Soluble guanylate cyclase stimulator, trans-4-methoxy-β-nitrostyrene, has a beneficial effect in monocrotaline-induced pulmonary arterial hypertension in rats

Article abstract of DOI:10.1016/j.ejphar.2021.173948

The soluble guanylate cyclase (sGC)/GMPc pathway plays an important role in controlling pulmonary arterial hypertension (PAH). We investigated whether the novel sGC stimulator trans-4-methoxy-β-nitrostyrene (T4MN), ameliorates monocrotaline (MCT)-induced

Full text of DOI:10.1016/j.ejphar.2021.173948

European Journal of Pharmacology 897 (2021) 173948
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Soluble guanylate cyclase stimulator, trans-4-methoxy-β-nitrostyrene, has a
beneficial effect in monocrotaline-induced pulmonary arterial hypertension
in rats
Karoline Gonzaga-Costaa, Alfredo Augusto Vasconcelos-Silvaa,
a
b
c
´
˜
´
Matyelle Jussara Rodrigues-Silva , Conceiçao da Silva Martins Rebouça , Gloria Pinto Duarte ,
d
a
a,*
˜
Rosivaldo Santos Borges , Pedro Jorge Caldas Magalhaes , Saad Lahlou
a
´
Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceara, CE, Brazil
b
´
Department of Morphology, Lab Nempi, School of Medicine, Federal University of Ceara, CE, Brazil
c Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, PE, Brazil
d
´
´
Department of Pharmacy, Federal University of Para, Belem, PA, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
The soluble guanylate cyclase (sGC)/GMPc pathway plays an important role in controlling pulmonary arterial
hypertension (PAH). We investigated whether the novel sGC stimulator trans-4-methoxy-β-nitrostyrene (T4MN),
ameliorates monocrotaline (MCT)-induced PAH. At Day 0, rats were injected with MCT (60 mg/kg, s. c.). Control
(CNT) rats received an equal volume of monocrotaline vehicle only (s.c.). Four weeks later, MCT-treated rats
were orally treated for 14 days with T4MN (75 mg/kg/day) (MCT-T4MN group) or its vehicle (MCT-V group),
and with sildenafil (SIL; 50 mg/kg) (MCT-SIL group). Compared to the CNT group, MCT treatment induced a
significant increase in both the Fulton index and RV systolic pressure but significantly reduced the maximum
relaxation induced by acetylcholine. Indeed, MCT treatment increased the wall thickness of small and larger
pulmonary arterioles. Oral treatment with T4MN and SIL reduced the Fulton index and RV systolic pressure
compared to the MCT-V group. Maximum relaxation induced by acetylcholine was significantly enhanced in
MCT-SIL group. Both T4MN and SIL significantly reduced the enhanced wall thickness of small and larger
pulmonary arterioles. Treatment with T4MN has a beneficial effect on PAH by reducing RV systolic pressure and
consequently right ventricular hypertrophy, and by reducing pulmonary artery remodeling. T4MN may represent
a new therapeutic or complementary approach for the treatment of PAH.
Endothelial dysfunction
Pulmonary arterial hypertension
Pulmonary vascular remodeling
Right ventricle hypertrophy
Soluble guanylate cyclase
Trans-4-methoxy-β-nitrostyrene
1. Introduction
complex and multifactorial. To date, three pharmacological targets have
been explored in drug therapy because they interfere with endothelin-1
`
Pulmonary arterial hypertension (PAH) is a severe, progressive dis-
ease of small pulmonary arteries, which if left untreated, usually cul-
minates in right heart failure and death. One of the main causes of PAH
is increased pulmonary vascular resistance (PVR), which results from
sustained vasoconstriction, excessive pulmonary vascular remodeling, in
situ thrombosis, and increased pulmonary vascular stiffness (Peacock,
1999; Steiner et al., 2005). From a hemodynamic point of view, PAH
corresponds to a sustained increase in mean pulmonary arterial pressure
above 25 mmHg at rest compared to normal values of 10–18 mmHg for a
healthy adult. However, mechanisms involved in PAH pathogenesis are
(ET-1), prostacyclin and nitric oxide (NO) signaling pathways (Galie
et al., 2013). This allowed development of approved drugs to inhibit the
ET-1 pathway (ET-1 receptor antagonists) or to activate the prostacyclin
(e.g., prostacyclin), or NO pathways (inhalation of NO, PDE5 inhibitors,
and soluble guanylate cyclase (sGC) stimulators and activators).
Endothelial dysfunction is reported as an important factor in the
early development of PAH (Morrell et al., 2009). The NO/sGC/cGMP
signaling pathway regulates vascular tone and pulmonary resistance in
PAH (Arnold et al., 1977; Evgenov et al., 2006) as well as vascular cell
proliferation, platelet aggregation, and leukocyte recruitment (vascular
´
* Corresponding author. Department of Physiology and Pharmacology, Laboratory of Cardiovascular Pharmacology; Federal University of Ceara, School of
´
Medicine, Rua Cel. Nunes de Melo 1315, Rodolfo Teofilo, Fortaleza, CE, 60430-270, Brazil.
E-mail addresses: lahlou562@gmail.com, saad.lahlou@ufc.br (S. Lahlou).
https://doi.org/10.1016/j.ejphar.2021.173948
Received 7 November 2020; Received in revised form 28 January 2021; Accepted 10 February 2021
Available online 17 February 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.

K. Gonzaga-Costa et al.
European Journal of Pharmacology 897 (2021) 173948
remodeling), which are involved in pathogenesis of PAH (Stasch et al.,
2011). To date, the sGC stimulator, riociguat (BAY 63–252), is currently
the only treatment available for two types of pulmonary hypertension:
idiopathic PAH and inoperable chronic thromboembolic pulmonary
for the Care and Use of Laboratory Animals of the Brazilian National
Council for Animal Experimentation. All animal experiments were
reviewed and approved by the Commission for the Ethical Use of Ani-
´
mals (CEUA) of the Federal University of Ceara (96/2016).
`
hypertension (Ghofrani et al., 2013; Galie et al., 2015; Khouri et al.,
2018).
2.3. Solutions and drugs
Nitroderivatives found in higher plants are rare. Previously, we
showed that 1-nitro-2-phenylethane (NPa), the unique nitro compound
The perfusion medium used was a fresh modified Krebs-Henseleit
solution (KHS, pH 7.4) of the following composition (in mM): NaCl
118; KCl 4.7; NaHCO3 25; CaCl2.2H2O 2.5; KH2PO4 1.18; MgSO4.7H2O
1.18; glucose 11. Salts were purchased from Merck (Darmstradt, Ger-
many) and Vetec (Rio de Janeiro, Brazil). Phenylephrine (PHE) hydro-
chloride, acetylcholine (ACh) chloride, sildenafil (SIL) and MCT were
purchased from Sigma. PHE and ACh were dissolved in distilled water
and then with KHS to achieve desired concentration in the bath cham-
ber. T4MN was dissolved in DMSO and ethanol, brought up to the
desired concentration using saline solution, and sonicated just before
use. MCT was initially dissolved in 0.5 N HCl, and the pH was adjusted to
7.4 with 0.5 N NaOH, brought up to the desired concentration using
saline solution. SIL was dissolved in distilled water.
˜
isolated from plants (Gottlieb and Magalhaes, 1960), induced vaso-
relaxation in isolated aortic and small resistance artery preparations
from both normotensive and spontaneously hypertensive rats (Inter-
aminense et al., 2013; Brito et al., 2013). This effect involves activation
of soluble guanylate cyclase (sGC)/cGMP in a nitric oxide (NO)-inde-
pendent manner, resulting in increased intracellular cGMP levels (Brito
et al., 2013). Conformational restriction of NPa resulted in formation of
1-nitro-2-phenylethene (NPe), a β-nitrostyrene derivative. NPe was
about 3.5 times more potent as a vasorelaxant agent than its parent drug,
NPa (Arruda-Barbosa et al., 2014).
In order to assess the influence of electronic structural modifications,
we synthesized several new nitroderivatives in which electron donors
were bonded in the aromatic ring of NPe. Little is known about the
pharmacological activities of the methoxy derivative, trans-4-methoxy-
β-nitrostyrene (T4MN; Fig. 1). Previously, we showed that T4MN dis-
played vasorelaxant activity in aortic rings through a mechanism similar
to that described for NPa (Arruda-Barbosa et al., 2017). In the present
study, we assessed whether T4MN treatment reverses PAH induced by
monocrotaline (MCT) in rats. This model of MCT-induced PAH is closely
mimics processes occurring (Campian et al., 2006).
2.4. Induction of pulmonary hypertension in rats and experimental design
PAH was induced by a single subcutaneous (s.c.) injection of MCT at
a dose of 60 mg/kg. Rats were randomly divided into three experimental
groups (n = 5–8 per group): Control (CNT) rats (received one single s. c.
Injection of MCT’s vehicle at Day 0, n = 5), MCT-V rats (received MCT at
Day 0 and from Day 29 after MCT injection were orally treated with
T4MN’s vehicle once a day for two weeks, n = 8), MCT-T4MN rats
(received MCT at Day 0 and from Day 29 after MCT injection were orally
treated with 75 mg/kg T4MN once a day for two weeks, n = 6), and
MCT-SIL (received MCT at Day 0 and from Day 29 after MCT injection
were orally treated with 50 mg/kg SIL once a day for two weeks, n = 6).
SIL was used as positive control (Yoshiyuki et al., 2016). The dose of 75
mg/kg of T4MN was based upon our previous investigation regarding
the anti-inflammatory activity of NPa in mice (Vale et al., 2013). Rats of
all groups were examined on Day 42 of the study. In all experimental
groups, body weight (BW) was determined daily throughout treatment
and the dose of T4MN was adjusted accordingly.
2. Materials and methods
2.1. Synthesis of trans-4-methoxy-β-nitrostyrene
Synthesis of T4MN or 1-((E)-2-nitro-vinyl)-(4-methoxy)-benzene was
´
performed at the Department of Pharmacy, Federal University of Para,
´
Belem, employing the Claisen–Schmitd’s procedure (Vogel, 1989; Ford
et al., 1994) with p-anisaldehyde and nitromethane as substrates (0.1
and 0.12 eq., respectively). The aromatic aldehyde was converted in a
‘onepot’ procedure, with a 96% yield, to the corresponding β-nitro-
styrene by treatment with 0.05 eq. of NaOH in methanol and water at
0–10 ◦C (1:2) (Rosini, 1991). The resulting precipitate was filtered and
dried under vacuum to yield the desired β-nitrostyrene derivative,
T4MN. The trans product is preferable to the cis form due to a stereo-
selective reaction that gives a low energy product. T4MN was crystal-
lized in ethanol as yellow solid-crystals, m. p. 86.6–88.2 ◦C (86–88 ◦C;
Sigma-Aldrich standard). The final product was identified by NMR (1H
and 13C NMR) and FT-IR spectroscopy and compared with properties in
the literature (Wang and Wang, 2002). IR νmax (cmꢀ 1) 806.25, 1624.06,
1456.26, 2900, 1600, 1550, 1498, 1375; 1H NMR (CD3SOCD3, 300
MHz) δ 7.87 (dd, 2 H), 7.52 (dd, 2 H), 7.47 (d, 1 H), 6.87 (d, 1 H), 3.76
(s, 3 H); 13C NMR (CD3SOCD3, 75 MHz) δ 162.89, 139.12, 135.09,
131.33, 122.48, 114.87, 55.50.
2.5. Invasive hemodynamic measurement
Rats were anesthetized with sodium pentobarbital (60 mg/kg, i. p.).
The degree of anesthesia was assessed by pinching the animal’s paw
with forceps and by rapid eye movements. The trachea was cannulated.
For recording right ventricle systolic pressure (RVSP), a polyethylene
catheter (PE-50) was inserted into the right jugular vein and advanced
until large positive pulsations appeared, indicating that it was placed in
the right ventricle (RV). This catheter was fixed with a suture and its
position was verified by postmortem examination. RVSP was measured
using a pressure transducer (Statham P23 DI) coupled to a data acqui-
sition system (PowerLab 8/30, model ML870, ADInstruments, Bella
Vista, NSW, Australia). RVSP tracings corresponding to an average of
15–20 pressure cycles were recorded and analyzed offline using Lab-
chart Reader version 8.1.5. At the end of the hemodynamic measure-
ment, rats were killed by exsanguination under anesthesia, and the lungs
and heart were quickly removed in a block and washed in KHS. The
thoracic aorta artery was removed to study vascular reactivity, and the
heart was removed to assess RV hypertrophy while the lungs were
removed to assess pulmonary arterial remodeling.
2.2. Animals
Adult male Wistar rats (240–300 g) from our breeding facility were
kept under conditions of constant temperature (22 ± 2 ◦C) with a 12 h
light/12 h dark cycle. Rats had access to normal rodent diet and tap
water ad libitum. All animals were handled in accordance with the Guide
2.6. Vascular reactivity
This series of experiments was conducted to evaluate whether
treatment with T4MN reverses endothelial dysfunction in the MCT-
induced PAH model. For this purpose, the thoracic aorta was removed
Fig. 1. Chemical structure of trans-4-methoxy-β-nitrostyrene.
2

K. Gonzaga-Costa et al.
European Journal of Pharmacology 897 (2021) 173948
and immersed in perfusion medium at room temperature. After removal
of adipose tissue and adhering connective tissue, the aorta was cut into
rings (approximately 2–3 mm in length) which were gently suspended
with two stainless-steel hooks passed through the vessel lumen in a 5-mL
variance (ANOVA), followed by Dunnett’s multiple comparison test as
appropriate.
3. Results
◦
organ bath containing KHS at 37 C. They were continuously aerated
with a mixture of 95% O2 and 5% CO2 (pH = 7.4). Isometric tension was
recorded using an isometric force transducer (ML870B60, AD In-
struments, Bella Vista, Australia) connected to an acquisition system
(PowerLab 8/30, ML870 model, ADInstruments, Bella Vista, NSW,
Australia). Tissues were equilibrated for 60 min under a resting tension
of 1 g. After the equilibration period, aortic rings underwent another 1 h
of stabilization during which MKHS was replaced every 15 min with
fresh solution to remove metabolites. Tissues were then challenged with
75 mM KCl to confirm tissue viability. Initially, each aortic ring was
contracted by exposure to 60 mM KCl and experiments were only con-
ducted when reproducible contractions were obtained. Aortic rings were
3.1. Rat survival
Survival curves of the four experimental groups are shown in Fig. 2.
It is noteworthy that, in the MCT-T4MN group, two rats died at the end
of 42 days of follow-up which may indicate increased survival time.
However, comparison of the survival rate of MCT-V with respect to
MCT-T4MN and MCT-SIL groups using the Logrank test did not reveal a
significant difference among the groups (P > 0.05).
3.2. Morphometric data
challenged with 1
μg ACh to evaluate integrity of the endothelium
As expected, RV weight to BW ratio (Fig. 3A) and Fulton index
(Fig. 3B), that is the right ventricle to left ventricle plus septum ratio,
were significantly increased in the MCT group compared to CNT rats (P
< 0.001), indicating the existence of RV hypertrophy. Both these
measured parameters were significantly reduced by oral treatment with
T4MN and SIL (Fig. 3; P < 0.05). The significant increase (Fig. 4; P <
0.01) of lung to BW ratio in the MCT group compared to CNT group
indicates the development of pulmonary inflammation, an effect that
was abolished in the MCT-SIL group (Fig. 4; P < 0.05), but not in the
MCT-T4MN group (Fig. 4; P > 0.05).
before any experiment. Preparations were considered to possess intact
endothelium, when the vasorelaxant response to ACh was greater than
75% from the plateau of PHE-induced contraction. The relaxing effects
of increasing ACh (10ꢀ 9 to 10ꢀ 5 M) concentrations on sustained PHE (1
μ
M) contractions were studied in endothelium-intact aortic rings from
the three experimental groups. Data were expressed as % reversal of
contraction induced by PHE.
2.7. Assessment of right ventricle hypertrophy
Right ventricular hypertrophy was assessed by determining the
Fulton index (Fulton et al., 1952). During dissection of the cardiac
chambers, the RV was isolated from the left ventricle (LV) and septum
(S), and the latter form a single set. The RV hypertrophy index was
calculated as RV/LV + S weight ratio and by the RV to BW ratio (Kaur
et al., 2015). The lungs were weighted and the lung to BW ratio was also
determined.
3.3. Hemodynamic parameters
RVSP was fourfold higher in MCT-V group (Fig. 5; P < 0.001) than
that recorded in the CNT group, indicating the presence of very severe
PAH. Oral treatment with T4MN or SIL for 14 days significantly reduced
(P < 0.01) the enhanced RVSP (Fig. 5A and B).
3.4. Reactivity vascular data
2.8. Pulmonary artery remodeling
In endothelium-intact aortic rings from the CNT group, increasing
concentrations of ACh induced concentration-dependent vasorelaxation
The right and left pulmonary lobes were cut longitudinally and
processed for dehydration, and embedding in paraffin blocks. Blocks
with an IC50 of 1.07 ± 0.33
μM (Fig. 6). This vasorelaxation was
were cut into 4-μm sections and stained with hematoxylin and eosin and
attenuated in the MCT-V group as evidenced by the enhanced IC50 value
of ACh-induced vasodilation (>10 M). Maximal relaxation responses
from all experimental groups are shown in Table 1. Oral treatment with
SIL, restored the IC50 (0.66 ± 0.21 M) (Fig. 6) as well as the maximal
examined using light microscopy (NIKON Eclipse E200, Japan) by an
investigator who was blinded to the treatment groups to qualitatively
assess vascular remodeling that occurs in the MCT-induced PAH model.
μ
μ
Diameters of the vessels analyzed ranged between <50 μm and 50–100
μ
m. Sixty vessels with comparable outer diameters were randomly
measured from each group. From the images scanned with ImagenJ
(5.0), measurements included the outer and inner circumferences of the
vessels, with areas delimited by the outer and inner elastic laminae,
respectively. Vessel diameter was calculated from the medial circum-
ference, and the vascular wall area was calculated by subtracting the
luminal circumference from the outer medial circumference. Wall
thickness was expressed as a percentage of the total vessel area, ac-
cording to the following formula: [(outer area) - (inner area)/outer
area]/100.
2.9. Statistical analysis
Results are expressed as means ± S.E.M.. Survival analysis was
performed using Kaplan-Meier survival analysis. Pairwise comparisons
between survival groups were performed using the Log-rank test. IC50
Fig. 2. Treatment with trans-4-methoxy-β-nitrostyrene failed to enhance sur-
vival rate. Survival rate was significantly decreased in MCT-injected animals
treated with vehicle (MCT-V) or with sildenafil (MCT-SIL). In rats injected with
MCT and treated orally with trans-4-methoxy-β-nitrostyrene (T4MN; 75 mg/kg/
day; MCT-T4MN group), two rats died on the last day (Day 42) of MCT treat-
ment. No rats in the control (CNT) group died. MCT: monocrotaline; s. c.:
subcutaneous; V; vehicle.
values, defined as the concentration (μM) of ACh required to produce a
half-maximal reduction of the PHE-induced contractile response, were
calculated by interpolation from semi-logarithmic plots. Statistical
analysis was performed using GraphPad Prism 5 (GraphPad Software,
Inc., La Jolla, CA). Significance (P < 0.05) of results was assessed by
paired Student’s t-test, Mann-Whitney U test, and one-way analysis of
3

K. Gonzaga-Costa et al.
European Journal of Pharmacology 897 (2021) 173948
arterioles in the four experimental groups. In pulmonary arterioles with
an external diameter <50 μm, wall thickness was significantly (Fig 7C; P
< 0.001) increased in the MCT-V group compared with CNT rats, indi-
cating MCT-induced pulmonary vascular remodeling. Oral treatment
with T4MN and SIL significantly reduced (P < 0.001) the enhanced wall
thickness of these vessels. In larger pulmonary arterioles (50–100 μm)
from MCT-injected rats, wall thickness was also enhanced (Fig 7D; P <
0.001), an effect that was also significantly reduced by T4MN and SIL
treatment (Fig. 7D; P < 0.01).
4. Discussion
In MCT-T4MN, survival time was increased with respect to MCT-V
and MCT-SIL groups (Fig. 2). However, comparison of survival rate of
MCT-V with respect to MCT-T4MN and MCT-SIL groups by means of the
Logrank test did not reveal a significant difference among the groups.
The apparent increased survival would have reflected beneficial effects
of T4MN treatment on cardiac and pulmonary vessel remodeling, and
ventricular hypertrophy as well as on increased RVSP. Given the vaso-
dilatory properties of T4MN through stimulation of the sGC/cGMP
pathway by the vascular endothelium (Arruda-Barbosa et al., 2017), we
investigated whether treatment with T4MN could normalize hemody-
namic changes and cardiac hypertrophy in this model of PAH. In our
experimental conditions, chronic treatment with T4MN for 14 days after
the establishment of PAH was able to prevent RV hypertrophy, reduce
pulmonary vascular remodeling, and improve RV function in animals
with PAH.
Fig. 3. Treatment with trans-4-methoxy-β-nitrostyrene reduced right ventricle
hypertrophy. Morphometric data collected in control (CNT) rats, those injected
only with monocrotaline (MCT; 60 mg/kg, s. c.) (MCT-V group) and those
injected with MCT and treated orally with trans-4-methoxy-β-nitrostyrene
(T4MN; 75 mg/kg/day) (MCT-T4MN group) or sildenafil (SIL; 50 mg/kg/day)
(MCT-SIL group) during 14 the days from Day 29 after MCT injection. In A,
right ventricle weight (RVW) to body weight (BW) ratio. In B, Fulton index, i.e.
the right ventricle to left ventricle plus septum weight ratio. Data are expressed
as means S.E.M. (n = 4–5 rats per group). *P < 0.001, with respect to CNT
group; #P < 0.05, ##P < 0.01 with respect to MCT-V group, by one-way ANOVA
followed by Dunnett’s test..
Experimental models of PAH are widely used to study and under-
stand the pathophysiological mechanisms of this disease, and the use of
monocrotaline (MCT) for its induction is a well-established model. MCT
induces injury to pulmonary capillaries with increased PVR and ven-
tricular afterload, progressively causing pathological remodeling of the
RV with the induction of hypertrophy, increased interstitial fibrosis,
endothelial dysfunction, and heart failure (Pacagnelli et al., 2016). The
experimental model of MCT-induced PAH employed in the current study
is valid and could be comparable to the clinical situation since,
compared to the CNT group, animals from the MCT-V group had right
ventricular hypertrophy, pulmonary edema, increased RVSP, pulmo-
nary vascular remodeling, and endothelial dysfunction.
Compared to the CNT group, animals treated with MCT showed an
increased Fulton index and RV to BW ratio, indicating development of
RV hypertrophy that may worsen RV systolic function. Oral T4MN, as
well as SIL, treatment reduces pathological MCT-induced cardiac ven-
tricular remodeling since the RV to BW ratio and Fulton index in the
MCT-T4MN and MCT-SIL groups were statistically reduced compared to
those recorded in MCT-V group. Regarding pulmonary edema in-
dicators, initial inflammation and alveolar damage followed later by
fibrosis and pulmonary vascular remodeling are changes of MCT-
promoted pulmonary histopathological findings, which within twenty-
one days, lead to an increase in capillary permeability, resulting in
interstitial and alveolar edema (Dumitrascu et al., 2008). Compared
with the CNT group, the lung to BW ratio in the MCT-V group was
significantly increased, as was expected. A 14-day SIL treatment
significantly reduced the MCT-induced increase in lung to BW ratio, an
effect that was not observed in PAH rats treated with T4MN.
Fig. 4. Treatment with trans-4-methoxy-β-nitrostyrene failed to alter lung hy-
pertrophy. Lung to body weight ratio in control (CNT) rats, those injected only
with monocrotaline (MCT; 60 mg/kg, s. c.) (MCT-V group) and those injected
with MCT and treated orally with trans-4-methoxy-β-nitrostyrene (T4MN; 75
mg/kg/day) (MCT-T4MN group) or sildenafil (SIL; 50 mg/kg/day) (MCT-SIL
group) during 14 days from the Day 29 after MCT injection. Data are expressed
as means S.E.M. (n = 4–5 rats per group). *P < 0.05, **P < 0.01 with respect to
CNT group; #P < 0.01 with respect to MCT-V group, by one-way ANOVA fol-
lowed by Dunnett’s test.
Endothelial dysfunction is reported as an important factor in the
early development of PAH (Morrell et al., 2009). In the present study,
evaluation of endothelial dysfunction was performed using thoracic
aorta although a distinct tissue sensitivity to MCT (aorta vs. pulmonary
aorta) has been reported (Gout et al., 1999). MCT significantly impaired
ACH-induced aortic preparations evidenced by a significant right shift of
the concentration-response curve and a reduction of Emax and increased
IC50 compared with the control group. This is in agreement with pre-
vious studies using thoracic aorta in PAH (Zapata-Sudo et al., 2012;
Steven et al., 2017). Sildenafil treatment significantly improved the
endothelial function; however, T4MN treatment failed to improve the
relaxation response to ACh (Table 1). However, oral treatment with
T4MN failed to reverse the endothelial dysfunction (Fig. 6) as the
enhanced maximal ACh relaxation evoked by T4MN did not reach sta-
tistical significance (Table 1).
3.5. Vascular morphology of pulmonary arterioles
Fig. 7A and B provide representative images of pulmonary terminal
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K. Gonzaga-Costa et al.
European Journal of Pharmacology 897 (2021) 173948
Fig. 5. Treatment with 1 trans-4-methoxy-β-nitrostyrene reduced the monocrotaline-induced increased right ventricle systolic pressure. In A, representative tracings
of right ventricle systolic pressure (RVSP) in control (CNT) rats, and in rats with monocrotaline (MCT)-induced pulmonary arterial hypertension that had been treated
orally for two weeks following Day 29 after MCT injection with trans-4-methoxy-β-nitrostyrene (T4MN; 75 mg/kg/day) (MCT-T4MN group) or its vehicle (MCT-V
group), or with sildenafil (SIL; 50 mg/kg/day) (MCT-SIL). In B, RVSP was fourfold higher than in the CNT group, indicating severe PAH. Oral treatment with T4MN
and SIL reduced the MCT-induced increased RVSP. Data are expressed as means ± S.E.M. (n = 4–5 rats per group). **P < 0.001 with respect to the CNT group; #P <
0.01 with respect to MCT-V group by one-way ANOVA followed by Dunnett’s test.
Table 1
Oral treatment with trans-4-methoxy-β-nitrostyrene failed to reverse the endo-
thelial dysfunction of aortic rings from monocrotaline (MCT)-treated rats. IC50
and maximum vasorelaxation (Rmax) to acetylcholine (10ꢀ 9 to 10ꢀ 5 M) in aortic
rings from control (CNT) rats and those with pulmonary arterial hypertension
(PAH) treated (MCT-T4MN) or not (MCT-V) with trans-4-methoxy-β-nitro-
styrene (T4MN) or those with PAH treated with sildenafil (MCT-SIL) used as
positive control.
Acetylcholine
Groups
IC50
(μ
M)
Rmax (%)
CNT
1.07 ± 0.33
>10
71.63 ± 4.08
35.92 ± 7.45a
47.05 ± 6.60a
69.19 ± 3.89b
MCT-V
MCT-T4MN
MCT-SIL
>10
0.66 ± 0.21
Fig. 6. Treatment with trans-4-methoxy-β-nitrostyrene failed to improve
endothelial function. Concentration-response curves for relaxation induced by
acetylcholine from control (CNT) rats, and in rats with monocrotaline (MCT)-
induced pulmonary arterial hypertension that had been treated orally for two
weeks following Day 29 after MCT injection with trans-4-methoxy-β-nitro-
styrene (T4MN; 75 mg/kg/day) (MCT-T4MN group) or its vehicle (MCT-V
group), or with sildenafil (SIL; 50 mg/kg/day) (MCT-SIL). Data are expressed as
means ± S.E.M. (n = 9–14 per group). *P < 0.01 vs. MCT group by two-
way ANOVA.
All values are means ± SEM (n = 9–14 per group). aP < 0.00 by Mann-Whitney U
test with respect to CNT. bP < 0.01 by Mann-Whitney U test with respect to MCT-
V.
remodeling), which are involved in pathogenesis of PAH (Stasch et al.,
2011). In our previous investigation performed in aortic preparations
from normotensive rats, we showed that T4MN induced potent vaso-
relaxation involving stimulation of sGC/cGMP pathway, leading to
increased cGMP levels (Arruda-Barbosa et al., 2017). The actions of
cGMP are attributed to activation of protein kinase G (PKG), which can
modulate different target effectors in vascular smooth muscle. Although
activation of PKG by cAMP requires nearly a 10-fold-higher cAMP
concentration than cGMP concentration, it is unlikely that it contributes
to the mediation of T4MN-induced vasorelaxation since this effect was
endothelial function and vascular tone as its enhancement of the
reduced Emax induced by MCT was insignificant. Nevertheless, protec-
tion offered by T4MN against PAH may result from vasodilatation,
which reducing pulmonary vascular resistance. The NO/sGC/cGMP
signaling pathway regulates vascular tone and pulmonary resistance in
PAH (Arnold et al., 1977; Evgenov et al., 2006) as well as vascular cell
proliferation, platelet aggregation, and leukocyte recruitment (vascular
unaltered by the adenylate cyclase inhibitor MDL-12,330
A
5

K. Gonzaga-Costa et al.
European Journal of Pharmacology 897 (2021) 173948
Fig. 7. Treatment with trans-4-methoxy-β-nitro-
styrene reduces pulmonary vascular remodeling
in monocrotaline-induced PAH. In A and B,
Representative images of lung sections stained
with hematoxylin and eosin in three different
groups: control (CNT) rats, monocrotaline
(MCT)-injected rats treated with vehicle (MCT-
V), MCT-injected rats treated with trans-4-
methoxy-β-nitrostyrene (MCT-T4MN), or MCT-
injected rats treated with sildenafil (MCT-SIL).
In A, pulmonary arterioles with an external
diameter <50
with an external diameter between 50 and 100
M. Images show vessels at 20× magnification.
In C, wall thickness of vessels with an external
diameter <50 M. In D, wall thickness of vessels
with an external diameter between 50 and 100
μM. In B, pulmonary arterioles
μ
μ
μ
M. Wall thickness was expressed as a percentage
of total area. All data are means ± S.E.M. (n = 60
images per group) *P < 0.001 vs. CNT; ##P <
0.001 vs. MCT group by one-way ANOVA fol-
lowed by Dunnett’s test.
6

K. Gonzaga-Costa et al.
European Journal of Pharmacology 897 (2021) 173948
(Arruda-Barbosa et al., 2017). In aortic rings, T4MN-activated PKG
mediates opening of potassium channels and reduction of intracellular
Ca2+ by partial inhibition of Ca2+ influx through either voltage-operated
calcium channels or intracellular Ca2+ stores. Such a mode of action is
consistent with the ability of T4MN to stimulate sGC. This stimulation
was also cridited for the vasorelaxant effects of T4MN in resistance
mesenteric artery preparations from spontaneously hypertensive rats
(Alves-Santos et al., 2019). Recently, we showed that T4MN evokes
endothelium-independent vasorelaxant effects in isolated rat pulmonary
artery, partially by inhibiting Ca2+ influx through L-type Ca2+ channels,
as well as by activating sGC and potassium channels (Arruda-Barbosa,
2021). Notably, the IC50 for vasorelaxant effects of T4MN in
endothelium-intact aortic rings pre-contracted with phenylephrine from
CRediT authorship contribution statement
Karoline Gonzaga-Costa: performed experiments regarding to in-
duction of pulmonary hypertension, assessment of right ventricle hy-
pertrophy, pulmonary artery remodeling, hemodynamic measurement
and statistical analysis of data; review & editing of the manuscript.
Alfredo Augusto Vasconcelos-Silva: performed vascular reativity ex-
´
periments, statistical analysis of data. Matyelle Jussara Rodrigues-
Silva: contributed to experiments of hemodynamic measurement, sta-
˜
tistical analysis of data. Conceiçao da Silva Martins Rebouça:
contributed to assessment of pulmonary artery remodeling, statistical
´
analysis of data. Gloria Pinto Duarte: Writing - review & editing,
Funding acquisition, contributed to funding aquisition, review & editing
of the manuscript. Rosivaldo Santos Borges: performed synthesis of
the nitroderivatives, contributed to funding aquisition, review & editing
normotensive rats was about 57.64
μM (Arruda-Barbosa et al., 2017), a
value that was of the same order of magnitude as that (48.60
μM)
˜
recorded for vasorelaxant effects of T4MN in pulmonary artery rings
(Arruda-Barbosa, 2021). Although this observation may suggest that the
pulmonary artery is no more sensitive than aorta, this sGC stimulator,
T4MN, could be a promising candidate not only as an antihypertensive
drug, but also for treatment of PAH.
of the manuscript. Pedro Jorge Caldas Magalhaes: Writing - review &
editing, Supervision, review & editing of the manuscript. Saad Lahlou:
Supervision, Conceptualization, "Writing - original draft, contributed to
funding aquisition, and review & editing.
MCT injection in rats results in enhanced RVSP and pulmonary vessel
wall thickness and PAH. Our results showed that after 14-day period
treatment, T4MN reduced the enhanced RVSP, as with the positive
control, SIL. T4MN reduced pulmonary vascular remodeling as evi-
Declaration of competing interest
The authors declare that they no conflict of interest regarding pub-
lication of this article.
denced by the decreased vessel wall thickness of small (<50
μm) and
larger (50–100
μm) pulmonary arterioles, as corroborated with hemo-
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