Angiogenic capacity of M1- and M2-polarized macrophages is determined by the levels of TIMP-1 complexed with their secreted proMMP-9

Abstract

Key Points• Acquisition of high angiogenesis-inducing capacity by human and murine macrophages requires their polarization toward the M2 phenotype.• M2-polarized macrophages shutdown their TIMP1 gene expression and initiate production of highly angiogenic TIMP-deficient proMMP-9.A proangiogenic function of tissue-infiltrating monocytes/macrophages has long been attributed to their matrix metalloproteinase-9 zymogen (proMMP-9). Herein, we evaluated the capacity of human monocytes, mature M0 macrophages, and M1-and M2-polarized macrophages to induce proMMP-9-mediated angiogenesis. Only M2 macrophages induced angiogenesis at levels comparable with highly angiogenic neutrophils previously shown to release their proMMP-9 in a unique form, free of tissue inhibitor of metalloproteinases-1 (TIMP-1). Macrophage differentiation was accompanied by induction of low-angiogenic, TIMP-1-encumbered proMMP-9. However, polarization toward the M2, but not the M1 phenotype, caused a substantial downregulation of TIMP-1 expression, resulting in production of angiogenic, TIMP-deficient proMMP-9. Correspondingly, the angiogenic potency of M2 proMMP-9 was lost after its complexing with TIMP-1, whereas TIMP-1 silencing in M0/M1 macrophages rendered them both angiogenic. Similar to human cells, murine bone marrow-derived M2 macrophages also shut down their TIMP-1 expression and produced proMMP-9 unencumbered by TIMP-1. Providing proof that angiogenic capacity of murine M2 macrophages depended on their TIMP-free proMMP-9, Mmp9-null M2 macrophages were nonangiogenic, although their TIMP-1 was severely downregulated. Our study provides a unifying molecular mechanism for high angiogenic capacity of TIMP-free proMMP-9 that would be uniquely produced in a pathophysiological microenvironment by influxing neutrophils and/or M2 polarized macrophages. (Blood. 2013;122(25):4054-4067) Introduction A strong link has been established between infiltrating leukocytes and various pathophysiological conditions involving tissue remodeling and transformation. [1][2][3] The leukocyte infiltrate can be represented by hematopoietic cells of different lineages, including lymphocytes, granulocytes, and macrophages. Tumor-associated macrophages (TAMs) have been implicated in cancer progression, 4,5 and high numbers of TAMs have been linked to poor prognosis in certain human malignancies. 6,7 A remarkable plasticity of macrophages allows them to acquire functionally distinct phenotypes. 8,9 Two major alternative phenotypes, namely M1 and M2, have been ascribed to tumor-suppressing and tumor-promoting TAMs, respectively, although a spectrum of activation states has been demonstrated in several settings. [10][11][12][13] In general, M1 macrophages are associated with an induction of strong immune response and tumoricidal activity. In contrast, M2 macrophages appear to suppress immune surveillance and enhance neovascularization.Macrophage-induced angiogenesis involves an angiogenic switch, 14,15 which is triggered by proteolytic release of directacting angiogenic ...

Liposome-induced immunosuppression and tumor growth is mediated by macrophages and mitigated by liposome-encapsulated alendronate

“…Similar to previously published findings [56], we also observed that PLN increased the number of TAMs, and this effect was prevented by PLN-alen, although these findings did not reach statistical significance probably due to the small sample size (Supplemental Fig. S5).…”

“…It was recently reported that PLN significantly enhanced tumor growth in a murine model of cancer and this was associated with diminished T-cell functionality and tumor infiltration [56], suggesting inhibition of antitumor immunity. We theorized that mechanisms of PLN-induced tumor growth involve immunosuppressive leukocytes such as tumor-associated macrophages (TAM) since macrophages have been implicated as key players in both the pharmacology of carrier-mediated therapies [10,37] and in cancer progression [21,43].…”

“…Spleens and tumors were excised and processed to obtain single cell suspensions as previously described [56]. Briefly, spleens were dissociated and passed through a 40 μm cell strainer, and red blood cells were lysed with ACK solution (K.D Medical, Columbia, Maryland).…”

“…It was reported that polystyrene nanoparticles enhanced tumor growth in murine cancer models through mechanisms involving complement C5a receptors and MDSCs, suggesting that interactions between nanoparticles and the immune system can have protumoral effects [46]. In addition, we have previously reported that a poly-ethylene glycol (PEG)-coated (pegylated) liposomal nanoparticle (PLN) carrier, similar to that used in patients, significantly enhanced tumor growth in mice bearing TC-1 tumors, a mouse model of human papilloma virus (HPV)-induced cancer [56]. In these studies, PLNs were administered as weekly intravenous injections at clinically relevant lipid doses (four doses of 47 nmoles/g or two doses of 85 nmoles/g).…”

“…The primary purpose of this study was to identify the subpopulation of immune cells which mediate the tumor promoting effects of particulate carrier systems, and to determine whether the tumor promoting and immunosuppressive effects of the carrier can be reversed with an immune modulatory and non-cytotoxic drug cargo. We chose PLN as the model carrier since we previously observed enhanced tumor growth with this nanoparticle [56]. We chose alendronate, an aminobisphosphonate, as the model drug cargo because PLN-encapsulated alendronate (PLN-alen) is not cytotoxic to leukocytes [51] and our formulation did not deplete tissue macrophages (Supplemental Fig.…”