Heart Vessels DOI 10.1007/s00380-013-0425-z

ORIGINAL ARTICLE

Cardioprotective effects of adipokine apelin on myocardial infarction Bao-Hai Zhang • Cai-Xia Guo • Hong-Xia Wang Ling-Qiao Lu • Ya-Jie Wang • Li-Ke Zhang • Feng-He Du • Xiang-Jun Zeng

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Received: 1 May 2013 / Accepted: 27 September 2013 Ó Springer Japan 2013

Abstract Angiogenesis plays an important role in myocardial infarction. Apelin and its natural receptor (angiotensin II receptor-like 1, AGTRL-1 or APLNR) induce sprouting of endothelial cells in an autocrine or paracrine manner. The aim of this study is to investigate whether apelin can improve the cardiac function after myocardial infarction by increasing angiogenesis in infarcted myocardium. Left ventricular end-diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP), left ventricular developed pressure (LVDP), maximal left ventricular pressure development (±LVdp/dtmax), infarct size, and angiogenesis were evaluated to analyze the cardioprotective effects of apelin on ischemic myocardium. Assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-20 -deoxyuridine incorporation, wound healing, transwells, and tube formation were used to detect the effects of apelin on proliferation, migration, and chemotaxis of cardiac microvascular endothelial cells. Fluorescein isothiocyanate-labeled bovine serum albumin penetrating through monolayered cardiac microvascular

endothelial cells was measured to evaluate the effects of apelin on permeability of microvascular endothelial cells. In vivo results showed that apelin increased ±LV dp/dtmax and LVESP values, decreased LVEDP values (all p \ 0.05), and promoted angiogenesis in rat heart after ligation of the left anterior descending coronary artery. In vitro results showed that apelin dose-dependently enhanced proliferation, migration, chemotaxis, and tube formation, but not permeability of cardiac microvascular endothelial cells. Apelin also increased the expression of vascular endothelial growth factor receptors-2 (VEGFR2) and the endothelium-specific receptor tyrosine kinase (Tie-2) in cardiac microvascular endothelial cells. These results indicated that apelin played a protective role in myocardial infarction through promoting angiogenesis and decreasing permeability of microvascular endothelial cells via upregulating the expression of VEGFR2 and Tie-2 in cardiac microvascular endothelial cells. Keywords Apelin  Myocardial infarction  Endothelial cell  Angiogenesis

B.-H. Zhang and C.-X. Guo contributed equally to this study. B.-H. Zhang  H.-X. Wang  L.-Q. Lu  L.-K. Zhang  X.-J. Zeng (&) The Department of Pathophysiology, Capital Medical University, Beijing 100069, China e-mail: [email protected] B.-H. Zhang  C.-X. Guo  F.-H. Du (&) Division of Cardiology, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China e-mail: [email protected] Y.-J. Wang Laboratory of Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, China

Introduction Myocardial infarction is a leading cause of cardiovascular morbidity and mortality. After infarction, the repair process will be switched on, which strongly depends on the vascular status. Rapid angiogenesis and residual vascular potency are important for rebuilding coronary collateral circulation and improving blood supply to the infarction zone and its periphery [1–3]. New capillary networks are formed from the pre-existing vessels by the proliferation and migration of previously differentiated endothelial cells [4].

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Apelin, an endogenous ligand for the G-protein-coupled receptor APLNR [5], is expressed in a variety of tissues, including myocardium and vascular endothelial cells [6, 7]. Apelin displayed positive inotropic effects on myocardium [8], and its expression in rat was altered after acute ischemic injury [9]. In vitro studies revealed that apelin and its natural receptor (APLNR) induced sprouting of endothelial cells, thus indicating its role in angiogenesis [10– 13]. Therefore, apelin might play an important role in myocardial repair after infarction. Our previous studies revealed that apelin administration attenuated myocardial injury induced by isoproterenol overdose and ischemia in rats [14, 15]. Recent studies demonstrated that exogenously administrated apelin protected the ischemic myocardium against ischemia/reperfusion injury [16–18]. However, the effects of apelin/ APLNR on angiogenesis after infarction remain elusive. So far, no study has evaluated the effect of apelin/APLNR on promoting angiogenesis in ischemic heart. Interaction between vascular endothelial growth factor (VEGF) and its receptor VEGFR2 has powerful angiogenic and permeability-inducing effects [19]. Tie-2 is the receptor of angiogenic factor angiopoietin-1 (Ang-1) [20]. Transgenic mice lacking either Ang-1 or Tie-2 showed embryonic lethality. Morphologic assessments of Tie-2 or Ang-1-deficient mice have shown diminished vessel branching, loosening of endothelial cell contacts, poorly organized subendothelial matrix, and generalized lack of recruitment of pericytes [21, 22]. Based on our research, we hypothesized that apelin displays its cardioprotective effects by promoting angiogenesis. Therefore, our aim was to observe the angiogenic effects of apelin on rat heart after infarction. We also proposed to investigate whether apelin promoted angiogenesis in rat heart through upregulating the expression of VEGFR2 and Tie-2 in cardiac microvascular endothelial cells.

with saline group (MI ? saline group, n = 8), and myocardial infarction with apelin group (MI ? apelin group, n = 8). The myocardial infarction model was carried out by ligation of left anterior descending coronary artery (LAD) as previously described [24]. Sham-operated rats underwent an identical surgical procedure except suture of the coronary artery. Apelin-13 (20 nmol/kg/day) or saline and 5-bromo-20 -deoxyuridine (BrdU) (50 mg/kg/day) (Sigma, St Louis, MO, USA) were intraperitoneally injected once a day for 28 days. Apelin was injected on the onset of myocardial infarction after the operation. BrdU was primarily injected 72 h after the surgery, to be incorporated into proliferating cardiac microvascular endothelial cells. Hemodynamic measurements were performed 28 days after the operation under pentobarbital sodium (30 mg/kg, intraperitoneally) anesthesia and controlled ventilation. A catheter filled with heparin saline (250 IU/ml) was inserted from the carotid artery to the left ventricular cavity. The heart rate, maximal left ventricular pressure development (±LVdp/dtmax), left ventricular end-systolic pressure (LVESP), and left ventricular end-diastolic pressure (LVEDP) were recorded on a PowerLab 4S (ADInstruments, Sydney, Australia) as previously described [15]. After the measurements, rats were euthanized and hearts were removed rapidly to measure cardiac angiogenesis and infarct size. Determination of infarct size Infarct size was determined by the methods described by Pfeffer et al. [25]. Five-micrometer sections were cut and stained with Masson’s trichrome. Infarct length was measured along the endocardial and epicardial perimeters of infarct area from each of the left ventricular (LV) sections, and the values from all specimens were summed. Total LV circumference was calculated as the sum of endocardial and epicardial segment lengths from all LV sections. Infarct size (%) was calculated as total infarct circumference divided by total LV circumference.

Materials and methods Immunohistochemical staining for angiogenesis Animals and experimental model [23] Male Wistar rats (220–250 g) were acquired from the Animal Department, Capital Medical University. The experimental protocol was approved by the Animal Care and Use Committee of Capital Medical University. Animals were maintained in a pathogen-free environment, in temperature-controlled rooms with a 12:12-h light/dark cycle, and had free access to a standard diet and tap water. Rats were randomly divided into four groups: sham-operated rats (SHAM group, n = 8), myocardial infarction group (MI group, n = 8), myocardial infarction

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Angiogenesis was determined by von Willebrand Factor (vWF) and BrdU staining [26]. Four weeks after LAD ligation, the hearts were harvested. Optimum cutting temperature compound (OCT)-embedded sections were fixed with 4 % paraformaldehyde in phosphate-buffered saline (PBS), and incubated in 2 mol/l HCl at 37 °C for 60 min. Sections were incubated in blocking solution with primary antibodies at 4 °C overnight and with secondary antibodies at room temperature for 2 h. Primary antibodies were as follows: mouse monoclonal anti-BrdU antibody (1:800; Sigma) and rabbit polyclonal anti-vWF antibody (1:200;

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Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were TR-conjugated goat antimouse immunoglobulin G (IgG) (1:200) and fluorescein isothiocyanate-conjugated goat antirabbit IgG (1:100) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Capillaries in the border of infarct areas were counted under a fluorescence microscope at 209 magnification. vWF-positive staining was considered to be the total microvasculars, and BrdU and vWF colocalized staining was considered to be the newly generated microvasculars. Fifteen fields from three sections of each rat were randomly selected for the counts. Eight rats in each group were evaluated for the newly formed capillaries. Isolation and culture of cardiac microvascular endothelial cells Cardiac microvascular endothelial cells were isolated according to a method described in a previous study [27], which is very similar to the procedures originally developed by Nishida et al. [28]. The medium was replaced every 3 days. The cultured cells were identified by morphologic observation and positive staining with antibodies to vWF. The third to fifth passages of cardiac microvascular endothelial cells were used in the following experiments.

Cell proliferation MTT assay Proliferation of cells was evaluated using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake assay, in which MTT uptake into the cells is converted into formazan by mitochondrial dehydrogenase. The MTT assay was performed as previously described [29]. First, cardiac microvascular endothelial cells were collected and seeded at a density of 8.0 9 103 cells/well into 96-well flat-bottomed culture plates. At 80 % confluence, cells were partially starved in M199 supplemented with 1 % fetal bovine serum (FBS) for 24 h. The cells were then exposed to various doses (10 pM, 1 nM, 100 nM, and 1 lM) of apelin-13 (Sigma) at 37 °C for 24 h. Then, 20 ll of MTT working solution (5 mg/ml in PBS) was added to each well of cultured cells and incubated for 4 h at 37 °C in humidified air supplemented with 5 % CO2. Afterward, the formazan was solubilized with 150 ll of dimethylsulfoxide (DMSO). The absorbance was detected at 490 nm with a microplate reader (Wellscan MK3; Labsystems Dragon, Vantaa, Finland). Each experiment was performed three times to validate the results.

BrdU incorporation assay The BrdU incorporation assay was used as a measure of cell proliferation [30]. Cardiac microvascular endothelial cells were partially starved in medium M199 supplemented with 1 % FBS for 24 h, then added with different concentrations of apelin-13 and BrdU (3000 lg/l) for 24 h at 37 °C in humidified air supplemented with 5 % CO2. Cardiac microvascular endothelial cells were fixed with 10 % formalin in PBS. Fixed cells were subsequently treated with 2 N HCl for 60 min and incubated with a mouse monoclonal anti-BrdU antibody (Sigma, St Louis, MO), then incubated with Donkey polyclonal secondary antibody to sheep IgG-H&L (FITC) (Abcam, Hong Kong). Cell proliferation was quantified by the average ratio (BrdUpositive staining cells/total cells per field) in five fields per dish under a fluorescence microscope at 209 magnification. The experiment was repeated at least three times. Cell motility assay The chemotactic motility was assessed in a modified Boyden chamber assay as described previously [31]. In brief, the Boyden chamber with an 8 lm-pore polycarbonate filter (Millipore, Billerica, MA, USA) was coated with 50 ll Matrigel (Becton–Dickinson, Frankin Lakes, NJ, USA) in 37 °C for 30 min. Then fresh medium containing different concentrations of apelin-13 was placed in the lower wells. Trypsin-harvested cardiac microvascular endothelial cells (2 9 104) in 100 ll medium were loaded into each insert and the chamber was incubated for 24 h. Nonmigrating cells on the upper surface of the filter were removed by wiping with a cotton swab. Cell motility was quantified by counting cells that migrated across the filter toward the lower surface in five fields per filter under a microscope at 209 magnification. The experiment was repeated at least three times. Scratch-wound assay for migration Scratch-wound assay was carried out as described previously [32]. Cardiac microvascular endothelial cells were seeded (6 9 104/well) into dishes and grown to 100 % confluence and then synchronized in M199 medium supplemented with 1 % FBS for 24 h. Confluent cell monolayers were wounded by pressing a sterile 1000-ll pipette tip down onto the plate to cut the cell sheet and to mark on the plate a sharp visible demarcation at the wound edge (wounds were approximately 1 mm wide). The wounded monolayers were washed three times in PBS to remove cell debris and incubated for 24 h at 37 °C. M199 medium with 1 % FBS (control group) or different concentrations of apelin-13 were added to the wells and incubated at 37 °C

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with 5 % CO2. Five representative images from each well of the scratched areas under each condition were photographed at 24 h to estimate the migration distance. The experiments were performed at least three times. In vitro tube-like formation assay Twenty-four-well plates were coated with Matrigel (Becton–Dickinson) at a final concentration of 10 mg/ml (300 ll) and placed at 37 °C for 30 min or until the mixture gelled following the manufacturer’s instructions. Trypsinharvested cells (6 9 104) suspended in 1000 ll of the fresh medium with apelin-13 (0 nM, 10 pM, 1 nM, 100 nM, 1 lM) were seeded onto Matrigel and incubated for 24 h. The networks of tubes formed in each well were photographed in five random regions under a light microscope at 109 magnification [33]. The total length of tubes per image was analyzed using Image-Pro Plus 6.0 software. The experiments were performed at least three times. Endothelial permeability assay Endothelial permeability to macromolecules was assessed by measuring passage of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA; Sigma) across the monolayer. The assay method is based on a published protocol [34]. Polycarbonate micropore membranes in hanging cell-culture inserts (0.4 lm PET; Millipore) were gelatinized (type II calf skin gelatin). Endothelial cells (1 9 105 in 0.50 ml of M199) were then seeded to the gelatinized membranes and cultured for 5–7 days (37 °C, 5 % CO2) to allow the cells to be confluent. In brief, the system consists of two compartments separated by a microporous polycarbonate membrane lined with the endothelial cell monolayer. The luminal (upper) compartment (0.7 ml) was suspended in the abluminal (lower) compartment (2.5 ml). Medium in both well and insert containing cardiac microvascular endothelial cell monolayers were partially starved in M199 medium supplemented with 1 % FBS. After 1 h, the medium in the insert was replaced with 500 ll of M199 medium with 1 % FBS containing 0.5 mg/ml FITC-BSA. The medium in the well was replaced with 600 ll of M199 medium with 1 % FBS containing various concentrations of apelin-13 (0 M, 10 pM, 1 nM, 100 nM, 1 lM). At 1, 2, 3, and 4 h, 200-ll aliquots were removed and the same amount of M199 medium with 1 % FBS containing various concentrations of apelin-13 was added. The fluorescence of the aliquots was measured using a fluorospectrometer at 488/525 nm absorption/emission wavelengths for FITCBSA, and the concentration of FITC-BSA was calculated by reference to a set of standard dilutions.

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Western blot analysis The expression of VEGFR-2 and Tie-2 were evaluated by Western blot assay as described previously [35]. Cells were seeded in six-well plates, grown to 90 % confluence, and starved in medium M199 supplemented with 1 % FBS for 24 h, followed by stimulation with different concentrations of apelin (0 M, 10 pM, 1 nM, 100 nM, 1 lM) for 24 h. Cells were washed with ice-cold PBS, lysed with lysis buffer (50 mM Tris–HCl pH 6.8, 1 % Tween 20, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM Na3VO4, 1 mM NaF, 4 % sodium dodecyl sulfate (SDS), 0.2 % bromophenol blue, 20 % glycerol, and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany)) and boiled for 5 min. Protein concentration was measured by using the bicinchoninic acid assay (Bio-Rad, Munich, Germany). Aliquots of samples (50 lg/lane) were resolved by 10 % SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. After blocking with 5 % nonfat milk, the membranes were probed with primary antibodies (VEGFR2, 1:2000 and Tie-2, 1:1000; Cell Signaling Technologies, Beverly, MA, USA) at 4 °C overnight, followed by incubation with corresponding horseradish peroxidase-conjugated secondary antibody (1:2000; Amersham Pharmacia Biotech, Freiburg, Germany). Equal loading was controlled with antimouse glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (1:5000; Sigma–Aldrich, Taufkirchen, Germany). Visualization was done using the enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech), according to the manufacturer’s specifications. Chemiluminescence was captured on a Kodak XAR film. Signals were quantified using a BioDoc Analyzer (Goettingen, Germany). Statistical analysis The results were expressed as mean values ± standard deviation (SD). Statistical differences were evaluated by one-way analysis of variance, and the least-significantdifference test was used to compare the difference between two groups. P \ 0.05 was considered statistically significant.

Results One of nine rats in the sham group died after sham surgery; six of the 30 rats died after MI surgery. All the rats survived the period of the experiment.

Heart Vessels Table 1 Heart function after ligation of left anterior descending coronary artery (LAD) (mean ± SD, n = 8) Sham Heart rate (beats/min)

MI

MI ? saline

MI ? apelin

365 ± 20

382 ± 29

356 ± 26

361 ± 17

?LVdp/dtmax (mmHg/s)

2189.2 ± 366.3

1281.1 ± 152.1*

1151.1 ± 194.4*

2029.7 ± 189.5#

-LVdp/dtmax (mmHg/s)

-2132.0 ± 366.6

-1317.9 ± 127.3*

-1154.6 ± 197.4*

-2050.8 ± 226.7#

LVESP (mmHg)

120.1 ± 6.4

84.2 ± 6.1*

78.9 ± 12.1*

LVEDP (mmHg)

7.3 ± 2.1

12.7 ± 2.5*

13.4 ± 5.5*

112.8 ± 5.2

71.5 ± 4.9*

65.5 ± 10.3*

LVDP (mmHg)

112.5 ± 9.5# 5.2 ± 3.8# 107.3 ± 8.4#

Infarction size: sum of the inside and outside perimeter of collagen staining divided by that of the whole heart LVdp/dtmax maximal left ventricular pressure development, LVESP left ventricular end-systolic pressure, LVEDP left ventricular end-diastolic pressure, LVDP left ventricular developed pressure * P \ 0.05 compared with sham group #

P \ 0.05 compared with MI group

Cardiac function in rats after LAD ligation with or without apelin Compared with the sham group, MI group showed markedly lower ?LV dp/dtmax, -LV dp/dtmax, and LVDP values by 41.5 % (P \ 0.05), 38.2 % (P \ 0.05), and 36.6 % (P \ 0.05), respectively; lower LVESP by 29.9 % and higher LVEDP by 73.9 %, with significance (P \ 0.05 for both). Similarly, the MI ? saline group showed markedly lower ?LV dp/dtmax, -LV dp/dtmax and LVDP values by 47.4 % (P \ 0.05), 45.8 % (P \ 0.05) and 41.9 % (P \ 0.05), respectively; lower LVESP by 34.3 % and higher LVEDP by 83.6 %, in comparison with the sham group (P \ 0.05 for both). Compared with the MI group, the MI ? apelin group increased ?LV dp/dtmax, -LV dp/dtmax and LVDP values by 58.4 % (P\0.05), 55.6 % (P\0.05) and 50.1 % (P \ 0.05), respectively; and increased LVESP by 33.6 % and decreased LVEDP by 71.2 % (P \ 0.05 for both). Heart rate was not significantly altered (P [ 0.05) (n = 8; Table 1, Fig. 1a–e). Cardiac infarction size after LAD

quantified. MI ? apelin increased angiogenesis to 56 % of total capillaries compared with the MI ? saline group, which was 9.5 % (P \ 0.05; Fig. 2). Compared with the sham group, MI and MI ? saline groups showed no difference in the newly formed capillaries (2 % in sham group, 10 % in MI group, P \ 0.05, n = 8; Fig. 2). Proliferating effects of apelin on cardiac microvascular endothelial cells We examined the proliferation of cardiac microvascular endothelial cells with MTT assay and BrdU incorporation assay. After exposure to apelin for 24 h, cardiac microvascular endothelial cells showed an increased cell proliferation in a dose-dependent manner, while only 1 lM apelin caused a significant increase in cell proliferation (n = 5, P \ 0.05; Fig. 3a, c). Effects of apelin on the migration of cardiac microvascular endothelial cells

There was no difference between MI and MI ? saline groups in the infarct size (n = 8, P [ 0.05, Fig. 1f). Compared with the MI ? saline group, the MI ? apelin group showed a significantly decreased infarct size (n = 8, P \ 0.05; Fig. 1f).

We examined the effects of apelin on the migration of cardiac microvascular endothelial cells using the scratchwound assay. After exposure to apelin for 24 h, cardiac microvascular endothelial cells migrated into the wound for different distances, while only 1 lM apelin caused a significant increase in the cell migration (n = 5, P \ 0.05; Fig. 4b).

The effects of apelin on angiogenesis in heart after LAD ligation

Chemotactic effects of apelin on cardiac microvascular endothelial cells

Cardiac sections were stained with the antibody against vWF, an endothelial cell marker, and BrdU, a proliferating marker, to evaluate the angiogenesis after LAD ligation. Proliferated capillaries, which were double stained with vWF and BrdU in the border zone of the infarct area, were

We measured chemotactic effects of apelin on cardiac microvascular endothelial cells using the transwell assay. Apelin promoted the cardiac microvascular endothelial cells passing through the transwell in a dose-dependent manner (n = 5, P \ 0.05; Fig. 4c).

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Heart Vessels Fig. 1 The effects of apelin on the heart function of anesthetized rats with or without LAD ligation. a Apelin increased the LVESP value of heart after 4 weeks of LAD ligation. b, c Apelin increased the ±LV dp/dtmax value of heart after 4 weeks of LAD ligation. d Apelin decreased the LVEDP value of heart after 4 weeks of LAD ligation. e Apelin increased the LVDP value of heart after 4 weeks of LAD ligation. f Apelin decreased the infarct size of heart after 4 weeks of LAD ligation. LAD left anterior descending coronary artery, LVESP left ventricular end-systolic pressure, LVEDP left ventricular end-diastolic pressure, LVDP left ventricular developed pressure; ±LV dp/ dtmax maximal left ventricular pressure development. Data represent the mean ± SD (n = 8, *P \ 0.05 vs control group)

Stimulation of tube formation by apelin Microvascular endothelial cells without apelin treatment formed a less complete tubular network after 24 h incubation. Upon treatment with various concentrations of apelin, the formed network was more complete, with longer tubule length (n = 5; Fig. 5a). The total length of all tubes formed in each sample was measured and summarized. Apelin stimulated the tube formation of microvascular endothelial cells in a dose-dependent manner with a significant promotion at the concentrations of 1 nM, 100 nM, and 1 lm (n = 5, P \ 0.05; Fig. 5b). Apelin decreases the permeability of cardiac microvascular endothelial cells Apelin has been reported to regulate vascular permeability [36]. We analyzed the endothelial permeability in the presence of various concentrations of apelin using FITCBSA. As shown in Fig. 5c, we found that apelin (10 pmol/l)

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significantly decreased the amount of FITC-BSA passing through the monolayer in comparison with the control (n = 5, P \ 0.05; Fig. 5c). Thus, we concluded that apelin was able to significantly decrease the permeability of cardiac microvascular endothelial cells. Apelin upregulates VEGFR2/Tie-2 expression in cardiac microvascular endothelial cells To examine the effects of apelin on VEGFR2 and Tie-2 expression, confluent cardiac microvascular endothelial cells were exposed to apelin-13 (0–1 lM) for 24 h. As Fig. 2 Promotive effects of apelin on angiogenesis in rat heart. After c LAD ligation for 28 days, apelin increased the newly generated microvasculars. Green vWF-positive staining, indicating total microvasculars. Red BrdU-positive staining, indicating newly generated cells. Yellow BrdU and vWF colocalized staining, indicating newborn vessels. LAD left anterior descending coronary artery, BrdU bromodeoxyuridine, vWF von Willebrand factor. Data represent the mean ± SD (n = 6, *P \ 0.05 vs sham group)

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Fig. 3 Proliferating effects of apelin on cardiac microvascular endothelial cells. a Cardiac microvascular endothelial cells were incubated with apelin-13 (0–1 lM) for 24 h. MTT assay showed that cell viability was increased significantly at the concentration of 1 lM apelin-13 compared with the control group. Data represent the mean ? SD (n = 5, *P \ 0.05 vs control group). b, c BrdU incorporation assay showed that apelin dose-dependently promoted the BrdU incorporation into cardiac microvascular endothelial cells. Data are expressed as the mean ? SD (n = 5, *P \ 0.05 vs control group)

shown in Fig. 6a, b, apelin dose-dependently increased the protein expression levels of Tie-2 at 24 h compared with the control (n = 3, P\0.05). Similarly, the effects of apelin on the expression of VEGFR2 were further investigated by treating cardiac microvascular endothelial cells with 0–1 lM apelin. Following 24 h exposure to apelin, VEGFR2 levels were significantly increased (n = 3, P \ 0.01; Fig. 6c, d).

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Fig. 4 Promotive effects of apelin on migration and chemotaxis of cardiac microvascular endothelial cells. a Scratch-wound assay for cardiac microvascular endothelial cells. Injury was performed by scraping the monolayered microvascular endothelial cells (denuded area was between lines of panels). After 24 h incubation in the presence or absence of apelin, cells that migrated to the wound edge were observed by light microscopy. Migration distance into the denuded area is demonstrated on the graph. b The decreased distance to the denuded area was significantly enhanced by apelin-13 at the concentration of 1 lM compared with the control group (apelin at concentration of 0 nM). Data are expressed as the mean ? SD (n = 5, *P\0.05 vs control group). c Chemotactic effect of apelin on cardiac microvascular cells. Cells were treated with various concentrations of apelin (0–1 lM) after starvation for 24 h. Cells passed through the transwells were counted to measure the chemotactic effects. Data are expressed as the mean ? SD (n = 5, *P \ 0.05 vs control group)

Discussion Studies have shown that the concentration of apelin in plasma was decreased in patients with first ST-elevation myocardial infarction [37]. This finding encouraged us to investigate the role of apelin on myocardial infarction. Excitingly, our results revealed that apelin elevated the

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Fig. 6 Western blot analysis of Tie-2 and VEGFR2 in cultured cardiac microvascular endothelial cells. a Images were derived from identical gels of Tie-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with each lane loaded with the same amount of protein samples (50 lg). Numbers indicate the expected molecular weight of bands. b Densitometry of Tie-2 levels normalized to GAPDH for each treatment condition in a. c Images were derived from identical gels of VEGFR2 and GAPDH, with each lane loaded with the same amount of protein samples (50 lg). Numbers indicate the expected molecular weight of bands. d Densitometry of VEGFR2 levels normalized to GAPDH for each treatment condition in c. Data are expressed as the mean ? SD (*P \ 0.01 vs control group)

Fig. 5 Effects of apelin on capillary-like formation and permeability of cardiac microvascular endothelial cells. a Cardiac microvascular endothelial cells without apelin treatment formed a less complete tubular network after 24 h incubation. Upon treatment with various concentrations of apelin, the formed network was more complete with longer tube length. b The total length of all tubes formed in each sample was measured and summarized. Apelin stimulated the tube formation of cardiac microvascular endothelial cells in a dosedependent manner, with a significant promotion at the concentrations of 1 nM, 100 nM, and 1 lM. c Graphs showing the effect of apelin on fluorescein isothiocyanate-labeled bovine serum albumin (FITCBSA) passing through cardiac microvascular endothelial cells monolayer at different time points. Apelin dose-dependently inhibited the permeability of cardiac microvascular endothelial cells. Data are expressed as the mean ± SD (n = 5, *P \ 0.05 vs control group)

values of ±LV dp/dtmax and LVESP, lowered the LVEDP value, decreased the infract size of myocardium after LAD ligation for 4 weeks, which suggest that apelin has a strong cardioprotective effect after myocardial infarction. These results were consistent with those reported for perfused rat heart [38], and indicate that apelin produces similar protective effects in the heart independently of the protocol. The potential ways [39, 40] to protect myocardial infarction are: (1) reducing the myocardial oxygen consumption by decreasing the preload or afterload of heart, (2) dilating the vessels in ischemic myocardium, and (3) promoting angiogenesis after ischemia in myocardium.

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Our results showed that BrdU- and vWF-positive microvessels in peripheral myocardium were increased by apelin treatment after LAD ligation for 4 weeks, meaning that apelin promoted angiogenesis in ischemic myocardium. Therefore, we assumed that apelin protects the heart from ischemic injury by promoting angiogenesis in the myocardium. Therefore, we measured the proliferating effects of apelin on cardiac microvascular endothelial cells. MTT and BrdU incorporation tests showed that apelin induced the proliferation of cardiac microvascular endothelial cells in a dose-dependent manner (Fig. 3a–c). Tubeformation assay also revealed that apelin was able to stimulate cardiac microvascular endothelial cells to form a capillary-like structure on matrigel. These findings clearly suggest that apelin might be a crucial factor in angiogenesis. To protect the ischemic myocardium by angiogenesis, endothelial cells have to migrate to where new capillaries are formed. We inspected the chemotactic and migratory effects of apelin on cardiac microvascular endothelial cells. The results showed that apelin accelerated wound healing of cardiac microvascular endothelial cells (Fig. 4b) and increased the number of migrating cells in the experiment (Fig. 4c). These results confirmed that apelin facilitated vessel formation in the myocardial ischemic region to protect the heart from ischemic injury. Myocardial edema and inflammation in the infarction area are considered to be causal in the development of cardiac dysfunction [41, 42]. Endothelial damage caused by ischemia leads to the increased vascular permeability [43], which increases not only water permeability but also protein leakage, and enhances inflammation. In this study, we found that apelin inhibited the permeability of cardiac microvascular cells. These results indicated that apelin might alleviate myocardial injury by inhibiting vascular permeability (Fig. 5). VEGFR2 and Tie-2 are important for angiogenesis and permeability in myocardial microvasculars. In the present study, we detected upregulation of VEGFR2 and Tie-2 by apelin in cardiac microvascular endothelial cells (Fig. 6). VEGFR2 can promote proliferation and chemotaxis, and induce permeability of endothelial cells by binding to VEGF [19]. Meanwhile, Tie-2 can inhibit vascular permeability, tighten pre-existing vessels [44], and play a critical role in angiogenesis of endothelial cells via binding to angiopoietin (Ang)-1 [45]. These results suggest that apelin promotes angiogenesis of cardiac microvascular endothelial cells by increasing the expression of VEGFR2 and Tie-2. Collectively, the results of the present study identified the myocardial protective role played by apelin in myocardial infarction, by promoting angiogenesis and inhibiting the permeability of cardiac microvasculars.

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Acknowledgments This project was supported by the Natural Science Foundation of China (Grant No. 30900573) and Beijing Talents Education Project.

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