VEGFR-3 signaling in macrophages: friend or foe in disease?

Lymphatic vessels have been increasingly appreciated in the context of immunology not only as passive conduits for immune and cancer cell transport but also as key in local tissue immunomodulation. Targeting lymphatic vessel growth and potential immune regulation often takes advantage of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling to manipulate lymphatic biology. A receptor tyrosine kinase, VEGFR-3, is highly expressed on lymphatic endothelial cells, and its signaling is key in lymphatic growth, development, and survival and, as a result, often considered to be "lymphatic-specific" in adults. A subset of immune cells, notably of the monocyte-derived lineage, have been identified to express VEGFR-3 in tissues from the lung to the gut and in conditions as varied as cancer and chronic kidney disease. These VEGFR-3+ macrophages are highly chemotactic toward the VEGFR-3 ligands VEGF-C and VEGF-D. VEGFR-3 signaling has also been implicated in dictating the plasticity of these cells from pro-inflammatory to anti-inflammatory phenotypes. Conversely, expression may potentially be transient during monocyte differentiation with unknown effects. Macrophages play critically important and varied roles in the onset and resolution of inflammation, tissue remodeling, and vasculogenesis: targeting lymphatic vessel growth and immunomodulation by manipulating VEGFR-3 signaling may thus impact macrophage biology and their impact on disease pathogenesis. This mini review highlights the studies and pathologies in which VEGFR-3+ macrophages have been specifically identified, as well as the activity and polarization changes that macrophage VEGFR-3 signaling may elicit, and affords some conclusions as to the importance of macrophage VEGFR-3 signaling in disease.

Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS.

The lymphatic vasculature is the natural pathway for the resolution of inflammation, yet the role of pulmonary lymphatic drainage function in sepsis-induced acute respiratory distress syndrome (ARDS) remains poorly characterized. In this study, indocyanine green-near infrared lymphatic living imaging was performed to examine pulmonary lymphatic drainage function in septic mouse models. We found that the pulmonary lymphatic drainage was impaired owing to the damaged lymphatic structure in sepsis-induced ARDS. Moreover, prior lymphatic defects by blocking vascular endothelial growth factor receptor-3 (VEGFR-3) worsened sepsis-induced lymphatic dysfunction and inflammation. Posttreatment with vascular endothelial growth factor-C (Cys156Ser) (VEGF-C156S), a ligand of VEGFR-3, ameliorated lymphatic drainage by rejuvenating lymphatics to reduce the pulmonary edema and promote draining of pulmonary macrophages and neutrophils to pretracheal lymph nodes. Meanwhile, VEGF-C156S posttreatment reversed sepsis-inhibited CC chemokine ligand 21 (CCL21), which colocalizes with pulmonary lymphatic vessels. Furthermore, the advantages of VEGF-C156S on the drainage of inflammatory cells and edema fluid were abolished by blocking VEGFR-3 or CCL21. These results suggest that efficient pulmonary lymphatic drainage is necessary for inflammation resolution in ARDS. Our findings offer a therapeutic approach to sepsis-induced ARDS by promoting lymphatic drainage function.

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 is required for button junction formation in lymphatic vessels.

Lymphatic capillaries develop discontinuous cell-cell junctions that permit the absorption of large macromolecules, chylomicrons, and fluid from the interstitium. While excessive vascular endothelial growth factor 2 (VEGFR2) signaling can remodel and seal these junctions, whether and how VEGFR3 can alter lymphatic junctions remains incompletely understood. Here, we use lymphatic-specific Flt4 knockout mice to investigate VEGFR3 signaling in lymphatic junctions. We show that loss of Flt4 prevents specialized button junction formation in multiple tissues and impairs interstitial absorption. Knockdown of FLT4 in human lymphatic endothelial cells results in impaired NOTCH1 expression and activation, and overexpression of the NOTCH1 intracellular domain in Flt4 knockout vessels rescues the formation of button junctions and absorption of interstitial molecules. Together, our data reveal a requirement for VEGFR3 and NOTCH1 signaling in the development of button junctions during postnatal development and may hold clinical relevance to lymphatic diseases with impaired VEGFR3 signaling.

VEGFR-3 signaling restrains the neuron-macrophage crosstalk during neurotropic viral infection.

Upon recognizing danger signals produced by virally infected neurons, macrophages in the central nervous system (CNS) secrete multiple inflammatory cytokines to accelerate neuron apoptosis. The understanding is limited about which key effectors regulate macrophage-neuron crosstalk upon infection. We have used neurotropic-virus-infected murine models to identify that vascular endothelial growth factor receptor 3 (VEGFR-3) is upregulated in the CNS macrophages and that virally infected neurons secrete the ligand VEGF-C. When cultured with VEGF-C-containing supernatants from virally infected neurons, VEGFR-3+ macrophages suppress tumor necrosis factor α (TNF-α) secretion to reduce neuron apoptosis. Vegfr-3ΔLBD/ΔLBD (deletion of ligand-binding domain in myeloid cells) mice or mice treated with the VEGFR-3 kinase inhibitor exacerbate the severity of encephalitis, TNF-α production, and neuron apoptosis post Japanese encephalitis virus (JEV) infection. Activating VEGFR-3 or blocking TNF-α can reduce encephalitis and neuronal damage upon JEV infection. Altogether, we show that the inducible VEGF-C/VEGFR-3 module generates protective crosstalk between neurons and macrophages to alleviate CNS viral infection.

Anatomy and function of the lymphatic vessels in the parietal pleura and their plasticity under inflammation in mice.

Inflammatory pleuritis often causes pleural effusions, which are drained through lymphatic vessels (lymphatics) in the parietal pleura. The distribution of button- and zipper-like endothelial junctions can identify the subtypes of lymphatics, the initial, pre-collecting, and collecting lymphatics. Vascular endothelial growth factor receptor (VEGFR)-3 and its ligands VEGF-C/D are crucial lymphangiogenic factors. Currently, in the pleura covering the chest walls, the anatomy of the lymphatics and connecting networks of blood vessels are incompletely understood. Moreover, their pathological and functional plasticity under inflammation and the effects of VEGFR inhibition are unclear. This study aimed to learn the above-unanswered questions and immunostained mouse chest walls as whole-mount specimens. Confocal microscopic images and their 3-dimensional reconstruction analyzed the vasculatures. Repeated intra-pleural cavity lipopolysaccharide challenge induced pleuritis, which was also treated with VEGFR inhibition. Levels of vascular-related factors were evaluated by quantitative real-time polymerase chain reaction. We observed the initial lymphatics in the intercostals, collecting lymphatics under the ribs, and pre-collecting lymphatics connecting both. Arteries branched into capillaries and gathered into veins from the cranial to the caudal side. Lymphatics and blood vessels were in different layers with an adjacent distribution of the lymphatic layer to the pleural cavity. Inflammatory pleuritis elevated expression levels of VEGF-C/D and angiopoietin-2, induced lymphangiogenesis and blood vessel remodeling, and disorganized the lymphatic structures and subtypes. The disorganized lymphatics showed large sheet-like structures with many branches and holes inside. Such lymphatics were abundant in zipper-like endothelial junctions with some button-like junctions. The blood vessels were tortuous and had various diameters and complex networks. Stratified layers of lymphatics and blood vessels were disorganized, with impaired drainage function. VEGFR inhibition partially maintained their structures and drainage function. These findings demonstrate anatomy and pathological changes of the vasculatures in the parietal pleura and their potential as a novel therapeutic target.

Blockade of VEGFR3 signaling leads to functional impairment of dural lymphatic vessels without affecting autoimmune neuroinflammation.

The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II-associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.

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.

Conjugated Bile Acids Promote Lymphangiogenesis by Modulation of the Reactive Oxygen Species-p90RSK-Vascular Endothelial Growth Factor Receptor 3 Pathway.

Conjugated bile acids (BA) are significantly elevated in several liver pathologies and in the metastatic lymph node (LN). However, the effects of BAs on pathological lymphangiogenesis remains unknown. The current study explores the effects of BAs on lymphangiogenesis. BA levels were elevated in the LN and serum of Mdr2-/- mice (model of sclerosing cholangitis) compared to control mice. Liver and LN tissue sections showed a clear expansion of the lymphatic network in Mdr2-/- mice, indicating activated lymphangiogenic pathways. Human lymphatic endothelial cells (LECs) expressed BA receptors and a direct treatment with conjugated BAs enhanced invasion, migration, and tube formation. BAs also altered the LEC metabolism and upregulated key metabolic genes. Further, BAs induced the production of reactive oxygen species (ROS), that in turn phosphorylated the redox-sensitive kinase p90RSK, an essential regulator of endothelial cell dysfunction and oxidative stress. Activated p90RSK increased the SUMOylation of the Prox1 transcription factor and enhanced VEGFR3 expression and 3-D LEC invasion. BA-induced ROS in the LECs, which led to increased levels of Yes-associated protein (YAP), a lymphangiogenesis regulator. The suppression of cellular YAP inhibited BA-induced VEGFR3 upregulation and lymphangiogenic mechanism. Overall, our data shows the expansion of the lymphatic network in presclerotic liver disease and establishes a novel mechanism whereby BAs promote lymphangiogenesis.

High VEGFR3 Expression Reduces Doxorubicin Efficacy in Triple-Negative Breast Cancer.

Due to the lack of specific targets, cytotoxic chemotherapy still represents the common standard treatment for triple-negative breast patients. Despite the harmful effect of chemotherapy on tumor cells, there is evidence that treatment could modulate the tumor microenvironment in a way favoring the propagation of the tumor. In addition, the lymphangiogenesis process and its factors could be involved in this counter-therapeutic event. In our study, we have evaluated the expression of the main lymphangiogenic receptor VEGFR3 in two triple-negative breast cancer in vitro models, resistant or not to doxorubicin treatment. The expression of the receptor, at mRNA and protein levels, was higher in doxorubicin-resistant cells than in parental cells. In addition, we confirmed the upregulation of VEGFR3 levels after a short treatment with doxorubicin. Furthermore, VEGFR3 silencing reduced cell proliferation and migration capacities in both cell lines. Interestingly, high VEGFR3 expression was significantly positively correlated with worse survival in patients treated with chemotherapy. Furthermore, we have found that patients with high expression of VEGFR3 present shorter relapse-free survival than patients with low levels of the receptor. In conclusion, elevated VEGFR3 levels correlate with poor survival in patients and with reduced doxorubicin treatment efficacy in vitro. Our results suggest that the levels of this receptor could be a potential marker of meager doxorubicin response. Consequently, our results suggest that the combination of chemotherapy and VEGFR3 blockage could be a potentially useful therapeutic strategy for the treatment of triple-negative breast cancer.

Vascular Endothelial Growth Factor A (VEGF-A) and Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) as Potential Biomarkers for Oral Squamous Cell Carcinoma: A Sri Lankan Study.

BACKGROUND: The incidence of oral squamous cell carcinoma (OSCC) is very high in South Asia and Vascular endothelial growth factor (VEGF) is one of the key factors essential for cancer growth. The importance of VEGF-A and VEGF Receptor 2(VEGFR-2) in oral cancer pathophysiology is yet to be decided. Vascular Endothelial Growth Factor A (VEGF-A) is the main factor concerned in angiogenesis in tumors, but its role in Oral Squamous Cell Carcinoma (OSCC) is still debatable. Our study aimed to determine the role of VEGF-A and VEGFR-2 in OSCC. METHODS: Blood from 30 patients with primary OSCC and 1:1 age-sex-matched controls was subjected to qPCR and ELISA to detect VEGF-A gene expression and serum level. Tumors of the 30 patients were investigated for VEGF Receptor-2 (VEGFR-2) expression and were analyzed using Image J software version 1.52 for DAB percentage (DAB-P) area and optical density (OD). RESULTS: VEGF-A relative gene expression among patients was 2.43-fold higher compared to the healthy control group. Well-differentiated had a 1.98-fold increment, while poorly differentiated had a 3.58-fold increment. Serum VEGF-A was significantly elevated among the patients compared to controls (458.7 vs 253.2, p=0.0225). Poorly differentiated had a higher serum VEGF concentration (1262.0±354.7pg/ml) compared with other two. Mean VEGFR-2 DAB-P level in OSCC was 42.41±5.61(p=0.15). Well-differentiated had a DAB-P of 41.20±5.32 while poorly differentiated had DAB-P 46.21±3.78. The mean OD in OSCC was 0.54±0.16. VEGFR-2 OD in well and poorly differentiated OSCC were 0.48±0.12 and 0.68±0.17, respectively. CONCLUSIONS: VEGF-A gene expression, serum levels, and tissue VEGFR-2 levels correlated linearly with the stage and grade of the tumor. This study justifies the value of VEGF-A as a potential biomarker in OSCC in early detection of OSCC. More studies are needed to accept the use of VEGF-A.

Signaling mechanisms underlying lymphatic vessel dysfunction in skin aging and possible anti-aging strategies.

Aging-related skin diseases are gradually increasing due to the imbalance of cutaneous homeostasis in the aging population. Skin aging-induced inflammation promotes systemic inflammation and may lead to whole-body aging. Lymphatic vessels play an important role in maintaining fluid and homeostasis balance. In intrinsically aged skin, the number of lymphatic vessels decrease and their functions decline, which is related to the reduced adhesion junctions between lymphatic endothelial cells, particularly VE-cadherin. VEGFC/VEGFR-3 signal pathway plays an important role in remodeling and expansion of lymphatic vessels; the downregulation of this pathway contributes to the dysfunction of lymphatic vessels. Meanwhile, we proposed some additional mechanisms. Decline of the pumping activity of lymphatic vessels might be related to age-related changes in extracellular matrix, ROS increase, and eNOS/iNOS disturbances. In extrinsically aged skin, the hyperpermeability of lymphatic vessels results from a decrease in endothelial-specific tight junction molecules, upregulation of VEGF-A, and downregulation of the VEGFC/VEGFR-3 signaling pathway. Furthermore, some of the Phyto therapeutics could attenuate skin aging by modulating the lymphatic vessels. This review summarized the lymphatic vessel dysfunction in skin aging and anti-aging strategies based on lymphatic vessel modulation.

Formation of the glomerular microvasculature is regulated by VEGFR-3.

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4 and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics, VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here, we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with the highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal interendothelial cell junctions, and poor attachment between glomerular endothelial cells and the basement membrane. In addition, we demonstrated that expression of the VEGFR-3 ligand VEGF-C by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, the results of our study support a VEGF-C-independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels, which has implications for clinical therapies that target this receptor.NEW & NOTEWORTHY Targeting VEGFR-3 in kidney lymphatics has been proposed as a method to treat kidney disease. However, expression of VEGFR-3 is not lymphatic-specific. We demonstrated developmental expression of VEGFR-3 in glomerular endothelial cells, with loss of Vegfr-3 leading to malformation of glomerular capillary loops. Furthermore, we showed that VEGFR-3 attenuates VEGFR-2 activity in glomerular endothelial cells independent of paracrine VEGF-C signaling. Together, these data provide valuable information for therapeutic development targeting these pathways.

The Effect of Lymphangiogenesis in Transplant Arteriosclerosis.

BACKGROUND: Transplant arteriosclerosis is a major complication in long-term survivors of heart transplantation. Increased lymph flow from donor heart to host lymph nodes has been reported to play a role in transplant arteriosclerosis, but how lymphangiogenesis affects this process is unknown. METHODS: Vascular allografts were transplanted among various combinations of mice, including wild-type, Lyve1-CreERT2;R26-tdTomato, CAG-Cre-tdTomato, severe combined immune deficiency, Ccr2KO, Foxn1KO, and lghm/lghdKO mice. Whole-mount staining and 3-dimensional reconstruction identified lymphatic vessels within the grafted arteries. Lineage tracing strategies delineated the cellular origin of lymphatic endothelial cells. Adeno-associated viral vectors and a selective inhibitor were used to regulate lymphangiogenesis. RESULTS: Lymphangiogenesis within allograft vessels began at the anastomotic sites and extended from preexisting lymphatic vessels in the host. Tertiary lymphatic organs were identified in transplanted arteries at the anastomotic site and lymphatic vessels expressing CCL21 (chemokine [C-C motif] ligand 21) were associated with these immune structures. Fibroblasts in the vascular allografts released VEGF-C (vascular endothelial growth factor C), which stimulated lymphangiogenesis into the grafts. Inhibition of VEGF-C signaling inhibited lymphangiogenesis, neointima formation, and adventitial fibrosis of vascular allografts. These studies identified VEGF-C released from fibroblasts as a signal stimulating lymphangiogenesis extending from the host into the vascular allografts. CONCLUSIONS: Formation of lymphatic vessels plays a key role in the immune response to vascular transplantation. The inhibition of lymphangiogenesis may be a novel approach to prevent transplant arteriosclerosis.

PTEN regulates invasiveness in pancreatic neuroendocrine tumors through DUSP19-mediated VEGFR3 dephosphorylation.

BACKGROUND: Phosphatase and tensin homolog (PTEN) is a tumor suppressor. Low PTEN expression has been observed in pancreatic neuroendocrine tumors (pNETs) and is associated with increased liver metastasis and poor survival. Vascular endothelial growth factor receptor 3 (VEGFR3) is a receptor tyrosine kinase and is usually activated by binding with vascular endothelial growth factor C (VEGFC). VEGFR3 has been demonstrated with lymphangiogenesis and cancer invasiveness. PTEN is also a phosphatase to dephosphorylate both lipid and protein substrates and VEGFR3 is hypothesized to be a substrate of PTEN. Dual-specificity phosphatase 19 (DUSP19) is an atypical DUSP and can interact with VEGFR3. In this study, we investigated the function of PTEN on regulation of pNET invasiveness and its association with VEGFR3 and DUSP19. METHODS: PTEN was knocked down or overexpressed in pNET cells to evaluate its effect on invasiveness and its association with VEGFR3 phosphorylation. In vitro phosphatase assay was performed to identify the regulatory molecule on the regulation of VEGFR3 phosphorylation. In addition, immunoprecipitation, and immunofluorescence staining were performed to evaluate the molecule with direct interaction on VEGFR3 phosphorylation. The animal study was performed to validate the results of the in vitro study. RESULTS: The invasion and migration capabilities of pNETs were enhanced by PTEN knockdown accompanied with increased VEGFR3 phosphorylation, ERK phosphorylation, and increased expression of epithelial-mesenchymal transition molecules in the cells. The enhanced invasion and migration abilities of pNET cells with PTEN knockdown were suppressed by addition of the VEGFR3 inhibitor MAZ51, but not by the VEGFR3-Fc chimeric protein to neutralize VEGFC. VEGFR3 phosphorylation is responsible for pNET cell invasiveness and is VEGFC-independent. However, an in vitro phosphatase assay failed to show VEGFR3 as a substrate of PTEN. In contrast, DUSP19 was transcriptionally upregulated by PTEN and was shown to dephosphorylate VEGFR3 via direct interaction with VEGFR3 by an in vitro phosphatase assay, immunoprecipitation, and immunofluorescence staining. Increased tumor invasion into peripheral tissues was validated in xenograft mouse model. Tumor invasion was suppressed by treatment with VEGFR3 or MEK inhibitors. CONCLUSIONS: PTEN regulates pNET invasiveness via DUSP19-mediated VEGFR3 dephosphorylation. VEGFR3 and DUSP19 are potential therapeutic targets for pNET treatment.

Vascular endothelial growth factor-C in activating vascular endothelial growth factor receptor-3 and chemokine receptor-4 in melanoma adhesion.

Vascular endothelial growth factor-C (VEGF-C) binds to receptor vascular endothelial growth factor receptor-3 (VEGFR-3) expressed on lymphatic endothelial and melanoma cells. Binding of VEGF-C to VEGFR-3 enhances receptor phosphorylation that activates mitogen-activated protein kinase (MAP-K) and phosphatidylinositol-3-kinase (PI3K). These signalling pathways regulate cell migration and adhesion in response to internal or external changes. In addition, the overexpression of VEGF-C upregulates chemokine receptor CXCR-4 in tumours (melanoma). CXCR-4 is expressed on cells of the immune system (natural killer cells) and facilitates the migration of leukocytes in response to the CXCL12 ligand. The latter is expressed by lymphatic endothelial cells and by stromal cells in the tumour microenvironment (TME). The gradient established between CXCR-4 expressed on tumour cells and CXCL12 produced by stromal and lymphatic endothelial cells enhances tumour cell metastasis. 3-(4-Dimethylamino-naphthalen-1-ylmethylene)-1, 3-dihydroindol-2-one, MAZ-51, is an indolinone-based synthetic molecule that inhibits the phosphorylation of the tyrosine kinase receptor VEGFR-3. CTCE-9908, a CXCR-4 antagonist derived from human CXCL12, hinders receptor phosphorylation and the subsequent signalling pathways that would be activated. VEGF-C is stimulated by transforming growth factor-beta 1 (TGF-ß1), which facilitates cell-cell and cell-matrix adhesion by regulating cadherins through the activation of focal adhesion kinase (FAK) and mediates paxillin upregulation. Increased VEGF-C protein levels stimulated by TGF-ß bound to VEGFR-3 impact on intracellular pathways that promote tumour cell adhesion. In addition, increased VEGF-C protein levels lead to enhanced CXCR-4 protein expression. Therefore, effective blocking of VEGR-3 and CXCR-4 may inhibit tumour cell metastasis by hampering intracellular proteins promoting adhesion.

ADSCs stimulated by VEGF-C alleviate intestinal inflammation via dual mechanisms of enhancing lymphatic drainage by a VEGF-C/VEGFR-3-dependent mechanism and inhibiting the NF-κB pathway by the secretome.

BACKGROUND: Adipose-derived stem cells (ADSCs) have provided promising applications for Crohn's disease (CD). However, the practical efficacy of ADSCs remains controversial, and their mechanism is still unclear. Based on the pathogenesis of dysregulated immune responses and abnormal lymphatic alterations in CD, vascular endothelial growth factor-C (VEGF-C) is thought to be a favourable growth factor to optimize ADSCs. We aimed to investigate the efficacy of VEGF-C-stimulated ADSCs and their dual mechanisms in both inhibiting inflammation "IN" and promoting inflammation "OUT" in the intestine. METHODS: Human stem cells isolated from adipose tissues were identified, pretreated with or without 100 ng/ml VEGF-C and analysed for the secretion of cell culture supernatants in vitro. Lymphatic endothelial cells (LECs) were treated with ADSCs-conditioned medium or co-cultured with ADSCs and VEGF-C stimulated ADSCs. Changes in LECs transmigration, and VEGF-C/VEGFR-3 mRNA levels were assessed by transwell chamber assay and qRT-PCR. ADSCs and VEGF-C-stimulated ADSCs were intraperitoneally injected into mice with TNBS-induced chronic colitis. ADSCs homing and lymphatic vessel density (LVD) were evaluated by immunofluorescence staining. Lymphatic drainage was assessed using Evans blue. Cytokines and growth factors expression was detected respectively by ELISA and qRT-PCR. The protein levels of VEGF-C/VEGFR-3-mediated downstream signals and the NF-κB pathway were assayed by western blot. Faecal microbiota was measured by 16S rRNA sequencing. RESULTS: ADSCs stimulated with VEGF-C released higher levels of growth factors (VEGF-C, TGF-ß1, and FGF-2) and lower expression of cytokines (IFN-γ and IL-6) in cell supernatants than ADSCs in vitro (all P < 0.05). Secretome released by VEGF-C stimulated ADSCs exhibited a stronger LEC migratory capability and led to elevated VEGF-C/VEGFR-3 expression, but these effects were markedly attenuated by VEGFR-3 inhibitor. VEGF-C-stimulated ADSCs homing to the inflamed colon and mesenteric lymph nodes (MLNs) can exert stronger efficacy in improving colitis symptoms, reducing inflammatory cell infiltration, and significantly enhancing lymphatic drainage. The mRNA levels and protein concentrations of anti-inflammatory cytokines and growth factors were markedly increased with decreased proinflammatory cytokines in the mice treated with VEGF-C-stimulated ADSCs. Systemic administration of VEGF-C-stimulated ADSCs upregulated the colonic VEGF-C/VEGFR-3 pathway and activated downstream AKT and ERK phosphorylation signalling, accompanied by decreased NF-κB p65 expression. A higher abundance of faecal p-Bacteroidetes and lower p-Firmicutes were detected in mice treated with VEGF-C-stimulated ADSCs (all P < 0.05). CONCLUSION: VEGF-C-stimulated ADSCs improve chronic intestinal inflammation by promoting lymphatic drainage and enhancing paracrine signalling via activation of VEGF-C/VEGFR-3-mediated signalling and inhibition of the NF-κB pathway. Our study may provide a new insight into optimizing ADSCs treatment and investigating potential mechanisms in CD.

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.

Babao Dan inhibits lymphangiogenesis of gastric cancer in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

Gastric cancer (GC) is one of the most common gastrointestinal malignancies in the world. Growing evidence emphasizes the critical role of long non-coding RNA (lncRNA) in GC tumorigenesis. The aim of the research was to elucidate the effect and mechanism of Babao Dan (BBD) on lymphangiogenesis of GC in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis. The present study investigated BBD significantly decreased the expression of lncRNA-ANRIL and VEGF-C in GC cells (AGS, BGC823, and MGC80-3) by using real-time quantitative polymerasechain reaction (RT-qPCR) and the secretion and expression of VEGF-C by (enzyme linked immunosorbent assay) ELISA and western blot (WB). BBD significantly inhibited the tumor xenograft of GC growth and the expression of lncRNA-ANRIL, VEGF-C, VEGFR-3 and LYVE-1 in vivo. BBD reduced serum VEGF-C level. In vitro, BBD inhibited the tube formation and decreased the cell viability, proliferation and migration of HLECs by using tube formation, MTT, Hoechst and Transwell assays. In addition, WB assay found that BBD decreased the expression levels of VEGF-C, VEGFR-3, matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9), and RT-qPCR assay found that the mRNA expression levels of lncRNA-ANRIL, VEGF-C, VEGFR-3, MMP-2, MMP-9, CDK4, Cyclin D1, and Bcl-2 were down-regulated, and the expression of p21 and Bax were increased. Taken together, these results demonstrated that BBD inhibited lymphangiogenesis of GC in vitro and in vivo via the lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.