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Pulmonary capillary endothelial metabolic dysfunctionSeverity in pulmonary arterial hypertension related to connective tissue disease versus idiopathic pulmonary arterial hypertension.

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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.
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;
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.
Submitted for publication July 17, 2007; accepted in revised
form December 14, 2007.
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
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
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.
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
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
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
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
body surface area (BSA), with results expressed as the FCSA/
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
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
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
Table 1.
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)
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‡
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‡
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 ⬍
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.
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 ⬍
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.
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
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
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).
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).
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
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-
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
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.
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
Christina Sotiropoulou for providing statistical expertise and
for performing part of the statistical analysis.
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.
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