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.

3-O-Acetyloleanolic acid inhibits angiopoietin-1-induced angiogenesis and lymphangiogenesis via suppression of angiopoietin-1/Tie-2 signaling.

Tumor angiogenesis and lymphangiogenesis are important processes in tumor progression and metastasis. The inhibitory effects of 3-O-acetyloleanolic acid (3AOA), a pentacyclic triterpenoid compound isolated from Vigna sinensis K., on tumor-induced angiogenesis and lymphangiogenesis in vitro and in vivo were studied. Angiopoietin-1 is an important angiogenic and lymphangiogenic factor secreted from colon carcinoma CT-26 cells under hypoxia conditions. 3AOA inhibited proliferation, migration, and tube formation of angiopoietin-1-treated human umbilical vein endothelial cells (HUVEC) and human lymphatic microvascular endothelial cells (HLMEC). 3AOA reduced angiogenesis and lymphangiogenesis in angiopoietin-1-stimulated Matrigel plugs. Also, 3AOA inhibited tumor growth and tumor-induced angiogenesis and lymphangiogenesis in an angiopoietin-1-induced CT-26 allograft colon carcinoma animal model. 3AOA inhibited activation of the angiopoietin-1 receptor Tie-2 and activation of the downstream signaling factors FAK, AKT, and ERK1/2 that are involved in the angiopoietin-1/Tie-2-signaling pathway. Thus, 3AOA has an inhibitory effect on angiogenesis and lymphangiogenesis induced by angiopoietin-1 both in vitro and in vivo, and the inhibitory effect of 3AOA is probably due to suppression of angiopoietin-1/Tie-2 signaling in HUVEC and HLMEC.

Hedyotis diffusa Willd. inhibits VEGF­C­mediated lymphangiogenesis in colorectal cancer via multiple signaling pathways.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignancies worldwide. For patients diagnosed with the presence of metastatic disease, surgery is not suitable for the majority of them. Lymphangiogenesis is a key factor during cancer metastasis and is regulated by vascular endothelial growth factor C (VEGF­C). Hedyotis diffusa Willd. (HDW) is a Chinese herb of the Rubiaceae family that reportedly inhibits tumor metastasis. However, its underlying anticancer mechanisms have not yet been elucidated. In the present study, we investigated the effects of an ethanol extract of HDW (EEHDW) on the migration capacity by wound healing and Transwell assays, and the effect on the VEGF­C expression in different CRC cell lines by western blot analysis and ELISA assays. A model of VEGF­C­stimulated human lymphatic endothelial cells (HLECs) was constructed. It was found that EEHDW suppressed lymphangiogenesis via the mediation of multiple pathways, which attenuated the migration of cells and their tube formation abilities. Multiple signaling pathways were found to be involved in the VEGF­C­mediated lymphangiogenesis. After EEHDW treatment in VEGF­C­stimulated HLECs, EEHDW was found to downregulate the expression levels of multiple signaling pathways. Taken together, these results indicate that EEHDW possesses significant anti­metastatic activities. Moreover, the suppressive effect of EEHDW on lymphangiogenesis, particularly via downregulation of VEGF­C, partly explains the potential molecular mechanism underlying the inhibitory effect of EEHDW on CRC metastasis.

Attenuated Joint Tissue Damage Associated With Improved Synovial Lymphatic Function Following Treatment With Bortezomib in a Mouse Model of Experimental Posttraumatic Osteoarthritis.

OBJECTIVE: To investigate the roles of the synovial lymphatic system in the severity and progression of joint tissue damage and functional responses of synovial lymphatic endothelial cells (LECs) to macrophage subsets, and to evaluate the therapeutic potential of the proteasome inhibitor bortezomib (BTZ) in a mouse model of experimental posttraumatic osteoarthritis (OA). METHODS: C57BL/6J wild-type mice received a meniscal ligamentous injury to induce posttraumatic knee OA. Lymphangiogenesis was blocked by a vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody. Synovial lymphatic drainage was examined by near-infrared imaging. Joint damage was assessed by histology. RNA-sequencing and pathway analyses were applied to synovial LECs. Macrophage subsets in the mouse synovium were identified by flow cytometry and immunofluorescence staining. M1 and M2 macrophages were induced from mouse bone marrow cells, and their effects on LECs were examined in cocultures in the presence or absence of BTZ. The effects of BTZ on joint damage, LEC inflammation, and synovial lymphatic drainage were examined. RESULTS: Injection of a VEGFR-3 neutralizing antibody into the joints of mice with posttraumatic knee OA reduced synovial lymphatic drainage and accelerated joint tissue damage. Synovial LECs from the mouse OA joints had dysregulated inflammatory pathways and expressed high levels of inflammatory genes. The number of M1 macrophages was increased in the knee joints of mice with posttraumatic OA, thereby promoting the expression of inflammatory genes by LECs; this effect was blocked by BTZ. Treatment with BTZ decreased cartilage loss, reduced the expression of inflammatory genes by LECs, and improved lymphatic drainage in the knee joints of mice with posttraumatic OA. CONCLUSION: Experimental posttraumatic knee OA is associated with decreased synovial lymphatic drainage, increased numbers of M1 macrophages, and enhanced inflammatory gene expression by LECs, all of which was improved by treatment with BTZ. Intraarticular administration of BTZ may represent a new therapy for the restoration of synovial lymphatic function in subjects with posttraumatic knee OA.

Shikonin Suppresses Lymphangiogenesis via NF-κB/HIF-1α Axis Inhibition.

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, promotes cancer growth and metastasis. Finding natural compounds with anti-lymphangiogenic activity will be useful for preventive treatment of lymphatic metastasis. Shikonin, an ingredient of a traditional Japanese and Chinese medicinal herb Lithospermum erythrorhizon, has been widely used in several pharmaceutical and cosmetic preparations, as well as in food colorants. Shikonin has been reported to inhibit lymphangiogenesis in vitro, but the mechanism of inhibition has not been determined. The aim of this study is to investigate the mechanism of anti-lymphangiogenesis of shikonin in primary human lymphatic endothelial cells (HMVEC-dLy). Shikonin, at non-toxic concentrations, significantly inhibited cord formation ability of lymphatic endothelial cells in a dose- and time-dependent manner. Western blotting analysis showed that shikonin decreased nuclear factor-kappaB (NF-κB) activation, as indicated by phosphorylation and nuclear translocation of NF-κB p65, and also reduced both mRNA and protein levels of hypoxia-inducible factor-1 (HIF-1)α. Use of an NF-κB inhibitor (BAY 11-7085) and HIF-1α small interfering RNA (siRNA) transfection revealed that NF-κB activation was upstream of HIF-1α expression, which controls cord formation by HMVEC-dLy. In addition, the reduction of vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3) mRNA levels were also found in HMVEC-dLy that treated with shikonin. In conclusion, shikonin inhibits lymphangiogenesis in vitro by interfering the NF-κB/HIF-1α pathway and involves in suppression of VEGF-C and VEGFR-3 mRNA expression.

Oxyresveratrol prevents murine H22 hepatocellular carcinoma growth and lymph node metastasis via inhibiting tumor angiogenesis and lymphangiogenesis.

The purpose of this study was to investigate the effects and mechanisms of oxyresveratrol (Oxyres) on hepatocellular carcinoma (HCC) in vitro and in vivo. The MTT and Transwell assays were performed to investigate the effects of Oxyres on cell proliferation and migration of two HCC cell lines, QGY-7701 and SMMC-7721 cells. H22 cells were subcutaneously injected into hind foot pads of 70 male mice to establish a lymph node metastasis model. These mice were randomly divided into seven groups as follows, control group, HCC group, Oxyres 20 mg/kg group, Oxyres 40 mg/kg group, Oxyres 60 mg/kg group, Resveratrol (Res) group, and Adriamycin (ADM) group. Oxyres, Res, and ADM were intraperitoneally injected daily for consecutive 21 days. Tumors and popliteal lymph node were isolated and embedded for histology analysis. Expressions of CD31 and vascular endothelial growth factor receptor-3 (VEGFR3) in tumors were detected by immunohistocehmistry. Expressions of vascular endothelial growth factor C (VEGF-C) were measured by Western blot. Oxyres significantly inhibited the proliferation and migration of QGY-7701 and SMMC-7721 cells. Oxyres significantly inhibited tumor growth (p < 0.001) and metastasis to sentinel lymph nodes (70%) in a dose-dependent manner. Oxyres showed a similar inhibition rate as Res. Oxyres also significantly decreased micro-blood vessel density and micro-lymphatic vessel density in tumors (p < 0.05). Expressions of CD31, VEGFR3, and VEGF-C of tumors were also inhibited by Oxyres (p < 0.05). Oxyres exerts anti-tumor effects against HCC through inhibiting both angiogenesis and lymph node metastasis, which suggests Oxyres be a potential therapeutic agent.

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.

Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat diet-induced solid tumor growth and lymph node metastasis.

Previously, we reported that high-fat-diet (HFD)-induced obesity stimulates melanoma progression in the B16F10 allograft model. In this study, we examined whether oleuropein (OL), the most abundant phenolic compound in olives, inhibits HFD-induced melanoma progression. Four-week-old male C57BL/6N mice were fed a HFD-diet with or without OL. After 16 weeks of feeding, B16F10-luc cells were subcutaneously injected and the primary tumor was resected 3 weeks later. OL suppressed HFD-induced solid tumor growth. In the tumor tissues, OL reduced HFD-induced expression of angiogenesis (CD31, VE-cadherin, VEGF-A, and VEGFR2), lymphangiogenesis (LYVE-1, VEGF-C, VEGF-D, and VEGFR3), and hypoxia (HIF-1α and GLUT-1) markers as well as HFD-induced increases in lipid vacuoles and M2 macrophages (MΦs). All animals were euthanized 2.5 weeks after tumor resection. OL suppressed HFD-induced increases in lymph node (LN) metastasis; expression of VEGF-A, VEGF-C, and VEGF-D in the LN; and M2-MΦs and the size of adipocytes in adipose tissues surrounding LNs. Co-culture results revealed that the crosstalk between B16F10s, M2-MΦs, and differentiated 3T3-L1 cells under hypoxic conditions increased the secretion of VEGF-A and -D, which stimulated tube formation and migration of endothelial cells (HUVECs) and lymphatic endothelial cells (LEC), respectively. Additionally, OL directly inhibited the differentiation of 3T3-L1 preadipocytes and tube formation by HUVECs and LECs. The overall results indicated that dietary OL inhibits lipid and M2-MΦ accumulation in HFD-fed mice, which contributes to decreases in VEGF secretion, thereby leading to inhibition of angiogenesis and lymphangiogenesis.

The role of fatty acid ß-oxidation in lymphangiogenesis.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid ß-oxidation, impairs lymphatic development. LECs use fatty acid ß-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid ß-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo.

Pien Tze Huang suppresses VEGF-C-mediated lymphangiogenesis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. The majority of patients are not suitable for surgery due to the presence of metastatic disease at the time of diagnosis, which has led to a high mortality rate for patients with CRC. Lymphangiogenesis, formation of new lymphatic vessels, plays an critical role in cancer progression particularly in cancer metastasis. Vascular endothelial growth factor-C (VEGF-C) has been previously demonstrated to play a pivotal role in cancer metastasis and therefore has become an attractive target for anticancer treatments. Pien Tze Huang (PZH) is a well-known traditional Chinese formula, which has exhibited significant therapeutic effects against CRC. However, the molecular mechanisms underlying its anticancer effects, particularly in regards to antimetastasis activity, still require further elucidation. In the present study, we evaluated the effects of PZH on cell migration and VEGF-C expression using various human CRC cell lines. Moreover, using a VEGF­C-stimulated human lymphatic endothelial cell (HLEC) model, we demonstrated that PZH suppresses lymphangiogenesis by attenuating cell migration and tube formation. This indicates that PZH possesses significant antimetastatic activity. Moreover, suppression of lymphangiogenesis by PZH via the downregulation of VEGF-C may be a potential molecular mechanism by which PZH inhibits metastasis in CRC.

Total saponins of panaxnotoginseng promotes lymphangiogenesis by activation VEGF-C expression of lymphatic endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lymphatic system plays an important role in maintaining the fluid homeostasis and normal immune responses, anatomic or functional obstruction of which leads to lymphedema, and treatments for therapeutic lymphangiogenesis are efficiency for secondary lymphedema. Total saponins of panaxnotoginseng (PNS) are a mixture isolated from Panaxnotoginseng (Burkill) F.H.Chen, which has been used as traditional Chinese medicine in China for treatment of cardio- and cerebro-vascular diseases. The aim of this study was to determine the effect and mechanism of PNS on lymphangiogenesis. METHODS: The Tg (fli1: egfp; gata1: dsred) transgenic zebrafish embryos were treated with different concentrations of PNS (10, 50, 100µM) for 48h with or without the 6h pretreatment of the 30µM Vascular endothelial growth factors receptor (VEGFR)-3 kinase inhibitor, followed with morphological observation and lympangiogenesis of thoracic duct assessment. The effect of PNS on cell viability, migration, tube formation and Vascular endothelial growth factors (VEGF)-C mRNA and protein expression of lymphatic endothelial cells (LECs) were determined. The role of phosphatidylinositol-3 (PI-3)-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2 pathways, c-Jun N-terminal kinase (JNK) and P38 mitogen activated protein kinases (MAPK) signaling in PNS-induced VEGF-C expression of LECs by using pharmacological agents to block each signal. RESULTS: PNS promotes lymphangiogenesis of thoracic duct in zebrafish with or without VEGFR3 Kinase inhibitor pre-impairment. PNS promotes proliferation, migration and tube formation of LECs. The tube formation induced by PNS could be blocked by VEGFR3 Kinase inhibitor. PNS induce VEGF-C expression of LEC, which could be blocked by ERK1/2, PI3K and P38MAPK signaling inhibitors. CONCLUSION: PNS activates lymphangiogenesis both in vivo and in vitro by up-regulating VEGF-C expression and activation of ERK1/2, PI3K and P38MAPK signaling. These findings provide a novel insight into the role of PNS in lymphangiogenesis and suggest that it might be an attractive and suitable therapeutic agent for treating secondary lymphedema or other lymphatic system impairment related disease.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

BACKGROUND: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. METHODS AND RESULTS: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. CONCLUSIONS: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

Ginsenoside Rg1 enhances lymphatic transport of intrapulmonary silica via VEGF-C/VEGFR-3 signaling in silicotic rats.

Ginsenoside Rg1, extracted mainly from Panax ginseng, has been shown to exert strong pro-angiogenic activities in vivo. But it is unclear whether ginsenoside Rg1 could promote lung lymphangiogenesis to improve lymphatic transport of intrapulmonary silica in silicotic rats. Here we investigated the effect of ginsenoside Rg1 on lymphatic transport of silica during experimental silicosis, and found that ginsenoside Rg1 treatment significantly raised the silicon content in tracheobronchial lymph nodes and serum to reduce the silicon level in lung interstitium, meanwhile increased pulmonary lymphatic vessel density by enhancing the protein and mRNA expressions of vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3). The stimulative effect of ginsenoside Rg1 on lymphatic transport of silica was actively correlated with its pro-lymphangiogenic identity. And VEGFR-3 inhibitor SAR131675 blocked these above effects of ginsenoside Rg1. These findings suggest that ginsenoside Rg1 exhibits good protective effect against lung burden of silica during experimental silicosis through improving lymphatic transport of intrapulmonary silica, which is potentially associated with the activation of VEGF-C/VEGFR-3 signaling pathway.

VEGF-C and VEGF-C156S in the pro-lymphangiogenic growth factor therapy of lymphedema: a large animal study.

INTRODUCTION: VEGF-C156S, a lymphangiogenesis-specific form of vascular endothelial growth factor C (VEGF-C), has been considered as a promising candidate for the experimental pro-lymphangiogenic treatment, as it lacks potential angiogenic effects. As a precursor to future clinical trials, the therapeutic efficacy and blood vascular side effects of VEGF-C and VEGF-C156S were compared in a large animal model of secondary lymphedema. Combination of lymphatic growth factor treatment and autologous lymph node transfer was used to normalize the lymphatic anatomy after surgical excision of lymphatic tissue. METHODS: Lymph vessels around the inguinal lymph node of female domestic pigs were destroyed in order to impair the normal lymphatic drainage from the hind limb. Local injections of adenoviruses (Ad) encoding VEGF-C or VEGF-C156S were used to enhance the regrowth of the lymphatic vasculature. AdLacZ (ß-galactosidase) and saline injections served as controls. RESULTS: Both VEGF-C and VEGF-C156S induced growth of new lymphatic vessels in the area of excision, although lymphangiogenesis was notably stronger after VEGF-C treatment. Also the transferred lymph nodes were best-preserved in the VEGF-C-treated pigs. Despite the enlargement of blood vessels following the VEGF-C therapy, no signs of sprouting angiogenesis or increased blood vascular permeability in the form of increased wound exudate volumes were observed. CONCLUSIONS: Our results show that VEGF-C provides the preferred alternative for growth factor therapy of lymphedema when compared to VEGF-C156S, due to the superior lymphangiogenic response and minor blood vessel effects. Furthermore, these observations suggest that activation of both VEGFR-2 and VEGFR-3 might be needed for efficient lymphangiogenesis.

Corosolic Acid Exhibits Anti-angiogenic and Anti-lymphangiogenic Effects on In Vitro Endothelial Cells and on an In Vivo CT-26 Colon Carcinoma Animal Model.

We describe the anti-angiogenic and anti-lymphangiogenic effects of corosolic acid, a pentacyclic triterpenoid isolated from Cornus kousa Burg. A mouse colon carcinoma CT-26 animal model was employed to determine the in vivo anti-angiogenic and anti-lymphangiogenic effects of corosolic acid. Corosolic acid induced apoptosis in CT-26 cells, mediated by the activation of caspase-3. In addition, it reduced the final tumor volume and the blood and lymphatic vessel densities of tumors, indicating that it suppresses in vivo angiogenesis and lymphangiogenesis. Corosolic acid inhibited the proliferation and tube formation of human umbilical vein endothelial cells and human dermal lymphatic microvascular endothelial cells. In addition, corosolic acid decreased the proliferation and migration of human umbilical vein endothelial cells stimulated by angiopoietin-1. Pretreatment with corosolic acid decreased the phosphorylation of focal adhesion kinase (FAK) and ERK1/2, suggesting that corosolic acid contains anti-angiogenic activity that can suppress FAK signaling induced by angiopoietin-1.

Cilostazol improves lymphatic function by inducing proliferation and stabilization of lymphatic endothelial cells.

BACKGROUND: Cilostazol, an inhibitor of phosphodiesterase type III, is an antiplatelet agent and vasodilator. Some clinical reports have suggested that this drug can improve progressive and refractory lymphedema. OBJECTIVE: In this study, we investigated whether cilostazol has the potential to proliferate lymphatic vessels and to improve lymphatic function using human lymphatic endothelial cells (LECs) and mouse lymphedema models. METHODS: Human LECs were counted at several time points while they were cultured in the presence of cilostazol and/or protein kinase A inhibitor. After receiving a diet including 0.1% cilostazol or control diet, skin tissue and lymphatic function of k-cyclin transgenic (kCYC(+/-)) mice, which have pernicious lymphatic dysfunction, was analyzed. A different lymphedema model was generated in wild type mice by excising circumferential tail skin to remove the superficial lymphatics. After oral administration of cilostazol, tail lymphedema was examined in this mouse model. RESULTS: Proliferation of LECs was promoted in a dose-dependent manner, which was partially inhibited by a protein kinase A inhibitor. Lymphatic vessel count increased in the cilostazol-treated kCYC(+/-) mice over that in the non-treated mice. Lymph flow improved in cilostazol-fed kCYC(+/-) mice as assessed by subcutaneous injection of Evans blue dye into the footpad. Oral administration of cilostazol also decreased lymphedema in a tail of wild type mice. CONCLUSION: Cilostazol promoted growth of human LECs and improved lymph flow and lymphedema in two different mouse lymphedema models. These results suggest that cilostazol would be a promising agent for the treatment of lymphedema.

VEGF and VEGFR genotyping in the prediction of clinical outcome for HCC patients receiving sorafenib: the ALICE-1 study.

Although new treatment modalities changed the global approach to hepatocellular carcinoma (HCC), this disease still represents a medical challenge. Currently, the therapeutic stronghold is sorafenib, a tyrosine kinase inhibitor (TKI) directed against the vascular endothelial growth factor (VEGF) family. Previous observations suggested that polymorphisms of VEGF and its receptor (VEGFR) genes may regulate angiogenesis and lymphangiogenesis and thus tumour growth control. The aim of our study was to evaluate the role of VEGF and VEGFR polymorphisms in determining the clinical outcome of HCC patients receiving sorafenib. From a multicentre experience 148 samples (tumour or blood samples) of HCC patients receiving sorafenib were tested for VEGF-A, VEGF-C and VEGFR-1,2,3 single nucleotide polymorphisms (SNPs). Patients' progression-free survival (PFS) and overall survival (OS) were analysed. At univariate analysis VEGF-A alleles C of rs25648, T of rs833061, C of rs699947, C of rs2010963, VEGF-C alleles T of rs4604006, G of rs664393, VEGFR-2 alleles C of rs2071559, C of rs2305948 were significant predictors of PFS and OS. At multivariate analysis rs2010963, rs4604006 and BCLC (Barcelona Clinic Liver Cancer) stage resulted to be independent factors influencing PFS and OS. Once prospectively validated, the analysis of VEGF and VEGFR SNPs may represent a clinical tool to better identify HCC patients more likely to benefit from sorafenib. On the other hand, the availability of more accurate predictive factors could help avoiding unnecessary toxicities to potentially resistant patients who may be optimal candidates for different treatments interfering with other tumour molecular pathways.

Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells.

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.

Impact of acetylsalicylic acid on tumor angiogenesis and lymphangiogenesis through inhibition of VEGF signaling in a murine sarcoma model.

Aspirin is a salicylate drug that is widely used, and recently it has been shown to influence the development of various types of cancers. Our previous study revealed that aspirin had an inhibitory effect on the growth of S180 sarcoma and 3AO human ovarian cancer cells. The present study utilized a murine S180 sarcoma model to investigate the molecular mechanisms involved in aspirin-induced tumor growth inhibition. Tumor-bearing mice were randomly divided into five groups with 10 mice in each group: i) control; ii) 5-fluorouracil (5-FU); iii) high-dose aspirin (250 mg/kg); iv) low-dose aspirin (50 mg/kg); and v) combination of 5-FU and aspirin (50 mg/kg). The effect of aspirin on tumor growth was observed by measuring tumor volume and evaluating the antitumor effect. Tumor histology and immunohistochemistry were performed to detect the microvessel density (MVD), lymphatic vessel density (LVD), and the expression levels of vascular endothelial growth factor A (VEGF-A) and VEGF-C. The expression of VEGF-A and VEGF-C was also confirmed and quantified by western blotting. We discovered significant growth delay in murine S180 sarcoma as a result of aspirin treatment. The inhibition rate of tumor growth induced by high-dose and low-dose aspirin was 33.5 and 22.2%, respectively (P<0.05). The expression of VEGF-A and VEGF-C in tumor tissues inhibited by aspirin was demonstrated by immunohistochemistry, and the MVD was decreased in a dose-dependent manner (p<0.05). Reduced LVD was particularly apparent in the high-dose aspirin group (p<0.05). Western blot data showed that the expression of both VEGF-A and VEGF-C was reduced after treatment with aspirin. In conclusion, the impact of aspirin-induced tumor growth delay of murine S180 sarcoma may correlate with the inhibition of angiogenesis and lymphangiogenesis by reducing VEGF-A and VEGF-C expression in tumor tissues.

Studies on lymphangiogenesis inhibitors from Korean and Japanese crude drugs.

Metastasis occurs when cancer cells detach from a tumor, travel to distant sites in the body and develop into tumors in these new locations. Most cancer patients die from metastases. Among the various forms of cancer metastasis, lymphatic metastasis is an important determinant in cancer treatment and staging. In this study, we investigated lymphangiogenesis inhibitors from crude drugs used in Japan and Korea. The three crude drugs Saussureae Radix, Psoraleae Semen and Aurantti Fructus Immaturus significantly inhibited the proliferation of temperature-sensitive rat lymphatic endothelial (TR-LE) cells in vitro. By a chromatographic method using bioassay-guided fractionation methods, costunolide (1) and dehydrocostus lactone (2) from S. Radix, p-hydroxybenzaldehyde (3), psoralen (4), angelicin (5), psoracorylifol D (6), isobavachalone (7), bavachinin (8) Δ(3),2-hydroxybakuchiol (9) and bakuchiol (10) from P. Semen and cis-octadecyl ferulate (11), (2R)-3ß,7,4'-trihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (12), (2S)-7,4'-dihydroxy-5-methoxy-8-(γ,γ-dimethylallyl)-flavanone (13) and umbelliferone (14) from A. F. Immaturus were obtained. Three compounds (compounds 11-13) from A. F. Immaturus were isolated for the first time from this medicinal plant. Among isolated compounds, ten compounds (compounds 1, 2, 6-12, 13) showed an inhibitory effect on the proliferation and the capillary-like tube formation of TR-LE cells. In addition, all compounds except compound 12 showed selective inhibition of the proliferation of TR-LE cells compared to Hela and Lewis lung carcinoma (LLC) cells. These compounds might offer clinical benefits as lymphangiogenesis inhibitors and may be good candidates for novel anti-cancer and anti-metastatic agents.