Deletion of Soluble Epoxide Hydrolase Attenuates Cardiac Hypertrophy via Down-Regulation of Cardiac Fibroblasts–Derived Fibroblast Growth Factor-2 Huanji Zhang, MD, PhD1,2,3; Tong Wang, MD, PhD1,2; Kun Zhang, MD1,2; Yu Liu, MD1,2; Feifei Huang, MD1,2; Xinhong Zhu, MD, PhD4; Yang Liu, MD1,2; Mong-Heng Wang, PhD5; Wanchun Tang, MD1,6; Jingfeng Wang, MD1,2; Hui Huang, MD, PhD1,2
Objective: Inhibition of soluble epoxide hydrolase (Ephx2) has been shown to play a protective role in cardiac hypertrophy, but the mechanism is not fully understood. We tested the hypothesis that deletion of soluble epoxide hydrolase attenuates cardiac hypertrophy via down-regulation of cardiac fibroblasts–derived fibroblast growth factor-2. Design: Prospective, controlled, and randomized animal study. Setting: University laboratory. Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, China. 2 Guangdong Province Key Laboratory of Arrhythmia and Electrophysiology, Guangzhou, China. 3 Department of Cardiology, Shenzhen Futian Hospital of Guangdong Medical College, Shenzhen, China. 4 Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China. 5 Department of Physiology, Georgia Regents University, Augusta, GA. 6 Weil Institute of Critical Care Medicine, Rancho Mirage, CA. Drs. Zhang, Wang, and Zhang contributed equally to this work. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ ccmjournal). Dr. T. Wang received grant support from the National Natural Science Foundation of China (NSFC) (81070125 and 81270213). Dr. M.-H. Wang received grant support from American Heart Association (AHA) Grant-in-Aid grant (AHASE0054). Dr. J. Wang received grant support from the NSFC (81270212). Dr. Huang received grant support from the NSFC (81170647, 91029742, 81370837, and 30973207), Fok YingTong Education Foundation for Young Teachers in the Higher Education Institutions of China (132030), Yat-Sen Scholarship for Young Scientists, the Science and Technology star of Zhujiang (Guangzhou), and Guangdong Province Key Laboratory of Computational Science at the Sun YatSen University. The remaining authors have disclosed that they do not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected]; [email protected] Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000226 1
Critical Care Medicine
Subjects: Male wild-type C57BL/6 mice and Ephx2 (–/–) mice. Interventions: Male wild-type or Ephx2 (–/–) mice were subjected to transverse aorta constriction surgery. Measurements and Main Results: Four weeks after transverse aorta constriction, Ephx2 (–/–) mice did not develop significant cardiac hypertrophy as that of wild-type mice, indicated by no changes in the ratio of heart weight/body weight and ventricular wall thickness after transverse aorta constriction. Cardiac fibroblast growth factor-2 increased in wild-type-transverse aorta constriction group but this did not change in Ephx2 (–/–)-transverse aorta constriction group, and the serum level of fibroblast growth factor-2 did not change in both groups. In vitro, cardiac fibroblasts were stimulated by angiotensin II to analyze the expression of fibroblast growth factor-2. The effect of increased fibroblast growth factor-2 from cardiac fibroblasts induced by angiotensin II was attenuated by soluble epoxide hydrolase deletion. ERK1/2, p38, and AKT kinase were involved in fibroblast growth factor-2 expression regulated by angiotensin II, and soluble epoxide hydrolase deletion lowered the phosphorylation of ERK1/2 not p38 or AKT to mediate fibroblast growth factor-2 expression. In addition, soluble epoxide hydrolase deletion did not attenuate cardiomyocytes hypertrophy induced by exogenous fibroblast growth factor-2. Conclusions: Our present data demonstrated that deletion of soluble epoxide hydrolase prevented cardiac hypertrophy not only directly to cardiomyocytes but also to cardiac fibroblasts by reducing expression of fibroblast growth factor-2. (Crit Care Med 2014; 42:e345–e354) Key Words: cardiac fibroblast; cardiac hypertrophy; fibroblast growth factor-2; soluble epoxide hydrolase
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response to a variety of extrinsic and intrinsic stimuli such as pressure or volume overload (1) and results in morphological, molecular, and functional changes including hypertrophy of cardiomyocytes (CMs), accumulation of extracellular matrix proteins, reexpression of fetal cardiac genes, and hyperplasia of cardiac fibroblasts (CFBs) (2). CFBs are the most prevalent cell type of nonmyocytes in the heart and are crucially involved in the cardiac hypertrophy process (3–5). The interaction between CFBs and CMs is essential for the progression of cardiac hypertrophy (6). The primary functions of CFBs are to maintain the integrity of cardiac extracellular matrix and generate fibroblast growth factor-2 (FGF-2) which can impact cardiac function and the fate of other cardiac cells (4). CFBs and CMs also synthesize and release FGF-2 in response to a hemodynamic stress (7). The release of FGF-2 results in an autocrine or paracrine effect mediated via FGF receptor signaling to regulate contraction-induced CMs injury and the hypertrophic response in CMs (8, 9). Animal models of cardiac hypertrophy have provided strong evidence that FGF-2 is essential for the manifestation of cardiac hypertrophy induced by pressure overload and angiotensin II (Ang II). It has been demonstrated that Ang II cannot induce hypertrophic response in CMs when CFBs lack FGF-2 expression and have a defective capacity for releasing growth factors (10). But the cell-cell interaction in the heart and local paracrine function of CFBs are not well established. Understanding the functions and mechanisms of CFBs in the development of cardiac hypertrophy is essential to carry out proper management in clinic. This may include prevention or reduction of the hypertrophic response or selective interference with CFBs elements of pathological signal transduction pathways (11, 12). Soluble epoxide hydrolase (Ephx2, sEH) is the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs) to the less bioactive dihydroxyeicosatrienoic acids (13, 14). EETs are produced from arachidonic acid via cytochrome P450 (CYP) epoxygenase (15). It is well known that EETs play an important role in cardiovascular protection by reducing ischemia reperfusion injury (16), producing anti-inflammatory effects, promoting angiogenesis, and reversing cardiac hypertrophy (17, 18). Inhibition or deletion of the sEH stabilizes EETs and enhances the beneficial effects of EETs, which has been demonstrated as a possible treatment for cardiac hypertrophy and high blood pressure (19–21). However, Morgan et al (22) found that sEH inhibition did not prevent cardiac hypertrophy and cardiac dysfunction following transverse aortic constriction (TAC) in rodents. Specifically, several potent and selective sEH inhibitors have been shown to have little to no effects on blood pressure (23, 24). These controversial results might be due to the use of different sEH inhibitors. Furthermore, it is not known whether the antihypertrophic effect of sEH inhibitors is mainly due to the effects of EETs or through the offtarget effects of sEH inhibitors. Although previous work indicated that inhibition of nuclear factor kappaB (NF-κB) was involved in anticardiac hypertrophic effect by the use of sEH inhibitors (21), the mechanism of e346
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sEH in cardiac hypertrophy is not fully understood. In addition, the antihypertrophic effect is not tested in Ephx2 (–/–) mouse model and the interaction between CMs and CFBs mediated by FGF-2 is often ignored. Therefore, in the present study, we try to further determine whether sEH deletion affects cardiac hypertrophy and whether sEH deletion affects CFBs via regulation of CFBs-derived FGF-2.
MATERIALS AND METHODS TAC in Mice Animal experiments were approved by the Committee on Ethics of Animal Experiments and conducted in accordance with the Guidelines for Animal Experiments, Sun Yat-Sen University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). A colony of mice with targeted deletion of the Ephx2 gene was obtained from Jackson Laboratory (Bar Harbor, ME) and maintained in house. These mice had been backcrossed to C57BL/6 mice for at least six generations. Phenotype of Ephx2 (–/–) mice and genotyping identification procedure had been previously described (25). Eight-week-old male Ephx2 (–/–) and wild-type (WT) mice weighing 25–30 g were anesthetized with a mixture of ketamine (8 mg/100 g) and xylazine (1.2 mg/100 g) administered intraperitoneally (26). TAC surgery was performed as described before (27). In sham-operated mice, the entire procedure was identical except for the constriction of the aorta. Four weeks after surgery, echocardiogram was performed by M-mode and 2D measurements. The mice were anesthetized intraperitoneally with 1% pentobarbital (80 mg/kg) to harvest the hearts and blood samples. Neonatal CMs and CFBs Culture and Treatment Primary culture of neonatal mice CMs and CFBs were described previously (28). Briefly, neonatal hearts were collected from 1- to 2-day-old C57BL/6 WT and Ephx2 (–/–) mice; the ventricles were enzymatically digested in 0.05% trypsin for 5 minutes and then in 0.06% collagenase I (GBICO, Grand Island, NY) for 2 hours in a thermostat shaker at 37°C and a speed of 60 rpm. Cells were plated in tissue culture dishes and maintained at 37°C in a 5% Co2 incubator. Afterward, the cells were differentially plated for 45 minutes to remove nonmyocytes. The enriched CMs fractions were cultured on plates, and nonmyocytes were cultured by competitive growth for 3 days to get an overwhelming majority of CFBs. Both cells were maintained in serum-free Dulbecco’s modified Eagle medium/ Ham’s F-12 medium for 24 hours before treatment. Immunohistochemical Staining and Immunofluorescence Confocal Microscopy CMs were stained by the primary antibody of cardiac troponin I (Abcam, Cambridge, UK; ab47003, 1:200), followed by the secondary antibody of goat anti-rabbit. Sliced tissues were stained by the primary antibody of FGF-2 (Santa Cruz Biotechnology, Dallas, TX; 1:50) and the secondary antibody is May 2014 • Volume 42 • Number 5
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horseradish peroxidase (HRP)-conjugated. Immunofluorescence labeling was performed as described previously (29) and imaged using a confocal laser scanning microscopy (Leica TCS SP2, Wetzlar, Germany).
loading control. The protein bands were visualized by the ECL detection system (Thermo, Boston, MA) and the densities of the bands were quantified and normalized against GAPDH by image software (Thermo).
Quantitative Real-Time Polymerase Chain Reaction Total RNA was isolated from hearts or CFBs with TRIzol reagent (Invitrogen, Carlsbad, CA). Quantitative real-time polymerase chain reaction (RT-PCR) of cDNA with the SYBR II Green QPCR system was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The nucleotide sequences of the primers from previous studies were as follows (30, 31): FGF-2: forward 5'-ATCACCTCGCTTCCCGCA-3' and reverse 5'-CTCGTCTTGGCCTTTTGG-3'; atrial natriuretic peptide (ANP): forward 5'-CTCGTCTTGGCCTTTTGG-3' and reverse 5'-TCGGGGAGGGAGCTAAGT-3'; β-myosin heavy chain (MHC): forward 5'-CTACAGGCCTGGGCTTACCT-3' and reverse 5'-TCTCCTTCTCAGACTTCCGC-3'; GAPDH: forward 5'-TATGTCGTGGAGTCTACTGG-3' and reverse 5'-AGTGATGGCATGGACTGTGG-3'.
Enzyme-Linked Immunosorbent Assay for the Quantitative Measurement of Mouse FGF-2 Concentrations in Serum and Cell Supernatants Mice blood was centrifuged at 1,500 rpm for 5 minutes to collect serum, and cell supernatants were collected from six-well plates. The enzyme-linked immunosorbent assay (ELISA) (Abcam, ab100670) employed an antibody specific for mouse FGF-2 coated on a 96-well plate. The procedure followed the company’s instruction.
Western Blot Analysis Cell lysates and heart extracts were resolved by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane; FGF-2 (Santa Cruz Biotechnology; 1:200), total-AKT, total-ERK1/2, total-p38, phospho-AKT, phospho-ERK1/2, and phospho-p38 (all from Cell Signaling Technology, Boston, MA; 1:1,000) proteins were detected by use of antibodies, respectively, and then by an HRPconjugated secondary antibody. The level of GAPDH (Cell Signaling Technology; 1:10,000) protein was also measured as a
Statistical Analysis Data were expressed as mean ± sd from at least three independent experiments. Myocyte cross-sectional area, tissue weights, quantitative RT-PCR of cardiac genes, and protein levels were compared by Student t test. Differences among groups in echocardiographic measurements were compared by analysis of variance test. Statistical differences were considered significant at a p value less than 0.05.
RESULTS sEH Deletion Attenuated Cardiac Hypertrophy in a Murine TAC Model We established a TAC model in male mice after a period of 4 weeks, resulting in a pressure overload–induced cardiac hypertrophy. Before operation, heart weight/body weight (HW/BW) and cardiac function measured by echocardiography were
Figure 1. Inhibition of cardiac hypertrophy in Ephx2 (–/–) mice. A and B, Transverse aorta constriction (TAC) increased heart size in wild-type (WT) group and it was attenuated in Ephx2 (–/–) group (morphology and hematoxylin and eosin). C, Heart weight/body weight ratio (mg/g) increased in WT-TAC mice compared with Ephx2 (–/–)-TAC mice. D, M-mode echocardiography showed evidence of increased wall thickness in WT-TAC mice but not in Ephx2 (–/–)-TAC mice. Data were expressed as mean ± sd (n = 6). *p < 0.01 versus sham-operated group; #p < 0.01 versus WT-TAC group.
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undifferentiated in the WT and Ephx2 (–/–) mice (19). The expected increase in heart size and the ratio of HW/BW were detected in WT-TAC mice (Fig. 1A–C) as previously described, indicating significant cardiac hypertrophy developed in WTTAC mice (32, 33). However, sEH deletion prevented this development of cardiac hypertrophy (Fig. 1A–C). We also assessed the chamber size and ventricular wall thickness in sham-operated, WT-TAC, and Ephx2 (–/–)-TAC mice by echocardiography. As shown in Table 1 and Figure 1D, a significant increase was found in left ventricular anterior and posterior wall thickness both in systolic and diastolic periods. And these effects were inhibited in Ephx2 (–/–)-TAC group. Both WT-TAC and Ephx2 (–/–)-TAC mice had a slight increase in left ventricular ejection fraction comparing with the sham-operated mice. However, no significant difference was found, and this implied that the cardiac function was not affected in fourth week (Table 1). sEH Deletion Down-Regulated Cardiac FGF-2 Expression in TAC We determined the cardiac protein levels of FGF-2 by both immunohistochemical and Western blot analyses. The WT and Ephx2 (–/–) mice showed the undifferentiated baseline in the protein levels of FGF-2 in hearts ( Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/CCM/A842). In sham-operated group, FGF-2 was highly expressed near epicardium and endocardium where CFBs are abundant, indicating that CFBs were the main source of FGF-2 in heart (Fig. 2A). Figure 2B showed the change of FGF-2 levels under higher magnification (40×). The levels of FGF-2 protein in hearts were significantly higher in WT-TAC group but returned to control levels in Ephx2 (–/–)-TAC group (Fig. 2C). We also measured the serum levels of FGF-2 to see whether it was affected by pressure overload. The results showed that FGF-2 did not change after TAC surgery and no significant difference was found among the three groups (Fig. 2D). These data suggested that FGF-2 in heart was up-regulated after TAC, while this only occurred in heart rather than in circulation (34). Table 1.
CFBs-Derived FGF-2 Induced by Ang II Was D ownRegulated by sEH Deletion In Vitro As the above findings indicated down-regulation of cardiac FGF-2 expression by sEH deletion in TAC, we tried to determine if elevated FGF-2 levels and cardiac hypertrophy were mediated by a direct effect of sEH deletion on CFBs. We used RT-PCR analysis to determine messenger RNA (mRNA) levels of FGF-2 after Ang II treatment. CFBs were either untreated or treated with 100 nM Ang II for various periods of time. In WT group, Ang II significantly increased FGF-2 mRNA levels in a time-dependent manner (Fig. 3A), with a maximum effect of 7.5-fold at 8 hours after treatment and return to baseline levels at 24 hours. The effect stimulated by Ang II was attenuated in Ephx2 (–/–) group when compared with that in WT group. We further used ELISA and Western blot analysis to determine the protein levels of FGF-2 expression after Ang II treatment in CFBs and cultivated CFBs supernatant (Fig. 3, B and C). Compared with the Ephx2 (–/–) group, Ang II caused a significant increase in the protein levels of FGF-2 both in CFBs and cultivated CFBs supernatant in WT group. sEH Deletion Did Not Attenuate CMs Hypertrophy Induced Directly by FGF-2 To test whether sEH deletion plays a similar role in FGF-2 induced maladaptive hypertrophy, we determined the response of isolated WT and Ephx2 (–/–) neonatal ventricular CMs by the treatment of FGF-2 at a dose of 50 ng/mL (35). Immunocytochemical and morphometric analysis revealed a significant increase in cell surface area after FGF-2 treatment in both WT and Ephx2 (–/–) groups. But it did not show any significant difference between the two groups (Fig. 4, A and B). We used RT-PCR analysis to determine the expressions of established markers of pathological cardiac hypertrophy after FGF-2 treatment. Expressions of fetal ANP and β-MHC significantly increased after FGF-2 treatment in both WT and Ephx2 (–/–) groups. However, no
Echocardiographic Data of Three Operated Groups After a 4-Week Follow-Up Ephx2(–/–) (n = 6)
Sham-Operated (n = 6)
Wild-Type-Transverse Aorta Constriction (n = 6)
Left ventricular anterior wall thickness in diastole (mm)
0.80 ± 0.09
1.04 ± 0.20a
0.82 ± 0.08b
Left ventricular anterior wall thickness in systole (mm)
1.00 ± 0.15
1.40 ± 0.22a
1.06 ± 0.14b
Left ventricular posterior wall thickness in diastole (mm)
0.72 ± 0.07
0.90 ± 0.05a
0.80 ± 0.08b
Left ventricular posterior wall thickness in systole (mm)
0.93 ± 0.15
1.22 ± 0.16a
0.93 ± 0.09b
Echocardiographic Assessment
Left ventricular mass (mg) left ventricular ejection fraction (%) Left ventricular internal dimension at end diastole (mm)
109.64 ± 25.40
155.02 ± 27.07a
116.03 ± 12.49b
46.83 ± 8.68
52.51 ± 7.60
47.80 ± 9.22
3.55 ± 0.28
4.13 ± 0.39
a
3.48 ± 0.32b
p < 0.05 versus sham-operated. p < 0.05 versus wild-type-transverse aorta constriction.
a
b
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Figure 2. Soluble epoxide hydrolase deletion down-regulated fibroblast growth factor (FGF)-2 expression in mouse transverse aorta constriction (TAC) model. A and B, Immunochemistry staining result showed that FGF-2 was highly expressed near epicardium and endocardium. In wild-type (WT)-TAC group, the FGF-2 was up-regulated significantly, but reduced in Ephx2 (–/–)-TAC group. B, The change of FGF-2 under higher magnifications (40×). C, Western blot result showed that cardiac FGF-2 was increased in WT-TAC group while attenuated in Ephx2 (–/–)-TAC group. D, Enzyme-linked immunosorbent assay result of serum FGF-2 showed no change of FGF-2 levels among three groups. *p < 0.05 versus sham-operated group; #p < 0.05 versus WT-TAC group. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
significant difference was found between these two groups (Fig. 4, C and D). These data showed that sEH deletion did not have the similar effect to attenuate cardiac hypertrophy induced by FGF-2 as induced by Ang II. sEH Deletion Down-Regulated CFB-Derived FGF-2 by Reducing the Phosphorylation of ERK1/2 Signal Pathway To understand the signal pathways involved in the synthesis of FGF-2, we used inhibitors of U0126 to ERK1/2, SB203580 to p38, and A6730 to AKT, respectively. After 24-hour stimulation with Ang II in WT CFBs, the expressions of FGF-2 were significantly elevated. However, when pretreated with U0126, SB203580, and A6730 for 1 hour, the up-regulation of FGF-2 induced by Ang II was significantly attenuated Critical Care Medicine
(Fig. 5A). The comparison of the effects among these three inhibitors showed that U0126 was the most effective inhibitor to block the synthesis of FGF-2 comparing with SB203580 and A6730 (Fig. 5A). Ang II increased phosphorylation of ERK1/2, p38, and AKT in CFBs in 1 hour (Fig. 5B–D). These results suggested that ERK1/2, p38, and AKT signal pathways were involved in the expression of FGF-2 mediated by Ang II. Furthermore, we compared the phosphorylation degree of ERK1/2, p38, and AKT stimulated by Ang II in CFBs between WT group and Ephx2 (–/–) group. The results showed that sEH deletion lowered the phosphorylation of ERK1/2 significantly (Fig. 5B). But no significant difference was found in p38 and AKT phosphorylation between the two groups (Fig. 5, C and D). www.ccmjournal.org
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Figure 3. Cardiac fibroblasts (CFBs)-derived fibroblast growth factor (FGF)-2 induced by angiotensin II (Ang II) was down-regulated by soluble epoxide hydrolase deletion in vitro. A, Real-time polymerase chain reaction result showed Ang II increased FGF-2 messenger RNA (mRNA) expression significantly. The elevation of FGF-2 was much higher in wild-type (WT) group than that in Ephx2 (–/–) group after Ang II stimulation. B and C, In CFBs and CFBs supernatant, Western blot and enzyme-linked immunosorbent assay results showed that Ang II induced a significant elevation in the protein levels of FGF-2 in WT group while attenuated in Ephx2 (–/–) group after Ang II stimulation. *p < 0.05 versus control; #p < 0.05 versus WT. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
DISCUSSION Our major findings were as follows: 1) Deletion of sEH prevented cardiac hypertrophy in a murine TAC model by down-regulation of the expression of FGF-2 in CFBs. 2) The expression of FGF-2 in CFBs was mediated by ERK1/2, p38, and AKT signal pathways. 3) The hypertrophic effect in CMs induced by FGF-2 was not attenuated by the deletion of sEH. 4) Deletion of sEH down-regulated the expression of FGF-2 in CFBs by lowering phosphorylation of ERK1/2. The mechanisms of cardiac hypertrophy are very complicated, in which multiple pathways, including Ang II and norepinephrine-mediated pathways, are involved (36). The detailed mechanism of antihypertrophy by sEH deletion is still unclear. The possible reasons for the protective effects by sEH deletion may be explained as follows. First, deletion of sEH lowers the blood pressure. Sinal et al (19) found that the mean systolic blood pressure was significantly decreased in Ephx2 (–/–) mice than in WT mice. Imig et al (20) discovered that sEH inhibition lowered arterial blood pressure in Ang II-induced hypertension. So, in Ephx2 (–/–)-TAC model, the improvement of cardiac hypertrophy may partly attribute e350
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to the reduction of blood pressure. Second, EETs play an anti-inflammatory role in TAC model in which the inflammatory response is activated. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokineinduced endothelial cell adhesion molecule expression and leukocyte adhesion to the vascular wall through the inhibition of transcription factor NF-κB and IkappaB kinase (37–39). Growth factors and cytokines are highly synthesized by different cells in heart during TAC because the local renin-angiotensin system is activated (40, 41). The cell-cell communication in cardiac hypertrophy is required for induction of hypertrophic responses in CMs (42). As we know, CFBs play an important role in regulating cardiac hypertrophy and cardiac function (3, 43). CFBs also have numerous other functions, including synthesis and deposition of extracellular matrix, secretion of growth factors and cytokines, and cell-cell communication with CMs (5). FGF-2 is a growth factor expressed both in CMs and CFBs. Our study showed that FGF-2 was highly expressed in epicardium and endocardium where CFBs are abundant, implying that CFBs were the main resource of FGF-2 in heart (44). This result was consistent with May 2014 • Volume 42 • Number 5
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Figure 4. Cardiomyocytes (CMs) hypertrophy induced by fibroblast growth factor (FGF)-2 was not attenuated by soluble epoxide hydrolase deletion. A, Surface area of isolated neonatal mouse CMs increased after FGF-2 treatment (50 ng/mL), as revealed by confocal immunocytochemical analysis with antibody to cardiac troponin I (green) and 4',6-diamidino-2-phenylindole (blue) to nuclei. B, Compared with that of untreated CMs, treatment with FGF-2 for 24 hr significantly increased cell surface area analyzed by morphometry. C and D, After FGF-2 treatment, the expressions of atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) messenger RNA (mRNA) were increased in CMs (mean ± sd; n = 3). *p < 0.05 versus control. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
previous information on the function of FGF-2 in epicardial and myocardial development (45, 46). Animal models have provided evidence that FGF-2 is essential for the manifestation of cardiac hypertrophy induced by pressure overload and Ang II (10, 47). However, due to the limitations that only low molecular weight (LMW) of the recombinant FGF-2 was available and FGF-2 antibody in the ELISA kit was nonspecific to recognize its isoforms, we did not distinguish the roles of individual isoforms of FGF-2 as many other investigations confirming the role of FGF-2 in cardiac hypertrophy (8, 35, 46). Recently, Kardami et al (12) suggested that cardiac hypertrophy was only associated with high molecular weight (HMW) rather than the LMW isoform of FGF-2. Both HMW FGF-2 and LMW FGF-2 have similar paracrine effects on proliferation, but HMW FGF-2 exerts a distinct intracrine effect on binucleation (48), which may be due to the different models of animals (46). Further investigation remains to be determined regarding the role of sEH deletion on the individual isoforms of FGF-2 in cardiac hypertrophy. Critical Care Medicine
In our study, Ang II directly stimulated the expression of FGF-2 from CFBs by activating phosphorylation of ERK1/2, AKT, and p38. The results demonstrated that FGF-2 was mediated via multiple signal pathways. However, we did not find that deletion of sEH reduced the phosphorylation of p38 and AKT except for ERK1/2, which conflicted with the previous studies that EETs activated ERK1/2 and phosphatidylinositol 3-kinase/AKT signal pathways (49, 50). The possible explanation is that deletion of sEH in vitro or in vivo has multiple cellular functions besides elevation of EETs (25). In addition, the sEH is a bifunctional enzyme with epoxide hydrolase and phosphatase activities, genetic deletion of sEH removes both activities, whereas EETs have relatively simple activity and may not reflect the whole function of sEH in vivo. More studies are needed to explore this problem. Our results also showed the lack of correlation between the serum levels of FGF-2 and the cardiac hypertrophy. This was in concordance with other reports describing the lack of association between the serum FGF-2 levels and left ventricular mass www.ccmjournal.org
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Figure 5. Soluble epoxide hydrolase deletion decreased angiotensin II (Ang II)-induced fibroblast growth factor (FGF)-2 in cardiac fibroblasts (CFBs) by reducing phosphorylation of ERK1/2. A, Pretreatment with U0126, SB203580, and A6730 for 1 hr attenuated the increase of wild-type (WT) C FBsderived FGF-2 induced by Ang II. B, The comparison between the WT and Ephx2 (–/–) groups after Ang II treatment. The phospho-ERK1/2 expressed higher in WT group than that in Ephx2 (–/–) group. C, The comparison of phosphorylation of p38 between the WT and Ephx2 (–/–) groups after Ang II treatment. No significant difference of phosphorylation of p38 was found between the two groups. D, The comparison of phosphorylation of AKT between the WT and Ephx2 (–/–) groups after Ang II treatment. No significant difference of phosphorylation of AKT was found between the two groups. Data were expressed as mean ± sd (n = 3). *p < 0.05 versus control; #p < 0.05 versus Ang II (individual treatment); **p < 0.05 versus SB203580 and A6730. KO = knockout, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
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in hypertensive patients (44, 51, 52). This might be due to the reason that FGF-2 acts mainly in the local area of its expression. The serum levels may not reflect the local concentration of FGF-2 in the heart. Meanwhile, we demonstrated that deletion of sEH did not prevent CMs hypertrophy induced by FGF2, indicating that deletion of sEH may only partly be involved in the process of cardiac hypertrophy or the upstream effect of FGF-2. In addition, in our study, although the cardiac function of WT-TAC and Ephx2 (–/–)-TAC groups was found to have a tendency of increase compared with that of sham-operated group, no significant difference was found among these groups which may be because of the relative low degree and the limited time of aortic constriction.
LIMITATIONS Although we found that deletion of sEH attenuated cardiac hypertrophy via down-regulation of CFBs-derived FGF-2, there are still several limitations in this study. First, we did not treat CMs with Ang II and observe the changes of FGF-2 so as to reveal the intracrine biological effect of FGF-2 directly. Second, the CFBs were not treated with the inhibitor of ERK, p38, and AKT alone which may influence the expressions of FGF-2 in some cases. Furthermore, we only explored the limited signal pathways, and the possible signaling mechanisms involved in the regulation of FGF-2 in CFBs need further study. In summary, our present data suggested that deletion of sEH not only prevented cardiac hypertrophy directly to CMs but also reduced expression of FGF-2 from CFBs. The present study provides a new evidence to understand the role of sEH in cardiac hypertrophy and uncovers the essential interaction between CFBs and CMs for the progression of cardiac hypertrophy. In future study, we will further explore the role of sEH in the cell-cell interaction in cardiac hypertrophy.
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1. Frey N, Katus HA, Olson EN, et al: Hypertrophy of the heart: A new therapeutic target? Circulation 2004; 109:1580–1589 2. Frey N, Olson EN: Cardiac hypertrophy: The good, the bad, and the ugly. Annu Rev Physiol 2003; 65:45–79 3. Porter KE, Turner NA: Cardiac fibroblasts: At the heart of myocardial remodeling. Pharmacol Ther 2009; 123:255–278 4. Fan D, Takawale A, Lee J, et al: Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in heart disease. Fibrogenesis Tissue Repair 2012; 5:15 5. Souders CA, Bowers SL, Baudino TA: Cardiac fibroblast: The renaissance cell. Circ Res 2009; 105:1164–1176 6. Manabe I, Shindo T, Nagai R: Gene expression in fibroblasts and fibrosis: Involvement in cardiac hypertrophy. Circ Res 2002; 91:1103–1113 7. Beenken A, Mohammadi M: The FGF family: Biology, pathophysiology and therapy. Nat Rev Drug Discov 2009; 8:235–253 8. Kaye D, Pimental D, Prasad S, et al: Role of transiently altered sarcolemmal membrane permeability and basic fibroblast growth factor release in the hypertrophic response of adult rat ventricular myocytes to increased mechanical activity in vitro. J Clin Invest 1996; 97:281–291 9. Clarke MS, Caldwell RW, Chiao H, et al: Contraction-induced cell wounding and release of fibroblast growth factor in heart. Circ Res 1995; 76:927–934
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10. Schultz JE, Witt SA, Nieman ML, et al: Fibroblast growth factor-2 mediates pressure-induced hypertrophic response. J Clin Invest 1999; 104:709–719 11. Hunter JJ, Chien KR: Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 1999; 341:1276–1283 12. Kardami E, Jiang ZS, Jimenez SK, et al: Fibroblast growth factor 2 isoforms and cardiac hypertrophy. Cardiovasc Res 2004; 63: 458–466 13. Wang YX, Ulu A, Zhang LN, et al: Soluble epoxide hydrolase in atherosclerosis. Curr Atheroscler Rep 2010; 12:174–183 14. Wang ZH, Davis BB, Jiang DQ, et al: Soluble epoxide hydrolase inhibitors and cardiovascular diseases. Curr Vasc Pharmacol 2013; 11:105–111 15. Yu Z, Xu F, Huse LM, et al: Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids. Circ Res 2000; 87:992–998 16. Kompa AR, Wang BH, Xu G, et al: Soluble epoxide hydrolase inhibition exerts beneficial anti-remodeling actions post-myocardial infarction. Int J Cardiol 2013; 167:210–219 17. Spector AA, Fang X, Snyder GD, et al: Epoxyeicosatrienoic acids (EETs): Metabolism and biochemical function. Prog Lipid Res 2004; 43:55–90 18. Spector AA: Arachidonic acid cytochrome P450 epoxygenase pathway. J Lipid Res 2009; 50(Suppl):S52–S56 19. Sinal CJ, Miyata M, Tohkin M, et al: Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation. J Biol Chem 2000; 275:40504–40510 20. Imig JD, Zhao X, Capdevila JH, et al: Soluble epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension. Hypertension 2002; 39:690–694 21. Xu D, Li N, He Y, et al: Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors. Proc Natl Acad Sci U S A 2006; 103:18733–18738 22. Morgan LA, Olzinski AR, Upson JJ, et al: Soluble epoxide hydrolase inhibition does not prevent cardiac remodeling and dysfunction following aortic constriction in rats and mice. J Cardiovasc Pharmacol 2013; 61:291–301 23. Shen HC, Ding FX, Deng Q, et al: Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas as highly potent soluble epoxide hydrolase inhibitors. Bioorg Med Chem Lett 2009; 19:5314–5320 24. Shen HC, Ding FX, Wang S, et al: Discovery of spirocyclic secondary amine-derived tertiary ureas as highly potent, selective and bioavailable soluble epoxide hydrolase inhibitors. Bioorg Med Chem Lett 2009; 19:3398–3404 25. Zhang W, Otsuka T, Sugo N, et al: Soluble epoxide hydrolase gene deletion is protective against experimental cerebral ischemia. Stroke 2008; 39:2073–2078 26. Wang D, Oparil S, Feng JA, et al: Effects of pressure overload on extracellular matrix expression in the heart of the atrial natriuretic peptide-null mouse. Hypertension 2003; 42:88–95 27. deAlmeida AC, van Oort RJ, Wehrens XH: Transverse aortic constriction in mice. J Vis Exp 2010; 38:pii:1729 28. Xu Y, Zhang Z, Timofeyev V, et al: The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: Novel insights from genetically altered mice. J Physiol 2005; 562:745–758 29. Savergnini SQ, Ianzer D, Carvalho MB, et al: The novel Mas agonist, CGEN-856S, attenuates isoproterenol-induced cardiac remodeling and myocardial infarction injury in rats. PLoS One 2013; 8:e57757 30. Kiprianova I, Schindowski K, von Bohlen und Halbach O, et al: Enlarged infarct volume and loss of BDNF mRNA induction following brain ischemia in mice lacking FGF-2. Exp Neurol 2004; 189:252–260 31. Bernardi S, Burns WC, Toffoli B, et al: Angiotensin-converting enzyme 2 regulates renal atrial natriuretic peptide through angiotensin-(1-7). Clin Sci (Lond) 2012; 123:29–37 32. Kolwicz SC Jr, Olson DP, Marney LC, et al: Cardiac-specific deletion of acetyl CoA carboxylase 2 prevents metabolic remodeling during pressure-overload hypertrophy. Circ Res 2012; 111:728–738 www.ccmjournal.org
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44. Santiago JJ, Ma X, McNaughton LJ, et al: Preferential accumulation and export of high molecular weight FGF-2 by rat cardiac non-myocytes. Cardiovasc Res 2011; 89:139–147 45. Lavine KJ, Ornitz DM: Fibroblast growth factors and Hedgehogs: At the heart of the epicardial signaling center. Trends Genet 2008; 24:33–40 46. Liao S, Bodmer J, Pietras D, et al: Biological functions of the low and high molecular weight protein isoforms of fibroblast growth factor-2 in cardiovascular development and disease. Dev Dyn 2009; 238:249–264 47. Pellieux C, Foletti A, Peduto G, et al: Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2. J Clin Invest 2001; 108:1843–1851 48. Pasumarthi KB, Kardami E, Cattini PA: High and low molecular weight fibroblast growth factor-2 increase proliferation of neonatal rat cardiac myocytes but have differential effects on binucleation and nuclear morphology. Evidence for both paracrine and intracrine actions of fibroblast growth factor-2. Circ Res 1996; 78:126–136 49. Zhang K, Wang J, Zhang H, et al: Mechanisms of epoxyeicosatrienoic acids to improve cardiac remodeling in chronic renal failure disease. Eur J Pharmacol 2013; 701:33–39 50. Zhang B, Cao H, Rao GN: Fibroblast growth factor-2 is a downstream mediator of phosphatidylinositol 3-kinase-Akt signaling in 14,15-epoxyeicosatrienoic acid-induced angiogenesis. J Biol Chem 2006; 281:905–914 51. Kieć-Wilk B, Stolarz-Skrzypek K, Sliwa A, et al: Peripheral blood concentrations of TGFβ1, IGF-1 and bFGF and remodelling of the left ventricle and blood vessels in hypertensive patients. Kardiol Pol 2010; 68:996–1002 52. Migliore T, Parrella LS, Caputi A, et al: Pathogenesis of hypertrophic cardiomyopathy. Impact of growth factors on left ventricular anatomy. Minerva Cardioangiol 2008; 56:13–20
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Objective: Inhibition of soluble epoxide hydrolase (Ephx2) has been shown to play a protective role in cardiac hypertrophy, but the mechanism is not fully understood. We tested the hypothesis that deletion of soluble epoxide hydrolase attenuates cardiac hypertrophy via down-regulation of cardiac fibroblasts–derived fibroblast growth factor-2. Design: Prospective, controlled, and randomized animal study. Setting: University laboratory. Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, China. 2 Guangdong Province Key Laboratory of Arrhythmia and Electrophysiology, Guangzhou, China. 3 Department of Cardiology, Shenzhen Futian Hospital of Guangdong Medical College, Shenzhen, China. 4 Department of Neurobiology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, China. 5 Department of Physiology, Georgia Regents University, Augusta, GA. 6 Weil Institute of Critical Care Medicine, Rancho Mirage, CA. Drs. Zhang, Wang, and Zhang contributed equally to this work. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ ccmjournal). Dr. T. Wang received grant support from the National Natural Science Foundation of China (NSFC) (81070125 and 81270213). Dr. M.-H. Wang received grant support from American Heart Association (AHA) Grant-in-Aid grant (AHASE0054). Dr. J. Wang received grant support from the NSFC (81270212). Dr. Huang received grant support from the NSFC (81170647, 91029742, 81370837, and 30973207), Fok YingTong Education Foundation for Young Teachers in the Higher Education Institutions of China (132030), Yat-Sen Scholarship for Young Scientists, the Science and Technology star of Zhujiang (Guangzhou), and Guangdong Province Key Laboratory of Computational Science at the Sun YatSen University. The remaining authors have disclosed that they do not have any potential conflicts of interest. For information regarding this article, E-mail: [email protected]; [email protected] Copyright © 2014 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins DOI: 10.1097/CCM.0000000000000226 1
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Subjects: Male wild-type C57BL/6 mice and Ephx2 (–/–) mice. Interventions: Male wild-type or Ephx2 (–/–) mice were subjected to transverse aorta constriction surgery. Measurements and Main Results: Four weeks after transverse aorta constriction, Ephx2 (–/–) mice did not develop significant cardiac hypertrophy as that of wild-type mice, indicated by no changes in the ratio of heart weight/body weight and ventricular wall thickness after transverse aorta constriction. Cardiac fibroblast growth factor-2 increased in wild-type-transverse aorta constriction group but this did not change in Ephx2 (–/–)-transverse aorta constriction group, and the serum level of fibroblast growth factor-2 did not change in both groups. In vitro, cardiac fibroblasts were stimulated by angiotensin II to analyze the expression of fibroblast growth factor-2. The effect of increased fibroblast growth factor-2 from cardiac fibroblasts induced by angiotensin II was attenuated by soluble epoxide hydrolase deletion. ERK1/2, p38, and AKT kinase were involved in fibroblast growth factor-2 expression regulated by angiotensin II, and soluble epoxide hydrolase deletion lowered the phosphorylation of ERK1/2 not p38 or AKT to mediate fibroblast growth factor-2 expression. In addition, soluble epoxide hydrolase deletion did not attenuate cardiomyocytes hypertrophy induced by exogenous fibroblast growth factor-2. Conclusions: Our present data demonstrated that deletion of soluble epoxide hydrolase prevented cardiac hypertrophy not only directly to cardiomyocytes but also to cardiac fibroblasts by reducing expression of fibroblast growth factor-2. (Crit Care Med 2014; 42:e345–e354) Key Words: cardiac fibroblast; cardiac hypertrophy; fibroblast growth factor-2; soluble epoxide hydrolase
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ardiac hypertrophy is an important cardiovascular pathological change that increases the risk of heart failure and cardiac death. It is a compensatory www.ccmjournal.org
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response to a variety of extrinsic and intrinsic stimuli such as pressure or volume overload (1) and results in morphological, molecular, and functional changes including hypertrophy of cardiomyocytes (CMs), accumulation of extracellular matrix proteins, reexpression of fetal cardiac genes, and hyperplasia of cardiac fibroblasts (CFBs) (2). CFBs are the most prevalent cell type of nonmyocytes in the heart and are crucially involved in the cardiac hypertrophy process (3–5). The interaction between CFBs and CMs is essential for the progression of cardiac hypertrophy (6). The primary functions of CFBs are to maintain the integrity of cardiac extracellular matrix and generate fibroblast growth factor-2 (FGF-2) which can impact cardiac function and the fate of other cardiac cells (4). CFBs and CMs also synthesize and release FGF-2 in response to a hemodynamic stress (7). The release of FGF-2 results in an autocrine or paracrine effect mediated via FGF receptor signaling to regulate contraction-induced CMs injury and the hypertrophic response in CMs (8, 9). Animal models of cardiac hypertrophy have provided strong evidence that FGF-2 is essential for the manifestation of cardiac hypertrophy induced by pressure overload and angiotensin II (Ang II). It has been demonstrated that Ang II cannot induce hypertrophic response in CMs when CFBs lack FGF-2 expression and have a defective capacity for releasing growth factors (10). But the cell-cell interaction in the heart and local paracrine function of CFBs are not well established. Understanding the functions and mechanisms of CFBs in the development of cardiac hypertrophy is essential to carry out proper management in clinic. This may include prevention or reduction of the hypertrophic response or selective interference with CFBs elements of pathological signal transduction pathways (11, 12). Soluble epoxide hydrolase (Ephx2, sEH) is the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs) to the less bioactive dihydroxyeicosatrienoic acids (13, 14). EETs are produced from arachidonic acid via cytochrome P450 (CYP) epoxygenase (15). It is well known that EETs play an important role in cardiovascular protection by reducing ischemia reperfusion injury (16), producing anti-inflammatory effects, promoting angiogenesis, and reversing cardiac hypertrophy (17, 18). Inhibition or deletion of the sEH stabilizes EETs and enhances the beneficial effects of EETs, which has been demonstrated as a possible treatment for cardiac hypertrophy and high blood pressure (19–21). However, Morgan et al (22) found that sEH inhibition did not prevent cardiac hypertrophy and cardiac dysfunction following transverse aortic constriction (TAC) in rodents. Specifically, several potent and selective sEH inhibitors have been shown to have little to no effects on blood pressure (23, 24). These controversial results might be due to the use of different sEH inhibitors. Furthermore, it is not known whether the antihypertrophic effect of sEH inhibitors is mainly due to the effects of EETs or through the offtarget effects of sEH inhibitors. Although previous work indicated that inhibition of nuclear factor kappaB (NF-κB) was involved in anticardiac hypertrophic effect by the use of sEH inhibitors (21), the mechanism of e346
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sEH in cardiac hypertrophy is not fully understood. In addition, the antihypertrophic effect is not tested in Ephx2 (–/–) mouse model and the interaction between CMs and CFBs mediated by FGF-2 is often ignored. Therefore, in the present study, we try to further determine whether sEH deletion affects cardiac hypertrophy and whether sEH deletion affects CFBs via regulation of CFBs-derived FGF-2.
MATERIALS AND METHODS TAC in Mice Animal experiments were approved by the Committee on Ethics of Animal Experiments and conducted in accordance with the Guidelines for Animal Experiments, Sun Yat-Sen University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). A colony of mice with targeted deletion of the Ephx2 gene was obtained from Jackson Laboratory (Bar Harbor, ME) and maintained in house. These mice had been backcrossed to C57BL/6 mice for at least six generations. Phenotype of Ephx2 (–/–) mice and genotyping identification procedure had been previously described (25). Eight-week-old male Ephx2 (–/–) and wild-type (WT) mice weighing 25–30 g were anesthetized with a mixture of ketamine (8 mg/100 g) and xylazine (1.2 mg/100 g) administered intraperitoneally (26). TAC surgery was performed as described before (27). In sham-operated mice, the entire procedure was identical except for the constriction of the aorta. Four weeks after surgery, echocardiogram was performed by M-mode and 2D measurements. The mice were anesthetized intraperitoneally with 1% pentobarbital (80 mg/kg) to harvest the hearts and blood samples. Neonatal CMs and CFBs Culture and Treatment Primary culture of neonatal mice CMs and CFBs were described previously (28). Briefly, neonatal hearts were collected from 1- to 2-day-old C57BL/6 WT and Ephx2 (–/–) mice; the ventricles were enzymatically digested in 0.05% trypsin for 5 minutes and then in 0.06% collagenase I (GBICO, Grand Island, NY) for 2 hours in a thermostat shaker at 37°C and a speed of 60 rpm. Cells were plated in tissue culture dishes and maintained at 37°C in a 5% Co2 incubator. Afterward, the cells were differentially plated for 45 minutes to remove nonmyocytes. The enriched CMs fractions were cultured on plates, and nonmyocytes were cultured by competitive growth for 3 days to get an overwhelming majority of CFBs. Both cells were maintained in serum-free Dulbecco’s modified Eagle medium/ Ham’s F-12 medium for 24 hours before treatment. Immunohistochemical Staining and Immunofluorescence Confocal Microscopy CMs were stained by the primary antibody of cardiac troponin I (Abcam, Cambridge, UK; ab47003, 1:200), followed by the secondary antibody of goat anti-rabbit. Sliced tissues were stained by the primary antibody of FGF-2 (Santa Cruz Biotechnology, Dallas, TX; 1:50) and the secondary antibody is May 2014 • Volume 42 • Number 5
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horseradish peroxidase (HRP)-conjugated. Immunofluorescence labeling was performed as described previously (29) and imaged using a confocal laser scanning microscopy (Leica TCS SP2, Wetzlar, Germany).
loading control. The protein bands were visualized by the ECL detection system (Thermo, Boston, MA) and the densities of the bands were quantified and normalized against GAPDH by image software (Thermo).
Quantitative Real-Time Polymerase Chain Reaction Total RNA was isolated from hearts or CFBs with TRIzol reagent (Invitrogen, Carlsbad, CA). Quantitative real-time polymerase chain reaction (RT-PCR) of cDNA with the SYBR II Green QPCR system was performed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The nucleotide sequences of the primers from previous studies were as follows (30, 31): FGF-2: forward 5'-ATCACCTCGCTTCCCGCA-3' and reverse 5'-CTCGTCTTGGCCTTTTGG-3'; atrial natriuretic peptide (ANP): forward 5'-CTCGTCTTGGCCTTTTGG-3' and reverse 5'-TCGGGGAGGGAGCTAAGT-3'; β-myosin heavy chain (MHC): forward 5'-CTACAGGCCTGGGCTTACCT-3' and reverse 5'-TCTCCTTCTCAGACTTCCGC-3'; GAPDH: forward 5'-TATGTCGTGGAGTCTACTGG-3' and reverse 5'-AGTGATGGCATGGACTGTGG-3'.
Enzyme-Linked Immunosorbent Assay for the Quantitative Measurement of Mouse FGF-2 Concentrations in Serum and Cell Supernatants Mice blood was centrifuged at 1,500 rpm for 5 minutes to collect serum, and cell supernatants were collected from six-well plates. The enzyme-linked immunosorbent assay (ELISA) (Abcam, ab100670) employed an antibody specific for mouse FGF-2 coated on a 96-well plate. The procedure followed the company’s instruction.
Western Blot Analysis Cell lysates and heart extracts were resolved by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane; FGF-2 (Santa Cruz Biotechnology; 1:200), total-AKT, total-ERK1/2, total-p38, phospho-AKT, phospho-ERK1/2, and phospho-p38 (all from Cell Signaling Technology, Boston, MA; 1:1,000) proteins were detected by use of antibodies, respectively, and then by an HRPconjugated secondary antibody. The level of GAPDH (Cell Signaling Technology; 1:10,000) protein was also measured as a
Statistical Analysis Data were expressed as mean ± sd from at least three independent experiments. Myocyte cross-sectional area, tissue weights, quantitative RT-PCR of cardiac genes, and protein levels were compared by Student t test. Differences among groups in echocardiographic measurements were compared by analysis of variance test. Statistical differences were considered significant at a p value less than 0.05.
RESULTS sEH Deletion Attenuated Cardiac Hypertrophy in a Murine TAC Model We established a TAC model in male mice after a period of 4 weeks, resulting in a pressure overload–induced cardiac hypertrophy. Before operation, heart weight/body weight (HW/BW) and cardiac function measured by echocardiography were
Figure 1. Inhibition of cardiac hypertrophy in Ephx2 (–/–) mice. A and B, Transverse aorta constriction (TAC) increased heart size in wild-type (WT) group and it was attenuated in Ephx2 (–/–) group (morphology and hematoxylin and eosin). C, Heart weight/body weight ratio (mg/g) increased in WT-TAC mice compared with Ephx2 (–/–)-TAC mice. D, M-mode echocardiography showed evidence of increased wall thickness in WT-TAC mice but not in Ephx2 (–/–)-TAC mice. Data were expressed as mean ± sd (n = 6). *p < 0.01 versus sham-operated group; #p < 0.01 versus WT-TAC group.
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undifferentiated in the WT and Ephx2 (–/–) mice (19). The expected increase in heart size and the ratio of HW/BW were detected in WT-TAC mice (Fig. 1A–C) as previously described, indicating significant cardiac hypertrophy developed in WTTAC mice (32, 33). However, sEH deletion prevented this development of cardiac hypertrophy (Fig. 1A–C). We also assessed the chamber size and ventricular wall thickness in sham-operated, WT-TAC, and Ephx2 (–/–)-TAC mice by echocardiography. As shown in Table 1 and Figure 1D, a significant increase was found in left ventricular anterior and posterior wall thickness both in systolic and diastolic periods. And these effects were inhibited in Ephx2 (–/–)-TAC group. Both WT-TAC and Ephx2 (–/–)-TAC mice had a slight increase in left ventricular ejection fraction comparing with the sham-operated mice. However, no significant difference was found, and this implied that the cardiac function was not affected in fourth week (Table 1). sEH Deletion Down-Regulated Cardiac FGF-2 Expression in TAC We determined the cardiac protein levels of FGF-2 by both immunohistochemical and Western blot analyses. The WT and Ephx2 (–/–) mice showed the undifferentiated baseline in the protein levels of FGF-2 in hearts ( Supplemental Fig. 1, Supplemental Digital Content 1, http://links.lww.com/CCM/A842). In sham-operated group, FGF-2 was highly expressed near epicardium and endocardium where CFBs are abundant, indicating that CFBs were the main source of FGF-2 in heart (Fig. 2A). Figure 2B showed the change of FGF-2 levels under higher magnification (40×). The levels of FGF-2 protein in hearts were significantly higher in WT-TAC group but returned to control levels in Ephx2 (–/–)-TAC group (Fig. 2C). We also measured the serum levels of FGF-2 to see whether it was affected by pressure overload. The results showed that FGF-2 did not change after TAC surgery and no significant difference was found among the three groups (Fig. 2D). These data suggested that FGF-2 in heart was up-regulated after TAC, while this only occurred in heart rather than in circulation (34). Table 1.
CFBs-Derived FGF-2 Induced by Ang II Was D ownRegulated by sEH Deletion In Vitro As the above findings indicated down-regulation of cardiac FGF-2 expression by sEH deletion in TAC, we tried to determine if elevated FGF-2 levels and cardiac hypertrophy were mediated by a direct effect of sEH deletion on CFBs. We used RT-PCR analysis to determine messenger RNA (mRNA) levels of FGF-2 after Ang II treatment. CFBs were either untreated or treated with 100 nM Ang II for various periods of time. In WT group, Ang II significantly increased FGF-2 mRNA levels in a time-dependent manner (Fig. 3A), with a maximum effect of 7.5-fold at 8 hours after treatment and return to baseline levels at 24 hours. The effect stimulated by Ang II was attenuated in Ephx2 (–/–) group when compared with that in WT group. We further used ELISA and Western blot analysis to determine the protein levels of FGF-2 expression after Ang II treatment in CFBs and cultivated CFBs supernatant (Fig. 3, B and C). Compared with the Ephx2 (–/–) group, Ang II caused a significant increase in the protein levels of FGF-2 both in CFBs and cultivated CFBs supernatant in WT group. sEH Deletion Did Not Attenuate CMs Hypertrophy Induced Directly by FGF-2 To test whether sEH deletion plays a similar role in FGF-2 induced maladaptive hypertrophy, we determined the response of isolated WT and Ephx2 (–/–) neonatal ventricular CMs by the treatment of FGF-2 at a dose of 50 ng/mL (35). Immunocytochemical and morphometric analysis revealed a significant increase in cell surface area after FGF-2 treatment in both WT and Ephx2 (–/–) groups. But it did not show any significant difference between the two groups (Fig. 4, A and B). We used RT-PCR analysis to determine the expressions of established markers of pathological cardiac hypertrophy after FGF-2 treatment. Expressions of fetal ANP and β-MHC significantly increased after FGF-2 treatment in both WT and Ephx2 (–/–) groups. However, no
Echocardiographic Data of Three Operated Groups After a 4-Week Follow-Up Ephx2(–/–) (n = 6)
Sham-Operated (n = 6)
Wild-Type-Transverse Aorta Constriction (n = 6)
Left ventricular anterior wall thickness in diastole (mm)
0.80 ± 0.09
1.04 ± 0.20a
0.82 ± 0.08b
Left ventricular anterior wall thickness in systole (mm)
1.00 ± 0.15
1.40 ± 0.22a
1.06 ± 0.14b
Left ventricular posterior wall thickness in diastole (mm)
0.72 ± 0.07
0.90 ± 0.05a
0.80 ± 0.08b
Left ventricular posterior wall thickness in systole (mm)
0.93 ± 0.15
1.22 ± 0.16a
0.93 ± 0.09b
Echocardiographic Assessment
Left ventricular mass (mg) left ventricular ejection fraction (%) Left ventricular internal dimension at end diastole (mm)
109.64 ± 25.40
155.02 ± 27.07a
116.03 ± 12.49b
46.83 ± 8.68
52.51 ± 7.60
47.80 ± 9.22
3.55 ± 0.28
4.13 ± 0.39
a
3.48 ± 0.32b
p < 0.05 versus sham-operated. p < 0.05 versus wild-type-transverse aorta constriction.
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Figure 2. Soluble epoxide hydrolase deletion down-regulated fibroblast growth factor (FGF)-2 expression in mouse transverse aorta constriction (TAC) model. A and B, Immunochemistry staining result showed that FGF-2 was highly expressed near epicardium and endocardium. In wild-type (WT)-TAC group, the FGF-2 was up-regulated significantly, but reduced in Ephx2 (–/–)-TAC group. B, The change of FGF-2 under higher magnifications (40×). C, Western blot result showed that cardiac FGF-2 was increased in WT-TAC group while attenuated in Ephx2 (–/–)-TAC group. D, Enzyme-linked immunosorbent assay result of serum FGF-2 showed no change of FGF-2 levels among three groups. *p < 0.05 versus sham-operated group; #p < 0.05 versus WT-TAC group. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
significant difference was found between these two groups (Fig. 4, C and D). These data showed that sEH deletion did not have the similar effect to attenuate cardiac hypertrophy induced by FGF-2 as induced by Ang II. sEH Deletion Down-Regulated CFB-Derived FGF-2 by Reducing the Phosphorylation of ERK1/2 Signal Pathway To understand the signal pathways involved in the synthesis of FGF-2, we used inhibitors of U0126 to ERK1/2, SB203580 to p38, and A6730 to AKT, respectively. After 24-hour stimulation with Ang II in WT CFBs, the expressions of FGF-2 were significantly elevated. However, when pretreated with U0126, SB203580, and A6730 for 1 hour, the up-regulation of FGF-2 induced by Ang II was significantly attenuated Critical Care Medicine
(Fig. 5A). The comparison of the effects among these three inhibitors showed that U0126 was the most effective inhibitor to block the synthesis of FGF-2 comparing with SB203580 and A6730 (Fig. 5A). Ang II increased phosphorylation of ERK1/2, p38, and AKT in CFBs in 1 hour (Fig. 5B–D). These results suggested that ERK1/2, p38, and AKT signal pathways were involved in the expression of FGF-2 mediated by Ang II. Furthermore, we compared the phosphorylation degree of ERK1/2, p38, and AKT stimulated by Ang II in CFBs between WT group and Ephx2 (–/–) group. The results showed that sEH deletion lowered the phosphorylation of ERK1/2 significantly (Fig. 5B). But no significant difference was found in p38 and AKT phosphorylation between the two groups (Fig. 5, C and D). www.ccmjournal.org
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Figure 3. Cardiac fibroblasts (CFBs)-derived fibroblast growth factor (FGF)-2 induced by angiotensin II (Ang II) was down-regulated by soluble epoxide hydrolase deletion in vitro. A, Real-time polymerase chain reaction result showed Ang II increased FGF-2 messenger RNA (mRNA) expression significantly. The elevation of FGF-2 was much higher in wild-type (WT) group than that in Ephx2 (–/–) group after Ang II stimulation. B and C, In CFBs and CFBs supernatant, Western blot and enzyme-linked immunosorbent assay results showed that Ang II induced a significant elevation in the protein levels of FGF-2 in WT group while attenuated in Ephx2 (–/–) group after Ang II stimulation. *p < 0.05 versus control; #p < 0.05 versus WT. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
DISCUSSION Our major findings were as follows: 1) Deletion of sEH prevented cardiac hypertrophy in a murine TAC model by down-regulation of the expression of FGF-2 in CFBs. 2) The expression of FGF-2 in CFBs was mediated by ERK1/2, p38, and AKT signal pathways. 3) The hypertrophic effect in CMs induced by FGF-2 was not attenuated by the deletion of sEH. 4) Deletion of sEH down-regulated the expression of FGF-2 in CFBs by lowering phosphorylation of ERK1/2. The mechanisms of cardiac hypertrophy are very complicated, in which multiple pathways, including Ang II and norepinephrine-mediated pathways, are involved (36). The detailed mechanism of antihypertrophy by sEH deletion is still unclear. The possible reasons for the protective effects by sEH deletion may be explained as follows. First, deletion of sEH lowers the blood pressure. Sinal et al (19) found that the mean systolic blood pressure was significantly decreased in Ephx2 (–/–) mice than in WT mice. Imig et al (20) discovered that sEH inhibition lowered arterial blood pressure in Ang II-induced hypertension. So, in Ephx2 (–/–)-TAC model, the improvement of cardiac hypertrophy may partly attribute e350
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to the reduction of blood pressure. Second, EETs play an anti-inflammatory role in TAC model in which the inflammatory response is activated. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokineinduced endothelial cell adhesion molecule expression and leukocyte adhesion to the vascular wall through the inhibition of transcription factor NF-κB and IkappaB kinase (37–39). Growth factors and cytokines are highly synthesized by different cells in heart during TAC because the local renin-angiotensin system is activated (40, 41). The cell-cell communication in cardiac hypertrophy is required for induction of hypertrophic responses in CMs (42). As we know, CFBs play an important role in regulating cardiac hypertrophy and cardiac function (3, 43). CFBs also have numerous other functions, including synthesis and deposition of extracellular matrix, secretion of growth factors and cytokines, and cell-cell communication with CMs (5). FGF-2 is a growth factor expressed both in CMs and CFBs. Our study showed that FGF-2 was highly expressed in epicardium and endocardium where CFBs are abundant, implying that CFBs were the main resource of FGF-2 in heart (44). This result was consistent with May 2014 • Volume 42 • Number 5
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Figure 4. Cardiomyocytes (CMs) hypertrophy induced by fibroblast growth factor (FGF)-2 was not attenuated by soluble epoxide hydrolase deletion. A, Surface area of isolated neonatal mouse CMs increased after FGF-2 treatment (50 ng/mL), as revealed by confocal immunocytochemical analysis with antibody to cardiac troponin I (green) and 4',6-diamidino-2-phenylindole (blue) to nuclei. B, Compared with that of untreated CMs, treatment with FGF-2 for 24 hr significantly increased cell surface area analyzed by morphometry. C and D, After FGF-2 treatment, the expressions of atrial natriuretic peptide (ANP) and β-myosin heavy chain (MHC) messenger RNA (mRNA) were increased in CMs (mean ± sd; n = 3). *p < 0.05 versus control. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
previous information on the function of FGF-2 in epicardial and myocardial development (45, 46). Animal models have provided evidence that FGF-2 is essential for the manifestation of cardiac hypertrophy induced by pressure overload and Ang II (10, 47). However, due to the limitations that only low molecular weight (LMW) of the recombinant FGF-2 was available and FGF-2 antibody in the ELISA kit was nonspecific to recognize its isoforms, we did not distinguish the roles of individual isoforms of FGF-2 as many other investigations confirming the role of FGF-2 in cardiac hypertrophy (8, 35, 46). Recently, Kardami et al (12) suggested that cardiac hypertrophy was only associated with high molecular weight (HMW) rather than the LMW isoform of FGF-2. Both HMW FGF-2 and LMW FGF-2 have similar paracrine effects on proliferation, but HMW FGF-2 exerts a distinct intracrine effect on binucleation (48), which may be due to the different models of animals (46). Further investigation remains to be determined regarding the role of sEH deletion on the individual isoforms of FGF-2 in cardiac hypertrophy. Critical Care Medicine
In our study, Ang II directly stimulated the expression of FGF-2 from CFBs by activating phosphorylation of ERK1/2, AKT, and p38. The results demonstrated that FGF-2 was mediated via multiple signal pathways. However, we did not find that deletion of sEH reduced the phosphorylation of p38 and AKT except for ERK1/2, which conflicted with the previous studies that EETs activated ERK1/2 and phosphatidylinositol 3-kinase/AKT signal pathways (49, 50). The possible explanation is that deletion of sEH in vitro or in vivo has multiple cellular functions besides elevation of EETs (25). In addition, the sEH is a bifunctional enzyme with epoxide hydrolase and phosphatase activities, genetic deletion of sEH removes both activities, whereas EETs have relatively simple activity and may not reflect the whole function of sEH in vivo. More studies are needed to explore this problem. Our results also showed the lack of correlation between the serum levels of FGF-2 and the cardiac hypertrophy. This was in concordance with other reports describing the lack of association between the serum FGF-2 levels and left ventricular mass www.ccmjournal.org
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Figure 5. Soluble epoxide hydrolase deletion decreased angiotensin II (Ang II)-induced fibroblast growth factor (FGF)-2 in cardiac fibroblasts (CFBs) by reducing phosphorylation of ERK1/2. A, Pretreatment with U0126, SB203580, and A6730 for 1 hr attenuated the increase of wild-type (WT) C FBsderived FGF-2 induced by Ang II. B, The comparison between the WT and Ephx2 (–/–) groups after Ang II treatment. The phospho-ERK1/2 expressed higher in WT group than that in Ephx2 (–/–) group. C, The comparison of phosphorylation of p38 between the WT and Ephx2 (–/–) groups after Ang II treatment. No significant difference of phosphorylation of p38 was found between the two groups. D, The comparison of phosphorylation of AKT between the WT and Ephx2 (–/–) groups after Ang II treatment. No significant difference of phosphorylation of AKT was found between the two groups. Data were expressed as mean ± sd (n = 3). *p < 0.05 versus control; #p < 0.05 versus Ang II (individual treatment); **p < 0.05 versus SB203580 and A6730. KO = knockout, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
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in hypertensive patients (44, 51, 52). This might be due to the reason that FGF-2 acts mainly in the local area of its expression. The serum levels may not reflect the local concentration of FGF-2 in the heart. Meanwhile, we demonstrated that deletion of sEH did not prevent CMs hypertrophy induced by FGF2, indicating that deletion of sEH may only partly be involved in the process of cardiac hypertrophy or the upstream effect of FGF-2. In addition, in our study, although the cardiac function of WT-TAC and Ephx2 (–/–)-TAC groups was found to have a tendency of increase compared with that of sham-operated group, no significant difference was found among these groups which may be because of the relative low degree and the limited time of aortic constriction.
LIMITATIONS Although we found that deletion of sEH attenuated cardiac hypertrophy via down-regulation of CFBs-derived FGF-2, there are still several limitations in this study. First, we did not treat CMs with Ang II and observe the changes of FGF-2 so as to reveal the intracrine biological effect of FGF-2 directly. Second, the CFBs were not treated with the inhibitor of ERK, p38, and AKT alone which may influence the expressions of FGF-2 in some cases. Furthermore, we only explored the limited signal pathways, and the possible signaling mechanisms involved in the regulation of FGF-2 in CFBs need further study. In summary, our present data suggested that deletion of sEH not only prevented cardiac hypertrophy directly to CMs but also reduced expression of FGF-2 from CFBs. The present study provides a new evidence to understand the role of sEH in cardiac hypertrophy and uncovers the essential interaction between CFBs and CMs for the progression of cardiac hypertrophy. In future study, we will further explore the role of sEH in the cell-cell interaction in cardiac hypertrophy.
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