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“Angiotensin II memory” contributes to the development of hypertension and vascular injury via activation of NADPH oxidase
Life Sciences 149 (2016) 18–24
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“Angiotensin II memory” contributes to the development of hypertension and vascular injury via activation of NADPH oxidase Wen-Jun Li a, Ying Liu c, Jing-Jing Wang b, Yun-Long Zhang c, Song Lai c, Yun-Long Xia c, Hong-Xia Wang a,⁎, Hui-Hua Li a,⁎ a b c
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China Department of Laboratory Animal Sciences, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China Department of Cardiology, Institute of Cardiovascular Diseases, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
a r t i c l e
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Article history: Received 30 October 2015 Accepted 9 February 2016 Available online 10 February 2016 Keywords: Angiotensin II Hypertension NADPH oxidases Free radicals
a b s t r a c t Aims: Activation of the rennin-angiotensin system plays a critical role in the development of hypertension and its complication. Our previous study has demonstrated that a cellular “memory” is involved in angiotensin II (Ang II)-induced cardiac hypertrophy. The aim of this study is to investigate the effect of reversal of high Ang II to normal condition on hypertension and vascular damage. Main methods: Wild-type male mice were randomly divided into five groups. The vascular function, inflammation, oxidative stress and angiogenesis were examined by aortic ring relaxation studies, histological analysis, real-time PCR and Western blot analysis. Key findings: We found that continuous high Ang II infusion for 3 weeks (Ang II 3w) significantly elevated blood pressure, increased aortic wall thickness, collagen deposition, inflammation, oxidative stress, vascular function and activation of p38 MAPK, JNK1/2, STAT3 and NF-κB pathways in mouse aorta compared with saline group. High Ang II exposure for 2 weeks followed by saline for 1 week (Ang II 2 + 1w) failed to reverse these alterations. This phenomenon was named “metabolic memory” (or persistent effect). However, addition of NADPH oxidase inhibitor apocynin during saline infusion (Ang II 2 + 1w + Apo) markedly ameliorated such deleterious effects. Significance: These results showed that we report the first that persistent effect or “metabolic memory” of angiotensin II through NADPH oxidase-mediated oxidative stress plays important roles in hypertension and vascular injury. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Hypertension is a major risk of death from stroke, ischemic heart disease, atherosclerosis, and other cardiovascular diseases [1]. The reninangiotensin system (RAS) has been known to play a critical role in the process of hypertension and other cardiovascular diseases [2]. Angiotensin II (Ang II) is a key factor to cause vasoconstriction, salt retention and inflammation. Ang II also stimulates NADPH oxidases to produce reactive oxygen species (ROS) that result in prolonged vascular injury and hypertension [3, 4]. Treatment of animals with angiotensin receptor blocker (ARB) markedly attenuates hypertension and renal arteriolar injury in stroke-prone spontaneously rats or Dahl salt-sensitive rats (DS rats) [5, 6]. Many clinical studies have also shown that intensive drug treatment with ACE inhibitors and ARB can reduce the progression
⁎ Corresponding authors at: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China. E-mail addresses: [email protected] (H.-X. Wang), [email protected] (H.-H. Li).
http://dx.doi.org/10.1016/j.lfs.2016.02.037 0024-3205/© 2016 Elsevier Inc. All rights reserved.
and incidence of hypertensive complications, including nephropathy, vascular disease, and heart failure and can also reduce the incidence of death [7]. Although extensive studies have been conducted, the cause of most cases of human hypertension remains unclear. Many clinical studies have demonstrated that “metabolic memory” plays an important role in the development of diabetes and its chronic complications [8]. Intensive glycemic control in patients with diabetes can reduce the incidence and progression of diabetic nephropathy and can also reduce the incidence of other complications [9, 10]. Indeed, a similar “cellular memory” phenomenon has been demonstrated in hypertension and its complication such as cardiac hypertrophy [11, 12]. Transient treatment of Ang II causes sustained hypertension after cessation of Ang II in spontaneously hypertensive rat (SHR) [13]. We also reported that high Ang II infusion results in the persistent cardiac hypertrophy and fibrosis after cessation of Ang II in wild-type mice [12]. More recently, Oguchi et al showed the “salt memory” can induce sustained elevation of blood pressure in hypertensive rat models [11]. However, it is unknown whether “metabolic memory” or persistent effect of Ang II plays a role in hypertension and vascular injury in a mouse model.
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In this study, we demonstrated that this phenomenon exists and plays a critical role in the development of hypertension and vascular damage in a mouse model. We also showed that activation of NADPH oxidase has been implicated in this process. 2. Materials and methods
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2.5. Vascular relaxation studies Isolated aortas were cut into 4-mm segments, then mounted on force transducers in organ chambers (Power lab, AD Instruments, Germany). Concentration-relaxation curves in response to increasing concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP) was recorded.
2.1. Animal model 2.6. Quantitative real-time RT-PCR The wild-type male mice (C57BL/6, 8–10 week-old) were randomly divided into 5 groups and then infused with saline or Ang II (1000 ng/kg/min, Sigma-Aldrich technology, St. Louis, MO) with or without apocynin (10 mg/kg/day, Sigma-Aldrich technology, St. Louis, MO) 2–3 weeks as described in our publication [12]. The groups included control (saline), Ang II 3w (Ang II for 3 weeks), Ang II 2 + 1w (Ang II for 2 weeks and then saline for 1 week), Ang II 2 + 1w + Apo (Ang II for 2 weeks and then saline for 1 week plus apocynin), Apo (apocynin for the last week). The Ang II 3w, Ang II 2 + 1w and Ang II 2 + 1w + Apo groups were implanted with osmotic pumps (Alzet MODEL 1007D; Cupertino, CA) and infused with Ang II at 1000 ng/kg/min in Ringer's solution (0.01 mmol/L acetic acid in saline) for 2 or 3 weeks. The Ang II 2 + 1w + Apo or Apo group animals were injected with apocynin (solved in DMSO, 10 mg/kg/day, intraperitoneally) during the last week [14]. Control mice received the same volume of DMSO. The systolic blood pressure (SBP) was measured by tail-cuff method as described [12, 15]. To avoid the variance in blood pressure measurement in mice caused by panic, mice were trained for 3 days before SBP was measured. All procedures were approved by the Animal Care and Use Committee of Capital medical University. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996).
Total RNA was extracted from fresh mouse aortas using TRIzol method (Invitrogen). RNA samples (1 μg) were reverse-transcribed to generate first-strand cDNA. Quantitative RT-PCR (qPCR) analysis was as described previously [19]. The primers of IL-1β, TNF-α, IL-6, MCP-1, ICAM-1, NOX1, P22phox and GAPDH were designed as described previously [12, 20, 21]. 2.7. Cell culture and treatment Vascular smooth muscle cells (VSMCs) were cultured in DMEM supplemented with 10% FBS, L-glutamine (330 mg/mL), penicillin (100 U/ mL), and streptomycin (100 mg/mL) at 37 °C under a humidified atmosphere of 95% air and 5%CO2 as described [22]. Cells were randomly divided into five groups. The Ang II 3d group was treated with Ang II (100 nmol/L) for 3 days. The Ang II 2 + 1d group was treated with Ang II (100 nmol/L) for 2 days, and then incubated fresh FCS-free DMEM for 1 day. The control group was incubated with fresh FCS-free DMEM for 3 days. The apocynin group was incubated with fresh FCSfree DMEM containing 200 μmol/L apocynin for 3 days [23, 24]. The Ang II 2 + 1d + Apo group was incubated with apocynin (200 μmol/ L) before treated with Ang II (100 nmol/L), 2 days later, the medium was replaced with fresh FCS-free DMEM containing 200 μmol/L apocynin for 1 day.
2.2. Histopathology and immunohistochemistry 2.8. Western blotting Mice were anesthetized with tribromoethanol (0.25 mg/g intraperitoneally), and artery tissues were perfused with normal saline and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm). Aortic sections were stained with hematoxylin and eosin (H&E), Masson trichrome and Verhoeff-Van Gieson (VVG) as described previously [16]. Immunohistochemical staining was performed with primary antibody against Mac-2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA). Digital photographs were taken at 10 × or 20 × magnification of over 10 random fields from each aorta. The images were evaluated in a blinded fashion by a pathologist, and were captured using a Nikon Labophot 2 microscope (Nikon, Tokyo, Japan).
VSMCs were lysed with lysis buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM EGTA, 1 mM β-glycerophosphate, 1%Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 4 μg/mL aprotinin,
2.3. Measurement of NADPH oxidase activity Sample preparation was performed by glass/glass homogenization, centrifugation at 8000g for 20 min and subsequent protein determination using the Bradford assay. The resulting particulate fraction was used for measurement of NADPH oxidase activity in aortic tissue as described [17]. NADPH oxidase activity in the aorta was measured by lucigenin (5 μM) ECL. The results were normalized for protein content and expressed as counts/mg/min after 30 min. 2.4. DHE staining Isolated aorta was cut into 3 mm rings and incubated in KrebsHepes-solution for 15 min at 37 °C, embedded in aluminium cups of about 1 mL of OCT resin (Tissue Tek, USA) and frozen in liquid nitrogen. Cryosections (8 μm) were stained with the superoxide-sensitive dye dihydroethidine (DHE, 1 μM in PBS) and incubated for 30 min at 37 °C. Green autofluorescence and red DHE fluorescence was detected using a Nikon Labophot 2 microscope (Nikon, Tokyo, Japan) [18].
Fig. 1. Effect of high Ang II infusion and apocynin on blood pressure in mice. Mice were randomly divided into five groups and treated as described under materials and methods: Control (Saline), Ang II 3w (1000 ng/kg/min, for 3 weeks), Ang II 2 + 1w (Ang II for 2 weeks and saline for 1 week), Ang II 2 + 1w + Apo (Ang II for 2 weeks and saline for 1 week plus Apo), and Apo (Apo for the last week). Apocynin was given intraperitoneally at day 14 (after angiotensin II infusion), and angiotensin II infusion at 1000 ng/kg/min was on day 0. Mouse systemic blood pressure (SBP) was measured by tail-cuff method 3 days before and after angiotensin II infusion. Histogram shows the mean blood pressure of the 5 groups during training and during days 21 of angiotensin II infusion. Data represent the mean ± SEM (n = 8–10). *P b 0.05 vs. vehicle control group; #P b 0.05 vs. Ang II 3w or Ang II 2 + 1w group.
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4 μg/mL leupeptin, 4 μg/mL pepstatin, and 1 mM PMSF). Western blot analysis was performed as previously described [25]. 40–50 μg protein lysates were separated by 10% SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies against p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, p65, phospho-p65, STAT3, phospho-STAT3 (dilution 1:800–1000, Cell Signaling Technology, Danvers, MA) and GAPDH (1:5000, Cell Signaling Technology, Danvers, MA), and then with horseradish peroxidaseconjugated secondary antibodies (1:1000, Cell Signaling Technology, Danvers, MA). As for the phospho-specific protein, we normalized the signal to the amount of total target protein and GAPDH.
(Ang II for 3w) significantly elevated SBP, increased collagen deposition, elastin damage, aortic thickening, infiltration of Mac-2-positive macrophages, the expression of proinflammatory cytokines (including IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1), NADPH oxidase activity, ROS production, and the expression of NADPH oxidase (including NOX1 and p22phox) in the aorta, and also markedly reduced endotheliumdependent and independent vasodilatation as compared with control (Figs. 1–4).
2.9. Statistical analysis
To determine whether high Ang II has a persistent effects (metabolic memory) on blood pressure and vascular remodeling, mice were infused with high Ang II (1000 ng/kg/min) for 2 weeks followed by saline for 1 week (Ang II 2 + 1w). We found that one week of saline injection that followed 2 weeks of high Ang II infusion did not significantly reduced high Ang II-induced effects, including hypertension, vascular injury, oxidative stress compared with mice treated with Ang II 3w (Figs. 1–4). These results suggest that there is a phenomenon of persistent effects (metabolic memory) of high Ang II in this process.
All data are expressed as mean ± SEM. Statistical analyses involved use of one-way ANOVA analysis followed by LSD for multiple comparisons within treatment groups with use of SPSS v13.0 (SPSS Inc., Chicago, IL). Analysis of dose-response curves for vascular studies was performed using ANOVA for repeated measures. P b 0.05 was considered statistically significant.
3.2. Effect of 2 weeks of high Ang II followed by 1 week of saline on hypertension and vascular injury
3. Results 3.1. Effect of high Ang II on blood pressure, vascular injury, inflammation, oxidative stress and vascular function To investigate whether high Ang II promoted the development of hypertension and vascular damage, wild-type male mice were continuously infused with saline or Ang II (1000 ng/kg/min) for 3 weeks, and the systolic blood pressure (SBP) was measured by the noninvasive tail-cuff method in the different groups. Ang II infusion for 3 weeks
3.3. Effect of 2 weeks of high Ang II followed by 1 week of saline plus apocynin on hypertension and vascular injury It is reported that ROS plays an important role in vascular injury and dysfunction induced by Ang II during hypertension [26]. To test whether NADPH oxidase mediates metabolic memory-induced responses, apocynin, a selective NADPH oxidase inhibitor, was injected during saline infusion period that followed 2 weeks of high Ang II infusion (Ang II 2 + 1w + Apo). Our results showed that one week of apocynin
Fig. 2. Effect of high Ang II infusion and apocynin on vascular injury in mice. Mice were treated as for Fig. 1. (A) Sections (5 μm) of the thoracic aortas were stained with Masson trichome to highlight collagen formation (blue) and quantification. (B) Sections of the thoracic aortas were stained with Van Gieson elastica stains to evaluate elastin structure (yellow). Data represent the means ± SEM (n = 5). *P b 0.05 vs. vehicle control group; #P b 0.05 vs. Ang II 3w or Ang II 2 + 1w group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effect of high Ang II infusion and apocynin on vascular inflammation in mice. Mice were treated as for Fig. 1. (A) Sections (5 μm) of thoracic aorta were stained with Hematoxylin and eosin (H&E) and measured as total wall area determined by planimetry. (B) Sections (5 μm) of thoracic aorta were stained with immunohistochemistry for Mac-2 and quantification of Mac-2 positive cells. (C) The mRNA expression of IL-1β, IL-6,TNFα, MCP-1 and ICAM-1 in the aorta was detected by qPCR analysis. (D) The expression of NOX1 and p22phox in the aorta was detected by qPCR analysis. Data represent the means ± SEM (n = 5). *P b 0.05 vs. vehicle control group; #P b 0.05 vs. Ang II 3w or Ang II 2 + 1w group.
injection that followed 2 weeks of high Ang II infusion not only markedly attenuated Ang II-induced activation of NADPH oxidase and ROS production (Fig. 4), but also markedly attenuated the induction of Ang IIinduced hypertension, aortic injury, inflammation and vascular dysfunction as compared with mice treated with Ang II 3w and Ang 2 + 1w groups (Figs. 1–4).
shown in Fig. 5A, treatment of VSMCs with Ang II (100 nmol/L) for 3 days significantly increased the levels of phosphorylated ERK1/2, p38 MAPK, JNK1/2, STAT3 and NF-κB/p65, and remained high levels in Ang II 2 + 1d group as compared with the saline control, but ERK phosphorylation did not changed (Fig. 5A). Notably, addition of apocynin markedly attenuated these affects, but not phosphorylated p38 MAPK (Fig. 5B). Apocynin alone had no significant influence (Fig. 5B).
3.4. Apocynin suppresses Ang II-induced activation of multiple signaling pathways in vitro
4. Discussion
Next, we attempted to clarify the mechanism by which apocynin inhibited “metabolic memory” of high Ang II. Previous studies demonstrated that Ang II stimulation induces ROS production and activation of several signaling pathways (MAPKs, STAT, and NF-κB), which promote vascular injury and inflammation during hypertension [4]. A
In the present study, using an Ang II infused mouse model, we presented several lines of evidence supporting the phenomenon of persistent effects (metabolic memory) of high Ang II as novel mediator of hypertension and vascular injury. We demonstrated that transient high Ang II infusion not only induces sustained elevation of blood
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Fig. 4. Effect of high Ang II infusion and apocynin on vascular NADPH oxidase activity, ROS production and vascular function. Mice were treated as for Fig. 1. (A) NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence in vascular tissue. (B) The mRNA expression of NOX1 in the aorta was detected by qPCR analysis. (C) The mRNA expression of p22phox in the aorta was detected by qPCR analysis. (D) ROS production was measured by DHE staining. Data are mean ± SEM (n = 5). (E) Effect of angiotensin II-induced hypertension on endothelium-dependent vasodilatation to acetylcholine (Ach, left). Relaxations to sodium nitroprusside (SNP) were examined as a measure of nonendotheliumdependent vasodilatation (right). *P b 0.05 versus control group; #P b 0.05 versus Ang II 2 + 1w group.
pressure, but also results in aortic remodeling, inflammation, activation of NADPH oxidase, and impaired vascular relaxation. However, inhibition of NADPH oxidase activity by apocynin markedly blocked this phenomenon. Hypertensive vascular injury is associated with impaired endothelial cell function and alterations of vascular smooth muscle cells (VSMCs), including contraction/relaxation, migration, apoptosis, deposition of
extracellular matrix and inflammation [27, 28]. Multiple cellular mechanisms and signaling pathways have been implicated in vascular injury. Activation of the RAS is crucial in the pathogenesis of hypertension and vascular injury [4]. Recent trials clearly suggest that antihypertensive treatment with RAS inhibitors prevents organ damage and reduces cardiovascular events through protective actions beyond blood pressure lowering in hypertensive patients [29, 30]. Studies have also
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Fig. 5. Effect of high Ang II infusion and apocynin on activation of MAPKs, STAT3 and NF-κB signaling pathways in human vascular smooth cells. (A) Human vascular smooth muscle cells (VSMCs) were cultured in normal medium (control), high (100 nmol/L) Ang II for 3 days (Ang II 3d), or high (100 nmol/L) Ang II for 2 days followed by normal medium for 1 day (Ang II 2 + 1d, the memory condition). (B) Cells were treated as in A in presence or absence of 200 μmol/L apocynin (Ang II 2 + 1d + Apo) during the last day of normalized Ang II or Apo alone. Western blot analysis was performed to detect protein levels of total and phosphorylated p38 MAPK, ERK, JNK1/2, STAT-3 and NF-κB. GADPH was used as a loading control. Data are mean ± SEM (n = 5). *P b 0.05 versus control group; #P b 0.05 versus Ang II 2 + 1d group.
demonstrated that even the early stage of hypertension, including prehypertension, is associated with an increased risk of cardiovascular events, such as stroke and coronary heart disease (CHD) [31, 32]. The transition from the early stage of preclinical hypertension to clinical hypertension enhances organ damage, including vascular remodeling [31, 33]. In animal experiments, transient Ang II treatment can result in sustained hypertension or cardiac hypertrophy and inflammation after cessation of Ang II in SHR or wild type mice [12, 13]. Interestingly, a similar phenomenon “salt memory” also plays a critical role in elevation of blood pressure in hypertensive rat models [11]. In this study, we further confirmed the phenomenon “metabolic memory” (persistent effects) in a mouse model, which plays an important role in the development of hypertension and vascular remodeling including wall thickness, collagen deposition, infiltration of inflammatory cells and expression of proinflammatory cytokines and oxidative stress (Figs. 1–4). Collectively, these data suggest that a “metabolic memory” retained in the development of hypertension and vascular injury.
Accumulating evidence suggests that vascular injury and remodeling play a major role in the pathogenesis of hypertension in different animal models [27]. One potential mechanism by which Ang II causes this process could involve an increase in oxidative stress-mediated signaling pathways, including MAPK pathway (including ERK, p38 MAPK and JNK), STAT3 and NF-κB [4]. Recent studies have demonstrated that increased ROS is involved in several models of experimental hypertension [34, 35]. Ang II can bind to angiotensin type 1 receptor (AT1R) and then stimulates NADPH oxidases to produce superoxide, which can react with the endogenous vasodilator nitric oxide (NO) thereby resulting in vasoconstriction [34, 35]. Apocynin is a natural organic compound and is used as a NADPH oxidase inhibitor in various cells. It also functions as an antioxidant in endothelial, vascular smooth muscle cells and cardiomyocytes [36, 37] and with antihypertensive effects attenuates Ang II or DOCA-salt hypertension [38]. Although it is known that a persisting activation of NADPH oxidase can mediate the “metabolic memory” in diabetes or cardiac hypertrophy [12, 38], its role in “Ang II
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memory”-mediated hypertension and vascular injury remain unclear. Our present results revealed that inhibitor apocynin significantly suppressed NADPH oxidase activity and ROS production, and attenuated “metabolic memory” induced hypertension and vascular dysfunction (Figs. 1-4). Moreover, apocynin treatment markedly inhibited activation of multiple signaling pathways (JNK1/2, STAT3 and NF-κB), suggesting that “metabolic memory” is linked to persistent activation of NADPH oxidase stimulated by Ang II. 5. Conclusions The present study provides novel evidence for the role of “metabolic memory” of high Ang II in the development of hypertension, vascular injury and dysfunction in a mouse model. Inhibition of NADPH oxidase by apocynin markedly attenuated these effects. Thus, our data for the first time demonstrated that “metabolic memory” of high Ang II contributed vascular injury and hypertension. Persistent activation of NADPH oxidase may be an important event during this process. Use of antioxidant therapy may offer significant benefits on hypertension and its complications. Acknowledgements This study was funded by grants from the 973 program (No. 2012CB517802), China National Natural Science Funds (No. 81330003 and 81400247). Author contributions H.X.W. and H.H.L. were responsible for conception, design and writing the article. W.J.L. did the experiment and analyzed the data. Y.L., J.J.W., Y.L.Z., S.L., and Y.L.X. collected the data. All authors have read and approved the final version of the manuscript. References [1] N.S. Beckett, R. Peters, A.E. Fletcher, et al., Treatment of hypertension in patients 80 years of age or older, N. Engl. J. Med. 358 (18) (2008) 1887–1898. [2] M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, et al., Role of the renin-angiotensin system in vascular diseases: expanding the field, Hypertension 38 (6) (2001) 1382–1387. [3] A.C. Montezano, R.M. Touyz, Oxidative stress, Noxs, and hypertension: experimental evidence and clinical controversies, Ann. Med. 44 (Suppl. 1) (2012) S2–16. [4] A.C. Montezano, A. Nguyen Dinh Cat, F.J. Rios, R.M. Touyz, Angiotensin II and vascular injury, Curr. Hypertens. Rep. 16 (6) (2014) 431. [5] H. Nakaya, H. Sasamura, M. Hayashi, T. Saruta, Temporary treatment of prepubescent rats with angiotensin inhibitors suppresses the development of hypertensive nephrosclerosis, J. Am. Soc. Nephrol. 12 (4) (2001) 659–666. [6] H. Nakaya, H. Sasamura, M. Mifune, et al., Prepubertal treatment with angiotensin receptor blocker causes partial attenuation of hypertension and renal damage in adult Dahl salt-sensitive rats, Nephron 91 (4) (2002) 710–718. [7] A. Holdiness, K. Monahan, D. Minor, R.D. de Shazo, Renin angiotensin aldosterone system blockade: little to no rationale for ACE inhibitor and ARB combinations, Am. J. Med. 124 (1) (2011) 15–19. [8] S. Roy, R. Sala, E. Cagliero, M. Lorenzi, Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory, Proc. Natl. Acad. Sci. U. S. A. 87 (1) (1990) 404–408. [9] M.A. Ihnat, J.E. Thorpe, C.D. Kamat, et al., Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling, Diabetologia 50 (7) (2007) 1523–1531. [10] M.A. Ihnat, J.E. Thorpe, A. Ceriello, Hypothesis: the ‘metabolic memory’, the new challenge of diabetes, Diabet. Med. 24 (6) (2007) 582–586. [11] H. Oguchi, H. Sasamura, K. Shinoda, et al., Renal arteriolar injury by salt intake contributes to salt memory for the development of hypertension, Hypertension 64 (4) (2014) 784–791. [12] H.X. Wang, H. Yang, Q.Y. Han, et al., NADPH oxidases mediate a cellular “memory” of angiotensin II stress in hypertensive cardiac hypertrophy, Free Radic. Biol. Med. 65 (2013) 897–907.
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“Angiotensin II memory” contributes to the development of hypertension and vascular injury via activation of NADPH oxidase Wen-Jun Li a, Ying Liu c, Jing-Jing Wang b, Yun-Long Zhang c, Song Lai c, Yun-Long Xia c, Hong-Xia Wang a,⁎, Hui-Hua Li a,⁎ a b c
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China Department of Laboratory Animal Sciences, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China Department of Cardiology, Institute of Cardiovascular Diseases, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China
a r t i c l e
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Article history: Received 30 October 2015 Accepted 9 February 2016 Available online 10 February 2016 Keywords: Angiotensin II Hypertension NADPH oxidases Free radicals
a b s t r a c t Aims: Activation of the rennin-angiotensin system plays a critical role in the development of hypertension and its complication. Our previous study has demonstrated that a cellular “memory” is involved in angiotensin II (Ang II)-induced cardiac hypertrophy. The aim of this study is to investigate the effect of reversal of high Ang II to normal condition on hypertension and vascular damage. Main methods: Wild-type male mice were randomly divided into five groups. The vascular function, inflammation, oxidative stress and angiogenesis were examined by aortic ring relaxation studies, histological analysis, real-time PCR and Western blot analysis. Key findings: We found that continuous high Ang II infusion for 3 weeks (Ang II 3w) significantly elevated blood pressure, increased aortic wall thickness, collagen deposition, inflammation, oxidative stress, vascular function and activation of p38 MAPK, JNK1/2, STAT3 and NF-κB pathways in mouse aorta compared with saline group. High Ang II exposure for 2 weeks followed by saline for 1 week (Ang II 2 + 1w) failed to reverse these alterations. This phenomenon was named “metabolic memory” (or persistent effect). However, addition of NADPH oxidase inhibitor apocynin during saline infusion (Ang II 2 + 1w + Apo) markedly ameliorated such deleterious effects. Significance: These results showed that we report the first that persistent effect or “metabolic memory” of angiotensin II through NADPH oxidase-mediated oxidative stress plays important roles in hypertension and vascular injury. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Hypertension is a major risk of death from stroke, ischemic heart disease, atherosclerosis, and other cardiovascular diseases [1]. The reninangiotensin system (RAS) has been known to play a critical role in the process of hypertension and other cardiovascular diseases [2]. Angiotensin II (Ang II) is a key factor to cause vasoconstriction, salt retention and inflammation. Ang II also stimulates NADPH oxidases to produce reactive oxygen species (ROS) that result in prolonged vascular injury and hypertension [3, 4]. Treatment of animals with angiotensin receptor blocker (ARB) markedly attenuates hypertension and renal arteriolar injury in stroke-prone spontaneously rats or Dahl salt-sensitive rats (DS rats) [5, 6]. Many clinical studies have also shown that intensive drug treatment with ACE inhibitors and ARB can reduce the progression
⁎ Corresponding authors at: Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China. E-mail addresses: [email protected] (H.-X. Wang), [email protected] (H.-H. Li).
http://dx.doi.org/10.1016/j.lfs.2016.02.037 0024-3205/© 2016 Elsevier Inc. All rights reserved.
and incidence of hypertensive complications, including nephropathy, vascular disease, and heart failure and can also reduce the incidence of death [7]. Although extensive studies have been conducted, the cause of most cases of human hypertension remains unclear. Many clinical studies have demonstrated that “metabolic memory” plays an important role in the development of diabetes and its chronic complications [8]. Intensive glycemic control in patients with diabetes can reduce the incidence and progression of diabetic nephropathy and can also reduce the incidence of other complications [9, 10]. Indeed, a similar “cellular memory” phenomenon has been demonstrated in hypertension and its complication such as cardiac hypertrophy [11, 12]. Transient treatment of Ang II causes sustained hypertension after cessation of Ang II in spontaneously hypertensive rat (SHR) [13]. We also reported that high Ang II infusion results in the persistent cardiac hypertrophy and fibrosis after cessation of Ang II in wild-type mice [12]. More recently, Oguchi et al showed the “salt memory” can induce sustained elevation of blood pressure in hypertensive rat models [11]. However, it is unknown whether “metabolic memory” or persistent effect of Ang II plays a role in hypertension and vascular injury in a mouse model.
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In this study, we demonstrated that this phenomenon exists and plays a critical role in the development of hypertension and vascular damage in a mouse model. We also showed that activation of NADPH oxidase has been implicated in this process. 2. Materials and methods
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2.5. Vascular relaxation studies Isolated aortas were cut into 4-mm segments, then mounted on force transducers in organ chambers (Power lab, AD Instruments, Germany). Concentration-relaxation curves in response to increasing concentrations of acetylcholine (ACh) and sodium nitroprusside (SNP) was recorded.
2.1. Animal model 2.6. Quantitative real-time RT-PCR The wild-type male mice (C57BL/6, 8–10 week-old) were randomly divided into 5 groups and then infused with saline or Ang II (1000 ng/kg/min, Sigma-Aldrich technology, St. Louis, MO) with or without apocynin (10 mg/kg/day, Sigma-Aldrich technology, St. Louis, MO) 2–3 weeks as described in our publication [12]. The groups included control (saline), Ang II 3w (Ang II for 3 weeks), Ang II 2 + 1w (Ang II for 2 weeks and then saline for 1 week), Ang II 2 + 1w + Apo (Ang II for 2 weeks and then saline for 1 week plus apocynin), Apo (apocynin for the last week). The Ang II 3w, Ang II 2 + 1w and Ang II 2 + 1w + Apo groups were implanted with osmotic pumps (Alzet MODEL 1007D; Cupertino, CA) and infused with Ang II at 1000 ng/kg/min in Ringer's solution (0.01 mmol/L acetic acid in saline) for 2 or 3 weeks. The Ang II 2 + 1w + Apo or Apo group animals were injected with apocynin (solved in DMSO, 10 mg/kg/day, intraperitoneally) during the last week [14]. Control mice received the same volume of DMSO. The systolic blood pressure (SBP) was measured by tail-cuff method as described [12, 15]. To avoid the variance in blood pressure measurement in mice caused by panic, mice were trained for 3 days before SBP was measured. All procedures were approved by the Animal Care and Use Committee of Capital medical University. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996).
Total RNA was extracted from fresh mouse aortas using TRIzol method (Invitrogen). RNA samples (1 μg) were reverse-transcribed to generate first-strand cDNA. Quantitative RT-PCR (qPCR) analysis was as described previously [19]. The primers of IL-1β, TNF-α, IL-6, MCP-1, ICAM-1, NOX1, P22phox and GAPDH were designed as described previously [12, 20, 21]. 2.7. Cell culture and treatment Vascular smooth muscle cells (VSMCs) were cultured in DMEM supplemented with 10% FBS, L-glutamine (330 mg/mL), penicillin (100 U/ mL), and streptomycin (100 mg/mL) at 37 °C under a humidified atmosphere of 95% air and 5%CO2 as described [22]. Cells were randomly divided into five groups. The Ang II 3d group was treated with Ang II (100 nmol/L) for 3 days. The Ang II 2 + 1d group was treated with Ang II (100 nmol/L) for 2 days, and then incubated fresh FCS-free DMEM for 1 day. The control group was incubated with fresh FCS-free DMEM for 3 days. The apocynin group was incubated with fresh FCSfree DMEM containing 200 μmol/L apocynin for 3 days [23, 24]. The Ang II 2 + 1d + Apo group was incubated with apocynin (200 μmol/ L) before treated with Ang II (100 nmol/L), 2 days later, the medium was replaced with fresh FCS-free DMEM containing 200 μmol/L apocynin for 1 day.
2.2. Histopathology and immunohistochemistry 2.8. Western blotting Mice were anesthetized with tribromoethanol (0.25 mg/g intraperitoneally), and artery tissues were perfused with normal saline and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm). Aortic sections were stained with hematoxylin and eosin (H&E), Masson trichrome and Verhoeff-Van Gieson (VVG) as described previously [16]. Immunohistochemical staining was performed with primary antibody against Mac-2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA). Digital photographs were taken at 10 × or 20 × magnification of over 10 random fields from each aorta. The images were evaluated in a blinded fashion by a pathologist, and were captured using a Nikon Labophot 2 microscope (Nikon, Tokyo, Japan).
VSMCs were lysed with lysis buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM EGTA, 1 mM β-glycerophosphate, 1%Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 4 μg/mL aprotinin,
2.3. Measurement of NADPH oxidase activity Sample preparation was performed by glass/glass homogenization, centrifugation at 8000g for 20 min and subsequent protein determination using the Bradford assay. The resulting particulate fraction was used for measurement of NADPH oxidase activity in aortic tissue as described [17]. NADPH oxidase activity in the aorta was measured by lucigenin (5 μM) ECL. The results were normalized for protein content and expressed as counts/mg/min after 30 min. 2.4. DHE staining Isolated aorta was cut into 3 mm rings and incubated in KrebsHepes-solution for 15 min at 37 °C, embedded in aluminium cups of about 1 mL of OCT resin (Tissue Tek, USA) and frozen in liquid nitrogen. Cryosections (8 μm) were stained with the superoxide-sensitive dye dihydroethidine (DHE, 1 μM in PBS) and incubated for 30 min at 37 °C. Green autofluorescence and red DHE fluorescence was detected using a Nikon Labophot 2 microscope (Nikon, Tokyo, Japan) [18].
Fig. 1. Effect of high Ang II infusion and apocynin on blood pressure in mice. Mice were randomly divided into five groups and treated as described under materials and methods: Control (Saline), Ang II 3w (1000 ng/kg/min, for 3 weeks), Ang II 2 + 1w (Ang II for 2 weeks and saline for 1 week), Ang II 2 + 1w + Apo (Ang II for 2 weeks and saline for 1 week plus Apo), and Apo (Apo for the last week). Apocynin was given intraperitoneally at day 14 (after angiotensin II infusion), and angiotensin II infusion at 1000 ng/kg/min was on day 0. Mouse systemic blood pressure (SBP) was measured by tail-cuff method 3 days before and after angiotensin II infusion. Histogram shows the mean blood pressure of the 5 groups during training and during days 21 of angiotensin II infusion. Data represent the mean ± SEM (n = 8–10). *P b 0.05 vs. vehicle control group; #P b 0.05 vs. Ang II 3w or Ang II 2 + 1w group.
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4 μg/mL leupeptin, 4 μg/mL pepstatin, and 1 mM PMSF). Western blot analysis was performed as previously described [25]. 40–50 μg protein lysates were separated by 10% SDS-PAGE and then transferred to PVDF membranes, which were incubated with primary antibodies against p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, p65, phospho-p65, STAT3, phospho-STAT3 (dilution 1:800–1000, Cell Signaling Technology, Danvers, MA) and GAPDH (1:5000, Cell Signaling Technology, Danvers, MA), and then with horseradish peroxidaseconjugated secondary antibodies (1:1000, Cell Signaling Technology, Danvers, MA). As for the phospho-specific protein, we normalized the signal to the amount of total target protein and GAPDH.
(Ang II for 3w) significantly elevated SBP, increased collagen deposition, elastin damage, aortic thickening, infiltration of Mac-2-positive macrophages, the expression of proinflammatory cytokines (including IL-1β, IL-6, TNF-α, MCP-1 and ICAM-1), NADPH oxidase activity, ROS production, and the expression of NADPH oxidase (including NOX1 and p22phox) in the aorta, and also markedly reduced endotheliumdependent and independent vasodilatation as compared with control (Figs. 1–4).
2.9. Statistical analysis
To determine whether high Ang II has a persistent effects (metabolic memory) on blood pressure and vascular remodeling, mice were infused with high Ang II (1000 ng/kg/min) for 2 weeks followed by saline for 1 week (Ang II 2 + 1w). We found that one week of saline injection that followed 2 weeks of high Ang II infusion did not significantly reduced high Ang II-induced effects, including hypertension, vascular injury, oxidative stress compared with mice treated with Ang II 3w (Figs. 1–4). These results suggest that there is a phenomenon of persistent effects (metabolic memory) of high Ang II in this process.
All data are expressed as mean ± SEM. Statistical analyses involved use of one-way ANOVA analysis followed by LSD for multiple comparisons within treatment groups with use of SPSS v13.0 (SPSS Inc., Chicago, IL). Analysis of dose-response curves for vascular studies was performed using ANOVA for repeated measures. P b 0.05 was considered statistically significant.
3.2. Effect of 2 weeks of high Ang II followed by 1 week of saline on hypertension and vascular injury
3. Results 3.1. Effect of high Ang II on blood pressure, vascular injury, inflammation, oxidative stress and vascular function To investigate whether high Ang II promoted the development of hypertension and vascular damage, wild-type male mice were continuously infused with saline or Ang II (1000 ng/kg/min) for 3 weeks, and the systolic blood pressure (SBP) was measured by the noninvasive tail-cuff method in the different groups. Ang II infusion for 3 weeks
3.3. Effect of 2 weeks of high Ang II followed by 1 week of saline plus apocynin on hypertension and vascular injury It is reported that ROS plays an important role in vascular injury and dysfunction induced by Ang II during hypertension [26]. To test whether NADPH oxidase mediates metabolic memory-induced responses, apocynin, a selective NADPH oxidase inhibitor, was injected during saline infusion period that followed 2 weeks of high Ang II infusion (Ang II 2 + 1w + Apo). Our results showed that one week of apocynin
Fig. 2. Effect of high Ang II infusion and apocynin on vascular injury in mice. Mice were treated as for Fig. 1. (A) Sections (5 μm) of the thoracic aortas were stained with Masson trichome to highlight collagen formation (blue) and quantification. (B) Sections of the thoracic aortas were stained with Van Gieson elastica stains to evaluate elastin structure (yellow). Data represent the means ± SEM (n = 5). *P b 0.05 vs. vehicle control group; #P b 0.05 vs. Ang II 3w or Ang II 2 + 1w group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. Effect of high Ang II infusion and apocynin on vascular inflammation in mice. Mice were treated as for Fig. 1. (A) Sections (5 μm) of thoracic aorta were stained with Hematoxylin and eosin (H&E) and measured as total wall area determined by planimetry. (B) Sections (5 μm) of thoracic aorta were stained with immunohistochemistry for Mac-2 and quantification of Mac-2 positive cells. (C) The mRNA expression of IL-1β, IL-6,TNFα, MCP-1 and ICAM-1 in the aorta was detected by qPCR analysis. (D) The expression of NOX1 and p22phox in the aorta was detected by qPCR analysis. Data represent the means ± SEM (n = 5). *P b 0.05 vs. vehicle control group; #P b 0.05 vs. Ang II 3w or Ang II 2 + 1w group.
injection that followed 2 weeks of high Ang II infusion not only markedly attenuated Ang II-induced activation of NADPH oxidase and ROS production (Fig. 4), but also markedly attenuated the induction of Ang IIinduced hypertension, aortic injury, inflammation and vascular dysfunction as compared with mice treated with Ang II 3w and Ang 2 + 1w groups (Figs. 1–4).
shown in Fig. 5A, treatment of VSMCs with Ang II (100 nmol/L) for 3 days significantly increased the levels of phosphorylated ERK1/2, p38 MAPK, JNK1/2, STAT3 and NF-κB/p65, and remained high levels in Ang II 2 + 1d group as compared with the saline control, but ERK phosphorylation did not changed (Fig. 5A). Notably, addition of apocynin markedly attenuated these affects, but not phosphorylated p38 MAPK (Fig. 5B). Apocynin alone had no significant influence (Fig. 5B).
3.4. Apocynin suppresses Ang II-induced activation of multiple signaling pathways in vitro
4. Discussion
Next, we attempted to clarify the mechanism by which apocynin inhibited “metabolic memory” of high Ang II. Previous studies demonstrated that Ang II stimulation induces ROS production and activation of several signaling pathways (MAPKs, STAT, and NF-κB), which promote vascular injury and inflammation during hypertension [4]. A
In the present study, using an Ang II infused mouse model, we presented several lines of evidence supporting the phenomenon of persistent effects (metabolic memory) of high Ang II as novel mediator of hypertension and vascular injury. We demonstrated that transient high Ang II infusion not only induces sustained elevation of blood
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Fig. 4. Effect of high Ang II infusion and apocynin on vascular NADPH oxidase activity, ROS production and vascular function. Mice were treated as for Fig. 1. (A) NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence in vascular tissue. (B) The mRNA expression of NOX1 in the aorta was detected by qPCR analysis. (C) The mRNA expression of p22phox in the aorta was detected by qPCR analysis. (D) ROS production was measured by DHE staining. Data are mean ± SEM (n = 5). (E) Effect of angiotensin II-induced hypertension on endothelium-dependent vasodilatation to acetylcholine (Ach, left). Relaxations to sodium nitroprusside (SNP) were examined as a measure of nonendotheliumdependent vasodilatation (right). *P b 0.05 versus control group; #P b 0.05 versus Ang II 2 + 1w group.
pressure, but also results in aortic remodeling, inflammation, activation of NADPH oxidase, and impaired vascular relaxation. However, inhibition of NADPH oxidase activity by apocynin markedly blocked this phenomenon. Hypertensive vascular injury is associated with impaired endothelial cell function and alterations of vascular smooth muscle cells (VSMCs), including contraction/relaxation, migration, apoptosis, deposition of
extracellular matrix and inflammation [27, 28]. Multiple cellular mechanisms and signaling pathways have been implicated in vascular injury. Activation of the RAS is crucial in the pathogenesis of hypertension and vascular injury [4]. Recent trials clearly suggest that antihypertensive treatment with RAS inhibitors prevents organ damage and reduces cardiovascular events through protective actions beyond blood pressure lowering in hypertensive patients [29, 30]. Studies have also
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Fig. 5. Effect of high Ang II infusion and apocynin on activation of MAPKs, STAT3 and NF-κB signaling pathways in human vascular smooth cells. (A) Human vascular smooth muscle cells (VSMCs) were cultured in normal medium (control), high (100 nmol/L) Ang II for 3 days (Ang II 3d), or high (100 nmol/L) Ang II for 2 days followed by normal medium for 1 day (Ang II 2 + 1d, the memory condition). (B) Cells were treated as in A in presence or absence of 200 μmol/L apocynin (Ang II 2 + 1d + Apo) during the last day of normalized Ang II or Apo alone. Western blot analysis was performed to detect protein levels of total and phosphorylated p38 MAPK, ERK, JNK1/2, STAT-3 and NF-κB. GADPH was used as a loading control. Data are mean ± SEM (n = 5). *P b 0.05 versus control group; #P b 0.05 versus Ang II 2 + 1d group.
demonstrated that even the early stage of hypertension, including prehypertension, is associated with an increased risk of cardiovascular events, such as stroke and coronary heart disease (CHD) [31, 32]. The transition from the early stage of preclinical hypertension to clinical hypertension enhances organ damage, including vascular remodeling [31, 33]. In animal experiments, transient Ang II treatment can result in sustained hypertension or cardiac hypertrophy and inflammation after cessation of Ang II in SHR or wild type mice [12, 13]. Interestingly, a similar phenomenon “salt memory” also plays a critical role in elevation of blood pressure in hypertensive rat models [11]. In this study, we further confirmed the phenomenon “metabolic memory” (persistent effects) in a mouse model, which plays an important role in the development of hypertension and vascular remodeling including wall thickness, collagen deposition, infiltration of inflammatory cells and expression of proinflammatory cytokines and oxidative stress (Figs. 1–4). Collectively, these data suggest that a “metabolic memory” retained in the development of hypertension and vascular injury.
Accumulating evidence suggests that vascular injury and remodeling play a major role in the pathogenesis of hypertension in different animal models [27]. One potential mechanism by which Ang II causes this process could involve an increase in oxidative stress-mediated signaling pathways, including MAPK pathway (including ERK, p38 MAPK and JNK), STAT3 and NF-κB [4]. Recent studies have demonstrated that increased ROS is involved in several models of experimental hypertension [34, 35]. Ang II can bind to angiotensin type 1 receptor (AT1R) and then stimulates NADPH oxidases to produce superoxide, which can react with the endogenous vasodilator nitric oxide (NO) thereby resulting in vasoconstriction [34, 35]. Apocynin is a natural organic compound and is used as a NADPH oxidase inhibitor in various cells. It also functions as an antioxidant in endothelial, vascular smooth muscle cells and cardiomyocytes [36, 37] and with antihypertensive effects attenuates Ang II or DOCA-salt hypertension [38]. Although it is known that a persisting activation of NADPH oxidase can mediate the “metabolic memory” in diabetes or cardiac hypertrophy [12, 38], its role in “Ang II
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memory”-mediated hypertension and vascular injury remain unclear. Our present results revealed that inhibitor apocynin significantly suppressed NADPH oxidase activity and ROS production, and attenuated “metabolic memory” induced hypertension and vascular dysfunction (Figs. 1-4). Moreover, apocynin treatment markedly inhibited activation of multiple signaling pathways (JNK1/2, STAT3 and NF-κB), suggesting that “metabolic memory” is linked to persistent activation of NADPH oxidase stimulated by Ang II. 5. Conclusions The present study provides novel evidence for the role of “metabolic memory” of high Ang II in the development of hypertension, vascular injury and dysfunction in a mouse model. Inhibition of NADPH oxidase by apocynin markedly attenuated these effects. Thus, our data for the first time demonstrated that “metabolic memory” of high Ang II contributed vascular injury and hypertension. Persistent activation of NADPH oxidase may be an important event during this process. Use of antioxidant therapy may offer significant benefits on hypertension and its complications. Acknowledgements This study was funded by grants from the 973 program (No. 2012CB517802), China National Natural Science Funds (No. 81330003 and 81400247). Author contributions H.X.W. and H.H.L. were responsible for conception, design and writing the article. W.J.L. did the experiment and analyzed the data. Y.L., J.J.W., Y.L.Z., S.L., and Y.L.X. collected the data. All authors have read and approved the final version of the manuscript. References [1] N.S. Beckett, R. Peters, A.E. Fletcher, et al., Treatment of hypertension in patients 80 years of age or older, N. Engl. J. Med. 358 (18) (2008) 1887–1898. [2] M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, et al., Role of the renin-angiotensin system in vascular diseases: expanding the field, Hypertension 38 (6) (2001) 1382–1387. [3] A.C. Montezano, R.M. Touyz, Oxidative stress, Noxs, and hypertension: experimental evidence and clinical controversies, Ann. Med. 44 (Suppl. 1) (2012) S2–16. [4] A.C. Montezano, A. Nguyen Dinh Cat, F.J. Rios, R.M. Touyz, Angiotensin II and vascular injury, Curr. Hypertens. Rep. 16 (6) (2014) 431. [5] H. Nakaya, H. Sasamura, M. Hayashi, T. Saruta, Temporary treatment of prepubescent rats with angiotensin inhibitors suppresses the development of hypertensive nephrosclerosis, J. Am. Soc. Nephrol. 12 (4) (2001) 659–666. [6] H. Nakaya, H. Sasamura, M. Mifune, et al., Prepubertal treatment with angiotensin receptor blocker causes partial attenuation of hypertension and renal damage in adult Dahl salt-sensitive rats, Nephron 91 (4) (2002) 710–718. [7] A. Holdiness, K. Monahan, D. Minor, R.D. de Shazo, Renin angiotensin aldosterone system blockade: little to no rationale for ACE inhibitor and ARB combinations, Am. J. Med. 124 (1) (2011) 15–19. [8] S. Roy, R. Sala, E. Cagliero, M. Lorenzi, Overexpression of fibronectin induced by diabetes or high glucose: phenomenon with a memory, Proc. Natl. Acad. Sci. U. S. A. 87 (1) (1990) 404–408. [9] M.A. Ihnat, J.E. Thorpe, C.D. Kamat, et al., Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling, Diabetologia 50 (7) (2007) 1523–1531. [10] M.A. Ihnat, J.E. Thorpe, A. Ceriello, Hypothesis: the ‘metabolic memory’, the new challenge of diabetes, Diabet. Med. 24 (6) (2007) 582–586. [11] H. Oguchi, H. Sasamura, K. Shinoda, et al., Renal arteriolar injury by salt intake contributes to salt memory for the development of hypertension, Hypertension 64 (4) (2014) 784–791. [12] H.X. Wang, H. Yang, Q.Y. Han, et al., NADPH oxidases mediate a cellular “memory” of angiotensin II stress in hypertensive cardiac hypertrophy, Free Radic. Biol. Med. 65 (2013) 897–907.
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