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Telmisartan use in rats with preexisting osteoporotics bone disorders increases bone microarchitecture alterations via PPARγ
Journal Pre-proof Telmisartan use in rats with preexisting osteoporotics bone disorders increases bone microarchitecture alterations via PPARγ Antonio Marcos Birocale, Antonio Ferreira de Melo, Jr., Pollyana Peixoto, Phablo Wendell Costalonga Oliveira, Leandro Dias Gonçalves Ruffoni, Liliam Masako Takayama, Breno Valentim Nogueira, Keico Okino Nonaka, Rosa Maria Rodrigues Pereira, José Martins de Oliveira, Jr., Nazaré Souza Bissoli PII:
S0024-3205(19)30817-3
DOI:
https://doi.org/10.1016/j.lfs.2019.116890
Reference:
LFS 116890
To appear in:
Life Sciences
Received Date: 31 July 2019 Revised Date:
11 September 2019
Accepted Date: 19 September 2019
Please cite this article as: A.M. Birocale, A. Ferreira de Melo Jr., P. Peixoto, P.W. Costalonga Oliveira, L.D. Gonçalves Ruffoni, L.M. Takayama, B.V. Nogueira, K.O. Nonaka, R.M. Rodrigues Pereira, José. Martins de Oliveira Jr., Nazaré.Souza. Bissoli, Telmisartan use in rats with preexisting osteoporotics bone disorders increases bone microarchitecture alterations via PPARγ, Life Sciences (2019), doi: https://doi.org/10.1016/j.lfs.2019.116890. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
1
Telmisartan use in rats with preexisting osteoporotics bone disorders
2
increases bone microarchitecture alterations via PPARγ
3
Authors: Antonio Marcos Birocale, MDa, Antonio Ferreira de Melo Jr, MDb,
4
Pollyana Peixoto, MDb, Phablo Wendell Costalonga Oliveira, PhDb, Leandro
5
Dias Gonçalves Ruffoni, PhDc, Liliam Masako Takayama, MDd, Breno Valentim
6
Nogueira, PhDe, Keico Okino Nonaka, PhDc, Rosa Maria Rodrigues Pereira,
7
PhDd, José Martins de Oliveira Jr., PhDf, Nazaré Souza Bissoli, PhDb*
8 9
a
Department of Health Integrated Education, Federal University of Espirito
10
Santo, Vitória, ES, Brazil
11
b
12
Vitória, ES, Brazil
13
c
14
Carlos, SP, Brazil
15
d
16
Paulo, SP, Brazil
17
e
18
Brazil
19
f
20
Brazil
Department of Physiological Sciences, Federal University of Espirito Santo,
Department of Physiological Sciences, Federal University of São Carlos, São
Department of Medical Clinic, Medicine College, University of São Paulo, São
Department of Morphology, Federal University of Espirito Santo, Vitoria, ES,
Laboratory of Applied Nuclear Physics, University of Sorocaba, Sorocaba, SP,
21 22
*
23
Nazaré Souza Bissoli ([email protected])
24
Phone number: 55 27 998806356; 55 27 33357333
25
Address: Department of Physiological Sciences; Centre for Health Sciences,
26
Federal University of Espirito Santo. Av. Marechal Campos 1468, 29042-755,
27
Vitória, ES – Brazil.
Corresponding author:
28
Telmisartan use in rats with preexisting osteoporotics bone disorders
29
increases bone microarchitecture alterations via PPARγ
30
Abstract
31
Aims: Telmisartan (TEL), an angiotensin II type I receptor blocker and PPARγ
32
partial agonist, has been used for to treat hypertension. It is known that PPARγ
33
activation induces bone loss. Therefore, we evaluate the effects of telmisartan
34
on
35
microarchitecture of femurs and lumbar vertebrae in SHR ovariectomized
36
animals, a model of hypertension in which preexisting bone impairment has
37
been demonstrated.
38
Main methods: SHR females (3 months old) were distributed into four groups:
39
sham (S), sham+TEL (ST), OVX (C) and OVX+TEL (CT). TEL (5mg/kg/day) or
40
vehicle were administered according to the groups. After the protocol, blood
41
pressure was measured and density, microarchitecture and biomechanics of
42
bone were analyzed. Western blotting analysis was performed to evaluate
43
PPARγ protein expression in the bones.
44
Key findings: Castration induced a deleterious effect on mineral density and
45
trabecular parameters, with telmisartan enhancing such effects. Telmisartan
46
increased PPARγ levels, which were at their highest when the treatment was
47
combined with castration. As to biomechanical properties, telmisartan reduced
48
the stiffness in the castration group (CT vs. S or C group), as well as resilience
49
and failure load in ST group (vs. all others groups).
PPARγ
protein
expression,
biomechanics,
density
and
bone
50
Significance: These results demonstrated that telmisartan compromised bone
51
density and microarchitecture in animals that shows preexisting osteoporotic
52
bone disorders, probably via mechanisms associated with increased PPARγ. If
53
this translates to humans, a need for greater caution in the use of telmisartan by
54
patients that have preexisting bone problems, as in the postmenopausal period,
55
may be in order.
56
Keywords: Telmisartan, PPARγ, Hypertension, Ovariectomy, Bone
57
INTRODUCTION
58
Osteoporosis is associated with loss of bone mass due to decreased tissue
59
continuity, with the rupture of the trabecular microarchitecture leading to
60
reduced connectivity, increased bone fragility and fracture risk. The concomitant
61
imbalance between osteoclastic and osteoblastic activities is exacerbated in the
62
postmenopausal phase, being a determining factor for osteoporosis during this
63
period [1]. There is evidence that both men and women – particularly those over
64
50 years old – may suffer from osteoporotic fractures during their lifetime and in
65
the Caucasian population 40% of women and 13% of men are thus affected [2].
66
Among the many determinants playing an important role on bone structure that
67
may lead to osteoporosis are renin angiotensin system (RAS), PPARγ
68
(peroxisome proliferator activated receptor gamma) protein expression levels in
69
the bone and the presence of female sex hormones.
70
The relationship between bones and the renin angiotensin system has been
71
investigated, with evidence showing that increased RAS activity has a
72
detrimental role on the bone tissue structure and metabolism, increasing
73
fracture risk [3, 4]. PPARγ is a superfamily of nuclear receptors for ligand-
74
activated transcription factors involved in the regulation of the metabolism of
75
glucose and lipids, in the immune response and promotion of cell differentiation
76
[5]. It has been found that increase PPARγ protein expression induced
77
microarchitecture deterioration in the bone structure [6-8]. The reduction of
78
estrogens in aging female is usually associated with reduced bone mass and
79
changes in bone microarchitecture, along with a high risk for fractures due to
80
the increased bone fragility associated with osteoporosis [9, 10].
81
Hypertension is a chronic disease in which the prevalence increases with age,
82
with the postmenopausal reduction of female sex hormones being considered a
83
risk factor for higher blood pressure [11-15]. The coexistence of hypertension
84
and significant bone loss in the femoral neck has been described, suggesting
85
an association between osteoporotic fractures and arterial hypertension [16].
86
Therefore, the evaluation of how antihypertensive agents can affect the bone is
87
highly relevant. Even more so because the osseous effects of antihypertensive
88
agents are controversial in the literature: they have been shown to preserve the
89
tissue [17-24] or favor its resorption, leading to bone loss [25, 26].
90
Osteoporotic bone disorders have been previously detected in spontaneously
91
hypertensive rats (SHR) [27], an animal model of postmenopausal hypertension
92
[28]. These animals were the chosen as a postmenopausal osteoporosis model
93
for the study of the effects of the antihypertensive drug telmisartan in both intact
94
and ovariectomized female [29]. Telmisartan, a dual drug, work as an
95
angiotensin II receptor blocker (ARB) and also as a partial agonist for PPARγ,
96
having been selected for our study because little is known regarding its effect
97
on osteoporosis associated with hypertension. Thus, in the present study we
98
propose to evaluate the microarchitecture, biomechanics and bone density, as
99
well as PPARγ protein levels, in bones of ovariectomized SHR females treated
100
with telmisartan for 8 weeks.
101
METHODS
102
Ethical approval
103
Adult female SHRs (3 months old) were provided by the animal care facility of
104
the central laboratory of the Federal University of Espírito Santo (UFES) and
105
were maintained on 12-h light/dark cycles at controlled conditions (temperature
106
and humidity), receiving water and a standard rat diet (Purina Labina, SP-Brazil)
107
ad libitum. All procedures were performed according to biomedical research
108
guidelines for the ethical care and use of animals in scientific research, having
109
been approved by our local Ethical Committee for Animal Use (Protocol
110
64/2012).
111
Experimental animals
112
At the time of ovariectomy, the animals were randomly assigned into the
113
following four groups: sham + vehicle (S), sham under telmisartan (ST),
114
castration + vehicle (C) and castration under telmisartan (CT). S and C groups
115
received
116
carboxymethylcellulose sodium-CMC-Na) and ST and CT groups received a
117
telmisartan dose dissolved in vehicle (5 mg/kg/day). All animals were weighed
118
weekly on a digital scale in order to adjust the doses of both drug and vehicle by
119
body mass, as well as to enable biometric analysis over the treatment period.
120
After the experimental protocol the animals were euthanized by a ketamine (90
121
mg/kg) and xylazine (10 mg/kg) combination; the bones were then removed (left
122
and right femur, left tibia and fifth lumbar vertebra) and dissected from soft
123
tissues, stored in saline and kept at -80° C for subsequent analysis. Hearts
daily
oral
gavage
for
8
weeks
with
vehicle
(0.5%
124
were excised and weighed tibia length was measured. Weight of heart were
125
related to tibia length to determine heart weight to tibia length ratio.
126
Ovariectomy surgery
127
The surgical procedure for castration (ovariectomy) was performed according to
128
the standard set by previous studies from our group, consisting of abdominal
129
incision in the midline under anesthesia with ketamine (70 mg / kg) and xylazine
130
(10 mg / kg). Animals belonging to the control groups (S and ST) underwent a
131
sham surgery. The onset of telmisartan or vehicle treatment occurred fifteen
132
days after the surgeries.
133
Non-invasive systolic blood pressure recordings
134
Forty-eight hours after the end of the treatment period, systolic blood pressure
135
(SBP) measurements were taken by the tail-cuff method coupled to an electro-
136
sphygmomanometer recorder (IITC Life Science - 23924 Victory Blvd,
137
Woodland Hills, CA). Results were expressed as the mean from three
138
independent measurements [30].
139
Analysis by Bone Densitometry by Dual Energy X-Ray Absorptiometry
140
(DXA)
141
Dual-energy X-ray absorptiometry (DXA) analysis was used to assess the BMD
142
of the spine vertebrae (L5) and total femur (total femur length including
143
diaphysis and epiphyses). The experiment was performed with the Discovery-A
144
SN: 80999 Hologic device (Bedford, MA, USA) in the high-resolution mode, with
145
the aid of the small animal software provided by the same manufacturer. The
146
accuracy of the DXA for assessing BMD was previously analyzed by measuring
147
the coefficient of variation, expressed as a percentage of the mean. The
148
coefficient of variation was 1.9% for the spine and 0.6% for the total femur,
149
indicating that the measurements were highly accurate [31-32].
150
Biomechanical Analysis
151
Bones were frozen at −80°C until the time of testing and then were thawed and
152
kept fully moist before the mechanical testing To evaluate biomechanical
153
properties, the left femora were tested in three-point bending using a universal
154
test machine (Instron, model 4444, Canton, Massachusetts, USA) with 100 kgf
155
capacity. The bones were supported by the ends in two rolls of 3 mm diameter
156
and at a distance of 21.7 mm. The central region of each bone underwent
157
tension loading [33].
158
A preload of 10 N was applied perpendicularly to the longitudinal axis of the
159
femur and, after a 1-min period of accommodation and stabilization, force was
160
applied at a constant speed of 0.5 cm/min until bone involvement and fracture.
161
The stress generated on the bone was determined by plotting the amount of
162
force applied over time with the aid of the software Instron (series IX). The
163
mechanical properties visualized in the graph were maximum load (N), stiffness
164
(N/mm), resilience (J) and failure load (N).
165
Morphometric analysis the trabecular structure by Micro-Computed
166
Tomography (µCT)
167
The evaluation of the femur trabecular structure parameters was done using
168
Bruker's micro CT equipment (model SkyScan 1174, Kontich, Belgium). The
169
system configurations were as follows: electric current of 730 µA and operating
170
voltage set at 45 kV. Several X-rays of the bone were taken at different angles,
171
generating measurements of the intensity of X-rays transmitted through the
172
bone sample. Femur samples were rotated 180 degrees, with an angular step
173
of 0.8 degrees, thus producing 225 radiographs (projections) per image, each
174
containing 1024×1024 pixels with a spatial resolution of 9.8 µm. A 0.5 mm-thick
175
aluminum filter was used at the outlet of the X-ray source. CT scans with the
176
same spatial resolution as the 2D radiographs were established.
177
Three-dimensional (3D) renderings of the proximal right femur region were
178
generated from the two-dimensional (2D) projections using appropriate
179
algorithms. Thus, for each femur sample the volume of data generated was
180
isotropic in relation to spatial resolution. The following parameters were
181
analysed: bone and tissue volumes, bone superface, trabecular thickness,
182
trabecular number and trabecular separation, porosity, number of closed pores
183
and connectivity.
184
Reconstruction of tomographic images was done with the aid of an appropriate
185
algorithm and of Bruker's NRecon ™ software (version 1.6.9.4, Kontich,
186
Belgium), which gathered all the radiographic projections at each angular
187
position [34].
188
Western blot analysis
189
PPARγ protein expression levels in the total femur of rats from the different
190
groups were detected by Western Blot. Femurs were excised, cleaned of all
191
muscle and connective tissue, and kept at -20 °C until processing. The samples
192
were homogenized in lysis buffer [Tris-HCL pH 7.4 (10 mM), PMSF (1 mM),
193
NaVO3 (1 mM), SDS (1 %), DTT (0.5 mM), EDTA (5 mM) and protease inhibitor
194
cocktail (1:100 dilution)]. Total protein content was determined using the
195
Bradford method. Samples containing 50 µg of protein were fractionated on a
196
10 % SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad
197
Laboratories, Hercules, CA, USA). After saturation with 5 % (w/v) nonfat dry
198
milk in TBS and 0.1 % (w/v) Tween 20 (TBST), the membranes were incubated
199
with Mouse anti-PPAR-γ ([1:200], Santa Cruz, Inc., USA) or Mouse anti-β-actin
200
([1:5000], Santa Cruz, Inc., USA). Membranes were washed five times with
201
TBST and incubated with the peroxidase-conjugated secondary antibodies
202
([1:5000], Sigma, USA). Western blot signals were quantified using Bio-Rad
203
Image Lab 5.2.1 software, and protein expression levels were normalized to β-
204
actin expression.
205
Statistical analysis
206
Data are expressed as means ± SEM and the statistical analysis was performed
207
using GraphPad Prism 6. The data were compared using two-way ANOVA
208
followed by post-hoc Fisher LSD’s test for inter-groups comparison. The data
209
were considered significant at p < 0.05.
210
RESULTS
211
SBP and Ponderal data
212
Baseline physiological parameters and the effects of telmisartan are presented
213
in table 1. SBP was found to be reduced in both telmisartan groups after the
214
experimental protocol period, with the CT group showing a greater reduction
215
than the ST group. The ovariectomy surgery led to the expected changes in
216
total body weight and uterine weight, with a significant increase in body weight
217
and a reduction in uterus weight having been observed.
218
Table
219
morphometric data, SBP, and biometric and biomechanical parameters.
220
Animals groups: S (sham), ST (sham with telmisartan use), C (castration),
221
CT (castration with telmisartan use).
1.
Effect
of
treatment
with
S
telmisartan
on
ponderal
and
ST
C
CT
Ponderal and morphometrics data and SBP (n=9) Final body weight (g)
199.66 ± 3.87
188.85 ± 2.34a,c
244.63 ± 5.03a
215.13 ± 2.96a,b,c
Uterine weight (g)
0.426 ± 0.035
0.360 ± 0.038c
0.122 ± 0.032a
0.093 ± 0.024a,b
Heart/tibia (g/cm)
0.236 ± 0.010
0.209 ± 0.007a,c
0.253 ± 0.005
0.203 ± 0.006a,c
190.8 ± 2.3
122.8 ± 2.8a,c
191.2 ± 3.4
111.2 ± 3.7a,b,c
3,28 ± 0,01
3,19 ± 0,02a,c
3,34 ± 0,01a
3,32 ± 0,0b
0,092 ± 0,004
0,081 ± 0,001a,c
0,092 ± 0,003
0,087 ± 0,003
Systolyc Blood Pressure (mmHg) Biometric data (n=7) Femur, cm Biomechanical data (n=7) Maximum Load (N) Stiffness (N/mm)
227,52 ± 20,51
192,67 ± 7,38
208,01 ± 7,93
173,83 ± 12,74a
Resilience (J)
0,049 ± 0,007
0,034 ± 0,003a,c
0.043 ± 0.004
0.047 ± 0.004b
Failure Load (N)
0,076 ± 0,004
0,054 ± 0,005a,c
0.072 ± 0.004
0.070 ± 0.003b
a
a
b
c
222
Data are expressed as the mean ± SEM. p < 0.05 vs. S group; p < 0.05 vs. ST group; p <
223
0.05 vs. C group. (two-way ANOVA and post hoc Fisher’s test)
224
Treatment with telmisartan reduced the final body weight of castrated and intact
225
animals when compared to their respective counterparts (ST vs. S and CT vs.
226
C). Heart weight and tibia length ratio were reduced upon treatment of both
227
castrated (CT) and intact (ST) groups when compared to intact sham animals
228
(S), with animals from the CT group presenting lower ratios than those who
229
went through castration but remained untreated (C).
230
Biometrical and biomechanical parameters
231
In relation to the effects of telmisartan on femoral biometrical and biomechanical
232
parameters (table 1), its use was found to cause damage to the femur’s length,
233
especially in ST group. The maximal load was reduced in the animals from ST
234
when compared to those of the non use telmisartan groups (S and C). There
235
was a reduction in stiffness in the animals from the ST and CT groups when
236
compared to those of the S group. Finally, resilience and failure load were lower
237
in animals from the ST group compared to those belonging to the others
238
groups. No differences were found in others parameters analyzed (data no
239
shown).
240
Bone mineral density
241
On the whole, when the BMD of femur and the fifth lumbar vertebra was
242
evaluated by DXA, a density reduction was observed in the bones of castrated
243
animals when compared to intact ones (Fig. 1). Notably, castration was able to
244
reduce bone density on its own, which was evidenced by the reduction in the
245
BMD of the animals in the C group when compared to those in the S group
246
(panels A, B and C). The treatment with telmisartan led to the worst outcome for
247
the proximal femur and the lumbar vertebra, regardless of castration.
248
Nevertheless, the combination of castration and treatment with telmisartan
249
resulted in a larger reduction in BMD when compared to animals who did not
250
receive the drug (CT versus S or C groups in panels A, B and C).
251 252
Figure 1. Effects of the 8-week treatment with telmisartan on bone mineral
253
density (BMD). (A) total femur, (B) fifth lumbar vertebrae, (C) femur proximal
254
region (R1), (C1) femur proximal region (R1) by micro-CT. Data are expressed
255
as the mean ± SEM (n = 7). *p < 0.05 vs. S group, &p < 0.05 vs. CT group, #p <
256
0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
257
The use of telmisartan in sham animals resulted in reduced femoral BMD, but
258
no alterations were observed in the lumbar vertebra (ST vs. S group).
259
BMD measurements from the proximal femoral region were also obtained using
260
the µCT method (figure 1, panel D). Yet again, although this parameter was
261
obtained through a technique other than DXA, a clear impairment was observed
262
in castrated animals (C group) in relation to sham ones (S group). Animals
263
treated with telmisartan (ST or CT) showed a reduction in BMD when compared
264
to their respective untreated groups (S or C). Finally, the association of
265
castration with telmisartan use (CT group) resulted in the lowest BMD when
266
compared to all others groups.
267
Morphometric analysis of trabecular microarchitecture
268
The analysis of bone parameters related to the integrity of the trabecular
269
microarchitecture by µCT is shown in the figures 2 and 3. Porosity, total pore
270
volume, bone surface density, percent bone volume, number of closed pores,
271
trabecular number and connectivity were parameters that were affected by the
272
interventions.
273
On the whole, we observed that ovariectomy (C group) caused a marked
274
deterioration in the microstructure of the trabecular region of proximal femur
275
when compared to intact animals (S group). Treatment with telmisartan
276
increased the deterioration in bone architecture of ovariectomized animals (CT
277
group) even further. Also, its use in sham animals (ST group) usually caused
278
damage in the bone microarchitecture when compared to intact, untreated
279
animals (S group).
280
In general, there was an increase of porosity and a reduction in the closed
281
number pores, bone surface density, percent bone volume, trabecular number
282
and in bone connectivity in animals from the castrated group treated with
283
telmisartan (CT versus all other groups).
284 285
Figure 2. Effects of 8-week treatment of the animals with telmisartan on
286
the microarchitectural characteristics. (A) Total porosity, (B) total volume of
287
pore space, (C) bone surface density, (D) bone volume percentual. Data are
288
expressed as the mean ± SEM (n = 5). &p < 0.05 vs. CT group, *p < 0.05 vs. S
289
group, #p < 0.05 vs. all others groups. (two-way ANOVA and post hoc Fisher’s
290
test).
291 292
Figure 3. Effects of 8-week treatment of the animals with telmisartan on
293
microarchitectural characteristics.
294
trabecular number, (C) connectivity, (D) connectivity density. Data are
295
expressed as the mean ± SEM (n = 5). *p < 0.05 vs. S group, &p < 0.05 vs. C
296
group, #p < 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s
297
test).
298
Western blot analysis (PPARγ protein expression)
299
Figure 4 shows the analysis of PPARγ protein expression, quantified by
300
densitometry and normalized by the expression of β-actin. PPARγ levels were
301
found to be increased in castrated animals (C group) and in those who went
302
through telmisartan treatment (ST and CT group), when compared to intact,
303
untreated animals (S group). It is worthy of note that the combination of
(A) Number of closed pores, (B)
304
castration and telmisartan use yielded the higher PPARγ expression levels (CT
305
group vs. ST and C groups).
306 307
Figure 4. Effects of 8-week treatment with telmisartan on PPARγ protein
308
(54 kDa) expression in total femur. The signal from each protein was
309
normalized by the respective amount of β-actin (42 kDa) even before calculating
310
the ratios. Data are expressed as mean ± SEM (n = 5). *p < 0.05 vs. S group, #p
311
< 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
312
DISCUSSION
313
In the present study, we provide evidence of increased expression of the protein
314
PPARγ associated with the occurrence of biomechanical and morphologic
315
defects in bones – femur and lumbar vertebrae – in intact and ovariectomized
316
SHR females (an animal model of both postmenopausal osteoporosis and
317
hypertension) by telmisartan use. Interestingly, rats from the ST group
318
resembled those from the castrated group (C) regarding bone loss and
319
increased PPARγ protein expression, and one should note that these
320
detrimental changes were even more pronounced in ovariectomized animals
321
treated with telmisartan (CT group). In spite of negative bone effects,
322
telmisartan efficiently reduced blood pressure and cardiac hypertrophy in
323
animals that were underwent treatment, which corroborates others studies
324
conducted in hypertensive rats [35, 36]. Such findings are quite important,
325
adding to the overall relevance of the data present here.
326
Bone microarchitecture deterioration (trabecular destruction and reduction in the
327
connectivity), which can happen as a result of drastic reductions on female sex
328
hormone levels, is considered a sensitive parameter to demonstrate trabecular
329
disconnectivity, therefore reducing the strength of the tissue and its ability to
330
resist to stress, increasing the risk of fractures [37]. The increased risk of
331
fractures observed during the postmenopausal phase is a consequence of the
332
rapid bone loss caused by an increase in bone turnover and a decrease in
333
tissue formation [38]. The consequent reduction in estrogen levels that
334
necessarily follow the castration performed in our model could account for the
335
worsening in the quality of bone microarchitecture observed here, corroborating
336
the aforementioned reports.
337
It has been put forward that the renin angiotensin system acts in bones through
338
the regulation of structural and metabolic aspects of this tissue by angiotensin II
339
[3, 4]. Specifically regarding the use of ARBs the data are controversial and not
340
conclusive [25, 39-41]. It has been reported that Angio II activates osteoclasts
341
decreasing bone density in ovariectomized SHRs, and the use of olmesartan
342
reverses this effect [39]. In contrast, telmisartan use in SHR male increased
343
bone detrimental effects [25] or did not change bone turnover markers in
344
patients with mild hypertension [40]. Also, losartan was unable to prevent bone
345
derangement in both hypertensive and normotensive male rats, suggesting a
346
lower participation of AT1 receptors in the effects of angiotensin II on bone
347
tissue [41]. These controversial results from different ARBs may be due to their
348
mode of action and the experimental doses and experimental design used.
349
Even though the mechanisms involved are not fully understood, it is known that
350
PPARγ agonists can cause bone loss in vitro and in rodent models [6-8, 42-44].
351
Treatment with PPARγ agonists – thiazolidinediones (TZDs) – has side effects
352
such as a significant risk of adverse effects on human bone, and PPARγ
353
activation by TZDs causes bone loss by suppressing differentiation and activity
354
of osteoblasts, while reducing bone formation and enhancing osteoclasts
355
differentiation [42, 44]. Another possible mechanism underlying the bone loss
356
related to the action of PPARγ agonists involves adipogenic cells differentiation
357
from mesenchymal stem cells (MSCs) at the expense of osteoblastogenesis
358
[45, 46]. Taken together, these data suggest that PPARγ agonists may increase
359
the risk of bone loss.
360
Our results suggest that the use of telmisartan, a PPARγ partial agonist, may
361
have induced bone loss mediated by the activation of PPARγ in this tissue, in
362
view of the higher levels of this protein found in bone of ovariectomized animals
363
and in those treated with telmisartan (ST and CT). However, one cannot rule
364
out th participation of other mechanisms.
365
In summary, telmisartan in ovariectomized SHRs – a model of oestrogen
366
deficiency in hypertensive rats – increased the levels of femoral PPARγ protein
367
expression and compromised trabecular bone microarchitecture by reducing
368
bone mass as evidenced by the shape and connectivity parameters observed
369
by DXA and micro-CT analysis.
370
CONCLUSION
371
In conclusion, these results demonstrated that the treatment of animals showing
372
preexisting osteoporotic bone disorders with telmisartan compromised bone
373
density and microarchitecture, probably via mechanisms associated with
374
increased PPARγ. Actual data may reveal a need for greater caution in the use
375
of telmisartan in patients that have preexisting bone alterations, as in the
376
postmenopausal period.
377
Whether these experimental findings translate into human clinical situations is
378
not yet known and additional studies employing specific imaging techniques,
379
such as densitometry and/or tomography, may be required to follow up patients
380
taking telmisartan.
381
Conflicts of interest
382
The authors declare that there are no conflicts of interest associated with this
383
manuscript.
384
Author contributions
385
The contributions of each author to the study were:
386
Antonio Marcos Birocalea,b, Antonio Ferreira de Melo Jra,b, Pollyana Peixotoa,
387
Phablo Wendell Costalonga Oliveiraa, Leandro Dias Gonçalves Ruffonia, Liliam
388
Masako Takayamaa, Breno Valentim Nogueiraa,b, Keico Okino Nonakaa,b, Rosa
389
Maria Rodrigues Pereiraa,b, José Martins de Oliveira Jr.a,b, Nazaré Souza
390
Bissolia,b
391
a
392
b
393
of data; drafting the article; revising it critically for important intellectual content;
394
and final approval of the version to be published.
395
Acknowledgements
396
This work was funded by Fundação de Amparo a Pesquisa do Espirito Santo –
397
FAPES [Grant number 033/2016] for the purchase of inputs and drugs.
398
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565
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567
Figure Captions
568
Figure 1. Effects of the 8-week treatment with telmisartan on bone mineral
569
density (BMD). (A) total femur, (B) fifth lumbar vertebrae, (C) femur proximal
570
region (R1), (C1) femur proximal region (R1) by micro-CT. Data are expressed
571
as the mean ± SEM (n = 7). *p < 0.05 vs. S group, &p < 0.05 vs. CT group, #p <
572
0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
573
Figure 2. Effects of 8-week treatment of the animals with telmisartan on
574
the microarchitectural characteristics. (A) Total porosity, (B) total volume of
575
pore space, (C) bone surface density, (D) bone volume percentual. Data are
576
expressed as the mean ± SEM (n = 5). &p < 0.05 vs. CT group, *p < 0.05 vs. S
577
group, #p < 0.05 vs. all others groups. (two-way ANOVA and post hoc Fisher’s
578
test).
579
Figure 3. Effects of 8-week treatment of the animals with telmisartan on
580
microarchitectural characteristics. (A) Number of closed pores, (B)
581
trabecular number, (C) connectivity, (D) connectivity density. Data are
582
expressed as the mean ± SEM (n = 5). *p < 0.05 vs. S group, &p < 0.05 vs. C
583
group, #p < 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s
584
test).
585
Figure 4. Effects of 8-week treatment with telmisartan on PPARγ protein
586
(54 kDa) expression in total femur. The signal from each protein was
587
normalized by the respective amount of β-actin (42 kDa) even before calculating
588
the ratios. Data are expressed as mean ± SEM (n = 5). *p < 0.05 vs. S group, #p
589
< 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
S0024-3205(19)30817-3
DOI:
https://doi.org/10.1016/j.lfs.2019.116890
Reference:
LFS 116890
To appear in:
Life Sciences
Received Date: 31 July 2019 Revised Date:
11 September 2019
Accepted Date: 19 September 2019
Please cite this article as: A.M. Birocale, A. Ferreira de Melo Jr., P. Peixoto, P.W. Costalonga Oliveira, L.D. Gonçalves Ruffoni, L.M. Takayama, B.V. Nogueira, K.O. Nonaka, R.M. Rodrigues Pereira, José. Martins de Oliveira Jr., Nazaré.Souza. Bissoli, Telmisartan use in rats with preexisting osteoporotics bone disorders increases bone microarchitecture alterations via PPARγ, Life Sciences (2019), doi: https://doi.org/10.1016/j.lfs.2019.116890. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
1
Telmisartan use in rats with preexisting osteoporotics bone disorders
2
increases bone microarchitecture alterations via PPARγ
3
Authors: Antonio Marcos Birocale, MDa, Antonio Ferreira de Melo Jr, MDb,
4
Pollyana Peixoto, MDb, Phablo Wendell Costalonga Oliveira, PhDb, Leandro
5
Dias Gonçalves Ruffoni, PhDc, Liliam Masako Takayama, MDd, Breno Valentim
6
Nogueira, PhDe, Keico Okino Nonaka, PhDc, Rosa Maria Rodrigues Pereira,
7
PhDd, José Martins de Oliveira Jr., PhDf, Nazaré Souza Bissoli, PhDb*
8 9
a
Department of Health Integrated Education, Federal University of Espirito
10
Santo, Vitória, ES, Brazil
11
b
12
Vitória, ES, Brazil
13
c
14
Carlos, SP, Brazil
15
d
16
Paulo, SP, Brazil
17
e
18
Brazil
19
f
20
Brazil
Department of Physiological Sciences, Federal University of Espirito Santo,
Department of Physiological Sciences, Federal University of São Carlos, São
Department of Medical Clinic, Medicine College, University of São Paulo, São
Department of Morphology, Federal University of Espirito Santo, Vitoria, ES,
Laboratory of Applied Nuclear Physics, University of Sorocaba, Sorocaba, SP,
21 22
*
23
Nazaré Souza Bissoli ([email protected])
24
Phone number: 55 27 998806356; 55 27 33357333
25
Address: Department of Physiological Sciences; Centre for Health Sciences,
26
Federal University of Espirito Santo. Av. Marechal Campos 1468, 29042-755,
27
Vitória, ES – Brazil.
Corresponding author:
28
Telmisartan use in rats with preexisting osteoporotics bone disorders
29
increases bone microarchitecture alterations via PPARγ
30
Abstract
31
Aims: Telmisartan (TEL), an angiotensin II type I receptor blocker and PPARγ
32
partial agonist, has been used for to treat hypertension. It is known that PPARγ
33
activation induces bone loss. Therefore, we evaluate the effects of telmisartan
34
on
35
microarchitecture of femurs and lumbar vertebrae in SHR ovariectomized
36
animals, a model of hypertension in which preexisting bone impairment has
37
been demonstrated.
38
Main methods: SHR females (3 months old) were distributed into four groups:
39
sham (S), sham+TEL (ST), OVX (C) and OVX+TEL (CT). TEL (5mg/kg/day) or
40
vehicle were administered according to the groups. After the protocol, blood
41
pressure was measured and density, microarchitecture and biomechanics of
42
bone were analyzed. Western blotting analysis was performed to evaluate
43
PPARγ protein expression in the bones.
44
Key findings: Castration induced a deleterious effect on mineral density and
45
trabecular parameters, with telmisartan enhancing such effects. Telmisartan
46
increased PPARγ levels, which were at their highest when the treatment was
47
combined with castration. As to biomechanical properties, telmisartan reduced
48
the stiffness in the castration group (CT vs. S or C group), as well as resilience
49
and failure load in ST group (vs. all others groups).
PPARγ
protein
expression,
biomechanics,
density
and
bone
50
Significance: These results demonstrated that telmisartan compromised bone
51
density and microarchitecture in animals that shows preexisting osteoporotic
52
bone disorders, probably via mechanisms associated with increased PPARγ. If
53
this translates to humans, a need for greater caution in the use of telmisartan by
54
patients that have preexisting bone problems, as in the postmenopausal period,
55
may be in order.
56
Keywords: Telmisartan, PPARγ, Hypertension, Ovariectomy, Bone
57
INTRODUCTION
58
Osteoporosis is associated with loss of bone mass due to decreased tissue
59
continuity, with the rupture of the trabecular microarchitecture leading to
60
reduced connectivity, increased bone fragility and fracture risk. The concomitant
61
imbalance between osteoclastic and osteoblastic activities is exacerbated in the
62
postmenopausal phase, being a determining factor for osteoporosis during this
63
period [1]. There is evidence that both men and women – particularly those over
64
50 years old – may suffer from osteoporotic fractures during their lifetime and in
65
the Caucasian population 40% of women and 13% of men are thus affected [2].
66
Among the many determinants playing an important role on bone structure that
67
may lead to osteoporosis are renin angiotensin system (RAS), PPARγ
68
(peroxisome proliferator activated receptor gamma) protein expression levels in
69
the bone and the presence of female sex hormones.
70
The relationship between bones and the renin angiotensin system has been
71
investigated, with evidence showing that increased RAS activity has a
72
detrimental role on the bone tissue structure and metabolism, increasing
73
fracture risk [3, 4]. PPARγ is a superfamily of nuclear receptors for ligand-
74
activated transcription factors involved in the regulation of the metabolism of
75
glucose and lipids, in the immune response and promotion of cell differentiation
76
[5]. It has been found that increase PPARγ protein expression induced
77
microarchitecture deterioration in the bone structure [6-8]. The reduction of
78
estrogens in aging female is usually associated with reduced bone mass and
79
changes in bone microarchitecture, along with a high risk for fractures due to
80
the increased bone fragility associated with osteoporosis [9, 10].
81
Hypertension is a chronic disease in which the prevalence increases with age,
82
with the postmenopausal reduction of female sex hormones being considered a
83
risk factor for higher blood pressure [11-15]. The coexistence of hypertension
84
and significant bone loss in the femoral neck has been described, suggesting
85
an association between osteoporotic fractures and arterial hypertension [16].
86
Therefore, the evaluation of how antihypertensive agents can affect the bone is
87
highly relevant. Even more so because the osseous effects of antihypertensive
88
agents are controversial in the literature: they have been shown to preserve the
89
tissue [17-24] or favor its resorption, leading to bone loss [25, 26].
90
Osteoporotic bone disorders have been previously detected in spontaneously
91
hypertensive rats (SHR) [27], an animal model of postmenopausal hypertension
92
[28]. These animals were the chosen as a postmenopausal osteoporosis model
93
for the study of the effects of the antihypertensive drug telmisartan in both intact
94
and ovariectomized female [29]. Telmisartan, a dual drug, work as an
95
angiotensin II receptor blocker (ARB) and also as a partial agonist for PPARγ,
96
having been selected for our study because little is known regarding its effect
97
on osteoporosis associated with hypertension. Thus, in the present study we
98
propose to evaluate the microarchitecture, biomechanics and bone density, as
99
well as PPARγ protein levels, in bones of ovariectomized SHR females treated
100
with telmisartan for 8 weeks.
101
METHODS
102
Ethical approval
103
Adult female SHRs (3 months old) were provided by the animal care facility of
104
the central laboratory of the Federal University of Espírito Santo (UFES) and
105
were maintained on 12-h light/dark cycles at controlled conditions (temperature
106
and humidity), receiving water and a standard rat diet (Purina Labina, SP-Brazil)
107
ad libitum. All procedures were performed according to biomedical research
108
guidelines for the ethical care and use of animals in scientific research, having
109
been approved by our local Ethical Committee for Animal Use (Protocol
110
64/2012).
111
Experimental animals
112
At the time of ovariectomy, the animals were randomly assigned into the
113
following four groups: sham + vehicle (S), sham under telmisartan (ST),
114
castration + vehicle (C) and castration under telmisartan (CT). S and C groups
115
received
116
carboxymethylcellulose sodium-CMC-Na) and ST and CT groups received a
117
telmisartan dose dissolved in vehicle (5 mg/kg/day). All animals were weighed
118
weekly on a digital scale in order to adjust the doses of both drug and vehicle by
119
body mass, as well as to enable biometric analysis over the treatment period.
120
After the experimental protocol the animals were euthanized by a ketamine (90
121
mg/kg) and xylazine (10 mg/kg) combination; the bones were then removed (left
122
and right femur, left tibia and fifth lumbar vertebra) and dissected from soft
123
tissues, stored in saline and kept at -80° C for subsequent analysis. Hearts
daily
oral
gavage
for
8
weeks
with
vehicle
(0.5%
124
were excised and weighed tibia length was measured. Weight of heart were
125
related to tibia length to determine heart weight to tibia length ratio.
126
Ovariectomy surgery
127
The surgical procedure for castration (ovariectomy) was performed according to
128
the standard set by previous studies from our group, consisting of abdominal
129
incision in the midline under anesthesia with ketamine (70 mg / kg) and xylazine
130
(10 mg / kg). Animals belonging to the control groups (S and ST) underwent a
131
sham surgery. The onset of telmisartan or vehicle treatment occurred fifteen
132
days after the surgeries.
133
Non-invasive systolic blood pressure recordings
134
Forty-eight hours after the end of the treatment period, systolic blood pressure
135
(SBP) measurements were taken by the tail-cuff method coupled to an electro-
136
sphygmomanometer recorder (IITC Life Science - 23924 Victory Blvd,
137
Woodland Hills, CA). Results were expressed as the mean from three
138
independent measurements [30].
139
Analysis by Bone Densitometry by Dual Energy X-Ray Absorptiometry
140
(DXA)
141
Dual-energy X-ray absorptiometry (DXA) analysis was used to assess the BMD
142
of the spine vertebrae (L5) and total femur (total femur length including
143
diaphysis and epiphyses). The experiment was performed with the Discovery-A
144
SN: 80999 Hologic device (Bedford, MA, USA) in the high-resolution mode, with
145
the aid of the small animal software provided by the same manufacturer. The
146
accuracy of the DXA for assessing BMD was previously analyzed by measuring
147
the coefficient of variation, expressed as a percentage of the mean. The
148
coefficient of variation was 1.9% for the spine and 0.6% for the total femur,
149
indicating that the measurements were highly accurate [31-32].
150
Biomechanical Analysis
151
Bones were frozen at −80°C until the time of testing and then were thawed and
152
kept fully moist before the mechanical testing To evaluate biomechanical
153
properties, the left femora were tested in three-point bending using a universal
154
test machine (Instron, model 4444, Canton, Massachusetts, USA) with 100 kgf
155
capacity. The bones were supported by the ends in two rolls of 3 mm diameter
156
and at a distance of 21.7 mm. The central region of each bone underwent
157
tension loading [33].
158
A preload of 10 N was applied perpendicularly to the longitudinal axis of the
159
femur and, after a 1-min period of accommodation and stabilization, force was
160
applied at a constant speed of 0.5 cm/min until bone involvement and fracture.
161
The stress generated on the bone was determined by plotting the amount of
162
force applied over time with the aid of the software Instron (series IX). The
163
mechanical properties visualized in the graph were maximum load (N), stiffness
164
(N/mm), resilience (J) and failure load (N).
165
Morphometric analysis the trabecular structure by Micro-Computed
166
Tomography (µCT)
167
The evaluation of the femur trabecular structure parameters was done using
168
Bruker's micro CT equipment (model SkyScan 1174, Kontich, Belgium). The
169
system configurations were as follows: electric current of 730 µA and operating
170
voltage set at 45 kV. Several X-rays of the bone were taken at different angles,
171
generating measurements of the intensity of X-rays transmitted through the
172
bone sample. Femur samples were rotated 180 degrees, with an angular step
173
of 0.8 degrees, thus producing 225 radiographs (projections) per image, each
174
containing 1024×1024 pixels with a spatial resolution of 9.8 µm. A 0.5 mm-thick
175
aluminum filter was used at the outlet of the X-ray source. CT scans with the
176
same spatial resolution as the 2D radiographs were established.
177
Three-dimensional (3D) renderings of the proximal right femur region were
178
generated from the two-dimensional (2D) projections using appropriate
179
algorithms. Thus, for each femur sample the volume of data generated was
180
isotropic in relation to spatial resolution. The following parameters were
181
analysed: bone and tissue volumes, bone superface, trabecular thickness,
182
trabecular number and trabecular separation, porosity, number of closed pores
183
and connectivity.
184
Reconstruction of tomographic images was done with the aid of an appropriate
185
algorithm and of Bruker's NRecon ™ software (version 1.6.9.4, Kontich,
186
Belgium), which gathered all the radiographic projections at each angular
187
position [34].
188
Western blot analysis
189
PPARγ protein expression levels in the total femur of rats from the different
190
groups were detected by Western Blot. Femurs were excised, cleaned of all
191
muscle and connective tissue, and kept at -20 °C until processing. The samples
192
were homogenized in lysis buffer [Tris-HCL pH 7.4 (10 mM), PMSF (1 mM),
193
NaVO3 (1 mM), SDS (1 %), DTT (0.5 mM), EDTA (5 mM) and protease inhibitor
194
cocktail (1:100 dilution)]. Total protein content was determined using the
195
Bradford method. Samples containing 50 µg of protein were fractionated on a
196
10 % SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad
197
Laboratories, Hercules, CA, USA). After saturation with 5 % (w/v) nonfat dry
198
milk in TBS and 0.1 % (w/v) Tween 20 (TBST), the membranes were incubated
199
with Mouse anti-PPAR-γ ([1:200], Santa Cruz, Inc., USA) or Mouse anti-β-actin
200
([1:5000], Santa Cruz, Inc., USA). Membranes were washed five times with
201
TBST and incubated with the peroxidase-conjugated secondary antibodies
202
([1:5000], Sigma, USA). Western blot signals were quantified using Bio-Rad
203
Image Lab 5.2.1 software, and protein expression levels were normalized to β-
204
actin expression.
205
Statistical analysis
206
Data are expressed as means ± SEM and the statistical analysis was performed
207
using GraphPad Prism 6. The data were compared using two-way ANOVA
208
followed by post-hoc Fisher LSD’s test for inter-groups comparison. The data
209
were considered significant at p < 0.05.
210
RESULTS
211
SBP and Ponderal data
212
Baseline physiological parameters and the effects of telmisartan are presented
213
in table 1. SBP was found to be reduced in both telmisartan groups after the
214
experimental protocol period, with the CT group showing a greater reduction
215
than the ST group. The ovariectomy surgery led to the expected changes in
216
total body weight and uterine weight, with a significant increase in body weight
217
and a reduction in uterus weight having been observed.
218
Table
219
morphometric data, SBP, and biometric and biomechanical parameters.
220
Animals groups: S (sham), ST (sham with telmisartan use), C (castration),
221
CT (castration with telmisartan use).
1.
Effect
of
treatment
with
S
telmisartan
on
ponderal
and
ST
C
CT
Ponderal and morphometrics data and SBP (n=9) Final body weight (g)
199.66 ± 3.87
188.85 ± 2.34a,c
244.63 ± 5.03a
215.13 ± 2.96a,b,c
Uterine weight (g)
0.426 ± 0.035
0.360 ± 0.038c
0.122 ± 0.032a
0.093 ± 0.024a,b
Heart/tibia (g/cm)
0.236 ± 0.010
0.209 ± 0.007a,c
0.253 ± 0.005
0.203 ± 0.006a,c
190.8 ± 2.3
122.8 ± 2.8a,c
191.2 ± 3.4
111.2 ± 3.7a,b,c
3,28 ± 0,01
3,19 ± 0,02a,c
3,34 ± 0,01a
3,32 ± 0,0b
0,092 ± 0,004
0,081 ± 0,001a,c
0,092 ± 0,003
0,087 ± 0,003
Systolyc Blood Pressure (mmHg) Biometric data (n=7) Femur, cm Biomechanical data (n=7) Maximum Load (N) Stiffness (N/mm)
227,52 ± 20,51
192,67 ± 7,38
208,01 ± 7,93
173,83 ± 12,74a
Resilience (J)
0,049 ± 0,007
0,034 ± 0,003a,c
0.043 ± 0.004
0.047 ± 0.004b
Failure Load (N)
0,076 ± 0,004
0,054 ± 0,005a,c
0.072 ± 0.004
0.070 ± 0.003b
a
a
b
c
222
Data are expressed as the mean ± SEM. p < 0.05 vs. S group; p < 0.05 vs. ST group; p <
223
0.05 vs. C group. (two-way ANOVA and post hoc Fisher’s test)
224
Treatment with telmisartan reduced the final body weight of castrated and intact
225
animals when compared to their respective counterparts (ST vs. S and CT vs.
226
C). Heart weight and tibia length ratio were reduced upon treatment of both
227
castrated (CT) and intact (ST) groups when compared to intact sham animals
228
(S), with animals from the CT group presenting lower ratios than those who
229
went through castration but remained untreated (C).
230
Biometrical and biomechanical parameters
231
In relation to the effects of telmisartan on femoral biometrical and biomechanical
232
parameters (table 1), its use was found to cause damage to the femur’s length,
233
especially in ST group. The maximal load was reduced in the animals from ST
234
when compared to those of the non use telmisartan groups (S and C). There
235
was a reduction in stiffness in the animals from the ST and CT groups when
236
compared to those of the S group. Finally, resilience and failure load were lower
237
in animals from the ST group compared to those belonging to the others
238
groups. No differences were found in others parameters analyzed (data no
239
shown).
240
Bone mineral density
241
On the whole, when the BMD of femur and the fifth lumbar vertebra was
242
evaluated by DXA, a density reduction was observed in the bones of castrated
243
animals when compared to intact ones (Fig. 1). Notably, castration was able to
244
reduce bone density on its own, which was evidenced by the reduction in the
245
BMD of the animals in the C group when compared to those in the S group
246
(panels A, B and C). The treatment with telmisartan led to the worst outcome for
247
the proximal femur and the lumbar vertebra, regardless of castration.
248
Nevertheless, the combination of castration and treatment with telmisartan
249
resulted in a larger reduction in BMD when compared to animals who did not
250
receive the drug (CT versus S or C groups in panels A, B and C).
251 252
Figure 1. Effects of the 8-week treatment with telmisartan on bone mineral
253
density (BMD). (A) total femur, (B) fifth lumbar vertebrae, (C) femur proximal
254
region (R1), (C1) femur proximal region (R1) by micro-CT. Data are expressed
255
as the mean ± SEM (n = 7). *p < 0.05 vs. S group, &p < 0.05 vs. CT group, #p <
256
0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
257
The use of telmisartan in sham animals resulted in reduced femoral BMD, but
258
no alterations were observed in the lumbar vertebra (ST vs. S group).
259
BMD measurements from the proximal femoral region were also obtained using
260
the µCT method (figure 1, panel D). Yet again, although this parameter was
261
obtained through a technique other than DXA, a clear impairment was observed
262
in castrated animals (C group) in relation to sham ones (S group). Animals
263
treated with telmisartan (ST or CT) showed a reduction in BMD when compared
264
to their respective untreated groups (S or C). Finally, the association of
265
castration with telmisartan use (CT group) resulted in the lowest BMD when
266
compared to all others groups.
267
Morphometric analysis of trabecular microarchitecture
268
The analysis of bone parameters related to the integrity of the trabecular
269
microarchitecture by µCT is shown in the figures 2 and 3. Porosity, total pore
270
volume, bone surface density, percent bone volume, number of closed pores,
271
trabecular number and connectivity were parameters that were affected by the
272
interventions.
273
On the whole, we observed that ovariectomy (C group) caused a marked
274
deterioration in the microstructure of the trabecular region of proximal femur
275
when compared to intact animals (S group). Treatment with telmisartan
276
increased the deterioration in bone architecture of ovariectomized animals (CT
277
group) even further. Also, its use in sham animals (ST group) usually caused
278
damage in the bone microarchitecture when compared to intact, untreated
279
animals (S group).
280
In general, there was an increase of porosity and a reduction in the closed
281
number pores, bone surface density, percent bone volume, trabecular number
282
and in bone connectivity in animals from the castrated group treated with
283
telmisartan (CT versus all other groups).
284 285
Figure 2. Effects of 8-week treatment of the animals with telmisartan on
286
the microarchitectural characteristics. (A) Total porosity, (B) total volume of
287
pore space, (C) bone surface density, (D) bone volume percentual. Data are
288
expressed as the mean ± SEM (n = 5). &p < 0.05 vs. CT group, *p < 0.05 vs. S
289
group, #p < 0.05 vs. all others groups. (two-way ANOVA and post hoc Fisher’s
290
test).
291 292
Figure 3. Effects of 8-week treatment of the animals with telmisartan on
293
microarchitectural characteristics.
294
trabecular number, (C) connectivity, (D) connectivity density. Data are
295
expressed as the mean ± SEM (n = 5). *p < 0.05 vs. S group, &p < 0.05 vs. C
296
group, #p < 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s
297
test).
298
Western blot analysis (PPARγ protein expression)
299
Figure 4 shows the analysis of PPARγ protein expression, quantified by
300
densitometry and normalized by the expression of β-actin. PPARγ levels were
301
found to be increased in castrated animals (C group) and in those who went
302
through telmisartan treatment (ST and CT group), when compared to intact,
303
untreated animals (S group). It is worthy of note that the combination of
(A) Number of closed pores, (B)
304
castration and telmisartan use yielded the higher PPARγ expression levels (CT
305
group vs. ST and C groups).
306 307
Figure 4. Effects of 8-week treatment with telmisartan on PPARγ protein
308
(54 kDa) expression in total femur. The signal from each protein was
309
normalized by the respective amount of β-actin (42 kDa) even before calculating
310
the ratios. Data are expressed as mean ± SEM (n = 5). *p < 0.05 vs. S group, #p
311
< 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
312
DISCUSSION
313
In the present study, we provide evidence of increased expression of the protein
314
PPARγ associated with the occurrence of biomechanical and morphologic
315
defects in bones – femur and lumbar vertebrae – in intact and ovariectomized
316
SHR females (an animal model of both postmenopausal osteoporosis and
317
hypertension) by telmisartan use. Interestingly, rats from the ST group
318
resembled those from the castrated group (C) regarding bone loss and
319
increased PPARγ protein expression, and one should note that these
320
detrimental changes were even more pronounced in ovariectomized animals
321
treated with telmisartan (CT group). In spite of negative bone effects,
322
telmisartan efficiently reduced blood pressure and cardiac hypertrophy in
323
animals that were underwent treatment, which corroborates others studies
324
conducted in hypertensive rats [35, 36]. Such findings are quite important,
325
adding to the overall relevance of the data present here.
326
Bone microarchitecture deterioration (trabecular destruction and reduction in the
327
connectivity), which can happen as a result of drastic reductions on female sex
328
hormone levels, is considered a sensitive parameter to demonstrate trabecular
329
disconnectivity, therefore reducing the strength of the tissue and its ability to
330
resist to stress, increasing the risk of fractures [37]. The increased risk of
331
fractures observed during the postmenopausal phase is a consequence of the
332
rapid bone loss caused by an increase in bone turnover and a decrease in
333
tissue formation [38]. The consequent reduction in estrogen levels that
334
necessarily follow the castration performed in our model could account for the
335
worsening in the quality of bone microarchitecture observed here, corroborating
336
the aforementioned reports.
337
It has been put forward that the renin angiotensin system acts in bones through
338
the regulation of structural and metabolic aspects of this tissue by angiotensin II
339
[3, 4]. Specifically regarding the use of ARBs the data are controversial and not
340
conclusive [25, 39-41]. It has been reported that Angio II activates osteoclasts
341
decreasing bone density in ovariectomized SHRs, and the use of olmesartan
342
reverses this effect [39]. In contrast, telmisartan use in SHR male increased
343
bone detrimental effects [25] or did not change bone turnover markers in
344
patients with mild hypertension [40]. Also, losartan was unable to prevent bone
345
derangement in both hypertensive and normotensive male rats, suggesting a
346
lower participation of AT1 receptors in the effects of angiotensin II on bone
347
tissue [41]. These controversial results from different ARBs may be due to their
348
mode of action and the experimental doses and experimental design used.
349
Even though the mechanisms involved are not fully understood, it is known that
350
PPARγ agonists can cause bone loss in vitro and in rodent models [6-8, 42-44].
351
Treatment with PPARγ agonists – thiazolidinediones (TZDs) – has side effects
352
such as a significant risk of adverse effects on human bone, and PPARγ
353
activation by TZDs causes bone loss by suppressing differentiation and activity
354
of osteoblasts, while reducing bone formation and enhancing osteoclasts
355
differentiation [42, 44]. Another possible mechanism underlying the bone loss
356
related to the action of PPARγ agonists involves adipogenic cells differentiation
357
from mesenchymal stem cells (MSCs) at the expense of osteoblastogenesis
358
[45, 46]. Taken together, these data suggest that PPARγ agonists may increase
359
the risk of bone loss.
360
Our results suggest that the use of telmisartan, a PPARγ partial agonist, may
361
have induced bone loss mediated by the activation of PPARγ in this tissue, in
362
view of the higher levels of this protein found in bone of ovariectomized animals
363
and in those treated with telmisartan (ST and CT). However, one cannot rule
364
out th participation of other mechanisms.
365
In summary, telmisartan in ovariectomized SHRs – a model of oestrogen
366
deficiency in hypertensive rats – increased the levels of femoral PPARγ protein
367
expression and compromised trabecular bone microarchitecture by reducing
368
bone mass as evidenced by the shape and connectivity parameters observed
369
by DXA and micro-CT analysis.
370
CONCLUSION
371
In conclusion, these results demonstrated that the treatment of animals showing
372
preexisting osteoporotic bone disorders with telmisartan compromised bone
373
density and microarchitecture, probably via mechanisms associated with
374
increased PPARγ. Actual data may reveal a need for greater caution in the use
375
of telmisartan in patients that have preexisting bone alterations, as in the
376
postmenopausal period.
377
Whether these experimental findings translate into human clinical situations is
378
not yet known and additional studies employing specific imaging techniques,
379
such as densitometry and/or tomography, may be required to follow up patients
380
taking telmisartan.
381
Conflicts of interest
382
The authors declare that there are no conflicts of interest associated with this
383
manuscript.
384
Author contributions
385
The contributions of each author to the study were:
386
Antonio Marcos Birocalea,b, Antonio Ferreira de Melo Jra,b, Pollyana Peixotoa,
387
Phablo Wendell Costalonga Oliveiraa, Leandro Dias Gonçalves Ruffonia, Liliam
388
Masako Takayamaa, Breno Valentim Nogueiraa,b, Keico Okino Nonakaa,b, Rosa
389
Maria Rodrigues Pereiraa,b, José Martins de Oliveira Jr.a,b, Nazaré Souza
390
Bissolia,b
391
a
392
b
393
of data; drafting the article; revising it critically for important intellectual content;
394
and final approval of the version to be published.
395
Acknowledgements
396
This work was funded by Fundação de Amparo a Pesquisa do Espirito Santo –
397
FAPES [Grant number 033/2016] for the purchase of inputs and drugs.
398
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Figure Captions
568
Figure 1. Effects of the 8-week treatment with telmisartan on bone mineral
569
density (BMD). (A) total femur, (B) fifth lumbar vertebrae, (C) femur proximal
570
region (R1), (C1) femur proximal region (R1) by micro-CT. Data are expressed
571
as the mean ± SEM (n = 7). *p < 0.05 vs. S group, &p < 0.05 vs. CT group, #p <
572
0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).
573
Figure 2. Effects of 8-week treatment of the animals with telmisartan on
574
the microarchitectural characteristics. (A) Total porosity, (B) total volume of
575
pore space, (C) bone surface density, (D) bone volume percentual. Data are
576
expressed as the mean ± SEM (n = 5). &p < 0.05 vs. CT group, *p < 0.05 vs. S
577
group, #p < 0.05 vs. all others groups. (two-way ANOVA and post hoc Fisher’s
578
test).
579
Figure 3. Effects of 8-week treatment of the animals with telmisartan on
580
microarchitectural characteristics. (A) Number of closed pores, (B)
581
trabecular number, (C) connectivity, (D) connectivity density. Data are
582
expressed as the mean ± SEM (n = 5). *p < 0.05 vs. S group, &p < 0.05 vs. C
583
group, #p < 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s
584
test).
585
Figure 4. Effects of 8-week treatment with telmisartan on PPARγ protein
586
(54 kDa) expression in total femur. The signal from each protein was
587
normalized by the respective amount of β-actin (42 kDa) even before calculating
588
the ratios. Data are expressed as mean ± SEM (n = 5). *p < 0.05 vs. S group, #p
589
< 0.05 vs. all others groups (two-way ANOVA and post hoc Fisher’s test).