhibitor calpastatin by caspase could potentiate the combined cytotoxic effects of calpains and caspases (Wang et al., 1998), but this explanation is unlikely because calpastatin immunoreactivity was unaltered in the first 24-h response to U-46619 (data not shown). Another potential mechanism of interaction between these protease systems could be through the release of cytochrome c in the cytosol following calpain-induced disruption of mitochondrial integrity, which in turn would predictably sequentially activate caspases-9 and -3 (Vindis et al., 2004). However, neither activation nor a role for caspases in U-46619-induced neurovascular endothelial cell death could be detected (Figs. 1, 2, 3C, and 4). Perhaps the explanation lies in the inactivation of caspases-9 and -3 by calpains, despite the release of cytochrome c, as reported for certain types of cell death (Chua et al., 2000) consistent with present observations (Fig. 6C).

Cell death induced by calpains is for the most part believed to occur by causing mitochondrial dysfunction and ensuing ATP depletion (Liu et al., 2004) by modifying the expression of pro- and anticytotoxic small proteins such as Bax and Bcl-2 (Gao et al., 2000) on mitochondrial membrane (Cory et al., 2003) following kinase activation (Nomura et al., 2003; Tsuruta et al., 2004) or proteolysis (Chen et al., 2001); calpain-dependent cell death can also take place through the direct presence of calpain (-like) activity at the mitochondria, which impairs the permeability transition pore function (Gores et al., 1998). Although we cannot rule out the latter possibility, our findings support a TXA₂-induced increase in Bax/Bcl-2 ratio at the mitochondria and associated loss of mitochondrial membrane polarity and cellular ATP depletion, which are prevented by calpain inhibitors (Fig. 5, A, B, and D).

Mechanisms for TXA₂-induced activation of calpain seem complex and somewhat unexpected. Other than in humans, in other species TXA₂ acts on a single receptor homologous to the human TP receptor (Kinsella, 2001). TP can couple to G_s, G_q/11, or G_12/13 (Walsh et al., 2000) but apparently not to G_i; correspondingly, pertussis toxin did not effect calpain activation. Stimulation of G_s would lead to an increase in cAMP, but the latter is primarily involved in cell survival, including of the central nervous system (Cui and So, 2004), and thus exerts effects opposite to those we observed (Figs. 1, 2, 3). G_12/13 effects are mostly mediated by Rho GTPases (Kurose, 2003), which are downstream of calpains (Sato and Kawashima, 2001). Conversely, our findings support coupling of TP to G_q/11, which leads to activation of phospholipase C to generate inositol triphosphate (Fig. 5B). But surprisingly, although calpains are well known to be activated by a rise in intracellular Ca²⁺ (Sato and Kawashima, 2001), U-46619 (and I-BOP) do not elicit Ca²⁺ transients in neurovascular endothelial cells (Lahaie et al., 1998; Fig. 5F). However, as long as intracellular Ca²⁺ concentrations are not depleted, other mechanisms partake in calpain activation (Fig. 5, A, C, and D-F) such as phosphorylation (Sato and Kawashima, 2001). Of relevance, diacylglycerol generated concurrently with inositol triphosphate during phospholipase C catalysis activates protein kinase C (Exton, 1993), which in turn can phosphorylate and activate calpain. Indeed, both the inhibitors of phospholipase C and PKC, U73122 [GenBank] and calphostin-C, respectively, prevented U-46619-induced activation of m-calpain (active fragment and activity) (Fig. 5, A and D) and ensuing endothelial cell death (Fig. 3C).

Endothelial cytotoxicity and ensuing impaired angiogenesis in response to TXA₂ has been reported in a number of studies (Beauchamp et al., 2001; Ashton et al., 2003; Brault et al., 2003; Ashton and Ware, 2004). However, endothelial cells of various origins respond differently; for example, dermal and aortic endothelial cells are not susceptible to TXA₂ (Beauchamp et al., 2001, 2002; Brault et al., 2003), whereas on cornea U-46619 induces angiogenesis in the presence (but not in the absence) of fibroblast growth factor (Daniel et al., 1999). Dissimilar actions of TXA₂ on different endothelial cells probably reflect the heterogeneity of endothelium as the same receptor couples to different signaling partners in different cells (Gudermann et al., 1996); notably, for example, one would expect distinct phenotypes and corresponding functions in glomerular and brain endothelium.

In summary, we have identified a major mechanism in neurovascular endothelial cell death in response to TXA₂, specifically (and principally) m-calpain. In this process, we have also uncovered a previously unreported role for calpain in endothelial cell death secondary to this important mediator of oxidant stress, namely, TXA₂ (Beauchamp et al., 2001, 2002; Brault et al., 2003). Because preservation of microvasculature especially in the salvageable ischemic penumbra is important, our findings provide an additional explanation for the efficacy of calpain inhibitors in ischemic encephalopathies and retinopathies (Tamada et al., 2002; Rami, 2003).

	Acknowledgements

 
We are thankful to H. Fernandez for technical assistance.

	Footnotes

 
This study was supported in parts by grants from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Québec, and the March of Dimes. C.Q. is a recipient of a scholarship from the Heart and Stroke Foundation of Canada. F.G. is a recipient of a Junior 1 scholarship from and a researcher of the Canada Foundation for Innovation. S.C. is a Canada Research Chair recipient.

doi:10.1124/jpet.105.093898.

ABBREVIATIONS: TXA₂, thromboxane A₂; TP, thromboxane receptor; Calp, calpain; U-46619, 5-heptenoic acid, 7-[6-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1]hept-5-yl]-[1R-[1,4,5(Z),6,(1E,3S)]]-9,11-dedioxy-9,11-methanolpoxy; IBOP, [1S-[1,2(Z),3(1E,3S^*),4]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.1.1]-hept-2-yl]-5-heptenoic acid; L670596, (-)-6,8-difluoro-9-p-methylsulfonyl-benzyl-1,2,3,4-tetrahydrocarbazol-1-yl-acetic acid; MTT, 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide; U73122 [GenBank] , 1-[6-((17-3-methoxyestro-1,3,5(10)-trien-17-yl)-amino)hexyl]-1H-pyrrole-2,5-dione; TRITC, tetramethylrhodamine B isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PI, propidium iodide; LDH, lactate dehydrogenase; SR, sulforhodamine; AMC, 7-amino-4-methylcoumarin; [Ca²⁺]_i, intracellular calcium concentration; PKC, protein kinase C; Z, N-benzyloxycarbonyl; Suc-LLVY-AMC, N-succinyl-Leu-Leu-Val-Tyr-7-AMC; DEVD, Asp-Glu-Val-Asp; fmk, fluoromethyl ketone; VAD, Val-Arg-Asp.

Address correspondence to: Dr. Sylvain Chemtob, Department of Pediatrics, Ophthalmology, and Pharmacology, Research Centre, Ste-Justine Hospital, 3175 Cote Ste-Catherine Montreal, Quebec H3T 1C5, Canada. E-mail: sylvain.chemtob{at}umontreal.ca

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