HIF‐1α‐induced up‐regulation of microRNA‐126 contributes to the effectiveness of exercise training on myocardial angiogenesis in myocardial infarction rats

Abstract Exercise training (ET) is a non‐drug natural rehabilitation approach for myocardial infarction (MI). Among the numerous beneficial effects of ET, myocardial angiogenesis is indispensable. In the present study, we investigated the role and mechanism of HIF‐1α and miR‐126 in ET‐induced MI myocardial angiogenesis which may provide new insights for MI treatment. Rat model of post‐MI and human umbilical vein endothelial cells (HUVECs) were employed for our research. Histomorphology, immunohistochemistry, quantitative real‐time PCR, Western blotting and small‐interfering RNA (siRNA) transfection were applied to evaluate the morphological, functional and molecular mechanisms. In vivo results showed that 4‐week ET could significantly increase the expression of HIF‐1α and miR‐126 and reduce the expression of PIK3R2 and SPRED1, while 2ME2 (HIF‐1α inhibitor) partially attenuated the effect of ET treatment. In vitro results showed that HIF‐1α could trigger expression of miR‐126 in HUVECs in both normoxia and hypoxia, and miR‐126 may be involved in the tube formation of HUVECs under hypoxia through the PI3K/AKT/eNOS and MAPK signalling pathway. In conclusion, we revealed that HIF‐1α, whose expression experiences up‐regulation during ET, could function as an upstream regulator to miR‐126, resulting in angiogenesis promotion through the PI3K/AKT/eNOS and MAPK signalling pathway and subsequent improvement of the MI heart function.

ET against diverse cardiovascular diseases, and ET is thus considered an acceptable method for the active role in the rehabilitation of MI hearts. 4 Exercise, a non-drug natural cardiac rehabilitation means, 5 can boast of its superiorities for its simple administration, easy application and lack of obvious side effects. Yet up to now, the mechanism of ET-induced MI cardiac protection has not been fully elucidated.
Myocardial angiogenesis has been recognized as one of the innovative therapeutic methods for the treatment of MI.
Angiogenesis refers to the generation of new blood vessels from pre-existing capillaries, 6 which is regulated by a wide range of regulators and signalling molecules. 7 Among the molecules involved in the angiogenesis induced by ET, hypoxia-inducible factor-1 α (HIF-1α) may play a crucial role. 8 HIF-1α is an important transcription factor that regulates angiogenesis through transcription of a series of hypoxia response genes archived with hypoxia response element. 9 Acute exercise is accompanied by reduced regional-and-systemic partial pressure of oxygen, which are stimulatory factors of HIF-1α. 10 Ten-week aerobic exercise has been proven to be able to significantly up-regulate the expression of HIF-1α in rat left ventricle. 11 Therefore, we speculated that HIF-1α would play important role in ET-induced myocardial angiogenesis and cardiac function improvement in MI heart. MicroRNAs (miRNAs; miRs) are small (18-25 nucleotides in length), single-stranded non-coding RNAs that act as post-transcriptional regulators of gene expression. It has been discovered that dozens of miRNAs, such as miR-221/222, miR-17-72 cluster, miR-126 and miR-93, are involved in various stages of angiogenesis. [12][13][14] Among them, miR-126, highly expressed in vascular endothelial cells, is considered a master regulator of physiological angiogenesis and vascular integrity. 15 miR-126 functions by directly repressing two negative regulators, phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) and Sprouty-related protein 1 (SPRED1). 16 PIK3R2 and SPRED1 have been shown to function as angiogenesis signalling via PIK3/Akt/eNOS and Ras/MAPK pathway, respectively. 17 Loss of miR-126 causes leaky vessels and haemorrhage in zebrafish during embryonic development. 18 Furthermore, targeted deletion of miR-126 in mice causes partial embryonic or perinatal lethality and the proliferation of ECs in embryos is significantly reduced. Our previous research found that ET could significantly up-regulate the expression of miR-126 of MI myocardium, suppress its target protein expression of PIK3R2/SPRED1 and promote angiogenesis in peri-infarct zone, 19 leaving the mechanism that ET-induced up-regulation of miR-126 unexplored fully. As it is well established that some miR-NAs are identified to be regulated under hypoxic conditions, 20 we then hypothesize that ET may contribute to myocardial angiogenesis against MI injury via the HIF-1α/miR-126 pathway.
In this study, rat model of MI and human umbilical vein endothelial cells (HUVECs) were employed to valid our hypothesis, and we explored the role of HIF-1α and miR-126 in ET-induced cardiac protection of MI hearts and its underlying mechanisms. Hopefully, these findings can provide new targets for the research on the rehabilitation mechanism of ischaemic heart diseases.

| Animal
A total of 50 male Sprague Dawley rats (204 ± 6 g, SPF) were purchased in the Laboratory Animal Centre of Xi'an Jiao tong University (Animal certificate No.: SCXK2012-201). Rats were housed five per cage in a temperature-controlled room with a 12-hour dark-light cycle and free access to water and standard rat chow. All experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Shaanxi Normal University.

| Surgical procedures and experimental design
Experimental MI models were induced by the ligation of the left anterior descending coronary artery (LAD) as previously research applied. 21 Briefly, the rats were anaesthetized by intraperitoneal (ip) injection of sodium pentobarbital (30 mg/kg body weight). LAD was ligated under the stereoscopic microscope (OLYMPUS SZX16), 2 mm beneath the left atrial appendage. Sham-operated rats had the same surgery, with threading yet no ligation. Four rats died during or after the operation, and two rats without ST elevation were eliminated. All post-operation rats were randomly assigned to five groups: sham-operated group (S, n = 8), myocardial infarction group (MI, n = 9), MI-with-ET group (MIE, n = 9), MIE group treated with HIF-1α inhibitor 2-methoxyestradiol (2ME2; MIE + 2ME2, n = 9) and MIE group treated with PBS (MIE + PBS, n = 9).

| Drug administration
The specific HIF-1α inhibitor 2-methoxyestradiol (2ME2; Selleck Chemicals) was dissolved in PBS with 10% dimethy1 sulfoxide (DMSO). 2ME2 was administered at 15 mg/kg by intraperitoneal injection 10 minutes before ET in MIE + 2ME2 group. 22 Rats in MIE + PBS group received the same volume of vehicle (10% DMSO in PBS). All rats in the two groups were weighed once a week to determine the next week dose.

| Hemodynamic measurement
The next day after the 4 weeks' training, all rats were anaesthetized as mentioned above, and then, the right carotid artery was isolated and cannulated with a pressure catheter. The tip of the catheter was advanced from the aorta to the left ventricular (LV) cavity. LV cardiac function was obtained via intraventricular catheter recordings

| Histomorphology analysis of collagen deposition
After the haemodynamic measurement, thoracotomy was conducted immediately and hearts were collected. For histological staining, heart samples were fixed in ice-cold 4% paraformaldehyde for 48 hours, routinely paraffin embedded sectioned (5 μm). Masson's trichrome staining was applied to evaluate myocardium collagen deposition, and collagen volume fraction (CVF) was measured. CVF was quantified by calculating the area percentage of collagen staining performed with Image-Pro plus 6.0 software. CVF = collagen area/total area. Five visions under a microscope of each sample were randomly chosen, and the average of them was taken for analysis.

| Tube formation assay
All required groups of HUVECs were prepared for culture. 24-well plate, pipette tips and matrix gels were pre-cooled the day before the test. 300 μL Matrigel matrix (BD Biosciences) was added to 24-well plate and put into incubator for gel formation. Subsequent to gel solidification (30 minutes), 500 μL of HUVECs suspension (2 × 10 5 /mL) was added to each well. The plate was incubated at 37°C in the corresponding incubator. After incubation for 4 hours, the branch points of the formed tubes were observed and quantitated at the 100 × magnification using an inverted microscope (DMIL LED; Leica). ImageJ software was used to analyse the tube lengths in 5 randomly selected visual fields.
F I G U R E 1 Experimental animal programmes. After the surgery, rats in S and MI groups will receive rest-treatment until the end of the whole experiment. Rats in MIE, MIE + 2ME2 and MIE + PBS groups will experience one-week surgery recovery and another week of adjustment exercise, followed by formal ET programme for next four weeks. S, sham-operated group; MI, myocardial infarction group; MIE, MI-with-ET group; MIE + 2ME2, MIE group treated with HIF-1α inhibitor 2-methoxyestradiol (2ME2); MIE + PBS, MIE group treated with PBS

| Western Blotting
The frozen hearts and HUVECs were lysed using RIPA lysis buffer, and then, protein concentration was determined using BCA kit. Signaling). GAPDH (1:10 000; Bioworld) was used as loading control to determine the relative expression level of the target protein. A full-feature instrument (Bio-Rad Chemidoc MP) was used for gels imaging and analysing.

| Quantitative real-time polymerase chain reaction
Total RNA from cardiac tissues and HUVECs were extracted using

| Statistical analysis
Data were expressed as mean ± standard deviation. The SPSS 20.0 was used for general statistical analysis. The differences between groups were performed with one-way ANOVA followed by Tukey's post hoc test. A two-sided P value less than .05 was accepted as statistically significant. Masson stained sections were observed and photographed under light microscope (BX51 OLYMPUS), and CVF was measured by Image-Pro plus 6.0 software. Histograms were plotted by GraphPad Prism 5.01 software.

| Effect of ET and ET combined 2ME2 on cardiac function and MI-induced ventricular pathological remodelling
To evaluate the effect of ET and ET combined 2ME2 on cardiac hemodynamics, ventricular function was performed in all groups. There was a significant reduction in LVSP, +dp/dt max and −dp/dt max in MI group compared with S group (P < .01, Figure 2A,C,D); however, LVEDP was significantly increased in the MI group (P < .01). 4-week ET significantly increased LVSP, +dp/dt max (P < .01) and decreased LVEDP (P < .01) compared with MI group (Figure 2A-C). ET combined 2ME2 significantly decreased LVSP (P < .05) and increased LVEDP (P < .05) compared with MIE group (Figure 2A-B). To investigate ET and ET combined 2ME2 on cardiac remodelling, we determined the myocardial fibrotic response in all groups. As shown in Figure 2E, the collagen fibres were blue, the cardiomyocytes red and the nucleuses blue violet. CVF was significantly increased in MI group compared with S group (P < .01). 4-week ET significantly reduced CVF compared with MI group (P < .01). CVF in MIE + 2ME2 group was significantly higher than that in MIE group (P < .05, Figure 2F). Together, these data have shown that cardiac function was impaired after MI; ET could improve the cardiac function of MI heart, while 2ME2 partially attenuated the cardiac protective effect of ET treatment.

| Effect of ET and ET combined 2ME2 on myocardial angiogenesis
To evaluate the effect on myocardial angiogenesis, we analysed endothelial cell proliferation by co-staining of PCNA + and vWF + , a key factor in DNA replication and a glycoprotein produced by endothelial cells respectively. In S group tissue, almost no PCNA + /vWF + cells were detected. But strong staining was detected in other four groups ( Figure 3A). Compared with MI group, 4-week ET induced more PCNA + / vWF + cells (P < .01). Whereas, the double stained cells were significantly reduced in MIE + 2ME2 group compared with MIE group (P < .01, Figure 3B). All the above results indicated that 4-week ET could promote myocardial angiogenesis in post-MI heart, while 2ME2 partially attenuated the protective effect of ET treatment.

| Effect of IE and IE combined 2ME2 on expression of HIF-1α, miR-126 and its targets
The protein expressions of cardiac HIF-1α were detected using Western blot for all groups. Compared with MI group, the expression of HIF-1α was significantly up-regulated in MIE group (P < .01).
Cardiac miR-126 expression was analysed by real-time polymerase chain reaction (RT-qPCR) for all groups. Figure 4B showed increased miR-126 expression (P < .01) in MIE groups compared with MI group.
miR-126 was significantly down-regulated (P < .01) in MIE + 2ME2 group compared with MIE group. As we have predicted, the change tendency in PIK3R2 and SPRED1 protein levels were inversely proportional to miR-126 expression ( Figure 4C-D). All the above results indicated that ET could increase the expression of HIF-1α and miR-126 and reduce the expression of PIK3R2 and SPRED1, which could be all attenuated by 2ME2.

| Effect of DMOG and 2ME2 on expression of HIF-1α, miR-126 and its targets in HUVECs
To explore whether HIF-1α might influenced miR-126 expression, HUVECs were treated with different concentrations of DMOG (activator of HIF-1α) and 2ME2 (inhibitor of HIF-1α) for 6 hours, and the expression level of miR-126 was determined using RT-qPCR.
We found that DMOG significantly increased the expression level of HIF-1α and miR-126 in a concentration-dependent manner in normoxia ( Figure 5A

| Effect of miR-126 on hypoxia-induced tube formation and angiogenic signalling pathway of HUVECs
To explore the role of miR-126 in HUVECs, we overexpressed and si-

| D ISCUSS I ON
The present study revealed that HIF-1α played an important role during ET-induced myocardial angiogenesis and cardiac function in MI rats, thus providing new insights on the rehabilitation mechanism of ischaemic heart diseases. Furthermore, miR-126, which can be triggered by HIF-1α, could promote the tube formation through Exercise training has received increasing attention as natural, non-drug cardioprotective stimuli that can induce prolonged or sustainable cardioprotective states. 5 Many factors contribute to the beneficial effects of exercise in maintaining cardiovascular system health and delaying the progress of heart diseases. 24 The beneficial effects of exercise may be associated with anabolic/ catabolic balance, calcium handling and myocardial fibrosis. [25][26][27] Recently, myocardial angiogenesis has been recognized as one of the innovative therapeutic methods for the treatment of MI. 28 Angiogenesis, in which new blood vessels form from the existing vascular network by proliferation and migration of endothelial cells, is an important rehabilitative strategy validated in multiple forms of cardiac disease animal models. 29 Our results showed that Interestingly, previous study had highlighted the critical role of miR-126, partly functioning by directly repressing negative regulators of the VEGF pathway, including the PIK3R2 and SPRED1. 16 Our recent research also found that ET could significantly up-regulate the expression of myocardium miR-126, suppress the expression of PIK3R2/SPRED1 and promote angiogenesis in peri-infarct zone. 19 Consequently, we hypothesized that there may be a functional interaction linking HIF-1α and miR-126. In the present study, DMOG and 2ME2 significantly up-regulated and down-regulated the expression levels of miR-126 in HUVECs in the concentration-dependent manner compared with the untreated group.
These findings indicated that HIF-1α could induce the expression of miR-126 in HUVECs.
Angiogenesis is a complex process precisely regulated by multiple signalling pathways and molecules. 38 Hundreds of genes involved in different steps of angiogenesis are independently responsive to hypoxia. miR-126, highly expressed in vascular endothelial cells, is considered a master regulator of physiological angiogenesis. 15 Vascularization of the injured myocardium can be found in wild-type post-MI mice. In contrast, there was a relative paucity of new vessels in the miR-126 null mice. 16  In summary, we demonstrated that HIF-1α, whose expression experienced up-regulation during ET, could function as an upstream regulator to miR-126, resulting in angiogenesis promotion through the PI3K/AKT/eNOS and MAPK signalling pathway and consequent improvement of the MI heart function. This study provides evidence for what we believe is a novel molecular pathway underlying the ET-induced cardiac repair. The importance of miR-126 may be considered as a potential candidate for therapeutic approach against angiogenesis-related disorders.

ACK N OWLED G EM ENTS
We highly appreciate the members for Institute of Sports and Exercise Biology participating in this study. We thank Mrs Shanshan Ding for her assistance in English editing.

CO N FLI C T O F I NTE R E S T
All authors approve final version of manuscript and declare that there is no conflict of interest.