Left ventricles were homogenized, and the cells were lysed as described previously [ 25 ].
Briefly, samples were diluted , and recombinant cytokines were reconstituted and serially diluted to make a low-PMT standard curve. The plate was analyzed with a Bio-Plex System Workstation. Test runs were performed before analyses of the included samples to optimize the Luminex analyses. All samples were analyzed in duplicate to enhance precision.
Tube formation was observed under a phase-contrast microscope and photographed.
The results were expressed as the mean tube length and normalized to the control. The experiment was repeated three times. All statistical analyses were performed using GraphPad Prism 6. Single-cell suspensions from digested hearts were analyzed for CD45 and CD11b expression. Gating strategy for identification of immune cells isolated from mouse hearts.
Neutrophils were one of the first cell types recruited to the site of infarction and peaked on day 1 after MI, with a more than fold increase compared with that in the sham operated hearts Fig. Despite such remarkable neutrophil accumulation, macrophages were still the most abundant cell type after infarction, reaching a peak on day 3 after MI Fig. Further analysis revealed that in the resting state, the vast majority of macrophages in the heart were M2-like macrophages, which were significantly reduced on day 1 after MI, then recovered to a level similar to that seen in the sham group on day 3 after MI and remained stable up to day 7 after MI Fig.
The NK cell content in the infarcted hearts did not differ from that of the sham-operated hears on day 1 after MI, but it significantly increased and reached a peak at day 3 after MI Fig. T-cells as well as T-helper infiltration significantly increased on day 1 after MI in the infarcted hearts compared with the sham-operated hearts and remained high up to day 3 after MI but decreased to a level statistically indistinguishable from that in the sham hearts on subsequent days Fig.
B-cells had a slight, statistically nonsignificant increase on day 3 after MI in infarcted hearts compared with the sham hearts Fig. It has been well documented that the adaptive immune system plays an important role in inflammation after infarction [ 6 , 9 , 11 , 26 ]. There is evidence suggesting crosstalk between the heart and MLNs [ 27 ]. B-cells were dominant over macrophages and T-cells in the MLNs; the number of B-cells had increased at day 1 after MI, reached a maximum at day 3 after MI, and then returned to the level seen in the sham group Fig.
To obtain a picture of the molecular component of the immune response in prolonged ischemia reperfusion, we analyzed both the heart and the serum from the same mouse. The levels of major cytokines in the infarcted hearts significantly changed in a time-dependent manner Fig. These results indicate that heart function worsens after CSA treatment, which is associated with a tendency toward left ventricular dilatation. Consistently, histological examination of the mouse hearts 4 weeks after the infarction revealed a larger scar area with a thinner infarcted wall in the CSA group than in the VEH group Fig.
Because pharmacological intervention with CSA began at day 1 after infarction, the first time point at which we made a comparison was day 3 after MI. These data suggest that CSA has no profound effects on the major cellular components of the immune response when given from day 1 after MI. The Y-axis except in the first graph shows the percentage of the chosen cell population among 1. At the same time, CSA significantly reduced the total cell count Fig. Because MCP-1 is a potent stimulator of angiogenesis in infarcted hearts [ 28 , 29 ] and IL is believed to possess antiangiogenic effects [ 30 ], we next examined whether CSA affects angiogenesis after MI by comparing the vascularization of infarcted hearts in the VEH and CSA groups.
Next, to exclude the possibility of a direct effect of CSA on endothelial cells, we used a capillary-like tube formation assay to evaluate the impact of CSA on in vitro angiogenesis. This finding provides new knowledge on the interaction of CSA with infarcted hearts and suggests that the caution might be taken when use of CSA in the acute phase of MI with or without the cell transplantation. However, the immune response caused by permanent myocardial ischemia in MI is notably delayed [ 11 , 22 ]. Hypothetically, reperfusion opens the routes for inflammatory cells to and from the site of injury and therefore promotes faster immune response and subsequently faster healing [ 3 ].
In this respect, knowledge of the normal immune response following prolonged ischemia is essential for molecular cardiology. Further study to determine the reperfusion time window for the optimal modulation of the immune response after MI is required. In a study by Yan et al. Analysis of CD the mannose receptor — a surrogate marker of M2-like macrophages — reveals that in the resting state, the vast majority of heart macrophages are CD high. This is consistent with the fact that resident cardiac macrophages present in the heart at steady state [ 31 , 32 ] and the fact that M2 is a default state for cardiac resident macrophages [ 11 , 31 , 32 ].
Furthermore, we found that on day 1 after prolonged ischemia, the vast majority of heart macrophages become CD low , but, as mentioned above, this occurred without a notable increase in macrophage number. Three days after infarction, when the immune response reaches its peak, most of the macrophages express CD However, it is impossible to reach any conclusion regarding macrophage functional switching based only on surface markers, and in this respect, our findings have serious limitations.
There are countless reports on the involvement of a variety of cytokines and chemokines in the immune response after MI [ 11 , 34 ]. However, a majority of studies have focused on the mRNA expression levels of the corresponding cytokines either in the serum or in the heart. Few studies have provided simultaneous assessments of cytokine protein levels in serum and in the heart. Regulation of the immune response after MI is extremely complex. However, many cytokines involved in this process are not necessarily its main orchestrators. In this regard, it is likely that the reaction of the serum represents a systemic response to MI, one that is nonspecific and includes cross-activation of multiple organs and systems, including the liver [ 35 ], the bone marrow [ 36 ], the spleen and other lymphoid organs [ 37 ], the kidney [ 38 ] and others.
Our data show that the systemic response ceases quickly and that a further inflammatory response is driven locally at the site of infarction. IL-6 is a potent metabolic and immune regulator that has different actions in multiple cell types, acting as both a proinflammatory cytokine and an anti-inflammatory myokine [ 39 ]. Moreover, activation of inflammation stimulates cardiomyocyte proliferation in the neonatal heart, and blockade of IL-6 or signal transducer and activator of transcription 3 STAT3 , its downstream effector, blocks heart regeneration after apical resection [ 40 ].
This makes IL-6 a possible candidate for an intermediary between systemic and local responses, as well as between the metabolic and inflammatory pathways, suggesting that IL-6 acts as a key initiator and regulator of metabolic and immune responses in MI [ 39 , 41 ]. ILp40 is another well-known cytokine that is able to induce immune responses and has antiangiogenic activity.
Deficiency of ILp40 improves cardiac repair after MI by promoting angiogenesis [ 30 ]. Our data show that ILp40 operates in later stages of inflammation starting on day 3 after MI.
Sex differences in the myocardial inflammatory response to acute injury.
It is possible that ILp40 is necessary to counterbalance the wide range of proangiogenic factors. Nevertheless, its function should be further clarified. Levels of monocyte chemoattractant protein-1 MCP-1, or CCL2 are significantly elevated in the heart from day 1 to 3 post-infarction. Moreover, its maximum levels in the heart coincide with massive macrophage infiltration.
KC has been recognized to play a critical role in the induction of systemic inflammation and tissue damage after trauma-hemorrhage [ 46 ]. RANTES plays an important role in determing infarct size and postinfarction heart failure in mice [ 47 ] and has prognostic value in humans [ 48 ]. Another notable finding in this study is that continuous administration of CSA in the acute phase of MI causes functional deterioration and adverse remodeling of infarcted mouse hearts in association with reduced vascularization. This finding runs counter to a substantial number of previous studies suggesting a protective role of CSA in MI [ 16 , 17 , 49 ].
In addition, CSA is administered during ischemia or immediately on reperfusion in the majority of studies on cardioprotection [ 50 , 51 , 52 ], whereas, in our study, CSA is administered from day 1 after MI, following echocardiographic assessment and proper randomization. Additionally, only a single injection of CSA is used in the majority of studies on cardioprotection by CSA [ 52 ], whereas we used continuous administration of CSA for 5 days to imitate immunosuppression similar to that used for nonautologous stem cell transplantation [ 12 ], though the single dose of CSA used in our study is comparable to that used in cardioprotective studies [ 52 ].
Our findings suggest that the negative effect of CSA on the infarcted heart might interfere with the benefits of stem cell transplantation when CSA is used along with cell transplantation in the acute phase of MI. The precise mechanism responsible for the negative effect of CSA on the infarcted heart needs to be explored in the future. This suggests that CSA administered from day 1 after infarction does not significantly influence the recruitment of immune cells to the infarction site.
Given that the main producers of inflammatory cytokines and chemokines are immune cells, our findings imply that CSA changes the function of immune cells recruited to the infarction site; this may be the reason for the deterioration of infarcted hearts after CSA treatment. At this time, cytokine levels in serum as well as neutrophil and T-cell infiltration of the infarcted hearts are peaking.
In addition, we found that CSA reduced the vascularization of infarcted hearts. IL, in turn, is widely recognized as a suppressor of angiogenesis [ 30 ]. Under such conditions, less IL is required to counterbalance MCP-1 and allow sufficient angiogenesis.
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However, these possibilities need to be confirmed by experimental data, and this is a limitation of the present study. Taking this into consideration, further study on the detailed mechanisms of CSA interference with immune response and angiogenesis after MI is highly desirable. Such knowledge is essential for the development of novel approaches, including pharmacotherapies, for ischemic heart disease. The latter may stem from the effect of CSA on the secretion of major cytokines involved in the immune response after infarction.
These findings suggest that studies using CSA in the acute phase of MI need to be conducted with caution.
Eur Heart J. Late percutaneous coronary intervention prevents left ventricular remodeling and improves clinical outcomes in patients with ST-elevation myocardial infarction. Clin Cardiol. Kim CB, Braunwald E. Potential benefits of late reperfusion of infarcted myocardium. The open artery hypothesis.
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