In recent years, the incidence rate and mortality rate of ischemic heart disease are increasing year by year. IRI often occurs in patients undergoing surgery for coronary artery disease, which can cause oxidative stress and trigger systemic inflammatory response, making patients face considerable surgical risk [20]. However, the molecular mechanism of IRI is not clear completely and needs to be further studied. Recent studies have found that PMN expression in peripheral blood increases after myocardial infarction, becoming the first cell to invade the heart [21]. In MI or heart failure, the expression level of SDF-1α is up-regulated, exerting stronger chemotaxis [22]. This is consistent with our study. Under the premise that there is no difference in basic information between PCI patients and the control group, compared with the control group, the expression levels of SDF-1α and PMN in the blood of PCI patients are increased, and both were statistical differences (P<0.05) (see Table 1). It shows that PMN and SDF-1α play an important role in the pathological mechanism of IRI.
In order to study how PMN induces myocardial damage after I/R, the specific mechanism of action of PMN and SDF-1α in IRI, the correlation between them, we established I/R mice model and H/R cells model.In I/R model mice, we found that the expression level of SDF-1α and CXCR4 in myocardial tissue increased significantly. It shows that SDF-1 and CXCR4 interact to form SDF-1/CXCR4 biological axis after IRI (Fig. 1A-D). As an important effector cell of inflammatory response, PMN can release MPO after activation, MPO is usually used as a marker of PMN aggregation [23]. Flow cytometry was used to detect the amount of water in cells positive for the PMN marker Ly6G to evaluate the migration of PMN. By testing the two indicators of MPO and Ly6G, we found that IRI promotes the massive migration and aggregation of PMN to myocardial tissue (Fig. 1E&F). Studies have shown that the SDF-1/CXCR4 signaling pathway is also an important pathway for chemotactic PMN migration [24]. Therefore, we speculate that the increased expression of SDF-1α can activate the SDF-1/CXCR4 signaling pathway, promote the migration and accumulation of PMN, lead to myocardial damage.In the H/R cell model, we further studied which cells secreted and enhanced SDF-1α expression under H/R conditions, we isolated and cultured primary mouse cardiomyocytes and cardiac fibroblasts. The supernatant of cultured cells was collected and tested by ELISA. It was found that the ability of H/R cardiomyocytes to secrete SDF-1α was increased significantly, while the ability of H/R cardiomyocyte fibroblasts to secrete SDF-1α did not change significantly (Fig. 1G). Through PCR and Western blot detection, it was found that SDF-1α, CXCR4 mRNA and protein expression in H/R cardiomyocytes increased, while H/R cardiomyocyte fibroblasts did not change significantly (Fig. 1E, F and H), indicating that H/R cardiomyocytes secrete and express SDF-1α, activate the SDF-1/CXCR4 signal pathway. Therefore, we speculate that the increased expression of SDF-1α in myocardial cells of I/R mice can promote PMN's damage. We have done the following research on the specific damage mechanism.
Studies have shown that PMN is the main source of reactive oxygen species (ROS) [25]. When a large amount of activated PMN is not cleared in time by the body, PMN extracellular trap net is attached to vascular endothelium, causing vascular endothelium apoptosis [26]. AMD3100, as a CXCR4 receptor blocker, can inhibit the interaction between SDF-1α and CXCR4 [27]. Therefore, by co-cultivating cardiomyocytes with PMN, we found that compared with the control group, the number of PMN migration increased in the H/R group (Fig. 2A), the recruitment of activated PMNs would cause cardiomyocyte apoptosis and oxidative stress levels. Both are elevated, but these changes can be blocked by AMD3100 (Fig. 2B-F). It shows that PMN migrates to cardiomyocytes through the SDF-1α/CXCR4 signaling pathway under H/R conditions and causes its damage. Then intervention on the migration of PMN and the impact on PMN cardiomyocytes may reduce the reperfusion injury.
In recent years, the rise of stem cell therapy has become a promising tool for the treatment of ischemic heart disease [28]. Stem cells recruited to ischemic tissue or injected into the infarcted heart may secrete various cytokines and adjust the local microenvironment, thereby enhancing the survival of cardiomyocytes, angiogenesis and heart regeneration [29]. Although many chemokines are involved in tissue inflammation and post-injury processes, SDF-1α is considered to be the main stem cell chemokine. SDF-1α combined with CXCR of stem cell plays an important role in regulating BM homing, reproliferation and mobilizing stem cells into peripheral blood [30]. In addition to participating in the mobilization and migration of stem cells, SDF-1α can also participate in ischemia-related signaling pathways to directly protect cardiomyocytes. Therefore, we have successfully constructed BMSCs (oe-SDF-1α) overexpressing SDF-1α (Fig. 3A-C), and co-cultured BMSCs and oe-SDF-1α with cardiomyocytes. By testing the migration ability of stem cells, cardiomyocyte apoptosis and oxidative stress levels (Fig. 4A-E) have found that oe-SDF-1α is more suitable as a tool cell for the treatment of IRI.
In view of the fact that the migration of BMSC and PMN to cardiomyocytes under H/R conditions is related to the SDF-1α/CXCR4 signaling pathway. We speculate that when myocardial ischemia occurs, the SDF-1α/CXCR4 pathway may eliminate dead cardiomyocytes by recruiting PMN on the one hand, and reduce the killing effect of PMN on normal cardiomyocytes by recruiting stem cells on the other hand, so as to promote the repair of heart injury. However, BMSC is only partially recruited to the site of myocardial injury in the treatment of ischemic heart disease, which has limitations [31]. It may be because in the inflammatory response, cathepsin G and elastase released by PMN can remove the essential N-terminal amino acid residues that interact between SDF-1α and CXCR4, thereby inactivating SDF-1 [31, 32]. And oe-SDF-1α can effectively improve this phenomenon. The results showed that compared with the H/R+'BMSC group, the H/R+'oe-SDF-1α group did not affect the migration of PMN to H/R cardiomyocytes (Fig. 5A&D), but it could reduce level of apoptosis in cardiomyocytes (Fig. 5B), reduce the level of ROS (Fig. 5C), reduce MPO activity (Fig. 5D), reduce the content of MDA (Fig. 5E), and enhance SOD activity (Fig. 5F) significantly. Interestingly, its effect on cardiomyocytes is equivalent to that of the (H/R+AMD3100)+'BMSC group, blocking PMN migration is equivalent. It shows that oe-SDF-1α can reduce the damage of PMN to cardiomyocytes without affecting the phagocytic function of PMN and the ability to generate NETosis.
There have been a large number of reports on the protective effects of BMSC treatment on various ischemic tissue damage in I/R animal models [33–37]. Pan et al. used adenoviruses overexpressing the SOD gene to infect BMSCs and transplanted them into the myocardium of MI mice. They found that the survival rate of BMSCs was greatly increased, the oxidative stress at the injured site was reduced, which has a potential cardioprotective effect [38]. However, BMSCs overexpressing SDF-1α are rarely studied in I/R therapy. In this study, we gave I/R mice intravenously with BMSC, oe-SDF-1α and CXCR4 antagonist AMD3100 to observe the effect of SDF-1α/CXCR4 signaling pathway in the treatment of myocardial IRI by oe-SDF-1α. We found that BMSC can improve cardiomyocytes (Fig. 6A), reduce the apoptotic rate of cardiomyocytes (Fig. 6B, E), inhibit the amount of PMN infiltration in myocardial tissue. Overexpression of SDF-1α in BMSC can promote BMSC to enter the site of myocardial injury (Fig. 6C and F), the number of PMN significantly decreases (Fig. 6D and G). Further promote its performance to improve cardiomyocytes and reduce the apoptotic rate of cardiomyocytes (Fig. 6A). Combined with the results of our in vitro experiments: oe-SDF-1α does not affect the migration of PMN to H/R cardiomyocytes. However, in I/R mice, oe-SDF-1α can reduce the amount of PMN, indicating that oe-SDF-1α can reduce PMN staying in the injury site of I/R mice. Although AMD3100 is used, it can prevent BMSC from accumulating to the site of myocardial injury. However, this study shows that AMD3100 cannot reduce the amount of PMN in the damaged myocardial tissue, suggesting that there may be other ways to promote the migration of PMN to the site of myocardial injury, which needs to be further studied.
Although the migration pathway of chemotactic PMN to the site of myocardial injury cannot be fully described, the role of PMN activation in the pathogenesis of cardiovascular disease is clear. PMN was recruited to the infarct area very early, they play a role by adhering to vascular endothelial cells through molecules of selectins, integrins and immunoglobulin superfamily [39]. Once adhered to the infarct area, PMN will release ROS, cytokines and proteolytic enzymes, resulting in the increase of MDA level and the decrease of SOD level [40]. The increase of MPO produced by PMN activation has a great adverse effect on left ventricular remodeling and function in the infarcted myocardium [41]. This is also applicable to IRI. Our study shows that compared with normal mice, the levels of ROS, MPO, and MDA in the myocardium of I/R mice increase, while the levels of SOD decrease (Fig. 7F-I) and SDF-1α in the blood increases (Fig. 7E). Although the exact mechanism of IRI is not very clear, it is currently believed to be related to the essence of IRI, in addition to the increased production of free radicals leading to oxidative stress and excessive activation of the inflammatory response [42].
The inflammatory response induced by myocardial IRI involves PMN infiltration of macrophages, thus exhibiting high expression of pro-inflammatory cytokines (such as IL-1β, IL-6 and TNF-α) and suppressing immune regulatory factors such as IL-10 [43]. TNF-α can promote inflammation damage, induce the synthesis of chemokines and adhesion molecules in the myocardium [44]. IL-1β mediates the recruitment and activation of inflammatory leukocytes, at the same time delays the activation of myofibroblasts. In vitro studies have shown that inhibiting IL-1β can reduce cardiomyocyte apoptosis [45]. IL-6 is also up-regulated in infarcted myocardium through IL-6 receptor β Subunit and activate JAK/STAT cascade to regulate inflammation [46]. IL-10 has a strong anti-inflammatory effect and can prevent excessive inflammation [47, 48]. Yang and his colleagues believe that the inflammatory response is enhanced after I/R in IL-10 knockout mice, which is manifested by increased PMN recruitment, increased plasma TNF-α levels, and increased ICAM-1 expression in tissues [49]. Therefore, we have confirmed from both oxidative stress and immune inflammation: injecting BMSCs overexpressing SDF-1α into the tail vein of I/R mice, on the one hand, promotes the accumulation of BMCS to the injury site, on the other hand, it can reduce the PMN in the myocardial injury site. It can reduce the level of ROS at the injured site, reduce the level of IL-1β, IL-6, and TNF-α in the serum, increase the level of IL-10, and play a role in inhibiting inflammation, thereby improving myocardial remodeling and function.