Pulmonary capillary endothelial metabolic dysfunctionSeverity in pulmonary arterial hypertension related to connective tissue disease versus idiopathic pulmonary arterial hypertension.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 58, No. 4, April 2008, pp 1156–1164 DOI 10.1002/art.23405 © 2008, American College of Rheumatology Pulmonary Capillary Endothelial Metabolic Dysfunction Severity in Pulmonary Arterial Hypertension Related to Connective Tissue Disease Versus Idiopathic Pulmonary Arterial Hypertension David Langleben,1 Stylianos E. Orfanos,2 Michele Giovinazzo,1 Andrew Hirsch,1 Murray Baron,1 Jean-Luc Senécal,3 Apostolos Armaganidis,2 and John D. Catravas4 Objective. Pulmonary endothelial dysfunction is intertwined with the development and progression of pulmonary arterial hypertension (PAH). Pulmonary endothelium is an active metabolic tissue in healthy human subjects. This study was undertaken to determine the effects of PAH on pulmonary endothelial angiotensin-converting enzyme (ACE) activity and to identify differences between common PAH types, i.e., PAH related to connective tissue disease (PAH-CTD) versus idiopathic PAH (IPAH). Methods. Nineteen patients with PAH-CTD, 25 patients with IPAH, and 23 control subjects were evaluated. The single-pass transpulmonary percent metabolism (%M) and hydrolysis (both reflecting enzyme activity per capillary) of an ACE synthetic substrate were determined. In addition, the calculated functional capillary surface area (FCSA), normalized to body surface area (BSA), was determined. Results. The %M values in patients with PAHCTD (mean ⴞ SEM 53.6 ⴞ 3.6%) were significantly reduced compared with those in control subjects (P < 0.01) and those in patients with IPAH (P < 0.03), but were similar between the IPAH and control groups (mean ⴞ SEM 66.2 ⴞ 3.6% and 74.7 ⴞ 2.7%, respectively). Substrate hydrolysis was also significantly reduced in patients with PAH-CTD. The FCSA/BSA was significantly reduced in patients with PAH-CTD (mean ⴞ SEM 1,068 ⴞ 118 ml/minute/m2) and in patients with IPAH (1,443 ⴞ 186 ml/minute/m2) compared with that in controls (2,948 ⴞ 245 ml/minute/m2; P < 0.01 for both). At a given cardiac index, the FCSA/BSA tended to be lower in the PAH-CTD group than in the IPAH group. Moreover, unlike in IPAH, a linear relationship between the FCSA/BSA and the diffusing capacity for carbon monoxide (DLCO) was observed in PAH-CTD (r ⴝ 0.54, P < 0.03). Conclusion. The metabolically functional pulmonary capillary bed appears to be reduced to an equal extent in PAH-CTD and IPAH. However, %M and hydrolysis appear to be reduced in PAH-CTD but not in IPAH, reflecting relatively diminished ACE activity on the pulmonary capillary endothelial cells of patients with PAH-CTD, and showing that pulmonary endothelial metabolic function differs between PAH types. This study also provides the first functional evidence that a Supported by the Bank of Montreal Center for the Study of Heart Disease in Women at Jewish General Hospital. Dr. Langleben’s work was supported by the Quebec Lung Association and the Canadian Institutes for Health Research (grants MOP-42476 and MOP67145); he is a Fonds de la Recherche en Sante du Quebec Senior Clinical Research Scholar. Dr. Orfanos’ work was supported by the University of Athens (grant 70/4/3517) and the Thorax Foundation. Dr. Baron’s work was supported by the Canadian Institutes for Health Research (grants M0P-82740 and SKI-83345); he is Director of the Canadian Scleroderma Research Group. Dr. Senécal’s work was supported by the Canadian Institutes for Health Research (grants MOP-68966 and MOP-159996) and Sclérodermie Québec; he holds the University of Montreal Scleroderma Research Chair. 1 David Langleben, MD, Michele Giovinazzo, BSc, Andrew Hirsch, MD, Murray Baron, MD: Sir Mortimer B. Davis Jewish General Hospital, and McGill University, Montreal, Quebec, Canada; 2 Stylianos E. Orfanos, MD, PhD, Apostolos Armaganidis, MD, PhD: University of Athens Medical School, and Attikon Hospital, AthensHaidari, Greece; 3Jean-Luc Senécal, MD: Hôpital Notre Dame du Centre Hospitalier de l’Université de Montréal, Montreal, Quebec, Canada; 4John D. Catravas, PhD: Medical College of Georgia, Augusta. Drs. Langleben and Orfanos contributed equally to this work. Dr. Langleben has received speaking fees, consulting fees, and research funding (less than $10,000 from each company) from Northern Therapeutics and Encysive. Address correspondence and reprint requests to David Langleben, MD, Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote St. Catherine Road, Montreal, Quebec H3T 1E2, Canada. E-mail: email@example.com. Submitted for publication July 17, 2007; accepted in revised form December 14, 2007. 1156 PULMONARY CAPILLARY ENDOTHELIAL METABOLIC DYSFUNCTION IN PAH TYPES reduced DLCO value in patients with PAH-CTD is related to the degree of FCSA loss. Pulmonary arterial hypertension (PAH) remains an incompletely understood and difficult clinical problem. Its pathogenesis includes vascular remodeling and obliteration, as well as variable components of vasoconstriction, thrombosis, and inflammation (1). The result is a progressive loss of vascular surface area, leading to a progressive increase in pulmonary vascular resistance and a relentless downhill course in untreated patients. Endothelial cell dysfunction is manifested as excessive proliferation and altered production of endothelial products, such as prostacyclin, nitric oxide, and endothelin 1 (1). The human lung microcirculation has a large capillary surface area and a high metabolic capacity (2,3). The entire cardiac output passes through this system, and a variety of circulating mediators are either cleared or metabolized before the blood reenters the systemic circulation (3). The surface of the capillary endothelium is the major site for conversion of angiotensin I to angiotensin II by the pulmonary capillary endothelium–bound (PCEB) angiotensin-converting enzyme (ACE) (PCEB-ACE; kininase II, EC 18.104.22.168). The fact that PCEB-ACE is uniformly distributed along the luminal endothelial surface suggests that its measurement might permit assessment of the functional capillary surface area (FCSA) in healthy and diseased lungs (4–7). Indeed, using the injection of a hemodynamically inactive radiolabeled tripeptide, 3H-benzoyl-Phe-AlaPro (3H-BPAP), that is specifically metabolized by ACE, we were able to measure the single-pass transpulmonary percent metabolism (%M) and hydrolysis of the substrate, which are reflections of enzyme activity per perfused microvessel. In addition, we were able to calculate the FCSA, which reflects ACE activity in the entire vascular bed (i.e., the total lung). We have previously validated measurements of PCEB-ACE activity and FCSA in subjects with healthy lungs, in patients with systemic sclerosis but no PAH, and in patients with acute lung injury (8–10). We have also previously evaluated the characteristics of PAH in a small number of patients, in order to demonstrate that reductions in PCEB-ACE activity are within the range of detection by this technique (8). There has, however, been no description of larger cohorts of patients with PAH and no analysis of variation between the types of PAH. Moreover, the relationship of loss of PCEB-ACE activity to hemodynamics or to the diffusing capacity for carbon 1157 monoxide (DLCO) has not been previously examined in patients with PAH. In the present study, we provide the first description of PCEB-ACE functional abnormalities in patient populations comprising 2 common types of PAH, the PAH related to connective tissue disease (PAH-CTD) and idiopathic PAH (IPAH). When the classification of PAH was initially developed, the inclusion of idiopathic disease and that related to CTD, as well as other etiologic types, into the overall PAH category was based on a presumption of similar histologic features and pathophysiologic mechanisms despite differing natural histories (11,12). However, the existence of differences in pulmonary endothelial function between the PAH etiologic types remains a possibility, and we therefore hypothesized that PCEB-ACE activity might differ between common PAH types. Furthermore, preserved pulmonary capillary endothelial ACE immunoreactivity and expression have previously been described in patients with IPAH, but the functional significance of this finding was unknown (13,14). Our data provide evidence that these preserved ACE levels are metabolically active. PATIENTS AND METHODS Subjects. The present study was conducted in compliance with the Helsinki Declaration. All subjects gave written informed consent in a protocol approved by the Hospital Research Ethics Committees. Nineteen patients with PAHCTD (3 with diffuse systemic sclerosis, 9 with limited systemic sclerosis, 1 with systemic lupus erythematosus, 2 with mixed CTD, 1 with polymyositis, and 3 with rheumatoid arthritis) (15) and 25 patients with IPAH were studied. Partial data from 2 of the patients with IPAH and 3 of the patients with PAH-CTD were presented briefly as part of our initial validation study (8). The diagnoses were established and all patients were evaluated according to the recommendations of the third World Symposium on Pulmonary Arterial Hypertension (11). All patients with PAH had a total lung capacity of more than 70% of predicted and had undetectable or mild interstitial disease on high-resolution computerized tomography of the lungs. For comparison purposes, the data from patients with PAH were compared with those from a concurrently collected control group of subjects undergoing elective cardiac catheterization for evaluation of coronary artery disease. The results for this control group have been published previously, in other studies in which these subjects also served as controls (8,9). No subject had a patent foramen ovale or anatomic shunt, as assessed by 2-dimensional Doppler echocardiography with the use of agitated saline as a contrast agent. Patients with PAH had not received ACE inhibitors or angiotensin II receptor antagonists, nor had they been exposed to prostaglandin, endothelin receptor antagonist, or phosphodiesterase inhibitor therapy for PAH. Catheterization and injection technique. Femoral venous and femoral arterial sheaths were inserted as part of the 1158 routine cardiac catheterization procedure. A catheter was advanced into the pulmonary artery. For the determination of PCEB-ACE activity, all patients received a bolus injection containing trace amounts of 3H-BPAP, through the pulmonary artery catheter into the right atrium. Cardiac output was determined using either the thermodilution technique or the Fick technique, immediately prior to injection of the substrate. Determination of PCEB-ACE activity. Using indicatordilution–type techniques under first-order reaction conditions, we estimated the %M and the hydrolysis of BPAP and calculated the kinetic parameters of this reaction. This method provides estimates of pulmonary endothelial ACE activity in the microvessels, which is almost exclusively capillary-based (i.e., PCEB-ACE activity) (8,16,17). One milliliter of a 3H-BPAP solution (30 Ci/1.2 ml of 0.9% saline, or 22.2 Ci/mmole) was used for each injection, followed by 5 ml of 0.9% saline flush. Simultaneously, femoral arterial blood was withdrawn through the sheath using a peristaltic pump (40 ml/minute; Cole-Palmer, Montreal, Quebec, Canada) into a fraction collector (Gilson, Middleton, WI), advancing at 1 tube per 1.2 seconds (0.8 ml blood/tube, n ⫽ 39 tubes). The tubes contained 1.75 ml of 0.9% saline with 5 mmoles/liter EDTA and 6.8 mmoles/liter 8-hydroxyquinoline 5-sulfonic acid to inhibit further activity of ACE in the blood, and heparin at 1,000 IU/liter. The samples were centrifuged (3,000 revolutions per minute for 10 minutes), and 0.5 ml of the supernatant was transferred into a scintillation vial containing 5 ml Ecolite. The total 3H-radiolabeled enzyme activity was then measured. To determine the radioactivity associated with metabolites, another 0.5 ml of the supernatant was transferred into a scintillation vial containing 2.5 ml HCl (0.12N). After the addition of 3 ml of 0.4% Omnifluor in toluene, with mixing by inversion, the 3H-radiolabeled enzyme activity (with 3Htoluene) was measured, after 48 hours of undisturbed equilibration in the dark. Slight technical differences, which did not alter the measurements, occurred for some of the controls (9). Using this technique, ⬃60% of the 3H-BPAP metabolite 3 H-benzoyl-Phe and ⱕ13% of the parent 3H-BPAP were extracted in the organic phase of the mixture (i.e., with toluene). The precise values were calculated by identically processing separate standard tubes containing either substrate or previously synthesized product. Estimations of enzyme indices. The methods applied to determine enzyme activity have previously been described in detail (8). PCEB-ACE activity was estimated as the %M of 3 H-BPAP, as described previously (8). In addition, the transpulmonary hydrolysis (v) was calculated as follows: v ⫽ [E] ⫻ tc ⫻ kcat/Km, where [E] is the enzyme concentration available for reaction, tc is the capillary transit time (i.e., enzyme-substrate reaction time), kcat is the catalytic rate constant, and Km is the Michaelis-Menten constant. To further assess PCEB-ACE activity, the data were analyzed using the integrated Henri-Michaelis-Menten equation as modified by Catravas and White (18). The FCSA (9) (originally termed Amax/Km [8,10]) was calculated as follows: Amax/Km ⫽ E ⫻ kcat/Km ⫽ PPF ⫻ v, where E is the total enzyme mass available for reaction and PPF is the pulmonary plasma flow (determined as cardiac output ⫻ [1 ⫺ hematocrit]). The FCSA was then normalized to LANGLEBEN ET AL body surface area (BSA), with results expressed as the FCSA/ BSA. The hydrolysis and %M values reflect ACE activity per capillary, whereas the FCSA reflects ACE activity per vascular bed and is a measure of the dynamically perfused (i.e., accessible to substrate) capillary bed in healthy human subjects. In a disease state that involves the lung vasculature, such as PAH, in which there are possible alterations in enzyme expression and/or kinetic constants, the FCSA is proportional to the enzyme mass available for reaction (calculated as dynamically perfused capillary bed ⫻ enzyme mass expressed on the endothelial surface) and the enzyme kinetic constants (8,9). Statistical analysis. Variables studied included clinical and hemodynamic characteristics, single-breath DLCO, hydrolysis and %M of 3H-BPAP, and FCSA/BSA. Group summary data are presented as the mean ⫾ SEM. For normally distributed data, comparisons of group data were performed using one-way analysis of variance (ANOVA), followed, where appropriate, by the Tukey-Kramer multiple comparison test. For nonnormally distributed data, the Kruskal-Wallis ANOVA test and, where appropriate, the Kruskal-Wallis multiple comparison Z-value test were performed. Correlations were assessed using least squares regression analysis, with determination of the Pearson’s r and, where appropriate, the Spearman’s rho, as well as their corresponding P values. To detect potential ageor sex-related effects on the observed differences in ACE metabolism between the PAH groups, a two-way analysis of covariance (ANCOVA) was performed, adjusting for age and sex, followed by the Bonferroni multiple comparison test. All P values were 2-sided, and P values less than 0.05 were considered significant. To detect a 10% absolute difference in mean %M values between the control and PAH groups, and to have a two-sided error probability (␣) of 0.05 and a probability of correctly rejecting the null hypothesis (power) of 0.8, it was determined that each experimental group (e.g., IPAH) would require a sample size of at least 25 patients (19). RESULTS Demographic and clinical characteristics of the subjects (Table 1). Distribution of the sexes differed between the groups. As has previously been described for patients with PAH, both PAH groups had a predominance of women, with 95% of the patients with PAHCTD and 76% of the patients with IPAH being female. Given that control subjects were being screened for coronary artery disease, the group comprised predominantly men, with only 43% of subjects being female. The mean age of the patients with IPAH was lower than that of the patients with PAH-CTD or that of the controls. There were no differences in the mean systemic blood pressure between the groups. Although there was a trend toward a higher mean right atrial pressure in both PAH groups as compared with the controls, this was only statistically significantly PULMONARY CAPILLARY ENDOTHELIAL METABOLIC DYSFUNCTION IN PAH TYPES Table 1. 1159 Demographic and clinical characteristics of the patient and control groups* Sex, no. male/no. female Age, years mSAP, mm Hg mRAP, mm Hg mPAP, mm Hg mPAWP, mm Hg Cardiac output, liters/minute SVR, dynes/second/cm5 PVR, dynes/second/cm5 IPAH (n ⫽ 25) PAH-CTD (n ⫽ 19) Control (n ⫽ 23) 6/19 44.8 ⫾ 2.7† 96.6 ⫾ 2.7 10.8 ⫾ 1.6‡ 56.5 ⫾ 3.1† 9.1 ⫾ 3.7 3.42 ⫾ 0.24‡ 2,216 ⫾ 152‡ 1,200 ⫾ 136‡ 1/18 55.2 ⫾ 2.9 97.2 ⫾ 2.7 9.1 ⫾ 1.5 47.0 ⫾ 2.3‡ 8.7 ⫾ 3.8 3.55 ⫾ 0.24‡ 2,088 ⫾ 120‡ 936 ⫾ 80‡ 13/10 57.6 ⫾ 2.6 91.9 ⫾ 2.6 5.1 ⫾ 0.7 17.0 ⫾ 0.9 10.1 ⫾ 4.3 5.86 ⫾ 0.25 119 ⫾ 272 96 ⫾ 8 * Except where indicated otherwise, values are the mean ⫾ SEM. IPAH ⫽ idiopathic pulmonary arterial hypertension; PAH-CTD ⫽ pulmonary arterial hypertension related to connective tissue disease; mSAP ⫽ mean systemic arterial pressure; mRAP ⫽ mean right atrial pressure; mPAP ⫽ mean pulmonary arterial pressure; mPAWP ⫽ mean pulmonary artery wedge pressure; SVR ⫽ systemic vascular resistance; PVR ⫽ pulmonary vascular resistance. † P ⬍ 0.05 versus PAH-CTD and control groups. ‡ P ⬍ 0.05 versus control group. § P ⬍ 0.05 versus PAH-CTD group. different between the patients with IPAH and the controls. The mean pulmonary artery wedge pressure was similar among all 3 groups. Both the IPAH and PAH-CTD groups had significant increases in the mean pulmonary arterial pressure, pulmonary vascular resistance, and systemic vascular resistance as compared with the values in the control group. The PAH-CTD group had a significantly lower mean pulmonary arterial pressure and showed a trend toward lower pulmonary vascular resistance as compared with the values in the IPAH group. There were no significant differences in the degree of hemodynamic or pulmonary function test abnormalities between the patients with limited systemic sclerosis and those with diffuse systemic sclerosis, although the numbers of patients for comparison were small (data not shown). None of the patients with diffuse systemic sclerosis had clinically evident renal involvement. The duration of symptoms was similar between patients with IPAH and those with PAH-CTD. Substrate metabolism and hydrolysis (Figure 1). As compared with the mean %M of BPAP in the control group (mean ⫾ SEM 74.7 ⫾ 2.7%), that in the IPAH group (66.2 ⫾ 3.6%) was not significantly different. However, patients in the PAH-CTD group had a significantly reduced mean %M (53.6 ⫾ 3.6%) as compared with that in the control group (P ⬍ 0.01). Moreover, the mean %M in the PAH-CTD group was significantly reduced as compared with that in the IPAH group (P ⬍ 0.03). This pattern was also seen for the measurement Figure 1. Single-pass transpulmonary percent metabolism (A) and hydrolysis (v) (B) of 3H-benzoyl-Phe-Ala-Pro (BPAP) in the 3 groups. Symbols represent individual subjects; bars show the mean ⫾ SEM for each group. ⴱ ⫽ P ⬍ 0.05 versus the other groups. IPAH ⫽ idiopathic pulmonary arterial hypertension; PAH-CTD ⫽ pulmonary arterial hypertension related to connective tissue disease. 1160 of BPAP hydrolysis. The mean value in the IPAH group (mean ⫾ SEM 1.26 ⫾ 0.13) did not differ significantly from that in the control group (1.51 ⫾ 0.11). However, the mean hydrolysis of BPAP in the PAH-CTD group (0.82 ⫾ 0.08) was reduced as compared with that in the controls (P ⬍ 0.01) and that in the IPAH group (P ⬍ 0.03). When the 12 patients with systemic sclerosis (diffuse or limited) were analyzed, both the mean ⫾ SEM %M (53.3 ⫾ 4.4%) and the mean ⫾ SEM BPAP hydrolysis (0.81 ⫾ 0.09) were lower than corresponding control levels (P ⬍ 0.01). By two-way ANCOVA, adjusting for age and sex, the differences in metabolism between the PAH groups (P ⫽ 0.03 for PAH-CTD versus IPAH) and between PAH-CTD and controls were conserved, indicating that the observed differences in metabolic activity among the groups were independent of the effects of age or sex. Functional capillary surface area (Figure 2). As might be expected in a disease that involves vascular remodeling and narrowing, the FCSA available for BPAP hydrolysis was decreased. As compared with that in controls (mean ⫾ SEM 2,948 ⫾ 245 ml/minute/m2), the mean ⫾ SEM FCSA/BSA in the PAH-CTD group (1,068 ⫾ 118 ml/minute/m2) was reduced to 36% of control levels (P ⬍ 0.01), and that in the IPAH group (1,443 ⫾ 186 ml/minute/m2) was decreased to 49% of control levels (P ⬍ 0.01). There was no significant difference in the mean FCSA/BSA between the PAHCTD group and the IPAH group. When the 12 patients with systemic sclerosis (diffuse or limited) were analyzed, the mean ⫾ SEM FCSA/BSA was 993 ⫾ 134 ml/minute/m2 (P ⬍ 0.01 versus controls). Figure 2. Functional capillary surface area normalized to body surface area (FCSA/BSA) in the 3 groups. Symbols represent individual subjects; bars show the mean ⫾ SEM for each group. ⴱ ⫽ P ⬍ 0.05 versus controls. See Figure 1 for other definitions. LANGLEBEN ET AL Figure 3. Functional capillary surface area normalized to body surface area (FCSA/BSA) versus the cardiac index in the 3 groups. Symbols represent individual subjects in the IPAH group (red triangles), PAH-CTD group (blue circles), and control group (green inverted triangles). The broken line is the line of identity for the graph, and correlation lines for each group are shown in their respective colors. Significant correlations between the FCSA/BSA and the cardiac index are evident in all 3 groups. See Figure 1 for other definitions. Relationship of functional capillary surface area and metabolic parameters to blood flow (Figures 3 and 4). There were moderate linear relationships between the FCSA/BSA and the cardiac index (Figure 3) in the control group (r ⫽ 0.51, P ⫽ 0.01), in the patients with IPAH (rho ⫽ 0.54, P ⬍ 0.01), and in the patients with PAH-CTD (r ⫽ 0.61, P ⬍ 0.01). The correlation line for the control group (slope ⫽ 0.98) was close to the line of identity, while that for the IPAH group (slope ⫽ 0.86) was almost parallel to the control line. However, the slope for the PAH-CTD group (0.46) was flatter than that for the other 2 groups. No relationships were observed between BPAP hydrolysis or %M and the cardiac output in any of the 3 groups studied (Figure 4). Relationship of functional capillary surface area to DLCO (Figure 5). When the FCSA/BSA was compared with the DLCO (% predicted) in the 2 PAH groups, the group of patients with IPAH did not demonstrate a relationship between these parameters (rho ⫽ 0.36, P not significant). However, the group of patients with PAH-CTD showed a stronger and significant linear PULMONARY CAPILLARY ENDOTHELIAL METABOLIC DYSFUNCTION IN PAH TYPES 1161 vascular function. Although there was some overlap between the groups, our data showed significant differences in pulmonary endothelial metabolic function between 2 of the major types of PAH, with more dysfunction evident in the patients with PAH-CTD. The subset of patients with PAH related to systemic sclerosis did not differ from the larger heterogeneous PAH-CTD group in any of the metabolic parameters. The study was powered to detect differences in substrate %M in the IPAH group as compared with that in the control group. Despite this, the IPAH group did not differ from the control group in the mean %M or in the mean substrate hydrolysis, whereas differences were observed in the smaller group of patients with PAHCTD. Neither age nor sex affected these group differences. Both of these metabolic parameters are reflec- Figure 4. Single-pass transpulmonary percent metabolism (A) and hydrolysis (v) (B) of BPAP versus cardiac output in the 3 groups of subjects. Symbols represent individual subjects in the IPAH group (red triangles), PAH-CTD group (blue circles), and control group (green inverted triangles). Correlation lines for each group are shown in their respective colors. None of the relationships are significant. See Figure 1 for definitions. relationship between the FCSA/BSA and the DLCO (r ⫽ 0.54, P ⬍ 0.03). DISCUSSION This is the first study to examine pulmonary endothelial metabolism in populations of patients who were carefully characterized using the current criteria for PAH (20). The presumption that the various types of PAH have similar disease features and pathophysiologic mechanisms has permitted clinical study of combined cohorts, thus providing adequate patient numbers for therapeutic trials (11,12). Moreover, that presumption has been proven correct, in that a therapy that is effective in one type of PAH is often effective in many of the other types (21). However, this lumping of the various types of PAH might obscure differences in Figure 5. Functional capillary surface area normalized to body surface area (FCSA/BSA) versus the diffusing capacity for carbon monoxide (DLCO) (% predicted) in the group of patients with IPAH (red triangles) (A) and patients with PAH-CTD (blue circles) (B). Symbols represent individual patients. Correlation lines for each group are shown in their respective colors. A significant relationship is evident in the PAH-CTD group (B), but not in the IPAH group (A). NS ⫽ not significant (see Figure 1 for other definitions). 1162 tions of enzyme activity per perfused microvessel, normally representing the highly predominant metabolic activity by the capillary endothelium (22). Our data thus imply that PCEB-ACE activity (as expressed using the enzyme activity parameters in the equation described in Patients and Methods) is preserved. These findings provide the first functional confirmation that the previously described preserved pulmonary capillary ACE expression in IPAH is enzymatically active (13,14). Those same previous studies (13,14) also described increased ACE expression in preacinar and intraacinar pulmonary arteries in patients with IPAH as compared with controls, but vessels of more than 20 m in diameter do not appear to present sufficient surface area to contribute significantly to single-pass ACE substrate hydrolysis (16,17). The mechanisms of increased ACE expression in the more proximal vessels in relation to the pathogenesis of PAH remain unknown. Although ACE is thought to contribute to a variety of vascular diseases, including atherosclerosis, renal disease, and, potentially, PAH (1,13,14,16), our data do not allow us to elaborate on its relative effects in the various types of PAH. Contrary to the results in the IPAH group, the PAH-CTD group had reduced substrate hydrolysis and %M. This could be due to alterations in either substrateenzyme reaction time (i.e., capillary transit time) or enzyme concentrations and/or kinetic constants (as shown in the equation described in Patients and Methods). The reaction time does not change over a wide range of cardiac output in healthy human subjects (22) but could be altered in a diseased, remodeled vascular bed. In that case, however, substrate hydrolysis should be expected to be inversely related to cardiac output (10,22). Figure 4 shows no inverse relationship for the %M or hydrolysis versus cardiac output, suggesting that capillary transit time does not contribute to the difference. Thus, unlike in patients with IPAH, patients with PAH-CTD appear to exhibit enzyme activity alterations at the level of endothelial cells, which could be attributed to either a reduction in enzyme mass and/or decreased kinetic constants. These alterations denote endothelial dysfunction and indicate a true difference in pulmonary capillary endothelial ACE metabolic activity between the 2 PAH types. We have previously reported reduced pulmonary endothelial ACE activity in systemic sclerosis, even in the absence of PAH and/or interstitial lung disease (9). Thus, pulmonary endothelial metabolic dysfunction may be a frequent feature of systemic sclerosis, regardless of disease severity, and it likely contrib- LANGLEBEN ET AL utes to the pathogenesis of PAH in affected patients. It is a troubling, but consistent, observation that despite having similar hemodynamic abnormalities, patients with PAH-CTD have a worse prognosis than those with IPAH (23,24). This may be due, in part, to the systemic nature of CTD, but it might also be due, in part, to the aforementioned more severe endothelial dysfunction associated with a more aggressive vasculopathy in the pulmonary circulation. The FCSA available for reaction was found to be decreased in both PAH types. The equation described in Patients and Methods presents the factors upon which the FCSA is contingent. The reduced number of perfused capillaries resulting from precapillary vascular obstruction and obliteration (14,25,26) appears to be the principal cause of a decreased FCSA in patients with IPAH. In contrast, the altered endothelial enzyme mass and/or kinetic constants (as suggested above for PAHCTD) along with the reduced number of perfused capillaries appear to contribute to the FCSA loss seen in patients with PAH-CTD. Perfused pulmonary FCSA normally increases with increasing blood flow, by recruitment of underused vasculature; this relationship is explored in Figure 3. The line for the control group was quite close to the line of identity. This line is similar to those generated in animal models (22,27) and is likely representative of the true surface area–flow relationship in healthy human subjects under physiologic resting conditions. The line for the IPAH group was nearly parallel to the line for the controls, suggesting that although the lungs of patients with IPAH are operating with a reduced number of perfused functional capillaries, this number would be close to the number of capillaries that would be perfused in a healthy lung under low flow states. In contrast, the PAH-CTD group had a flatter slope, suggesting that at levels of blood flow that are closer to normal, less FCSA is available, due either to more extensive remodeling or to less ACE expression or activity. However, the hemodynamic abnormalities were, if anything, milder in those with PAH-CTD, which does not suggest worse remodeling as a factor. In addition, kinetic constants are not affected by hemodynamics in normal animals (7,28). If this were the case in the PAH-CTD group, lower endothelial ACE expression would be the likelier possibility. The single-breath DLCO is contingent upon having a normal alveolar capillary interface and a normal density of perfused capillaries available for diffusion (29). None of our patients had evidence of more than mild interstitial disease. In patients with CTD and PULMONARY CAPILLARY ENDOTHELIAL METABOLIC DYSFUNCTION IN PAH TYPES minimal or no interstitial lung disease, a reduction in the DLCO may indicate the onset of PAH, presumably due to loss of perfused microvessels (30,31). However, it has previously been impossible to test the relationship of capillary surface area loss to DLCO levels in vivo in humans with PAH and to examine differences between PAH types. Although abnormalities in the DLCO are well recognized in IPAH (32) and were seen in our patients with IPAH, we did not find a significant relationship in the IPAH group. In fact, patients with IPAH had a wide variance in the FCSA/BSA in relation to the DLCO. However, results from the PAH-CTD group showed a significant relationship between the FCSA/BSA and the DLCO, with a lower DLCO value being related to a lower FCSA/BSA. Thus, we provide the first direct in vivo confirmation that the reduction in DLCO seen in patients with PAH-CTD is at least partly related to loss of FCSA. In summary, the results of our study identify etiology-specific patterns of pulmonary capillary endothelial metabolic dysfunction in patients with PAH, with a greater degree of dysfunction in patients with PAHCTD, in whom a combination of reduced PCEB-ACE activity and FCSA loss occurs, in contrast to patients with IPAH, who seem to experience only loss of FCSA. Future studies, particularly in patients with the CTD variety of PAH, should explore whether detection of endothelial dysfunction early in the disease course can be used to predict subsequent development of accelerated PAH. The correlation of endothelial metabolic abnormalities as an index of endothelial dysfunction with the degree and duration of response to PAH therapy would also be of interest. We extend the findings of previous histologic studies of ACE expression in IPAH by demonstrating the functional significance of this feature, and provide in vivo confirmation of the relationship between the DLCO and the loss of FCSA in PAH-CTD. Our techniques should be considered as specific research tools rather than as widely useful clinical or diagnostic assays. They nevertheless provide quantifiable indices of pulmonary endothelial function, display functional differences between PAH etiologic types, and may be applicable to analysis of disease progression and response to therapy. ACKNOWLEDGMENTS We thank the physicians, nurses, and technicians of the cardiac catheterization laboratories and the staff of the coronary care and intensive care units of Jewish General Hospital for their patient and generous assistance. We also thank Ms 1163 Christina Sotiropoulou for providing statistical expertise and for performing part of the statistical analysis. AUTHOR CONTRIBUTIONS Drs. Langleben and Orfanos had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Langleben, Orfanos, Giovinazzo, Hirsch. Acquisition of data. Langleben, Orfanos, Giovinazzo, Hirsch, Baron, Senécal, Catravas. Analysis and interpretation of data. Langleben, Orfanos, Giovinazzo, Hirsch, Baron, Armaganidis, Catravas. Manuscript preparation. Langleben, Orfanos, Giovinazzo, Hirsch, Baron, Senécal, Armaganidis, Catravas. Statistical analysis. Langleben, Orfanos. REFERENCES 1. Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, Lang IM, et al. Cellular and molecular pathobiology of pulmonary arterial hypertension. J Am Coll Cardiol 2004;43: 13–24S. 2. Hassoun PM, Fanburg BL, Junod AF. Metabolic functions. In: Crystal RG, West JB, editors. The lung: scientific foundations. New York: Raven Press; 1991. p. 313–27. 3. Orfanos SE, Mavrommati I, Korovesi I, Roussos C. Pulmonary endothelium in acute lung injury: from basic science to the critically ill. Intensive Care Med 2004;30:1702–14. 4. Ryan JW, Catravas JD. Angiotensin converting enzyme as an indicator of pulmonary microvascular function. In: Hollinger MA, editor. Focus on pulmonary pharmacology and toxicology. Boca Raton (FL): CRC Press; 1991. p. 183–210. 5. Ryan JW, Ryan US. Processing of endogenous polypeptides by the lung [review]. Annu Rev Physiol 1982;44:241–55. 6. Ryan JW. Assay of pulmonary endothelial surface enzymes in vivo. In: Ryan US, editor. Pulmonary endothelium in health and disease. New York: Marcel Dekker; 1987. p. 161–88. 7. Orfanos SE, Chen XL, Ryan JW, Chung AY, Burch SE, Catravas JD. Assay of pulmonary microvascular endothelial angiotensinconverting enzyme in vivo: comparison of 3 probes. Toxicol Appl Pharmacol 1994;124:99–111. 8. Orfanos SE, Langleben D, Khoury J, Schlesinger RD, Dragatakis L, Roussos C, et al. Pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in humans. Circulation 1999;99:1593–9. 9. Orfanos SE, Psevdi E, Stratigis N, Langleben D, Catravas JD, Kyriakidis M, et al. Pulmonary capillary endothelial dysfunction in early systemic sclerosis. Arthritis Rheum 2001;44:902–11. 10. Orfanos SE, Armaganidis A, Glynos C, Psevdi E, Kaltsas P, Sarafidou P, et al. Pulmonary capillary endothelium-bound angiotensin-converting enzyme activity in acute lung injury. Circulation 2000;102:2011–8. 11. Simonneau G, Galie N, Rubin LJ, Langleben D, Seeger W, Dominghetti G, et al. Clinical classification of pulmonary hypertension. J Am Coll Cardiol 2004;43:5–12S. 12. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 2004;351:1425–31. 13. Schuster DP, Crouch EC, Parks WC, Johnson T, Botney MD. Angiotensin converting enzyme expression in primary pulmonary hypertension. Am J Respir Crit Care Med 1996;154:1087–91. 14. Orte C, Polak JM, Haworth SG, Yacoub MH, Morrell NW. Expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension. J Pathol 2000;192:379–84. 15. Scussel-Lonzetti L, Joyal F, Raynauld JP, Roussin A, Rich E, 1164 16. 17. 18. 19. 20. 21. 22. 23. Goulet JR, et al. Predicting mortality in systemic sclerosis: analysis of a cohort of 309 French Canadian patients with emphasis on features at diagnosis as predictive factors for survival. Medicine 2002;81:154–67. Catravas JD, Orfanos SE. Pathophysiologic functions of endothelial angiotensin converting enzyme. In: Born GV, Schwartz CJ, editors. Vascular endothelium: physiology, pathology and therapeutic options. Stuttgart: Schattauer; 1997. p. 193–204. Horvath IG, Cziraki A, Parkerson JB, Kahn SU, Catravas JD. Effect of acute coronary occlusion on the size of the dynamically perfused coronary capillary bed in the dog. Microvasc Res 1998; 56:95–103. Catravas JD, White RE. Kinetics of pulmonary angiotensinconverting enzyme and 5⬘ nucleotidase in vivo. J Appl Physiol 1984;57:1173–81. Dupont WD, Plummer WD Jr. Power and sample size calculations: a review and computer program. Control Clin Trials 1990; 11:116–28. Barst RJ, McGoon M, Torbicki A, Sitbon O, Krowka MJ, Olschewski H, et al. Diagnosis and differential assessment of pulmonary arterial hypertension. J Am Coll Cardiol 2004;43:40–7S. Badesch DB, Abman SH, Ahearn GS, Barst RJ, McCrory DC, Simonneau G, et al. Medical therapy for pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 2004;126:35–62S. Orfanos SE, Erhart IC, Barman S, Hofman WF, Catravas JD. Endothelial ectoenzyme assays estimate perfused capillary surface area in the dog lung. Microvasc Res 1997;54:145–55. McLaughlin VV, Presberg KW, Doyle RL, Abman SH, McRory DC, Fortin T, et al. Prognosis of pulmonary arterial hypertension: LANGLEBEN ET AL 24. 25. 26. 27. 28. 29. 30. 31. 32. ACCP evidence-based clinical practice guidelines. Chest 2004;126: 78–92. Kawut SM, Taichman DB, Archer-Chicko CL, Palevsky HI, Kimmel SE. Hemodynamics and survival in patients with pulmonary arterial hypertension related to systemic sclerosis. Chest 2003;123: 344–50. Qing F, McCarthy TJ, Markham J, Schuster DP. Pulmonary angiotensin-converting enzye (ACE) binding and inhibition in humans. Am J Respir Crit Care Med 2000;161:2019–25. Pietra GG, Capron F, Stewart S, Leone O, Humbert M, Robbins IM, et al. Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 2004;43:25–32S. Dupuis J, Goresky CA, Ryan JW, Rouleau JL, Bach GG. Pulmonary angiotensin-converting enzyme substrate hydrolysis during exercise. J Appl Physiol 1992;72:1868–86. Orfanos SE, Catravas JD. Metabolic functions of the pulmonary endothelium. In: Yacoub MH, Pepper J, editors. Annual review of cardiac surgery. London: Current Science; 1993. p. 52–9. Single-breath carbon monoxide diffusing capacity (transfer factor). Am J Respir Crit Care Med 1995;152:2185–98. Stupi AM, Steen VD, Owens GR, Barnes EL, Rodnan GP, Medsger TA Jr. Pulmonary hypertension in the CREST syndrome variant of systemic sclerosis. Arthritis Rheum 1986;29:515–24. Steen V, Medsger TA Jr. Predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited cutaneous involvement. Arthritis Rheum 2003;48:516–22. Sun XG, Hansen JE, Oudiz RJ, Wasserman K. Pulmonary function in primary pulmonary hypertension. J Am Coll Cardiol 2003;41:1028–35.