Divergent impact of gestational diabetes mellitus between the thoracic and
abdominal rat aorta: Influence of endothelium and angiotensin II receptors
Cecilia Tufino, ˜ Miriam Vanegas, Ruth Velazquez ´ Nev´
arez, Cleva Villanueva Lopez, ´
Rosa Amalia Bobadilla Lugo *
Secci´
on de Estudios de Posgrado e Investigacion, ´ Escuela Superior de Medicina, Instituto Polit´ecnico Nacional, Plan de San Luis y Díaz Miron, ´ Col. Santo Tomas, ´ M´exico,
11340, D.F, Mexico
ARTICLE INFO
Keywords:
AT1 receptors
AT2 receptors
Gestational diabetes mellitus
Thoracic and abdominal aorta
Endothelium
ABSTRACT
Gestational diabetes mellitus (GDM) affects 5–10% of pregnancies and increases the risk of fetal and maternal
adverse outcomes. Interestingly, the vascular response to AngII is decreased by pregnancy while the response is
increased by diabetes. It remains unclear how GDM affects vascular tone and how angiotensin II receptors
contribute to these changes. In this work, we sought to establish the vascular impact of a hypercaloric diet￾induced GDM through changes in AT1 and AT2 receptor’s expression. Female rats fed for 7 weeks with stan￾dard (SD) or hypercaloric (HD) diet were divided at week 4. Half of the rats of each group were mated to become
pregnant and those fed with a HD developed GDM. AngII-induced vasoconstriction was measured in thoracic or
abdominal aorta rings using a conventional isolated organ bath and AT1 and AT2 receptors were searched by
immunohistochemistry. Experiments where conducted on the pregnant standard diet group (PSD) and the
pregnant hypercaloric-gestational diabetes mellitus group (PHD-GDM). Vasoconstriction was reduced in the
thoracic aorta (P < 0.05 vs PSD) but increased in the abdominal aorta of PHD-GDM rats (P < 0.05 vs PSD).
Blockade of AT2 receptors using PD123319 decreased vasoconstriction, particularly in the abdominal aorta of
PHD-GDM animals (P < 0.05 vs PSD). PHD-GDM increased AT1 receptors expression (P < 0.05 vs PSD). Also,
PHD-GDM reverted physiologic hypoglycemia and hypotension of healthy pregnancy. Findings provide new
insight into the hypercaloric diet induced damage on the vasculature during pregnancy.
1. Introduction
Obesity and overweight during pregnancy have an increased prev￾alence that is strongly related to complications such as gestational dia￾betes mellitus (GDM) and preeclampsia (Ramírez Torres, 2005; Wellen
and Hotamisligil, 2003). GDM defined by the American Diabetes Asso￾ciation as “any degree of glucose intolerance with an onset or first
recognition during pregnancy” affects 5–10% of pregnancies worldwide
(Schmidt et al., 2001; Setji et al., 2005).
Endocrine, paracrine or intracellular components of the renin￾angiotensin system (RAS) (Ferrao ˜ et al., 2017) produce angiotensin II
(AngII), a peptide with several physiological functions -fluid and elec￾trolyte balance-as well as physiopathological contributions in insulin
resistance, diabetes and hypertension (Boudina and Abel, 2007; Sen￾anayake et al., 2018; Velazquez-Roman et al., 2011). Physiological
adaptation to pregnancy includes an increase of plasma AngII (Mishra
et al., 2018), blood volume and cardiac output. Paradoxically, blood
pressure is decreased and the response to vasoconstrictors is blunted
during pregnancy (Irani and Xia, 2008; Mishra et al., 2018). Also,
normal pregnancy entails increased serum insulin as well as insulin
resistance (Catalano et al., 1999).
Changes in the relative expression and function of AngII main re￾ceptors AT1 and AT2 have been held responsible for the contradictory
vascular and metabolic effects of AngII during pregnancy. AT1 receptors
have a determinant role in vasoconstriction, hypertension (Nyby et al.,
2007) and insulin resistance (Sowers et al., 2004). Furthermore, evi￾dence supports AT2 endothelial receptors exert a counterforce to AT1
receptors by inducing a direct or bradykinin mediated NO dependent
vasodilation Chow and Allen, 2016; Yayama and Okamoto, 2008) and
by improving insulin sensitivity (Munoz ˜ et al., 2017; Pinaud et al.,
2007). In accordance, an increased Ang II-induced vascular contraction
has been described in the presence of AT2 receptor antagonists (Baten￾burg et al., 2004; Hannan et al., 2003). Likewise, studies using AT2 re￾ceptor agonists and antagonists suggest AT2 receptors have
* Corresponding author.
E-mail address: [email protected] (R.A. Bobadilla Lugo).
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

https://doi.org/10.1016/j.ejphar.2021.173981

Received 18 September 2020; Received in revised form 15 February 2021; Accepted 23 February 2021
European Journal of Pharmacology 899 (2021) 173981
2
anti-apoptotic, anti-inflammatory, cardioprotective (Qi et al., 2012) and
antifibrotic effects (Namsolleck et al., 2014). Besides, an AT2 receptors
upregulation has been considered as part of a natural protective or
contra regulatory system (Kaschina et al., 2017) for certain pathological
conditions such as hypertension, vascular injury, and inflammation (Lee
et al., 2008; Namsolleck et al., 2014). Moreover, AT2 receptors seem to
be responsible of the vasodilatation and remodeling of vessels near the
uterus (Mishra et al., 2018; Osol and Cipolla, 1993; Pulgar et al., 2011)
and AT2 receptor-dependent vasodilatation (Stennett et al., 2009), can
be involved in the characteristic hypotension of pregnancy (Mishra
et al., 2018; Stennett et al., 2009).
Studies about the impact of GDM on the mother’s vasculature are
scarce; nevertheless, there is some evidence of GDM’s deleterious effect
in the thoracic aorta (Tufino ˜ et al., 2014). Also, there is evidence of
regional differences along the rodent aorta segments both in nonpreg￾nant and pregnant animals (Gregg et al., 1995). Indeed, there are reports
about an increased participation of endothelial-derived hyperpolarizing
factor (EDHF) in the abdominal segment of this conductance vessel
(Bobadilla et al., 2005; Oloyo et al., 2012) that definitely has an
important influence on uteroplacental blood flow.
It is not clear if GDM damages vascular function particularly in the
abdominal aorta segment. We hypothesized GDM increases abdominal
aorta vasoconstriction through changes in AT1/AT2 receptor relative
participation. Therefore, the aim of the current work was to evaluate the
AT2 receptor-mediated changes in vasoconstriction produced by
hypercaloric diet-induced GDM comparing the thoracic and the
abdominal segment of the rat aorta.
2. Materials and methods
2.1. Animals
12 weeks old female Wistar rats of 250 ± 15 g, kept in controlled
conditions of light (12 h light/dark cycle) and humidity were used for
this experiment. Rats were fed a standard diet (SD) rat chow (3.1 kcal/g)
or to a high-calorie diet (HD) (6.3 kcal/g) over 7 weeks. HD was pre￾pared with 33% ground commercial rat chow, 33% full fat sweetened
condensed milk (Nestle), 7% sucrose and 27% water (Holemans et al.,
2004). Both diets and water were provided ad libitum. At the end of the
4th week rats were randomized and half of each group were separately
mated to become pregnant. Day one of pregnancy was considered when
spermatozoa were found in a vaginal smear. Gestational diabetes mel￾litus (GDM) model (n = 8) was confirmed when hypercaloric diet
pregnant (PHD) rats showed an altered glucose tolerance test. Pregnant
fed standard diet (PSD) (n = 8) rats conformed control group. For
immunohistochemistry, aortas were obtained from additional animals
(n = 3–4) for each experimental group (with a total of n = 16).
All procedures were approved by the Official Mexican Norm (NOM-
062-ZOO-1969) for animal handling and were approved by the local
Ethical and Research Committee of the Escuela Superior de Medicina del
Instituto Polit´ecnico Nacional (CICUAL-03/21-06-2017).
2.2. Records in the whole animal
Weight was recorded weekly and blood pressure was obtained at the
beginning of the protocol and at the end of weeks 4 and 7. Glucose
tolerance test was evaluated at the end of protocol.
Blood pressure was recorded by the indirect tail-cuff plethysmog￾raphy method (Letica 5007; PanLab, Barcelona). Previously trained rats
were placed into appropriate traps and systolic blood pressure was
measured inside a room free from noise and light, previously warmth to
32 ◦C. The mean of systolic blood pressure after 3 consecutive successful
measurements was considered.
Glycemia was determined by a drop of blood from the tail tip on a
reactive strip and a glucose meter Accu-Chek Roche ®. For glucose
tolerance test (GTT) glycemia was measured before (min 0) and after the
intraperitoneal administration of 1 g/kg glucose. Blood samples were
taken 5, 10, 15, 30, 45, 60, 90, and 120 min after glucose. Glucose
tolerance was evaluated by comparing the area under the curve (AUC)
obtained from each group.
2.3. Isolated aorta rings
At the end of the protocol, animals were killed under anesthesia and
the aorta excised and cleaned from surrounding connective tissue. The
portion from the heart to the diaphragm was considered thoracic
segment and the portion under the renal arteries to the iliac bifurcation
was considered the abdominal aorta. Isolated arteries were cut into rings
(3 mm long). Half of the rings from each segment were left intact and the
endothelium was removed by gently rubbing off the intimal surface with
a metal device from the other half. Contraction was evaluated using a
conventional isolated organ bath technique (Grobe et al., 2018; Lautner
et al., 2017; Mahmoud and El Bassossy, 2014; Xie et al., 2015). Rings
were placed in tissue chambers filled with 10 mL Krebs-Henseleit solu￾tion of the following composition (mM): NaCl 118, KCl 4.8, CaCl2 2.5
MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11.7, EDTA 0.026. Aortic
rings were suspended between two nikrom wire hooks; one hook was
fixed to the bottom of the tissue bath chamber, and the other hook was
connected to a 50G-TSD125C force transducer linked to a
general-purpose DA100C amplifier and then to a data acquisition system
(Biopac System Inc. Santa Barbara, CA, USA®), in order to record the
isometric tension developed by the vessel. Aortic rings were stretched
under 3 g of resting tension and allowed to equilibrate for 45 min in a
temperature-controlled, water-jacketed tissue bath filled with 10 ml
Krebs solution continuously bubbled with 95% O2 – 5% CO2 at 37 ◦C.
After tissue equilibration, in order to determine the functionality of
smooth muscle, contraction was induced with 80 mM KCl. Then rings
were stimulated three times with phenylephrine (1 × 10− 6
M). The third
time, contracted vessel was allowed to plateau and exposed to acetyl￾choline (ACh) 10− 7 – 10− 6 M to assess the presence of endothelium.
Rings were considered to have endothelium if relaxation was ≥80%.
Afterwards, aortic rings were stimulated with increasing concentrations
of AngII (10− 12 -10− 6 M). Individual concentration-response curves
were further analyzed using a nonlinear regression curve (best-fit
sigmoidal dose-response curve), and determined the effective concen￾tration producing half the maximal contraction (EC50) as well as the
pD2 value (pD2 = -log EC50). Also, the AngII concentration-contraction
curves were evaluated in the presence of the AT2 receptor antagonist
PD123319 (10− 7 M).
2.4. Immunohistochemistry
At the end of protocol, animals were killed and aortas excised,
cleaned and separated in its thoracic and abdominal segment. Vessels
were fixed in a 3% paraformaldehyde solution for 3 days and then
included in paraffin to be ready for immunohistochemistry procedure as
previously described (Grobe et al., 2018; Zalatnai et al., 2019). Briefly
vessels were 5 μm thick sectioned using a Lauca microtome. Cuts were
placed in a water bath (40 ◦C) for 1 min and then “caught” on a slide
previously treated with poly-L-Lysine. Slides were warmth (60 ◦C),
rehydrated and warmth again (100 ◦C) with the antigen recuperator
(Antigen Retrieval Citra Plus 10x, Biogen, cat. HK080 –9 K). Endogenous
peroxidases were blocked with 3% peroxide incubation followed by a
3-fold wash with PBS-tween. Tissue sections were incubated overnight
(4 ◦C) in a wet chamber with AT1 and AT2 receptor antibodies (rabbit
polyclonal AT1, Santacruz Biotechnology and rabbit polyclonal AT2
from Santacruz Biotechnology) in a 1:100 dilution with the antibody
diluent (DAKO). The excess of primary antibody was cleansed with 3
PBS-Tween washes. Slides were incubated for 20 min with a drop of link
from the LSAB + System-HRP kit (BIOTINYLATED LINK UNIVERSAL;
STREPTAVIDIN + HRP, cat. K0690) at room temperature and then
washed with PBS-tween to remove excess. Each slide was exposed to
C. Tufino ˜ et al.
European Journal of Pharmacology 899 (2021) 173981
3
100 μl of chromogen for 20 s and the excess removed with distilled
water. Negative control slides were run simultaneously with no primary
antibody.
Contratinction was performed with hematoxylin and subsequently
washed with distilled water, and dehydrated with 70% and 80%, 90%
and 96%, absolute alcohol and xylene. A coverslip was placed using 2
drops of Entellan (Merk mounting medium). After a 24 h period for
drying, slides were observed under the microscope and analyzed using
image-analysis software Gen 5 integrated optical density program.
2.5. Analysis and statistics
Each of the four experimental groups for isolated vessels contraction
included 6–8 animals. Data are expressed as the mean ± S.E.M. pD2
(-Log EC50) and Emax values were obtained by non-linear regression
analysis from concentration-response curves. Immunohistochemistry
was performed in three experimental groups: non pregnant, pregnant SD
and pregnant HD. Statistical evaluation of the data, when comparing
each point of the concentration-response curve, was carried out by two￾way (ANOVA), with Bonferroni test for comparison of means. pD2 (-Log
EC50) and Emax values were compared by using unpaired Student’s “t”
test.
Weight gain and glucose tolerance curve data were expressed as the
mean ± S.E.M. and tested by two-way analysis of variance (ANOVA)
statistical analysis. Statistical differences in the expression of receptors
were evaluated using one-way analysis of variance (ANOVA), unpaired
t-tests, S.E.M. and Tukey test. For all comparisons, a significant differ￾ence was considered if P < 0.05 using Prism Graph Pad version 6.0
software.
3. Results
3.1. Body weight and glucose blood levels
At the end of the protocol, pregnant rats fed with HD (PHD-GDM),
had a significant increase in weight compared to those fed with SD (PSD)
(125.62 ± 5.75 vs. 67.11 ± 4.23 g PHD-GDM vs. PSD respectively) (P <
0.05) (Fig. 1).
Plasma glucose levels were also increased in the PHD-GDM group
(78.11 ± 3.23 vs. 68.1 ± 4.75 mg/dL P < 0.05 PHD-GDM vs. PSD
respectively). Nevertheless, PHD-GDM values did not surpass those of
non-pregnant animals, this means that the present GDM model only
slightly modifies basal glucose levels.
3.2. Blood pressure
Systolic BP was increased in PHD-GDM rats (98.11 ± 3.23 vs 120.5
± 5.6 mmHg P < 0.05 PSD vs. PHD-GDM) (Fig. 2) at the end of protocol.
3.3. Glucose tolerance
A glucose tolerance test (GTT) was conducted at the end of preg￾nancy. GTT was impaired in PHD-GDM compared to PSD animals:
(1151 ± 20.1 vs 1585 ± 21 arbitrary units of area under the curve P <
0.05, PSD vs. PHD-GDM respectively). Values were significantly
different at 15, 30, 45, 60, 75, 90 and 120 min. Each point represents the
mean ± S.E.M. of 5 PSD and 7 PHD-GDM independent animals per
group. *P < 0.001 (Fig. 3). This altered glucose tolerance in the PHD
group, is a defining feature for the GDM model.
3.4. Isolated vessels
Smooth muscle conditions were evaluated with 80 mM KCl induced
contraction. We found there were no significant differences between
PSD and PHD-GDM groups with (3.57 ± 0.29 vs. 3.15 ± 0.20 g
respectively) or without endothelium (3.42 ± 0.28 vs. 3.18 ± 0.30 g
respectively) (data not shown).
3.5. Vasoconstriction
Response to angiotensin II.
Vascular response to Ang II was evaluated with concentration￾response curves (10− 12 -10− 6 M) ran in thoracic and abdominal aorta
rings both with endothelium and without endothelium from PSD (10
rings per segment obtained from 5 animals) and PHD-GDM (14 rings per
segment obtained from 7 animals).
PHD-GDM decreased Ang II-induced vasoconstriction in the thoracic
aorta but increased the response in the abdominal segment of the vessel
(Fig. 4 A and B, Table 1). These effects were not observed in
endothelium-denuded rings of either group (Fig. 4 C and D, Table 1).
3.6. AT2 receptors participation
In PSD animals, PD123319 (10− 7 M) increased thoracic and
abdominal aorta AngII-induced contraction (10− 12-10− 6 M). (Fig. 5 A
and B, Table 2). The increased contraction was not observed in
endothelium-denuded vessels (Fig. 5 C & D).
In contrast, in PHD-GDM rats, PD123319 (10− 7 M) did not modify
AngII-induced contraction of the thoracic aorta, moreover, PD123319
(10− 7 M) decreased the Ang II induced response of the abdominal
segment (Fig. 6 A and B). These effects were not present in endothelium￾Fig. 1. Percentage of weight gain. Standard diet SD (o) or hypercaloric diet HD
(Δ). Rats were pregnant from week 4–7. Results are the mean ± S.E.M. *P <
0.05 versus SD.
Fig. 2. Systolic blood pressure. Pregnant standard diet PSD (gray) vs. pregnant
hypercaloric diet-gestational diabetes mellitus PHD-GDM (gouged). Results are
the mean ± S.E.M. *P < 0.05 versus PSD.
C. Tufino ˜ et al.
European Journal of Pharmacology 899 (2021) 173981
4
denuded vessels (data not shown).
3.7. Immunohistochemistry
Both AT1 and AT2 receptor’s expression was found in the
endothelium, smooth muscle and adventitia of the thoracic and
abdominal aorta of both experimental groups.
PHD-GDM increased the expression levels of AT1 receptors of both
the thoracic (P < 0.06) and of the abdominal (*P < 0.01) segment of the
aorta (Fig. 7 A, B). In contrast, AT2 receptors’ expression was not
significantly increased by PHD-GDM treatment (Fig. 8 A, B).
Fig. 3. Blood glucose values obtained after IP administration of 1 g/kg of
glucose. Pregnant standard diet PSD (o), pregnant hypercaloric diet – gesta￾tional diabetes mellitus rats PHD-GDM (Δ). Results are the mean ± S.E.M. *P <
0.05 versus PSD of determinations in 5 PSD and 7 PHD-GDM rats.
Fig. 4. Angiotensin II induced contraction of
isolated rat aorta from pregnant standard diet
PSD (o), or pregnant hypercaloric diet – gesta￾tional diabetes mellitus PHD-GDM (Δ) groups.
Rings with endothelium: (A) thoracic or (B)
abdominal. Rings without endothelium: (C)
thoracic or (D) abdominal. Graphs were con￾structed using the percentage of contraction
regarding KCl maximal effect (100%). The data
are mean ± S.E.M. of 10 and 14 rings per con￾dition obtained from 5 and 7 animals respec￾tively. *P < 0.05 versus PSD.
Table 1
Percentage of Emax respect to 80 mM KCL induced contraction and pD2 values
from the response to concentration-response curves to angiotensin II (10− 12
-10− 6 M) of isolated aorta rings with or without endothelium from pregnant
standard diet (PSD) rats, or pregnant hypercaloric diet-gestational diabetes
mellitus rats (PHD-GDM).
Aorta rings with endothelium
PSD PHD
Thoracic Abdominal Thoracic Abdominal
EMAX (%) 13.57 ± 1.711 6.682 ±
0.8766
4.334 ±
0.5733
25.20 ±
2.006
pD2 (lOG
M)
8.271 ±
0.4084
7.872 ±
0.2979
16.51 ± 1.389 7.83 ±
0.2406
Aorta rings without endothelium
PSD PHD
Thoracic Abdominal Thoracic Abdominal
EMAX (%) 7.585 ±
0.5733
15.43 ±
1.2431
21.11 ± 1.625 5.928 ±
0.834
pD2 (lOG
M)
11.34 ±
0.4117
7.243 ±
0.4179
8.246 ±
0.5174
17.3 ±
1.2361
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European Journal of Pharmacology 899 (2021) 173981
5
4. Discussion
Physiological adaptations to pregnancy include a significant incre￾ment of plasma insulin concentration (up to 250%) and the development
of insulin resistance (IR). IR is also associated with overweight (Wellen
and Hotamisligil, 2003), which is an important risk factor for developing
GDM. GDM is defined as “a carbohydrate intolerance with varying de￾grees of severity, which is recognized for the first time during preg￾nancy” (American Diabetes Association, 2016; Schmidt et al., 2001).
GDM has been associated with pregnancy complications and with long
term vascular and metabolic disturbances for both mother and child
(Barbour et al., 2014; Bellamy et al., 2009; Metzger and Contreras M,
2008; Sullivan et al., 2012). Nevertheless, information about the acute
impact of gestational diabetes mellitus on mother’s vasculature is
scarce. Present work showed that while the glucose tolerance test was
impaired in PHD-GDM rats at term, blood glucose stayed within normal
values. However, such subtle metabolic disarrangement impacted blood
pressure enough as to abolish end-pregnancy physiological hypotension.
We found the PHD based GDM model raised blood pressure and
impacted aorta contractility in an endothelium and segment dependent
way. PHD-GDM increased abdominal aorta maximal contraction but had
the opposite effect on the thoracic segment. Indeed, diminished
contraction of the thoracic aorta of GDM animals is in agreement with
previous reports studying the contractility of the thoracic aorta from
male specimens with diabetes mellitus (Lee et al., 2008; Sartoretto et al.,
2019; Tufino ˜ et al., 2014). The reduced contractility has been attributed
to an early compensatory release of endothelium-derived vasodilator
mediators in response to metabolic stress. However, this cannot explain
PHD-GDM’s impact in the abdominal aorta, where PHD-GDM seems to
affect the endothelium by tilting the vasoconstrictor/vasodilator bal￾ance towards vasoconstriction.
Fig. 5. Angiotensin II induced contractions in isolated rat aorta form pregnant standard diet (PSD) group in the absence (o) or in the presence of PD123319 (10-7 M)
(Δ). Aorta rings with endothelium: (A) thoracic or (B) abdominal. Rings without endothelium: (C) thoracic or (D) abdominal. Graphs were constructed using the
percentage of contraction respect to KCl maximal effect (100%). The data are mean ± S.E.M. of 12 rings per condition obtained from 6 animals per condition.
Table 2
AT2 receptor antagonist PD123319 effect on angiotensin II vascular response of
pregnant rats. Percentage of Emax respect to 80 mM KCL induced contraction
and pD2 values from the response to concentration-response curves to angio￾tensin II (10− 12 -10− 6 M) of isolated aorta rings with or without endothelium
from pregnant standard diet (PSD) rats in absence or presence of 10 mM
PD123319.
Aorta rings with endothelium
PSD PSD + PD 123319
Thoracic Abdominal Thoracic Abdominal
EMAX (%) 9.407 ±
0.976
4.33 ± 0.1250 19.506 ±
1.246
9.633 ± 0.971
pD2 (lOG
M)
7.918 ±
0.493
7.872 ±
0.2979
8.453 ±
0.453
7.55 ± 0.541
Aorta rings without endothelium
PSD PSD + PD 123319
Thoracic Abdominal Thoracic Abdominal
EMAX (%) 9.193 ±
0.573
13.52 ± 1.232 11.031 ±
0.974
23.298 ±
6.421
pD2 (lOG
M)
11.34 ±
0.4117
7.267 ± 0.625 9.469 ±
0.933
7.907 ± 0.45
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European Journal of Pharmacology 899 (2021) 173981
6
Fig. 6. Angiotensin II induced contractions in isolated rat aorta form hypercaloric diet-gestational diabetes mellitus rats (PHD- GDM) group in the absence (o) or in
the presence of PD123319 (10-7 M) (Δ). Aorta rings with endothelium: (A) thoracic or (B) abdominal. Graphs were constructed using the percentage of contraction
respect to KCl maximal effect (100%). The data are mean ± S.E.M. of 12 rings per condition obtained from 6 animals per condition. *P < 0.05 versus PSD.
Fig. 7. (A) Representative images of AT1R
expression in the thoracic and abdominal aorta
following PSD or PHD-GDM treatment (a =
endothelium, b = smooth muscle, c = adven￾titia); (A and C) thoracic and abdominal aorta
from PSD, (B and D) thoracic and abdominal
aorta from PHD-GDM rats. Expression levels of
AT1 R in (B) thoracic (C) abdominal aorta. Re￾sults are mean ± S.E.M. of 8 images obtained
form 4 animals per condition and segment. *P <
0.01 versus PSD, ***P < 0.001 versus PSD.
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European Journal of Pharmacology 899 (2021) 173981
7
Even when abdominal aorta contractility studies are scarce, differ￾ences regarding endothelium-derived vasoactive mediators between
aorta segments have been reported in pregnant (Bobadilla et al., 2005;
Gregg et al., 1995) or in non-pregnant rodents (Lindesay et al., 2018).
Indeed, increased production of NO toward the distal aorta was
observed in pregnant guinea pigs (Gregg et al., 1995) as well as
increased participation of endothelium-derived hyperpolarizing factor
(EDHF) in the abdominal aorta of pregnant rats (Bobadilla et al., 1997).
These findings were explained as a physiological adaptation of the
vascular segment proximal to the uteroplacental territory. Therefore, we
can assume that the results are influenced by the intrinsic characteristics
of the abdominal aorta along with pregnancy and GDM.
Contraction of the thoracic aorta of the control group (PSD) was
increased in the presence of AT2 receptors’ antagonist PD123319. The
effect was not observed when the endothelium was removed, high￾lighting the inhibitory role of endothelial AT2 receptors. The presence of
PD123319 did not increase PHD-GDM thoracic aorta contraction,
probably because PHD-GDM increases AT1 receptors-dependent
contractility. Differences in the behavior of the two segments of the
aorta were corroborated by the results obtained in the abdominal
segment, in which antagonism of AT2 receptors slightly decreased the
contraction of PHD-GDM vessels. Lastly, results show PHD-GDM deeply
modifies aorta contractility mechanisms probably by changing the
functionality of AngII receptors both in endothelium and smooth
muscle.
Increased plasmatic or local concentrations of AngII and a change in
the functionality of its main receptors can explain our findings. Indeed,
stimulation of AngII AT1 receptors is related to vasculopathy and organ
dysfunction in hypertension (Masi et al., 2019) and diabetes mellitus
(Wilcox et al., 2019) and both AT1 and AT2 receptors have been found
increased in the thoracic aorta of hypertensive/diabetic rats (Romer￾o-Nava et al., 2016). Serum AngII has been found increased during rat
pregnancy (Mishra et al., 2018) and chronic hyperinsulinemia has also
been associated with increases of AngII, which has a role in the
Fig. 8. (A) Representative images of AT2R expression in the thoracic and abdominal aorta following PSD or PHD-GDM treatment: (a = endothelium, b = smooth
muscle, c = adventitia); (A and C) thoracic and abdominal aorta from PSD, (B and D) thoracic and abdominal aorta from PHD-GDM. Expression levels of AT2 R in (B)
thoracic (C) abdominal rat aorta. Results are mean ± S.E.M. of 8 images obtained form 4 animals per condition and segment n.s.d.
C. Tufino ˜ et al.
European Journal of Pharmacology 899 (2021) 173981
8
maintenance of the vicious cycle of vascular damage (Surapongchai
et al., 2017).
AT1 and AT2 receptors were detected by immunohistochemistry in
the three layers (endothelium, smooth muscle and adventitia) of the two
segments of rat aorta. PHD-GDM increased expression of AT1 receptors
while AT2 receptors of both aorta segments showed a non-significant
increase. Findings are in accordance with previous work describing
AT1 (Ramchandran et al., 2006) and AT2 receptor (Toedebusch et al.,
2018) expression in human and rodent endothelium and vascular
smooth muscle cells. Additionally, comparable effects of pregnancy on
AngII vascular receptors have been reported (Mishra et al., 2018).
Nevertheless, the increased AT1 receptors expression produced by GDM
may represent an important mechanism of vascular damage and is a
significant finding of this work.
The role of vascular AT2 receptors has raised controversy. Endothe￾lial AT2 receptors have proved to mainly stimulate NO-induced vaso￾dilation (Padia and Carey, 2013) and studying pregnant rat aortas,
Stenett et al. described AT2 receptor agonist CGP 42112 A produced
vasorelaxation involving increased expression of eNOS (Stennett et al.,
2009). Indeed, it has been postulated that under physiological condi￾tions AT2 receptors function as counter-regulators (ying-yang hypothe￾sis) to the actions of AngII on AT1 receptors (limiting the contraction)
(Karpe et al., 2012). Besides, overexpression of AT2 receptors has been
observed in pathologies of the blood vessels or when a biological process
that includes angiogenesis and tissue remodeling occurs, as in pregnancy
(Jones et al., 2008; Munoz ˜ et al., 2017; Tsutsumi et al., 1999). Then,
considering present results, AT2 receptors seem to effectively counteract
the effects of AT1 receptors as observed on the PHD-GDM thoracic aorta,
but this effect is not extended to the abdominal segment.
Likewise, results suggest the PHD-GDM model induces changes in the
function of AngII receptors, particularly on the abdominal aorta. One of
these changes can be dimerization: it has been proposed that AngII
promotes the dimerization of receptors. AngII binding to any of its re￾ceptors induces their homodimerization and/or heterodimerization,
which, in turn, induces different cellular responses (Bellot et al., 2015;
Ferrao ˜ et al., 2017).
A non-standard role of AT2 receptors as a consequence of GDM can
also be considered. Indeed, Schinzari et al. (2011) found that PD123319
blunts the vasoconstriction response to AngII, suggesting some AT2 re￾ceptors contribute to AngII contractile effects and cooperate in the
maintenance of vasoconstrictor tone. Changes in AT1 and AT2 receptor
functionality like a cross-talk between the 2 receptor subtypes (AT1 and
AT2) (Padia and Carey, 2013) can also be contemplated. Also, research
has described a “switch” in the classic role of the AT1 and/or AT2 re￾ceptors (Funke-Kaiser et al., 2010; Schinzari et al., 2011). Indeed,
classical roles for AT1-AT2 receptors have been challenged by studies
demonstrating vasoconstrictor effects mediated by AT2 receptors
(Pinaud et al., 2007; Touyz et al., 1999) or vasodilator effects mediated
by endothelium AT1 receptors (Ramchandran et al., 2006), supporting
the heterogeneity of AT2-mediated hemodynamic responses.
Relative quantification of receptor expression in each of the three
layers of the aorta was not feasible in this work. Nevertheless, results
showed PHD-GDM changed the relative proportion of AngII receptors
along the aorta. Then, AT2 receptors may be contributing to vasocon￾striction in the PHD-GDM abdominal segment probably through an
atypical AT2 receptor behavior, but the exact molecular mechanism of
damage requires further investigation.
In conclusion we report that rats with a PHD-GDM experimental
model remain normotensive and normoglycemic while developing
impaired results in the glucose tolerance test. PHD-GDM damaged aorta
contractility and increased AT1 receptors’ expression mainly in the
abdominal segment of the aorta. Also, we found evidence of a differ￾ential response between the thoracic and abdominal aorta to the PHD￾GDM challenge. Findings provide new insight into the hypercaloric
diet induced damage on the vasculature during pregnancy. This evi￾dence is particularly relevant since overweight-induced damage can be
dismissed during regular women’s pregnancy medical control. Even
when more deep mechanistic studies are needed, findings contribute to
elucidate the role of AT2 vascular receptors in the pathophysiology of
this frequent pregnancy complication.
Funding
This work was partially supported by a Secretara de Investigacin y
Posgrado Instituto Politcnico Nacional, Mexico grant number
20201027, and a scholarship from Consejo Nacional de Ciencia Y Tec￾nologa, Mexico.
CRediT authorship contribution statement
Cecilia Tufino: ˜ Investigation, Data curation, Writing – review &
editing, writing. Miriam Vanegas: Investigation, Data curation, Soft￾ware. Ruth Velazquez Narvaez: Investigation, Data curation. Cleva
Villanueva Lopez ´ Villanueva: Validation, Writing – review & editing,
review, Visualization. Rosa Amalia Bobadilla Lugo: Conceptualiza￾tion, Formal analysis, Writing – review & editing, writing, Supervision,
Funding acquisition.
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