Blockade of angiotensin AT₁ receptors prevents arterial remodelling and stiffening in iron‐overloaded rats

Abstract

Background and Purpose

Damage to the vasculature caused by chronic iron‐overload in both humans and animal models, is characterized by endothelial dysfunction and reduced compliance. In vitro, blockade of the angiotensin II AT₁ receptors reversed functional vascular changes induced by chronic iron‐overload. In this study, the effect of chronic AT₁ receptor blockade on aorta stiffening was assessed in iron‐overloaded rats.

Experimental Approach

Male Wistar rats were treated for 15 days with saline as control group, iron dextran 200 mg·kg⁻¹·day⁻¹, 5 days a week (iron‐overload group), losartan (20 mg·kg⁻¹·day⁻¹ in drinking water), and iron dextran plus losartan. Mechanical properties of the aorta were assessed in vivo. In vitro, aortic geometry and biochemical composition were assessed with morphometric and histological methods.

Key Results

Thoracoabdominal aortic pulse wave velocity (PWV) increased significantly, indicating a decrease in aortic compliance. Co‐treatment with losartan prevented changes on PWV, β‐index, and elastic modulus in iron‐overloaded rats. This iron‐related increase in PWV was not related to changes in aortic geometry and wall stress. but to increased elastic modulus/wall stress ratio, suggesting that a change in the composition of the wall was responsible for the stiffness. Losartan treatment also ameliorated the increase in aorta collagen content of the iron‐overload group, without affecting circulating iron or vascular deposits.

Conclusions and Implications

Losartan prevented the structural and functional indices of aortic stiffness in iron‐overloaded rats, implying that inhibition of the renin–angiotensin system would limit the vascular remodelling in chronic iron‐overload.

Abbreviations

Ang II

angiotensin II

DBP

diastolic BP

EM

elastic modulus

MCSA

medial cross‐sectional area

PWV

pulse wave velocity

RAS

renin–angiotensin system

SBP

systolic BP

WS

wall stress

What is already known

• Vascular changes due to iron‐overload are characterized by endothelial dysfunction and reduced compliance.

What does this study add

• In rats, iron‐overload increased aortic PWV and collagen without changing vascular tone or geometry.

• Co‐administration of the AT₁ receptor antagonist, losartan, prevented aortic stiffening and fibrosis.

What is the clinical significance

• To date, there is no treatment for vasculopathies that follow iron‐overloading.

• AT₁ receptor blockade has the potential to act as adjuvant therapy in patients with iron‐overload.

1. INTRODUCTION

Iron is an essential metal for cellular homeostasis participating in important physiological processes, such as oxygen binding and transport through haemoglobin and myoglobin, mitochondrial respiration, DNA synthesis and many oxidation–reduction reactions (Aisen, Enns, & Wessling‐Resnick, 2001; Emerit, Beaumont, & Trivin, 2001; Kakhlon & Cabantchik, 2002). Iron levels in the body should be rigorously regulated, and, as there are no known mechanisms regulating iron excretion, the accumulation of this metal may damage several tissues, including the cardiovascular system (Siddique & Kowdley, 2012).

It is well documented by experimental and clinical studies that increased oxidative stress plays a key role in the cardiomyopathy following iron‐overloading (Bartfay & Bartfay, 2000; Cheng & Lian, 2013), and, although the relationship between iron deposit and cardiovascular damage has been known since the 1960s, iron‐overload vasculopathy has been poorly studied. Only a few clinical studies have suggested a relationship between iron stores and changes in BP (Cash et al., 2013), endothelial dysfunction (Gaenzer et al., 2002; Kukongviriyapan et al., 2008), and arterial stiffness (Cheung, Chan, & Ha, 2002; Detchaporn et al., 2012; Gedikli et al., 2007; Valenti et al., 2015). However, because these clinical studies have been based on patients of different ages, uncontrolled comorbidities, different iron levels, and diverse aetiology leading to iron‐overload (i.e., haemochromatosis, β‐thalassaemia, and sickle cell disease), the effects of chronic iron‐overload per se on vascular structure and function still remain under investigation.

In this regard, we identified that, in the rat model of iron‐overloading, there is an impairment of the vascular function associated with increased production of ROS and reduced https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2509 bioavailability in conductance (Marques et al., 2015) and resistance arteries (Bertoli et al., 2018; Ribeiro Júnior, Marques, Nunes, Stefanon, & dos Santos, 2017). These studies also suggested that vascular dysfunction induced by iron‐overload may be a result of localized changes in https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2504 (Ang II), as in vitro blockade of the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=34 reversed functional changes (Bertoli et al., 2018; Marques et al., 2015). Moreover, Ribeiro Júnior et al. (2017) demonstrated that chronic iron‐overload induces not only functional but also structural changes in the artery characterized by vascular remodelling, collagen deposition, and increased aortic pulse wave velocity (PWV).

Thus, because chronic inhibition of the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2413–angiotensin system (RAS) has been successfully used in experimental and clinical studies in the prevention or reversal of structural remodelling and arterial stiffness (Ng, Hildreth, Avolio, & Phillips, 2011; Salum et al., 2014; Shahin, Khan, & Chetter, 2012; Zhu et al., 2019) and the functional changes induced by iron loading were related to increased local Ang II activation, we aimed to test the effects of chronic AT₁ receptor blockade on the arterial structural and mechanical changes induced by iron‐overload in rats.

2. METHODS

2.1. Animals and treatments

All animal care and experimental procedures were conducted in accordance with the Brazilian Guidelines for the Care and Use of Animals for Scientific and Educational Purposes and were approved by the Institutional Ethics Committee on Animal Use (007/2013 CEUA‐UFES). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology. Male Wistar rats with an average age of 3 months (200–250 g) were used. The animals were provided by the animal facilities of the Health Sciences Center of the Federal University of Espirito Santo. During treatment, the rats were kept in cages with free access to water and food under conditions of controlled temperature and humidity and under a 12‐hr light‐dark cycle.

The first set of experiment was conducted to test whether short‐term iron‐overload could change aortic PWV. Rats were randomized into two groups: control and iron‐overload. The iron‐overload group received daily i.p. injections of iron dextran at 200 mg·kg⁻¹, 5 days a week for 2 weeks, while the control group received saline isotonic solution for the same period and the same enforcement regime as the iron group. The parenteral administration of iron dextran in rats has been used by our group (Bertoli et al., 2018; Marques et al., 2015; Ribeiro Júnior et al., 2017) and others (El‐Sheikh, Ameen, & AbdEl‐Fatah, 2018; Gong et al., 2016; Ishizaka et al., 2002; Lou et al., 2009), and it has been shown to be an appropriate model that mimics clinical situations of iron‐overload such as haemosiderosis or haemochromatosis. In addition, in a second set of experiments, to evaluate the effect of AT₁ receptor blockade on the aortic mechanics during iron‐overload, another sample of rats were randomized into four groups: control, iron‐overload, control treated with https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=590, or iron‐overload treated with losartan. Iron‐dextran injections were given concomitantly with losartan, which was orally administered in drinking water at concentrations adjusted to deliver the required dose (20 mg·kg⁻¹ daily). All analyses were carried out after 2 weeks of treatment, with the operator and analyst blinded to the treatment groups or conditions.

2.2. Haemodynamic study

Animals were anaesthetized with urethane (1.2 g·kg⁻¹, i.p.), placed on a heated operating table (37°C), 100% oxygen provided through a mask, and the left jugular vein dissected for drug infusion and anaesthetic supplementation. Next, the left common carotid and left femoral arteries were carefully isolated, and two polyethylene catheters PE‐10 (25 mm length, 0.28 mm ID, 0.61 mm OD, Clay Adams, Parsippany, NJ, USA) fused to a PE‐50 (35 mm length, 0.58 mm ID, 0.96 mm OD, Clay Adams, Parsippany, NJ, USA) were inserted to simultaneously measure pressure pulses at the thoracic and abdominal portions of the aorta respectively. For this, each arterial catheter filled with heparinized 0.9% NaCl (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4214 50 U·ml⁻¹) was connected to a low‐volume pressure transducer (TSD104A; BIOPAC Systems, Santa Barbara, CA, USA; https://www.biopac.com/, https://scicrunch.org/resources/Any/record/nlx_144509-1/SCR_014829/resolver?q=Biopac%20Systems&l=Biopac%20Systems).

2.2.1. Aortic PWV

PWV was assessed as previously described (Cosson et al., 2007) using 10‐min recordings after stabilization. PWV (cm·s⁻¹) was calculated as the distance between the two cannula tips (measured in situ following killing) divided by the pulse wave transit time. To correct for the effects of BP, PWV was normalized to systolic BP (PWV/SBP ratio), and β‐index was calculated by normalizing to diastolic BP (2.11·PWV²·DBP⁻¹).

2.2.2. Vasopressor response to phenylephrine

To assess whether PWV changes in this iron‐overload model could be due to an increased vascular tone or vasoconstriction, after a 10‐min stabilization period, the vascular reactivity to https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=485 was tested in rats with iron‐overload and compared with control values. Vasopressor responses were elicited by injecting increasing doses of phenylephrine (0.03 to 100 μg·kg⁻¹) administered as 10‐ to 15‐μl bolus injections via the jugular catheter, and central BP and pulse rate were measured. Pulse rate was determined from the intra‐beat interval and expressed as beats per minute.

2.3. In vitro vascular reactivity of the aorta

The control of vascular tone was evaluated in vitro as previously described (Marques et al., 2015). Segments (4 mm in length) of the thoracic aorta from the control and iron‐overload groups were mounted in an organ bath with Krebs–Henseleit solution (in mM: NaCl 118, KCl 4.7, NaHCO₃ 23, CaCl₂–2H₂O 2.5, KH₂PO₄ 1.2, MgSO₄–7H₂O 1.2, glucose 11, and EDTA 0.01) at a temperature of 37°C gassed with a mixture of 95% O₂ and 5% CO₂ (pH 7.4) and kept at a baseline tension of 1.0 g (optimal resting tension). Isometric tension was recorded using an isometric force–displacement transducer (TSD125C; BIOPAC Systems; https://www.biopac.com/, https://scicrunch.org/resources/Any/record/nlx_144509-1/SCR_014829/resolver?q=Biopac%20Systems&l=Biopac%20Systems) attached to an acquisition system (MP100, BIOPAC System, Inc.; https://www.biopac.com/, https://scicrunch.org/resources/Any/record/nlx_144509-1/SCR_014829/resolver?q=Biopac%20Systems&l=Biopac%20Systems) and connected to a computer with a analysis software (AcqKnowledge 3.7.5, Santa Barbara, CA, USA; http://www.biopac.com/product/acqknowledge-software/, https://scicrunch.org/resources/Any/record/nlx_144509-1/SCR_014279/resolver?q=Biopac%20Systems&l=Biopac%20Systems).

Aortic rings were initially exposed to 75‐mM KCl (30 min) to check their functional integrity and the maximum developed tension. After a washout period of 60 min, concentration–response curves to phenylephrine were constructed by cumulative addition (0.1 nM to 0.3 mM). To analyse the influence of the endothelium on the vascular responses, endothelial cells were mechanically removed, in some experiments, by rubbing the lumen with a needle. The absence of endothelium was confirmed by the inability of 10‐μM https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=294 to induce relaxation. Also, the effects of the non‐specific NOS inhibitor, N^ω‐nitro‐l‐arginine methyl ester (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5213, 100 μM) on the phenylephrine‐elicited response were investigated. For this, this inhibitor was added 30 min before the concentration–response curve to phenylephrine was generated. The vasoconstrictor responses were normalized to the contraction induced by 75‐mM KCl of the respective aortic ring and expressed as a percentage of this contraction. For each concentration–response curve, the maximum effect (E _max) and the negative log of concentrations producing 50% of E _max (‐LogEC₅₀) were calculated using non‐linear regression analysis with sigmoidal dose–response fitting (GraphPad Prism 6 Software, San Diego, CA, USA; http://www.graphpad.com/, https://scicrunch.org/resources/Any/search?q=SCR_002798&l=SCR_002798).

2.4. Aorta histomorphometry, wall stress, and elastic modulus

At the end of the second set of experiments, rats from the four groups were anaesthetized, and haemodynamic measurements were made, as described in Section 2.2.1, and the rats then killed with a KCl overdose. Thereafter, through a thoracotomy, the aortic tree was perfused (4% https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4196) for 30 min at the baseline central aortic mean BP for the individual animal, in order to maintain aortic diameters at the distension corresponding to in situ conditions (Cantini et al., 2001; Cordaillat et al., 2011). Segments (1 cm) of descending thoracic aorta and abdominal aorta were excised and embedded in a freezing medium (Killik‐OCT EasyPath; Erviegas Ltda, SP, Brazil). Three 10‐μm‐thick sections were assembled on glass slides and stained with haematoxylin‐eosin for measurement of artery dimensions, Picrosirius Red for collagen staining, and Von Kossa for calcium deposits. Colour images were captured with a microscope (Leica, 10× or 40× objectives), using a digital camera. Quantitative and qualitative analyses were performed with ImageJ Software (https://imagej.nih.gov/ij/, https://scicrunch.org/resources/Any/search?q=SCR_003073&l=SCR_003073) by researchers blinded to the experimental group.

The medial cross‐sectional area (MCSA, in mm²) was calculated as follows: π divided by 4·(D _o ² − D _i ²), where D _o and D _i are outer and inner diameter (in mm) respectively. Medial thickness (h, in mm) was calculated from h = (D _o − D _i) divided by 2. Elastic modulus (EM, in 10⁶ dyne·cm⁻²) was calculated from the Moens–Korteweg equation (EM = PWV²·Di·ρ divided by h) and wall stress (WS, in 10⁶ dyne·cm⁻²) was derived from the Lamé equation (WS = central aortic mean BP multiplied by Di and divided by 2h), using with ρ = blood density, 1.05 g·cm⁻³. EM/WS ratio was used as an isobaric index of intrinsic aortic wall stiffness (Cordaillat et al., 2011).

2.5. Serum iron levels and tissue deposits

Serum iron and tissue deposits were evaluated in the four groups: control and iron‐overload, treated with placebo or losartan. The serum iron concentration was measured in duplicate by a modified Goodwin colorimetric method with the use of commercial colorimetric kit (Bioclin, Belo Horizonte, Brazil). Iron was released from transferrin in an acid medium (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=3637) and reduced to its ferrous state by the action of hydroxylamine. Subsequently, it reacted with ferrozine leading to the formation of a violaceous complex measured at 540 nm and expressed as μmol·L⁻¹. Tissue iron deposits were visualized in sections of the aorta and liver stained with Prussian blue (Mallory's method).

2.6. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). All data were analysed blindly in terms of the animal group and/or condition. Results are expressed as the mean ± SEM. For each data set, the Grubbs' test (α 0.05) was used to identify and exclude an outlier (there was only one outlier identified in the in vitro vascular reactivity assay after incubation with L‐NAME). Additionally, data were tested for homogeneity of variance by Levene's test, while normality was assessed by D'Agostino–Pearson omnibus normality test. Then, differences were analysed using Student's t test or the Mann–Whitney U test for two samples, one‐ or two‐way ANOVA followed by a Tukey post hoc or Kruskal–Wallis test followed by Dunn's test for multiple comparisons when appropriate. For all inferences, significance was considered when P < .05. Statistical analysis and graph construction were performed using 6 (GraphPad Prism 6 Software, San Diego, CA, USA; http://www.graphpad.com/, https://scicrunch.org/resources/Any/search?q=SCR_002798&l=SCR_002798).

2.7. Materials

Iron dextran was purchased from Fabiani Ltda (Ferrodex®, Fabiani Ltda, SP, Brazil). Unless otherwise stated, all other salts and reagents were of analytical grade and were obtained from Sigma‐Aldrich or Merck.

2.8. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos, et al., 2019; Alexander, Fabbro, et al., 2019).

3. RESULTS

3.1. Two weeks of iron injection induces systemic overload with changes in the PWV, but not in vascular tone control

The animals injected with iron dextran for 2 weeks have reduced body weight (Table 1) and hyperpigmentation of the skin and internal organs such as liver and spleen, all typical characteristics of iron‐overload. There was also increases in liver and spleen weight but no changes on cardiac weight.

Table 1.

General characteristics and haemodynamic parameters of control and iron‐injected rats (mean ± SEM)

	Control	Iron‐overload
Body weight	(n = 19)	(n = 19)
Initial body weight (g)	249 ± 8	255 ± 5
Final body weight (g)	308 ± 9 #	278 ± 6 *#
Biometry	(n = 14)	(n = 14)
Tibia length (mm)	35.42 ± 0.49	34.86 ± 0.46
Liver weight (g)	12.29 ± 0.55	15.69 ± 0.45*
Liver/tibia ratio (g·mm−1)	0.364 ± 0.014	0.461 ± 0.013*
Spleen weight (g)	0.614 ± 0.056	0.856 ± 0.048*
Spleen/tibia ratio (g·mm−1)	0.018 ± 0.001	0.024 ± 0.001*
Heart weight (g)	0.949 ± 0.041	1.005 ± 0.060
Heart/tibia ratio (g·mm−1)	0.029 ± 0.001	0.028 ± 0.001
Haemodynamics	(n = 14)	(n = 13)
Mean BP (mmHg)	83.4 ± 3.0	78.8 ± 3.2
Pulse pressure (mmHg)	40.6 ± 2.5	42.5 ± 2.1
Pulse wave velocity (cm·s−1)	492.8 ± 16.4	643.7 ± 47.1*

^* P < .05 versus control,

^# P < .05 versus initial value; Student's t test.

Although no significant differences were found in the haemodynamic parameters evaluated in anaesthetized animals, the iron‐overload regime used here caused a significant increase in PWV, compared with that of the control rats (Table 1). To assess whether PWV was associated with altered vascular tone, the vasomotor responses were evaluated in vitro and in vitro in both groups (Figure 1). There was an increase in systolic and diastolic pressures associated with a parallel reduction of heart rate, following phenylephrine injections in vivo (Figure 1a–c), but these responses were equal in magnitude between the control and iron‐overload groups. Similarly, our data from isolated aortic rings confirmed that responsivity to phenylephrine was not changed by iron‐overload (Figure 1D and Table S1). Moreover, both endothelium removal (Figure 1e,f) and L‐NAME incubation (Figure 1h,i) enhanced the phenylephrine responses in thoracic aortas from both groups, but these changes occurred to an equal extent in arteries from the control and those from the iron‐overload groups (Figure 1g).

Figure 1.

Iron treatment did not change pressor responses in vivo or aortic rings reactivity and endothelial function in vitro. Dose–response curves were obtained in vivo to study the effects of phenylephrine on changes in SBP (a), DBP (b), and pulse rate (c) in controls and iron‐dextran injected groups. Moreover, concentration–response curves to phenylephrine were obtained in vitro to study the reactivity in endothelium‐intact aortic rings isolated from both groups (d) and after endothelium removal (e,f) or after incubation with L‐NAME 100 μM (h,i). Data shown are means ± SEM, and the number of animals used is indicated in parentheses. *P < .05, significantly different from control maximal responses, and the differences in the area under the concentration–response curves (dAUC) after endothelium removal or L‐NAME incubation between controls and iron‐overload; Student's t test

3.2. Effects of AT₁ receptor blockade on the aortic haemodynamics and PWV of iron‐overloaded rats

Treatment with losartan did not alter the hypercorated pattern of animals concomitantly submitted to iron‐dextran injections, although it reduced weight gain in all groups. Moreover, BP and heart rate were not influenced by treatment with losartan in the control group, but these parameters were decreased in the iron‐overload group treated with losartan (Table 2).

Table 2.

Effects of losartan on the aortic haemodynamics and geometry of iron‐injected rats (mean ± SEM)

	Control		Iron‐overload
	Vehicle	Losartan	Vehicle	Losartan
Aortic haemodynamics	(n = 14)	(n = 6)	(n = 13)	(n = 12)
Systolic BP (mmHg)	107.1 ± 2.6	105.5 ± 4.2	100.4 ± 2.6	91.5 ± 3.4 *
Diastolic BP (mmHg)	67.2 ± 4.3	60.5 ± 4.4	63.9 ± 2.4	54.5 ± 4.0
Heart rate (bpm)	305 ± 7	291 ± 10	304 ± 9	280 ± 12
Aortic geometry	(n = 5)	(n = 5)	(n = 5)	(n = 5)
Inner diameter (mm)	1.59 ± 0.03	1.72 ± 0.07	1.70 ± 0.07	1.60 ± 0.01
Outer diameter (mm)	1.75 ± 0.02	1.88 ± 0.07	1.86 ± 0.09	1.73 ± 0.01
Medial thickness × 10−3 (mm)	78.9 ± 3.4	74.1 ± 3.1	74.9 ± 8.1	69.8 ± 4.1
Medial cross‐sectional area × 103 (mm2)	458 ± 17	411 ± 26	449 ± 64	331 ± 11 *, #
Wall‐to‐lumen ratio × 10−3	48.7 ± 1.2	45.8 ± 4.4	44.1 ± 4.2	43.4 ± 2.7

Note. Wall‐to‐lumen ratio was calculated by dividing medial thickness by inner diameter.

^* P < .05, significantly different from controls; two‐way ANOVA and Tukey's post hoc test.

^# P < .05 significantly different from iron‐overload without losartan; two‐way ANOVA and Tukey's post hoc test.

The increase in PWV induced by iron‐overload, as absolute values or in relation to SBP and HR, was completely prevented by AT₁ receptor blockade (Figure 2a,b). In addition, due to the effect of losartan, especially on DBP, the β‐index was calculated. As shown in Figure 2c, co‐treatment with losartan totally prevented the increase of this pressure‐independent parameter of aortic stiffness found in rats injected with iron alone.

Figure 2.

Losartan prevented changes in the aortic mechanics of iron‐overloaded rats. (a) Thoracoabdominal aortic pulse wave velocity (PWV) normalized by systolic BP (SBP), (b) heart rate (HR), or (c) calculated β‐index from the control and iron‐overload groups treated or not with losartan. (d) Wall stress, (e) elastic modulus, and EM/WS ratio (f) calculated from the experimental groups (see Section 2 for calculation details). Data are expressed as the means ± SEM and the number of animals used is indicated in parentheses. *P < .05, significantly different as indicated; two‐way ANOVA and Tukey's post hoc test

3.3. Effects of AT₁ receptor blockade on the aortic structural and mechanical properties of iron‐overloaded rats

MCSA was smaller in iron‐overloaded rats treated with losartan, compared with controls or iron‐overloaded rats given vehicle. However, as shown in Table 2, medial thickness and inner or outer diameters were not different in thoracic aortas from iron‐overloaded rats compared to controls, and these parameters were not modified by treatment with losartan. As a result, the media‐to‐lumen ratio was similar between all groups, more evidence that no hypertrophic remodelling occurred in aortas using this model. Because aortic wall thickness, lumen dimension, and central mean BP did not change, WS was not significantly different in iron‐overloaded rats, and losartan had no effects as well (Figure 2d). However, there were substantial increases in EM and EM/WS ratio of the aortas from rats injected with iron dextran, and losartan efficiently prevented these stiffness indexes (Figure 2e,f????????). Of note, losartan had no effects on the control group.

Although there were no indications of hypertrophy, aortas from iron‐treated rats exhibited greater deposition of collagen in the aortic wall, than arteries from the control group, as evaluated by the fibres stained with Picrosirius Red (Figure 3a). Importantly, treatment with losartan reduced the area occupied by collagen fibre deposition in the iron‐overloaded group to levels, similar to those of the control group (Figure 3b). As shown in Figure 3c,d, there were no differences between calcium deposits in the aortic wall from groups.

Figure 3.

Increases of total aortic collagen content, due to iron‐overload was prevented by losartan. Representative wall pictures at 100× and high‐magnification photomicrography at 400× showing Picrosirius Red‐stained fibres (a) and Von Kossa‐stained aortic sections (c) in the control and iron‐overload groups treated or not with losartan. Summary graphs represent the collagen content expressed as relative collagen area (b) calcification area (d) of the vascular wall. Data are the means ± SEM, and the number of animals used is indicated in parentheses. *P < .05, significantly different as indicated; using two‐way ANOVA and Tukey's post hoc test

3.4. Serum iron levels and tissue deposits are not influenced by AT₁ receptor blockade

Serum iron was elevated in both iron‐overloaded groups, compared with their corresponding controls, without differences in the groups treated with vehicle or losartan (Figure 4c). Moreover, to investigate whether the treatment with losartan could alter the tissue pattern of iron deposition, sections of the aorta and liver were stained with Prussian blue. Such staining indicated iron‐positive plots on the vascular wall layers including medial and endothelial spots and significant iron deposition in the adventitia of the iron‐overloaded group, with or without losartan. There were no iron‐positive plots in the aortas of the rats not injected with iron (Figure 4b). Furthermore, there was strong deposition of non‐haem iron, only in the livers from iron‐treated rats, which was not influenced by treatment with losartan (Figure 4a).

Figure 4.

Iron deposits in the liver and aorta, and serum iron levels. Representative microscopic photographs of Prussian blue staining in the liver at 100× and 400× indicate severe iron deposition in the hepatic parenchyma and in Kupffer cells, forming agglomerates in the samples from iron‐overloaded rats (arrows), whereas in control animals, there are no deposits at any magnification (a). Iron deposits evaluated in photomicrography of the thoracic aortas from all groups (b). The high‐magnification photomicrography at 400× shows iron deposition not only in the adventitia but also into the medial (asterisks) and endothelial layers (arrows) of the aortas from iron‐overload groups treated or not with losartan. Serum iron levels were assessed in all groups, reinforcing that neither the tissue levels nor the circulating iron was modified by treatment with losartan (c). Data are expressed as the means ± SEM, and the number of animals used is indicated in parentheses. *P < .05 significantly different as indicated; two‐way ANOVA and Tukey's post hoc test

4. DISCUSSION

In this study, we have demonstrated that iron‐overloading induced changes in aortic structure and mechanics in the rat and that these changes were prevented by chronic block of AT₁ receptors. Using a moderate duration of iron‐overload i.e., 2 weeks, there was evidence of aortic stiffness, as characterized by increased aortic PWV and EM/WS ratio. Clinical studies have shown increased PWV in iron‐overloaded patients due to β‐thalassaemia major (Detchaporn et al., 2012; Gedikli et al., 2007; Ulger, Aydinok, Gurses, Levent, & Ozyurek, 2006) or haemochromatosis (Cash et al., 2013). Furthermore, Valenti et al. (2015) identified an association between hyperferritinemia and aortic stiffness in hypertensive subjects, and Merchant et al. (2016) described that arterial stiffness increased significantly as cardiac iron‐overload increased in β‐thalassaemia patients receiving regular blood transfusions. On the other hand, Stakos et al. (2009) described that, in the absence of cardiac iron‐overload, patients with β‐thalassaemia major demonstrated aortic stiffening. Also, β‐thalassaemia patients receiving iron chelation and, therefore, without elevated serum iron, exhibit increased PWV (Cheung et al., 2002), indicating that under these conditions, the underlying mechanism for vascular remodelling could be other than iron‐overload. Thus, a direct cause–effect relationship between iron per se and arterial stiffness has not yet been clinically established, as all trials were conducted in individuals with co‐morbidities that were not controlled. In animal models, Wistar male rats, after 4 weeks of iron‐overload, exhibited functional, structural, and mechanical changes in mesenteric resistance arteries, accompanied by an increase in the aortic PWV (Ribeiro Júnior et al., 2017). There was evidence of inward hypertrophic remodelling associated with increased collagen deposition in the small arteries, which could be responsible for enhancing vascular stiffness due to iron‐overload. In the present study, we found that after 2 weeks of iron‐dextran injections using a double‐dose, there was increased aortic PWV, but unchanged BP and vascular tone control. This could be intriguing because increasing evidence indicates that chronic iron‐overload is associated with endothelial dysfunction (Bertoli et al., 2018; Day et al., 2003; Marques et al., 2015; Ribeiro Júnior et al., 2017). However, in our present study, we assessed vascular structure and function at 15 days of iron‐overload, and, according to both in vivo and in vitro protocols, there was no impairment of the vascular tone control and endothelium, and NO modulations were preserved as well. In this way, our results reinforce the hypothesis that iron‐overload could indeed stiffen the arterial tree, regardless of changes in vascular tone or endothelial function. In addition, because aortic wall thickness is unchanged in this regimen, changes in wall composition should explain the decreased elasticity after iron‐overload.

Notwithstanding, the major finding in the present study was the ability of AT₁ receptor blockade to prevent the increase in aortic stiffness in a rat model of iron‐overload, as indicated by lower PWV, β‐index, and EM/WS ratio compared to rats treated with vehicle. Increased aortic PWV, an indicator of arterial stiffness, is a strong and independent predictor of cardiovascular risk not only in patients with vascular diseases (Alvim et al., 2013; Alvim, Santos, Bortolotto, Mill, & Pereira, 2017; Baldo et al., 2018; Georgianos, Sarafidis, & Lasaridis, 2015; Mitchell, 2009) but also in patients with haemochromatosis (Cash et al., 2013). This increased risk could be due to a loss of the ability of a stiffer aorta to buffer pulsatile changes in BP. In fact, for both man and experimental animals, the loss of aortic distensibility increases BP and pulsatile pressure and places vulnerable tissues at risk of microvascular damage, by reducing capillary proliferation and increasing thrombosis, atherosclerosis, and vasomotor dysfunction (Alvim et al., 2017; Mitchell, 2009; Safar, 2018). In our study, we did not identify significant changes in the BP or pulse pressure of the rats subjected to iron‐overload, which suggests that altered control of vascular tone remained normal despite the signs of vascular stiffness. Because in previous studies using iron administration for 4 weeks we identified clear vascular dysfunction characterized by loss of endothelial modulation and increased vasocontractile response in both the aorta and resistance vessels, it is possible that iron‐overload duration (i.e., treatment time) might have been a differential factor. In other words, the overload regimen used here caused aortic remodelling with increased wall collagen deposition but still without hypertrophy or evident endothelial dysfunction.

In our model, the thoracic aortas from iron‐overload rats have no changes on medial thickness, inner diameter, MCSA, or wall‐to‐lumen ratio. However, absolute PWV or normalized to BP and heart rate was elevated in this rat model, and losartan completely prevented these changes. In addition, the iron‐overload rats showed 2.2‐fold greater EM/WS ratio, indicating relative stiffening, whereas the rats co‐treated with losartan showed values similar to those of controls. It is likely that these pressure‐independent mechanical changes in the aorta may be due to structural abnormalities such as a medial wall calcification, decrease in elastin, or an increase in collagen deposition, all possible ways of promoting arterial stiffness.

In this regard, some reports have indicated that increased iron in vitro enhances calcification of both endothelial (Nanami et al., 2005) and smooth muscle cells (Kawada et al., 2018) through inflammation and oxidative stress. In addition, it has been proposed that aortic calcification may be causally linked to arterial stiffness and increased PWV in humans (Guo et al., 2017; Sekikawa et al., 2012; Tsao et al., 2014) and a rat model of vascular disease (Ng et al., 2011). However, our data indicate that this short‐term protocol of iron‐overload was unable to induce significant calcification of the rat aorta, and treatment with losartan did not modify this parameter.

Interestingly, this reduced distensibility of the aorta from iron‐overloaded rats was accompanied by collagen deposition in aortic walls, but no changes on medial thickness or MCSA. The coexistence of hypertrophic and matrix deposition processes and haemodynamic changes is not the rule, and all features can be separately identified in different models of vasculopathy. For example, in a rat model of chronic kidney disease, the aortic collagen fraction was significantly increased, and cellular hypertrophy was only modest, whereas BP was normal but EM/WS was markedly elevated (Amann et al., 1997). To provide a reliable representation of wall stiffness independently of the artery geometry, EM is generally expressed as a function of WS. Thus, we could affirm that, in the iron‐overload group, the aorta is significantly stiffer at a comparable WS imposed by the aortic BP. Similarly, the EM/WS ratio was reduced in the iron‐overloaded rats co‐treated with losartan, whereas no difference was found between the control groups treated or not with the AT₁ receptor antagonist.

It is known that the compliance of large arteries depends not only on geometry and muscular tone, but also on the balance between distensible interstitial components, such as elastin, and less distensible elements, such as collagen and calcification (Intengan & Schiffrin, 2000). Especially in smaller arteries and arterioles, it is also determined by other mechanisms modulating smooth muscle tone, including endothelial function and NO availability. Thus, the endothelial dysfunction induced by iron‐overload could increase peripheral vascular resistance and, therefore, potentially change the elastic properties of the aortic wall (Alvim et al., 2017; Lacolley et al., 2017). However, as discussed above, because the vasoresponses to phenylephrine both in vivo and in vitro were not different between control and iron‐overload rats, a possible contribution of endothelial dysfunction or altered smooth muscle cell tone to the vessel wall stiffness is unlikely.

Regarding the underlying mechanisms by which iron‐overload may induce these changes on the vasculature, in addition to growth factors commonly related to inflammation and the reduced NO bioavailability evident in this model, Ang II appears to be involved in both remodelling and dysfunction of arteries from different sites (Bertoli et al., 2018; Marques et al., 2015; Ribeiro Júnior et al., 2017). As identified in the protocols for vascular reactivity under in vitro AT₁ receptor blockade, these authors suggested that reduced NO and elevated ROS, due to AT₁ receptor activation could play a role in the genesis of vascular functional and structural remodelling in the rat model of iron‐overload. In fact, the mechanism by which drugs that inhibit the RAS attenuate or reverse vascular remodelling is related to the modulation of growth‐promoting, pro‐oxidant, and pro‐inflammatory actions of Ang II and thereby improving the arterial compliance, all independent of BP reduction (Brassard, Amiri, & Schiffrin, 2005; Janić, Lunder, & Sabovič, 2014; Neves, Cunha, Cunha, Gismondi, & Oigman, 2018; Ng et al., 2011; Shahin et al., 2012; Zhu et al., 2019). Consistent with these findings, the blockade of AT₁ receptors in vivo, was capable of preventing aortic deposition of collagen and stiffening also in the iron‐overload model.

As discussed above, excess iron enhances oxidative stress, which in turn triggers molecular mechanisms that could lead to vascular remodelling and stiffening. Thus, elevated iron deposits in the aorta could be the key mechanism for the development of the mechanical changes found here, that is, by increasing collagen deposition. However, our results demonstrate that the blockade of AT₁ receptors had clearly evident effects in preventing the increase of vascular collagen and stiffening, despite not attenuating serum and tissue iron‐overload, thereby reinforcing the role of the RAS in this vascular remodelling. In fact, Ishizaka et al. (2002, 2005) have suggested an interplay between Ang II and iron‐overload in cardiac and perivascular fibrosis, showing that, while iron chelation reduces, iron‐overload intensified, collagen deposition in the heart and aorta of Ang II‐infused rats. Because it is well known that the RAS influences other important neurohumoral and local mediators also for cardiac remodelling, further studies are required to delineate the relative contribution of Ang II for the iron‐overload cardiomyopathy.

In summary, in the iron‐overload model used here, there was an increase in aortic stiffness as shown by the elevation of PWV, β‐index and EM/WS ratio, associated with increased collagen deposition, which was prevented by the co‐administration of losartan. It is noteworthy that this is the first preclinical indication of the AT₁ receptors as key mediators of cardiovascular disease in chronic iron‐overload, suggesting a therapeutic potential of inhibition of the RAS in these patients. Thus, our study should stimulate further experimental investigations regarding other benefits of this approach on the cardiovascular phenotype of iron‐overload and encourage clinical research to test its role as adjuvant therapy.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

V.B.M., R.A.A., and L.S. designed the research; H.G.F., J.A.G.M., S.C.E.G., T.B., R.A.A. and V.B.M. performed experiments; H.G.F., J.A.G.M., S.C.E.G., V.B.M., R.A.A., T.B., and L.S. analysed data; V.B.M., R.A.A., and L.S. wrote and revised the manuscript.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14207, and https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14206, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1. Concentration‐response parameters for the agonist effect of phenylephrine on the vasoconstriction of aorta segments isolated from controls and iron‐overloaded rats.

Fidelis HG, Mageski JGA, Goes SCE, et al. Blockade of angiotensin AT₁ receptors prevents arterial remodelling and stiffening in iron‐overloaded rats. Br J Pharmacol. 2020;177:1119–1130. 10.1111/bph.14904