Pheophorbide a identified in an Eupatorium perfoliatum extract is a novel lymphatic vascular activator.

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.

Angiotensin II Induces Cardiac Edema and Hypertrophic Remodeling through Lymphatic-Dependent Mechanisms.

Cardiac lymphatic vessel growth (lymphangiogenesis) and integrity play an essential role in maintaining tissue fluid balance. Inhibition of lymphatic lymphangiogenesis is involved in cardiac edema and cardiac remodeling after ischemic injury or pressure overload. However, whether lymphatic vessel integrity is disrupted during angiotensin II- (Ang II-) induced cardiac remodeling remains to be investigated. In this study, cardiac remodeling models were established by Ang II (1000 ng/kg/min) in VEGFR-3 knockdown (Lyve-1Cre VEGFR-3f/-) and wild-type (VEGFR-3f/f) littermates. Our results indicated that Ang II infusion not only induced cardiac lymphangiogenesis and upregulation of VEGF-C and VEGFR-3 expression in the time-dependent manner but also enhanced proteasome activity, MKP5 and VE-cadherin degradation, p38 MAPK activation, and lymphatic vessel hyperpermeability. Moreover, VEGFR-3 knockdown significantly inhibited cardiac lymphangiogenesis in mice, resulting in exacerbation of tissue edema, hypertrophy, fibrosis superoxide production, inflammation, and heart failure (HF). Conversely, administration of epoxomicin (a selective proteasome inhibitor) markedly mitigated Ang II-induced cardiac edema, remodeling, and dysfunction; upregulated MKP5 and VE-cadherin expression; inactivated p38 MAPK; and reduced lymphatic vessel hyperpermeability in WT mice, indicating that inhibition of proteasome activity is required to maintain lymphatic endothelial cell (LEC) integrity. Our results show that both cardiac lymphangiogenesis and lymphatic barrier hyperpermeability are implicated in Ang II-induced adaptive hypertrophic remodeling and dysfunction. Proteasome-mediated hyperpermeability of LEC junctions plays a predominant role in the development of cardiac remodeling. Selective stimulation of lymphangiogenesis or inhibition of proteasome activity may be a potential therapeutic option for treating hypertension-induced cardiac remodeling.

Functional Investigation of Meningeal Lymphatic System in Experimental Intracerebral Hemorrhage.

BACKGROUND: Promotion of hematoma resolution in a timely manner reduces intracerebral hemorrhage (ICH) brain injury induced by toxic blood components and subsequent neuroinflammation. The meningeal lymphatic system is responsible for clearance of macromolecules and pathogenic substances from the central nervous system; however, its role in intraparenchymal hematoma clearance and ICH outcomes is unknown. In the present study, we aimed to understand the contribution of the meningeal lymphatic system to ICH pathologies and to test whether pharmacological enhancement of meningeal lymphatic function promotes hematoma resolution and brain recovery after ICH. METHODS: Immunofluorescence of whole-mount meninges was used to measure complexity and coverage level of meningeal lymphatic vasculature following ICH induction. Fluorescent microbeads and PKH-26-labeled erythrocytes were used to evaluate drainage function of the meningeal lymphatic system. Visudyne treatment, deep cervical lymph node ligation, and VEGF (vascular endothelial growth factor)-C injection were performed to manipulate meningeal lymphatic function. Neurobehavioral performance and hematoma volume were assayed by the cylinder test and histological measurements. Iron deposition, residual erythrocytes, neuronal loss, and astrogliosis were assessed by immunohistochemistry and antibody-based fluorescence staining. RESULTS: Meningeal lymphangiogenesis and enhanced lymphatic drainage occurred during the late phase of ICH. Ablation and blockage of meningeal lymphatic vessels impeded hematoma clearance, whereas pharmacological enhancement of their function reduced hematoma volume, improved behavioral performance, and reduced brain residual erythrocytes, iron deposition, neuronal loss, and astroglial activation. CONCLUSIONS: Early enhancement of meningeal lymphatic function is beneficial for ICH recovery. Targeting the meningeal lymphatic system is therefore a potential therapeutic approach for treating ICH.

Molecular mechanisms of cyclic phosphatidic acid-induced lymphangiogenic actions in vitro.

The lymphatic system plays important roles in various physiological and pathological phenomena. As a bioactive phospholipid, lysophosphatidic acid (LPA) has been reported to function as a lymphangiogenic factor as well as some growth factors, yet the involvement of phospholipids including LPA and its derivatives in lymphangiogenesis is not fully understood. In the present study, we have developed an in-vitro lymphangiogenesis model (termed a collagen sandwich model) by utilizing type-I collagen, which exists around the lymphatic endothelial cells of lymphatic capillaries in vivo. The collagen sandwich model has revealed that cyclic phosphatidic acid (cPA), and not LPA, augmented the tube formation of human dermal lymphatic endothelial cells (HDLECs). Both cPA and LPA increased the migration of HDLECs cultured on the collagen. As the gene expression of LPA receptor 6 (LPA6) was predominantly expressed in HDLECs, a siRNA experiment against LPA6 attenuated the cPA-mediated tube formation. A synthetic LPA1/3 inhibitor, Ki16425, suppressed the cPA-augmented tube formation and migration of the HDLECs, and the LPA-induced migration. The activity of Rho-associated protein kinase (ROCK) located at the downstream of the LPA receptors was augmented in both the cPA- and LPA-treated cells. A potent ROCK inhibitor, Y-27632, suppressed the cPA-dependent tube formation but not the migration of the HDLECs. Furthermore, cPA, but not LPA, augmented the gene expression of VE-cadherin and ß-catenin in the HDLECs. These results provide novel evidence that cPA facilitates the capillary-like morphogenesis and the migration of HDLECs through LPA6/ROCK and LPA1/3 signaling pathways in concomitance with the augmentation of VE-cadherin and ß-catenin expression. Thus, cPA is likely to be a potent lymphangiogenic factor for the initial lymphatics adjacent to type I collagen under physiological conditions.

Verteporfin-Loaded Mesoporous Silica Nanoparticles' Topical Applications Inhibit Mouse Melanoma Lymphangiogenesis and Micrometastasis In Vivo.

Photodynamic therapy (PDT) has been pointed out as a candidate for improving melanoma treatment. Nanotechnology application in PDT has increased its efficacy by reducing side effects. Herein, mesoporous silica nanoparticles (MSNs) conjugated with verteporfin (Ver-MSNs), in use with PDT, were administered in mice to evaluate their efficacy on lymphoangiogenesis and micrometastasis in melanoma. Melanoma was induced in mice by the subcutaneous injection of B16-F10 cells. The mice were transcutaneously treated with MSNs, Ver-MSNs, or glycerol and exposed to red light. The treatment was carried out four times until day 20. Lymphangiogenesis and micrometastasis were identified by the immunohistochemical method. Lymphoangiogenesis was halved by MSN treatment compared with the control animals, whereas the Ver-MSN treatment almost abolished it. A similar reduction was also observed in lung micrometastasis. PDT with topically administrated Ver-MSNs reduced melanoma lymphoangiogenesis and lung micrometastasis, as well as tumor mass and angiogenesis, and therefore their use could be an innovative and useful tool in melanoma clinical therapy.

VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Low-intensity pulsed ultrasound therapy suppresses coronary adventitial inflammatory changes and hyperconstricting responses after coronary stent implantation in pigs in vivo.

BACKGROUNDS: We demonstrated that coronary adventitial inflammation plays important roles in the pathogenesis of drug-eluting stent (DES)-induced coronary hyperconstricting responses in pigs in vivo. However, no therapy is yet available to treat coronary adventitial inflammation. We thus developed the low-intensity pulsed ultrasound (LIPUS) therapy that ameliorates myocardial ischemia by enhancing angiogenesis. AIMS: We aimed to examine whether our LIPUS therapy suppresses DES-induced coronary hyperconstricting responses in pigs in vivo, and if so, what mechanisms are involved. METHODS: Sixteen normal male pigs were randomly assigned to the LIPUS or the sham therapy groups after DES implantation into the left anterior descending (LAD) coronary artery. In the LIPUS group, LIPUS (32 cycles, 193 mW/cm2) was applied to the heart at 3 different levels (segments proximal and distal to the stent edges and middle of the stent) for 20 min at each level for every other day for 2 weeks. The sham therapy group was treated in the same manner but without LIPUS. At 4 weeks after stent implantation, we performed coronary angiography, followed by immunohistological analysis. RESULTS: Coronary vasoconstricting responses to serotonin in LAD at DES edges were significantly suppressed in the LIPUS group compared with the sham group. Furthermore, lymph transport speed in vivo was significantly faster in the LIPUS group than in the sham group. Histological analysis at DES edges showed that inflammatory changes and Rho-kinase activity were significantly suppressed in the LIPUS group, associated with eNOS up-regulation and enhanced lymph-angiogenesis. CONCLUSIONS: These results suggest that our non-invasive LIPUS therapy is useful to treat coronary functional abnormalities caused by coronary adventitial inflammation, indicating its potential for the novel and safe therapeutic approach of coronary artery disease.

Cardiac macrophage subsets differentially regulate lymphatic network remodeling during pressure overload.

The lymphatic network of mammalian heart is an important regulator of interstitial fluid compartment and immune cell trafficking. We observed a remodeling of the cardiac lymphatic vessels and a reduced lymphatic efficiency during heart hypertrophy and failure induced by transverse aortic constriction. The lymphatic endothelial cell number of the failing hearts was positively correlated with cardiac function and with a subset of cardiac macrophages. This macrophage population distinguished by LYVE-1 (Lymphatic vessel endothelial hyaluronic acid receptor-1) and by resident macrophage gene expression signature, appeared not replenished by CCR2 mediated monocyte infiltration during pressure overload. Isolation of macrophage subpopulations showed that the LYVE-1 positive subset sustained in vitro and in vivo lymphangiogenesis through the expression of pro-lymphangiogenic factors. In contrast, the LYVE-1 negative macrophage subset strongly expressed MMP12 and decreased the endothelial LYVE-1 receptors in lymphatic endothelial cells, a feature of cardiac lymphatic remodeling in failing hearts. The treatment of mice with a CCR2 antagonist during pressure overload modified the proportion of macrophage subsets within the pathological heart and preserved lymphatic network from remodeling. This study reports unknown and differential functions of macrophage subpopulations in the regulation of cardiac lymphatic during pathological hypertrophy and may constitute a key mechanism underlying the progression of heart failure.

Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

Dysregulation of Amphiregulin stimulates the pathogenesis of cystic lymphangioma.

Along with blood vessels, lymphatic vessels play an important role in the circulation of body fluid and recruitment of immune cells. Postnatal lymphangiogenesis commonly occurs from preexisting lymphatic vessels by sprouting, which is induced by lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C). However, the key signals and cell types that stimulate pathological lymphangiogenesis, such as human cystic lymphangioma, are less well known. Here, we found that mouse dermal fibroblasts that infiltrate to sponges subcutaneously implanted express VEGF-D and sushi, Von Willebrand factor type A, EGF, and pentraxin domain containing 1 (SVEP1) in response to PDGFRß signal. In vitro, Pdgfrb knockout (ß-KO) fibroblasts had reduced expression of VEGF-D and SVEP1 and overproduced Amphiregulin. Dysregulation of these three factors was involved in the cyst-like and uneven distribution of lymphatic vessels observed in the ß-KO mice. Similarly, in human cystic lymphangioma, which is one of the intractable diseases and mostly occurs in childhood, fibroblasts surrounding cystic lymphatics highly expressed Amphiregulin. Moreover, fibroblast-derived Amphiregulin could induce the expression of Amphiregulin in lymphatic endothelial cells. The dual source of Amphiregulin activated EGFR expressed on the lymphatic endothelial cells. This exacerbation cascade induced proliferation of lymphatic endothelial cells to form cystic lymphangioma. Ultimately, excessive Amphiregulin produced by fibroblasts surrounding lymphatics and by lymphatic endothelial cells per se results in pathogenesis of cystic lymphangioma and will be a fascinating therapeutic target of cystic lymphangioma.

Protective roles of the TIR/BB-loop mimetic AS-1 in alkali-induced corneal neovascularization by inhibiting ERK phosphorylation.

Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 µL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45+ immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45+ cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn.

Subconjunctival injections of dimethyl fumarate inhibit lymphangiogenesis and allograft rejection in the rat cornea.

Corneal lymphangiogenesis induced by macrophages played a critical role in corneal allograft rejection (CGR). However, there are few Food and Drug Administration (FDA)-approved drugs that target lymphangiogenesis. The aim of our study is to evaluate the effects of dimethyl fumarate (DMF) on corneal allograft survival in rats. Penetrating corneal transplantation was performed in rats. Subconjunctival injections of dimethyl fumarate (20 µg) were administered at the end of the operation and postoperative day 3 to day 11. The clinical signs of corneal allografts were evaluated. Immunohistochemistry, quantitative real-time PCR (qPCR), flow cytometry and western blot were performed respectively. The effects and mechanism of DMF on RAW264.7 cells were determined by qPCR, enzyme-linked immunosorbent assay (ELISA), and western blot in vitro. The results showed that subconjunctival injections of DMF could significantly inhibit corneal lymphangiogenesis and CGR with decreased corneal macrophage infiltration compared with the vehicle group. Moreover, DMF could reduce the mRNA expression of monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and vascular endothelial growth factor-C (VEGF-C) in the corneal grafts and RAW264.7 macrophages by inhibiting NF-κB activation. Furthermore, compared with the vehicle group, the number of dendritic cells in the ipsilateral cervical lymph nodes of the DMF-treated group was decreased significantly. Collectively, our findings showed that DMF could suppress CGR by inhibiting the macrophage-induced corneal lymphoangiogenesis.

Inhibition of NADPH oxidase 4 attenuates lymphangiogenesis and tumor metastasis in breast cancer.

Lymphangiogenesis is thought to contribute to promote tumor cells to enter lymphatic vessels and plant at a secondary site. Endothelial cells are the cornerstone of the generation of new lymphatic vessels. NADPH oxidase 4 (Nox4) is the most abundant one of NADPH oxidases in endothelial cells and the most studied one in relevance with cancer. Our purpose is to analyze the relationship between Nox4 and lymphangiogenesis and find out whether the newborn lymphatic vessels lead to cancer metastasis. We first explored the expression of Nox4 in lymphatic endothelial cells of primary invasive breast tumors and human normal mammary glands using GEO databases and found that Nox4 was upregulated in primary invasive breast tumors samples. In addition, its high expression correlated with lymph node metastasis in breast cancer patients. Nox4 could increase the tube formation and lymphatic vessel sprouting in a three-dimensional setting. In vivo, inhibition of Nox4 in 4T1 tumor-bearing mice could significantly decrease the tumor lymphangiogenesis and metastasis. Nox4 may increase tumor lymphangiogenesis via ROS/ERK/CCL21 pathway and attract CCR7-positive breast cancer cells to entry lymphatic vessels and distant organs. In conclusion, our results show that Nox4 is a factor that promotes lymphangiogenesis and is a potential target of antitumor metastasis.

Thymosin ß4 released from functionalized self-assembling peptide activates epicardium and enhances repair of infarcted myocardium.

The epicardium plays an important role in cardiomyogenesis during development, while it becomes quiescent in adult heart during homeostasis. This study investigates the efficiency of thymosin ß4 (Tß4) release with RPRHQGVM conjugated to the C-terminus of RADA16-I (RADA-RPR), the functionalized self-assembling peptide (SAP), to activate the epicardium and repairing the infarcted myocardium. Methods: The functionalized SAP was constituted with self-assembling motif, Tß4-binding site, and cell adhesive ligand. Myocardial infarction (MI) models of the transgenic mice were established by ligation of the left anterior descending coronary artery. At one week after intramyocardial injection of Tß4-conjugated SAP, the activation of the epicardium was assessed. At four weeks after implantation, the migration and differentiation of epicardium-derived cells (EPDCs) as well as angiogenesis, lymphangiogenesis and myocardial regeneration were examined. Results: We found that the designer RADA-RPR bound Tß4 and adhered to EPDCs and that Tß4 released from the functionalized SAP could effectively activate the epicardium and induce EPDCs to differentiate towards cardiovascular cells as well as lymphatic endothelial cells. Moreover, SAP-released Tß4 (SAP-Tß4) promoted proliferation of cardiomyocytes. Furthermore, angiogenesis, lymphangiogenesis and myocardial regeneration were enhanced in the MI models at 4 weeks after delivery of SAP-Tß4 along with attenuation of adverse myocardial remodeling and significantly improved cardiac function. Conclusions: These results demonstrate that sustained release of Tß4 from the functionalized SAP can activate the epicardium and effectively enhance the repair of infarcted myocardium. We believe the delivery of SAP-Tß4 may be a promising strategy for MI therapy.

The mineral dust-induced gene, mdig, regulates angiogenesis and lymphangiogenesis in lung adenocarcinoma by modulating the expression of VEGF-A/C/D via EGFR and HIF-1α signaling.

Mineral dust­induced gene (mdig) is a novel lung cancer­related oncogene. The aim of this study was to explore the effects of mdig on angiogenesis and lymphangiogenesis by vascular endothelial growth factor (VEGF) in lung adenocarcinoma. mdig­overexpressing A549, H1299 and 293T cells, mdig­silenced A549, human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were cultured under normoxic and hypoxic conditions. Protein expression levels of mdig, epidermal growth factor receptor (EGFR), phospho(p)­EGFR Tyr1068, hypoxia­inducible factor­1α (HIF­1α), VEGF­A/C/D and VEGF­R1/R2/R3 were assessed using western blotting. mRNA expression levels of mdig, EGFR and HIF­1α were measured using RT­qPCR. Tube formation and xenograft tumor experiments were performed to examine the mechanism of mdig in angiogenesis and lymphangiogenesis. Protein expression levels of EGFR, HIF­1α and VEGF­A/C/D were significantly upregulated in cells cultured under hypoxic conditions compared with those cultured under normoxic conditions, whereas the levels of mdig were decreased. Protein expression levels of EGFR, p­EGFR and VEGF­A/R1/R2 were significantly increased in the mdig­overexpressing cells, whereas the levels of HIF­1α and VEGF­C/D/R3 were decreased compared with those in control cells, all of which were reversed in mdig­silenced cells. Tumor volumes and density of angiogenesis in the mdig­overexpressing group were significantly increased compared with those in the control group, whereas the density of lymphangiogenesis was decreased. No tumors formed in the mdig­silenced group after 3 weeks of assessment in vivo. Protein expression levels of EGFR, p­EGFR, VEGF­A and angiogenesis density were significantly reduced in the mdig­overexpressing cells treated with an EGFR inhibitor, whereas the levels of HIF­1α, VEGF­C/D and the lymphangiogenesis density were significantly increased in mdig­overexpressing cells treated with a HIF­1α agonist. All changes in protein expression were reversed in EGFR agonist and HIF­1α inhibitor treated mdig­silenced cells. In conclusion, mdig is an oxygen­sensitive protein that promotes tumor growth and angiogenesis by activating the EGFR/p­EGFR/VEGF­A/VEGF­R1/R2 pathway and inhibits lymphangiogenesis by blocking the HIF­1α/VEGF­C/D/VEGF­R3 pathway.

Roles of Thromboxane Receptor Signaling in Enhancement of Lipopolysaccharide-Induced Lymphangiogenesis and Lymphatic Drainage Function in Diaphragm.

[Figure: see text].

Qingjie Fuzheng Granule suppresses lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway.

SCOPE: To investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. METHODS: The effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. RESULTS: QFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. CONCLUSION: QFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

Lymphatic Vasculature: An Emerging Therapeutic Target and Drug Delivery Route.

The lymphatic system has received increasing scientific and clinical attention because a wide variety of diseases are linked to lymphatic pathologies and because the lymphatic system serves as an ideal conduit for drug delivery. Lymphatic vessels exert heterogeneous roles in different organs and vascular beds, and consequently, their dysfunction leads to distinct organ-specific outcomes. Although studies in animal model systems have led to the identification of crucial lymphatic genes with potential therapeutic benefit, effective lymphatic-targeted therapeutics are currently lacking for human lymphatic pathological conditions. Here, we focus on the therapeutic roles of lymphatic vessels in diseases and summarize the promising therapeutic targets for modulating lymphangiogenesis or lymphatic function in preclinical or clinical settings. We also discuss considerations for drug delivery or targeting of lymphatic vessels for treatment of lymphatic-related diseases. The lymphatic vasculature is rapidly emerging as a critical system for targeted modulation of its function and as a vehicle for innovative drug delivery.

Effects of 6,8-Diprenylgenistein on VEGF-A-Induced Lymphangiogenesis and Lymph Node Metastasis in an Oral Cancer Sentinel Lymph Node Animal Model.

BACKGROUND: The major determining factor of prognosis of oral squamous cell carcinoma is cervical lymph node metastasis. 6,8-Diprenylgenistein (6,8-DG), an isoflavonoid isolated from Cudrania tricuspidata has been reported to have anti-microbial and anti-obesity activities. However, its effects on lymphangiogenesis and lymph node metastasis in oral cancer have not yet been reported. METHODS: To investigate the in vitro inhibitory effects of 6,8-DG on VEGF-A-induced lymphangiogenesis, we performed the proliferation, tube formation, and migration assay using human lymphatic microvascular endothelial cells (HLMECs). RT-PCR, Western blot, immunoprecipitation, ELISA and co-immunoprecipitation assays were used to investigate the expression levels of proteins, and mechanism of 6,8-DG. The in vivo inhibitory effects of 6,8-DG were investigated using an oral cancer sentinel lymph node (OCSLN) animal model. RESULTS: 6,8-DG inhibited the proliferation, migration and tube formation of rhVEGF-A treated HLMECs. In addition, the in vivo lymphatic vessel formation stimulated by rhVEGF-A was significantly reduced by 6,8-DG. 6,8-DG inhibited the expression of VEGF-A rather than other lymphangiogenic factors in CoCl2-treated SCCVII cells. 6,8-DG inhibited the expression and activation of VEGFR-2 stimulated by rhVEGF-A in HLMECs. Also, 6,8-DG inhibited the activation of the lymphangiogenesis-related downstream signaling factors such as FAK, PI3K, AKT, p38, and ERK in rhVEGF-A-treated HLMECs. Additionally, 6,8-DG inhibited the expression of the hypoxia-inducible factor (HIF-1α), which is involved in the expression of VEGF-A in CoCl2-treated SCCVII cells, and 6,8-DG inhibited VEGF-A signaling via interruption of the binding of VEGF-A and VEGFR-2 in HLMECs. In the VEGF-A-induced OCSLN animal model, we confirmed that 6,8-DG suppressed tumor-induced lymphangiogenesis and SLN metastasis. CONCLUSION: These data suggest that 6,8-DG inhibits VEGF-A-induced lymphangiogenesis and lymph node metastasis in vitro and in vivo. Furthermore, the inhibitory effects of 6,8-DG are probably mediated by inhibition of VEGF-A expression in cancer cells and suppression of the VEGF-A/VEGFR-2 signaling pathway in HLMEC. Thus, 6,8-DG could be novel and valuable therapeutic agents for metastasis prevention and treatment of oral cancer.

Live Imaging of Intracranial Lymphatics in the Zebrafish.

RATIONALE: The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. OBJECTIVE: Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. METHODS AND RESULTS: Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. CONCLUSIONS: This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.