CD226 deletion improves post-infarction healing via modulating macrophage polarization in mice

Macrophages are essential for wound repair after myocardial infarction (MI). CD226, a member of immunoglobulin superfamily, is expressed on inflammatory monocytes, however, the role of CD226 in infarct healing and the effect of CD226 on macrophage remain unknown. Methods: Wild type and CD226 knockout (CD226 KO) mice were subjected to permanent coronary ligation. CD226 expression, cardiac function and ventricular remodeling were evaluated. Profile of macrophages, myofibroblasts, angiogenesis and monocytes mobilization were determined. Results: CD226 expression increased in the infarcted heart, with a peak on day 7 after MI. CD226 KO attenuated infarct expansion and improved infarct healing after MI. CD226 deletion resulted in increased F4/80+ CD206+ M2 macrophages and diminished Mac-3+ iNOS+ M1 macrophages accumulation in the infarcted heart, as well as enrichment of α-smooth muscle actin positive myofibroblasts and Ki67+ CD31+ endothelial cells, leading to increased reparative collagen deposition and angiogenesis. Furthermore, CD226 deletion restrained inflammatory monocytes mobilization, as revealed by enhanced retention of Ly6Chi monocytes in the spleen associated with a decrease of Ly6Chi monocytes in the peripheral blood, whereas local proliferation of macrophage in the ischemic heart was not affected by CD226 deficiency. In vitro studies using bone marrow-derived macrophages showed that CD226 deletion potentiated M2 polarization and suppressed M1 polarization. Conclusion: CD226 expression is dramatically increased in the infarcted heart, and CD226 deletion improves post-infarction healing and cardiac function by favoring macrophage polarization towards reparative phenotype. Thus, inhibition of CD226 may represent a novel therapeutic approach to improve wound healing and cardiac function after MI.


Figure S1
Figure S1 A, Representative immunoblots showing CD226 expression in infarcted heart tissue at different time points after MI. B, Representative images of immunofluorescence staining for CD226 of WT hearts 7 days after sham surgery or MI. C. Representative images of immunofluorescence co-staining for CD226 (red) and Mac-3 (green) in the infarcted hearts of WT and CD226KO mice 7 days after MI. DAPI was used for nuclear staining (blue). Scale bars, 20 μm.         zone, and attenuated interstitial fibrosis in the border zone of CD226 KO mice, when compared to WT mice 5 weeks after MI. More noticeably, fibrotic tissue of WT infarct border zone was mainly composed of loosely assembled and fragmented fibers, whereas, closely assembled and well aligned fibers were predominant in CD226 KO mice. Scale bars as indicated.

Figure S10
Figure S10 Immunohistochemical staining for CD68 + macrophages in the infarct zone and border zone of WT and CD226 KO mice at day 7 after MI. Scale bars as indicated. Representative images from one of five samples are shown.

Figure S11
Figure S11 Immunohistochemical staining for CD68, Mac-3 and Ly6G in the infarct border zone of serial sections at day 7 after MI. Mac-3 + macrophages showed a similar observation with CD68 + macrophages. Ly6G + neutrophil infiltration was rarely observed in the ischemic heart at day 7 after MI. Scale bars, 50 μm.

Figure S12
Figure S12 Serial heart sections were used for immunohistochemical CD68 staining, Masson trichrome and picrosirius red staining. Increased accumulation of CD68 + macrophages corresponded to reduced interstitial fibrosis and well-aligned collagen fibers in the infarct border zone, as observed in CD226 KO mice. Scale bars as indicated. Representative images from one of five samples are shown.

Figure S18
A B Figure S18 A, Immunofluorescence staining for F4/80 (red) and Ki67 (green) in the infarcted hearts of WT and CD226 KO mice 7 days after MI. DAPI was used for nuclear staining (blue). B, quantification data of F4/80 + Ki67 + proliferating macrophages in the infarcted hearts, and no significant difference between WT and CD226 KO mice was noted (n=5-6 mice /each group). Scale bars as indicated.

Mouse model of MI surgery
Adult male CD226 KO and WT mice at the age of 10-12 weeks were subjected to permanent ligation of the left anterior descending (LAD) coronary artery as described previously. 1 Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg) and then intubated using a 20-gauge intravenous catheter with a blunt end and ventilated with air with a small-animal respirator (tidal volume, 0.3 ml; rate, 150 breaths/ minute; Harvard Apparatus, USA). The chest wall was shaved, and a left thoracotomy was performed in the fourth intercostal space. The left ventricle and the LAD coronary artery were visualized using a microscope and the LAD artery was permanently ligated with a 7-0 monofilament suture at 1-2 mm distal to its emergence from under the left atrium. Infarction was confirmed by the presence of myocardial blanching in the ischemic area and ST segment elevation on the electrocardiogram. The thoracotomy was closed with 5-0 silk sutures. The endotracheal tube was removed once spontaneous respiration resumed, and animals were placed on a warm pad maintained at 37 ℃ until they were completely awake. Sham-operated animals underwent the same procedure without coronary artery ligation. Mice that did not survive the recovery from anesthesia and mice that died within 24 hours after surgery were excluded from the experiment.

Echocardiography and hemodynamics
Serial transthoracic echocardiographic measurements at baseline, 1 week, and 5 weeks after MI surgery was performed with a VEVO 2100 platform (Visual Sonics, Toronto, Canada) as described previously. 2 Mice were anesthetized by inhalation of isoflurane (1-2%) in a 100% oxygen mix, and heart rate was maintained at approximately 400-500 bpm in all mice during the echocardiographic examination to minimize data deviation.

Hemodynamic measurements and pressure-volume loop analysis
Invasive cardiac catheterization studies were performed 5 weeks after coronary artery ligation, using a 1.4 F pressure-volume conductance catheter (SPR-839, Millar Instruments) as described previously. 3 Mice were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg) with spontaneous respiration. The catheter was inserted into the right carotid artery and guided into the LV. Hemodynamic and volume parameters including Heart rate, LV end-systolic pressure, LV end-diastolic pressure, maximum rate of isovolumic pressure development (±dP/dt max ), LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were measured and analyzed using a Power Lab System (AD Instruments Inc.). Pressure-volume loop data recorded at steady-state and during injection of hypertonic saline were analyzed for the calibration of parallel conductance volume (Vp). LV volume was calculated for each mouse from conductance volume corrected by the relative Vp.
No less than fifty sequential beats were averaged for each measurement. At the end of the experiments, anaesthetized animals were sacrificed by cervical dislocation. Hearts were rapidly excised and prepared for histological analysis and protein extraction.

Morphometric analysis
Heart tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm -thick sections.
Sections were stained with hematoxylin and eosin, Masson trichrome and picrosirius red to determine the infarct size, collagen volume fraction and morphological effects. Infarct size was represented by scar circumference expressed as the ratio of scar circumference to the total LV circumference, including the septum, as described previously. 1 The wall thickness of the infarct scars at the papillary muscle and apical levels were measured as well. Cardiac fibrosis in infarct border zone was analyzed on Masson trichrome-or picrosirius red-stained sections, and collagen volume fraction was calculated as the ratio of the total area of interstitial fibrosis to the total area of interest. Myocyte/fibrosis ratio in the left ventricular free wall were determined and expressed as the ratio of myocyte area to fibrosis area as previously reported. 4 Collagen content in the infarcted region was quantitatively assessed with ImagePro software and expressed as the percentage of the area of the infarct, as described previously. 5 All these parameters were assessed in 5 randomly chosen high-power fields (×200) in each section. Results from all slides obtained in the same heart were averaged, and counted as n=1.

Immunohistochemistry and immunofluorescence
Paraffin-embedded sections (5 µm) were deparaffinized and rehydrated followed by heat mediated antigen retrieval. For immunohistochemical analysis, sections were pretreatment with 0.3% hydrogen peroxide for 20 min to inhibit endogenous peroxidase activity. Subsequently, sections were blocked with 3% BSA or serum for 30 min and incubated with the following primary antibodies: rat anti-CD68 (Clone: FA

Bone marrow-derived macrophages (BMDM) and in vitro macrophages stimulation
The tibias and femora were flushed with precooled PBS containing 2% FBS, and bone marrow cells were collected and strained through a 100 µm-nylon mesh, followed centrifugation at 1200 rpm for 5 min and resuspended in RBC Lysis buffer, then cultured in DMEM medium (Gibco) with recombinant murine M-CSF  6 The cells were incubated with fresh media for the unstimulated negative controls. The cells were harvested for RNA isolation.

RNA isolation and quantitative real-time polymerase chain reaction (PCR)
Total RNA was extracted using the TRIzol Reagent (Invitrogen) according to the manufacturer's instruction.
RNA levels were quantified using the NanoDrop ND-1000 Spectrophotometer (Thermo Scientific). Reverse transcription of equal RNA content (0.5 µg) was performed using the PrimeScript® RT Master Kit (Takara), and real-time quantitative PCR was conducted using SYBR® Premix Ex Taq™Ⅱ(Takara) in accordance with the manufacturer's protocol. The sequences of primer pairs were designed using Primer Premier 5.0 software and are described in Supplemental Table 3. The gene levels were normalized to the reference gene GAPDH and the data were reported as 2-∆Ct values±SEM.

Protein digestion, iTRAQ labeling, LC-MS/MS analysis and protein quantification analysis
Each sample was prepared with 30 μ L protein solution, and adjusted to the final concentration of was 100mM with DTT (Dithiotheritol), following heated in boiling water for 5 min and cooled to room temperature, 200 μL UA buffer was added, and centrifuged at 14000g for 15 min twice. Then the sample was alkylated using IAA (Iodoacetamide; 100mM IAA in UA) for 30 minutes in dark at room temperature, followed by adding UA buffer and dissolution buffer in succession and centrifugation. It was then incubated with trypsin buffer (4 μg Trypsin in 40 μL Dissolution buffer) at 37 ℃ for 16 hours. The filtrate was collected and peptide fragment was quantified with Nano Drop 2000. Digested peptides (100 μg) were labelled with iTRAQ reagents according to manufacturer's protocol (Applied Biosystems, USA). The labeled proteins were analyzed by nano LC-MS/MS using Q Exactive equipped with an Easy n-LC 1000 HPLC system (Thermo Scientific) by Genechem Company (Shanghai, China). The raw data and protein quantification data were analyzed with software Mascot 2.5 and Proteome Discovery version 2.1, and we set an arbitrary threshold of 1.2-fold to declare differences.

Statistical analysis
Data were analyzed with GraphPad Prism-5 statistic software. All values are presented as the mean±SEM of n independent experiments. One-way ANOVA was conducted across all investigated groups first. Post hoc tests were then performed with Bonferroni correction. For the iTRAQ data analysis, comparisons between 2 groups were performed using unpaired 2-tailed Student's t-test. Values of P < 0.05 were taken as statistically significant.