Moderation of skeletal muscle reperfusion injury by a sLex-glycosylated complement inhibitory protein

Constantinos Kyriakides1, Yong Wang1, William G. Austen Jr.1, Joanne Favuzza1, Lester Kobzik2, Francis D. Moore Jr.1, and Herbert B. Hechtman1

Departments of 1 Surgery and 2 Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of the sialyl Lewisx (sLex)-decorated version of soluble complement receptor type 1 (sCR1) in moderating skeletal muscle reperfusion injury, by antagonizing neutrophil endothelial selectin interaction and complement activation, is examined. Mice underwent 2 h of hindlimb ischemia and 3 h of reperfusion. Permeability index (PI) was assessed by extravasation of 125I-labeled albumin. Neutrophil depletion and complement inhibition with sCR1 reduced permeability by 72% (PI 0.81 ± 0.10) compared with a 42% decrease (PI 1.53 ± 0.08) observed in neutropenic mice, indicating that part of the complement-mediated injury is neutrophil independent. sCR1sLex treatment reduced PI by 70% (PI 0.86 ± 0.06), an additional 20% decrease compared with sCR1 treatment (PI 1.32 ± 0.08). Treatment with sCR1sLex 0.5 and 1 h after reperfusion reduced permeability by 63% (PI 0.09 ± 0.07) and 52% (PI 1.24 ± 0.09), respectively, compared with the respective decreases of 41% (PI 1.41 ± 0.10) and 32% (PI 1.61 ± 0.07) after sCR1 treatment. Muscle immunohistochemistry stained for sCR1 only on the vascular endothelium of sCR1sLex-treated mice. In conclusion, sCR1sLex is more effective than sCR1 in moderating skeletal muscle reperfusion injury.

ischemia; inflammation; complement activation; neutrophil; selectins


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LOWER TORSO ISCHEMIA occurs during aortic or peripheral vascular surgery, after trauma, or as result of an embolic or thrombotic event. Therapeutic attempts to restore blood flow, whether by surgery or thrombolysis, result in a reperfusion injury locally to the muscle and in a remote injury to other organs, such as the lungs (15). The muscle injury is characterized by endothelial damage and permeability edema (14).

Both polymorphonuclear leukocytes (PMNs) and the complement system have been shown to be important mediators of reperfusion injury (14, 28, 29). Neutrophil adhesion to the microvascular endothelium is thought to be a prerequisite in the sequence of events leading to the release of cytotoxic proteases and oxygen-derived free radicals and PMN diapedesis and sequestration within postischemic tissues (14). The adhesion sequence of events starts with leukocyte rolling and tethering on the activated endothelial cell, which is facilitated by the selectin family of adhesion molecules (24). Two adhesion molecules expressed during endothelial cell activation are P- and E-selectin. These can bind to glycoprotein counterreceptors expressing sialyl Lewisx (sLex), such as P-selectin glycoprotein ligand 1 (PSGL-1), which is present on neutrophils and other leukocytes (7, 16, 22). Experimental treatment with soluble oligosaccharides containing the sLex moiety has been shown to moderate local injury as effectively as PMN immunodepletion after hindlimb ischemia-reperfusion (28).

Blockade of complement activation with a soluble recombinant form of complement receptor type 1 (sCR1) has been shown to reduce the infarct size after myocardial ischemia (30). Similar observations were also described in a murine model of lower torso and gut ischemia where reperfusion injury was significantly reduced with sCR1 (29, 31). sCR1 is a single-chain glycoprotein consisting of 30 homologous domains known as short consensus repeats (SCR), followed by transmembrane and cytoplasmic domains (10, 11). Groups of seven SCRs form long homologous repeats (LHRs), which have been designated LHR-A, -B, -C, and -D for the most common human allotype of sCR1. sCR1 was prepared by deleting the cytoplasmic and transmembrane domains while retaining LHR-A, -B, -C, and -D (30, 32). This recombinant molecule blocked the assembly of the convertases responsible for cleavage of C3 and C5 and subsequent activation of the complement system and served as a cofactor in the proteolysis of C3b and C4b by factor I (30, 32).

In this study of hindlimb ischemia-reperfusion, the protective effects of the sLex-decorated version of sCR1 are examined. It is hypothesized that treatment with sCR1sLex presents a novel means by which anti-selectin activity is combined with a complement inhibitory action, leading to better protection against local skeletal muscle reperfusion injury compared with sCR1. Furthermore, the potential of sCR1sLex to localize its action on the activated vascular endothelium where selectin upregulation occurs is investigated. Finally, the possibility of treatment after reperfusion is studied.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Description of recombinant complement inhibitory proteins. sCR1 and sCR1sLex were provided by AVANT Immunotherapeutics (Needham, MA). sCR1 was produced in the Chinese hamster ovary cell line DUKX B11 and purified as previously described (30). This molecule possesses no sLex on its N-linked oligosaccharides (21). For sCR1sLex production, the expression plasmid coding for sCR1 was used (30). The plasmid pTCSLDHFR*, coding for a mutant mouse dihydrofolate reductase with an abnormally low affinity for methotrexate, was derived from pSV2-DHFR* and was cloned into pTCSLneo by direct substitution of the neomycin resistance gene (23). The Chinese hamster ovary cell line, Lec11, which expresses the alpha -(1-3)-fucosyltransferase activity necessary for synthesis of sLex-related oligosaccharides, was contransfected with pTCSLDHFR* together with the plasmid coding for sCR1 (2, 25). Clones grown in medium containing methotrexate were selected for production of high concentrations of sCR1sLex. Carbohydrate analysis of the purified glycoproteins indicated sLex glycosylation of sCR1sLex (19).

Animals. Male C57BL/6 mice aged 8-12 wk and weighing 25-30 g, purchased from Taconic Farms (Germantown, NY), were used for all experiments.

Inhibition of complement activation in vivo. Mice that did not undergo ischemia were anesthetized with intraperitoneal pentobarbital sodium (60 mg/kg), and the classical and alternative complement pathways were examined after injection with varying doses of sCR1 or sCR1sLex. The animals were killed with intraperitoneal pentobarbital sodium (90 mg/kg) 5 min after injection, and blood was aspirated from the right ventricle through a midline sternotomy and allowed to clot for 10 min at room temperature before being centrifuged at 4°C and 850 g for 10 min. The extracted serum was frozen in dry ice immediately and was stored at -70°C. At a later date, the serum was defrosted and centrifuged at 2,500 g for 5 min before assay. Most of the assays developed to measure complement activity in serum make use of sensitized sheep red blood cells. However, antibody-sensitized sheep red blood cells are resistant to hemolysis by the mouse classical complement pathway (9) and thus are not sensitive enough to determine mouse complement activity (3). To overcome these problems, a new assay specific to the mouse classical complement pathway was developed using purified mouse IgG2 antibody to rabbit red blood cells (26).

Classical complement pathway for hemolytic activity. Fresh citrated whole rabbit blood was washed three times with 0.9% saline and suspended in gelatin veronal buffer (GVB++; Sigma), resulting in a concentration of 1.2 × 108 red blood cells/ml. Serially diluted mouse serum in GVB++ (40 µl) was added to 25 µl of rabbit red blood cell solution and 25 µl of purified mouse IgG2 (diluted 64 times). The plate was shaken for 30 s and incubated for 1.5 h at 37°C. After centrifugation for 10 min at 850 g, 60 µl of each supernatant were collected. Absorbance was determined at 405 nm. To estimate the medium control and 100% lysis value, 65 µl of GVB++ and deionized water were added to 25 µl of rabbit red blood cell solution, respectively.

Alternative complement pathway hemolytic activity. Sera were diluted in Mg-EGTA (7 mmol/l of Mg2+, 10 mmol/l of EGTA). EGTA selectively chelates Ca2+, preventing activation of the classical pathway while allowing activation of the alternative pathway in the presence of Mg2+. Serially diluted serum (40 µl) was added to 25 µl of rabbit red blood cell solution and 25 µl of Mg-EGTA. The plate was shaken for 30 s and incubated for 1.5 h at 37°C. The rest of the procedure was performed as described above for the classical pathway.

Calculation. The lysis percentage was calculated as (sample OD - medium control OD)/(100% lysis OD - medium control OD), where OD is optical density. The data were plotted according to the transformation of the Von Krogh equation to determine the complement pathway hemolytic activity and the alternative complement hemolytic activity titer per milliliter of undiluted mouse serum (9).

Hindlimb model of ischemia-reperfusion injury. Mice were anesthetized with intraperitoneal pentobarbital sodium and underwent 2 h of hindlimb ischemia followed by 3 h of reperfusion. After a 2-min period of hindlimb elevation to minimize retained blood, bilateral rubber bands (Latex O-Rings) were applied above the greater trochanter using the McGivney Hemorrhoidal Ligator (Miltex Instrument). Sham mice did not undergo ischemia. Five minutes before rubber band release, animals received 1 µCi of 125I-labeled albumin (ICN, Irvine, CA) in 0.3 ml of PBS via tail vein injection. Hydration was maintained by intravenous infusion of 0.1 ml of 0.9% saline during each hour of reperfusion. Mice were maintained in a supine position and were kept anesthetized by intermittent intraperitoneal pentobarbital sodium injections. The mice were covered throughout the experiment to maintain body temperature. Experimental mortality was <10% in mice undergoing ischemia-reperfusion and 0 in animals undergoing sham injury. After death by an intraperitoneal pentobarbital sodium overdose, blood was aspirated from the right ventricle through a midline sternotomy, and its gamma radioactivity was counted (Packard, Downers Grove, IL). Muscle was harvested from both hindlimbs, its radioactivity was measured, and the muscle was dried to a constant weight in a gravity convection oven (Precision Scientific Group, Chicago, IL) at 90°C for 72 h. Extravasation of 125I-albumin was used to assess the hindlimb vascular permeability index (PI), which was determined by the ratio of radioactivity per gram of dry muscle to radioactivity per gram of blood.

PMN depletion. Mice were rendered neutropenic by a tail vein injection of rabbit anti-mouse PMN antibody (20 mg/kg; Accurate, Westbury, NY) 16 h before ischemia. Venous blood samples for total leukocyte counts and differentials were taken before ischemia and at the time of death.

Complement inhibition with sCR1 and sCR1sLex. Both classical and alternative pathways of complement activation were inhibited by an intravenous bolus of sCR1 or sCR1sLex administered 5 min before reperfusion. These agents were given at predetermined doses shown to effectively inhibit complement activation in vivo. Other groups of mice were treated with an intravenous bolus of CR1 or sCR1sLex 0.5, 1, or 2 h after reperfusion.

Immunohistological analysis. A group of mice that underwent ischemia as described above was killed with intraperitoneal pentobarbital sodium 0.5 and 1 h after reperfusion and hindlimb muscle was harvested. During this reperfusion period, vascular P-selectin is thought to be significantly upregulated (20). Immunohistological localization of sCR1 was performed on cryostat sections of snap-frozen hindlimb muscle after paraformaldehyde-methanol fixation using a peroxidase-antiperoxidase method as previously detailed (12). The primary antibody used was rabbit polyclonal IgG antibody to human sCR1 (AVANT Immunotherapeutics) at 2 µg/ml. Localization was visualized with the chromogen diaminbenzidine, and slides were counterstained with hematoxylin for microscopy.

Statistical analysis. Results are presented as means ± SE in the text and Figs. 1-5. Groups were subjected to one-way ANOVA, and, when significance was found, Student's t-test with the Bonferroni correction for multiple comparisons was applied. Percentage reduction in PI was calculated after subtraction of the background value determined in animals that had not undergone ischemia (sham).


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Fig. 1.   A: dose response of soluble complement receptor type 1 (sCR1) and sCR1 sialyl Lewisx (Lex) in inhibiting the in vivo classical complement pathway hemolytic activity (CH50) of mouse serum. sCR1 and sCR1sLex demonstrated a 10% difference in their ability to inhibit complement activation when given at 1 mg/kg. At doses of 5-20 mg/kg, both agents were similar in their capacity to inhibit complement activation (n = 6/time point). *P < 0.05. B: dose response of sCR1 and Lex in inhibiting the in vivo alternative complement pathway hemolytic activity (APCH50) of mouse serum. sCR1 and sCR1sLex demonstrated a 9% difference in their ability to inhibit complement activation when given at 1 mg/kg. At doses of 5-20 mg/kg, both agents were similar in their capacity to inhibit complement activation (n = 6/time point). *P < 0.05.



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Fig. 2.   Role of neutrophil depletion and complement inhibition in moderating skeletal muscle reperfusion injury. Sham mice did not undergo hindlimb ischemia. Neutrophil depletion moderated permeability by 42%. Permeability index in sCR1-treated mice that were also neutrophil depleted was 72% less than the injured untreated group. *P < 0.05 compared with injured untreated mice. dagger P < 0.05 compared with neutrophil-depleted group.



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Fig. 3.   Comparison of sCR1 and Lex in moderating skeletal muscle reperfusion injury. All groups underwent ischemia. The respective permeability indexes after treatment with 10 mg/kg sCR1 and 10 mg/kg sCR1sLex were 51 and 62% less compared with the untreated group. Treatment with 20 mg/kg sCR1sLex reduced permeability by 70%, significantly more than the 50% reduction noted in animals treated with 20 mg/kg sCR1. *P < 0.05 compared with untreated mice. dagger P < 0.05 compared with sCR1-treated group.



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Fig. 4.   Role of delayed treatment with sCR1 and Lex after reperfusion (R). All groups underwent ischemia. The respective permeability indexes after treatment with 20 mg/kg sCR1 at 0.5 and 1 h after reperfusion were 41 and 32%, significantly less than the untreated group, respectively. Permeability index after treatment with 20 mg/kg sCR1sLex at 0.5 and 1 h after reperfusion was moderated by 63 and 52%, respectively, significantly less than the sCR1-treated groups. No significant reduction in permeability was observed after treatment at 2 h after reperfusion for either agent. *P < 0.05 compared with injured untreated mice. dagger P < 0.05 compared with sCR1-treated group.



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Fig. 5.   Immunostaining for sCR1 in mouse hindlimb after 0.5 h of reperfusion. The 1-h immunohistochemistry is identical to 0.5 h and therefore is not shown. sCR1 was localized on the vascular endothelium of Lex-treated mice but not in animals treated with sCR1.

Animals in this study were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School and those prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council [Department of Health, Education, and Human Services, publication no. 85-23 (National Institutes of Health), revised 1985].


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibition of complement activation in vivo. Having established the presence of sLex tetrasaccharide in the Lec11 glycoproteins as indicated above, it was important to examine the effects of such glycosylation on complement inhibitory activation. At 1 mg/kg, sCR1 was marginally more effective than sCR1sLex in inhibiting complement activation, indicating a possible effect of differing glycosylation in these assays (21). At doses of 5-20 mg/kg, sCR1 and sCR1sLex exhibited very similar complement inhibitory properties for both classical and alternative pathways, with both agents reaching a plateau at 10 mg/kg, providing >99% complement inhibition. These data indicate that sCR1 and sCR1sLex are potent inhibitors of complement activation (Fig. 1, A and B).

Moderation of injury by PMN depletion and complement inhibition. Reperfusion of the ischemic skeletal muscle resulted in vascular injury, manifested by the extravasation of radiolabeled albumin. PI in mice after 3 h of reperfusion (n = 26) was 2.53 ± 0.11, significantly higher than sham PI of 0.13 ± 0.02 (n = 10, P < 0.05). Treatment with the anti-neutrophil antibody achieved an 89% neutropenia (mean absolute PMN count of 102 ± 37 vs. 893 ± 106 cells/µl in neutrophil-replete mice, P < 0.05). After reperfusion, neutropenic mice (n = 19) had a PI of 1.53 ± 0.08, representing a 42% reduction in permeability (P < 0.05). Complement antagonism with 10 mg/kg sCR1 in neutropenic mice (mean absolute PMN count of 111 ± 74 cells/µl, n = 20) reduced permeability by 72%, PI 0.81 ± 0.10 (P < 0.05), indicating that at least part of the complement-mediated injury is PMN independent (Fig. 2).

Moderation of injury by sCR1sLex. PI after treatment with 10 mg/kg sCR1 before reperfusion (n = 18) was 1.31 ± 0.07, representing a 51% reduction in injury (P < 0.05). Similarly, PI after treatment with 10 mg/kg sCR1sLex was reduced by 62% (1.04 ± 0.08, P < 0.05). PI of 1.32 ± 0.08 was not further moderated (50%) in animals treated with 20 mg/kg sCR1 (n = 19). However, treatment with 20 mg/kg sCR1sLex (n = 20) reduced injury by 70%, PI 0.86 ± 0.06, significantly better that the respective sCR1-treated group (P < 0.05; Fig. 3).

Effects of treatment after reperfusion. The reduction in PI observed in mice treated 0.5 h (n = 19) and 1 h (n = 20) after reperfusion with 20 mg/kg sCR1sLex of 0.90 ± 0.07 (63%) and 1.24 ± 0.09 (52%), respectively, was significantly more than the respective decreases in PI of 1.41 ± 0.10 (41%, n = 18) and 1.61 ± 0.07 (32%, n = 20) seen in the 20 mg/kg sCR1-treated groups (P < 0.05). Injury was not significantly reduced after treatment with 20 mg/kg sCR1sLex (n = 15) or 20 mg/kg sCR1 (n = 16) at 2 h into reperfusion (PI of 2.21 ± 0.13 and 2.22 ± 0.12, respectively; Fig. 4).

Immunohistological analysis. Hindlimb muscle samples snap-frozen in optimal cutting temperature compound 0.5 and 1 h after reperfusion were stained for human sCR1. sCR1 was localized on the vascular endothelium of sCR1sLex-treated mice but not in animals treated with sCR1 (Fig. 5).


    DISCUSSION
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ABSTRACT
INTRODUCTION
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A central role for the neutrophil in mediating the local events of reperfusion injury after tissue ischemia has been documented in a number of organs, including skeletal muscle (13, 14, 28). In this study, neutropenic mice had a 42% reduction in local injury. Support for the dependence of this model on neutrophil-endothelial selectin interaction is provided by our recent observations in mice treated with recombinant PSGL-Ig fusion protein, a known P- and E-selectin antagonist, where injury was moderated to the same extent as after PMN immunodepletion (14). In addition, the role of the tetrasaccharide sLex as a potent endothelial selectin blocker and neutrophil antagonist has been previously documented in a rat model of lower torso ischemia-reperfusion, where protection from local injury was shown to be dose dependant (28).

The dependence of skeletal muscle reperfusion injury on complement activation is also confirmed in these studies, with a 50% reduction in permeability in mice treated with sCR1. This is in accord with published reports in which experimental complement inhibition with sCR1 has ameliorated local reperfusion injury of the hindlimb, gut, and heart (14, 29-31). In general, activation of complement can induce injury by several mechanisms. First, released anaphylatoxins C3a and C5a enhance activation of neutrophils and endothelial cells, leading to vascular leakage (5, 16). Second, covalently deposited iC3b on endothelial cell membranes acts as a chemoactivator signaling for a neutrophil oxidative burst via CD11b (17). Third, integration of the membrane attack complex in the endothelial cell membrane can induce endothelial selectin upregulation and act as a pore, leading to unchecked ion flux that in turn could lead to second messenger signaling, enzyme activation, and potential osmotic lysis (4, 8). Weiser et al. (27), using a rat model of hindlimb ischemia, have demonstrated the presence of C5b-9 on reperfused skeletal muscle, an event associated with an increase in vascular permeability. This proinflammatory role of C5b-9 has been recently documented in our mouse model of hindlimb ischemia-reperfusion (14). In addition, PMN activity was found to be at least in part independent of C5, suggesting dissociation in the mechanisms by which neutrophils and complement cause injury (14). These observations were also confirmed in a mouse gut model of ischemia-reperfusion, whereby intestinal neutrophil sequestration was equally marked in both C5-sufficient and C5-deficient animals (1). Our observation of a 72% reduction in permeability after combined PMN depletion and complement inhibition with sCR1, representing 30% more protection than just neutrophil depletion, further supports the concept of a complement-mediated injury independent of neutrophil chemoactivation. Thus the combined action of an agent that could offer both complement and neutrophil antagonism would be anticipated to be more effective than just complement inhibition (6).

In this study, ischemic mice treated with sCR1sLex demonstrated a 70% reduction in permeability, representing 20% more protection from injury than animals treated with sCR1, and similar to the protection seen in neutropenic mice treated with sCR1. This increased efficacy of sCR1sLex compared with sCR1 in protecting against skeletal muscle reperfusion injury is likely related to its ability to block the interaction of P- and E-selectin with circulating neutrophils by binding to these selectin adhesion molecules on the reperfused vascular endothelium (14, 19). This observation is further supported by evidence provided from in vitro studies that demonstrate sCR1sLex but not sCR1 binding cell surface E-selectin and blocking P-selectin-mediated cellular adhesion (21). In addition to the possibility that sCR1sLex may compete with the ability of neutrophils to bind to their selectin counterreceptors on endothelial cells, it may also allow for complement inhibition to be focused at the site of the activated endothelium, as indicated by our immunohistochemistry data.

Finally, the protective effects of sCR1 and sCR1sLex were investigated after delayed treatment at various time points during reperfusion. Delayed complement inhibition with sCR1 has been associated with progressively increased permeability, although significant protection of 41 and 32% was observed after treatment at 0.5 and 1 h into reperfusion, respectively, compared with the untreated group. These protective effects of sCR1 were lost when delivery was given at 2 h into the reperfusion period. Delayed treatment with sCR1sLex at 0.5 and 1 h into reperfusion provided 63 and 52% reduction in permeability, respectively, significantly better than treatment with sCR1. As with sCR1, infusion of sCR1sLex at 2 h into reperfusion failed to significantly moderate injury compared with the untreated group. These data suggest that there appears to be a therapeutic window of up to 1 h after reperfusion where injury can be moderated by both complement and neutrophil antagonism.

In conclusion, our data indicate that, in skeletal muscle, ischemia-reperfusion complement-mediated local injury is at least in part neutrophil independent. The sLex-decorated version of sCR1 appears to be a more potent agent in moderating this reperfusion injury than sCR1. sCR1sLex has the ability to localize on the activated vascular endothelium, which may compete with neutrophil endothelial selectin interactions and offer inhibition of local complement activation. Finally, delayed complement and endothelial selectin antagonism in the reperfusion period can moderate permeability edema.


    ACKNOWLEDGEMENTS

We thank Dr. Henry Marsh, Jr., and Dr. Carolyn Pettey of AVANT Immunotherapeutics, Inc., Needham, MA, for generous donation of sCR1 and sCR1sLex used in the experiments and Amy C. Imrich for performing the immunohistochemistry.


    FOOTNOTES

This work was supported in part by General Medical Sciences Grants GM-07560, GM-35141, and GM-52585, The Brigham Surgical Group, Inc., and The Trauma Research Foundation.

Address for reprint requests and other correspondence: H. B. Hechtman, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115 (E-mail: HHechtman{at}Partners.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 August 2000; accepted in final form 14 February 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 281(1):C224-C230
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