New Model of Macrophage Acquisition of the Lymphatic Endothelial Phenotype

Abstract

BackgroundMacrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions.Methodology/Principal FindingsTime-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b+/VEGFR-3+ LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels.Conclusions/SignificanceWe present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the “RAW model” mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.

Cyclooxygenase-2–dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer

“…Whether COX-2 inhibition is also acting directly on the lymphatic vasculature is unanswered by our current studies and merits further investigation. Furthermore, macrophages are an additional cell type thought to play crucial roles in both normal and tumor-associated lymphangiogenesis (30,(42)(43)(44). Since macrophages are increased in the postpartum involuting gland (4,45), we also recognize the importance of determining whether macrophages are driving lymphangiogenesis in the postpartum mammary gland.…”

Inflammation-associated lymphangiogenesis: a double-edged sword?

“…Although this remains the mainstream theory, some recent evidence suggests that circulating progenitor cells might be incorporated directly into the growing lymphatic vessels and transdifferentiate into LECs (lymphovasculogenesis; Figure 1 and refs. [15][16][17][18][19][20]. Macrophage transdifferentiation was reported in an experimental orchitis model (15), LPS-induced peritonitis model (19), corneal injury model (16), and in a human transplanted kidney (17).…”

“…[15][16][17][18][19][20]. Macrophage transdifferentiation was reported in an experimental orchitis model (15), LPS-induced peritonitis model (19), corneal injury model (16), and in a human transplanted kidney (17). Interestingly, under normal conditions the lymphatic endothelial progenitor cells appear only in minute quantities, but inflammatory stimuli lead to remarkable increases (18).…”

“…Interestingly, under normal conditions the lymphatic endothelial progenitor cells appear only in minute quantities, but inflammatory stimuli lead to remarkable increases (18). It is notable that all of these observations supporting the presence of lymphatic endothelial progenitor cells and lymphovasculogenesis have been made under inflammatory conditions (16)(17)(18)(19). Nevertheless, the concepts of lymphovasculogenesis and macrophage transdifferentiation remain unclear and require further examination.…”

Novel Identification of LYVE-1 Positive Macrophages in Rheumatoid Synovial Tissue

“…Furthermore, associations between CD68+ macrophage populations and metastatic lymph node cancers have also been observed [13]. Experiments to establish the localisation of transplanted LECPs have shown that they are rapidly integrated into lymphatic vessels [11] but that there is a low incorporation frequency of them into new vessels of between 2% -5% [14], although one peritonitis model study reported a 50% incorporation of LECPs [15]. However, some of these studies have also shown that LECPs may be play a vital role in both the initiation of vessel formation and the maintenance of these vessels as they are present for a minimum of six months in the tissue under investigation [16].…”