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\begin{document}

\title{Angiotensin-converting enzyme 2 activator ameliorates severe pulmonary
hypertension in a rat model of left pneumonectomy combined with VEGF
inhibition}



\author[1]{I-Chen Chen}%
\author[2]{Jao-Yu Lin}%
\author[2]{Yi-Ching Liu}%
\author[3]{Chee-Yin Chai}%
\author[4]{Jwu-Lai Yeh}%
\author[2]{Jong-Hau Hsu}%
\author[4]{Bin-Nan Wu}%
\author[5]{Zen-Kong Dai}%
\affil[1]{kaohsiung medical university hospital}%
\affil[2]{Kaohsiung Medical University Hospital}%
\affil[3]{Kaohsiung Medical University Chong-Ho memorial Hospital}%
\affil[4]{School of Medicine,Kaohsiung Medical University}%
\affil[5]{Kaohsiung Medical University}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background Pulmonary artery hypertension (PAH) is a life-threatening and
deteriorating disease with no promising therapy available currently due
to its diversity and complexity. An imbalance between vasoconstriction
and vasodilation has been proposed as the mechanism of PAH.
Angiotensin-converting enzyme 2 (ACE2), which catalyzes the hydrolysis
of the vasoconstrictor angiotensin (Ang) II into the vasodilator
Ang-(1-7), has been shown to be an important regulator of blood pressure
and cardiovascular diseases. Herein we hypothesized diminazene aceturate
(DIZE), an ACE2 activator, could ameliorate the development of PAH and
pulmonary vascular remodeling. Methods A murine model of PAH was
established using left pneumonectomy (PNx) on day 0 followed by
injection of a single dose of the VEGF receptor-2 inhibitor SU5416
(25mg/kg) subcutaneously on day 1. All hemodynamic and biochemical
measurements were done at the end of the study on day 42. Animals were
divided into 4 groups (n= 6/group): (1) sham-operated group, (2)
vehicle-treatment group (SuPNx42), (3) early treatment group
(SuPNx42/DIZE1-42) with DIZE at 15 mg/kg/day, subcutaneously from day 1
to day 42, and (4) late treatment group (SuPNx42/DIZE29-42) with DIZE
from day 29-42. Results In both the early and late treatment groups,
DIZE significantly attenuated the mean pulmonary artery pressure,
pulmonary arteriolar remodeling, and right ventricle brain natriuretic
peptide (BNP), as well as reversed the overexpression of ACE while
up-regulating the expression of Ang-(1-7) when compared with the
vehicle-treatment group. In addition, the early treatment group also
significantly decreased plasma BNP and increased the expression of eNOS.
Conclusions ACE2 activator has therapeutic potentials for preventing and
attenuating the development of PAH in an animal model of left
pneumonectomy combined with VEGF inhibition. Activation of ACE2 may thus
be a useful therapeutic strategy for the treatment of human PAH.%
\end{abstract}%



\sloppy


\textbf{Angiotensin-converting enzyme 2 activator ameliorates severe
pulmonary hypertension in a rat model of~left pneumonectomy combined
with VEGF~inhibition}

I-Chen Chen, MD \textsuperscript{1,2,3}, Jao-Yu Lin,
MD\textsuperscript{4}, Yi-Ching Liu, MD \textsuperscript{1}, Chee-Yin
Chai, MD, PhD \textsuperscript{5}, Jwu-Lai Yeh,
PhD\textsuperscript{3,6}, Jong-Hau Hsu, MD, PhD, \textsuperscript{1,2},
Bin-Nan Wu, PhD \textsuperscript{3,6} and Zen-Kong Dai, MD,
PhD,\textsuperscript{1,2,3*}

\textsuperscript{1} Department of Pediatrics, Kaohsiung Medical
University Hospital, Kaohsiung, Taiwan

\textsuperscript{2} Department of Pediatrics, School of Medicine,
College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

\textsuperscript{3} Graduate Institute of Medicine, College of Medicine,
Kaohsiung Medical University, Kaohsiung, Taiwan

\textsuperscript{4} Department of Surgery, Kaohsiung Medical University,
Kaohsiung, Taiwan

\textsuperscript{5} Department of Pathology, School of Medicine, College
of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

\textsuperscript{6} Department of Pharmacology, School of Medicine,
College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

Correspondence: Zen-Kong Dai

Zen-Kong Dai, MD, PHD

Department of Pediatrics, Kaohsiung Medical University, 100 Shih-Chuan
1st Road, Sun-Ming District, Kaohsiung 807, Taiwan

Tel: 886-7-3121101-6507 E-mail: zenkong@kmu.edu.tw Fax: 886-7-3208316

Running title: ACE2 activator ameliorates pulmonary artery hypertension

\textbf{Summary}

\textbf{Background}

Pulmonary artery hypertension (PAH) is a~life-threatening and
deteriorating disease with no promising therapy available currently due
to its diversity and complexity. An imbalance between vasoconstriction
and vasodilation has been proposed as the mechanism of PAH.
Angiotensin-converting enzyme 2 (ACE2), which catalyzes the hydrolysis
of the vasoconstrictor angiotensin (Ang) II into the vasodilator
Ang-(1-7), has been shown to be an important regulator of blood pressure
and cardiovascular diseases. Herein we hypothesized diminazene aceturate
(DIZE), an ACE2 activator, could ameliorate the development of PAH and
pulmonary vascular remodeling.

\textbf{Methods}

A murine model of PAH was established using left pneumonectomy (PNx) on
day 0 followed by injection of a single dose of the VEGF receptor-2
inhibitor SU5416 (25mg/kg) subcutaneously on day 1. All hemodynamic and
biochemical measurements were done at the end of the study on day 42.
Animals were divided into 4 groups (n= 6/group): (1) sham-operated
group, (2) vehicle-treatment group (SuPNx\textsubscript{42}), (3) early
treatment group (SuPNx\textsubscript{42}/DIZE\textsubscript{1-42}) with
DIZE at 15 mg/kg/day, subcutaneously from day 1 to day 42, and (4) late
treatment group (SuPNx\textsubscript{42}/DIZE\textsubscript{29-42}) with
DIZE from day 29-42.

\textbf{Results} In both the early and late treatment groups, DIZE
significantly attenuated the mean pulmonary artery pressure, pulmonary
arteriolar remodeling, and right ventricle brain natriuretic
peptide\textbf{(} BNP), as well as reversed the overexpression of ACE
while up-regulating the expression of Ang-(1-7) when compared with the
vehicle-treatment group. In addition, the early treatment group also
significantly decreased plasma BNP and increased the expression of eNOS.

\textbf{Conclusions} ACE2 activator has therapeutic potentials for
preventing and attenuating the development of PAH in an animal model of
left pneumonectomy combined with VEGF inhibition. Activation of ACE2 may
thus be a useful therapeutic strategy for the treatment of human PAH.

\textbf{Key words: pulmonary artery hypertension; left pneumonectomy;
SU5416; ACE2 activator; diminazene aceturate; ACE2-Ang (1-7)-Mas axis}

\textbf{Introduction}

Pulmonary hypertension (PH) is a progressive and presently incurable
disorder, which complicates the majority of cardiovascular and
respiratory diseases.\textsuperscript{1,2} The etiology of PH is
classified by WHO into the following five distinct groups: (1) pulmonary
artery hypertension (PAH), (2) PH due to left heart disease, (3) PH due
to lung disease and/or hypoxia, (4) chronic thromboembolic pulmonary
hypertension, and (5) unclear multifocal
mechanisms.\textsuperscript{3}All of these groups share a mean, resting,
pulmonary arterial pressure (PAP) of greater than 20
mmHg.\textsuperscript{4} The pathogenesis of PH is complexed and
multifactorial; proliferative vasculopathy, pulmonary vascular
remodeling, vascular constriction, and endothelial cell dysfunction have
been proven in several previous studies.\textsuperscript{5,6}

As rodent models are beneficial for understanding the pathophysiology
of~PH~and for testing experimental therapies, more and more rodent
models have been developed and many of them have exhibited
characteristics mimicking WHO's classification of human
PH.\textsuperscript{6} Traditionally, rat models of pulmonary artery
hypertension (PAH) include monocrotaline (with or without pneumonectomy
and/or hypoxia), chronic hypoxia together with SU5416
(3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one; a VEGF
(Vascular endothelial growth factor) receptor-2 inhibitor, and the
fawn-hooded rat (FHR).\textsuperscript{7-9} A major advantage of the
SU5416 hypoxia models over that of the monocrotaline hypoxia one is the
remodeling of neointimal occlusive lesion, which accumulates unique
tumorlets of endothelial cells and obliterates medium-sized precapillary
arteries. It has also been shown that the SU5416 hypoxia model developed
pulmonary arterial changes resembling plexiform-like lesions, which are
characteristic features of human PAH, by increasing apoptosis of
endothelial cells followed by converting them into apoptosis resistant
and phenotypically altered endothelial
cells.\textsuperscript{10}Recently, a newly developed rodent PAH model
that combined left pneumonectomy with SU5416 was
reported.\textsuperscript{11} Unlike the traditional SU5416 hypoxia
model in which a partial reversal of PAH is seen upon returning to
normoxia, this new animal model does not reverse hypoxia-induced
vasoconstriction and hemoconcentration under similar conditions. Rather,
the right ventricular systolic pressure increased gradually over time, a
feature that may favor the assessment of drug effects in preclinical
trials.\textsuperscript{11}

The Renin-angiotensin system (RAS) has been implicated in playing a
causative role in PH.\textsuperscript{12,13} There are two opposing arms
in RAS: the presser arm, composed of angiotensin converting enzyme
(ACE), angiotensin II (Ang II) produced from Ang I by ACE, and the Ang
II type 1 (AT1) receptor as the main protein mediating the biological
actions of Ang II; and the vasodilator arm, consisting angiotensin
converting enzyme 2 (ACE2), Ang-(1-7) generated through hydrolysis of
Ang II by ACE2, and the Mas receptor as the protein conveying the
vasodilatory, antiproliferative, antifibrotic, and antithrombotic
effects of Ang-(1-7).\textsuperscript{12,13,14} Therefore, an increase
in the levels of Ang-(1-7) should have beneficial effects for PAH.
However, targeting the ACE2/Ang (1-7)/Mas receptor pathway in PAH is
still under scrutiny.

ACE2 has exhibited its modulating ability in the balance between
vasoconstriction and vasodilation in many
experiments.\textsuperscript{15,16} Recently, diminazene aceturate
(DIZE), a Food and Drug Administration (FDA) approved anti-trypanosomal
drug, has been demonstrated to exert off-target effect on activating
ACE2.\textsuperscript{17,18} This effect of DIZE has been confirmed
through the analysis of cleavage of the vasoconstrictor peptide Ang II,
the most physiologically relevant natural substrate for
ACE2.\textsuperscript{17}

On the basis of the benefits demonstrated by DIZE in other PH
models,\textsuperscript{18-20} we aimed to evaluate its effects on a rat
model of PAH induced by left pneumonectomy combined with SU5416. We
reasoned that in this model there could be~an up-regulated expression of
ACE, leading to the imbalance between vasoconstriction and vasodilation.
Treatment with DIZE would increase the levels of ACE2, and hence, the
vasodilatory peptide Ang-(1-7) to prevent and ameliorate the development
of PAH.

\textbf{Methods}

\textbf{Animal Model of PAH}

All protocols had been approved by the animal research committee of
Kaohsiung Medical University (IACUC approval NO: 104226). Male Wistar
rats weighing approximately 220 g were purchased from National Animal
Center, Taiwan. after arriving at the Kaohsiung Medical University
vivarium, the rats were acclimated for at least one week before being
used in the experiment. They were housed in a room on a 12-hr light/dark
cycle under controlled temperature of 22.1 and relative humidity of
55\%, and they were provided with normal chow and water ad libitum. On
the day of experiment, animals were anesthetized with a mixture of
sodium pentobarbitone (20 mg/kg, i.p.) and ketamine (40 mg/kg, i.m.)
under orotracheally ventilated rodent respirators (Harvard, South
Natick, MA) and lateral thoracotomy in the third intercostal space was
performed. Left lung of rats was resected and closed by clip ligation
(Ligaclip multiple clip applier, medium size, Ethicon Endo-Surgery), and
the animals were suture to prevent pneumothorax. The chest retractor was
then removed after 15 minutes, and the thymus was moved back to its
physiological position. The chest cavity and skin were closed. The
entire procedure took less than 30 min for each rat. After operation,
rats were extubated and received an additional injection of sterile
saline (10 ml). On the following day, SU5416 (25 mg/kg) was injected
subcutaneously in surviving rats. Sham-operated animals underwent
identical operation without pneumonectomy (PNx) and received sterile
saline (20 mg/kg) subcutaneously, instead. The day of SU5416 injection
was designated as day 0. All animals were individually housed in a 12-h
dark/light cycle-controlled room and fed a regular rat diet. The
procedure was also shown in supplemental figure 1.

\textbf{Study design}

All rats were randomized (n = 6 / group) to undergo the sham operation
(group 1) or SU5416 injection with left pneumonectomy (SuPNx). The SuPNx
rats were further randomized for subcutaneous treatment with saline
(SuPNx\textsubscript{42}) (group 2) or DIZE (Santa Cruz Biotechnology,
CA, USA) at a dose of 15 mg/kg/day from day 1 to day 42
(SuPNx\textsubscript{42}/DIZE\textsubscript{1--42}) in the early
treatment protocol (group 3) or from day 29 to day 42
(SuPNx\textsubscript{42}/DIZE\textsubscript{29--42}) in the late
treatment protocol (group 4). On day 42, all rats were ventilated and
sacrificed after checking hemodynamic data. The lungs and heart from
each rat were rapidly perfused with sterile saline under a pressure of
100-cm H\textsubscript{2}O prior to removal. One-half of the lung tissue
was homogenized for protein extraction, and the other half was kept in
10\% formaldehyde and froze in 4\selectlanguage{ngerman}°C for histopathologic analysis.

\textbf{Hemodynamics Measurements}

The method was described in our previous published
study.\textsuperscript{21,22} Briefly, a PE-50 catheter
(Beckton-Dickinson, Sparks, MD, USA) was inserted into the femoral
artery using a cut-down procedure for recording pressure. Thereafter, a
left parasternal thoracotomy was performed. Another catheter was
inserted closely to the main pulmonary artery via the right ventricular
outflow tract. Pulmonary and femoral arterial pressures were recorded
simultaneously.

\textbf{Plasma and right ventricle brain natriuretic peptide (BNP)}

Plasma samples (1.0 mL) and homogenous right ventricle (RV) were
expressed in v/v for TFA and homogenate in w/v and then centrifuged at
2000 × g for 15 min at 4°C. Enzyme-immunoassay kits (Phoenix
Pharmaceuticals, Burlingame, CA, USA) were used to measure plasma and RV
BNP contents according to the manufactural protocol.

\textbf{Histopathologic and immunohistochemical Analysis}

Lung tissues which were immersed in formalin for at least 24 hr were
analyzed for histopathology. Hematoxylin-Eosin (H-E) staining was used
for analysis of the pulmonary arterioles in terms of medial wall
thickness under a microscope at a magnification of 400X. The methods
were described in our previous published
study.\textsuperscript{21,22}and the medial wall thickness of each
arteriole was expressed as follows:

Percent wall thickness = (medial thickness × 2 /external diameter) ×
100.

Verhoeff-van Gieson staining was performed for evaluating the severity
of neointimal formation using a modified scoring system published
before.\textsuperscript{23-25} The degree of vascular remodeling was
determined by measuring 30 transversally cut vessels (outer diameter:
50-100 \selectlanguage{greek}µ\selectlanguage{english}m) from each rat and expressing the thickness of the combined
intimal and medial layers as a percentage of total vessel diameter. The
percentage of vascular occlusion of these 30 vessels was categorized as
Grade 0: no evidence of luminal obstruction; Grade 1: the presence of
partial (\textless{}50\%) luminal occlusion; and Grade 2: the present of
luminal occlusion greater than 50\%. An average Grade score for 30
vessels was calculated for each animal. Afterwards, a quantitative
analysis of luminal obstruction in 30 consecutive small pulmonary
arteries from all rats in each group was performed.

In addition, double immunohistochemical staining was performed to
present the smooth muscle actin (SMA) by using formalin-fixed,
paraffin-embedded tissue. Experiments were performed by double staining
polymer detection systems (TADS03, BioTnA, Taiwan) using rabbit
polyclonal anti-SMA (1:100, Cell signaling, MA, USA) and anti-PCNA
(Proliferating Cell Nuclear Antigen)(1:100, Cell signaling, MA, USA)
antibodies.

\textbf{Western blot analysis of pulmonary eNOS, ACE, ACE2, and MAS}

Lung tissues(100 mg) were homogenized in 1 ml of RIPA buffer (1\% Triton
X-100, 15 mM HEPES-NaOH {[}pH 7.5{]}, 0.15 mM NaCl, 1\% sodium
deoxycholate, 0.1\% SDS, 1 mM sodium orthovanadate, 10 mM EDTA, and
0.5\% protease inhibitor cocktail {[}Sigma, St. Louis, MO, USA{]}) and
centrifuged at 15,000 \selectlanguage{ngerman}× g for 20 min at 4°C. Proteins in the supernatant
(100 \selectlanguage{greek}µ\selectlanguage{english}g) were subjected to SDS-PAGE in 10\% polyacrylamide gels and were
transferred onto PVDF membranes (Pall, Port Washington, NY, USA). Each
membrane was blocked with 5\% non-fat dry milk in Tris-buffered saline
(TBS), probed with anti-actin (1:10,000) (Upstate Biotechnology, Lake
Placid, NY, USA), anti-eNOS ( 1:1000, Millipore, Milford, MA, USA)
anti-ACE, anti-ACE2 (1:500 dilution; Millipore, Billerica, MA, USA), or
anti-MAS antibodies (1:1,000 dilution; Millipore), and then incubated
with horseradish peroxidase-conjugated secondary antibody. (Leadgene
Biomedical, TW) Signals were detected using the Western Lighting\selectlanguage{ngerman}®
chemiluminescent kit (Millipore) according to the manufacturer's
specifications.

\textbf{Ang II and Ang-(1--7) measurements}

RV and lung supernatants were acidified with 0.6\% trifluoroacetic acid
(TFA) to obtain a 10\% (w/v) homogenate. The samples were centrifuged at
2,000 × g for 15 min at 4°C, and dried under a steam of nitrogen at
60°C. Enzyme-immunoassay kits (Phoenix Pharmaceuticals, CA, USA) were
used to measure the Ang II and Ang-(1--7) contents in the RV and lung.

\textbf{Statistical analysis}

The results obtained from ELISA and Western blots were analyzed by
densitometry and expressed as means ± standard error of the mean. All
data from the four groups were analyzed using one-way analysis of
variance (ANOVA) followed by Tukey's test. Comparison of the beneficial
efficacy of DIZE between the early treatment and the late treatment
protocols was conducted by two-way ANOVA (n= 6 rats/group). A P value
\textless{} 0.05 was considered statistically significant.

\textbf{Results}

\textbf{DIZE reduced RV systolic pressure and RV and plasma BNP}

Significant increases in the right ventricular systolic pressure (RVSP)
were observed in the SuPNx\textsubscript{42} groups when compared with
the sham-operated rats (Figure 2A). Both early
(SuPNx\textsubscript{42}/DIZE\textsubscript{1--42}) and late DIZE
treatment (SuPNx\textsubscript{42}/DIZe\textsubscript{29--42}) groups
decreased the RVSP when compared with the vehicle treatment group
(Figure 2A). The Fulton index, weight ratio of right ventricle to left
ventricle plus septum (RV/LV+S), in the SuPNx42 rats was significantly
increased than the sham-operated rats, which indicated the right
ventricle was hypertrophied in the SuPNx42 animals (Figure 2B). However,
the RV/LV+S ratio in either the early or late DIZE treatment group was
not different when compared to SuPNx\textsubscript{42} group (Figure
2B). There were no significant changes among all 4 groups in the lung or
body weight (Figures 2C and 2D).

Plasma BNP, an index of heart failure, was elevated significantly in the
SuPNx\textsubscript{42} group compared with the sham-operated rats. In
the early treatment group
(SuPNx\textsubscript{42}/DIZE\textsubscript{1--42}), DIZE resulted in
significant decreases in plasma BNP compared to that of vehicle-treated
SuPNx\textsubscript{42} rats, but no significant changes were seen in
the late treatment group
(SuPNx\textsubscript{42}/DIZE\textsubscript{29--42}) (Figure 3A).

Similar to the plasma BNP, RV BNP was also elevated significantly in
SuPNx\textsubscript{42} group compared with the sham-operated rats. Both
the early treatment group
(SuPNx\textsubscript{42}/DIZE\textsubscript{1--42}) and the late
treatment group (SuPNx\textsubscript{42}/DIZE\textsubscript{29--42})
displayed significant decreases in the levels of RV BNP compared to that
found in the vehicle-treated SuPNx\textsubscript{42} rats (Figure 3B).

\textbf{DIZE attenuated pulmonary vascular remodeling and smooth muscle
neointimal proliferation}

Using H-E staining, the medial wall thickness of pulmonary arterioles
(50--100 \selectlanguage{greek}μ\selectlanguage{english}m in diameter) was found to increase significantly in the
SuPNX\textsubscript{42} rats compared with that in sham-operated rats
(Figures 4A and 4D). Early DIZE treatment
(SuPNx\textsubscript{42}/DIZE\textsubscript{1-42}) significantly
attenuated the medial wall thickness of pulmonary arterioles, when
compared with the vehicle-treated rats (SuPNx\textsubscript{4})(Figures
4A and 4D). However, no significant changes in medial wall thickness
were observed in the
SuPNx\textsubscript{42}/DIZE\textsubscript{29-42}group when compared
with that of the SuPNx\textsubscript{42} rats.

In addition, Verhoeff-van Gieson staining was carried out for evaluating
the severity of neointimal formation (Figure 4B). The distribution of
vascular lesions and an average vascular occlusion score (VOS) are
presented in Figure 4E. The SuPNx\textsubscript{42} rats developed
severe pulmonary vascular remodeling with a VOS of 1.27\selectlanguage{ngerman}± 0.74. In the
early treatment group with DIZE, the VOS was significant decreased
(0.87± 0.54) compared to the SuPNx\textsubscript{42} rats
(\emph{p\textless{}0.05} ). There was also a trend of decrease in the
late treatment group with a VOS of 1.07 ± 0.79 when compared to the
SuPNx\textsubscript{42} rats\textsubscript{.}

\textbf{DIZE Regulated the expressions of eNOS, ACE, and ACE2/MAS Axis
in SuPNx Rats}

Figure 5A shows a representative pulmonary expression of eNOS, ACE,
ACE2, MAS by Western blot analysis. SuPNX\textsubscript{42} has the
trend to upregulated pulmonary eNOS expression when compared with the
sham-operated rats. Pulmonary expression of eNOS was significantly
elevated in the early treatment protocol when compared with the
SuPNx\textsubscript{42} groups, but this was not seen in the late
treatment protocol (Figure 5B).

Compared to the sham-operated rats, the ratio of pulmonary expression of
ACE was elevated in the SuPNx\textsubscript{42} group. Significant
decreases in ACE protein expression in both early and late DIZE
treatment groups were noted when compared with the vehicle-treated
SuPNx\textsubscript{42} rats. (Figure 5C)

Pulmonary expression of ACE2 was increased in the early treatment group
(SuPNX\textsubscript{42}/DIZE\textsubscript{1--42}) compared to the
SuPNx\textsubscript{42} rats. No significant differences were found
among the sham\textsubscript{42}, SuPNx\textsubscript{42}, and
SuPNX\textsubscript{42}/DIZE\textsubscript{29--42} groups (Figure 5D).

The Mas is a G protein-coupled receptor with its vasoprotective effect
in RAS system.\textsuperscript{14} In western blot analysis, there were
no significant changes in Mas expression among all four groups. (Figure
5E)

\textbf{DIZE upregulated RV and pulmonary Ang-(1-7) levels}

The RV and pulmonary tissue Ang-(1-7) levels were not changed in the
SuPNx\textsubscript{42} group when compared to the sham-operated rats
(Figures 6A and 6B). However, there was a significant increase of RV
Ang-(1-7) in both the early and late DIZE treatment groups when compared
with the vehicle-treated SuPNx\textsubscript{42} rats (Figures 6A and
6B). In addition, early DIZE treatment also increased the levels of
pulmonary Ang-(1-7) when compared with the SuPNx\textsubscript{42} group
(Figure 6B). No significant differences were found among the
sham\textsubscript{42}, SuPNx\textsubscript{42}, and
SuPNx\textsubscript{42}/DIZE\textsubscript{29--42} groups (Figure 6B).

\textbf{Discussion}

SU5416 was the first VEGF receptor 2 inhibitor to enter clinical
development for cancer therapy.\textsuperscript{26} It has been shown
that inhibiting the VEGF- and endothelial-dependent proliferation will
result in structural changes known as plexiform
lesions.\textsuperscript{10,26-29} Allowing for the selection of an
apoptosis resistant, proliferating endothelial cell phenotype by the
combination of blockade of VEGF receptor 2 and hypoxia, severe PAH will
develop.\textsuperscript{26} In this study, we reasoned that a pulmonary
insult such as pneumonectomy followed by injection with SU5416 will
yield similar results. In fact, our SuPNx model was shown to cause
severe angio-obliterative PAH associated with increased cell
proliferation and proapoptotic signaling, resulting in neointimal and
medial remodeling.\textsuperscript{11} In addition, unlike the hypoxia
model where partial reversibility of pulmonary hypertension is seen
after returning to normoxia, the SuPNx model is independent of hypoxic
vasoconstriction and hemoconcentration.\textsuperscript{11}

In our study, the RVSP in the SUPNx\textsubscript{42} rats increased
significantly, which may favor the assessment of drug effects in
preclinical trials. The RVSP improved significantly in both the early
and late DIZE treatment protocols, suggesting the effectiveness of
hemodynamic changes by DIZE in PAH. However, although the Fulton index
did increase in the SuPNx\textsubscript{42} rats, there were only trends
of improvement with DIZE in either the early or the late treatment
protocols. It is possible that, due to the severe pathologic changes in
this chronic PAH model, the observed hypertrophy in cardiomyocytes was
irreversible, leading to heart failure.

In addition to endothelin, RAS has also been implicated as a causative
factor in PAH.\textsuperscript{1} Ang II, a principal effector peptide
of the RAS, can exert deleterious effects on the pulmonary vasculature
resulting in vasoconstriction, proliferation, and inflammation, all of
which are contributable to the development of PAH. However, it is
difficult to measure the plasma and tissue levels of Ang II due to its
very short half-life (16 ± 1 s in mice).\textsuperscript{30} In
contrast, ACE, which catalyzes the conversion of Ang I~to Ang II, is
abundant in the small pulmonary arteries and is therefore more easily to
be detected \textsuperscript{31}. Thus, measuring ACE, instead of Ang II
levels in the lung, provides a more practical method for assessing the
associated hemodynamic changes. In our study, there were no significant
changes in pulmonary Ang II levels among the four animal groups.
However, the expression of pulmonary ACE was increased in
SuPNx\textsubscript{42} rats and ameliorated by DIZE in both early and
late treatment group, suggesting that ACE may be a representative marker
in in this animal model of PAH.

In the RAS, ACE/Ang II/AT1 constitutes the vasopressor arm, which is
counterbalanced by the ACE2/Ang-(1-7)/Mas receptor
axis.\textsuperscript{32} By converting Ang II to the vasodilatory
peptide Ang-(1--7), ACE2 provides a negative feedback on the RAS and
protects the major organs such as heart and kidneys from being damaged
by excessive Ang II generated during the development of
PAH.\textsuperscript{33,34} Interestingly, in the present study, the RV
and pulmonary levels of Ang-(1-7) were not significantly altered in the
SuPNx\textsubscript{42} group when compared with the sham-operated rats.
However, the RV levels of Ang-(1-7) were significantly elevated in both
the early and late DIZE treatment groups, and the pulmonary levels of
this peptide were also significantly increased in the early DIZE
treatment group.

It should be noted that, besides the action of ACE2, Ang-(1-7) can also
be formed by other biochemical pathways. It can be generated from
hydrolysis of angiotensin I by neprilysins (NEPs) or cleavage of the
Ang-(1-9) by ACE.\textsuperscript{35-38}~ With respect to its
metabolism, Ang-(1-7) can be subsequently degraded by ACE to form
Ang-(1-5), by dipeptidyl peptidase 3 (DPP3) to produce Ang-(3-7) and
Ang-(5-7), or by aminopeptidase A (APA) to generate
Ang-(2-7).\textsuperscript{38} Affecting the activity of any of the
aforementioned enzymes will undoubtedly result in a change of the levels
of Ang-(1-7). To this end, it is speculated other alternative pathways
may also influence the formation of Ang-(1-7) in our animal model.
Further studies are needed to fully elucidate the involved biochemical
pathways.

It has been reported that Ang-(1--7) can stimulate the releases of
endothelial derived nitric oxide (eNOS) and vasodilator prostaglandins
as well as potentiate the vasodilatory effect of
bradykinin.\textsuperscript{39-41} Consistent with these results, eNOS
was significantly elevated in our early DIZE treatment group when
compared to SuPNx\textsubscript{42} rats (Figure 5B), inferring that
Ang-(1-7) may stimulate eNOS release in this animal model. The increased
expression of eNOS could contribute to the lowering of pulmonary artery
pressure observed herein (Figure 2A).

In summary, our model of hypertensive pulmonary vascular disease in
pneumonectomized, SU5416-injected rats resemble the neointimal
proliferation and vascular occlusion by smooth muscle cells that occurs
in human PAH. The efficacy of DIZE in the early and late treatment group
suggests its ability to rescue animals from established hypertensive
pulmonary vascular disease. However, the exact mechanisms by which DIZE
exerts its beneficial effects remain to be investigated. From our
results, it is likely that attenuation of PAH by DIZE involves a
combination of antiproliferative effects on pulmonary vascular smooth
muscle cells through production of Ang-(1-7), suppression of the growth
of vascular smooth muscle cells, and also induction of endothelial cell
eNOS expression.\textsuperscript{41,42}

\textbf{Conclusion}

The present rat model of PAH showed elevated ACE protein levels in the
lung as well as histopathological findings such as Increased intimal and
medial fractions accompanied by obliterative lesions. DIZE, an ACE2
activator, reduced RVSP, medial wall thickness, and intimal fraction in
pulmonary arterioles. In addition, it upregulates pulmonary expression
of the ACE2/Ang-(1--7)/Mas receptor axis, leading to a therapeutic
effect in this rat model of PAH. Our study showed that DIZE may be a
potential agent for the treatment of PAH.

\textbf{Competing interests}

The authors state no conflict of interest.

\textbf{Acknowledgments}

This study was supported by grants from Ministry of Science and
Technology, R.O.C (MOST 106-2314-B-037 -078 and 105-2314-B-037 -011) and
Kaohsiung Medical University Hospital (KMUH105-5R36 and KMUH106-6R43).

The authors thank Mr. Chao-Hsiung Chen and Chia-An Yeh for technical
support.

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\textbf{Figure legends}

\textbf{Figure 1.}

The design of animal study. In the early treatment protocol, there were
sham-operated rats, SU5416 combined with left pneumonectomy rats
(SuPNx\textsubscript{42}), and SuPNx\textsubscript{42} rats given with
15 mg DIZE /kg/day from day 1 to day 42
(SuPNx\textsubscript{42}/DIZE\textsubscript{1-42}). In the late
treatment protocol, there were sham-operated rats (sham42),
SuPNx\textsubscript{42}, and a SuPNx\textsubscript{42} rats given with
15 mg DIZE /kg/day from day 29 to day 42
(SuPNx\textsubscript{42}/DIZE\textsubscript{29-42})

SuPNx: Su5416 with left pneumonectomy

DIZE: diminazene aceturate

\textbf{Figure 2} Comparison of (A) right ventricle systolic pressure
(RVSP), (B) Fulton index, the weight ratio of right ventricle to left
ventricle plus septum, (RV/LV+S), (C) the lung weight and (D) the body
weight among the sham\textsubscript{42}, SuPNx\textsubscript{42},
SuPNx\textsubscript{42}/DIZE\textsubscript{1-42} and
SuPNx\textsubscript{42}/DIZE\textsubscript{29-42} groups. RVSP levels
were significantly higher in the SuPNx\textsubscript{42} group (45.17 ±
4.62 mmHg) than in sham-operated rats. (18.37 ± 1.36 mmHg,\emph{p}
\textless{}0.001) Both DIZE treatment groups
(SuPNx\textsubscript{42}/DIZE\textsubscript{1--42} and
SuPNx\textsubscript{42}/DIZE\textsubscript{29--42}) showed significant
decreases in RVSP when compared with that of the
SuPNx\textsubscript{42}rats. (early, 25.10 ± 1.38 mmHg, \emph{p}
\textless{}0.01; late 31.23 ± 4.06 mmHg, \emph{p} \textless{}0.05) The
Fulton index was significantly increased in the SuPNx\textsubscript{42}
group (0.55 ± 0.06 ) than in sham-operated group (0.35 ± 0.04, \emph{p}
\textless{}0.05), but no significant changes were observed in the
SuPNx\textsubscript{42}/DIZE\textsubscript{1-42} and
SuPNx\textsubscript{42}/DIZE\textsubscript{29-42} groups when compared
with the SuPNx\textsubscript{42} group. There was no significant change
in either the lung or body weight in all groups. Values represent the
mean ± SEM. *\emph{P} \textless{}0.05, **\emph{P} \textless{}0.01,
***\emph{P} \textless{}0.001

\textbf{Figure 3} (A) The plasma brain natriuretic peptide (BNP) and (B)
RV BNP levels. The plasma BNP level was significantly higher in the
SuPNx\textsubscript{42} rats than in sham-operated rats.( 130.2 ± 19.76
vs. 59.50 ± 7.74 pg/ml; \emph{P} \textless{}0.001) Treatment with DIZE
for 42 days significantly lowered the plasma BNP levels when compared
with that of the vehicle-treated group.(31.14 ± 2.83 vs. 130.2 ± 19.76
pg/ml; \emph{P} \textless{}0.05) There was a trend that the
SuPNx\textsubscript{42}/DIZE\textsubscript{29--42} rats had a decreased
plasma BNP level when compared with the SuPNx\textsubscript{42} rats,
but the difference was not statistically significant. The RV BNP was
significantly higher in SuPNx\textsubscript{42} rats than that of the
sham\textsubscript{42} rats. (103.4 ± 10.01 vs. 31.05 ± 2.57
pg/ml;\emph{P} \textless{}0.0001) Both DIZE treatment groups had
significantly lowered RV BNP than in the SuPNx\textsubscript{42} rats.
(early: 46.82 ± 6.62 pg/ml vs. 103.4± 10.01 pg/ml, \emph{P}
\textless{}0.0001; late: 61.83 ± 5.10 pg/ml vs. 103.4± 10.01 pg/ml,
\emph{P} \textless{}0.001) Values represent the mean ± SEM. *\emph{P}
\textless{}0.05; ***\emph{P} \textless{}0.001, ****\emph{P}
\textless{}0.0001

\textbf{Figure 4} The representative results of Hematoxylin-eosin
stating of lung tissue (A), Verhoeff-van Gieson staining (B), and double
immunohistochemical staining of smooth muscle actin (green) and PCNA
(brown) (C). (D) The ratio of medial wall thickness of pulmonary
arterioles. The value for the sham\textsubscript{42} group was set at
1.0. The muscular layer of pulmonary arterioles (50--100 \selectlanguage{greek}μ\selectlanguage{english}m in diameter)
was significantly thicker in the SuPNx\textsubscript{42} rats than in
sham\textsubscript{42},
SuPNx\textsubscript{42}/DIZE\textsubscript{1-42}, but not
SuPNx\textsubscript{42}/DIZE\textsubscript{29--42} rats. (E) the
vascular occlusion score (VOS). The VOS of early treatment group
(SuPNx\textsubscript{42}/DIZE\textsubscript{1-42}) was decreased
compared to SuPNx\textsubscript{42}.( 1.27 \selectlanguage{ngerman}± 0.74 vs. 1.07 ± 0.56)
(magnification, 200x. Values represent the mean ± SEM.
*P\textless{}0.05, **P\textless{}0.01.

\textbf{Figure 5} (A) A representative Western blot of eNOS, ACE, ACE2,
and the MAS receptor in sham\textsubscript{42},
SuPNx\textsubscript{42,}SuPNx\textsubscript{42}/DIZE\textsubscript{1-42}
and SuPNx\textsubscript{42}/DIZE\textsubscript{29-42} rats. (B) The
normalized eNOS/actin ratio revealed the expression of eNOS was higher
in the SuPNx\textsubscript{42}/DIZE\textsubscript{1--42} rats than in
SuPNx\textsubscript{42} rats. (C) The normalized ratio of ACE/actin
showed that the expression of ACE was higher in
SuPNx\textsubscript{42}rats than in sham\textsubscript{42} rats. DIZE
administration decreased ACE expression in the early treatment
(SuPNx\textsubscript{42}/DIZE\textsubscript{1--42}) and the late
treatment (SuPNX\textsubscript{42}/DIZE\textsubscript{29--42}) groups.
(D) The normalized ratio of ACE2/actin revealed that an increase in the
early treatment group
(SuPNX\textsubscript{42}/DIZE\textsubscript{1--42}) compared to the
SuPNx\textsubscript{42} rats. No significant differences were found
among the sham\textsubscript{42}, SuPNx\textsubscript{42}, and
SuPNX\textsubscript{42}/DIZE\textsubscript{29--42} groups. (E) The
normalized MAS/actin ratios were not changed in the all groups. The
value for the sham\textsubscript{42} group was set at 1.0 in Figures 5B
-- 5E. Values represent the mean ± SEM *\emph{P} \textless{}0.05,
**\emph{P} \textless{}0.01

\textbf{Figure 6} (A) The levels of RV Ang-(1-7). The level of RV
Ang-(1-7) was significantly higher in the
SuPNx\textsubscript{42}/DIZE\textsubscript{1--42} and
SuPNx\textsubscript{42}/DIZE\textsubscript{29--42} rats than in
SuPNx\textsubscript{42} rats. (B) The levels of pulmonary Ang-(1-7). The
expression of pulmonary Ang-(1-7) was significantly higher in
SuPNx\textsubscript{42}/DIZE\textsubscript{1--42} rats than in
SuPNx\textsubscript{42} rats. Values represent the mean ± SEM. *\emph{P}
\textless{}0.05 ***P\textless{}0.001, ****P\textless{}0.0001

\textbf{Figure S1}

The rat model of left pneumonectomy followed by SU5416 injection.
Remodeling of neointimal occlusive lesions was observed in this animal
model. It has plexiform-like lesions by allowing for the endothelial
cell hyperproliferation, a distinct feature shown in human PAH.\selectlanguage{english}
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