The Role of VEGF-C Precursors in the Macrophage-Mediated Activation of VEGFR3 Signaling in Lymphatic Endothelium In Vitro

 

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Background: Lung transplantation (LTx) displays the worst long-term survival of all solid-organ transplantations due to the development of Bronchiolitis Obliterans Syndrome (BOS). Vascular endothelial growth factor receptor 3 (VEGFR-3) signaling block was recently reported to improve the chronic rejection rate in experimental heart transplantation. Vascular endothelial growth factor C (VEGF-C) can be released by macrophages (Mθ), binds to its receptor VEGFR-3 on lymphatic endothelial cells (LEC), and activates the VEGFR-3 signaling pathway leading to LEC proliferation. Unknown remains how VEGF-C release is regulated in Mθ after LTx and which role VEGFR-3 signaling plays during BOS development.

Method: The murine Mθ cell line RAW264.7 as well as primary bone marrow derived Mθ (BMDMs) were stimulated with lipopolysaccharide (LPS), LPS, and interferon-γ (INF) or INF-γ for 6 and 24 hours to mimic the ischemia–reperfusion injury (IRI). To elucidate the exact signaling pathway, p65 translocation or signal transducer and activator of transcription 1 (STAT1) were inhibited. Expression levels of Vegf-C and the VEGF-C processing molecules Adamts3 and Ccbe1 were determined by RT-qPCR, Western blot (WB) and flow cytometry. Conditioned medium (CM) of LPS stimulated Mθ, containing VEGF-C, was transferred on the LEC cell line SVEC4.10 and activation of the VEGFR-3 signaling pathway was evaluated by extracellular-signal regulated kinase phosphorylation (pERK) in WB.

Results: Vegf-C expression increased strongly upon LPS stimulation in all Mθ. Addition of INF-γ downregulated this increase at 6 and 24 hours. INF-γ alone decreased Vegf-C expression at 6 hours. P65 inhibition abolished the LPS induced increase of Vegf-C expression, whereas STAT1 inhibition lowered the downregulating effect of INF-γ. Ccbe1 as well as Adamts3 were expressed in Mθ, with exactly the same or similar expression pattern like Vegf-C, respectively. pErk levels of SVEC4.10 cells increased upon stimulation with Mθ conditioned medium.

Conclusion: Expression of Vegf-C, Ccbe1 and Adamts3 in Mθ was upregulated upon LPS in a p65 dependent manner and downregulated upon INF-γ in a STAT1 dependent manner. Mθ CM activated VEGFR3 signaling in LECs, shown by increased pERK levels. In summary, we describe molecular pathways of LEC activation in an in vitro model of IRI. Modulation of LECs by directly targeting Vegf-C expression in an in vivo model of LTx and its impact on the development of alloimmune response might be a promising target in the future.

Publication History

Article published online:
03 February 2022

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