Bone defects caused by trauma, infection, cancer, congenital diseases and other factors are common diseases endangering human health (1–3). Bone repair remains a great challenge in orthopedics because of poor vascularization. Vascularization is essential for bone defect repair, and the degree of bone repair is directly proportional to the degree of vascularization at the bone defect site (4, 5). Therefore, vascularization is a basic research problem that needs to be solved urgently.
Macrophages are the key regulators of inflammation-related immunity and have multiple functions (e.g., angiogenesis and injury repair) (6, 7). Importantly, macrophages have excellent plasticity, and they may respond to various stimuli effectively via their polarization into different phenotypes (8, 9). Classic macrophages (M1) are activated by microbial agents (lipopolysaccharides and interferon-γ) that have proinflammatory effects. M1 macrophages secrete major inflammatory cytokines, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α, and upregulate enzyme-inducible nitric oxide synthase (iNOS) (10, 11). The other type of macrophages (M2) are alternately activated by IL-4 or IL-13 and having anti-inflammatory effects. M2 macrophages upregulate the enzymes arginase (Arg)-1 and chitinase-like protein 3 (Chil3/Ym1) and produce cytokines, including IL-10 and transforming growth factor (TGF)-β (12–14). It is worth noting that M2 macrophages can also promote angiogenesis by secreting high levels of platelet-derived growth factor-BB (PDGF-BB) (15).
The proangiogenic properties of macrophages are not shared among all subgroups because M2 macrophages have been shown to secrete more angiogenic growth factors and cytokines than M1 macrophages do in vitro (16). Although it has been reported that M1 macrophages can also produce proangiogenic cytokines, such as vascular endothelial growth factor (VEGF), to promote angiogenesis, the blood vessels formed only by stimulation with VEGF are immature, leak, and are easily degraded (17). PDGF-BB expressed and secreted by M2 macrophages can recruit pericytes and mesenchymal stem cells to remodel the initial vascular buds, stabilize neovessels, and coordinate osteoblast differentiation (18, 19). The role of M2 macrophages in promoting angiogenesis was first evident in the occurrence and development of malignant tumors. It has been widely recognized that tumor-associated macrophages (TAMs) promote tumor angiogenesis and the formation of vessels at the tumor site (20, 21). The above studies show that M2 macrophages are clearly the prevalent phenotype for angiogenesis during bone repair. Therefore, the enhancement of the M2 phenotype is considered a means to promote vascularization. However, the role of nontumor-associated M2 macrophages in angiogenesis and their production are currently poorly understood.
M2 macrophages are induced by activating various signaling pathways. Signal transducer and activator of transcription (STAT) 3, STAT6, interferon regulatory factor (IRF) 4, and peroxisome proliferator-activated receptor ɣ (PPARɣ) are important transcription factors involved in M2 polarization (22–24). In addition, IL-4/13 is a cytokine known to induce polarization of M2 macrophages through the Janus kinase (JAK)1/STAT6 pathway, which is of great significance to tissue repair, elimination of apoptotic cells, and inhibition of inflammatory responses (25, 26). IL-4 activates JAK phosphorylation by binding to IL-4Rα on the membrane, and JAK further phosphorylates STAT6, allowing it to enter the nucleus. After nuclear entry, STAT6 binds to the nuclear receptor PPARγ to mediate M2 polarization (27). This pathway is a key signaling node for the activation of M2 macrophages (26).
WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH2) is a modified hexapeptide identified by screening synthetic peptide libraries (28). Formyl peptide receptor (FPR), which belongs to the G protein-coupled receptor family, is a known high-affinity receptor for WKYMVm. FPR is mainly expressed in phagocytic cells, such as neutrophils, monocytes, and macrophages, and plays an important role in host defense and inflammation. It has three subtypes, namely, FPR1, FPR2/ALX, and FPR3 (28). It is interesting that WKYMVm, by binding to FPR2, shows immunomodulatory capabilities, especially immunosuppressive activity, under different physiological and pathological conditions (29, 30). This process is initiated by FPR2 interacting with agonists to activate other signaling molecules, such as JAK, phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mitogen-activated protein kinases (P38, ERK, and JNK), thus ultimately promoting cell migration, phagocytosis, and inflammatory cytokine secretion (31, 32). However, the effect of WKYMVm on macrophages, key immune regulators, has hardly been studied, and the underlying mechanism is still unclear. Studies have found that in human lung cancer cells, CaLu-6, WKYMVm can bind to FPR2 to mediate epidermal growth factor receptor (EGFR) transactivation and further trigger the JAK1/STAT6 pathway (28). Therefore, it is reasonable to speculate that WKYMVm may modulate macrophages through the JAK1/STAT6 pathway.
In this study, it was hypothesized that WKYMVm can polarize macrophages to the M2 type, thus indirectly affecting remodeling and forming new blood vessels for bone repair. The results from this work show that WKYMVm induces polarization of M2 macrophages in vivo and in vitro, leading to angiogenesis. Additionally, the molecular mechanism underlying these phenomena was found to be based on the activation of the JAK1/STAT6 pathway mediated by WKYMVm. This study suggests that WKYMVm exerts a regulatory effect on the macrophage phenotype for angiogenesis, thus providing new ideas for promoting vascularization in the repair of bone defects.