Evidence for Angiotensin-Converting Enzyme– and Chymase-Mediated Angiotensin II Formation in the Interstitial Fluid Space of the Dog Heart In Vivo
Background—We have previously demonstrated that angiotensin II (Ang II) levels in the interstitial fluid (ISF) space of the heart are higher than in the blood plasma and do not change after systemic infusion of Ang I. In this study, we assess the enzymatic mechanisms (chymase versus ACE) by which Ang II is generated in the ISF space of the dog heart in vivo.
Methods and Results—Cardiac microdialysis probes were implanted in the left ventricular (LV) myocardium (3 to 4 probes per dog) of 12 anesthetized open-chest normal dogs. ISF Ang I and II levels were measured at baseline and during ISF infusion of Ang I (15 μmol/L, n=12), Ang I+the ACE inhibitor captopril (cap) (2.5 mmol/L, n=4), Ang I+the chymase inhibitor chymostatin (chy) (1 mmol/L, n=4), and Ang I+cap+chy (n=4). ISF infusion of Ang I increased ISF Ang II levels 100-fold (P<0.01), whereas aortic and coronary sinus plasma Ang I and II levels were unaffected and were 100-fold lower than ISF levels. Compared with ISF infusion of Ang I alone, Ang I+cap (n=4) produced a greater reduction in ISF Ang II levels than did Ang I+chy (n=4) (71% versus 43%, P<0.01), whereas Ang I+cap+chy produced a 100% decrease in ISF Ang II levels.
Conclusions—This study demonstrates for the first time a very high capacity for conversion of Ang I to Ang II mediated by both ACE and chymase in the ISF space of the dog heart in vivo.
Many lines of evidence have demonstrated that angiotensin II (Ang II) is formed locally in cardiovascular tissues and that augmented formation of Ang II participates in the paracrine regulation of tissue remodeling and ventricular function. Interpretation of the origins of Ang II in heart has recently been complicated by the finding of multiple Ang II–forming pathways in cardiac tissue.1 In particular, a serine protease with extremely high affinity for Ang I, “chymostatin-sensitive angiotensin-generating enzyme” (heart chymase), has been found in the human heart, purified, cloned, and sequenced.2 Heart chymase is insensitive to ACE inhibition, has catalytic activity for conversion of Ang I to Ang II that is 20-fold higher than that of ACE, and does not degrade kinins.3 Studies in dog and human heart have demonstrated that chymase accounts for >90% of Ang II formation in heart tissue extracts in vitro,4 5 6 whereas ACE is responsible for >80% of Ang II formation across the coronary vascular bed in vivo.7 8
This difference in intracardiac Ang II formation in vitro and in vivo may be related to the distribution and compartmentalization of chymase and ACE in the heart. In situ hybridization and electron microscope–immunocytochemical studies in the human heart have demonstrated that mast cells and other interstitial cell types synthesize and store chymase and that active enzyme is present in the extracellular matrix of the myocardium and vessel walls.9 ACE is bound to the cell membranes of endothelial cells, with its catalytic site exposed to the luminal surface.10 Intravascular Ang I is more accessible to ACE than to chymase, and ACE is primarily responsible for Ang II generation in the coronary vascular bed in vivo. In contrast, generation of Ang II in cardiac tissue extracts in vitro reflects a summation of catalytic activity of enzymes located within cells, bound to membranes, and within the interstitial compartment.
We used the microdialysis technique to dissect the relative contributions of ACE and chymase to Ang II formation in the interstitial fluid (ISF) space of the dog heart in vivo. We have shown that Ang I and Ang II levels were 100-fold higher in the ISF versus intravascular space.11 These levels were not affected by intravenous administration of Ang I or captopril, suggesting that Ang II production and/or degradation in the heart is compartmentalized and mediated by different enzymatic mechanisms in the interstitial and intravascular spaces. The microdialysis technique has several advantages over traditional methods of sampling blood or tissue for assays of biologically active substances in vitro. First, the ISF tissue samples provide the ability to monitor neurohormones and autacoids in the fluid bathing myocytes and interstitial cells within the heart, whereas circulating neurohormones/autacoids may not reflect local changes in that organ. Second, it is now appreciated that the concentration of neurohormones (ie, Ang II) in the circulation may differ from that in the ISF space, which is closer to target receptors. Third, the molecular-weight cutoff of the dialysis membrane can function as a barrier separating small and large molecules and can help separate peptides and cytokines of interest from degrading enzymes. Fourth, mast cells, which are a major site of chymase production, may be disrupted by tissue preparation and may release chymase. Thus, chymase-related Ang I conversion may be overestimated in tissue extracts in vitro and may have less significance in vivo.
Accordingly, we used the microdialysis technique to infuse Ang I substrate and the enzyme inhibitors captopril and chymostatin directly into the ISF space of the dog heart in vivo to test the hypotheses that Ang II is produced in the ISF and that chymase-mediated Ang II formation predominates over ACE-mediated Ang II production in the ISF space.
Twelve adult mongrel dogs (25 to 30 kg) were screened to rule out Ehrlichia canis, E platys, and Dirofilaria immitis and underwent general anesthesia with pentobarbital and mechanical ventilation with a Harvard ventilator. A median sternotomy was performed, and the heart was exposed and suspended in a pericardial cradle. An 8F sheath was inserted into the right carotid artery and positioned in the ascending aorta. Descending thoracic aortic pressure was continuously monitored with a 4F microtip Millar catheter (Millar Instruments) inserted through a femoral artery cutdown. A 7F catheter with multiple side holes was inserted into the coronary sinus through the left jugular vein. A Doppler coronary flow probe (Transonic Systems Inc) was inserted around the left anterior descending coronary artery (LAD) distal to the takeoff of the diagonal branch for coronary flow measurement during all drug infusions. Four microdialysis probes were inserted into the left ventricular myocardium in the region perfused by the LAD at the base, middle, and apical regions of the anterior wall of the left ventricle. This was done by inserting a curved 25-gauge needle through the myocardium and threading one end of the probe inlet tubing through the needle. The needle was then withdrawn and the probe pulled through the tissue, placing the dialysis membrane fully inside the muscle, as previously performed in our laboratory.11
ISF for measurement of Ang I and Ang II concentrations was collected from the microdialysis probes over a 60-minute collection period during 4 phases: (1) baseline; (2) ISF infusion of Ang I (15 μmol/L) in all 12 dogs; (3) ISF infusion of the selective ACE inhibitor captopril (2.5 mmol/L, n=4), the selective chymase inhibitor chymostatin (1 mmol/L, n=4), and captopril+chymostatin (n=4); and (4) ISF infusion of Ang I (15 μmol/L)+captopril (2.5 mmol/L, n=4), ISF infusion of Ang I+chymostatin (1 mmol/L, n=4), and ISF infusion of Ang I+captopril+chymostatin (n=4) (Figure 1⇓). Blood samples for measurement of Ang I and Ang II concentrations in plasma were collected from the aorta and coronary sinus after each infusion period. Heart rate and systemic arterial pressure were continuously recorded, and the LAD blood flow was recorded at 1-minute intervals throughout the protocol.
We have previously demonstrated high-capacity Ang II formation by a chymostatin-inhibitable mechanism (presumably due to heart chymase) in heart tissue extracts in vitro.4 8 We hypothesized that because chymase is present in the interstitium, chymase-mediated Ang II formation catalyzed by chymase should be substantial in the ISF space of the dog heart in vivo. Thus, our intent was to reproduce our in vitro assay conditions established in heart tissue extracts by infusion of Ang I substrate alone and Ang I plus inhibitors into the ISF space of the dog heart in vivo. We chose 15 μmol/L Ang I as the initial dose to infuse into the microdialysis probes, which was 40-fold lower than the 600 μmol/L Ang I dose used in our in vitro experiments.4 8 Our choices for doses of captopril and chymostatin ISF infusions were also based on our in vitro studies.4 8 For the in vivo experiments, we chose chymostatin 1 mmol/L and captopril 2.5 mmol/L, 10 and 25 times the in vitro doses of chymostatin and captopril, respectively.
Dose-Response Relationships of Captopril and Chymostatin Infusions Into the ISF
To establish a dose response for these inhibitors in the in vivo animal model, we infused 0.1, 1.0, and 10 mmol/L doses of captopril+Ang I (15 μmol/L, n=2 dogs, 4 probes per dog) and chymostatin+Ang I (15 μmol/L, n=2 dogs, 4 probes per dog) into the ISF according to the following protocol: (1) Ang I infusion for 45 minutes, (2) washout with saline for 45 minutes, (3) Ang I+inhibitor for 45 minutes, (4) Ang I+inhibitor for 45 minutes, and (5) Ang I+inhibitor for 45 minutes. Collections for Ang I and Ang II were made at the end of each 45-minute interval.
The cardiac microdialysis technique is similar to that developed by Van Wylen and coworkers12 and subsequently used in our laboratory.11 Each microdialysis probe (Clirans, Terumo Corp) is a semipermeable-membrane probe with a molecular-weight cutoff of 35 kDa and an ID of 200 μm, which is connected to methyl-deactivated silica capillary tubing (OD, 0.17 mm). Thus, each microdialysis probe consists of a single 200-μm dialysis fiber and 2 hollow tubes inserted, adjusted, and sealed within the dialysis fiber such that the distance between the ends of the silica tubes is 4 cm. The probe is perfused by a precision infusion syringe pump (BAS) at a flow rate of 2.5 μL/min. In each animal, 3 or 4 microdialysis probes were implanted into the left ventricular midmyocardium in the region perfused by the LAD at the base, middle, and apex of the left ventricular anterior wall. After insertion of the microdialysis probes, the inflow capillary tube of each probe was connected via the larger deactivated silica tube to a gas-tight glass syringe filled with normal saline and perfused at 2.5 μL/min. The effluent, or dialysate, is collected from the outflow silica tube in small plastic tubes with 50 μL of acetic acid (5 mol/L) and frozen (−80°C) until biochemical analysis.
Cardiac microdialysis is based on the principle that, as the dialysate solution passes through the microdialysis fiber, diffusion occurs between the fluid within the fiber and the ISF surrounding the fiber.12 The dialysate concentration is therefore an estimate of intramyocardial ISF concentration. However, at the flow rates used in the microdialysis experiments in vivo, it is unlikely that complete equilibration occurs between the normal saline within the fiber and the cardiac ISF in the vicinity of the fiber. Therefore, we performed in vitro experiments to estimate recovery from our microdialysis probes by the method described by Van Wylen and coworkers.12 Assuming that all probes have the same area available for diffusion, the recovery (determined by comparing the concentration in the dialysis probe effluent with that of the medium, ie, the percent recovery) depends primarily on the perfusion rate through the dialysis fiber. A recovery of 17% was used in the final calculation of ISF values, as validated by in vitro experiments previously performed in our laboratory.11 Furthermore, to document the stability of the preparation in vivo over time, we have previously determined the in vivo recovery of the stable compound acetaminophen.11 The percent recovery of acetaminophen was 17±3% at the beginning and end of perfusion, demonstrating that the recovery of this stable compound was unchanged over time in our experiment in vivo.
Biochemical Analyses: Angiotensin Peptide Levels
ISF and plasma Ang I and Ang II concentrations were determined by a method recently described from our laboratory that combines solid-phase extraction (SPE), high-performance liquid chromatography (HPLC), and radioimmunoassay (RIA).13 AG50WX4 (200- to 400-mesh) cation exchange resin was used in an SPE procedure for extraction of peptides from plasma samples. The recovery from the SPE procedure has been determined in our laboratory by use of both labeled and unlabeled Ang peptides.14 When 125I-Ang I (1.4×107 cpm) and 125I-Ang II (9×106 cpm) were used, recoveries were 93±2% (n=6) and 91±2% (n=6), respectively. With 0.5, 1.0, or 1.5 μmol unlabeled Ang I and II, recoveries were 91±9% (n=6) and 90±1% (n=6), respectively. Separation was performed by reverse-phase HPLC on a phenyl silica gel column with an eluent consisting of 20% acetonitrile in 0.1 mol/L ammonium phosphate buffer, pH 4.9. Each HPLC fraction was 300 μL. Aliquots (100 μL) of each relevant fraction of column effluent were subjected to RIA immediately after collection. Elution of standard Ang peptides under isocratic conditions revealed clear resolution of Ang I, II, and III and Ang1-7 and Ang3-8 peptides. RIA of relevant peaks revealed detectable levels of Ang I and II in all plasma and ISF samples examined. Antibodies to Ang I and II were raised in our laboratory in New Zealand White rabbits immunized against peptides conjugated to poly-l-lysine, as previously described.13 Cross-reactivity of anti–Ang I antiserum with Ang II and of anti–Ang II antiserum with Ang I was <0.5%. The sensitivity of the RIA for Ang I was 4 pg/mL; for Ang II, 2 pg/mL.
All data are presented as mean±SEM. ANOVA with Newman-Keuls post hoc comparison was used to compare hemodynamics and Ang I and Ang II levels at baseline and during infusions of Ang I, Ang I+captopril, Ang I+chymostatin, and Ang I+captopril+chymostatin into the ISF. A value of P<0.05 was required for significance.
Infusion of Ang I into the ISF did not change mean arterial pressure (115±2 to 122±3 mm Hg [SEM]) or heart rate (125±2 to 124±2 bpm). In addition, mean arterial pressure and heart rate did not change during infusions of Ang I+captopril, Ang I+chymostatin, or Ang I+captopril+chymostatin (Figure 2⇓). LAD blood flow did not change from a baseline value of 20±0.6 mL/min during any of the 4 phases of the protocol.
Dose-Response Relationships of Captopril and Chymostatin Infusions Into the ISF
All doses of captopril (0.1, 1.0, and 10 mmol/L) produced similar inhibition of Ang II (75% to 83%) over 3 sequential sampling periods, each 45 minutes in duration (n=2, 8 probes). However, 1 mmol/L chymostatin produced slightly greater inhibition than 0.1 mmol/L chymostatin (34% versus 23%) (n=2, 6 probes). Doses of chymostatin >2.5 mmol/L resulted in precipitation of perfusate in the microdialysis probes. For both captopril and chymostatin, maximum inhibition was achieved after the first 45-minute interval, and ISF Ang II levels remained unchanged for the next two 45-minute intervals, suggesting that our infusion and collection periods were sufficiently long to achieve steady-state inhibition. In these additional experiments, plasma renin activity was measured in the aorta and coronary sinus to rule out a systemic cause for the increase in plasma Ang I. Aortic and coronary sinus plasma renin activity was normal at baseline (1.8±0.5 and 1.0±0.2 ng · mL−1 · h−1, respectively) and did not change over the 4-hour protocol.
Plasma and ISF Ang Peptide Levels
Infusion of Ang I into the ISF space did not affect coronary sinus and aortic plasma Ang peptide levels; however, there was a tendency for coronary sinus and aortic plasma Ang I levels to increase during ISF infusion of Ang I+captopril and during ISF infusion of Ang I+captopril+chymostatin (Figure 3⇓). Aortic and coronary sinus plasma Ang II levels were unaffected by ISF infusion of Ang I, Ang I+captopril, Ang I+chymostatin, and Ang I+captopril+chymostatin (Figure 4⇓).
ISF Ang II increased 100-fold after Ang I infusion (P<0.01) (Figures 5 through 7⇓⇓⇓). Ang I+captopril produced a 1.2-fold increase in Ang I (P<0.01) and a 71% reduction in Ang II levels (P<0.05) compared with peptide levels achieved during Ang I infusion alone (Figure 5⇓). Ang I+chymostatin produced a 1.1-fold increase in Ang I (P<0.01) and a 43% reduction in Ang II (P<0.05) levels compared with Ang I infusion alone (Figure 6⇓). The reduction in Ang II levels was greater during Ang I+captopril than during Ang I+chymostatin infusions (71% versus 43%, P<0.01). Ang I+captopril+chymostatin produced a 1.3-fold increase in Ang I (P<0.01) and a 100% reduction in Ang II levels compared with Ang I infusion alone (Figure 7⇓). Infusion of inhibitors alone (captopril, chymostatin, and captopril+chymostatin) resulted in expected trends of an increase in Ang I and a decrease in Ang II compared with basal levels (Figures 5 through 7⇓⇓⇓, respectively).
This study gives the first direct evidence that Ang I is converted to Ang II in the ISF by the combined action of ACE and chymase. These results further demonstrate that the Ang I–converting enzymes in the ISF are of high capacity, that Ang II generation via ACE predominates over Ang II generation via chymase, and that Ang II generated in the ISF does not readily enter the vascular compartment.
All of the components of the renin-angiotensin system, including renin, angiotensinogen, ACE, and Ang I and Ang II, have been identified in the heart by biochemical, molecular biological, and immunohistochemical techniques,15 16 17 supporting our finding of local production of Ang II in the ISF. Studies in the isolated Langendorff-perfused rat heart have demonstrated that perfusion with Ang I leads to the appearance of Ang II in the coronary effluent and perfusion with renin leads to the appearance of Ang I and Ang II,18 reflecting Ang peptide formation across the coronary vascular bed. Using a modification of the isolated perfused rat heart in which the coronary effluent and ISF transudate were collected separately, de Lannoy and coworkers19 demonstrated that Ang I was formed in the ISF compartment during combined renin and angiotensinogen perfusion. Furthermore, Ang I from the intravascular compartment contributed little to the Ang I in the ISF, and Ang I from the ISF contributed little to the Ang I in the coronary effluent. Subsequent studies by the same group in the pig in vivo demonstrated that most cardiac Ang II is produced at tissue sites by conversion of in situ–synthesized rather than blood-derived Ang I.20 Taken together, these studies support our findings that enzymes located in the interstitium of the heart have a very large capacity for converting Ang I to Ang II.
Our results demonstrate that de novo formation of Ang II in the ISF after microinfusion of Ang I directly into the interstitial space did not affect coronary sinus or aortic Ang I and II plasma levels, confirming a compartmentalization of Ang II formation in the heart. However, there was a tendency for increases in plasma Ang I after infusion of Ang I+captopril and Ang I+captopril+chymostatin into the ISF space. We have previously demonstrated that intravenous infusion of captopril results in movement of captopril into the ISF space within 60 minutes.11 Because the infusion of Ang I alone into the ISF did not result in a significant increase in plasma Ang I, we interpret our data as suggesting that captopril infused into the ISF subsequently entered the intravascular space, resulting in the tendency for increases in plasma Ang I by inhibiting endothelial ACE. However, aortic and coronary sinus Ang II levels did not change significantly during any phase of the protocols. In a subset of 4 additional dogs, we measured plasma renin activity in the aorta and coronary sinus during ISF infusions of Ang I and Ang I plus inhibitors. In these experiments, aortic and coronary sinus plasma renin activity levels were unchanged over the 4-hour protocol. Thus, the increase in plasma Ang I was most likely due to spillover of captopril into the plasma.
The finding that ACE predominated over chymase-mediated Ang II formation in the interstitium of the heart was unexpected, because chymase is complexed to the extracellular matrix and has a higher catalytic activity for conversion of Ang I to Ang II than ACE in heart tissue extracts in vitro.4 5 6 However, Erdos and Skidgel21 22 reported that ACE is not only bound to cell membranes but is also present in soluble form in multiple body fluids, including seminal fluid, amniotic fluid, and lymph. Our results suggest that soluble ACE may be present in the ISF space of the heart and may be readily accessible to Ang I substrate. The lower-than-expected chymase-mediated Ang II formation in the cardiac interstitium could also be explained by the presence of endogenous chymase inhibitors in the ISF space of the heart. It was recently shown that Ang II formation from chymase in human heart tissue extracts in vitro was inhibited by endogenous protease inhibitors present in the ISF from skin.23 It is well established that Ang II formation from chymase cannot be detected in the plasma, in part because of the presence of protease inhibitors in plasma.6 In a similar fashion, the presence of endogenous protease inhibitors in the ISF of the heart could account for reduced chymase-mediated Ang II generation in the ISF space of our dogs in vivo.24 25 Nevertheless, infusion of captopril+chymostatin resulted in complete inhibition of Ang II formation compared with Ang I infusion alone, suggesting that both enzymes are important mechanisms for Ang II formation in the ISF of the heart. However, the relative contributions of these enzymes to Ang II formation in pathophysiological states remains an open question.
In vivo studies have suggested that Ang II formation resulted predominantly from chymase-like activity under ischemic conditions in the dog heart.26 Increases in Ang II levels from the anterior interventricular vein during ischemia were suppressed by the serine protease inhibitors nafamostat and chymostatin and were unaffected by captopril. In addition, we have previously shown that chronic mitral regurgitation in the dog results in an increase in cardiac mast cells, which correlates positively with the increase in cardiac chymase activity.27 Thus, the relative contributions of ACE and chymase-like activity to intracardiac Ang II formation in vivo may change, depending on the pathophysiological condition of the heart, especially in the presence of myocardial ischemia and/or inflammation, which may increase mast cell infiltration and degranulation in the cardiac interstitium.
The major problem with the microdialysis technique is its potential for causing tissue damage. This was addressed in the heart by Van Wylen and coworkers.12 These investigators found that adenosine levels were elevated immediately after implantation but rapidly declined within the first 20 minutes after implantation. A rapid decline in ISF concentrations of adenosine and other compounds has also been reported after implantation of microdialysis probes in the brain,28 suggesting that the initial effects of tissue damage dissipate rapidly. Thus, we routinely discard the ISF collection from the first 20 minutes. Furthermore, in our previous study, we found no change in ISF Ang I or Ang II levels over the three 1-hour collection periods in 6 dogs (3 to 4 probes per dog), suggesting that our elevated Ang II levels were not a result of acute tissue damage.11 In the present investigation, Ang II levels rapidly decreased below baseline as Ang I levels increased above baseline during infusion of inhibitors alone (phase 3). Furthermore, during infusion of Ang I plus captopril or chymostatin (phase 4), Ang II levels decreased as Ang I levels increased above levels achieved during Ang I infusion alone (phase 2). Finally, Ang II levels did not differ from baseline during infusion of Ang I plus captopril and chymostatin (phase 4). All of these directional changes in ISF Ang peptide levels argue against an effect of myocardial tissue damage on our results.
The present investigation demonstrates for the first time that Ang II generated in the ISF space of the dog heart does not affect systemic hemodynamics or Ang peptide levels in the coronary vascular bed. The predominance of ACE- over chymase-mediated Ang II formation in the cardiac interstitium supports the many clinical trials that demonstrate beneficial effects of ACE inhibitors in the prevention and treatment of cardiac hypertrophy and failure. Nevertheless, our studies do demonstrate substantial Ang II formation from chymase, which may increase during pathophysiological states and during chronic ACE inhibitor therapy, when Ang I levels are increased and are shunted to chymase. Furthermore, Ang II formation in the ISF is a regulatable process, supporting the hypothesis that Ang II formed locally in the ISF of the heart can exert an important effect on myocytes and fibroblasts independent of the circulating renin-angiotensin system. Future studies will investigate the relative roles of ACE versus chymase in Ang II production in the ISF space of the heart during acute and chronic hemodynamic stresses. This approach will provide a direct assessment of the milieu to which cardiomyocytes and interstitial cells are exposed in vivo and further insight into the enzymatic mechanisms of Ang II formation in the interstitial and intravascular spaces.
This study was supported by the Office of Research and Development, Medical Service, Department of Veteran Affairs (L.J.D.) and National Heart, Lung, and Blood Institute grants RO1-HL-54816 (L.J.D.) and (2T32-HL-O7457) (S.O.). The authors wish to acknowledge the expert secretarial assistance of Linda Arthur in the preparation of the manuscript.
Reprint requests to Louis J. Dell’Italia, MD, University of Alabama at Birmingham, Department of Medicine, Division of Cardiology, 834 MCLM, 1918 University Blvd, Birmingham, Alabama.
- Received October 5, 1998.
- Revision received February 8, 1999.
- Accepted February 12, 1999.
- Copyright © 1999 by American Heart Association
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