Targeting Lymphatic Vessel Activation and CCL21 Production by Vascular Endothelial Growth Factor Receptor-3 Inhibition Has Novel Immunomodulatory and Antiarteriosclerotic Effects in Cardiac Allografts

Abstract

Background— Lymphatic network and chemokine-mediated signals are essential for leukocyte traffic during the proximal steps of alloimmune response. We aimed to determine the role of lymphatic vessels and their principal growth signaling pathway, vascular endothelial growth factor (VEGF)-C/D/VEGFR-3, during acute and chronic rejection in cardiac allografts. Methods and Results— Analysis of heterotopically transplanted rat cardiac allografts showed that chronic rejection increased VEGF-C + inflammatory cell and hyaluronan receptor-1 (LYVE-1) + lymphatic vessel density. Allograft lymphatic vessels were VEGFR-3 + , contained antigen-presenting cells, and produced dendritic cell chemokine CCL21. Experiments with VEGFR-3/LacZ mice or mice with green fluorescent protein–positive bone marrow cells as cardiac allograft recipients showed that allograft lymphatic vessels originated almost exclusively from donor cells. Intraportal adenoviral VEGFR-3-Ig (Ad.VEGFR-3-Ig/VEGF-C/D-Trap) perfusion was used to inhibit VEGF-C/D/VEGFR-3 signaling. Recipient treatment with Ad.VEGFR-3-Ig prolonged rat cardiac allograft survival. Ad.VEGFR-3-Ig did not affect allograft lymphangiogenesis but was linked to reduced CCL21 production and CD8 + effector cell entry in the allograft. Concomitantly, Ad.VEGFR-3-Ig reduced OX62 + dendritic cell recruitment and increased transcription factor Foxp3 expression in the spleen. In separate experiments, treatment with a neutralizing monoclonal VEGFR-3 antibody reduced arteriosclerosis, the number of activated lymphatic vessels expressing VEGFR-3 and CCL21, and graft-infiltrating CD4 + T cells in chronically rejecting mouse cardiac allografts. Conclusions— These results show that VEGFR-3 participates in immune cell traffic from peripheral tissues to secondary lymphoid organs by regulating allograft lymphatic vessel CCL21 production and suggest VEGFR-3 inhibition as a novel lymphatic vessel–targeted immunomodulatory therapy for cardiac allograft rejection and arteriosclerosis.

SAR131675, a Potent and Selective VEGFR-3–TK Inhibitor with Antilymphangiogenic, Antitumoral, and Antimetastatic Activities

“…It is well established that macrophages and also TAMs express VEGFR-3 (28)(29)(30). Moreover, VEGFR-3 inhibition decreased dendritic cell recruitment to the spleen and resulted in prolonged rat cardiac allograft survival (50). Here we have shown that SAR131675 strongly reduced TAM infiltration in the 4T1 and the RIP1.Tag2 models.…”

“…In the autophosphorylation assay in HEK cells, SAR131675 inhibited VEGFR-1 autophosphorylation with an IC 50 of about 1 mmol/L and VEGFR-2 with an IC 50 of about 280 nmol/L (Table 1). These results confirmed that SAR131675 moderately inhibits VEGFR-2 and has very little effect on VEGFR-1, showing a good selectivity for VEGFR-3, which is not the case for sunitinib.…”

Ischemia–Reperfusion Injury Enhances Lymphatic Endothelial VEGFR3 and Rejection in Cardiac Allografts

“…We previously showed that lymphatic endothelial hyaluronan receptor 1-positive (LYVE-1þ) vessels express LEC transcription factor Prox-1 but not rat endothelial cell antigen-1, which is specific for vascular endothelial cells (19). We found that cold and warm ischemia had no effect on the number of LYVE-1þ lymphatic vessels ( Figure 1A) or the proportion of VEGFR3, ICAM-1 and VCAM-1 expression in LYVE-1þ vessels in the donor hearts ( Figure 1B) after ischemia.…”

“…Increased lymphangiogenesis is consistently associated with episodes of acute and chronic rejection of kidney, heart and lung transplants (8,(15)(16)(17). Furthermore, VEGFR3 signaling promotes adaptive immunity after cornea transplantation (7,9,18) and regulates the initiation of alloimmune response in cardiac allografts (19); however, the exact mechanisms of these phenomena are still unclear.…”

Increased Production of Vascular Endothelial Growth Factor-D and Lymphangiogenesis in Acute Kawasaki Disease

“…35-37 VEGF receptor-3 is expressed in LEC, dendritic cells, and macrophages in corneal transplant and cardiac allograft. 35, 37 It has recently been shown that VEGF-D and VEGF receptor-3 are critical in antigen clearance and inflammation resolution through mobilization of inflammatory cells from inflamed tissue to the draining lymph nodes in models of skin inflammation and arthritis. 38- 40 In these cases, the lymphangiogenic growth factors are secreted from infiltrated macrophages, local inflamed tissue and draining lymph nodes.…”