2 Vascular Biology Center, 1 Department of Pharmacology and Toxicology, 3 Department of Pediatrics, and 4 Department of Pathology, Medical College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
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We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White (n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [3H]benzoyl-Phe-Ala-Pro (BPAP) and the 5'-nucleotidase (NCT) substrate [14C]AMP were estimated, and the first-order reaction parameter Amax/Km, where Amax is the product of enzyme mass and the catalytic rate constant and Km is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and Amax/Km were reduced in WHHL (1.69 ± 0.16 vs. 2.9 ± 0.44 and 599 ± 69 vs. 987 ± 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP Km and Amax values were estimated separately under mixed-order reaction conditions. No differences in Km values were found (9.79 ± 1 vs. 9.9 ± 1.31 µM), whereas WHHL rabbit Amax was significantly decreased (5.29 ± 0.88 vs. 7.93 ± 0.8 µmol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.
Watanabe heritable hyperlipidemic; 5'-nucleotidase; pulmonary circulation; endothelium
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INTRODUCTION |
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FUNCTIONAL INJURY OF THE ENDOTHELIUM appears to be an early marker that underlies the onset of atherosclerosis (36), a pathology that predisposes to stroke and myocardial infarction and carries high morbidity and mortality. Endothelial cell (EC) dysfunction may exist long before structural changes can be detected (35). A number of anatomic (vessel branch points and flow dividers) and physical (shear stress, patterns of blood flow) factors and other factors such as hypercholesterolemia, hypertension, and hyperglycemia may promote endothelial injury and, consequently, development of atherosclerosis (5).
Hypercholesterolemia, particularly increases in the low-density lipoprotein (LDL) fraction, activates several EC signals and induces endothelial responses that eventually lead to vascular injury (14). In this respect, a cholesterol-rich diet induces the expression of vascular cell adhesion molecule-1 and, subsequently, recruits mononuclear leukocytes into the arterial wall (24), attenuates endothelium-dependent aortic relaxations (34), increases the production of superoxide anion (27), and enhances vascular wall angiotensin-converting enzyme (ACE) activity in the femoral artery, abdominal aorta, and the aortic arch (26). Native LDL generates epoxyeicosatrienoic acid species that could account for inflammation and increased thrombosis (33) and induces the production of von Willebrand factor and prostacyclin (17) while it increases monocyte adhesion to endothelial cells (1), thus enhancing one of the earliest events of the atherogenic process. Oxidized LDL impairs endothelium-dependent relaxation (46) and receptor-mediated nitric oxide (NO) activity (23) while it increases the production of endothelin (4).
The Watanabe heritable hyperlipidemic (WHHL) rabbit is a strain of rabbits that exhibits hypercholesterolemia, elevated plasma LDL levels, severe atherosclerosis, and cutaneous xanthomas. The abnormality is inherited as a single-gene mutation (48). WHHL rabbits lack the LDL receptors on cultured cells of different tissues including the endothelium (3), thus being the animal model of human homozygous familial hypercholesterolemia, suitable for studying the effects of long-term hyperlipidemia on ECs. In this respect, endothelium-dependent relaxation to acetylcholine was found to be impaired in WHHL rabbits due to reduced endothelial NO production (18), whereas superoxide-induced inactivation of NO might also occur (45) . Endothelial dysfunction appears to be age related (21) and possibly related, in part, to EC loss in the atherosclerotic lesion (22), whereas the aforementioned EC dysfunction may be induced by oxidized LDL occurring in vivo (20). Although WHHL rabbit pulmonary artery is susceptible to atherosclerosis, as evidenced by the presence of related lesions (15), to our knowledge, there are no in vivo studies investigating pulmonary endothelial function in this animal strain.
Pulmonary endothelium is a major metabolic organ necessary for the adequate homeostasis of both the pulmonary and systemic circulation (28). Healthy pulmonary endothelium, among others, promotes antiaggregation and hemofluidity; secretes and/or degrades several hormones and vasoactive peptides such as angiotensin II, NO, endothelins, and prostaglandins; processes lipids; and interacts with blood components such as leukocytes, monocytes, and platelets (16, 28). Many of the aforementioned functions are catalyzed by enzymes located on the luminal endothelial surface (i.e., ectoenzymes) Two such ectoenzymes are ACE and 5'-nucleotidase (NCT). ACE catalyzes the conversion of angiotensin I to vasoconstrictor angiotensin II and breaks down the vasodilator and inflammatory mediator bradykinin (38). NCT is responsible for the dephosphorylation of extracellular AMP to the vasodilator/antithrombogenic adenosine (39). Both enzymes are uniformly distributed along the luminal pulmonary endothelial surface, although NCT appears to be localized preferentially within the membrane caveolae (40). Due to their location, the activities of both enzymes may be estimated in vivo by means of indicator-dilution techniques (8, 32).
The use of these techniques in various animal models has provided evidence that pulmonary capillary endothelium-bound (PCEB) ectoenzyme, especially ACE, dysfunction is an early and sensitive marker of various types of acute pulmonary endothelial injury (11, 29, 32, 37). In this study, we tested the hypothesis that PCEB ectoenzyme activity, an index of pulmonary endothelial function, is altered when exposed to long-term hyperlipidemia. Using the rabbit heart bypass model in vivo (47), we measured PCEB ACE and NCT activities under conditions of controlled pulmonary blood flow and compared the kinetic parameters of the aforementioned ectoenzymes in WHHL rabbits, the animal model of chronic hyperlipidemia, with those of normolipidemic New Zealand White rabbits, a commonly used control animal model for WHHL rabbits in atherosclerosis-related studies.
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METHODS |
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Seven male homozygous WHHL rabbits 12-18 mo old and 8 age- and sex-matched New Zealand White (NZ) rabbits (control rabbits) were used in this study. WHHL rabbits were purchased from the National Institutes of Health (Genetic Resources Section, National Center for Research Resources, Bethesda, MD). All animals were housed in individual cages on a 12:12-h dark-light cycle, with Purina rabbit chow and water provided ad libitum.
Anesthetized rabbit heart bypass model in vivo.
The rabbits were anesthetized with a mixture of urethan (200 mg/ml) and
Dial (50 mg/ml) administered via the marginal ear vein at volumes
individually adjusted to produce surgical anesthesia. Each animal was
then placed on an operating table, and local anesthesia of the neck was
induced by infiltration of 1% procaine. Through a midline incision,
the trachea was isolated, intubated, and connected to an animal
respirator (Harvard Apparatus). Respiratory rate and tidal volume were
adjusted to maintain normal arterial pH and
PCO2 and supranormal
PO2 values (IL 213 Micro blood gas
analyzer). A polyethylene cannula was then inserted into the left
carotid artery, advanced to the level of the ascending aorta, and
connected through a pressure transducer to a Gould physiograph;
systemic arterial pressure was then recorded. A second cannula was
inserted into the right jugular vein. Two milliliters of venous blood
were withdrawn, divided into aliquots, and centrifuged at
3,000 rpm for 10 min, and the obtained serum and plasma were stored at
20°C for subsequent cholesterol (Stanbio Laboratory), ACE,
and renin activity (6) determinations. Pancuronium bromide (1 mg) was
then administered intravenously to the animal. The chest was then
opened via a midsternal incision. The pleura and pericardium were
removed, silk sutures (000) were placed around the aorta and main
pulmonary artery, and purse sutures were drawn through the right and
left ventricles. Blood-filled catheters were quickly inserted into the
right and left atria via small incisions in the ventricle wall, aorta,
and main pulmonary artery and tied in place. Heparin (5,000 IU) was then administered through the carotid catheter.
The atrial catheters emptied into two reservoirs connected to an
extracorporeal peristaltic pump (Sarns-3M) equipped with two flexible
Tygon tubes; the outflow from the pump supplied the aortic and
pulmonary arterial catheters with arterial and venous blood,
respectively. Blood withdrawn immediately before the experiment from
two donor rabbits was used to prime the reservoirs, pump, and tubing,
with additional volume provided with Krebs buffer containing 5%
dextran (mol wt 70,000) if needed. Interruption of blood circulation
never lasted >3 min. After surgery was completed, blood flow was kept
constant at 400 ml/min, a value comparable to the normal cardiac output
of a 3- to 4-kg rabbit at rest (47).
Determination of endothelial ectoenzyme function. One determination of the single-pass transpulmonary utilizations of the synthetic ACE substrate [3H]benzoyl-Phe-Ala-Pro (BPAP) and the natural NCT substrate [14C]AMP and the calculation of the apparent kinetic parameters of these reactions were performed by means of indicator dilution-type techniques at a pulmonary blood flow of 400 ml/min under first-order reaction conditions, i.e., at trace substrate concentrations [Michaelis-Menten constant (Km)]. A second determination of BPAP hydrolysis was performed 10 min later under mixed-order reaction conditions.
Immediately before the determination of enzyme function under first-order reaction conditions, a 1.2-ml saline solution was prepared containing 0.5 mg of indocyanine green and 30 nmol of dipyridamole to prevent cellular uptake of [14C]adenosine, the product of [14C]AMP metabolism by NCT. To this solution, 2 µCi of [3H]BPAP (22.2 Ci/mmol) and 0.5 µCi of [14C]AMP (50 mCi/mmol) were added. A 0.9-ml aliquot from the aforementioned isotope mixture was injected rapidly as a bolus into the pulmonary arterial catheter. Blood was simultaneously withdrawn from the left atrial catheter at a rate of 57 ml/min by means of a peristaltic pump (Coleman Instruments) into a Gilson Escargot fraction loader equipped with 13 × 100-mm borosilicate tubes advancing at the rate of 1 tube/0.6 s (0.57 ml blood/tube). The tubes contained 2 ml of "stop" solution (normal saline containing 5 mM EDTA and 6.8 mM 8-hydroxyquinoline-5-sulfonic acid for ACE inhibition plus 1 mM AMP and 0.2 mM dipyridamole to prevent further AMP metabolism and [14C]adenosine uptake into erythrocytes, respectively). Nineteen blood samples were thus collected. For mixed-order reaction conditions, each 0.9-ml aliquot contained indocyanine green and [3H]BPAP in amounts described above plus 630 nmol of nonradioactive BPAP to obtain the higher circulating substrate concentrations needed. Samples were collected as above except that sample tubes advanced at the rate of 1 tube/0.2 s (0.19 ml blood/tube); 45 blood samples were thus collected. All determinations were performed at an expiratory airway pressure of 0 mmHg, i.e., under zone III conditions. Arterial pH, PO2, PCO2, and hematocrit were determined immediately after each indicator-dilution experiment. After the blood samples were collected, each tube was mixed gently and centrifuged at 3,000 rpm for 10 min. A 0.5-ml aliquot of the supernatant was transferred into 7-ml polyethylene scintillation vials (Fisher Scientific), and total 3H and 14C radioactivity was measured in the presence of 5 ml of Ecoscint (National Diagnostics). For determination of the radioactivity due to metabolites, different procedures were followed for each substrate (30). For [3H]BPAP, a 0.5-ml aliquot of the supernatant was transferred into a 7-ml polyethylene scintillation vial containing 2.5 ml of HCl (0.12 N). Three milliliters of 0.4% omnifluor in toluene were added to the vials, the samples were mixed, and the radioactivity was measured 48 h later. In this way, 61% of the hydrolysis product [3H]benzoyl-Phe (BPhe) and <8% of the substrate were extracted in the organic phase of the mixture. All values were corrected for the distribution of substrate- and product-associated radioactivity between the two phases. For [14C]AMP, a 0.5-ml aliquot of the supernatant was transferred into a disposable 5-ml chromatography column containing 2.1 ml of Dowex 50 2 × 8,400 mesh (ClCalculations under first-order reaction conditions.
Transpulmonary utilization (v) of [3H]BPAP and
[14C]AMP was measured by applying the
integrated Henri-Michaelis-Menten equation under first-order reaction
conditions (43) as proposed by Ryan and Catravas (37)
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(1) |
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(2) |
Calculations under mixed-order reaction conditions.
Under mixed-order reaction conditions, the Hanes-Wolf transformation of
the integrated Michaelis-Menten equation (7) was used to estimate
individual values for apparent Km and
Amax values, with linear regression of the data
obtained from both first- and zero (i.e., mixed)-order reaction
determinations according to the equation
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(3) |
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(4) |
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(5) |
Determination of vascular tissue ACE activity. At the end of the in vivo experiments, pulmonary arteries and descending thoracic aortas were removed and immediately stored in liquid nitrogen. Later on, they were homogenized, incubated with trace amounts of [3H]BPAP, and further processed with the protocol described by Chen et al. (12) with minor modifications.
Histology. Segments from the same sites in each pulmonary artery and descending thoracic aorta were excised, fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, and embedded in Spurr's resin for both light and electron microscopy.
Statistics. All data are means ± SE; they were processed and analyzed with the aid of a Dell microcomputer with ANOVA followed by Newman-Keuls multiple range test and Student's t-test where appropriate. Differences were considered significant at P < 0.05.
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RESULTS |
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Systolic (SAP), diastolic (DAP) and mean systemic (MAP) arterial
pressures of both animal groups recorded immediately after carotid
artery cannulation and before sternotomy are presented in Fig.
1. WHHL rabbits exhibited higher SAP (163 ± 8 vs. 137 ± 3 mmHg in NZ rabbits; P < 0.01) and MAP
(136 ± 7 vs. 118 ± 4 mmHg in NZ rabbits; P < 0.05) than
control rabbits; DAP was not significantly different in WHHL rabbits
(123 ± 8 vs. 109 ± 4 mmHg in NZ rabbits).
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Serum cholesterol as well as plasma ACE and renin activities measured
from blood samples obtained at the beginning of each experiment and
hemodynamic parameters recorded during enzyme activity determinations
with the rabbits on total heart bypass are presented in Table
1. WHHL rabbits exhibited significantly
higher cholesterol and renin activity values than NZ control rabbits,
with no differences in plasma ACE activity. No differences were
observed in mean systemic and pulmonary arterial pressures or in
hematocrit and arterial blood gas values obtained immediately after
each enzyme activity determination (Table 1).
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Enzyme activity parameters estimated under first-order reaction
conditions are presented in Fig. 2. BPAP
transpulmonary hydrolysis (expressed as utilization) was lower in WHHL
rabbits compared with NZ control rabbits, whereas no differences were
observed in AMP dephosphorylation (expressed as utilization) between
the two groups (1.69 ± 0.16 vs. 2.90 ± 0.44 and 1.72 ± 0.31 vs.
1.44 ± 0.50 in WHHL and NZ rabbits for ACE and NCT, respectively;
P < 0.05; Fig. 2A). Similarly,
Amax/Km values of BPAP were
lower in WHHL rabbits, whereas similar
Amax/Km AMP values were
observed in the two groups (599 ± 69 vs. 987 ± 153 and 609 ± 112 vs. 494 ± 184 ml/min in WHHL and NZ rabbits for ACE and NCT,
respectively; P < 0.05; Fig. 2B).
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To investigate whether the decreased BPAP
Amax/Km values in WHHL rabbits
resulted from lower Amax, i.e., lower PCEB ACE mass available for reaction, or higher Km, i.e., lower
enzyme-substrate affinity, further experiments were performed, and
Km and Amax were estimated
under mixed-order reaction conditions from linear regression analysis
of Eq. 3. Plots from two representative animals are presented
in Fig. 3, whereas the
corresponding indicator-dilution curves used for the estimations, under
first-order (A and B) and mixed-order (C and
D) reaction conditions are presented in Fig. 4. Km values of PCEB
ACE for BPAP were similar between WHHL and control rabbits, whereas
Amax was lower in WHHL rabbits (9.79 ± 1 vs. 9.9 ± 1.31 µM and 5.29 ± 0.88 vs. 7.93 ± 0.8 µmol/min in WHHL and
NZ rabbits, respectively; P < 0.05; Fig.
5).
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BPAP %M and 3H mean transit times in both animal strains
under first- and mixed-order reaction conditions are presented in Table
2. Significant differences were noted in
BPAP %M among all four subgroups compared with each other (ANOVA and
Newman-Keuls multiple range test), whereas no differences were observed
in the corresponding transit times (ANOVA). Tissue ACE activity was higher in both NZ rabbit pulmonary artery and descending thoracic aorta
compared with corresponding WHHL rabbit vessels (P < 0.05; Table 2).
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Histological examination of segments of pulmonary artery and descending
thoracic aorta of NZ rabbits showed, as expected, normal histology,
with no lesions in either vessel. Descending thoracic aortas from WHHL
rabbits, however, showed extensive coverage, with atherosclerotic
lesions ranging from fatty streaks to complicated fibrous plaques with
relatively acellular, lipid-rich necrotic cores containing cholesterol
clefts and covered by fibrous caps (Fig.
6A). No comparable atherosclerotic
lesions were found in the pulmonary arteries of WHHL rabbits despite
extensive examination. A few atypical punctate lesions consisting of a
few subendothelial foam cells and focal necrosis of surrounding tissue,
sometimes extending into the inner media, were observed (Fig.
6B).
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DISCUSSION |
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WHHL rabbits possess an inheritable mutation of one gene similar to that in homozygous human familial hypercholesterolemia; this mutation makes the LDL receptor completely dysfunctional; consequently, WHHL exhibits unprovoked hypercholesterolemia, with the main plasma cholesterol mass found in LDL and severe atherosclerosis (2). Functional injury of the endothelium appears to be the first step in the atherogenic process (36), with hypercholesterolemia, and, more particularly, LDL being a major noxious stimulus of the EC dysfunction (14). This is also the case for WHHL rabbits where adhesion of leukocytes and platelets to activated ECs appear to be the initiative atherogenic factors, followed by accumulation of lipids in the aortic wall (2). Numerous studies (18, 20-22, 25, 45) have provided evidence of endothelial dysfunction in various WHHL rabbit systemic vessels. Despite the fact that rabbits (42), including WHHL rabbits (15), are susceptible to pulmonary arterial atherosclerosis, there are no in vivo studies assessing pulmonary endothelial function in this animal model.
In this study, we estimated PCEB ACE and NCT activities in vivo by means of indicator-dilution techniques in WHHL and NZ control rabbits. We chose to study the activities of the aforementioned ectoenzymes because numerous studies (11, 29, 31, 32, 37) performed in animals, and more recently in humans, have shown that ectoenzyme dysfunction is an early and sensitive marker of lung vascular injury. In addition, differences in PCEB ectoenzyme activity, if present, might have significant biological implications on the pulmonary and systemic vascular homeostasis in this animal model of chronic hypercholesterolemia.
Interpretation of measures of pulmonary enzyme activity in vivo is complicated by factors that interfere with the enzyme-catalyzed reactions. For example, changes in pulmonary blood flow, shunting, capillary recruitment (or derecruitment), or left atrial pressure elevation could alter enzyme concentration or enzyme reaction time, thus affecting substrate utilization (Eq. 1). To distinguish between frank endothelial enzyme activity differences and apparent differences due to hemodynamic influences, we utilized the rabbit heart bypass model in vivo that allows for controlled hemodynamic conditions.
Anesthetized WHHL rabbits exhibited high systemic arterial pressure (SAP and MAP) compared with control values (Fig. 1). This is in agreement with a previous report on WHHL rabbit hypertension (19). When rabbits were placed on total heart bypass, WHHL and NZ rabbits exhibited similar pressure values. This anticipated difference probably relates to the normalization of stroke volume to cardiac output in both groups under heart bypass (cardiac output = 400 ml/min). The serum cholesterol of WHHL rabbits was significantly higher than the control value (Table 1) and similar to previously reported values for animals of similar age (13).
BPAP transpulmonary hydrolysis was decreased in WHHL rabbits compared with that in control animals, denoting decreased PCEB ACE activity in this animal group, whereas no differences occurred in AMP dephosphorylation (Fig. 2). The lack of differences in AMP dephosphorylation implies that NCT activity is preserved in WHHL rabbits and probably reflects similar NCT concentrations, capillary transit times, and kinetic constants in both groups because it is highly unlikely that some or all of the parameters in Eq. 1 would have changed both reciprocally and proportionally. Consequently, the observed decrease in hydrolysis of the concomitantly injected BPAP should not be related to changes in capillary transit time, i.e., reaction time, and was probably related to either decreased PCEB ACE concentration, different kinetic constants, or a combination of both.
The fact that there were no differences observed in the Amax/Km values of AMP between WHHL and control rabbits, taken together with the aforementioned absence of differences in AMP dephosphorylation (i.e., similar kinetic constants), implies similar perfused capillary surface areas between the two groups (Eq. 2). Consequently, the observed decrease in Amax/Km of the concomitantly administered BPAP should not be related to changes in perfused surface area and should most probably reflect either decreased expression of PCEB ACE mass or different ACE kinetic constants (Eq. 2).
To differentiate whether the aforementioned decreased Amax/Km of BPAP in WHHL rabbits is related to a decrease in enzyme mass, i.e., decreased Amax, or decreased ACE affinity, i.e., increased Km, additional determinations of BPAP hydrolysis were performed under mixed-order reaction conditions. The almost identical values of Km suggest similar enzyme-substrate affinities in both groups (43), whereas the Amax reduction in WHHL rabbits suggests a lower PCEB ACE mass available for reaction in this animal model (Fig. 3). This finding appears to differentiate the PCEB ACE activity reduction in WHHL rabbits from models of acute lung injury, such as phorbol 12-myristate 13-acetate induced (11), where increases in Km, i.e., decreases in ACE affinity for BPAP, occurred. This probably suggests that noxious stimuli that induce acute pulmonary endothelial dysfunction might exert their action by altering either the enzyme molecule or the plasma membrane properties, whereas chronic hyperlipidemia might induce a decrease of otherwise functional PCEB ACE.
The fact that PCEB NCT function is preserved in our model is in agreement with a previous report (10) on unchanged NCT activity in other hypercholesterolemic animal tissues, suggesting that NCT is less susceptible to injury induced by chronic hyperlipidemia. This might be partly related to NCT localization within the EC membrane caveolae (38, 40), a fact that probably makes NCT less susceptible to plasma-borne lipids.
The observed decrease in PCEB ACE mass and decreased tissue ACE activity appear to be in contrast with a report (26) on increased tissue ACE activity in atherosclerotic vessels. Interestingly, data on increased tissue ACE activity are derived from animals fed atherogenic diets rather than the genetic hyperlipidemic model used in our study. Consequently, this discrepancy may also reflect a difference between animal strains.
The decrease in PCEB ACE mass observed in WHHL rabbits could be considered the result of overt endothelial injury secondary to chronic exposure of the pulmonary endothelium to high circulating levels of cholesterol. However, alternative explanations may exist: WHHL rabbits exhibited high systemic blood pressure (Fig. 1) and increased plasma renin activity (Table 1) compared with those in NZ control rabbits, suggesting an overactive renin-angiotensin system. The operation of a feedback regulatory system on pulmonary ACE by angiotensin II in the rat has been previously observed (41). It is therefore possible that the observed decrease in PCEB ACE mass in the WHHL rabbits might be the result of PCEB ACE downregulation via this feedback regulatory system in an effort to maintain lower arterial pressure. Consistent with this alternative explanation is the absence of any differences in plasma ACE activity between the two animal groups (Table 1) because it is well known that it is the PCEB rather than the plasma ACE that is responsible for the synthesis of circulating angiotensin II and is also the locus of the antihypertensive action of ACE inhibitors. Another possibility could be that pulmonary endothelial ACE expression is downregulated because of a reciprocal regulation of systemic and pulmonary ACE because increased systemic vessel activity has been reported in rabbits fed atherogenic diets (26). The fact, however, that in our study tissue ACE activities from both the pulmonary artery and descending thoracic aorta area also decreased in WHHL rabbits makes this alternative explanation less likely, although ACE activity determinations in several WHHL rabbit systemic arteries should be performed before a definite conclusion is drawn. Furthermore, the reduction in ACE activity seen in both the pulmonary artery and thoracic aorta of WHHL rabbits in this study cannot be attributed to the presence of atherosclerotic lesion formation in the arterial beds because the aortas showed extensive lesions ranging from moderate to severe, whereas the pulmonary arteries showed no typical atherosclerotic lesions. At best, the small punctate lesions seen occasionally in the pulmonary arteries are roughly analogous to the type I (initial) lesion (isolated macrophage foam cells) as defined by the American Heart Association Committee on Atherosclerotic Lesions (44). Even accepting this possibility, the underlying necrosis is atypical of lesions at the initiating stage, and only a few such foci were found.
In summary, our studies indicate differences in pulmonary endothelial function between WHHL and NZ rabbits as evidenced by the observed decrease in PCEB ACE activity in the former in vivo. Contrary to most acute lung injury models studied, PCEB ACE activity reduction appears to be related to a decrease in enzyme mass rather than to alterations in enzyme affinity and could be related to either a direct effect of chronic hypercholesterolemia or a downregulation of ACE, among others, that might counterbalance the hypertension observed in this animal strain.
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ACKNOWLEDGEMENTS |
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The technical assistance of George W. Forbes is gratefully acknowledged.
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FOOTNOTES |
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This study was supported in part by the American Heart Association-Georgia Affiliate (S. E. Orfanos) and National Heart, Lung, and Blood Institute Grants HL-31422 (J. D. Catravas) and HL-57930 (R. G. Gerrity).
Present address of S. E. Orfanos and C. Glynos: Critical Care Department, Evangelismos General Hospital, University of Athens Medical School, Athens, Greece.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. D. Catravas, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912 (E-mail: jcatrava{at}mail.mcg.edu).
Received 19 March 1999; accepted in final form 12 January 2000.
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