Abstract
Acute respiratory distress syndrome (ARDS) is an often fatal condition for which a genetic predisposition is postulated, although no specific genes have been identified to date. Angiotensin converting enzyme (ACE) has a potential role in the pathogenesis of ARDS via effects on pulmonary vascular tone/permeability, epithelial cell survival, and fibroblast activation. Forty-seven percent of the variance in plasma ACE activity is accounted for by the ACE insertion/deletion (I/D) polymorphism, the D allele being associated with higher activity. We therefore hypothesized that the presence of the D allele would be associated with the development of ARDS. Ninety-six white patients fulfilling American/European Consensus Committee criteria for ARDS were genotyped for the ACE polymorphism together with individuals from three comparison groups: 88 white patients with non-ARDS respiratory failure ventilated in the intensive care unit (ICU), 174 ICU patients undergoing coronary artery bypass grafting, and 1,906 individuals from a general population group. DD genotype frequency was increased in the patients with ARDS compared with the ICU (p = 0.00008), coronary artery bypass grafting (p = 0.0009), and general population group (p = 0.00004) control groups and was significantly associated with mortality in the ARDS group (p < 0.02). These data suggest a potential role for renin–angiotensin systems in the pathogenesis of ARDS and for the first time implicate genetic factors in the development and progression of this syndrome.
Acute respiratory distress syndrome (ARDS) remains an important cause of mortality in the intensive care unit (ICU) for which there are no specific therapies. Factors predicting the onset or severity of this syndrome are poorly understood, but the low incidence of ARDS in the relatively large group of patients at risk of developing this syndrome suggests the involvement of genetic factors (1). No specific genes have been identified to date.
There is considerable evidence to support the existence of local renin–angiotensin systems in a number of human tissues, including the lung (2), in which renin–angiotensin system components are produced independent of circulating precursors. In such systems, angiotensin converting enzyme (ACE) is a key enzyme for the generation of angiotensin (AT)-II from AT-I, but also degrades bradykinin, hence its alternative name—Kininase II. Both these molecules and related peptides have been shown to exert multiple cellular effects.
The early stages of ARDS are characterized by a high permeability pulmonary edema, alveolar epithelial cell loss, and neutrophil infiltration, which may progress to significant alveolar and interstitial remodelling. Experimental evidence suggests that activation of a pulmonary renin–angiotensin system might influence the pathogenesis of ARDS via such mechanisms as vascular permeability (3), vascular tone (4), and fibroblast activity (5) and by reducing alveolar epithelial cell survival (6).
In patients with ARDS, circulating ACE is often reduced (7); however, this may reflect loss of enzyme release from a damaged pulmonary vascular endothelium and may not be representative of activity in other lung compartments. For example, an elevation in bronchoalveolar lavage ACE has been reported despite a reduction in circulating concentrations (8). Similarly, the transpulmonary gradient and circulating concentrations of AT-II are increased in patients with ARDS (9), and activation of the circulating renin–angiotensin system, with a preservation of the transpulmonary gradient, was also found in another study of critically ill patients (10). In patients with smoke inhalation, despite a reduction in circulating ACE, ACE in bronchoalveolar lavage was elevated (11).
Large interindividual differences in plasma ACE concentrations exist, but concentrations are similar within families (12), suggesting a strong genetic influence. The human ACE gene (DCP1), located on chromosome 17q23, contains a restriction fragment length polymorphism (13) consisting of the presence (insertion, I) or absence (deletion, D) of a 287-bp alu repeat sequence in intron 16 (14). Amongst 80 healthy white individuals, the I/D polymorphism accounted for 47% of the variance in plasma ACE, being highest in those with the DD genotype (13). Tissue ACE concentrations appear to be similarly influenced. ACE activity in those of DD genotype is thus 75 and 39% higher in plasma and T-lymphocytes, respectively, than in those of II genotype (15). It is believed that this intronic (noncoding) polymorphism may be a marker for another genetic locus/loci with more functional significance, although extensive haplotyping of the region has excluded the promoter region and a number of exons (16).
Associations between the D allele and the development and/or progression of sarcoidosis (17), asthma (18), and berylliosis (19) have been described but are contradicted by others (20). No studies have been published regarding patients with ARDS or indeed any critical illness. In the present study, we hypothesized that the D allele (associated with higher ACE activity) would be associated with susceptibility and outcome in ARDS. To ensure that the specificity of any association observed was with ARDS rather than with critical illness, three comparison populations were recruited: patients admitted to the ICU with non-ARDS respiratory failure (ICU), patients admitted to ICU having undergone coronary artery bypass grafting (CABG), and a healthy population group (POP).
This study was reviewed and approved by the Ethics Committee of University College London Hospitals. All patients over 18 years of age fulfilling the joint American/European Consensus Committee criteria for ARDS (21) admitted to the ICU of the University College London Hospitals were considered eligible for the study. Patients were excluded from the study if they had a previous history of ARDS. Baseline clinical and demographic data including lung injury score, the Acute Physiology and Chronic Health Evaluation score (22), and Simplified Acute Physiology Score (23) were calculated. Patients were classified as survivors if they were discharged alive from the ICU and no longer required mechanical ventilation.
In addition, 88 patients admitted to the ICU over the same period for non-ARDS respiratory failure requiring invasive mechanical ventilation and 174 patients admitted to ICU following CABG, but without respiratory failure, were recruited as comparison groups. This was to ensure that any associations observed were specific to the development of ARDS and not merely to conditions associated with admission to the ICU or to respiratory failure. All patients were identified prospectively and within 24 hours of admission. Genotype and allele frequencies in all patient groups were also compared with 1,906 healthy British men (POP). A significant influence of ethnic background on ACE allele frequency has been observed and for this reason only white patients were studied in all groups.
DNA was extracted from whole blood samples by modified phenol–chloroform extraction. ACE genotype was determined by three-primer polymerase chain reaction amplification as described previously (24), performed by staff blinded to all subject data. This method yields amplification products of 84 bp for the D allele and 65 bp for the I allele, and products were visualized on polyacrylamide gels. II genotype was confirmed by repeat polymerase chain reaction in the absence of a primer for the I allele. All samples were randomized, genotyped, and independently replicated by two independent researchers.
Differences in demographic and clinical data between groups were assessed by Student's t test. For genotypes, frequencies between ARDS and comparison groups were compared by chi-squared test. Confidence intervals were also calculated for D allele frequency. For all tests, a p value of 0.05 or less was taken as significant.
A total of 96 patients fulfilling American/European Consensus Committee criteria for ARDS was enrolled and a sample for DNA extraction obtained from each patient. The main reason for nonentry into the study was failure to obtain consent within a 24-hour period. The most common diagnoses in the study ARDS group were pneumonia (27%), sepsis (26%), and trauma (11%).
Eighty-eight patients were recruited with non-ARDS respiratory failure requiring mechanical ventilation in the ICU. Patient demographics in the ARDS and ICU groups were not statistically different (Table 1)
ARDS (n = 96) | ICU Control (n = 88) | CABG Control (n = 174) | |
|---|---|---|---|
| Age* | 50.3 (17–91) | 53.6 (18–80) | 53.6 (22–78) |
| Male/female | 61/35 | 56/32 | 112/62 |
| APACHE II score† | 19.6 ± 4.3 | 18.8 ± 6.4 | N/A |
| SAPS 2 score† | 34.1 ± 14.4 | 35.6 ± 14.9 | N/A |
| PaO2/FiO2† | 132 ± 19 | 161 ± 55† | N/A |
| Lung injury score† | 3.3 ± 0.6 | 2.5 ± 0.9 | N/A |
| ICU mortality, % | 37 | 24 | N/A |
For all samples, genotype distribution was in Hardy–Weinberg equilibrium (χ2 = 0.03, p = 0.85 for ARDS; χ2 = 0.64, p = 0.21 for CABG; χ2 = 0.10, p = 2.57 for ICU; and χ2 = 0.03, p = 0.87 for POP). ACE genotype and allele frequencies in the ICU and CABG groups were not statistically different from the healthy population sample. There was no statistically significant difference in severity of illness scores or length of ICU stay in the ARDS group by ACE genotype (Table 2)
Genotype | |||||
|---|---|---|---|---|---|
| II | ID | DD | |||
| APACHE II score | 20.1 ± 6.4 | 19.6 ± 6.55 | 19.4 ± 4.3 | ||
| SAPS 2 | 35.7 ± 15.8 | 35.1 ± 15.8 | 38.1 ± 12.3 | ||
| PaO2/FiO2 | 127 ± 15 | 125 ± 22 | 126 ± 17 | ||
| Lung injury score | 3.2 ± 0.8 | 3.2 ± 0.6 | 3.2 ± 0.7 | ||
| Length of ICU stay, d | 18.3 ± 15.2 | 18.5 ± 12.8 | 19.5 ± 15.4 | ||
Genotype* | p (genotype)† | Frequency of D Allele (95% CI) | |||
|---|---|---|---|---|---|
| II | ID | DD | |||
| ARDS | 9 (0.09) | 43 (0.45) | 44 (0.46) | 0.69 (0.61–0.75) | |
| CABG | 40 (0.23) | 90 (0.52) | 44 (0.25) | 0.0009 | 0.51 (0.46–0.56) |
| ICU | 31 (0.35) | 36 (0.41) | 21 (0.24) | 0.00008 | 0.44 (0.37–0.52) |
| Healthy population | 459 (0.24) | 949 (0.50) | 498 (0.26) | 0.00004 | 0.51 (0.49–0.53) |
This is the first description of a specific allele association with the development of ARDS and with outcome. The strength of this association suggests a major role for renin–angiotensin systems in the development and progression of this syndrome.
It is encouraging that no association was seen in the ICU comparison group, which was similarly matched in terms of clinical severity. This implies that renin–angiotensin system activation is linked to processes specific to ARDS and not merely to critical respiratory illness. Given the relatively broad clinical criteria used to diagnose ARDS, it is perhaps surprising that such a strong association was observed. However, it may be that such observations themselves guide changes in the classification of patients in the future via an improved understanding of pathobiology. Therefore, we might at least conclude that patients diagnosed with ARDS in our institution appear to share common pathologic events in which renin–angiotensin systems are involved.
Patients undergoing coronary bypass surgery were chosen as a comparison group because they are exposed to a relatively controlled injury, are admitted to the ICU, and share similar comorbid conditions. They thus form a comparatively homogeneous and readily available in-patient population. Although in principle such patients are at risk of ARDS, only two such patients progressed to ARDS during the study period. A group with a higher incidence of ARDS would have been of value; however, the recruitment of such patients, although avoiding lead-time/survival bias and the exclusion of comorbid conditions, is fraught with difficulties, requiring a large and inclusive study. The possibility remains, however, that the association with the ACE genotype may still reside with a condition such as sepsis that might predispose to ARDS rather than with ARDS itself. Against this is the fact that a number of patients in the ICU comparison group fulfilled criteria for the systemic inflammatory response syndrome or sepsis at some point during their stay, yet there was no association with ACE in this group. In fact there was a nonsignificant trend toward the II genotype in these individuals. There is likely to be considerable overlap between these syndromes of critical illness, and the collection of additional comparison groups is planned.
The ICU and CABG comparison groups were collected prospectively over the same time period as the patients with ARDS, reducing the possibility that differences in genotype frequency were the result of chance variation in recruitment. The comparison groups were not specifically age/sex matched, and although post hoc analysis demonstrated no significant differences in age/sex demographics, the lack of strict age/sex matching may have contributed to variations in genotype frequency. However, in the large number of studies performed to date, no association has been observed between ACE genotype and either age or sex.
The determination of plasma ACE activity was not possible in this study due to ethical restrictions on the number of samples collected. Although clearly a limitation, we would suggest that a relationship between plasma ACE and ACE genotype in patients with ARDS is unlikely. First, the observed association with ACE genotype is more likely to depend on the activity of ACE within tissues rather than the circulation (no correlation has been observed previously between tissue and circulating ACE activity). Furthermore, circulating ACE derives largely from the pulmonary endothelium, and in the context of widespread pulmonary endothelial damage/activation, such as that occurring in ARDS, many additional factors will influence the production/expression of membrane-bound ACE and its release into the circulation. Thus, any relationship between genotype and circulating ACE is likely to be significantly disrupted, resulting in a high degree of variability in such measurements. We plan to assess ACE activity in bronchoalveolar lavage fluid and recovered inflammatory cells (which might better reflect ACE activity within the lung) as part of ongoing studies.
The association between the D allele and the development of ARDS implies a role for renin–angiotensin system activation in the earliest stages of this syndrome. Local renin–angiotensin systems independent of circulating precursors have been described for most tissues studied. Current evidence suggests a similar system in the lung (2, 5, 25, 26). Thus, increased ACE activity might influence ARDS pathogenesis via effects in a number of tissues and cell types—both pulmonary and nonpulmonary.
In the lung, the pulmonary circulation is a potentially important target for renin–angiotensin system activation. ACE inhibitors attenuate pulmonary vasoconstriction in normal humans and patients with cor pulmonale (4), as do type-1 angiotensin receptor antagonists (27). Infusion of AT-I (28) or AT-II (3) can produce pulmonary edema independent of catecholamine release. AT-II may, therefore, also influence microvascular permeability
Wang and colleagues have identified AT-II as a pro-apoptotic factor for alveolar epithelial cells in vitro (26). The loss of an intact epithelial barrier—with implications for the movement of fluid and cells between the vascular, interstitial, and alveolar spaces—is another early event in ARDS that might be influenced by a renin–angiotensin system.
A profound association was observed between the D allele and mortality. Both the processes influencing the development of ARDS and/or additional mechanisms may be involved. For example, fibroproliferation could have a significant effect on outcome in ARDS (5, 29). We have shown previously that AT-II is a mitogen for lung fibroblasts (5). Furthermore, both ACE inhibitors and type-1 angiotensin receptor antagonists successfully attenuate collagen deposition and interstitial fibrosis in a number of experimental models of lung injury (30, 31). In patients with left ventricular dysfunction, ACE inhibition leads to improved gas transfer and ventilation perfusion coupling by an as yet unknown mechanism (32). By implication, increased ACE activity might produce opposite effects in patients with acute respiratory failure. Finally, ACE is expressed in activated alveolar macrophages (33) and lymphocytes (13). In activated pulmonary macrophages, ACE inhibition reduced free radical expression; however, its role in modulating the inflammatory response has not been clearly defined (33).
Alternatively, renin–angiotensin systems may influence ARDS pathogenesis via more global effects in other tissues. For example, local systems in skeletal/cardiac muscle or the vascular wall might modulate the response to systemic inflammation or tissue hypoxia (34, 35) or regulate the mobilization and use of metabolic substrate (36). All such hypotheses are worthy of further study.
ACE is an important enzyme for the generation of AT-II but is also the key enzyme for the degradation of bradykinin, which in turn could lead to alterations in nitric oxide and prostaglandin production. Other substrates for ACE have also been identified including the enkephalins, neurotensin, cholecystokinin, substance P, and the gonadotrophin luteinizing hormone releasing hormone (37, 38). The delineation of key effector molecules upstream of ACE activity in ARDS is thus an important future aim.
Functional genetic polymorphisms in other renin–angiotensin system components have been described, including angiotensin receptors (39), bradykinin receptors (40), and Ao (41). One might expect the effect of alleles associated with increased type-1 angiotensin receptor or Ao expression to be additive with those of ACE if AT-II is the major biologic correlate of the association with the D allele. If, however, some other action of ACE underlies this association, a relationship with these genotypes may be absent. This logic could be extended to other components of the extended renin–angiotensin system to further delineate the biologic pathways involved.
The current findings might be applied clinically in a number of ways. In principle, the administration of an ACE inhibitor would modulate the effective phenotype (ACE activity) of a patient from that of a DD individual to that of an ID or II individual. Thus, ACE inhibitors might lower the risk of developing ARDS in an at-risk group or reduce the severity of disease and improve outcome in those with established lung injury. Knowing the genotype of a patient could also help target therapy, such that only patients with a D allele or DD genotype are given an ACE inhibitor to prevent the onset of ARDS. Further study is required to delineate the precise mechanisms involved and, potentially, to facilitate the design of effective clinical trials.
In conclusion, we provide the first evidence of a genetic influence in ARDS, suggesting an important role for ACE. Genetic studies in patients with this syndrome are likely to be of value in elucidating mechanisms of lung injury and in identifying new therapeutic targets.
:
The authors are grateful to the patients and staff of the UCLH and Middlesex Intensive Care Units for their participation in this study.
Supported by the Wellcome Trust, the British Heart Foundation, and the Medical Research Council. S. W. is an Oliver Prenn Research Fellow.
| 1. | Nelson S, Belknap SM, Carlson RW, Dale D, DeBoisblanc B, Farkas S, Fotheringham N, Ho H, Marrie T, Movahhed H, et al. A randomized controlled trial of filgrastim as an adjunct to antibiotics for treatment of hospitalized patients with community-acquired pneumonia. CAP Study Group. J Infect Dis 1998;178:1075–1080. |
| 2. | Campbell DJ, Kladis A, Valentijn AJ. Effects of Losartan on angiotensin and bradykinin peptides and angiotensin converting enzyme. J Cardiovasc Pharmacol 1995;26:233–240. |
| 3. | Yamamoto T, Wang L, Shimakura K, Sanaka M, Koike Y, Mineshita S. Angiotensin II-induced pulmonary edema in a rabbit model. Jpn J Pharmacol 1997;73:33–40. |
| 4. | Kiely DG, Cargill RI, Wheeldon NM, Coutie WJ, Lipworth BJ. Haemodynamic and endocrine effects of type 1 angiotensin II receptor blockade in patients with hypoxaemic cor pulmonale. Cardiovasc Res 1997; 33:201–208. |
| 5. | Marshall RP, Puddicombe A, Cookson WO, Laurent GJ. Adult familial cryptogenic fibrosing alveolitis in the UK. Thorax 2000;55:143–146. |
| 6. | Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, Uhal BD. Angiotensin II induces apoptosis in human and rat alveolar epithelial cells. Am J Physiol 1999;276:L885–L889. |
| 7. | Fourrier F, Chopin C, Wallaert B, Mazurier C, Mangalaboyi J, Durocher A. Compared evolution of plasma fibronectin and angiotensin-converting enzyme levels in septic ARDS. Chest 1985;87:191–195. |
| 8. | Idell S, Kueppers F, Lippmann M, Rosen H, Niederman M, Fein A. Angiotensin converting enzyme in bronchoalveolar lavage in ARDS. Chest 1987;91:52–56. |
| 9. | Wenz M, Steinau R, Gerlach H, Lange M, Kaczmarczyk G. Inhaled nitric oxide does not change transpulmonary angiotensin II formation in patients with acute respiratory distress syndrome. Chest 1997;112:478–483. |
| 10. | Wiberg-Jorgensen F, Klausen NO, Hald A, Qvist J, Giese J, Damkjaer NM. Pulmonary angiotensin II production in respiratory failure. Clin Physiol 1983;3:59–67. |
| 11. | Brizio-Molteni L, Piano G, Warpeha RL, Solliday NH, Molteni A, Angelats J, Lewis N, Patejak-Radwanski H. Angiotensin-1-converting enzyme activity as index of pulmonary damage in thermal injury with or without smoke inhalation. Ann Clin Lab Sci 1992;22:1–10. |
| 12. | Cambien F, Alhenc-Gelas F, Herbeth B, Andre JL, Rakotovao R, Gonzales MF, Allegrini J, Bloch C. Familial resemblance of plasma angiotensin-converting enzyme level: the Nancy Study. Am J Hum Genet 1988;43:774–780. |
| 13. | Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin-1-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990;86:1343–1346. |
| 14. | Tiret L, Rigat B, Visvikis S. Evidence from combined segregation analysis that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels. Am J Hum Gen 1992;51:197–205. |
| 15. | Costerousse O, Allegrini J, Lopez M, Alhenc-Gelas F. Angiotensin-I converting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in T-lymphocytes. Biochem J 1993;290:33–40. |
| 16. | Keavney B, McKenzie CA, Connell JM, Julier C, Ratcliffe PJ, Sobel E, Lathrop M, Farrall M. Measured haplotype analysis of the angiotensin-I converting enzyme gene. Hum Mol Genet 1998;7:1745–1751. |
| 17. | Furuya K, Yamaguchi E, Itoh A, Hizawa N, Ohnuma N, Kojima J, Kodama N, Kawakami Y. Deletion polymorphism in the angiotensin I converting enzyme (ACE) gene as a genetic risk factor for sarcoidosis. Thorax 1996;51:777–780. |
| 18. | Benessiano J, Crestani B, Mestari F, Klouche W, Neukrich F, Hacein-Bey S, Genevieve D, Aubier M. High frequency of a deletion polymorphism of the angiotensin-converting enzyme gene in asthma. J Allergy Clin Immunol 1997;99:53–57. |
| 19. | Maier LA, Raynolds MV, Young DA, Barker EA, Newman LS. Angiotensin-1 converting enzyme polymorphisms in chronic beryllium disease. Am J Respir Crit Care Med 1999;159:1342–1350. |
| 20. | Tomita H, Ina Y, Sugiura Y, Sato S, Kawaguchi H, Morishita M, Yamamoto M, Ueda R. Polymorphism in the angiotensin-converting enzyme (ACE) gene and sarcoidosis. Am J Respir Crit Care Med 1997; 156:255–259. |
| 21. | Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, Lamy M, Legall JR, Morris A, Spragg R. The American-European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994;149:818–824. |
| 22. | Knaus WA, Draper EA, Wagner DP, Zimmerman JE. APACHE II: a severity of disease classification system. Crit Care Med 1985;13:818–829. |
| 23. | Le Gall JR, Lemeshow S, Saulnier F. A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA 1993;270:2957–2963. |
| 24. | Montgomery H, Clarkson P, Barnard M, Bell J, Brynes A, Dollery C, Hajnal J, Hemingway H, Mercer D, Jarman P, et al. Angiotensin-converting-enzyme gene insertion/deletion polymorphism and response to physical training. Lancet 1999;353:541–545. |
| 25. | Song L, Wang D, Cui X, Shi Z, Yang H. Kinetic alterations of angiotensin-II and nitric oxide in radiation pulmonary fibrosis. J Environ Pathol Toxicol Oncol 1998;17:141–150. |
| 26. | Wang R, Ramos C, Joshi I, Zagariya A, Pardo A, Selman M, Uhal BD. Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides. Am J Physiol 1999;277: L1158–L1164. |
| 27. | Cargill RI, Lipworth BJ. Lisinopril attenuates acute hypoxic pulmonary vasoconstriction in humans. Chest 1996;109:424–429. |
| 28. | Xu ZH, Shimakura K, Yamamoto T, Wang LM, Mineshita S. Pulmonary edema induced by angiotensin I in rats. Jpn J Pharmacol 1998;76:51–56. |
| 29. | Chesnutt AN, Matthay MA, Tibayan FA, Clark JG. Early detection of type III procollagen peptide in acute lung injury: pathogenetic and prognostic significance. Am J Respir Crit Care Med 1997;156:840–845. |
| 30. | Molteni A, Ward WF, Ts'ao C, Solliday NH, Dunne M. Monocrotaline-induced pulmonary fibrosis in rats: amelioration by captopril and penicillamine. Proc Soc Exp Biol Med 1985;180:112–120. |
| 31. | Ward WF, Molteni A, Ts'ao C, Hinz JM. Captopril reduces collagen and mast cell accumulation in irradiated lung. Int J Radiat Oncol Biol Phys 1990;19:1405–1409. |
| 32. | Guazzi M, Agostoni P. Angiotensin-converting enzyme inhibition restores the diffusing capacity for carbon monoxide in patients with chronic heart failure by improving the molecular diffusion across the alveolar capillary membrane. Clin Sci (Colch) 1999;96:17–22. |
| 33. | Suzuki M, Teramoto S, Katayama H, Ohga E, Matsuse T, Ouchi Y. Effects of angiotensin-converting enzyme (ACE) inhibitors on oxygen radical production and generation by murine lung alveolar macrophages. J Asthma 1999;36:665–670. |
| 34. | Watanabe T, Yanagishita T, Konno N, Geshi E, Katagiri T. Reversal of early metabolic dysfunction in hypertensive rat left-ventricular myocytes by angiotensin-converting enzyme inhibition. Jpn Heart J 1997; 38:503–514. |
| 35. | Gvozdjakova A, Simko F, Kucharska J, Braunova Z, Psenek P, Kyselovic J. Captopril increased mitochondrial coenzyme Q10 level, improved respiratory chain function and energy production in the left ventricle in rabbits with smoke mitochondrial cardiomyopathy. Biofactors 1999;10:61–65. |
| 36. | Dietze GJ, Wicklmayr M, Rett K, Jacob S, Henriksen EJ. Potential role of bradykinin in forearm muscle metabolism in humans. Diabetes 1996;45:S110–S114. |
| 37. | Johnson AR, Erdos EG. Metabolism of vasoactive peptides by human endothelial cells in culture: angiotensin I converting enzyme (kininase II) and angiotensinase. J Clin Invest 1977;59:684–695. |
| 38. | Skidgel RA, Erdos EG. Cellular carboxypeptidases. Immunol Rev 1998; 161:129–141. |
| 39. | Bonnardeaux A, Davies E, Jeunemaitre X, Fery I, Charru A, Clauser E, Tiret L, Cambien F, Corvol P, Soubrier F. Angiotensin II type 1 receptor gene polymorphisms in human essential hypertension. Hypertension 1994;24:63–69. |
| 40. | Braun A, Kammerer S, Bohme E, Muller B, Roscher AA. Identification of polymorphic sites of the human bradykinin B2 receptor gene. Biochem Biophys Res Commun 1995;211:234–240. |
| 41. | Caulfield M, Lavender P, Farrall M, Munroe P, Lawson M, Turner P, Clark AJ. Linkage of the angiotensinogen gene to essential hypertension. N Engl J Med 1994;330:1629–1633. |
