Download Anti-toll like receptor 4 (TLR4) therapy diminishes cardiac

International Journal of Cardiology 187 (2015) 243–245
Contents lists available at ScienceDirect
International Journal of Cardiology
journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Anti-toll like receptor 4 (TLR4) therapy diminishes cardiac remodeling
regardless of changes in blood pressure in spontaneously hypertensive
rats (SHR)
Cinthya Echem a, Gisele Facholi Bomfim b, Graziela Scalianti Ceravolo c, Maria Aparecida Oliveira a,
Rosângela Aparecida Santos-Eichler a, Luiz Roberto Bechara a, Mariana Matera Veras d,
Paulo Hilário Nascimento Saldiva d, Julio Cesar Ferreira a, Eliana Hiromi Akamine a, Zuleica Bruno Fortes a,
Ana Paula Dantas e, Maria Helena Catelli de Carvalho a,⁎
a
Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
Institute of Health Sciences, Federal University of Mato Grosso, Sinop, Brazil
Department of Physiology Sciences, State University of Londrina, Londrina, Brazil
d
School of Medicine, University of São Paulo, São Paulo, Brazil
e
Group of Atherosclerosis and Coronary disease, Institut Clinic del Torax, Institut 12 d'Investigaciones Biomédiques August Pi I Sunyer (IDIBAPS), Barcelona, Spain
b
c
a r t i c l e
i n f o
Article history:
Received 6 February 2015
Accepted 17 March 2015
Available online 18 March 2015
Keywords:
Arterial hypertension
Toll-like receptor 4 (TLR4)
Cardiac hypertrophy
Cardiac fibrosis
Cardiac inflammation
Spontaneously hypertensive rats (SHR)
With the increasing global incidence of high blood pressure, arterial
hypertension is becoming the main risk factor for cardiac remodeling
and consequent end-organ damage [1]. Recent evidences have associated hypertension with a chronic low-grade inflammation and augmented levels of circulating pro-inflammatory molecules [2]. Among the proinflammatory pathways, toll-like receptors (TLRs) have been emerging
as key regulators of chronic inflammation and associated disease,
including cardiovascular disease. TLRs are important sources of proinflammatory molecules and they can recognize several damageassociated molecular patterns that are up-regulated during hypertension [3]. We recently described, for the first time, that hypertension is
associated with increased TLR4 expression in mesenteric arterioles,
kidneys and heart of adult spontaneously hypertensive rats (SHR)
⁎ Corresponding author at: Department of Pharmacology, Institute of Biomedical
Sciences, University of São Paulo, Avenida Professor Lineu Prestes 1524, São Paulo, SP
CEP 05508-900, Brazil.
E-mail address: [email protected] (M.H.C. Carvalho).
http://dx.doi.org/10.1016/j.ijcard.2015.03.190
0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
compared to pre-hypertensive SHR [4], as well as to normotensive
Wistar [5]. Nevertheless, there is no direct evidence showing the role
of TLR4 and TLR4-dependent signaling pathways in hypertensionassociated cardiac remodeling, which is the aim of this study.
Male adult SHR (21-week-old) and age-matched Wistar rats were
studied after a period of 15-day treatment with anti-TLR4 antibody or
non-specific IgG antibody, as previously described [5]. This study was
conducted in accordance with the ethical principles in animal research
adopted by Brazilian College of Animal and experimentation. Left ventricles sections were stained with hematoxylin–eosin or picro-sirius to
determine cardiomyocyte area, diameter and collagen deposition.
mRNA and protein expression were determined by conventional qPCR
and western blot techniques. Arterial blood pressure and heart rate
were measured by carotid artery cannulation. Left ventricular developed pressure was measured by Langendorff under basal conditions
and after isoproterenol stimulation. Analyses of mean ± SEM values
were performed using two-way ANOVA or multiple t tests (post-hoc
Sidak–Bonferroni), where appropriate. Data analysis was carried out
using IBM SPSS 20 software. Values of p b 0.05 were considered statistically significant.
In this study, we provide the first direct evidence that TLR4 plays a
key role in hypertension-associated cardiac remodeling. Our main findings demonstrate that anti-TLR4 treatment reduces TLR4 expression
and TLR-dependent signaling pathways in left ventricles of hypertensive rats, leading to decrease in cardiomyocyte hypertrophy and fibrosis.
The classic paradigm of hypertension-associated cardiac hypertrophy is that thickening of left ventricle occurs as a compensatory mechanism to minimize wall stress in response to elevated blood pressure
[1]. In fact, with established hypertension, left ventricles of adult SHR
(21-week-old) display augmented cardiomyocytes area and diameter
as well as increased collagen deposition compared to age-matched normotensive Wistar rats (Fig. 1A) These morphological changes were
paralleled by increased TLR4 mRNA expression in adult SHR in reference
to age-matched Wistar (39 ± 8.5%, p b 0.05) or young SHR (28 ± 13%,
244
C. Echem et al. / International Journal of Cardiology 187 (2015) 243–245
Fig. 1. (A) Histological analysis of cardiomyocyte area, cardiomyocyte diameter, and collagen deposition. Shown are representative images of hematoxylin and eosin (top) or pico-sirius
(bottom) staining of left ventricles of SHR and Wistar rats treated with anti-TLR4 antibody or non-specific IgG antibody. Bar graphs show the results of calculated area (μm2) and diameter
(μm) of cardiomyocytes, or the relative area stained with picro-sirius for collagen deposition. (B) mRNA levels of ANP, BNP, skeletal α-actin, collagen type I and collagen type III in left
ventricles of SHR treated with anti-TLR4 antibody or non-specific IgG. mRNA levels are expressed as 2−ΔΔCt using β-actin as internal control and in reference to SHR IgG groups. Each
data represents the mean ± SEM derived from 4 to 10 independent experiments. *p b 0.05.
p b 0.05). No differences in TLR4 expression were observed when comparing young (6-week-old) and adult (21-week-old) Wistar rats, reinforcing our hypothesis that there is a positive correlation between
hypertension and increased TLR4.
Accordingly, our next step aimed to determine whether the antiTLR4 therapy could counteract the hypertension associated cardiac remodeling. We found that anti-TLR4 therapy markedly reverses cardiomyocyte hypertrophy as well as decreases collagen deposition in left
ventricles of 21-week-old SHR to levels observed in age-matched
normotensive rats (Fig. 1A). No differences were found in salinetreated rats versus non-specific IgG antibody treatment (data not
shown), confirming the efficacy and specificity of anti-TLR4 therapy to
promote an anti-remodeling effect in the heart. The finding that antiTLR4 therapy inhibits hypertensive cardiac remodeling without affecting blood pressure in SHR (Table 1) was intriguing These results suggest
that changes in ventricle morphology during hypertension can be
trigged or maintained by mechanisms that are parallel and independent
of pressure overload. Corroborating our findings, Ha et al. [6] have described a decrease of cardiomyocyte area following pressure overload
in TLR4 knockout normotensive mice.
Cardiac hypertrophy is also associated with induction of genes
expressed during embryonic development, such as atrial natriuretic
peptide (ANP), brain natriuretic peptide (BNP) and skeletal α-actin
[7]. The precise mechanism that leads to this up-regulation remains unclear, but it is believed to be an adaptive process to protect the stressed
heart [7]. Reduction in expression of those genes was observed after
anti-TLR4 treatment in 21-week-old SHR (Fig. 1B). This response was
paralleled by decreased expression of collagen types I and III, which
may reinforce the protective role of anti-TLR4 therapy in reversing
hypertension-induced cardiac remodeling. It is known that excessive
collagen deposition increases ventricular stiffness and negatively affects
cardiac contractility properties [1]. Nonetheless, no changes in the left
Table 1
Hemodynamic parameters and Left ventricular developed pressure.
Animal
SBP
(mm Hg)
DBP
(mm Hg)
Heart rate
(bpm)
LVDP basal
(mm Hg)
LVDP isoproterenol
(mm Hg)
Wistar IgG
Wistar anti-TLR4
SHR IgG
SHR anti-TLR4
133.3 ± 3.8
132.7 ± 5.8
208.8 ± 3.4*
211.5 ± 4.1*
99.7 ± 3.4
95.2 ± 5.8
152.7 ± 2.8*
149.8 ± 2.8*
324.1 ± 15.2
335.4 ± 11.7
367.6 ± 25.4
360.6 ± 6.6
131.3 ± 5.8
117.4 ± 7.0
119.7 ± 7.3
121.1 ± 6.4
301.1 ± 3.9
273.3 ± 17.9
307.1 ± 5.8
300.9 ± 13.3
SBP: systolic blood pressure; DBP: diastolic blood pressure; LVDP: left ventricular developed pressure. *p b 0.05 vs Wistar. Data represented as mean ± SEM (n = 4–13).
C. Echem et al. / International Journal of Cardiology 187 (2015) 243–245
245
Fig. 2. (A) Protein expression of TLR4 and its effector MYD88 and (B) protein expressions of pro-inflammatory cytokines TNF-α and IL-1β
in left ventricles of SHR treated with anti-TLR4 antibody or non-specific IgG antibody. Shown are representative images of immunoblots and bar graphs of densitometric analysis of pulled
data. Each data represents the mean ± SEM derived from 7 to 10 independent experiments. *p b 0.05 vs SHR IgG.
ventricular developed pressure were observed in SHR at the time point
of this study (Table 1). These results are in accordance with Bing et al.
[8], who showed that cardiac function of SHR can be preserved until
1 year of age despite hypertrophy.
We further demonstrated that TLR4 antagonism reduces TLR4 expression and TLR4-dependent pro-inflammatory signaling pathways in left
ventricles of hypertensive rats (Fig. 2A). Initially we observed that antiTLR4 treatment decreases MyD88 expression (Fig. 2A), a key adaptor
molecule that interacts with TLR4 in the cell plasma membrane and mediates TLR4-signaling towards inflammation [9]. We also found that antiTLR4 treatment decreases cardiac phosphorylation and activation of NFκB (phospho NF-κB p65/total NF-κB: SHR 101 ± 1.6 vs SHR Anti-TLR4
92 ± 2.6, p b 0.05), as well as the expression of pro-inflammatory cytokines IL-1β and TNF-α (Fig. 2B). It has been established that inflammation
contributes to cardiac remodeling [1]. The absence of IL-1β reduces left
ventricular dilatation following myocardial infarction [10], and TNF-α
over-expression leads to changes in collagen composition [11].
In summary, our results show for the first time that TLR4 plays a key
role in hypertensive cardiac remodeling, via an inflammatory mechanism that increases cytokines (IL-1β and TNF-α), and which, moreover,
is independent on changes in blood pressure levels. These findings shed
new lights into the molecular mechanism that controls cardiac remodeling and suggests that targeting TLR4 may have major therapeutic implications to prevent hypertension-associated end-organ damage.
Conflict of interest
The authors report no relationships that could be construed as a conflict of interest.
Acknowledgments
The authors are grateful to Sonia Maria Leite, Marta Rodrigues da
Silva and Manoel Rocha for their technical support. This study was
supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) (10/13829-5), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (479949/2012-0) and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES/DGU 269/12).
Ana Paula Dantas is supported by a grant from Instituto de Salud Carlos
III, (RIC RD12/0042/0006) and Programa Hispano-Brasileño de
Cooperación Interuniversitaria (HBP-2011-0054 PC).
The authors report no disclosure.
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