MicroRNA-210 promotes angiogenesis in acute myocardial infarction
- Authors:
- Published online on: February 20, 2018 https://doi.org/10.3892/mmr.2018.8620
- Pages: 5658-5665
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Copyright: © Fan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Ischemic heart disease is considered to be a leading cause of morbidity and mortality in humans, particularly those suffering from acute myocardial infarction (AMI). These patients are considered to be more susceptible to complications, including cardiogenic shock, or the development of chronic heart failure (CHF), although emergency revascularization has been widely used (1,2). It is likely that the loss of blood flow to the myocardium, primarily due to the occlusion of coronary arteries, may lead to myocardiocyte death and myocardial remodeling. As a result, improving myocardial recovery following AMI may exert positive effects in reducing the incidence of associated complications and leading to improved clinical outcomes.
MicroRNAs (miRNAs/miRs) are non-protein coding RNAs with a length of 22–24 nucleotides, which are able to interact with the 3′-untranslated regions of target mRNAs and inhibit transcription or translation (3). It has been previously reported that a number of miRNAs, including miRNA-34a, miRNA-208a and miRNA-495, may be important gene regulators for remodeling or inducing angiogenesis in response to AMI, and these beneficial effects have made non-coding RNAs potentially important therapeutic tools for preventing the appearance of CHF following AMI (4–6).
miRNA-210 serves an important role in response to hypoxic conditions and may be upregulated by hypoxia-inducing factors, and resultant alterations of cellular processes, including apoptosis, angiogenesis and metastasis, have been observed (7). Recently, Zeng et al (8) established ischemic brain models in mice and demonstrated that overexpression of miRNA-210 was able to enhance the microvessel density (MVD) in brain samples using immunohistochemistry analysis. Similar results were additionally reported by Yang et al (9), who demonstrated that miRNA-210 promoted hepatocellular carcinoma angiogenesis. In addition, Wang et al (10,11) reported different results pertaining to the effects of miRNA-210 in AMI, although the same AMI rat models were used. It may be noted that the earlier formation of collateral circulation may reduce the risk of cardiomyocyte death and myocardial remodeling following AMI and facilitate improved clinical outcomes. However, these controversial data raised questions regarding the role of miRNA-210 in AMI. Therefore, the present study was designed to investigate the efficacy of miRNA-210 in AMI, and sought to elucidate the potential associated mechanisms.
Materials and methods
Animal experiments
Animals
A total of 48 Sprague-Dawley rats (4-week old; male) weighing 220–240 g were obtained from the Animal Center of Nanjing Medical University (Nanjing, China). All the rats were housed in the laboratory animal room maintained at a temperature of 20±2°C with a relative humidity of 50–70% and on a regular 12 h light-dark cycle, with access to standard chow and water for 1 week prior to any operative procedures. The protocol of the animal experiments was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Rat model of AMI
The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 ml/kg) and a tracheotomy was subsequently performed, supported by a small animal ventilator (ALC-V8S; Shanghai Alcott Biotech Co., Ltd., Shanghai, China). The establishment of the AMI models was achieved by ligating the proximal left anterior descending (LAD) coronary artery with a 6-0 silk suture. A successful procedure was confirmed if the followed criteria were fulfilled: i) Observation of rapid discoloration over the anterior surface; and ii) electrocardiogram exhibiting persistent ST segment elevation ≥0.2 mV in two or more contiguous limb leads three times (reexamined every 10 min after the ligation). At the end of the surgery, all rats received an intramuscular injection of penicillin sodium (400,000 units) to prevent potential infection.
Animal groups and myocardial transfection with lentiviral vectors in vivo
The animals were randomly divided into 4 groups: i) Sham-operated (Sham; 12 rats) rats received all surgical procedures except for ligation of the LAD coronary artery; ii) AMI and treatment with negative control vector (AMI + NV; 7 rats); iii) AMI (AMI; 8 rats); and iv) AMI and treatment with lentivirus-mediated miRNA-210 agonist (AMI + LV-miR-210 agonist; 9 rats). The myocardial transfection in vivo was performed via intravenous injection with LV-miR-210 agonist (precursor-hsa-miR-210; Shanghai Genechem Co., Ltd., Shanghai, China) or negative control vector (hu6-MCS-Ubiquitin-EGFP-IRES-puromycin; Shanghai Genechem Co., Ltd.). The forward primer sequence for hsa-miR-210 was 5′-GGAAAGGACGAAACACCGGGGACAAGAGAGGAGTGGCTCTG-3′, and the reverse primer sequence was 5′-TGTCTCGAGGTCGAGAATTAAAAAACTAGTGGCCCACTACCCTGTC-3′, which were amplified into the 301-bp product as described previously (4). The negative control vector and LV-miRNA210 agonist were added to PBS to a final volume of 0.5 ml, which was applied to each rat, while untreated rats received PBS (0.5 ml) only.
Cardiac function assessment
A total of 4 weeks post-surgery, echocardiography using the Vevo2100 system (Fujifilm VisualSonics, Inc., Toronto, ON, Canada) with a high-frequency (30 MHz) MS-400 transducer was performed to evaluate the cardiac functions of the rats noninvasively. Prior to echocardiography, 3% isoflurane was used to induce anesthesia and maintenance with 1.5% isoflurane was applied during the procedure. The thickness of interventricular septum in systole and diastole, left ventricle internal dimension in systole (LVIDs) and diastole (LVIDd), thickness of LV posterior wall in systole (LVPWs) and diastole (LVPWd), left atrium (LA) internal dimension, LV volume in systole (LV VOLs) and diastole (LV VOLd), LV mass and LV mass corrected were measured. In addition, the LV fractional shortening percentage (FS) and LV ejection fraction percentage (EF) were calculated as followed: FS (%)=[(LVIDd−LVIDs)/LVIDd] ×100; EF (%)=[(LV VOLd-LV VOLs)/LV VOLd] ×100.
Preparation of tissue samples
Heart tissues were quickly removed from the sacrificed rats following the assessment of cardiac function. Each of the tissue samples was divided into two parts which were prepared for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis (temporarily stored at −80°C) and immunohistochemical analysis (fixed in 4% paraformaldehyde at 20°C, for 2 h) separately.
RT-qPCR analysis
In order to measure the expression levels of miRNA-210 and associated genes, heart samples were analyzed via RT-qPCR. Total RNA was extracted using TRIzol reagent (Zoonbio Biotechnology Co., Ltd., Nanjing, China) and transcribed into cDNA using the TransScript miRNA RT Enzyme Mix (TransGen Biotech Co., Ltd., Beijing, China; www.transbionovo.com/). The amplification of cDNA was subsequently performed following the protocol (95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 20 sec) of the SYBR Premix Ex Taq II kit (Takara Biotechnology Co., Ltd., Dalian, China) using an ABI7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Human GAPDH was used as an internal control. The expression levels of miRNA-210, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were measured. The primer sequences of relative genes were as follows: GAPDH forward, 5′-GCAAGTTCAACGGCACAG-3′ and reverse, 5′-GCCAGTAGACTCCACGACAT-3′; HGF forward, 5′-GGGGCTACACTGGATTGA-3′ and reverse, 5′-GCCTTGATGGTGCTGACT-3′; miRNA-210 forward, 5′-AGCCACTGCCCACAGCACACTG-3′ and reverse, 5′-CAGTGCAGGGTCCGAGGTATT-3′; and VEGF forward, 5′-CTCACCAAAGCCAGCACAT-3′ and reverse, 5′-TTCTCCGCTCTGAACAAGG-3′. The comparative Cq method (2−ΔΔCq) was used to analyze the data (12).
Immunohistochemistry
The paraffin-embedded heart samples were sliced into 4–5 µm thicknesses following dehydration, and were stained with hematoxylin-eosin (H&E; hematoxylin solution and 1% eosin solution, at room temperature for 10 min each) and prepared for Masson's trichrome staining (Bouin's solution, at 56°C for 1 h at room temperature, Weigert hematoxylin working fluid for 20 min, then dying with ponceau red solution for 5 min, rinsing with distilled water for 30 sec, slides were subsequently soaked in 1% phosphotungstic solution and dying with aniline blue solution, for 8 min each, at room temperature). MVD counting was performed on the basis of platelet endothelial cell adhesion molecule (CD31) immunohistochemistry. After deparaffinization [twice in xylene, 5 min each, followed by three sequential (90, 75 and 50%) solutions of ethanol, 5 min each], heart tissues were soaked in citrate buffer (0.01 mol/l, pH 6.0) to recover antigenicity. Subsequently, slides were incubated with 0.5% hydrogen peroxide-methanol solution for 10 min at room temperature. After rinsing with distilled water, rabbit anti-CD31 polyclonal antibody (1:100; cat. no. BA2966; Wuhan Boster Biological Technology, Ltd., Wuhan, China) was applied to sections as the primary antibody for 2 h at room temperature and the secondary antibody solution (1:500; cat. no. K5007; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was added to induce horseradish peroxidase binding, maintained at room temperature for 30 min, following the methods described by Mineo et al (13). For each sample, at least 2 fields were selected for analysis. All the images were obtained using a light microscope (magnification, ×200; Nikon Corporation, Tokyo, Japan), among which three areas with highest MVD in each section were selected for counting and the average of these vascular counts (mean intensity=integral optical density sum/area) was recorded as the MVD level in each case, quantified using image analysis software (Image Pro Plus 6.0; Media Cybernetics, Inc., Rockville, MD, USA).
Cell experiments
Cell culture and regent exposure
Human umbilical vein endothelial cells (HUVECs) cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells cultured under normal oxygen levels were maintained at 37°C and supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in a humidified chamber with 5% CO2 and 95% air (labeled as the normoxia condition). The fresh culture medium was changed every day. Simultaneously, parallel cultured cells under hypoxic conditions were cultured as previously described (14).
Small interfering RNA (siRNA) transfection
The cells cultured under hypoxic condition were divided into two groups when they reached 50% confluence and were transfected with specific siRNA (The sequences were as follows: Upstream primer, GCUACAAGAAACCGCCUAUTT; downstream primer, AUAGGCGGUUUCUUGUAGCTT) for silencing HGF expression (100 nM/1.5×105 cells; labeled siHGF) or scrambled probe, as the negative control (both Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Lipofectamine RNAiMax (Invitrogen; Thermo Fisher Scientific, Inc.) was used, according to the manufacturer's protocol. Following 48 h of incubation, further western blot analyses were performed to evaluate the transfection efficiency and detect alterations in CD31 protein expression levels in these cells.
Western blot analysis
Each of heart samples was homogenized in lysis buffer with protease inhibitors (p0013c; Beyotime Institute of Biotechnology, Haimen, China) and centrifuged at 16,200 × g for 15 min at a temperature of 4°C. To determine the protein concentration, the Pierce Bicinchoninic Acid protein assay (Thermo Fisher Scientific, Inc.) was used. Extracted protein samples were separated using 8–15% SDS-PAGE and subsequently transferred to polyvinylidene difluoride membranes (40 µg protein/lane). The membranes were blocked with 5% skimmed milk in TBS for 1 h at 37°C. Rabbit polyclonal antibodies against HGF (1:400; cat. no. BA0911; Wuhan Boster Biological Technology, Ltd.) and β-myosin heavy chain (MHC) (1:500; cat. no. ab50967; Abcam, Cambridge, UK) were diluted with blocking buffer, in which the membranes were maintained overnight at 4°C and washed with TBS-Tween-20 four times for 5 min each the following day. The membranes were subsequently incubated in blocking buffer with added secondary antibodies (1:5,000; cat. no. ABP103; Zoonbio Biotechnology Co., Ltd.) for 2 h at room temperature. Rabbit anti-rat β-actin (1:2000; cat. no. 8457; Cell Signaling Technology, Inc., USA) was applied as an internal control.
Following 48 h of incubation, transfected cells were lysed with radioimmunoprecipitation buffer (p0013c; Beyotime Institute of Biotechnology) containing protease and phosphatase inhibitors, and centrifuged at 13,800 × g for 20 min at 4°C. The remainder of the protocol was as described above. However, the protein expression levels of HGF and CD31 (1:1,000; cat. no. 77699; Cell Signaling Technology, Inc., Danvers, MA, USA) were detected in the cells and GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) was used as the internal control. The Syngene Bio Imaging Device (Syngene Europe, Cambridge, UK) was used to visualize immunoreactive protein bands.
Statistical analysis
Statistical analysis was performed using the SPSS 22.0 software package (IBM Corp., Armonk, NY, USA) and all data are expressed as the mean ± standard deviation. One-way analysis of variance followed by Bonferroni corrections was used for comparisons between multiple groups. P<0.05 (two-tailed) was considered to indicate a statistically significant difference.
Results
AMI increases myocardial miRNA-210 expression
As depicted in Fig. 1A, myocardial miRNA-210 expression was increased significantly in the AMI + PBS subgroup compared with the sham group (P=0.011) at 4 weeks post-surgery. The expression level of miRNA-210 in the AMI + miR-210 agonist group was additionally significantly increased, compared with the other three subgroups (P<0.001 vs. Sham + PBS, AMI + NV and AMI + PBS, respectively). Notably, the comparison between the AMI + PBS group and AMI + NV group demonstrated a significant decrease in miRNA-210 expression in the latter group (P=0.038) and no statistical difference between the Sham + PBS group and the AMI + NV group was observed (P=0.799).
Overexpression of miRNA-210 up-regulates HGF expression
An apparent upregulation of HGF was observed in the AMI + miR-210 agonist subgroup compared with the other three subsets (P=0.02 vs. Sham + PBS; P=0.049 vs. AMI + NV; P=0.048 vs. AMI + PBS; Fig. 1B), which was principally driven by the overexpression of miRNA-210. However, the AMI-induced overexpression of miRNA-210 did not promote myocardial HGF expression (P=0.921 vs. Sham group) and the comparisons between multiple groups demonstrated no significant differences in the expression of VEGF (Fig. 1C). In addition, similar results in terms of protein expression levels exhibited an upregulation of HGF associated with miRNA-210 overexpression (Fig. 2). The attenuating increase of myocardial β-MHC protein expression induced by AMI was observed following treatment with miR-210 agonists, particularly compared with the AMI + NV group. These findings indicated that HGF may be a target gene of miRNA-210 in AMI.
Role of HGF in HUVEC proliferation
As presented in Fig. 3A, the protein expression level of HGF in HUVECs was decreased markedly following transfection with siRNA, demonstrating the suppressive effect of siRNA on HGF expression. Additionally, simultaneously increased CD31 and HGF protein expression was observed in the cells under hypoxic condition (Fig. 3B). When the HGF expression was silenced through transfection with siRNA, the level of CD31 protein expression in cells under hypoxic conditions was decreased, as depicted in Fig. 3C, indicating that HGF may be an important stimulator of HUVEC growth and proliferation.
Alterations induced by overexpression of miRNA-210 in histology and cardiac function
MVD in heart samples
MVD was measured in myocardial samples (Fig. 4). Compared with the sham group (Fig. 4A), a markedly decreased optical intensity of CD31 cells was noted in the myocardium in the AMI + NV subgroup (P=0.045; Fig. 4B and E). By contrast, the optical intensity of CD31 cells in the infarcted area of the heart treated with the miR-210 agonist was significantly increased compared with the AMI + NV group (P=0.029; Fig. 4D and E), whereas no statistical significance was observed when compared with the Sham group (P=0.996). The comparison between the AMI + NV group and AMI + PBS group indicated no significant difference (P=0.716; Fig. 4C and E). Therefore, better promoting angiogenesis effects with respect to over expression of miRNA-210 were observed.
Histopathological observation of rat heart tissue
As presented in the images of H-E and Masson's trichrome staining, the diffuse, edematous myocardial fibers were severely broken, taking the form of deranged cellular structures among which a large number of collagen fibers had been accumulated in the AMI + NV group and the AMI + PBS group, compared with the Sham group (Fig. 5A-C). Improved results were observed with miRNA-210 agonists, indicating reduced accumulation of collagen fibers and decreased destruction of myocardial fibers (Fig. 5D).
Cardiac function changes
Echocardiography was performed on the rats to evaluate alterations in cardiac structure and function at 4 weeks post-surgery. Compared with the Sham group, the cardiac contractility was markedly decreased in the AMI + NV group, which was primarily derived from the enlarged LVD and LV VOL in systole, resulting in a decrease in LVEF and LVFS. By contrast, the hearts receiving the miRNA-210 agonist exhibited improved cardiac contractility compared with the AMI + NV groups at the same points, and improved results in LVEF and LVFS were acquired, while no significant difference compared with the Sham group was observed. The characteristics of alterations in cardiac structure and function among the rats are summarized in Fig. 6 and Table I.
Discussion
The resulting myocardiocyte death and myocardial remodeling post-AMI, derived from the occlusion of associated coronary arteries which may completely block blood flow to the myocardium, has been hypothesized to be the most important risk factor for the development of heart failure among these patients (2). Therefore, reperfusion therapy strategies have been widely applied in the clinic and demonstrated to be beneficial, regardless of emergency revascularization or thrombolytic therapy (15). The concept of using gene therapy to stimulate the regenerative abilities of the body for damaged tissues had been proposed as a potential therapeutic strategy for AMI. A number of miRNAs have been reported to act as potential therapeutic tools, primarily as regulators of target gene expression inducing therapeutic angiogenesis or attenuating remodeling in response to ischemic states (4–6,14,16). miRNA-210 serves a role in response to hypoxic conditions, and has been observed to be an important regulator in angiogenesis. Wang et al (10) reported that the knockdown of miRNA-210 expression led to improved cardiac function in AMI, while the opposite results were described by Wang et al (11), in which protective effects of the upregulation of miRNA-210 and VEGF were observed in AMI, which were hypothesized to be derived from the promotion of myocardial angiogenesis. These controversial data, in addition to the unclear mechanisms, called the role of miRNA-210 in AMI into question. As a result, the present study was performed and the principal finding was that overexpression of miRNA-210 induced by agonists exerted positive effects on promoting angiogenesis in AMI through the potential mechanism of upregulating HGF expression, which may serve a role in reducing remodeling following AMI and lead to better clinical outcomes in terms of improved cardiac contractility.
HGF, similar to VEGF, has been demonstrated to be an important stimulator of angiogenesis, exerting promotive effects on endothelial cell (EC) growth, proliferation, migration and differentiation (17–19). Gorin et al (17) demonstrated that HGF was exhibited a high angiogenic potential due to the direct targeting of ECs, and the results of the present study were consistent with these findings. Among the rats receiving miRNA-210 agonists in the present study, the upregulation of HGF induced by miRNA-210 overexpression exhibited promotive effects on angiogenesis in in vivo and in vitro experiments, as confirmed by CD31 immunohistochemistry. In addition, the increased proliferation and migration of ECs associated with HGF expression was indicated by Kaga et al (18). The underlying mechanism was reported to be associated with enhanced intracellular signaling molecules, including focal adhesion kinase and RAC-α serine/threonine-protein kinase, with respect to the regulation of cytoskeletal remodeling, cellular migration and morphogenesis (20). The results of the present study indicated that angiogenesis in AMI was induced by miRNA-210 overexpression via direct targeting of HGF, instead of VEGF, which was different from the observations of certain previous studies (10,11). The balance between proliferation and migration in ECs and vascular smooth muscle cells (VSMCs) may be considered to serve an important role in angiogenesis. Basic fibroblast growth factor (bFGF) has been reported to upregulate aquaporin 1 mRNA expression in HUVECs, thereby influencing vascular permeability (21). However, bFGF promoted the production of inflammatory factors, including C-C motif chemokine 2 and interleukin-8, as the activation of nuclear factor-κB and the number of VSMCs was additionally increased, whereas HGF and VEGF did not (18). HGF has been suggested to be an anti-inflammatory gene, due to its antioxidant effect in VSMCs (22,23). To the best of our knowledge, the acute phase of AMI is frequently complicated by a severe inflammatory response, leading to negative effects on the proliferation and migration of ECs; this may explain the results indicated in the present study, as the anti-inflammatory effect of HGF appeared to facilitate the balance between proliferation and migration in ECs and VSMCs, and subsequently to promote angiogenesis in AMI.
In order to examine the potential mechanisms of miRNA-208a with respect to myocardial remodeling post-AMI, Shyu et al (4) performed experiments in vivo and in vitro, and demonstrated that miRNA-208a served an important role in the development of myocardial fibrosis following AMI, primarily through its regulatory effects on endoglin expression, in addition to potent targeting effects on β-MHC. In the present study, improved cardiac functions were observed on the basis of improved cardiac contractility among the rats receiving miRNA-210 agonists, as demonstrated by echocardiography and western blot analysis of β-MHC. Left ventricular remodeling post-AMI may lead to a temporary compensatory enhancement of left ventricular systolic function, indicating a marked increase in β-MHC protein expression, which may increase myocardial oxygen demand and result in worse clinical outcomes. The results of the present study illustrated the attenuating increase of β-MHC protein expression, and were consistent with the results described by Wang et al (24); therefore, this may be an effective interpretation of the antifibrotic effect associated with microvascular formation induced by miRNA-210 overexpression, which was primarily due to the improved blood flow following maturation of new blood vessels, forming complete collateral circulation to increase the supply of oxygenated blood to myocardial cells.
In conclusion, the present study provided evidence to support the protective effects of overexpression of miRNA-210 in AMI, which were considered to be due to the promotion of angiogenesis in the infarcted myocardium by targeting HGF expression and inducing improved left ventricular remodeling post-AMI. Therefore, the overexpression of miRNA-210 may be suggested to be an advantageous therapeutic tool for treating AMI in order to obtain improved clinical outcomes.
Acknowledgements
The present study was supported by the Jiangsu Provincial Special Program of Medical Science (grant no. BL2013001).
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