Abstract
Myocardial infarction (MI) triggers an inflammatory reaction, in which macrophages are of key importance for tissue repairing. Infiltration and/or migration of macrophages into the infarct area early after MI is critical for infarct healing, vascularization and cardiac function. Hydrogen sulfide (H2S) has been demonstrated to possess cardioprotective effects post MI and during the progress of cardiac remodeling. However, the specific molecular and cellular mechanisms involved in macrophage recruitment by H2S remain to be identified. In this study, the NaHS (exogenous sources of H2S) treatment exerted an increased infiltration of macrophages into the infarcted myocardium at early stage of MI cardiac tissues in both wild type (WT) and cystathionine-γ-lyase-knockout (CSE-KO) mice. And NaHS accelerated the migration of macrophage cells in vitro. While, the inhibitors not only significantly diminished the migratory ability in response to NaHS, but also blocked the activation of phospho-Src, -Pyk2, -FAK397 and -FAK925. Furthermore, NaHS induced the internalization of integrin β1 on macrophage surface, but, integrin β1 silencing inhibited macrophage migration and Src signaling activation. These results indicate that H2S may have the potential as an anti-infarct of MI by governing macrophage migration, which was achieved by accelerating internalization of integrin β1 and activating downstream Src-FAK/Pyk2-Rac pathway.
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Introduction
Myocardial infarction (MI) occurs, resulting in an inadequate substrate or oxygen supply of the downstream myocardium and further leading to cardiomyocytes deterioration1. These processes are mediated by a wide array of inflammatory reactions or factors2. Monocytes/macrophages, the major source of the inflammatory factors, are of central importance for healing after MI3,4. Macrophages reside in both healthy and injured heart and increase in number during disease5,6. And macrophage is a primary responder cell type that involved in the regulation of post-MI wound healing at multiple levels2,4.
Induced by MI, macrophages migrate into the infarct zone, initiate intracellular signaling, which localizes the inflammatory response to clean the debris and subsequently induce scar formation7. The infiltration of macrophages into the infarct area early after MI, is critical for infarct healing, vascularization and cardiac function8. Early depletion of infiltrating macrophages impaire wound healing, provoke adverse left ventricular (LV) remodeling and increase mortality after MI9. Injection of human activated macrophage suspension early after rat MI, promotes recruitment of resident macrophages and accelerates vascularization, tissue repair and improves cardiac remodeling and function10. Therefore, manipulating macrophage migration and function could be a promising therapeutic strategy in optimizing the process of infarct repair.
Hydrogen sulfide (H2S), as a gasotransmitter, has been demonstrated to possess cardioprotective function in various models of cardiac injury11,12. CSE (cystathionine-γ-lyase) is the predominant H2S-generating enzyme in the cardiovascular system and its deficiency significantly attenuates endogenous H2S and results in exacerbating myocardial ischemia/reperfusion injury13,14. Our previous studies, together with others, have demonstrated that H2S plays diverse roles in protecting against cardiovascular diseases such as atherosclerosis, myocardial ischemia and heart failure15,16,17,18,19. Some mechanisms are considered to contribute to the cardioprotection of H2S, such as protecting cells against oxidative stress by increasing glutathione, promoting the translocation of the nuclear transcription factor Nrf2 to induce the activation of numerous detoxifying genes20. However, the influence of H2S in macrophage migration and the contribution to infarct repair have not been clarified. Thus, the objective of this study was to elucidate the impact of H2S on macrophage infiltration and/or migration after MI and investigate the involved mechanisms, to offer a new dimension to our understanding of macrophage recruitment post MI.
Methods
Reagents and antibodies
NaHS was administered instead of H2S. NaHS and the Pyk2 inhibitor (PF431396) were obtained from Sigma-Aldrich; The FAK specific inhibitor (PF573228), the Src inhibitor (PP2) and the Rac1 inhibitor (NSC23766) were purchased from Selleck Chemicals. CES-siRNA (sc-142618) was obtained from Santa Cruz. Rhodamine was obtain from Cytoskeleton and DAPI from Beyotime; Following antibodies were used: anti-Rac, anti-Fak, anti-p-FAK397, anti-p-FAK925, anti-Src, anti-p-Src, anti-β-actin, anti-Integrin β1, anti-Integrin β3, anti-Cavenolin-1,anti-p-Pyk2 (Cell Signaling); anti-CSE (Santa Cruz); Anti-Integrin β1-FITC, anti-Galectin-3 (eBbioscience); anti-CD68 (Biolegend); Lactate dehydrogenase (LDH) assay from BeyotimeInstitute of Biotechnology.
Animals
Mice devoid of CSE (KO mice) were normal in growth rate and reproduction, but had markedly reduced endogenous H2S production. WT and KO mice used in this study were littermates obtained via heterozygous breeding. The animal procedures were performed in accordance with the Animal Management Rules of the local authorities and were approved by the ethics committee of Experimental Research, Shanghai Medical College, Fudan University.
MI Models
The left coronary artery was ligated permanently to induce myocardial infarction model as previously reported17.
Immunohistochemical analysis
The hearts excised from sacrificed animals were fixed in 4% paraformaldehyde, sectioned at 5 μm following embedded in paraffin. The immunohistochemical staining was performed with an EnVision Kit (Dako, Carpinteria, CA). Antibody specific for macrophages (anti-CD68, Biolegend and Galectin-3, eBioscience) was used to selectively detect macrophages.
Cell line culture
The murine macrophage cell line, RAW264.7, was purchased from American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FBS (HyClone, Logan, UT), at 37 °C in 5% CO2. The cells were treated with various concentrations of NaHS diluted in the RPMI 1640 medium (50, 100 and 200 mM) for different time points. Pharmacological inhibitors (5 μM of PF431396 or PF573228, 10 μM of PP2 and 25 μM of NSC23766) were treated for 1 hour before NaHS was added.
Isolation of macrophages from bone marrow
Bone marrow-derived macrophages (BMMs) were isolated using standard protocols21,22. After differentiation for 7 days in RPMI-1640 containing 10 ng/ml recombinant murine M-CSF, cells were either untreated or incubated with NaSH for Co-culturing.
Neonatal Mouse Primary Cardiomyocyte isolation
Primary cardiomyocytes were obtained from the ventricles of One- to two-day-old neonatal mice according to the method described23. The isolated primary cardiomyocytes were seeded into a 24-well plateat the density of 1*106/ml then subjected to hypoxia for 4 h, in accordance with the technique described24.
Co-culturing of BMMs and Cardiomyocyte
For transwell co-culturing, the 0.4-mm-pore size transwell insertscontaining 2*105 BMMs were placed into the 24-well plate with cardiomyocytes that were hypoxia initially. Co-culture system was incubated for 12 h.
Lactate dehydrogenaseassays
Lactate dehydrogenase (LDH) was detected to evaluate the severity of cardiomyocyte injury. LDH released in the culture medium was measured using commercial kits, according to the manufacturer’s instruction.
Real-time PCR
For CSE gene expression analysis, total RNA was extracted from RAW264.7 cells using TRIzol (Takara) method. cDNAs were synthesized with the RevertAidtm First Strand cDNA Synthesis Kit #1622 (Fermentas). The primer sequences for the CSE were previously described13.
Immunofluorescence and confocal microscopy
RAW264.7 cells plated onto glass coverslips were treated with various concentrations of NaHS for indicated time. The cells were fixed by 4% paraformaldehyde and blocked with 5% BSA (Amresco), following permeabilization by 0.5% Triton X-100. The cells were incubated with Rhodamine-conjugated phalloidin (red) for F-actin staining and DAPI (blue) for nucleus staining, or with primary antibody against integrin β1 followed by the incubation of appropriately labeled secondary antibodies. Confocal laser scanning was carried out with Zeiss710 confocal imaging system.
Migration
The migration of RAW264.7 cells exposed to NaHS was determined by Transwell assays using polycarbonate transwell filters (Corning, 8 μm). Cells incubated in the presence or absence of NaHS, or pharmacological inhibitors were seeded into the upper compartment (containing 1% FBS, while the lower compartment contains 10% FBS only). The cells were allowed to migrate for 6 h before they were fixed in cold methanol. The non-migratory cells in the upper compartment were removed with a cotton swab and the migrated cells were stained with 0.4% crystal violet (Sigma). For each experiment, the number of transmigrated cells in five random fields on the underside of the filter was counted and photographed and three independent filters were analyzed.
RNA interference
The siRNA to CSE (sc-142618) was obtained from Santa Cruz Biotechnology. And the siRNA to mouse integrin β1 were chemically synthesised by Shanghai GenePharma Co., Ltd. The siRNA sequences for integrin β1 were designed as follows: 5′-CAG AGA CAUUACUCAGAUdTdT-3′ (forward) and 5′-AUC UGA GUA AUG UCU UCU G dTdT-3′ (re-verse). A scrambled small RNA sequence was used as a negative control. The siRNAs were transfect into RAW264.7 cells in OPTI-MEM I Reduced SerumMedium (Gibco) for 24 or 48 h. The medium could be changed 6–8 h after transfection.
Protein isolation
Total proteins or cell surface proteins of cells were extracted by M-PER Mammalian Protein Extraction Reagent (Pierce) and Pierce Cell Surface Protein Isolation Kit (Thermo Scientific) respectively according to the manufacturer’s instructions. Membrane proteins were extracted by Membrane and Cytosol Protein Extraction Kit (Beyotime). The protein concentration was quantified using a BCA protein assay kit (Beyotime).
Western blot
The extracted proteins were separated and transferred to nitrocellulose membranes. The membranes were blocked, following immunoblotted with appropriate antibodies. Then, the blots were developed by ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore). And the immunoreactivity was visualized with a chemoluminescence reagent.
Statistical analyses
Quantitative data were presented as mean ± SEM. The calculations were performed with GraphPad Prism 6.0 software. Differences between two groups were analyzed by two-tailed Student’s t-tests, or assessed by one way analysis of variance with Tukey’s post-hoc test when more than two groups were compared. The differences were considered significant with P < 0.05.
Results
NaHS increased the infiltration of macrophages early after MI
Macrophage infiltrate into the myocardium is required for proper healing of infracted myocardium8. To identify the role of NaHS in cardiac remodeling, the recruitment of macrophages into the heart following MI was investigated by immunohistochemical analysis of CD68 and galectin-3, the macrophage markers. As shown in Fig. 1, the density of CD68 immunostaining in KO (CSE knock out)-MI mice was apparently less than that in WT-MI mice. In both WT and KO mice, NaHS treatment significantly increased the density of CD68 staining. Interestingly, the expression of the macrophage marker was increased by NaHS treatment in WT infarct LV at day 3, peaked at day 5 and weakened at day 8 post-MI. Moreover, in the KO mice, the NaHS treatment rescued the decrease of infiltrating macrophages caused by CES depletion and exhibited a similar time course for CD68 staining. Similarly, the immunostaining tendency of galectin-3 was consistent with CD68 (Supplemental Fig. 1). These findings indicate that H2S promotes macrophage infiltration in the early, but not later stage of MI.
To test the potent cardioprotective efficacy of NaHS, the Lactate dehydrogenase (LDH) level in BMMs cell supernatant was determined in NaHS treatment assay. First, the isolated neonatal mouse primary cardiomyocytes were impaired by hypoxia and LDH in supernatant was significantly increased, indicating a severity of cardiomyocyte injury (Fig. 2a). Then, BMMs, isolated from bone marrow of WT or KO mice, were co-cultured with the cardiomyocytes in the NaHS presence or not. As shown in Fig. 2b, although the LDH level was not apparently diminished in WT-BMMs and KO-BMMs groups, NaHS treated BMMs abrogated the LDH level largely compared to control group, demonstrating that macrophage infiltration triggered by NaHS antagonizes the damage of cardiomyocytes induced by hypoxia.
NaHS enhanced the migratory ability of macrophages
Cellular infiltration is suggested to be the migration of cells from their original sources. Given that NaHS preserved the cardiac function following MI through the increased infiltration of macrophages, it is still unclear whether and how NaHS stimulates macrophages to move from the border zone into the core of an infarct. To address these issues, the effect of NaHS on actin cytoskeleton was analyzed by immunofluorescent microscopy in the murine macrophage cells, RAW264.7. As shown in Fig. 3a, NaHS treatment resulted in significant rearrangement of the cellular actin cytoskeleton with the elongated lamellipodia, indicating a higher migration capability. The transwell assay also confirmed that NaHS dose-dependently increased the number of migratory RAW264.7 cells (Fig. 3b,c).
It is well known that Src/FAK axis played a pivotal role in macrophage mobilization and Pyk2 and Rac activation were involved in the destabilization of endothelial cell contacts25. The Src-mediated FAK phosphorylation on several tyrosine residues plays an essential role in macrophage locomotion26. Thus, to ascertain the involvement of this signaling pathway in H2S-promoted macrophage migration, the phosphorylated status of Src, Pyk2 and FAK and activation of Rac in RAW264.7 cells were examined. As expected, significant increases in phospho-Src, -Pyk2, -FAK397 and -FAK925 were observed in RAW264.7 cells exposed to NaHS for 0.5, 1 or 2 h. However, total Src and FAK were unaltered. Additionally, membrane bound Rac, the active form of Rac in the down-stream of Src/FAK signaling, was also increased under NaHS treatment for 3 h, while the loading control Caveolin-1 in cell membrane was not altered (Fig. 3d). Thus, those results indicate that H2S indeed accelerates the migration of macrophages and the Src-FAK/Pyk2-Rac signaling is involved in the process.
NaHS triggered macrophage migration through Src-FAK/Pyk2-Rac axis
To substantiate the Src-FAK/Pyk2-Rac axis is responsible for H2S triggered migration, the RAW264.7 cells were pre-incubated with the Pyk2 inhibitor PF431396, the FAK specific inhibitor PF573228, the Src inhibitor PP2, or the Rac inhibitor NSC23766 and then NaHS was added and incubated for 6 h. The migratory ability of the treated cells was determined by transwell assay. As shown in Fig. 4a,b, NaHS accelerated the migration of RAW264.7 cells, which were diminished by all the four inhibitors. Western blot results were also consistent with the results of transwell assay, revealed that PF431396 suppressed the NaHS-induced activation of Pyk2. Additionally, PF573228 significantly decreased the NaHS triggered phosphorylation of Fak, but had a weak effect on phosphorylated Src. PP2 completely blocked the phosphorylation of Src, Pyk2 and FAK induced by NaHS (Fig. 4c). These results confirm that the Src-FAK/Pyk2-Rac signaling is critical in H2S-induced macrophages migration and also indicate that the direct target of H2S is in the up-stream of this signaling pathway.
NaHS promoted the internalization of macrophage surface integrin β1
To pinpoint the direct target of H2S in triggering macrophage migration, the levels of integrin β1 and β3 in the surface of RAW264.7 cells with or without NaHS treatment were examined. The total level of integrin β1 or β3 did not change within the time course of NaHS treatment (Supplemental Fig. 2). While, the internalization of surface integrin β1 dramatically increased under NaHS treatment. And the surface integrin β1 decreased much more quickly in NaHS treated cells than in control cells. However, only a weak effect was observed on the internalization of integrin β3 (Fig. 5a). In addition, flow cytometric analysis also revealed that the surface integrin β1 was decreased much more quickly following NaHS treatment (Fig. 5b). To further verify the effect of NaHS on the internalization of integrin β1, confocal microscopic analyses were performed. As shown in Fig. 5c, within the indicated time course of treatment, the labeled integrin β1 was gradually internalized in the NaHS groups, but mainly presented on the plasma membrane in the untreated control. Collectively, these findings, on one side, suggest a function of H2S in regulating the internalization of macrophage surface integrin β1; on the other side, indicate that H2S may activate the Src-FAK/Pyk2-Rac signaling via an integrin β1-dependent pathway.
Integrin β1 silencing inhibited macrophage migration and Src-FAK/Pyk2 signaling activation induced by NaHS
To ascertain that integrin β1 is the target of H2S for promoting macrophages migration through activation of Src-FAK/Pyk2-Rac signaling, the integrin β1-specific-siRNA was introduced into RAW264.7 cells. The transfection efficiency was confirmed by western blot and the data showed that integrin β1 expression in the siRNA-β1 group was reduced significantly (Supplemental Fig. 3). Next, migratory capacity of the siRNA-β1 transfected cells in response to NaHS was evaluated by transwell assay. As shown in Fig. 5d, the increased migratory capacity of H2S treated macrophages was significantly impaired by integrin β1-siRNA. Furthermore, the silence of integrin β1 also inhibited the activation of Src-FAK/Pyk2 signaling induced by NaHS (Fig. 5e). Thus, these results confirm that integrin β1-Src-FAK/Pyk2-Rac signaling played a pivotal role in H2S-induced macrophage migration.
Endogenous CSE knockdown diminished macrophage migration, integrin β1 internalization and Src-FAK/Pyk2 signaling activation
To further explore the influence of H2S on macrophages migration, endogenous CSE was knocked down by specific siRNA in RAW264.7 cells before the NaHS treatment. Compared to siRNA-NC, the siRNA-CSE group significantly reduced the mRNA level of CSE (Fig. 6a). When the endogenous CSE was down-regulated, the NaHS induced migration of macrophages was abolished (Fig. 6b,c). Additionally, CSE silencing also abrogated the activation of phospho-Src, -Pyk2 and -FAK induced by NaHS. Further, although endogenous CSE knockdown did not alter the total amount of integrin β1, increased the cell surface integrin β1 (Fig. 6d). Taken together, these findings suggest that H2S indeed promotes macrophage migration via accelerating internalization of integrin β1 and activating the down-stream Src-FAK/Pyk2-Rac signaling.
Discussion
Up to date, therapeutic effects of clinically applicable drugs for MI are still not satisfactory1,27. H2S as a novel gaseous signaling molecule has been reported a potent cardioprotective effect16,17. Our previous study showed that increasing endogenous H2S by a water-soluble modulator S-Propargyl-Cysteine ameliorated ischemic conditions through angiogenesis promotion19. In our recent study, we also proved that decrease of H2S by CSE KO aggravated cardiac dysfunction and increased mortality post-MI; while, an improved cardiac remodeling and function accompanied with decreased mortality was observed in both WT mice and CSE KO-MI mice treated with different concentrations of NaHS, suggesting that a significant efficacy of H2S for MI treatment (unpublished data).
Macrophages digest debris and dominate inflammation resolution following MI28. At the early stage after MI, depletion of infiltrating macrophage aggravates pathological infarct healing, resulting in an increased left ventricular dilatation and wall thinning9. Acute MI alters the macrophage phenotype and supply chain29. While, increase in macrophage recruitment accelerates infarct repair and improves cardiac remodeling and function10. In the present study, by immunostaining with the antibodies against CD68 and galectin-3, the markers of macrophage30,31, we found that H2S could in vivo increase the infiltration of macrophages early after MI, suggesting a protective role of H2S in early post-MI inflammation and the subsequent healing process32.
Accentuation, prolongation, or expansion of the post infarction inflammatory response leads to a worse remodeling or dysfunction in MI33,34,35. Within the initial stage, MI, on one side, results in the migration of macrophages into the infarcted myocardium, initiating intracellular signaling; on the other side, macrophages responding to signals, could also move from the border zone into the core of an infarct2. The phase and rate of macrophage infiltration is orchestrated by a wide range of factors. And yet, the specific signals and mechanism responsible for macrophage migration remain unclear. In our present study, increased infiltrating macrophages raised an important question of how H2S triggers the migration of macrophages in the cardiac wound.
Previous report by Frangogiannis showed that macrophage infiltration and migration into the myocardium post-MI involved upregulation of both integrins and adhesion molecules36. Integrins, especially β1 and β3, are highly expressed isoforms in macrophages and have been reported to play important roles in receiving ECM signals and regulating cell migration37,38,39. Integrin trafficking, including internalization, is the crucial step in cell communication38. Downregulation of integrin β1 at the plasma membrane is a potential mechanism for enhancing cell motility40. Thus, through controlling integrin β1 internalization and recycling, junctional adhesion molecule could promote neutrophil chemotaxis41. Here, by biotin-based experiments, we found that H2S dramatically induced the internalization of integrin β1, but not integrin β3; and silence of integrin β1 inhibited the migration of macrophages, indicating that integrin β1 might be the possible target of H2S in regulating macrophage migration.
The internalization of integrin β1 can activate down-stream FAK/Src signaling, which is an important pathway involving in cell migration. The expression or activation of Src, a cellular nonreceptor tyrosine kinase in the downstream of integrin signaling, could reflect the migratory ability of macrophage42. As a substrate of Src, FAK serves as an adaptor that connects integrins with actin cytoskeleton43. Upon integrin activation, FAK is phosphorylated at tyrosine residues Tyr397 or/and Tyr925, which in turn activates the binding site Src42,43,44. Recently, Liu at al proved that integrins, phospho-FAK and phospho-Src participate in MI microenvironment improvement for myocardial repair45. By analogy to the related FAK kinase, Pyk2 is important in macrophage activation46. Additionally, evidence also shows that through controlling phosphorylation of Pyk2 and paxillin, Pyk2 regulates the polarization, migration and spreading of macrophage47. Rac, a member of Rho GTPase family, its activation is also related to the cell polarization and migration in macrophages48. In this study, the phospho-Src, -Pyk2, -FAK397 and -FAK925 were significantly upregulated upon H2S treatment and the downstream Rac was activated relatively later. Inhibitors for FAK, Src, Pyk2 and Rac could effectively diminish the migratory ability of macrophages stimulated by H2S and block the activation of related signaling molecules in response to H2S. Those results hint that H2S triggers macrophage migration via the Src-FAK/Pyk2-Rac axis.
In conclusion, our results revealed that H2S treatment exerted an increased infiltration of macrophages into the infarcted myocardium at early stage of MI in both WT mice and CSE-KO mice and the increased macrophage infiltrating antagonized the damage of cardiomyocytes induced by hypoxia. The favorable effects of H2S on the infarcted myocardium can be attributed to the promotion of infiltrating macrophages. Further, we present evidence for a novel mechanism that macrophage migration triggered by H2S is mediated by integrin β1-Src-FAK/Pyk2-Rac pathway. Thus, activation of macrophages by H2S supplement can be a promising therapeutic strategy for MI treatment.
Additional Information
How to cite this article: Miao, L. et al. Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction. Sci. Rep. 6, 22363; doi: 10.1038/srep22363 (2016).
Change history
02 February 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41598-022-06168-w
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Acknowledgements
This study was supported by the Key Program of National Nature Science Foundation of China (No. 81330080) and the Key Program of Shanghai Committee of Science and Technology in China (No. 14JC1401100).
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L.M., X.S. and Y.-Z.Z. designed the research; L.M., X.X. and H.X. performed the experiments. L.M. wrote the manuscript. X.X. and H.X. performed array analysis. Y.-Z.Z. and X.S. supervised the work and helped write the manuscript.
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Miao, L., Xin, X., Xin, H. et al. Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction. Sci Rep 6, 22363 (2016). https://doi.org/10.1038/srep22363
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DOI: https://doi.org/10.1038/srep22363
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