Abstract
Purpose: :
Activation and infiltration of myeloid derived cells are central to retinal vascular remodeling and regression. Their role in pathological angiogenesis remains ill defined. Given that environmental conditions and signals macrophages receive resulting in a paradigm of classical and alternative activation, we require greater understanding under which conditions macrophage soluble VEGF receptor-1 (sFlt-1) expression or VEGF expression occurs.
Methods: :
Bone marrow derived macrophages (BMDMs) or RAW264.7 cells were used. Cells were conditioned with either IFN-γ or LPS (classical; M1), IL-4 (alternative; M2) or PGE2 (pro-angiogenic; VEGFhi), and sFlt-1 mRNA and protein expression were determined. Specific sFlt-1/VEGF binding was confirmed by inhibition of VEGF ELISA and by modified binding assay with specific VEGF capture antibody paired with sFlt-1 detection antibody. HUVEC proliferation was also performed to elicit bioactivity of induced sFlt-1 under varying conditions. Specificity of sFlt-1 inhibition was confirmed in sFlt-1 siRNA knockdown as well as after IL-4 depletion following IL-4 conditioning (IL-4 deficient).
Results: :
Although IL-4 antagonizes IFN-γ or LPS-induced M1 phenotype in macrophages, in all conditions sFlt-1 was generated. IL-4-induced sFlt-1 expression was predominant with respect to dose and kinetics. IL-4-conditioned media perturbed VEGF ELISA, where specific sFlt-1 binding to VEGF was detected by our modified functional binding assay, demonstrating that IL-4-triggered sFlt-1 had specific affinity to VEGF. IL-4-conditioned media significantly inhibited HUVEC proliferation, as did IL-4 deficient conditioned media. Soluble Flt-1 knockdown in macrophages conditioned with IL-4 restored HUVEC proliferation. Whilst sFlt-1 was increased, VEGF level was suppressed in IL-4-conditioned macrophage, contrary to that seen in PGE2 conditioning and despite both conditions inducing M2 phenotype (arginase-1hi).
Conclusions: :
We have shown that IL-4 is the major inducer of macrophage sFlt-1, inferring that Th2 stimulation induces an anti-angiogenic response from macrophages. The results also counter the paradigm that the M1/M2 classification of macrophage activation is sufficiently robust when interrogating macrophage contribution to angiogenesis. Both IL-4 and PGE2 induced an M2 macrophage phenotype (arginase-1hi), yet generated an anti-angiogenic (sFlt-1hi and VEGFlo) and pro-angiogenic (sFlt-1lo and VEGFhi) phenotype, respectively.
Keywords: retinal neovascularization • vascular endothelial growth factor • wound healing