Reduced lung endothelial angiotensin-converting enzyme activity in Watanabe hyperlipidemic rabbits in vivo

Stylianos E. Orfanos1, James B. Parkerson1,2, Xilin Chen2, Eugene L. Fisher3, Constantinos Glynos1, Andreas Papapetropoulos1,2, Ross G. Gerrity4, and John D. Catravas1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

Systemic and pulmonary arterial, airway, and left atrial pressures were continuously recorded on a Gould physiograph (2400) through Statham pressure transducers referenced to the level of the left ventricle. Left atrial pressure could be modified during the experiment by means of an adjustable clamp and was kept between 1 and 3 mmHg (for a detailed description of the model, see Ref. 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 (Cl-) anion-exchange resin on glass wool support. [14C]adenosine was eluted with 3 × 1 ml of 1 mM NaCl in 20% ethanol into a 7-ml polyethylene scintillation vial, and radioactivity was measured in 6 ml of Ecoscint. In this way, 95% of the dephosphorylated compound (i.e., [14C]adenosine) and <5% of [14C]AMP were eluted. In all instances, radioactivity was corrected for quenching, counterefficiency, and cross spill between the 3H and 14C channels.

Calculations 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)
v = ln([S<SUB>0</SUB>]/[S]) = ([E] × <IT>t</IT><SUB>c</SUB> × <IT>k</IT><SUB>cat</SUB>)/<IT>K</IT><SUB>m</SUB> (1)
where [E], tc, and kcat are the capillary enzyme concentration, reaction time (capillary mean transit time), and catalytic rate constant, respectively, and [S0] and [S] reflect initial and final substrate concentrations, respectively, in the effluent arterial plasma, with [S0] estimated (in dpm/ml) as [BPAP] + [BPhe] or [AMP] + [adenosine], where [BPAP], [BPhe], [AMP], and [adenosine] are the concentration of BPAP, BPhe, AMP, and adenosine, respectively; and [S] as the surviving substrate concentration, i.e., [BPAP] or [AMP].

Data were further analyzed utilizing the integrated Henri-Michaelis-Menten equation (43) as modified by Catravas and White (9)
<IT>A</IT><SUB>max</SUB>/<IT>K</IT><SUB>m</SUB> = (<IT>E</IT> × <IT>k</IT><SUB>cat</SUB>)/<IT>K</IT><SUB>m</SUB> = F<SUB>p</SUB> × v (2)
where Amax is the enzyme mass (E) × the catalytic rate constant (kcat) by definition (9) and Fp is the pulmonary plasma flow. Under normal conditions, Km and kcat remain constant (30) and Amax/Km is proportional to the available enzyme mass and hence, for enzymes like ACE and NCT that are evenly distributed along the luminal pulmonary endothelial surface area, proportional to the surface area of the perfused capillary bed (30).

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
1/ln([S<SUB>0</SUB>]/[S]) = (F<SUB>p</SUB>/<IT>A</IT><SUB>max</SUB>) × {([S<SUB>0</SUB>] − [S])/ln([S<SUB>0</SUB>]/[S])}

+ F<SUB>p</SUB> × (<IT>K</IT><SUB>m</SUB>/<IT>A</IT><SUB>max</SUB>) (3)
where y =1/ln([S0]/[S]), x = ([S0- [S])/ln([S0]/[S]), slope = Fp/Amax, y-intercept = Fp × (Km/Amax), and x-intercept = -Km.

[3H]BPAP percent transpulmonary metabolism (%M) and mean 3H transit time (<OVL><IT>t</IT></OVL>) were additionally calculated under first- and mixed-order reaction conditions as (9)
%M = 100 × {([S<SUB>0</SUB>] − [S])/[S<SUB>0</SUB>]} (4)
and
<OVL><IT>t</IT></OVL> = ∫<IT>t</IT>[S<SUB>0</SUB>]d<IT>t</IT>&cjs0823;  ∫[S<SUB>0</SUB>]d<IT>t</IT> (5)
where <OVL><IT>t</IT></OVL> calculations were corrected for catheter transit time.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Systolic (SAP), mean (MAP), and diastolic (DAP) arterial pressures in Watanabe heritable hyperlipidemic (WHHL) and New Zealand White rabbits. Values are means ± SE. Significant difference between the 2 groups: * P < 0.05; ** P < 0.01 (by Student's t-test).

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Serum cholesterol, plasma ACE, and renin activity on experimental day and hemodynamic parameters during enzyme determinations in NZ White and WHHL rabbits

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).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   A: single-pass transpulmonary benzoyl-Phe-Ala-Pro (BPAP) hydrolysis and AMP dephosphorylation both expressed as transpulmonary utilization (v) in WHHL and New Zealand White rabbits. Values are means ± SE. * Significant difference between the 2 groups: P < 0.05 (by Student's t-test). B: Amax/Km, where Amax is the product of enzyme mass and the catalytic rate constant and Km is the Michaelis-Menten constant, of BPAP and AMP in WHHL and New Zealand White rabbits. Values are means ± SE. * Significant difference between the 2 groups: P < 0.05 (by Student's t-test).

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).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3.   Hanes-Wolf plots obtained from the 2 representative animals shown in Fig. 4 with data obtained from both first- and zero (i.e., mixed)-order reaction determinations. Km equals x-intercept. Amax was calculated as ratio of pulmonary plasma flow (Fp) to slope. [S0], initial substrate concentration; [S], final substrate concentration; -Km, x-intercept.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Arterial outflow fractional concentration curves of 2 representative animals: a New Zealand White (A and C) and a WHHL (B and D), under first-order (A and B) and mixed-order (C and D) reaction conditions. In all cases, BPAP percent transpulmonary metabolism (%M) was estimated from samples where total 3H counts/min (cpm) were >10 × background cpm.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Km and Amax values of pulmonary capillary endothelium-bound ACE for BPAP in WHHL and New Zealand White rabbits. Values are means ± SE. * Significant difference between the 2 groups, P < 0.05 (by Student's t-test).

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   BPAP %M, and 3H mean transit time under first- and mixed-order reaction conditions and pulmonary artery and descending thoracic aorta tissue ACE activities in NZ White and WHHL rabbits

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).



View larger version (15992K):
[in this window]
[in a new window]
 
Fig. 6.   A: a 1-µm plastic section through an atherosclerotic lesion in descending thoracic aorta of a WHHL rabbit showing a fibro-fatty plaque equal in thickness to underlying media (M). Note a relatively acellular lipid core immediately internal to the media containing foam cells (FC) and cholesterol clefts (C) and a fibrous cap (F) showing smooth muscle cells aligned parallel to endothelial layer. Original magnification, ×100. B: a 1-µm plastic section through an atypical punctate lesion in pulmonary artery of a WHHL rabbit. A few scattered FCs are present beneath endothelium, and a small necrotic lesion (L) devoid of cells and elastica is present, extending into the inner media. Original magnification, ×200. Sections were stained with methylene blue, azure II, and basic fuchsin stain.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

The technical assistance of George W. Forbes is gratefully acknowledged.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alderson, LM, Endermann G, Lindsey S, Pronzuk A, Hoover RL, and Hayes KC. LDL enhances monocyte adhesion to endothelial cells in vitro. Am J Pathol 123: 334-342, 1986[Abstract].

2.   Aliev, G, and Burnstock G. Watanabe rabbits with heritable hypercholesterolemia: a model of atherosclerosis. Histol Histopathol 13: 797-817, 1998[ISI][Medline].

3.   Baker, DP, Lenten BJ, Fogelman AM, Edwards PA, Kean C, and Berliner JA. LDL, scavenger, and beta-VLDL receptors on aortic endothelial cells. Arteriosclerosis 4: 248-255, 1984[Abstract].

4.   Boulanger, CM, Tanner FC, Bea ML, Hahn AW, Werner A, and Luscher TF. Oxidized low density lipoproteins induce mRNA expression and release of endothelin from human and porcine endothelium. Circ Res 70: 1191-1197, 1992[Abstract].

5.   Callow, AD. The clinical profile of atherosclerosis. In: Vascular Endothelium. Physiological Basis of Clinical Problems II, edited by Catravas JD, Callow AD, and Ryan US.. New York: Plenum, 1993, p. 89-98.

6.   Carr, AA, and Prisant LM. The calcium antagonist isradipine and its effect on blood pressure related to plasma renin activity. Am J Hypertens 3: 354-359, 1990[ISI][Medline].

7.   Catravas, JD. Michaelis-Menten kinetics of pulmonary endothelial angiotensin converting enzyme in the conscious rabbit. In: Kinins IV, edited by Greenbaum LM, and Magnolius HS.. New York: Plenum, 1986, p. 445-451.

8.   Catravas, JD, and Orfanos SE. Pathophysiologic functions of endothelial angiotensin-converting enzyme. In: Vascular Endothelium. Physiology, Pathology, and Therapeutic Opportunities, edited by Born GVR, and Schwartz CJ.. Stuttgart, Germany: Schattauer, 1997, p. 193-204.

9.   Catravas, JD, and White RE. Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo. J Appl Physiol 57: 1173-1181, 1984[Abstract/Free Full Text].

10.   Chen, WJ, Lin-Shiau SY, Huang HC, and Lee YT. Decrease in myocardial Na(+)-K(+)-ATPase activity and ouabain binding sites in hypercholesterolemic rabbis. Basic Res Cardiol 92: 1-7, 1997[ISI][Medline].

11.   Chen, XL, Orfanos SE, and Catravas JD. Effects of indomethacin on PMA-induced pulmonary endothelial enzyme dysfunction. Am J Physiol Lung Cell Mol Physiol 262: L153-L162, 1992[Abstract/Free Full Text].

12.   Chen, XL, Orfanos SE, Ryan JW, Chung AYK, Hess DC, and Catravas JD. Species variation in pulmonary endothelial aminopeptidase P activity. J Pharmacol Exp Ther 259: 1301-1307, 1991[Abstract].

13.   Chinellato, A, Banchieri N, Pandolfo L, Ragazzi E, Froldi G, Norido F, Caparrotta L, and Fassina G. Aortic response to relaxing agents in Watanabe heritable hyperlipidemic (WHHL) rabbits of different age. Atherosclerosis 89: 223-230, 1991[ISI][Medline].

14.   Haller, H. Risk factors for cardiovascular disease and the endothelium. In: Vascular Endothelium. Physiology, Pathology, and Therapeutic Opportunities, edited by Born GVR, and Schwartz CJ.. Stuttgart, Germany: Schattauer, 1997, p. 273-286.

15.   Hansen, BF, Mortensen A, Hansen JF, Ibsen P, Fradsen H, and Nordestgaad BG. Atherosclerosis in Watanabe heritable hyperlipidemic rabbits. Evaluation by macroscopic, microscopic and biochemical methods and comparison of atherosclerosis variables. APMIS 102: 177-190, 1994[ISI][Medline].

16.   Hassoun, PM, Fanburg BL, and Junod AF. Metabolic functions. In: The Lung: Scientific Foundations, edited by Crystal RG, and West JB.. New York: Raven, 1991, vol. 1, p. 313-327.

17.   Holland, JA, Pritchard KA, Rogers NJ, and Stemerman MB. Perturbation of cultured human endothelial cells by atherogenic levels of LDL. Am J Pathol 132: 474-478, 1988[Abstract].

18.   Kagota, S, Yamaguchi Y, Shinozuka K, and Kunimoto M. Mechanisms of impairment of endothelium-dependent relaxation to acetylcholine in Watanabe heritable hyperlipidemic rabbit aortas. Clin Exp Pharmacol Physiol 25: 104-109, 1998[ISI][Medline].

19.   Katsuda, S, Hosomi H, Shiomi M, and Watanabe Y. Effect of atherosclerosis on mean and daily variation of arterial pressure in conscious WHHL rabbits. J Vet Med Sci 54: 669-673, 1992[ISI][Medline].

20.   Kita, T, Ishii K, Yokode M, Kume N, Nagano Y, Arai H, and Kawai C. The role of oxidized low density lipoprotein in the pathogenesis of atherosclerosis. Eur Heart J 11, Suppl E: 122-127, 1990[ISI][Medline].

21.   Kitagawa, S, Yamaguchi Y, Sameshima E, and Kunitomo M. Differences in endothelium-dependent relaxation in various arteries from Watanabe heritable hyperlipidemic rabbits with increasing age. Clin Exp Pharmacol Physiol 21: 963-970, 1994[ISI][Medline].

22.   Kolodgie, FD, Virmani R, Rice HE, and Mergner WJ. Vascular reactivity during the progression of atherosclerotic plaque. A study in Watanabe heritable hyperlipidemic rabbits. Circ Res 66: 1112-1126, 1990[Abstract].

23.   Kugiyama, K, Kerns SA, Morrissett JD, Roberts R, and Henry PD. Impairment of endothelium-dependent arterial relaxation by lysolethicin in modified low-density lipoproteins. Nature 344: 160-162, 1990[ISI][Medline].

24.   Li, H, Cybulsky MI, Gimbrone MJ, and Libby P. An atherogenic diet rapidly induces VCAM-1, a cytokine-regulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler Thromb 13: 197-204, 1993[Abstract].

25.   Milner, P, Loesch A, and Burnstock G. Plasticity of expression of endothelial vasoactive agents. In: Vascular Endothelium. Physiology, Pathology, and Therapeutic Opportunities, edited by Born GVR, and Schwartz CJ.. Stuttgart, Germany: Schattauer, 1997, p. 243-260.

26.   Mitani, H, Bandoh T, Kimura M, Totsuka T, and Hayashi S. Increased activity of vascular ACE related to atherosclerotic lesions in hyperlipidemic rabbits. Am J Physiol Heart Circ Physiol 271: H1065-H1071, 1996[Abstract/Free Full Text].

27.   Ohara, Y, Peterson TE, and Harrison DG. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91: 2546-2551, 1993[ISI][Medline].

28.   Orfanos, SE, and Catravas JD. Metabolic functions of the pulmonary endothelium. In: Annual Review of Cardiac Surgery (6th ed.), edited by Yacoub M, and Pepper J.. London: Current Science, 1993, p. 52-59.

29.   Orfanos, SE, Chen XL, Burch SE, Ryan JW, Chung AYK, and Catravas JD. Radiation-induced early pulmonary endothelial ectoenzyme dysfunction in vivo: effect of indomethacin. Toxicol Appl Pharmacol 124: 112-122, 1994[ISI][Medline].

30.   Orfanos, SE, Chen XL, Ryan JW, Chung AYK, Burch SE, and Catravas JD. Assay of pulmonary microvascular endothelial angiotensin-converting enzyme in vivo: comparison of three probes. Toxicol Appl Pharmacol 124: 99-111, 1994[ISI][Medline].

31.   Orfanos, SE, Langleben D, Khoury J, Schlesinger RD, Dragatakis L, Roussos C, Ryan JW, and Catravas JD. Pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in humans. Circulation 99: 1593-1599, 1999[Abstract/Free Full Text].

32.   Pitt, BR, Lister G, and Gillis CN. Hemodynamic effects on lung metabolic function. In: Pulmonary Endothelium in Health and Disease, edited by Ryan US.. New York: Dekker, 1987, vol. 32, p. 65-87. (Lung Biol Health Dis Ser)

33.   Pritchard, KA, Wong YKP, and Stemerman MB. Atherogenic concentrations of low-density lipoprotein enhance endothelial cell generation of epoxyeicosatrienoic products. Am J Pathol 136: 1383-1391, 1990[Abstract].

34.   Ragazzi, E, Chinellato A, DeBiasi M, Pandolfo L, Prosdocimi M, Norido F, Caparrotta L, and Fassina G. Endothelium-dependent relaxation, cholesterol content and high energy metabolite balance in Watanabe hyperlipidemic rabbit aorta. Atherosclerosis 80: 125-134, 1989[ISI][Medline].

35.   Reddy, KG, Nair RN, Sheehan HM, and Hodgson JM. Evidence that selective endothelial dysfunction may occur in the absence of angiographic or ultrasonic atherosclerosis in patients with risk factors for atherosclerosis. J Am Coll Cardiol 23: 833-843, 1994[ISI][Medline].

36.   Ross, R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362: 801-809, 1993[ISI][Medline].

37.   Ryan, JW, and Catravas JD. Angiotensin converting enzyme as an indicator of pulmonary microvascular function. In: Focus on Pulmonary Pharmacology and Toxicology, edited by Hollinger MA.. Boca Raton, FL: CRC, 1991, p. 183-210.

38.   Ryan, JW, and Ryan US. Processing of endogenous polypeptides by the lung. Annu Rev Physiol 44: 241-255, 1982[ISI][Medline].

39.   Ryan, JW, and Smith US. Metabolism of adenosine-5'-monophosphate during circulation through the lungs. Trans Assoc Am Physicians 84: 297-306, 1971[Medline].

40.   Ryan, US, and Ryan JW. Correlations between the fine structure of the alveolar-capillary unit and its metabolic activities. In: Metabolic Functions of the Lung, edited by Bakhle YS, and Vane JR.. New York: Dekker, 1977, vol. 4, p. 197-232. (Lung Biol Health Dis Ser)

41.   Schunkert, H, Ingelfinger JR, Hirsch AT, Pinto Y, Remme WJ, Jacob H, and Dzau VJ. Feedback regulation of angiotensin converting enzyme activity and mRNA levels by angiotensin II. Circ Res 72: 312-318, 1993[Abstract].

42.   Schwenke, DC. Comparison of aorta and pulmonary artery: I. Early cholesterol accumulation and relative susceptibility to atheromatous lesions. Circ Res 81: 338-345, 1997[Abstract/Free Full Text].

43.   Segel, IH. Enzyme Kinetics. New York: Wiley, 1975.

44.   Stary, HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, and Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation 92: 1355-1374, 1995[Abstract/Free Full Text].

45.   Tagawa, H, Tomoike H, and Nakamura M. Putative mechanisms of the impairment of endothelium-dependent relaxation of the aorta with atheromatous plaque in heritable hyperlipidemic rabbits. Circ Res 68: 330-337, 1991[Abstract].

46.   Tanner, FC, Noll G, Boulanger CM, and Luscher TF. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries. Role of scavenger receptor and endothelium-derived nitric oxide. Circulation 83: 2012-2020, 1991[Abstract].

47.   Toivonen, HJ, and Catravas JD. Effects of blood flow on lung ACE kinetics: evidence of microvascular recruitment. J Appl Physiol 71: 2244-2254, 1991[Abstract/Free Full Text].

48.   Watanabe, Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit). Atherosclerosis 36: 261-268, 1980[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 278(6):L1280-L1288
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Orfanos, S. E.
Articles by Catravas, J. D.
Articles citing this Article
PubMed
PubMed Citation
Articles by Orfanos, S. E.
Articles by Catravas, J. D.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online