Published online before print May 24, 2007, doi: 10.1161/CIRCRESAHA.107.148270
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© 2007 American Heart Association, Inc.
Integrative Physiology |
Flow Antagonizes TNF-
Signaling in Endothelial Cells by Inhibiting Caspase-Dependent PKC
Processing
From the Cardiovascular Research Institute and Department of Medicine (G.G., J.A., A.M., W.L., C.Y., B.C.B.), University of Rochester, NY; Bristol Heart Institute (A.C.N.), University of Bristol, United Kingdom; and the Department of Pediatrics (A.R.), University of Rochester School of Medicine, NY.
Correspondence to Bradford C. Berk, MD, PhD, University of Rochester, Cardiovascular Research Institute, Box 679, 601 Elmwood Avenue, Rochester, NY 14642. E-mail Bradford_Berk{at}urmc.rochester.edu
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Abstract |
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Unidirectional laminar flow is atheroprotective, in part by inhibiting cytokine-mediated endothelial cell (EC) inflammation and apoptosis. Previously, we showed that flow inhibited TNF-
signaling by preventing activation of JNK. Recently, PKC
was identified as the PKC isoform most strongly regulated by flow pattern, with increased PKC activity in regions of disturbed flow versus unidirectional flow. Interestingly, PKC is cleaved by caspases after TNF- stimulation to generate a 50-kDa truncated form (CAT, catalytic domain of PKC) with a higher kinase activity than the full-length protein. We hypothesized that flow would inhibit TNF-–mediated PKC cleavage and thereby CAT formation. We found that PKC activity was required for TNF-–mediated JNK and caspase-3 activation in ECs. PKC was rapidly cleaved to generate CAT in cultured bovine and human aortic ECs and in intact rabbit vessels stimulated with TNF-. This truncated form of PKC enhanced JNK and caspase-3 activation. Interestingly, PKC cleavage was prevented by inhibitors of PKC, JNK, and caspase activities, suggesting that these enzymes, via regulating CAT formation, modulate caspase-3 activity in ECs. Finally, we found that flow reduced caspase-dependent processing of PKC and caspase-3 activation. These results define a novel role for PKC as a shared signaling mediator for flow and TNF-, and important for flow-mediated inhibition of proinflammatory and apoptotic events in ECs.
Key Words: endothelial cells • atypical PKC • caspase • TNF- • flow
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Introduction |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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Atherosclerosis is a focal inflammatory disease that develops preferentially in vascular areas exposed to low and disturbed flow within the vasculature (such as bifurcations or curvatures).1 In contrast, straight vascular segments that experience unidirectional flow appear relatively resistant to atherogenesis.2 Many findings suggest that some of these protective effects of flow are mediated via inhibition of tumor necrosis factor (TNF)-
signaling in endothelial cells (ECs).3,4 TNF- is a proinflammatory cytokine, produced by activated leukocytes and ECs during vascular injury.5 Binding of TNF- to the TNF receptor-1 (TNFR1) is followed by a rapid transcription of cell surface adhesion molecules (CAM) such as intercellular cell adhesion molecule-1 (ICAM-1)6 as well as induction of apoptosis7 in vivo and in vitro.8 Previous reports from our group and others showed that unidirectional flow, via inhibition of cytokine-mediated signaling3910 prevents EC apoptosis1112 and CAM expression.13 However, molecular and signaling events governing this interplay between flow and cytokine signaling are not fully understood.
Protein kinase C (PKC) enzymes are serine/threonine kinases that phosphorylate several effectors proteins in a cell- and stimulus-specific manner.14 Among the PKC family, the atypical PKC has recently emerged as an important isoform in ECs. PKC, promotes the adhesive phenotype of ECs via the regulation of nuclear factor kappa-B (NF
B)-dependent ICAM-1 expression.6,15 A recent study demonstrated a correlation between PKC activity and flow pattern in pig arteries,16 with lower PKC activity in ECs exposed to unidirectional flow compared with disturbed flow.16 Prolonged exposure to disturbed flow induces CAM expression in ECs in vitro and in vivo.17 Together, these observations suggest that PKC activity, differentially regulated by flow versus TNF-, could be an important determinant of atherogenesis susceptibility via regulation of inflammatory pathways.
To date, the role of PKC in TNF-–induced apoptosis has not been defined in ECs. Previous work showed that TNF- and cycloheximide (CHX) treatment induced PKC (72 kDa) processing into a shorter form, named CAT (catalytic domain of PKC, 50 kDa), in HeLa cells.18 This caspase-dependent processing occurs at 3 aspartate residues (Asp 210, 222, and 239) and promotes relief of the autoinhibitory state by separating the kinase domain (aa 268 to 335) from the pseudosubstrate auto-inhibitory sequence (aa 116 to 122).19 Such caspase-dependent processing was shown to increase PKC activity. Thus, CAT exhibits substantially higher kinase activity than the full-length protein.18 Little is known about the functional significance of this caspase-mediated processing of PKC.
In this study, we investigated the role of PKC in ECs challenged by a death stimulus. We report that PKC activity mediates TNF-–induced c-jun N-terminal kinase (JNK) and caspase-3 activation. Indeed, CAT potentiates JNK and caspase-3 activation. Interestingly, PKC is cleaved in TNF/CHX-treated ECs, whereas flow inhibits this caspase-dependent processing. Overall, our study defines PKC as an important protein in the interplay between prosurvival and proapoptotic signals in ECs.
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Methods and Materials |
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Antibodies, siRNA, Adenovirus, and Reagents
Mouse anti-actin (C-2), rabbit anti-PKC
(C-20), goat anti-platelet endothelial cell CAM-1 (PECAM-1) (M-20), rabbit and mouse anti-hemagglutinin (HA) antibodies (Y-11 and F-7) were from Santa Cruz. Anti-active JNK was bought from Promega and the mouse anti-smooth muscle actin (SM-actin) from DAKO. The phospho-specific antibody p-PKC (Thr 410), the rabbit anti-caspase-3, the rabbit anti-cleaved caspase 3 were from Cell Signaling. CHX and ZVAD-fmk were from Sigma. Myristoylated membrane-permeable PKC peptide inhibitor (PKC pseudosubstrate, PKC-PS), PKC inhibitors (Go6850 and Go6976), and JNK inhibitor were from Calbiochem. TNF- was purchased from Roche. PKC siRNAs predesigned and prevalidated from Ambion and control siRNA from Dharmacon. The adenoviral kinase dead form of PKC was a gift from Dr Newby20 and the PKC constructs from Dr Soh.21 Peroxynitrite was prepared as described.22
Cell Culture and Transfection
Bovine aortic endothelial cells (BAECs) were isolated as previously described23 and used from passage 4 to 12. Cells were grown in media-199 supplemented with 10% fetal clone III serum (Hyclone), 1% MEM amino acids (Gibco), 1% MEM vitamins (Cellgro), and antibiotics (100 U/mL penicillin, 68.6 mol/L streptomycin; Gibco). Human umbilical vein endothelial cells (HUVECs) were obtained from collagenase digested umbilical veins24 and collected in M200 medium supplemented. For chemical treatment, cells were grown to post confluence and pretreated with PKC inhibitors (PKC-PS, 30 µmol/L; Go6850, 10 nmol/L; G06976, 10 nmol/L), JNK inhibitor (SB600125, 10 µmol/L), or caspase inhibitor (ZVAd-fmk, 100 µmol/L) for 30 minutes. For transient expression experiments, 80% confluent cells were transfected using Opti-MEM and Lipofectamine 2000 as previously.12 After transfection, Opti-MEM was replaced with complete media and cells were treated 1 or 2 days later. For adenoviral infection, HUVECs were coinfected with 50 MOI of an adenovirus that drives expression of a dominant negative form of PKC under control of Tet regulator (Tet-OFF) and with an adenovirus that produces the Tet regulator under control of a constitutive CMV promoter.20 After treatment or transfection, cells were treated with TNF- (10 ng/mL, 30 minutes) for JNK activation or TNF- (10 ng/mL)/CHX (10 µg/mL) for 1, 3, or 6 hours to follow caspase-3 activity, caspase-3, and PKC cleavages.
Flow stimulation
Confluent cells were exposed to laminar flow as previously described by our laboratory12 in a cone and plate viscometer (24 hours, shear stress=12 dyn/cm2).
Rabbit Aortas: TNF/CHX Treatment and EC Isolation
Animal experiments were performed according to the guidelines of the National Institutes of Health and American Heart Association for the care and use of laboratory animals and were approved by the University of Rochester Animal Care Committee. Male New Zealand White rabbits were anesthetized with ketamine (50 mg/kg IV) and xylazine (2 mg/kg IV). Aortic segments were opened longitudinally and treated with TNF/CHX for 6 hours. After treatment, rabbit aortas were washed twice with cold PBS. To harvest ECs, lysis buffer (20 mmol/L Tris-HCl, pH 8.0, 0.05% Triton X-100, 150 mmol/L NaCl, 2 mmol/L EDTA, 50 mmol/L sodium fluoride, 2 mmol/L sodium orthovanadate, and protease inhibitor; Sigma) was applied to the endothelial surface on ice and collected.3 The remaining tissue was harvested for vascular smooth muscle cells (VSMCs) by freezing in liquid N2 and homogenizing in lysis buffer.
Cell Lysis and Western Blot Analysis
After stimulation, cells were washed twice with PBS and harvested on ice in lysis buffer (50 mmol/L NaPyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 µmol/L Na3VO4, 10 mmol/L Hepes pH 7.4, 0.1% Triton X100, protease inhibitor cocktail; Sigma). After freezing/thawing cycle, cells were centrifuged at 14 000g for 10 minutes and supernatants collected. Protein concentrations were determined by Bradford assays (Biorad). Total cell lysates were loaded on SDS-Page, electrotransfered into nitrocellulose membrane followed by blocking 1 hour at room temperature in Odyssey blocking buffer (Licor Odyssey). Membranes were incubated overnight at 4°C with primary antibodies followed by 4 washes in PBS/0.1%Tween20. For immunodetection, secondary antibody-conjugated to Alexa fluor (680 to 780 nm) were incubated 1 hour at room temperature followed by 4 washes in PBS/0.1% Tween-20. The LiCor scanner (Odyssey) was used for detection. Alternatively, horseradish peroxidase-conjugated secondary antibody was used and detected by chemiluminescence (Kit ECL, Amersham).
Apoptosis Assays
To induce apoptosis, ECs were treated with TNF- (10 ng/mL)/CHX (10 µg/mL) for 6 hours. Apoptosis was assayed by measuring caspase-3 activity (Apopto-alert Kit, BD) or the presence of the active cleaved form of caspase-3 (17/19kDa) detected by Western blot.
PKC Kinase Activity Assays
PKC activity was measured by immunoprecipitation and in vitro kinase assays as previously described20 using a specific PKC substrate (Calbiochem).
Statistical Analysis
Data are shown as mean±STDEV for 3 to 4 separate experiments. Differences were analyzed with 1-way analysis of variance (ANOVA) or Student t test. Values of P<0.05 were considered statistically significant.
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Results |
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PKC
Is a Positive Regulator of JNK in ECsWe used chemical and genetic approaches to study the role of PKC
in TNF-–mediated JNK activation, assayed by Western blot using an antibody that recognizes selectively the dually phosphorylated active form of JNK (pJNK). First, we determined the effects of a myristoylated cell permeable inhibitor of PKC (PKC pseudosubstrate, PKC-PS) known to inhibit PKC activity.25 Pretreatment of HUVECs with PKC-PS reduced TNF-–induced JNK activation by 62.7±2.2% (Figure 1A and 1B, compare lane 3 versus lane 4). To exclude the involvement of other PKC isoforms, we used a pharmacological approach to inhibit conventional and novel PKC isoforms. Inhibitors such as Go6976 that blocks PKC and ß, and Go6850 that blocks PKC , ß, 
, 
, 
did not affect significantly TNF-–induced JNK activation (Figure 1C and 1D). These findings suggest a specific role for PKC in JNK signaling.
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To confirm the specific role of PKC in TNF-–induced JNK, we used 2 approaches to inhibit PKC expression (with siRNA, Figure 1E and 1F) or activity (using a dominant negative kinase dead form of PKC, Ad.KD.PKC; Figure 1G and 1H). Endogenous PKC expression was decreased by 39.0±3.6% in PKC siRNA-transfected HUVECs compared with control cells (not shown). PKC expression was unaffected by PKC siRNA suggesting a specific inhibition of the PKC isoform (not shown). PKC depleted HUVECs showed a significant reduction in JNK activation compared with control cells (Figure 1F, 38.6±6.2% decrease compared with control cells).
To investigate the importance of PKC activity, we used an adenoviral kDa.PKC (mutation of Lys to Trp at residue 275, Ad.KD.PKC). This adenoviral form acts as a dominant negative mutant, ie, prevents endogenous PKC activity and phosphorylation. Its expression is under control of the tetracycline element (Tet-OFF system), so the addition of doxycycline turns off expression of KD.PKC and permits the activation and phosphorylation of endogenous PKC (Figure 1G and 1H, Tet-OFF). Inhibiting PKC activity reduced JNK activation in TNF-–treated ECs compared with control cells by 39±4.2% (Figure 1G and 1H, compare lane 2 to lane 4).
PKC, via JNK Regulation, Is a Positive Regulator of Caspase-3 Activity
Because of the well-established role of JNK in apoptosis, we investigated whether PKC is involved in EC apoptosis. Caspase-3 acting downstream of caspase-8 has been proposed to be the most important enzyme in the apoptotic process. Caspase-3 activation can be followed by Western blot via detection of the cleaved active form (at Asp175) or by activity assays. Inhibiting PKC activity (Figure 2A and 2B, PKC-PS) decreased cleaved caspase-3 abundance by 85±0.6% compared with control cells. It is important to note that JNK inhibitor (SB600125) reduced caspase-3 cleavage (Figure 2A and 2B, by 49±1.7% compared with vehicle-treated cells), but did not block it completely, suggesting the presence of parallel pathways. The dominant negative form of PKC (Ad.KD.PKC) reduced caspase-3 cleaved form abundance and activity by 41.7±3.5% (Figure 2D). Phosphorylation of PKC (p-PKC) was followed to assure that Ad.kDa.PKC was turned off thus allowing endogenous PKC phosphorylation.
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Death Stimuli Induce Caspase-Dependent Cleavage of PKC in Cultured ECs
Previous reports showed that TNF/CHX treatment induced cleavage of PKC (72kDa) to generate CAT in HeLa cells.19 Because PKC activity and function appear to be cell specific,26,27 we initially investigated whether this processing also occurs in ECs. ECs are resistant to TNF-–induced apoptosis, but can be rendered susceptible to TNF-–mediated cell death by inhibiting protin synthesis with CHX.28 First, we observed that exogenous PKC (HA-tagged PKC-wt, supplemental Figure I, available online at http://circres.ahajournals.org) was cleaved in BAECs, after TNF/CHX treatment, into a 50-kDa product (cleaved CAT form) that runs at an apparent molecular weight similar to both endogenous and transfected CAT (supplemental Figure I). No difference in cellular localization was observed between these 2 forms of PKC (data not shown). The formation of this cleaved form was time-dependent and maximal after TNF/CHX treatment concurrent with caspase-3 cleavage and persistent activation of JNK (Figure 3A). Next, we confirmed the presence of an endogenous cleaved product of PKC (50 kDa, cleaved) in TNF/CHX treated cells (Figure 3B). In contrast, treatment with TNF- alone, that does not induce caspase activation in ECs,26 did not induce cleavage of PKC (not shown), suggesting that a death stimulus is necessary for this cleavage to occur. As a physiologically relevant stimulus we used peroxynitrite (ONOO–), a reactive species generated from nitric oxide and superoxide anion, to induce EC apoptosis. Previous studies showed that peroxynitrite mediates apoptosis via caspase-3 activation.29 Treatment of HUVECs with ONOO– increased PKC cleavage (Figure 3C). Such cleavage of PKC was previously shown to correlate with a 3-fold increase in PKC activity.18,19 We observed a 3-fold increase of PKC activity (using a PKC specific substrate) after TNF/CHX treatment (Figure 3D).
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CAT Enhanced JNK and Caspase-3 Activation
The finding that TNF/CHX induces PKC cleavage in ECs suggested that CAT might participate in TNF- signaling. To investigate the effects of this truncated form, we transfected CAT in BAECs and examined its effects on JNK and caspase-3 activation. First, we observed that transfection of CAT increased TNF-–mediated JNK activation compared with control cells (Figure 4A and 4B, 1.9±0.2-fold increase of JNK activation compared with control cells). Transfection of CAT increased caspase-3 cleavage (Figure 4C, compare lane 2 to lane 4) and activity (Figure 4D) by 2.2±0.4-fold compared with control cells, consistent with its positive effect on JNK activation. Thus, an increase of PKC activity in TNF/CHX treated ECs, via overexpression of CAT, potentiates JNK and caspase-3 activation.
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A PKC/JNK/Caspase-3 Pathway Regulates PKC Cleavage
Because CAT, which results from caspase-dependent processing, potentiates caspase-3 activation, we investigated whether PKC, JNK, and caspase-3 regulated CAT formation. This concept implies that inhibiting any element of this apoptotic pathway should reduce PKC cleavage. To test our hypothesis, we used chemical inhibitors to block PKC, JNK, or caspase activities (Figure 5A). We showed that inhibiting PKC with PKC-PS reduced PKC cleavage in BAECs challenged with TNF/CHX for 6 hours (Figure 5B and 5C by 52±0.9% compared with control cells). Inhibiting JNK by SB600125 or caspases by ZVAD-fmk, also significantly reduce PKC cleavage (Figure 5B and 5D by 64±0.8% and 62±1.4%, respectively). These results suggest the existence of a positive feedback pathway whereby PKC cleavage enhances caspase-3 activation, which in turn amplifies PKC cleavage.
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PKC Cleavage in Intact Vessels
Next, we investigated the presence of CAT ex vivo in ECs of rabbit aorta. Lysates specifically enriched for ECs were prepared as previously described by our laboratory.3 Previous experiments showed that TNF- elicits minimal apoptotic effects on VSMCs. In fact, contractile VSMC express low levels of TNF-R130 and proteins involved in TNF- signaling, such as JNK.3 Using PECAM-1 and SM-actin as cell type specific markers for ECs and VSMCs, respectively, we first verified the purity of our cell preparations (Figure 6A). Next we showed that CAT form was present in ECs isolated from rabbit aortas after TNF/CHX treatment (Figure 6B and 6C). As in BAECs, treatment with PKC-PS and ZVAD-fmk blocked PKC cleavage in rabbit aortas. These observations show for the first time the presence of the cleaved PKC form ex vivo in ECs challenged by a death stimulus.
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Flow Prevents TNF/CHX-Induced PKC Cleavage and Reduces Proapoptotic Effects of CAT
Because unidirectional laminar flow antagonizes TNF- signaling,3,31 we determined whether flow could prevent PKC cleavage. First, we overexpressed PKC and analyzed the effect of flow on TNF/CHX-induced PKC cleavage (Figure 7A and 7B). PKC cleavage was dramatically inhibited by preexposure to flow (Figure 7A). Indeed, preexposure to flow significantly decreased CAT formation (Figure 7B, decrease of 46.6±9.9% compared with TNF/CHX alone). As previously reported, we found that preexposure of flow inhibited TNF/CHX-induced caspase-3 cleavage (not shown). Second, we followed endogenous PKC cleavage in HUVECs. As shown in Figure 7C and 7D, preexposure to flow decreased TNF/CHX-induced endogenous PKC cleavage by almost 2-fold.
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Finally, we measured the effect of flow on TNF/CHX-mediated caspase-3 activation in BAECs overexpressing CAT (Figure 8A and 8B). As shown in Figure 8A and 8B, preexposure to flow reduced the proapoptotic effect of CAT by 61% (Figure 8B, compare lane 2 to lane 3). Overall, our data show that flow inhibits CAT formation and downstream signaling events leading to caspase-3 activation (Figure 8C).
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Discussion |
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The major finding of the present study is that flow-mediated inhibition of PKC
processing is an important mechanism for prosurvival signals induced by flow. Our data demonstrate that PKC cleavage is an essential step in TNF-–induced EC apoptosis via regulation of JNK and caspase-3. This appears generally important for EC apoptosis because peroxynitrite also stimulated PKC cleavage. Interestingly, caspase-dependent PKC cleavage is significantly reduced by exposure to unidirectional flow thereby preventing activation of apoptotic pathways (Figure 8C). In combination with previous reports suggesting an important role for PKC in regulation of EC dysfunction,15,16 our data support a key role for inhibition of PKC in the antiapoptotic effects of unidirectional flow.
It has become clear that caspase-mediated activation of PKC is involved in transduction and amplification of apoptotic signals. Both atypical and novel PKC are substrates for caspases,32 and such cleavage also generates catalytically active carboxyl-terminal fragments.32 For example, PKC is a putative substrate of caspase-3 activated by such cleavage and described to promote cell death.32 The role of PKC in regulation of apoptosis appears cell type and stimulus specific.26,27 For example, sustained activation of PKC in macrophages33 and in cardiac myocytes34 increased apoptosis. In contrast, PKC mediates survival effects of low doses of ceramide in PC12 cells.35 A recent report shows that PKC participates in HOCl signaling in HUVECs, known to induce EC apoptosis.36 Here, we show that CAT increases caspase-3 cleavage and activation in ECs challenged by a death stimulus. These findings highlight the possibility of positive feedback between PKC and caspase-3, whereby PKC cleavage enhances caspase-3 activation and further PKC cleavage (Figure 8C). Using chemical inhibitors, we found that such a positive feedback loop occurred in response to TNF/CHX treatment. A similar feedback loop between PKC cleavage and caspase-3 was previously described for PKC.32
We also found in ECs that PKC is a positive regulator of JNK activation by TNF-. Activation of JNK is associated with the proinflammatory and apoptotic effects of TNF-. JNK activation induces apoptosis by regulating downstream targets such as Bcl-2. We showed that CAT, which exhibits a higher kinase activity, potentiates JNK activation under TNF- stimulation. However, we did not observe basal activation of caspase-3 or JNK in CAT overexpressing ECs. Among several explanations, it is possible that detection of caspase-3 or JNK activation was not sensitive enough, especially because EC transfection efficiency is low and cells that highly express CAT may die. Second, CAT may require other TNF-–dependent signals to be activated for its effects on caspase-3 and JNK to manifest. Third, Smith et al37 reported that the catalytic domain of PKC is intrinsically inactive and dependent on the transphosphorylation of its activation loop. Indeed, recombinant CAT from bacteria lacked detectable kinase activity. These previous observations highlight the importance of an additional stimulus to activate CAT and may explain why basally CAT has no effect by itself on JNK or caspase-3 activation.
In addition, our observations show that JNK inhibitor did not completely block caspase-3 activation or PKC cleavage suggesting the involvement of parallel pathways. It is relevant to note that PKC has been implicated as a negative regulator of Akt38 known to suppress the JNK pathway by phosphorylating and negatively regulating apoptosis signal-regulating kinase (ASK1)39 and MAP kinase kinase 4 (MKK4).40 Because our data suggest the potential involvement of additional pathways, further work is needed to identify CAT substrates in ECs challenged by a death stimulus.
Interestingly, we observed that PKC cleavage was prevented by preexposure of ECs to flow. Several studies showed that flow exerts prosurvival effects on ECs by inhibiting TNF- signaling.31,11 In particular, flow reduced caspase activation by preventing cytokine or oxidative stress signaling.11 Here, we show that flow reduces CAT-mediated caspase-3 activation, suggesting that flow affects signaling events downstream of CAT. It is relevant to note that previous reports from our laboratory showed that flow inhibits TNF-–induced JNK activation in vitro and ex vivo.9,31 We believe that flow-mediated inhibition of JNK or caspases breaks the feedback thus reducing caspase activation and PKC cleavage. In agreement with this concept, direct JNK inhibition reduced CAT abundance and caspase-3 cleavage in ECs challenged by a death stimulus.
Finally, it may be relevant to note that PKC cleavage induces the loss of a specific conserved region of PKC that contains a PB1 domain.27 This protein–protein interaction domain is known to mediate interactions of PKC with partners such as p62 and the polarity gene Par6. In fact, PKC is recruited to the polarity gene complex Par6/Par3 via its PB1 domain to participate in cell polarity and orientation.41 It is relevant to note that flow induces EC alignment and elongation in the direction of parallel to the shear stress. Thus, we may speculate that PKC cleavage impairs EC alignment in presence of TNF/CHX, whereas unidirectional flow favors EC alignment, in part by maintaining PKC integrity.
In conclusion, we found that PKC cleavage, associated with increased kinase activity, enhances JNK and caspase-3 activation. Importantly, unidirectional flow interferes with PKC processing, thereby reducing formation of the PKC truncated form and preventing its proapoptotic signals. Our study suggests that limiting PKC activity, via inhibition of its cleavage, is an essential event in flow-mediated prosurvival effects.
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Acknowledgments |
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We thank Dr Mike Massett for critical reading of this manuscript and Marlene Matthews for peroxynitrite preparation.
Sources of Funding
This work was supported by NIH Grant # HL64639 (to B.C.B.) and NIH Grant # HL-77789 and AHA Grant-in-Aid 0455783T (to J.A.).
Disclosures
None.
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Footnotes |
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Original received January 9, 2007; revision received May 15, 2007; accepted May 16, 2007.
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