1) This study found that in a rat model of pulmonary hypertension (PH), female rats developed more pronounced thickening of small pulmonary arteries compared to male rats. However, only male rats showed infiltration of inflammatory cells in the small pulmonary arteries and associated fibrosis. 2) While peak right ventricular systolic pressure was similar between males and females, male rats had worse survival and more severe right ventricular fibrosis and dysfunction. 3) The results suggest that in PH, females experience greater pulmonary artery remodeling but better survival, while pulmonary and cardiac fibrosis in males leads to more severe right ventricular failure and lower survival rates.

1. O R I G I N A L R E S E A R C H
The sexual dimorphism associated with pulmonary hypertension
corresponds to a fibrotic phenotype
Olga Rafikova,1,a
Ruslan Rafikov,1,a
Mary Louise Meadows,1
Archana Kangath,1
Danny Jonigk,2
Stephen M. Black1
1
Pulmonary Vascular Disease Program, Vascular Biology Center, Medical College of Georgia, Georgia Regents University,
Augusta, Georgia, USA; 2
Institute of Pathology, Hannover Medical School, Hanover, Germany; a
These authors contributed
equally to this study.
Abstract: Although female predominance in the development of all types of pulmonary hypertension (PH)
is well established, many clinical studies have confirmed that females have better prognosis and higher sur-
vival rate than males. There is no clear explanation of why sex influences the pathogenesis and progression
of PH. Using a rat angioproliferative model of PH, which closely resembles the primary pathological changes
observed in humans, we evaluated the role of sex in the development and progression of PH. Female rats
had a more pronounced increase in medial thickness in the small pulmonary arteries. However, the infiltra-
tion of small pulmonary arteries by inflammatory cells was found only in male rats, and this corresponded to
increased myeloperoxidase activity and abundant adventitial and medial fibrosis that were not present in
female rats. Although the level of right ventricle (RV) peak systolic pressure was similar in both groups, the
survival rate in male rats was significantly lower. Moreover, male rats presented with a more pronounced
increase in RV thickness that correlated with diffuse RV fibrosis and significantly impaired right cardiac func-
tion. The reduction in fibrosis in female rats correlated with increased expression of caveolin-1 and reduced
endothelial nitric oxide synthase–derived superoxide. We conclude that, in the pathogenesis of PH, female
sex is associated with greater remodeling of the pulmonary arteries but greater survival. Conversely, in males,
the development of pulmonary and cardiac fibrosis leads to early and severe RV failure, and this may be an
important reason for the lower survival rate among males.
Keywords: caveolin-1, nitric oxide synthase, oxidative stress, superoxide.
Pulm Circ 2015;5(1):184-197. DOI: 10.1086/679724.
Pulmonary hypertension (PH) is characterized by the pro-
gressive elevation of pulmonary pressure, remodeling of
the pulmonary arteries (intimal and medial hypertrophy,
plexiform lesion formation, and fibrosis), and the eventual
development of right ventricle (RV) failure. Despite the ex-
pansion of pharmacological therapies in past decade, PH
leads to more than 15,000 deaths and more than 250,000
hospital visits per year.1,2
There is a strong sexual dimor-
phism in all types of PH, with the female-to-male ratio vary-
ing between 2 ∶ 1 and 4 ∶ 1.3,4
Female sex hormones and
their metabolites have been postulated to be responsible
for the development of PH in females.5,6
However, the
available animal data suggest that estrogens exert a protec-
tive effect on the pulmonary vasculature, in contradiction
with the well-documented female predominance in clini-
cal studies, known as the “estrogen paradox.”7,8
Estrogens
exert their effects through both genomic and nongenomic
signaling pathways and have been shown to suppress en-
dothelin 1 production,9
increase the production of nitric
oxide (NO) and prostacyclin, and attenuate both vasocon-
striction and vascular remodeling.10-12
At the same time,
estrogens have been shown to promote pulmonary vascu-
lar remodeling through increased expression of the angio-
genic factor, vascular endothelial growth factor (VEGF);13,14
induce the proliferation of vascular cells;15,16
and possess
prothrombotic properties,17,18
especially in conditions where
the vascular endothelium is damaged.19
Thus, overall, the
available data regarding the role of sex hormones in the
development of PH are conflicting.
In spite of the fact that females are more prone than
males to the development of PH, they have a better sur-
vival prognosis than males. Indeed, male sex has been re-
Address correspondence to Dr. Stephen M. Black, Vascular Biology Center, Georgia Regents University, 1459 Laney Walker Boulevard, CB-3211,
Augusta, GA 30912, USA. E-mail: sblack@gru.edu.
Submitted March 28, 2014; Accepted August 18, 2014; Electronically published February 10, 2015.
© 2015 by the Pulmonary Vascular Research Institute. All rights reserved. 2045-8932/2015/0501-0016. $15.00.

2. ported to be one of the primary independent predictors
of death.20
Recent analysis of the French, Spanish, and UK
registries and of US patients enrolled in the Registry to
Evaluate Early and Long-Term Pulmonary Arterial Hyper-
tension Disease Management (REVEAL) identified a nega-
tive prognosis associated with male sex.20-23
However, there
is no clear explanation of this observation. Because RV func-
tion is the primary determinant of survival in PH,24
it
is possible that the cardioprotective properties of female
sex hormones play a role in the increased survival rate of
females with PH. Indeed, sexual dimorphism in various
types of heart disease is well established,25
although the mo-
lecular mechanisms are considered to be complex and go
beyond the estrogen alone.25-28
Nevertheless, the determi-
nation of these mechanisms is critical to allow the devel-
opment of differential approaches to PH therapy in males
and females. Therefore, in this study, we used a rat angio-
proliferative model of severe PH, originally developed by
Voelkel and colleagues,29
the most representative model
of human PH, to study the role of sex in the development
and progression of PH.
MATERIAL AND METHODS
Animals
All experimental procedures were approved by Georgia
Regents University. Sprague Dawley rats (200–250 g) were
used in this study. All animals were obtained from Charles
River (Wilmington, MA) and were housed in the Georgia
Regents University animal care facility for at least 1 week
before being used in this experiment. Animals were kept
in a 12-hour light∶ dark cycle at an ambient temperature of
22°C and received standard rodent food and water ad lib.
Rat model of PH
We used the rat model of occlusive-angioproliferative PH
initially described by Voelkel and colleagues.29
As previ-
ously described, this model mimics many of the clinical
and histopathological changes seen in human PH, such
as occlusive remodeling of the small pulmonary arteries
with prominent medial hypertrophy (“concentric lesions”)
and formation of complex glomeruloid-like vascular struc-
tures (“plexiform lesions”), and it exhibits a progressive
disease character even after reexposure to normoxia.29,30
Before beginning the study, male and female rats were ran-
domly allocated to either the control group, which received
the vehicle for SU5614, or the SU group, which was in-
jected with a single dose of the VEGF receptor 2 antago-
nist, SU5416 (SU; 20 mg/kg SC). Rats were then placed in
Plexiglas chambers (BioSpherix, Redfield, NJ) for 4 weeks.
Levels of O2 were maintained at 10% ± 0.5%, and the cham-
bers were continuously flushed with nitrogen and oxygen
to maintain low CO2 concentrations (<0.5%). The O2 and
CO2 concentrations were continuously monitored (PROOX
110 BioSpherix oxygen controller and LB-2 CO2 analyzer,
Sensormedics). After 4 weeks, the animals were returned
to normoxic conditions for an additional 10 weeks. The con-
trol rats were kept under normoxic conditions for the du-
ration of the study (14 weeks).
Echocardiographic measurements
The Vevo 2100 imaging system (VisualSonics, Toronto,
Canada) was used to perform Doppler echocardiography.
Baseline parameters were determined on day 0 and then
every 4 weeks thereafter. For the Doppler echocardiogra-
phy analyses, rats were anesthesitized using an isoflu-
rane chamber and laid supine with all legs taped to electro-
cardiogram electrodes for monitoring heart rate. The rat’s
chest was treated with depilatory cream (Nair; Carter-Horner,
Mississauga, Ontario, Canada), and a warm ultrasound gel
was spread over the chest. Transthoracic two-dimensional,
M-mode, and Doppler imaging were performed using 25-
MHz transducer. RV free wall (RVFW) thickness was mea-
sured during end diastole using right parasternal long-axis
view. These images were obtained from the right side of
the rat, with the ultrasonic beam positioned perpendicu-
larly to the wall of the mid-third of the RV. Pulsed-wave
Doppler was used to measure pulmonary artery accelera-
tion time (PAAT). The Doppler sample volume was cen-
trally positioned within the main pulmonary artery, just
distal from the pulmonary valve with the beam oriented par-
allel to the flow. The echocardiographic parameters were
measured offline using tool section of the VisualSonic Vevo
2100 System by a reader blinded to the sex or treatment of
the rat. Measurements were averaged on 15 cardiac cycles.
Invasive hemodynamic measurements
Fourteen weeks after experiment setup, subsets of ani-
mals were anesthetized (Inactin, 100 mg/kg i.p.) and instru-
mented for measurement of RV and systemic blood pres-
sure. Briefly, a PE-240 polyethylene tube was inserted into
the trachea to facilitate breathing. A customized pressure
transducer catheter (SPR-513, Millar Instruments, Hous-
ton, TX), connected to a Millar Transducer Control Unit
TC-510 and PL3504 PowerLab 4/35 data acquisition sys-
tem (ADInstruments, Colorado Springs, CO), was inserted
into the RV via the right jugular vein and right atrium. For
systemic blood pressure measurement, the right carotid
artery was cannulated with a fluid-filled PE-50 polyethyl-
ene catheter connected to a blood pressure transducer
and, through a bridge amplifier, to the PowerLab system.
A 30-minute stabilization period was permitted before a
30-minute recording of RV pressure and of RV functional
Pulmonary Circulation Volume 5 Number 1 March 2015 | 185

3. parameters was conducted. At the end of pressure record-
ing, the trachea catheter was connected to a Harvard Ro-
dent Ventilator (Model 683; Harvard Apparatus, South Na-
tick, MA), the thorax was opened, and lungs were flushed
with saline (0.9% sodium chloride) via a needle inserted
into the RV to remove the blood from pulmonary vessels.
Animals were euthanized by anesthetic overdose, and heart
and lungs were dissected and weighed.
Assessment of RV hypertrophy
The RV free wall was separated from the left ventricle
(LV) and ventricular septum (S). RV hypertrophy was ex-
pressed as a wet weight of RV to LV + S (RV/LV + S). At
the end of analysis, RV and LV + S were immersed in
10% buffered formalin for at least 72 hours before being
embedded for histological analysis.
Histological and morphometric analysis
of lung vessels and RV
The left lung from all animals was fixed in formalin and
embedded in paraffin. A series of 4-μM tissue sections were
dewaxed and stained with hematoxylin and eosin or Mas-
son trichrome staining for histological and morphometric
assessment. For morphometric analysis of pulmonary ves-
sels (medial wall thickness), the pulmonary arteries were
categorized depending on the external diameter, as de-
scribed elsewhere.31
Category I included arteries with an
external diameter between 30 and 100 μm, and category II
included arteries with an external diameter between 101
and 200 μm. Ten transversely sectioned pulmonary arter-
ies within each category per each animal were digitally
captured at 40× using an Olympus IX70 microscope and
analyzed using CellSens Standard software. The total cross-
sectional vessel area (TA) and the lumen area (LA) were
measured, and vascular wall thickness was determined as
the percentage of vascular cross-section area (TA − LA)
from total vessel cross-sectional area. To assess the level of
fibrotic changes (collagen deposition) in pulmonary arteries
with external diameter 50–200 μm, the 4-μm pulmonary
tissue sections stained with Masson trichrome were ran-
domly scored by a blinded observer. Ten random images
from each animal were scaled from 0 to 3, with 0 = no
blue staining in pulmonary artery media (no fibrosis), 1 =
mild blue staining in pulmonary artery media (mild fibro-
sis), 2 = moderate blue staining in pulmonary artery me-
dia (developed pulmonary artery media fibrosis), and 3 =
strong pulmonary artery media blue staining (severe pul-
monary artery fibrosis with loss of smooth muscle cells).
To detect fibrosis in RV tissue, RV sections were stained
with Masson trichrome staining and scored as described
elsewhere32
in a blinded manner. The cardiac fibrosis score
was as follows: 0 = no fibrosis; 1 = localized small amount
of fibrosis; 2 = mild patch fibrosis; 3 = moderate diffuse
fibrosis; 4 = severe diffuse fibrosis.
Immunofluorescence
Lung paraffin sections (4 μm in width) were exposed to
60°C for 30 minutes, deparaffinized in xylene, and run
through graded alcohol to distilled water. Endogenous
peroxidase was quenched with 0.3% H2O2 for 5 min-
utes, followed by two rinses with distilled water. Slides
were pretreated with Target pH6 (Dako), rinsed in distilled
water, incubated in Power Block (Biogenex Laboratories),
rinsed in distilled water, placed in phosphate-buffered sa-
line (PBS) for 5 minutes, incubated with primary antibody
(anti-myeloperoxidase; Cappel ICN Pharmaceuticals) 1∶2,000
for 30 minutes at room temperature, followed by two rinses in
PBS, incubated with secondary peroxidase-labeled polymer
conjugated to goat anti-rabbit immunoglobulin G (Envision +,
Dako) for 30 minutes, and rinsed in PBS. Bound antibody was
detected with the DAB+ substrate kit, (Dako). Hematoxylin
was used for counter stain.
Myeloperoxidase activity in peripheral lung tissue
Myeloperoxidase (MPO) activity was measured by using
fluorometric assay kit (BioVision) according to the man-
ufacturer’s protocol. Briefly, whole-lung tissues were pul-
verized in liquid nitrogen using a mortar and pestle, then
homogenized in 4 volumes of MPO assay buffer (supplied
in the assay kit) and centrifuged at 13,000 g for 10 min-
utes to remove the insoluble material. For the MPO reac-
tion, 46 μL of MPO assay buffer was mixed with 2 μL of
MPO substrate solution and 2 μL of MPO probe. The re-
action mixture was then mixed with 50 μL of sample (or
the positive control) and read using an excitation wave-
length of 485 nm and an emission wavelength of 528 nm
at T0 and again after incubation at room temperature (pro-
tected from light) for 60 minutes (T60). The amount of
relative fluorescence units (RFUs) generated in 60 minutes
(T60−T0) was then determined (ΔRFU). Protein concentra-
tions in lung lysates were determined using the Bradford
technique. MPO activity was then calculated as picomoles
fluorescein per 60 minutes per milligram of protein, equiv-
alent to picomoles per minute per milligram of protein,
equivalent to microunits per milligram of protein.
Western blot analysis
Heart tissue was lysed and centrifuged at 6,000 g, and
the supernatant was collected as previously described.33,34
Cell extracts were separated on 4%–20% polyacrylamide
186 | Sex differences in PH Rafikova et al.

4. gradient gels and electrophoretically transferred to polyvi-
nylidene fluoride membrane (Bio-Rad), then blocked with
1% bovine serum albumin in Tris-buffered saline. The
membranes were probed using an antibody specific to
Caveolin-1 (BD Biosciences). Reactive bands were visual-
ized using chemiluminescence (ThermoFisher) using a
Kodak 440CF image station. Band intensity was quantified
using AlphaView SA image processing software. Protein
loading was normalized by reprobing gels with a mono-
clonal antibody specific to β-actin (Sigma).
Measurement of superoxide levels in tissue
To detect superoxide generation in heart tissue, we per-
formed electron paramagnetic resonance (EPR) measure-
ments. Fresh-frozen tissues were pulverized in liquid ni-
trogen to form a fine powder. Then 4–6 mg of tissue
powder was dissolved in 300 μL of EPR buffer (PBS sup-
plemented with 5 μM diethyldithiocarbamate and 25 μM
desferrioxamine; Sigma-Aldrich) at 0°C in a temperature-
controlled cooling shaker (UltraCruz, Santa Cruz, CA) to
generate a suspension of tissue particles containing intact,
not lysed, cells. We found that, at 0°C, this tissue suspen-
sion does not generate a measurable amount of superox-
ide (data not shown). The dissolved powder was then split
equally (75 μL) between four 1.5-mL eppendorf tubes con-
taining only the spin probe, 1-Hydroxy-3-methoxycarbonyl-
2,2,5,5-tetramethylpyrrolidine-HCl (CMH; 5 mg/mL), or
CMH and the nitric oxide synthase inhibitor, ethyliso-
thiourea (ETU; 1 mM). Each tube was then incubated for
2 minutes at 0°C. Thirty microliters of tissue suspension
from each tube was then transferred to a glass capillary
for 60 minutes at room temperature. Superoxide radical
generation was then determined at room temperature us-
ing a Magnettech M100 instrument (Magnettech, Ger-
many). Amplitudes of the EPR spectra were analyzed us-
ing ANALYSIS 2.0 software (Magnettech). To quantitate
the amount of superoxide per milligram of protein, we per-
formed a standard reaction of the superoxide-generating
enzyme xanthine oxidase in the presence of xanthine and
CMH. A reaction curve was generated with 1 mU of xan-
thine oxidase in the presence of xanthine (1 mM in PBS,
pH 7.4, Sigma) in EPR reaction buffer containing CMH.
This generated 1 nM/min superoxide generation rate over
a 1-hour period at room temperature. Final superoxide gen-
eration in the tissue was then expressed as picomoles per
minute per milligram of tissue.
Statistical analysis
Statistical calculations were performed using the GraphPad
Prism software, version 4.01. The mean value (± standard
error) was calculated for all samples, and significance was
determined by either the unpaired or paired t test or anal-
ysis of variance (ANOVA). For ANOVA, Newman-Keuls
post hoc testing or Bonferroni testing to compare the se-
lected pairs of columns was used. A value of P < 0.05 was
considered significant. For survival curve, the log-rank test
for trend was used.
RESULTS
Our experimental protocol produced a significant differ-
ence in the survival rate between the male and female
groups (Fig. 1). This observation is in accordance with
clinical data, showing that, although females develop PH
more often than males, they survive longer than males
after PH has been diagnosed.20-23
By measuring the mean arterial pressure at the end
of the study, we found the systemic blood pressure to be
similar in all four groups (female, male, control, and
SU5416-treated groups; Fig. 2A). However, there was a
significant decrease in PAAT, assessed by echocardiogra-
phy, in both females and males after 4 weeks of hypoxia
(Fig. 2B), with only modest progression during return to
normoxia. Because PAAT is thought to provide the most
accurate estimation of pulmonary systolic blood pressure,35
we conclude that there are no significant differences be-
tween the sexes in the development and progression of
PH. This result was further supported using invasive he-
Figure 1. Sex-dependent differences in the survival rate of rats
with pulmonary hypertension (PH). The survival curve of male
and female rats during the 14-week study is shown. No deaths
occurred in control groups (Cont) during this period of time.
The survival rate in male but not female rats with PH was signif-
icantly different from that in controls. The survival rate in males
with PH was also significantly different from that in female rats
with PH. N = 6–10 rats in each group. Asterisk indicates P < 0.05
versus controls; dagger indicates P < 0.05 versus female rats with
PH (log-rank test for trends).
Pulmonary Circulation Volume 5 Number 1 March 2015 | 187

5. modynamic measurements of RV peak systolic pressure
(RVPSP) 14 weeks after SU injection (Fig. 2C). It should
be noted that our data are in conflict with a previous re-
port that indicated that the SU5416/hypoxia model in-
duces a greater increase in RVPSP in female rats.6
How-
ever, the conflicting data may be attributed to the use
of different doses of SU5416. The high dose of SU5416
(200 mg/kg) used previously may promote greater apopto-
tic damage to the pulmonary endothelial cells. Because fe-
males are known to be more sensitive to endothelial dam-
age,19
the more severe conditions induced by a high dose of
SU6416 could result in female rats developing worse PH.
The development of PH was associated with the remod-
eling of pulmonary arteries in both male and female rats
(Fig. 3). However, females presented with more severe pro-
liferation of the pulmonary parenchyma and small pulmo-
nary arteries (Fig. 3D). To confirm that females have more
pronounced pulmonary vascular cell proliferation, the vas-
cular wall area was measured and calculated as a percent-
age of the total vessel cross-sectional area. The ratios of
areas were used, because the thickness of the vessel wall
proved to be unreliable due to the irregularity in shape of
remodeled vessels.36
Although we found a significant in-
crease in vascular wall thickness in the pulmonary arteries
between 30–100 μm and 101–200 μm in males, there was
significantly greater thickening in female rats with PH
(Fig. 3E).
The development of PH was associated with perivas-
cular infiltration of the small pulmonary arteries by inflam-
matory cells in male (Fig. 4A) but not female (Fig. 4B) rats
with PH. To confirm the more pronounced inflamma-
tory component in males, lung sections were stained with
an anti-MPO antibody, and total MPO activity in periph-
eral lung tissue was also measured. We identified high
levels of MPO staining in the perivascular area of small
pulmonary arteries only in male rats with PH (Fig. 4E).
The pulmonary arteries of female rats (Fig. 4F) exhibited
MPO staining findings comparable to those in control
rats (Fig. 4C, 4D). Total MPO activity was also significantly
increased in lung tissue prepared from male rats with PH
(Fig. 4G). MPO activity did not significantly increase in
female rats with PH (Fig. 4G).
PH also induced an abundant adventitial and medial
fibrosis in male (Fig. 5A) but not female (Fig. 5B) rats.Figure 2. Systemic and pulmonary pressure in male and female
rats with pulmonary hypertension (PH). The systemic pressure
was evaluated by measuring the mean arterial pressure (MAP) in
the right carotid artery. No significant differences were found
between groups (A). Echocardiography identified a significant
decrease in pulmonary artery acceleration time (PAAT) in both
male and female rats with PH starting at week 4 of the study (B).
The RV peak systolic pressure (RVPSP) was also significantly
increased in both male and female rats at week 14 of the study
(C). Results are expressed as mean ± SEM; N = 5–8 rats in each
group. Asterisk indicates P < 0.05 versus control group (Cont).
188 | Sex differences in PH Rafikova et al.

6. The accumulation of collagen in the adventitia and me-
dia of small pulmonary arteries was confirmed by Masson
trichrome staining (Fig. 5C–5F) and scored in a blinded
manner (Fig. 5G). We identified significant deposition of
matrix proteins in the pulmonary vascular wall of male
rats with PH, with partial substitution of the smooth mus-
cle layer with fibrotic tissue (Fig. 5E). In female rats with
PH, medial hypertrophy was accompanied by only mild
adventitial fibrosis with no signs of medial fibrotic changes
(Fig. 5F). Scoring confirmed that the accumulation of con-
nective tissue in the male rats with PH was significantly
higher than that in female rats with PH. Although PH-
induced fibrotic changes in pulmonary arteries are well
described,36-38
this is the first evidence of the predomi-
nance of the development of lung fibrosis in the male sex,
with females being resistant to fibrotic remodeling of the
pulmonary arteries.
To evaluate the effect of sex on the development of RV
hypertrophy, we monitored the thickness of RV free wall
every 4 weeks by echocardiography. Our data indicate that
female rats with PH develop mild RV hypertrophy only
during their exposure to hypoxia (during the first 4 weeks
of the study), whereas RV thickness was not further al-
tered during the return to normoxia (Fig. 6A). In contrast,
the male rats with PH showed a continuous increase in
RV thickness under both hypoxia and normoxia conditions,
and by week 12, male rats with PH presented with approxi-
mately twice the level of RV hypertrophy compared with
females. These data were confirmed by measuring the RV/
LV+S wet weight ratio (Fulton index) at the end of study
(Fig. 6B). Together, our data suggest that female sex exerts
a cardioprotective effect.
To further investigate RV function, we studied RV dia-
stolic pressure, RV contractility, and RV relaxation. In fe-
male rats with PH, the RV diastolic pressure was un-
changed compared with controls (Fig. 6C). In contrast,
male rats with PH presented with RV diastolic dysfunction.
Indeed, the male group had a positive RV peak diastolic
pressure (Fig. 6C). The dP/dtmin values in males were also
significantly less negative than in females (Fig. 6E). Tak-
ing into account the similar increase in systolic RV pres-
sure in males and females with PH, the difference in dP/
dtmin suggests that the RV relaxation in males is signifi-
cantly impaired compared with that in females. We also
identified a nonsignificant trend of reduced RV contractility
in males with PH (Fig. 6D), which may correlate with the
development of RV systolic dysfunction.
To further investigate the mechanism underlying RV
failure in males, we evaluated fibrotic changes. Masson tri-
chrome staining revealed severe fibrosis in the RV of male
rats with PH (Fig. 7C), whereas the deposition of collagen
in the RV of female rats with PH was on a level with that
observed in control rats (Fig. 7A, 7B, 7D). To assess the
level of collagen deposition in each RV tissue, tissue sec-
tions were evaluated by a blinded observer. Scoring con-
firmed that fibrotic changes are apparent only in the RV
of male rats with PH (Fig. 7E). The level of fibrotic tis-
sue in female rats with PH was similar to that in controls
(Fig. 7E). Taken together, our data suggest that PH in
males is associated with the development of severe RV fail-
Figure 3. Female rats with pulmonary hypertension (PH) pre-
sent with more severe angioproliferative changes in the lung.
Representative images from hematoxylin-and-eosin-stained pul-
monary arteries of control (Cont) male (A), control female (B),
PH male (C), and PH female rats (D) are shown. Quantitative
analysis of the vascular wall thickness (%VT) was significantly
higher in both categories of pulmonary arteries examined (30–
100 μm and 101–200 μm) in female rats with PH (E). Ten pul-
monary arteries per each animal were analyzed. Results are ex-
pressed as mean ± SEM; N = 4 rats in each group. Asterisk in-
dicates P < 0.05 versus Cont group. Dagger indicates P < 0.05
versus male PH group. The black bar corresponds to 100 μm.
Pulmonary Circulation Volume 5 Number 1 March 2015 | 189

7. ure, whereas female rats with PH present with mild RV hy-
pertrophy, a completely preserved RV morphology, and pre-
served RV function.
It has been recently shown that caveolin-1 expression
is reduced in male rats in both the monocrotaline and
sugen/hypoxia-induced rat models of PH.39
To investi-
gate whether differences in caveolin-1 expression could
explain the differences in survival between males and fe-
males, we examine caveolin-1 protein levels in the heart.
Our data indicate that control female rats had higher lev-
els of caveolin-1 protein compared with control male rats
(Fig. 8A). In addition, caveolin-1 protein levels were pre-
served in female rats with PH, whereas caveolin-1 protein
levels were decreased significantly in male rats with PH
(Fig. 8A). It is widely accepted that increased oxidative
stress leads to activation of tissue fibrosis.40-42
Thus, we
determined whether the sexual dimorphism associated
with caveolin protein levels would modulate endothelial
nitric oxide synthase (eNOS)-mediated superoxide gener-
ation (i.e, eNOS uncoupling). Our data indicate that there
was a trend toward decreased eNOS-derived superoxide
generation in the hearts of control female rats compared
with control male rats (Fig. 8B), although this did not
reach statistical significance. Furthermore, there was no
significant increase in eNOS-dependent superoxide pro-
duction in the hearts of female rats with PH (Fig. 8B).
Figure 4. The inflammatory response is more pronounced in male rats with pulmonary hypertension (PH). Perivascular infiltration by
inflammatory cells was primarily found in male (A; arrows) but not female (B) rats with PH. Immunohistochemical analysis of the
lungs using a myeloperoxidase (MPO) antibody (brown) revealed significant accumulation of MPO staining in the perivascular area in
male (E; arrows) but not female rats with PH (F). Neither male (C) nor female control (Cont) rats (D) exhibited significant MPO
staining. A significant increase in MPO activity, measured in peripheral lung tissue, was also found in male rats with PH (G). Results
are expressed as mean ± SEM; N = 5–8 rats in each group. Asterisk indicates P < 0.05 versus Cont group; dagger indicates P < 0.05
versus male rats that were injected with a single dose of the vascular endothelial growth factor receptor 2 antagonist, SU5416. The
black bar corresponds to 100 μm.
190 | Sex differences in PH Rafikova et al.

8. However, there was a threefold increase in eNOS-derived
superoxide generation in male rats with PH (Fig. 8B).
DISCUSSION
During the last decade, significant advances in our un-
derstanding of the pathology of PH have been achieved.
The previously accepted “vasoconstriction” paradigm has
transformed into our current concept of pulmonary vas-
cular injury with subsequent proliferation of endothelial
and smooth muscle cells that results in the obliteration
of smooth pulmonary arteries and the formation of com-
plex vascular structures known as plexiform lesions. How-
ever, our study highlights that these pathological changes
are primarily associated with females with PH and that
males with PH present with a significantly lower level of
vascular proliferation. This may be due to the effects of
female sex hormones, because estrogens have been shown
to be damaging to the pulmonary vasculature,5
especially
when an imbalance between “good” and “bad” estrogen
metabolites exists.6
Therefore, estrogens may contribute to
the initial pulmonary endothelial damage, which, in turn,
is essential for the consequent vascular remodeling. In
addition, the ability of estradiol (E2) to trigger angiogen-
esis through the up-regulation of pro-angiogenic VEGF
gene transcription,13,14
and through stimulation of endo-
thelial and smooth muscle proliferation,15,16
has been pre-
Figure 5. Male rats with pulmonary hypertension (PH) present with fibrotic changes in the adventitia and media of pulmonary
arteries. Remodeling of the pulmonary arterial wall consisted of striking fibroproliferative changes in male rats with PH (A; arrows)
and predominantly medial hypertrophy, without evidences of fibrosis, in female rats with PH (B). Masson trichrome staining identi-
fied low levels of extracellular matrix proteins in the adventitia of pulmonary arteries in control (Cont) male (C) and Cont female rats
(D) as well as female rats with PH (F). However, abundant collagen accumulation was observed in the adventitia and media of
pulmonary arteries from male rats with PH (E). Ten random fields (100×) for each animal were scored by a blinded observer. Scoring
identified significant fibrotic tissue accumulation in male rats with PH and only mild fibrosis in female rats with PH (G). Results are
expressed as mean ± SEM; N = 6 rats in each group. Asterisk indicates P < 0.05 versus Cont group. Dagger indicates P < 0.05 versus
male rats that were injected with a single dose of the vascular endothelial growth factor receptor 2 antagonist, SU5416. The black bar
corresponds to 100 μm.
Pulmonary Circulation Volume 5 Number 1 March 2015 | 191

9. viously described. In contrast, testosterone appears to be
protective for pulmonary vasculature and has been dem-
onstrated to be a more potent pulmonary artery vasodila-
tor than E2.43,44
Taken together, our data and previously
published research indicate that females are more prone
to develop angioproliferative pulmonary vascular disease
than males. However, given the fact that males have a
worse survival rate, it is possible that the extent of the
vascular proliferative changes may play an important role
in the development of PH but may not determine the
progression of the disease or the survival time length.
Inflammation has been reported to be a prominent pro-
cess in human PH and may contribute to progression of
disease.45,46
Accumulation of inflammatory cells around
Figure 6. Right ventricle (RV) hypertrophy and function. The thickness of the RV free wall was measured by echocardiography. In
male rats with pulmonary hypertension (PH), there was a time-dependent increase in RV thickness (A), whereas female rats had
only a mild increase in RV hypertrophy during exposure to hypoxia (first 4 weeks) with no subsequent increase in RV thickness
(A). The wet weight ratio of RV wall normalized on left ventricle plus septum (LV+S)—Fulton index—was measured at 14 weeks.
The Fulton index was significantly higher in male rats with PH (B). Attenuation in the RV peak diastolic pressure (RVPDP) was
also observed only in male rats with PH (C). RV contractility and relaxation were evaluated by measuring RV dP/dtmax and dP/
dtmin, respectively. RV contractility was significantly increased in both male and female rats with PH (D). However, the increase in
RV relaxation was significantly impaired only in male rats (E). Results are expressed as mean ± SEM; N = 5–8 rats in each group.
Asterisk indicates P < 0.05 versus control (Cont) group. Dagger indicates P < 0.05 versus male rats that were injected with a single
dose of the VEGF receptor 2 antagonist, SU5416.
192 | Sex differences in PH Rafikova et al.

10. remodeled pulmonary arteries as well as in adventitia, me-
dia, and plexiform lesions is well described.45,47,48
In our
study, we found that the male sex is primarily associated
with inflammatory cell infiltration, whereas females did
not appear to develop a perivascular inflammatory re-
sponse. There are several lines of evidence suggesting the
importance of T and B cells in development of PH in
animal models and in patients with PH.45,47,49
However,
the contribution of monocytes and macrophages to PH is
less understood. By measuring the expression and activity
of MPO, which is an important product of activated neu-
trophils and macrophages, we were able to demonstrate
that macrophages represent an important component of
the inflammatory cell infiltrate. MPO has a number of
biological effects that may contribute to pulmonary vascu-
lar injury and remodeling. These include the induction of
severe oxidative stress and oxidative tissue damage, pro-
longation of inflammatory reactions, and propagation of
vascular remodeling.50-52
Therefore, our finding that the
male sex correlates with the accumulation of activated
neutrophils and macrophages in the perivascular area of
small pulmonary arteries coupled with increased MPO ac-
tivity may be important and suggests that, in males, PH
therapy may need to include an anti-inflammatory strategy.
The recruitment of leukocytes in the vessel wall of lungs
could also have a significant impact on the function and
structure of pulmonary arteries, because activated macro-
phages and neutrophils can release reactive oxygen spe-
cies that not only can induce damage to the surrounding
cells but also can act as an NO “sink”. This would then
compromise NO induced vasodilation and also potentially
the vasoprotective properties of NO that include antithrom-
botic, anti-inflammatory, and antiproliferative actions. The
oxidative and nitrative stress induced by active leukocytes
could also stimulate the proliferative status in smooth mus-
cle cells and fibroblasts through posttranslational activa-
tion of cell survival pathways53
or through the release of
various growth factors.54
We also found that caveolin-1
protein levels were decreased only in the hearts of male
rats with PH, and this correlated with increased oxidative
stress due to eNOS uncoupling. Although caveolin-1 bind-
ing to eNOS is known to inhibit NO generation, these in-
teractions have also been recently shown to attenuate eNOS
uncoupling.55
Indeed, the loss of caveolin-1 is sufficient
to stimulate the development of PH in mice exposed to
chronic hypoxia.56
Thus, it is possible that maintaining ade-
quate caveolin-1 protein levels protects female rats against
developing fibrosis. However, the mechanism behind this
ability to maintain caveolin-1 expression is unresolved and
will need further study.
The proliferation of resident pulmonary artery adventi-
tial fibroblasts and the attraction of circulating mononu-
clear cells with fibroblast-like properties to the sites of
tissue injury are capable of inducing the production and
accumulation of extracellular matrix proteins, especially
collagen, in pulmonary arteries.54
In this study, we found
a correlation between leukocyte accumulation in the peri-
vascular area of pulmonary vessels and the development
of fibroproliferative changes in the adventitia and media
of pulmonary arteries in male but not female rats with
Figure 7. Severe myocardial fibrosis in the right ventricle (RV) of
male rats with pulmonary hypertension (PH). Myocardial tis-
sues, subjected to Masson trichrome staining, revealed signifi-
cant accumulation of fibrotic tissue in the RV of male rats with
PH (C), whereas the RV of female rats with PH (D) did not differ
from controls (Cont; A, B). Five random fields (40×) for each
animal were scored by a blinded observer. Scoring confirmed the
development of severe RV fibrosis only in male rats with PH (E).
Results are expressed as mean ± SEM; N = 6 rats in each group.
Asterisk indicates P < 0.05 versus Cont group. Dagger indicates
P < 0.05 versus male PH group. The white bar corresponds to
200 μm.
Pulmonary Circulation Volume 5 Number 1 March 2015 | 193

11. PH. From these data, we conclude that this inflammatory
component may be an important factor in both vascular
remodeling and the development of pulmonary fibrosis,
although the known ability of male sex hormones to in-
duce direct fibrotic changes in lungs should also be con-
sidered.57-59
The profound accumulation of these extracel-
lular matrix proteins in the adventitia and media would
severely increase pulmonary artery stiffness. Therefore, we
speculate that the increase in pulmonary vascular resis-
tance in males with PH would be additionally exacerbated
by a decrease in compliance (increase in stiffness). Both
the narrowing and stiffening of the pulmonary arteries con-
tribute to RV afterload. Therefore, the ventricles of males
with PH would have enhanced workloads, compared with
the ventricles of females. Indeed, we found that the level
of RV hypertrophy in male hearts significantly exceeded
that observed in female hearts. In addition, male rats with
PH developed high levels of RV fibrosis, which could also
develop as a result of increased RV overload. It is well es-
tablished that the cardiac fibrosis that develops after myo-
cardial death in the hypertensive state is mainly attributable
to increased afterload. Although there was an equal pres-
sure load in both sexes during our study, the additional
increase in pulmonary artery stiffness may be the factor
that tips the balance in males toward RV failure. At the
same time, the severity of the fibrotic changes in the RV
of males with PH, coupled with previously published evi-
dence that the pressure load itself is insufficient for devel-
opment of RV failure,60
suggests that other unknown mech-
anisms contribute to cardiac remodeling in males. Indeed,
the sexual dimorphism associated with the development of
cardiac fibrosis has been postulated clinically,25
with more
severe fibrosis observed in males. The possible mechanisms
are traditionally attributed to sex hormones, although the
data are limited and controversial. Thus, it is possible that
the protective properties of estrogens or the deleterious
role of androgens may be involved. In addition, a recent
analysis of sex-dependent genes expressed in response to
cardiac pressure overload revealed an important correla-
tion, with genes activated in females being related to mito-
chondria and metabolism, whereas in males, the upregu-
lated genes were related to matrix biosynthesis.27
These
data suggest that the female heart may have a better adap-
tation to maintain energy metabolism even in the pres-
ence of a decreased oxygen supply. This could potentially
explain why we observed only mild RV hypertrophy and
no fibrotic changes in female rats with PH. In contrast,
the stimulation of matrix-regulating genes and genes with
function related to cell growth, morphology, and differen-
tiation may explain the development of severe RV hyper-
trophy and fibrosis in males with PH. The abundant accu-
mulation of collagens in RV myocardial tissue would, in
turn, lead to cardiac stiffness, and this could explain the
Figure 8. Caveolin-1 protein levels are decreased in male rats
with pulmonary hypertension (PH). Western blot analysis dem-
onstrated that caveolin-1 protein levels were higher in control
(Cont) female rates compared with Cont male rats (A). Caveolin-
1 protein levels were significantly reduced in male but not fe-
male rats with PH (A). Endothelial nitric oxide synthase (eNOS)-
mediated superoxide production was also significantly increased
in the hearts of male but not female rats with PH (B). Results
are expressed as mean ± SEM; N = 3–4 rats in each group.
Asterisk indicates P < 0.05 versus Cont group. Dagger indicates
P < 0.05 versus male PH group.
194 | Sex differences in PH Rafikova et al.

12. development of the diastolic dysfunction we observed in
males and has also been found in humans.
In conclusion, there is a strong sexual dimorphism in
progression of PH. PH in female rats is primarily associ-
ated with angioproliferative changes in the pulmonary ar-
teries in the absence of inflammatory and fibrinogenic
processes in either the lung or heart. This correlates with
preserved RV function and better survival rates. In con-
trast, in males with PH, inflammatory cell infiltration of
the pulmonary arteries was associated with severe fibrosis
in adventitia and media, profound myocardial fibrosis, im-
paired diastolic function, and lower survival rates. On the
basis of these data, we conclude that, although the prolif-
erative changes in pulmonary arteries play an important
role in the development of PH, other pathological pro-
cesses, such as inflammation and fibrosis, may also be a
significant determinant of survival. This conclusion is in
agreement with earlier clinical studies and may explain
the phenomena of the poor survival rate of male patients
with PH. Our data also provide a rationale for introducing
sex-specific therapies for PH. Our data suggest that, in the
female population, the primary focus should be on anti-
proliferative drugs, whereas in male patients, therapies
may also need to include anti-inflammatory and antifi-
brinogenic strategies.
Source of Support: This research was supported in part by
National Institutes of Health grants HL60190 (to SMB),
HL67841 (to SMB), and P01HL0101902; by an Entelligence
Actelion Young Investigator Award for Research Excellence in
Pulmonary Hypertension (to OR); and by a Scientist Develop-
ment grant from the American Heart Association National Of-
fice (to RR). OR was also supported in part by F32HL103136.
DJ is supported by the “Deutsche Forschungsgemeinschaft”
(30743/2-1).
Conflict of Interest: None declared.
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