Lymphangiogenic responses of lymphatic endothelial cells to steady direct-current electric fields.

Lymphangiogenesis plays pivotal roles in diverse physiological and pathological conditions. Steady direct-current electric fields (DC EFs) induce vascular endothelial behaviors related to angiogenesis have been observed. This study investigated the effects of DC EFs on the lymphangiogenic response of lymphatic endothelial cells (LECs). We demonstrated that EFs stimulation induced directional migration, reorientation, and elongation of human LECs in culture. These lymphangiogenic responses required VEGF receptor 3 (VEGFR-3) activation and were mediated through the PI3K-Akt, Erk1/2, and p38 MAPK signaling pathways in relation to the reorganization of the actin cytoskeleton. Our results indicate that endogenous EFs may play a role in lymphangiogenesis in vivo, and VEGFR-3 signaling activation may be involved in the cellular function of LECs driven by EFs.

VEGFR3 suppression through miR-1236 inhibits proliferation and induces apoptosis in ovarian cancer via ERK1/2 and AKT signaling pathways.

Vascular endothelial growth factor receptor 3 (VEGFR3) is expressed in cancer cell lines and exerts a critical role in cancer progression. However, the signaling pathways of VEGFR3 in ovarian cancer cell proliferation remain unclear. This study aimed to demonstrate the signaling pathways of VEGFR3 through the upregulated expression of miR-1236 in ovarian cancer cells. We found that the messenger RNA and protein of VEGFR3 were expressed in the ovarian cancer cell lines, but downregulated after microRNA-1236 (miR-1236) transfection. The inhibition of VEGFR3, using miR-1236, significantly reduced cell proliferation, clonogenic survival, migration, and invasion ability in SKOV3 and OVCAR3 cells (p < 0.01). The flow cytometry results indicated that the rate of apoptotic cells in SKOV3 (38.65%) and OVCAR3 (41.95%) cells increased following VEGFR3 inhibition. Moreover, VEGFR3 stimulation (using a specific ligand, VEGF-CS) significantly increased extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation (p < 0.01), whereas VEGFR3 suppression reduced p-ERK1/2 (67.94% in SKOV3 and 93.52% in OVCAR3) and p-AKT (59.56% in SKOV3 and 78.73% in OVCAR3) compared to the VEGF-CS treated group. This finding demonstrated that miR-1236 may act as an endogenous regulator of ERK1/2 and AKT signaling by blocking the upstream regulator of VEGFR3. Overall, we demonstrated the important role of the miR-1236/VEGFR3 axis in ovarian cancer cell proliferation by regulating the ERK1/2 and AKT signaling that might be an effective strategy against ovarian cancer.

Effects of plant-based medicinal food on postoperative recurrence and lung metastasis of gastric cancer regulated by Wnt/ß-catenin-EMT signaling pathway and VEGF-C/D-VEGFR-3 cascade in a mouse model.

BACKGROUND: The plant-based medicinal food (PBMF) is a functional compound extracted from 6 medicinal and edible plants: Coix seed, L. edodes, A. officinalis L., H. cordata, Dandelion, and G. frondosa. Our previous studies have confirmed that the PBMF possesses anti-tumor properties in a subcutaneous xenograft model of nude mice. This study aims to further investigate the effects and potential molecular mechanisms of the PBMF on the recurrence and metastasis of gastric cancer (GC). METHODS: Postoperative recurrence and metastasis model of GC was successfully established in inbred 615 mice inoculated with mouse forestomach carcinoma (MFC) cells. After tumorectomy, 63 GC mice were randomly divided into five groups and respectively subject to different treatments for 15 days as below: model control group, 5-Fu group, and three doses of PBMF (43.22, 86.44, 172.88 g/kg PBMF in diet respectively). The inhibition rate (IR) of recurrence tumor weights and organ coefficients were calculated. Meanwhile, histopathological changes were examined and the metastasis IR in lungs and lymph node tissues was computed. The mRNA expressions related to the canonical Wnt/ß-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and lymphangiogenesis were detected by RT-qPCR in recurrence tumors and/or lung tissues. Protein expressions of ß-catenin, p-ß-catenin (Ser33/37/Thr41), GSK-3ß, p-GSK-3ß (Ser9), E-cadherin, and Vimentin in recurrence tumors were determined by Western Blot. LYVE-1, VEGF-C/D, and VEGFR-3 levels in recurrence tumors and/or lung tissues were determined by immunohistochemistry staining. RESULTS: The mRNA, as well as protein expression of GSK-3ß were up-regulated and the mRNA expression of ß-catenin was down-regulated after PBMF treatment. Meanwhile, the ratio of p-ß-catenin (Ser33/37/Thr41) to ß-catenin protein was increased significantly and the p-GSK-3ß (Ser9) protein level was decreased. And PMBF could effectively decrease the mRNA and protein levels of Vimentin while increasing those of E-cadherin. Furthermore, PBMF markedly reduced lymphatic vessel density (LVD) (labeled by LYVE-1) in recurrence tumor tissues, and mRNA levels of VEGF-C/D, VEGFR-2/3 of recurrence tumors were all significantly lower in the high-dose group. CONCLUSIONS: PBMF had a significant inhibitory effect on recurrence and lung metastasis of GC. The potential mechanism may involve reversing EMT by inhabiting the Wnt/ß-catenin signaling pathway. Lymphatic metastasis was also inhibited by PBMF via down-regulating the activation of the VEGF-C/D-VEGFR-2/3 signaling cascade.

Exosomes derived from human umbilical cord mesenchymal stem cells regulate lymphangiogenesis via the miR-302d-3p/VEGFR3/AKT axis to ameliorate inflammatory bowel disease.

BACKGROUND: Exosomes released from human umbilical cord mesenchymal stem cell (hucMSC-Ex) have been revealed to hold great potential for the development of new treatment approaches for various diseases, including inflammatory bowel disease (IBD). Lymphatic vessels are vital for IBD development and progression to colorectal cancer (CRC), as an occluded conduit for lymphatic fluid to return to the blood. OBJECTIVE: The mechanism involved remains largely unexplored. Here, we investigate the therapeutic effect of hucMSC-Ex in a mouse model of IBD during the modulation of lymphangiogenesis. METHODS: We established a dextran sulfate sodium (DSS)-induced IBD model in BALB/c mice and observed the influence of hucMSC-Ex on tissue repair, intestinal lymphatic function, changes in lymphangiogenesis, and infiltration of macrophages. We also evaluated the functional changes of human lymphatic endothelial cells (hLECs) in vitro to determine the mechanism by which hucMSC-Ex regulate lymphangiogenesis. Finally, we identified key molecules in hucMSC-Ex by sequencing, database comparison, and cell validation. RESULTS: Results showed that hucMSC-Ex alleviates IBD in mice by improving intestinal lymphatic drainage, inhibiting lymphangiogenesis, and infiltration of macrophages. Mechanistically, the miRNA sequencing results showed that miR-302d-3p was highly expressed in hucMSC-Ex and played an important role in inhibiting lymphangiogenesis by targeting Fms-related receptor tyrosine kinase 4 (FLT4). At the same time, the phosphorylation of AKT was inhibited and vascular endothelial growth factor receptor 3 (VEGFR3) was reduced. CONCLUSION: Collectively, our study suggests that hucMSC-Ex can regulate lymphangiogenesis via the miR-302d-3p/VEGFR3/AKT axis to ameliorate IBD. Our findings identify VEGFR3 as a potential therapeutic target in IBD, where tightly regulated lymphangiogenesis is crucial in its pathogenesis and progression.

Inhibition of lymphangiogenesis impairs antitumour effects of photodynamic therapy and checkpoint inhibitors in mice.

Photodynamic therapy (PDT) has been shown to destroy tumour-associated lymphatic vessels. Therefore, we sought to investigate the functional outcomes of PDT-mediated damage to the lymphatic vessels. We observed that PDT with verteporfin, completely but transiently, blocks the functional lymphatic drainage in the orthotopic mammary tumour models. Sustained inhibition of lymphatic vessels regeneration induced by lenalidomide or the soluble form of vascular endothelial growth factor receptor 3 (sVEGFR3) that neutralises lymphangiogenic vascular endothelial growth factor C (VEGF-C), significantly impaired antitumour efficacy of PDT. Antilymphangiogenic compounds also significantly inhibited the ability of intratumourally inoculated dendritic cells (DCs) to translocate to local lymph nodes and diminished the number of tumour-infiltrating interferon-γ-secreting or tumour antigen-specific CD8+ T cells. Lenalidomide also abrogated antitumour effects of the combination immunotherapy with PDT and anti-programmed death-ligand 1 (PD-L1) antibodies. Altogether, these findings indicate that PDT-mediated damage to the lymphatic vessels negatively affects development of antitumour immunity, and that drugs that impair lymphatic vessel regeneration might not be suitable for the use in combination with PDT.

Vascular endothelial growth factor receptor-3 is a novel target to improve net ultrafiltration in methylglyoxal-induced peritoneal injury.

Appropriate fluid balance is important for good clinical outcomes and survival in patients on peritoneal dialysis. We recently reported that lymphangiogenesis associated with fibrosis developed in the peritoneal cavity via the transforming growth factor-ß1-vascular endothelial growth factor-C (VEGF-C) pathway. We investigated whether VEGF receptor-3 (VEGFR-3), the receptor for VEGF-C and -D, might be a new target to improve net ultrafiltration by using adenovirus-expressing soluble VEGFR-3 (Adeno-sVEGFR-3) in rodent models of peritoneal injury induced by methylglyoxal (MGO). We demonstrated that lymphangiogenesis developed in these MGO models, especially in the diaphragm, indicating that lymphangiogenesis is a common feature in the peritoneal cavity with inflammation and fibrosis. In MGO models, VEGF-D was significantly increased in the diaphragm; however, VEGF-C was not significantly upregulated. Adeno-sVEGFR-3, which was detected on day 50 after administration via tail vein injections, successfully suppressed lymphangiogenesis in the diaphragm and parietal peritoneum in mouse MGO models without significant effects on fibrosis, inflammation, or neoangiogenesis. Drained volume in the peritoneal equilibration test using a 7.5% icodextrin peritoneal dialysis solution (the 7.5% icodextrin peritoneal equilibration test) was improved by Adeno-sVEGFR-3 on day 22 (P<0.05) and day 50 after reduction of inflammation (P<0.01), indicating that the 7.5% icodextrin peritoneal equilibration test identifies changes in lymphangiogenesis. The solute transport rate was not affected by suppression of lymphangiogenesis. In human peritoneal dialysis patients, the dialysate to plasma ratio of creatinine positively correlated with the dialysate VEGF-D concentration (P<0.001). VEGF-D mRNA was significantly higher in the peritoneal membranes of patients with ultrafiltration failure, indicating that VEGF-D is involved in the development of lymphangiogenesis in peritoneal dialysis patients. These results indicate that VEGFR-3 is a new target to improve net ultrafiltration by suppressing lymphatic absorption and that the 7.5% icodextrin peritoneal equilibration test is useful for estimation of lymphatic absorption.

VEGF-trap aflibercept significantly improves long-term graft survival in high-risk corneal transplantation.

BACKGROUND: Graft failure because of immune rejection remains a significant problem in organ transplantation, and lymphatic and blood vessels are important components of the afferent and efferent arms of the host alloimmune response, respectively. We compare the effect of antihemangiogenic and antilymphangiogenic therapies on alloimmunity and graft survival in a murine model of high-risk corneal transplantation. METHODS: Orthotopic corneal transplantation was performed in hemevascularized and lymph-vascularized high-risk host beds, and graft recipients received subconjunctival vascular endothelial growth factor (VEGF)-trap, anti-VEGF-C, sVEGFR-3, or no treatment, beginning at the time of surgery. Fourteen days after transplantation, graft hemeangiogenesis and lymphangiogenesis were evaluated by immunohistochemistry. The frequencies of Th1 cells in regional lymphoid tissue and graft-infiltrating immune cells were evaluated by flow cytometry. Long-term allograft survival was compared using Kaplan-Meier curves. RESULTS: VEGF-trap significantly decreased graft hemangiogenesis as compared to the control group and was most effective in reducing the frequency of graft-infiltrating immune cells. Anti-VEGF-C and sVEGFR3 significantly decreased graft lymphangiogenesis and lymphoid Th1 cell frequencies as compared to control. VEGF-trap (72%), anti-VEGF-C (25%), and sVEGFR-3 (11%) all significantly improved in the 8-week graft survival compared to control (0%), although VEGF-trap was significantly more effective than both anti-VEGF-C (P < 0.05) and sVEGFR-3 (P < 0.05). CONCLUSION: In a clinically relevant model of high-risk corneal transplantation in which blood and lymphatic vessels are present and treatment begins at the time of transplantation, VEGF-trap is significantly more effective in improving long-term graft survival as compared to anti-VEGF-C and sVEGFR-3, but all approaches improve survival when compared to untreated control.

Soluble vascular endothelial growth factor receptor-3 suppresses allosensitization and promotes corneal allograft survival.

PURPOSE: To investigate the effect of VEGF-C and VEGF-D blockade via soluble VEGFR-3 (sVEGFR-3) on T cell allosensitization, corneal neovascularization, and transplant survival. METHODS: Corneal intrastromal suture placement and allogeneic transplantation were performed on BALB/c mice to evaluate the effect of sVEGFR-3 on corneal neovascularization. Soluble VEGFR-3 trap was injected intraperitoneally to block VEGF-C/D (every other day starting the day of surgery). Immunohistochemical staining of corneal whole mounts was performed using anti-CD31 (PECAM-1) and anti-LYVE-1 antibodies to quantify the levels of hem- and lymphangiogenesis, respectively. Mixed lymphocyte reaction (MLR) was performed to assess indirect and direct host T cell allosensitization and the frequencies of IFN-γ-producing T cells in the draining lymph nodes were assessed using flow cytometry. Graft opacity and survival was evaluated by slit-lamp biomicroscopy. RESULTS: Treatment with sVEGFR-3 resulted in a significant blockade of lymphangiogenesis 2 weeks post-transplantation and significantly prolonged corneal allograft survival compared to the control group at 8 weeks post-transplantation (87.5 % vs. 50 %), and this was associated with significant reduction in the frequencies of allosensitized T cells and decreased frequencies of IFN-γ-producing CD4 T cells. CONCLUSIONS: Soluble VEGFR-3 suppresses corneal lymphangiogenesis and allograft rejection and may offer a viable therapeutic modality for corneal neovascularization and corneal transplantation.

Soluble vascular endothelial growth factor receptor 3 is essential for corneal alymphaticity.

Corneal transparency is a prerequisite for optimal vision and in turn relies on an absence of blood and lymphatic vessels, which is remarkable given the cornea's proximity to vascularized tissues. Membrane-bound vascular endothelial growth factor receptor 3 (VEGFR-3), with its cognate ligand vascular endothelial growth factor C (VEGF-C), is a major mediator of lymphangiogenesis. Here, we demonstrate that the cornea expresses a novel truncated isoform of this molecule, soluble VEGFR-3 (sVEGFR-3), which is critical for corneal alymphaticity, by sequestering VEGF-C. sVEGFR-3 binds and sequesters VEGF-C, thereby blocking signaling through VEGFR-3 and suppressing lymphangiogenesis induced by VEGF-C. sVEGFR-3 knockdown leads to lymphangiogenesis and hemangiogenesis in the mouse cornea, while overexpression of sVEGFR-3 inhibits lymphangiogenesis and hemangiogenesis in a murine suture injury model. Pax6(+/-) mice spontaneously develop corneal and lymphatic vessels and are deficient in sVEGFR-3. sVEGFR-3 suppresses hemangiogenesis by blocking VEGF-C-induced phosphorylation of VEGFR-2. Overexpression of sVEGFR-3 leads to a 5-fold increase in corneal transplant survival in mouse models. sVEGFR-3 holds promise as a molecule to control and regress lymphatic-vessel-based dysfunction. Therefore, sVEGFR-3 has the potential to protect the injured cornea from opacification secondary to infection, inflammation, or transplant rejection.