VEGF-D Research Articles

Sonic hedgehog promotes endothelial differentiation of bone marrow mesenchymal stem cells via VEGF-D.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been proved to be capable of differentiating into endothelial cells (ECs), however, the differentiation efficiency is rather low. Sonic hedgehog (Shh), an important factor in vascular development and postnatal angiogenesis, exerted promotional effect on new vessel formation in the ischemic animal models. Therefore, the current study aims to investigate whether Shh could induce the endothelial differentiation of BMSCs both in vitro and in vivo, as well as the mechanism of differentiation induction. The current study over-expressed Shh in BMSCs by lentivirus transduction. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analysis was performed to determine the angiogenic factors in both control BMSCs and Shh over-expressed BMSCs. Immunocytochemistry was also conducted to examine the EC markers. Angiogenesis was determined by in vitro tube-forming assay on Matrigel and in vivo Matrigel plug in severe combined immunodeficient (SCID) mice. Last, mRNA sequencing analysis was used to elaborate the underlying mechanisms. Loss of function study was performed by vascular endothelial growth factor D (VEGF-D) siRNA. Shh expression was increased by about 3,000-fold and 5,000-fold at 3 days-transfection and 7 days-transfection, respectively. Patched 1 (Ptch1), the receptor for Shh, had a two-fold increase after transduction. The angiogenic factors such as hepatocyte growth factor (HGF), angiopoietin-1 (Ang-1), insulin-like growth factor 1 (IGF1) and vascular endothelial growth factor A (VEGF-A) had at least a 1.5-fold increase after transduction. Expression of EC-lineage markers, CD31 and VE-cadherin, on Shh-overexpressed BMSCs were increasingly detected by immunocytostaining. Angiogenesis of BMSCs could be efficiently induced by Shh overexpression in the in vitro tube-formation assay and in vivo Matrigel plug. Additionally, mRNA sequencing analysis revealed that Shh activation upregulated the expression of several pro-angiogenic factors, like Angptl4, Egfl6, VEGF-D. Loss of function study by VEGF-D siRNA confirmed that Shh enhanced the angiogenic ability of BMSCs via VEGF-D. This study demonstrated that Shh could promote endothelial differentiation of BMSCs via VEGF-D.

Histone deacetylase 4 shapes neuronal morphology via a mechanism involving regulation of expression of vascular endothelial growth factor D

Nucleo-cytoplasmic shuttling of class IIa histone deacetylases (i.e HDAC4, -5, -7, and -9) is a synaptic activity- and nuclear calcium-dependent mechanism important for epigenetic regulation of signal-regulated gene expression in hippocampal neurons. HDAC4 in particular has been linked to the regulation of genes important for both synaptic structure and plasticity. Here, using a constitutively nuclear-localized, dominant-active variant of HDAC4 (HDAC4 3SA), we demonstrate that HDAC4 accumulation in the nucleus severely reduces both the length and complexity of dendrites of cultured mature hippocampal neurons, but does not affect the number of dendritic spines. This phenomenon appeared to be specific to HDAC4, as increasing the expression of HDAC3 or HDAC11, belonging to class I and class IV HDACs, respectively, did not alter dendritic architecture. We also show that HDAC4 3SA decreases the expression of vascular endothelial growth factor D (VEGFD), a key protein required for the maintenance of dendritic arbors. The expression of other members of the VEGF family and their receptors was not affected by the nuclear accumulation of HDAC4. VEGFD overexpression or administration of recombinant VEGFD, but not VEGFC, the closest VEGFD homologue, rescued the impaired dendritic architecture caused by the nuclear-localized HDAC4 variant. These results identify HDAC4 as an epigenetic regulator of neuronal morphology that controls dendritic arborization via the expression of VEGFD.

Characterization of lymphangioleiomyomatosis patients with discordance between spirometric and diffusion measurements of pulmonary function.

Background: A subset of lymphangioleiomyomatosis (LAM) patients present with normal FEV1 and FVC but with reduced DLCO. Patients with an isolated reduction in DLCO in other diseases appear to be at higher risk for pulmonary hypertension and worse survival but this has not been previously described in LAM patients. Objective: To characterize the prevalence and clinical progression of LAM patients who present with discordantly low DLCO. Methods: This was a retrospective cohort study of LAM patients in two centers in the United States and Brazil. Discordant DLCO was defined as FEV1 >80% predicted, FVC >80% predicted, and DLCO<80% predicted. We compared the rate of decline in pulmonary function, pulmonary artery to aorta (PA-A) ratio, and VEGF-D levels in patients with concordant and discordant DLCO. Results: The overall prevalence of discordant DLCO was 26.0%. Patients with discordant DLCO did not have a higher rate of yearly decline in FEV1 (-1.0±0.6 vs -1.0±0.6, p=0.50), FVC (-1.0±0.7 vs -0.3±0.8, p=0.54), or DLCO (-2.2±0.9 vs -1.6±0.6, p=0.79). They did not have higher rates of PA-A ratio>1 (23.3% vs 20.1%, p=1.00). Patients with discordant DLCO did not have higher levels of VEGF-D (1214±1256 pg/mL vs 1706±1214 pg/mL, p=0.07). Conclusions: LAM patients who present with a discordantly low DLCO do not appear to have different rates of decline in pulmonary function. Additional biological and radiographic markers are needed to more fully characterize this population. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 206-212).

Computational Design Of A Mimic Against Neurodegenerative Diseases Based On VEGF‐D

The basis of this research is the use of a computational approach to design drugs based on the tertiary structure of the protein VEGF‐D. Vascular Endothelial Growth Factor‐D has been discovered to have a profound role in neuronal growth in which it demonstrates its ability to repair memory and improve cognitive functions. This discovery of VEGF‐D's new function has fascinated researchers as it shows promising results of being a possible cure against neurodegenerative diseases such as Alzheimer's however, its inability to bypass the blood brain barrier has intrigued our collaborators to develop a set of peptides that may induce the same reaction as VEGF‐D. The purpose of this research is to analyze the structural and functional details of VEGF‐D complexed to its receptor VEGFR‐3 at atomic level, design smaller drugs that have the potential to act as VEGF‐D, and identify the best modeled peptide that interacts most favorably with the receptor to present to our collaborators for experimental testing. This work involves the designing of 14 small peptides for which molecular dynamic simulations were performed to investigate the behavior of the peptides along the trajectory. Information obtained from dynamics simulation denote peptide 56 as the most suitable candidate to induce the same reaction as VEGF‐D.Support or Funding InformationNew York College Of Technology, NSFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional improvements in patients with lymphangioleiomyomatosis after sirolimus: an observational study

BackgroundSirolimus has been shown to be effective in patients with lymphangioleiomyomatosis (LAM). We wish to summarize our experience using sirolimus and its effectiveness in LAM patients.MethodsWe analyzed data from 98 patients who were diagnosed with definite or probable sporadic LAM based on the European Respiratory Society diagnosis criteria for LAM in 2010 at Peking Union Medical College Hospital and who had received sirolimus during January 2007 to June 2015. The data before and after the initiation of sirolimus therapy included pulmonary function tests, arterial blood gas analysis, 6-min walking distance (6MWD), size of chylous effusion and renal angiomyolipomas (AML), St. George’s Respiratory Questionnaires (SGRQ) and vascular endothelial growth factor-D (VEGF-D) levels. Serum levels of sirolimus and adverse events were collected.ResultsMedian follow-up was 2.5 years. Most patients had forced expiratory volume in 1 s (FEV1) values less than 70% predicted or symptomatic chylothorax. The mean changes before and after the initiation of sirolimus were − 31.12 ± 30.78 mL/month and 16.11 ± 36.00 mL/month (n = 18,p = 0.002) for FEV1 change, and − 0.55 ± 0.60 mmHg/month and 0.30 ± 1.19 mmHg/month (n = 17, p = 0.018) for PaO2 change. 6MWD improved from 358.8 ± 114.4 m to 415.6 ± 118.6 m (n = 46, p = 0.004) and SGRQ total score from 57.2 ± 21.0 to 47.5 ± 22.8 (n = 50, p < 0.001). The median VEGF-D concentration decreased to 1609.4 pg/mL from 3075.6 pg/mL after sirolimus therapy (n = 41, p < 0.001). Patients with sirolimus trough levels of 5–9.9 ng/mL had an increase in FEV1 (p < 0.05). Sixty-five percent of patients (13/20) had almost complete resolution of chylous effusions. The most frequent adverse events were mouth ulcers, menstrual disorder, hyperlipidemia and acneiform rash, all were mild.ConclusionLong-term use of sirolimus is safe in patients with LAM. LAM patients with FEV1 less than 70% predicted and symptomatic chylothorax are suitable for receiving sirolimus therapy. The maintaining serum trough levels of sirolimus are recommended between 5 to 9.99 ng/mL.

The nationwide screening project on plasma angiogenesis-related mediators for treatment selection of optimal antiangiogenic inhibitors in metastatic colorectal cancer: GI-SCREEN CRC-Ukit.

TPS885 Background: Antiangiogenic treatments are a proven useful tool to improve clinical outcomes in patients (pts) with metastatic colorectal cancer (mCRC). Recently, higher levels of vascular endothelial growth factor-D (VEGF-D) are a potential predictive biomarker for ramucirumab efficacy on overall and progression-free survival in mCRC (Tabernero J, et al. ESMO 2017). However, there are limited data on mCRC associating the efficacy of antiangiogenic therapy with angiogenesis-related mediators, such as VEGF family members and their receptors, and with dynamic transition among these mediators during clinical courses. In addition, immune-related factors such as interferon gamma (IFNγ) and transforming growth factor β1 (TGFβ1) may be associated with the regulation of angiogenesis. Methods: This prospective longitudinal study aims to investigate the association between plasma angiogenesis-related mediators and clinical outcomes in mCRC. The key eligibility criteria include pts with mCRC who will receive one of the following regimens: first-line chemotherapy plus bevacizumab, first-line chemotherapy plus anti-EGFR antibody (either cetuximab or panitumumab), second-line FOLFIRI plus ramucirumab, second-line FOLFIRI plus aflibercept, or second-line chemotherapy plus bevacizumab. Plasma is collected twice in pairs from all pts pre- and post-treatment. Comprehensive measurements of plasma angiogenesis-related mediators, such as placental growth factor (PlGF), hepatocyte growth factor (HGF), IL-6, IL-8, angiopoietin-2, neuropillin-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), thrombospondin-2 (TSP-2), osteopontin (OPN), sVEGFR-1, sVEGFR-2, sVEGFR-3, VEGF-A, VEGF-D, as well as key targets associated with immunotherapy such as IFNγ and TGFβ1 are analyzed in parallel by the multiplex assay with Luminex® technology. The target sample size is 1,000. This study was initiated in September 2017. Clinical trial information: UMIN000028616.

Emerging Roles for VEGF-D in Human Disease.

Blood vessels and lymphatic vessels are located in many tissues and organs throughout the body, and play important roles in a wide variety of prevalent diseases in humans. Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that can promote the remodeling of blood vessels and lymphatics in development and disease. Recent fundamental and translational studies have provided insight into the molecular mechanisms by which VEGF-D exerts its effects in human disease. Hence this protein is now of interest as a therapeutic and/or diagnostic target, or as a potential therapeutic agent, in a diversity of indications in cardiovascular medicine, cancer and the devastating pulmonary condition lymphangioleiomyomatosis. This has led to clinical trial programs to assess the effect of targeting VEGF-D signaling pathways, or delivering VEGF-D, in angina, cancer and ocular indications. This review summarizes our understanding of VEGF-D signaling in human disease, which is largely based on animal disease models and clinicopathological studies, and provides information about the outcomes of recent clinical trials testing agonists or antagonists of VEGF-D signaling.

EFFICACY OF INTRAVITREAL AFLIBERCEPT IN MACULAR TELANGIECTASIA TYPE 1 IS LINKED TO THE OCULAR ANGIOGENIC PROFILE.

To evaluate intravitreal aflibercept in macular telangiectasia Type 1 (MacTel 1) patients and measure their ocular angiogenic profile. Eight subjects with MacTel 1 refractory to bevacizumab, ranibizumab, or laser therapy and switched to aflibercept were included. Best-corrected visual acuity, central macular thickness, and cystic areas quantified on optical coherence tomography B-scans were assessed during 12 months. Perifoveal capillary densities were measured on optical coherence tomography angiography. Aqueous humor was sampled from six patients and eight control subjects undergoing cataract extraction. Growth factors were quantified using a multiarray immunoassay. Over 12 months, patients received 6.6 ± 1.4 (range, 5-8) intravitreal aflibercept injections. Twelve months after switching to aflibercept, best-corrected visual acuity increased by ≥5 letters in 5 of 8 patients, compared with preaflibercept levels. Mean best-corrected visual acuity improved from 79.6 (∼20/50) to 88.0 (∼20/35) Early Treatment Diabetic Retinopathy Study letters (P = 0.042), and central macular thickness decreased from 434 ± 98 μm to 293 ± 59 μm (P = 0.014). Compared with control subjects, the profile of angiogenic factors in MacTel 1 eyes revealed no difference in vascular endothelial growth factor-A levels but significantly higher levels of placental growth factor (P = 0.029), soluble vascular endothelial growth factor receptor-1 (sFlt-1; P = 0.013), vascular endothelial growth factor-D (P = 0.050), and Tie-2 (P = 0.019). Placental growth factor levels inversely correlated with both superficial and deep capillary plexus densities on optical coherence tomography angiography (P = 0.03). The clinical response to aflibercept coupled to the angiogenic profile of MacTel 1 eyes support the implication of the placental growth factor/Flt-1 pathway in MacTel 1.

Sirolimus for the treatment of lymphangioleiomyomatosis

ABSTRACTIntroduction: Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease that is associated with recurrent pneumothorax, abdominal tumors including lymphangioleiomyomas and angiomyolipomas, chylous fluid accumulations and progressive respiratory failure. Sirolimus has been shown to slow the progression of disease.Areas covered: In this review, the current evidence, indications, precautions, and practical use of sirolimus therapy in patients with LAM is discussed.Expert opinion: In LAM, the lung becomes diffusely infiltrated with smooth muscle-like cells containing mechanistic target of rapamycin complex (mTOR) activating mutations in tuberous sclerosis genes which drive destructive parenchymal remodeling. LAM cells arise from an unknown source, migrate to the lung and express vascular endothelial growth factor D (VEGF-D). In the laboratory, mTOR inhibitors block destructive LAM cell behaviors including abnormal growth, migration and metalloproteinase production and induce tumor regression in animal models. The MILES trial demonstrated that sirolimus, stabilizes lung function, reduces VEGF-D and improves measures of functional performance. Based on MILES, sirolimus was approved by the United States Food and Drug Administration for the treatment of LAM in 2015. Available mTOR inhibitor therapies are suppressive, requiring chronic or even lifelong exposure for continued benefit. Multiple trials are underway to refine the use of mTOR inhibitors in LAM.

Long-Term Effect of Sirolimus on Serum Vascular Endothelial Growth Factor D Levels in Patients With Lymphangioleiomyomatosis

Sirolimus reduces serum levels of vascular endothelial growth factor D (VEGF-D); the size of chylous effusions, lymphangioleiomyomas, and angiomyolipomas; and stabilizes lung function in patients with lymphangioleiomyomatosis (LAM). To determine whether reductions in VEGF-D levels are sustained over time, as well as parallel changes in lung function and lymphatic disease, we evaluated 25 patients with LAM and measured VEGF-D levels, lung function, and extent of lymphatic disease before and during sirolimus therapy. Treatment with sirolimus stabilized FEV1 and diffusion capacity for carbon monoxide (Dlco) over a period of 4.5 ± 1.6 years, caused resolution of lymphatic disease, and reduced the size of angiomyolipomas and VEGF-D levels (3,720 ± 3,020 pg/mL to 945 ± 591 pg/mL; P< .0001). Yearly changes in FEV1 %predicted and Dlco %predicted were reduced from -7.4%± 1.4%to -0.3%± 0.5%(P< .001) and -6.4%± 0.9%to -0.4%± 0.5%(P< .001), respectively. Lower VEGF-D levels correlated with sirolimus therapy (P<.001), but no significant relationship was observed between reduction in VEGF-D levels and FEV1 and Dlco during sirolimus therapy. The magnitude of VEGF-D decline was not related to the effect on lung function. Patients with lymphatic disease had higher serum VEGF-D levels, a greater reduction in VEGF-D levels, and better long-term sustained improvement in lung function during sirolimus therapy than did those without lymphatic disease. Sirolimus therapy stabilizes lung function over many years of therapy while producing a sustained reduction in VEGF-D levels in patients with elevated levels preceding therapy. An association was not demonstrated between the magnitude of VEGF-D decline and the beneficial effect of sirolimus on lung function. Persistent improvement in lung function was observed in patients with lymphatic disease.

Single-Arm Resistance Training Study to Determine the Relationship between Training Outcomes and Muscle Growth Factor mRNAs in Older Adults Consuming Numerous Medications and Supplements.

Determine if the muscle mRNA levels of three growth factors (insulin-like growth factor-1 [IGF1], ciliary neurotropic factor [CNTF], and vascular endothelial growth factor-D [VEGFD]) are correlated with muscle size and strength gains from resistance exercise while piloting a training program in older adults taking medications and supplements for age-associated problems. Single-arm prospective study. US Veterans Affairs hospital. Older (70±6 yrs) male Veterans (N=14) of US military service. Thirty-five sessions of high-intensity (80% one-rep max) resistance training including leg press, knee curl, and knee extension to target the thigh muscles. Vastus lateralis biopsies were collected and body composition (DEXA) was determined pre- and post-training. Simple Pearson correlations were used to compare training outcomes to growth factor mRNA levels and other independent variables such as medication and supplement use. Average strength increase for the group was ≥ 25% for each exercise. Subjects averaged taking numerous medications (N=5±3) and supplements (N=2±2). Of the growth factors, a significant correlation (R>0.7, P≤0.003) was only found between pre-training VEGFD and gains in lean thigh mass and extension strength. Mass and strength gains were also correlated with use of α-1 antagonists (R=0.55, P=0.04) and pre-training lean mass (R=0.56, P=0.04), respectively. Muscle VEGFD, muscle mass, and use of α-1 antagonists may be predisposing factors that influence the response to training in this population of older adults but additional investigation is required to determine if these relationships are due to muscle angiogenesis and blood supply.

Vascular endothelial growth factor-D modulates oxidant-antioxidant balance of human vascular endothelial cells.

Vascular endothelial growth factor‐D (VEGF‐D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF‐D stimulates the expression of proteins involved in cell–matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up‐regulated expression of proteins that form the antioxidant barrier of the cell in VEGF‐D‐treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF‐D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF‐D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF‐D‐induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser‐2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.

Sulfatase 2 facilitates lymphangiogenesis in breast cancer by regulating VEGF-D.

In our previous studies, sulfatase 2 (Sulf2) was found to upregulate vascular endothelial growth factor-D (VEGF-D) expression in breast cancer. As VEGF-D plays an important role in lymphangiogenesis, we hypothesized that Sulf2 facilitates lymphangiogenesis in breast cancer by regulating VEGF-D. To evaluate the functions of Sulf2 on lymphangiogenesis in breast cancer, proliferation, apoptosis, cell cycle, cell mobility and tube-formation of lymphatic endothelial cells (LECs) were measured in vitro. Lymphangiogenesis in nude mouse ears and breast cancer xenografts were examined in vivo. Furthermore, the expression levels of related signaling pathway genes were screened and verified in LECs. We found that Sulf2 significantly increased the mobility and tube formation of the LECs, inhibited cisplatin-induced LEC apoptosis, but had no effect on cell proliferation and the cell cycle. Moreover, recombinant Sulf2 (rSulf2) combined with VEGF-D further promoted the proliferation, cell cycle, mobility and tube-like structure formation in the LECs, and at the same time inhibited cisplatin-induced apoptosis especially in the late stage. Sulf2 also significantly increased the density of lymphatic vessels in mouse ears and breast cancer xenografts in vivo. AKT1 was also shown to be upregulated and activated by Sulf2. Our results confirmed that Sulf2 facilitated lymphangiogenesis in breast cancer cells by regulating VEGF-D and that the AKT1-related signaling pathway was involved.

Effect of uterine histotroph on embryo development in pigs*

The aim of this study was to investigate the effect of uterine histotroph on embryo development and the expression of cysteine-rich protein 2 (CRP2), coatomer subunit gamma-2 (G2COP), myoglobin (MYG), vascular endothelial growth factor D (VEGFD), collagen alpha 4 chain (COL4) and galactoside 3-L-fucosyltransferase 4 (FUT4) proteins in porcine embryo during pre-implantation. Uterine histotroph (UH) was collected from uterine horn on corpus albican phase, and embryos were cultured in porcine zygote medium with UH for 168 hours. Cleavage and blastocyst formation of embryo were detected at 168 hours after in vitro fertilization. And CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were observed using confocal laser microscope. In results, embryo cleavage rate was not significantly changed by UH, but blastocyst rate was significantly (P<0.05) decreased in UH-treated embryos. Moreover, CRP2, G2COP, MYG, VEGFD, COL4 and FUT4 proteins were expressed in blastomere. CRP2 in embryo was significantly overexpressed (P<0.05), but not G2COP, MYG, VEGFD, COL4 and FUT4 proteins. In summary, UH on corpus albican phase was increased CRP2 protein in embryo, and inhibited blastocyst formation in preimplantation porcine embryos, suggesting that CRP2 may play an interrupter on embryo development in pigs.

The effects of β-elemene on rabbit VX2 laryngeal carcinoma and factors associated with tumor growth and metastasis

To investigate the inhibition effect of β-elemene on the growth and metastasis of laryngeal carcinoma and the underlying mechanism. Sixty-six rabbits were vaccinated with suspension of VX2 cancer tissues to establish a rabbit laryngeal carcinoma model and then they were randomly treated with the injection of 0.9% sodium chloride solution (as a control), cisplatin, or β-elemene from the seventh day after vaccination. The rabbits were sacrificed three weeks after vaccination. The laryngeal tumor was dissected. and the volume of tumor and the inhibitory rate of tumor growth were measured. The expressions of PCNA (proliferation cell nuclear antigen), Bcl-2 (B-cell lymphoma-2) and VEGF-D (vascular endothelial growth factor D) in tumor were semiquantitatively examined by immunohistochemistry. The inhibition rates of tumor growth in β-elemene-treated group and the cisplatin-treated group were 48.5% and 51.4%, respectively. The expressions of Bcl-2 in β-elemene-treated group and cisplatin-treated group were significantly lower than the control (P<0.05), but with no significant difference between β-elemene-treated group and cisplatin-treated group. The expression of PCNA in β-elemene-treated group was significantly decreased compared to control group (P<0.05). The expression of VEGF-D in β-elemene-treated group was lower than that in control group or the cisplatin-treated group (P<0.05). β-elemene can inhibit the growth of rabbit VX2 laryngeal tumor, which may be associated with the inhibition of factors related to tumor growth and metastasis.

Elevated VEGF-D Modulates Tumor Inflammation and Reduces the Growth of Carcinogen-Induced Skin Tumors

Vascular endothelial growth factor D (VEGF-D) promotes the lymph node metastasis of cancer by inducing the growth of lymphatic vasculature, but its specific roles in tumorigenesis have not been elucidated. We monitored the effects of VEGF-D in cutaneous squamous cell carcinoma (cSCC) by subjecting transgenic mice overexpressing VEGF-D in the skin (K14-mVEGF-D) and VEGF-D knockout mice to a chemical skin carcinogenesis protocol involving 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate treatments. In K14-mVEGF-D mice, tumor lymphangiogenesis was significantly increased and the frequency of lymph node metastasis was elevated in comparison with controls. Most notably, the papillomas regressed more often in K14-mVEGF-D mice than in littermate controls, resulting in a delay in tumor incidence and a remarkable reduction in the total tumor number. Skin tumor growth and metastasis were not obviously affected in the absence of VEGF-D; however, the knockout mice showed a trend for reduced lymphangiogenesis in skin tumors and in the untreated skin. Interestingly, K14-mVEGF-D mice showed an altered immune response in skin tumors. This consisted of the reduced accumulation of macrophages, mast cells, and CD4+ T-cells and an increase of cytotoxic CD8+ T-cells. Cytokine profiling by flow cytometry and quantitative real time PCR revealed that elevated VEGF-D expression results in an attenuated Th2 response and promotes M1/Th1 and Th17 polarization in the early stage of skin carcinogenesis, leading to an anti-tumoral immune environment and the regression of primary tumors. Our data suggest that VEGF-D may be beneficial in early-stage tumors since it suppresses the pro-tumorigenic inflammation, while at later stages VEGF-D-induced tumor lymphatics provide a route for metastasis.

TNF-alpha promotes lymphangiogenesis and lymphatic metastasis of gallbladder cancer through the ERK1/2/AP-1/VEGF-D pathway.

BackgroundTumor necrosis factor-alpha (TNF-α), a key player in cancer-related inflammation, was recently demonstrated to be involved in the lymphatic metastasis of gallbladder cancer (GBC). Vascular endothelial growth factor D (VEGF-D) is a key lymphangiogenic factor that is associated with lymphangiogenesis and lymph node metastasis in GBC. However, whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC remains undetermined.MethodsThe expression of VEGF-D in patient specimens was detected by immunohistochemistry and the relationship between VEGF-D in the tissue and TNF-α in the bile of the matching patients was analyzed. The VEGF-D mRNA and protein levels after treatment with exogenous TNF-α in NOZ, GBC-SD and SGC-996 cell lines were measured by real-time PCR and ELISA. The promoter activity and transcriptional regulation of VEGF-D were analyzed with the relative luciferase reporter assay, mutant constructs, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, RNA interference and Western blotting. Inhibitors of JNK, p38 MAPK and ERK1/2 were used to explore the upstream signaling effector of AP-1. We used lentiviral vector expressing a VEGF-D shRNA construct to knockdown VEGF-D gene in NOZ and GBC-SD cells. The role of the TNF-α-VEGF-D axis in the tube formation of human dermal lymphatic endothelial cells (HDLECs) was determined using a three-dimensional coculture system. The role of the TNF-α - VEGF-D axis in lymphangiogenesis and lymph node metastasis was studied via animal experiment.ResultsTNF-α levels in the bile of GBC patients were positively correlated with VEGF-D expression in the clinical specimens. TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo.ConclusionTaken together, our data suggest that TNF-α can promote lymphangiogenesis and lymphatic metastasis of GBC through the ERK1/2/AP-1/VEGF-D pathway.

Death receptor 6 (DR6) is required for mouse B16 tumor angiogenesis via the NF-κB, P38 MAPK and STAT3 pathways.

Although death receptor 6 (DR6) is aberrantly expressed in certain cancer cell lines, its function, signaling pathway and potential clinical significance in tumor progression are not well characterized. We report here that knocking down DR6 in the mouse B16 cell line has no effect on B16 cell death in vitro but suppresses xenograft B16 tumor growth by preventing tumor blood vessel formation in vivo. Deficiency of DR6 changes cytokine expression and secretion; in particular, it inhibits the proinflammatory cytokine interleukin-6 (IL-6), which is able to induce the expression of the angiogenesis-related factors: vascular endothelial growth factor-A, platelet-derived growth factor-β, vascular endothelial growth factor-D and platelet-derived growth factor receptor-α. Further experiments demonstrate that DR6-dependent angiogenesis is involved in the IL-6/P38 MAPK and IL-6/STAT3 pathways. Our novel findings demonstrate for the first time that DR6 expression in B16 cells facilitates tumor growth by accelerating tumor angiogenesis. Moreover, these results suggest that DR6 is involved in three important intracellular pathways that lead to homeostatic angiogenesis in tumor growth.

Change of uterine histroph proteins during follicular and luteal phase in pigs

The aim of this study was to examine protein expression patterns of uterine histroph (UH) during the follicular phase (FP) and luteal phase (LP) in pigs. Forty-nine common proteins were identified from FP and LP samples; five were significantly down-regulated (>1.5-fold), while 15 were significantly up-regulated (>1.5-fold) in LPUH compared with FPUH (P<0.05). The 20 differentially-expressed proteins are involved in cell proliferation, cell responses, translation, transport, and metabolism and their molecular functions include nucleic acid binding, oxygen activity, enzymatic activity, growth activity, iron binding, and redox binding. Protein expression of vascular endothelial growth factor D (VEGFD), coatomer subunit gamma-2 (G2COP), collagen alpha 4 chain (COL4), cysteine rich protein 2 (CRP2), myoglobin (MYG), and galactoside 3-L-fucosyltransferase 4 (FUT4) was analyzed by Western blotting. These proteins were significantly higher in LPUH compared to FPUH (P<0.05). These data expand our understanding of changes in the intrauterine environment during the pre-implantation period in pigs.

Vascular endothelial growth factor-D mediates fibrogenic response in myofibroblasts.

Vascular endothelial growth factor (VEGF)-D is a crucial mediator of angiogenesis. Following myocardial infarction (MI), cardiac VEGF-D and VEGF receptor (VEGFR)-3 are significantly upregulated. In addition to endothelial cells, myofibroblasts at the site of MI highly express VEGFR-3, implicating the involvement of VEGF-D in cardiac fibrogenesis that promotes repair and remodeling. The aim of the current study was to further explore the critical role of VEGF-D in fibrogenic response in myofibroblasts. Myofibroblast proliferation, migration, collagen synthesis, and degradation were investigated in cultured cardiac myofibroblasts subjected to VEGF-D with/without VEGFR antagonist or ERK inhibitor. Vehicle-treated cells served as controls. Myofibroblast proliferation and migration were detected by BrdU assay and Boyden Chamber method, respectively. Expression of type I collagen, metalloproteinase (MMP)-2/-9, tissue inhibitor of MMP (TIMP)-1/-2, and ERK phosphorylation were evaluated by Western blot analyses. Our results revealed that compared to controls, (1) VEGF-D significantly increased myofibroblast proliferation and migration; (2) VEGF-D significantly upregulated type I collagen synthesis in a dose- and time-dependent manner; (3) VEGFR antagonist abolished VEGF-D-induced myofibroblast proliferation and type I collagen release; (4) VEGF-D stimulated MMP-2/-9 and TIMP-1/-2 synthesis; (5) VEGF-D activated ERK phosphorylation; and (6) ERK inhibitor abolished VEGF-D-induced myofibroblast proliferation and type I collagen synthesis. Our in vitro studies have demonstrated that VEGF-D serves as a crucial profibrogenic mediator by stimulating myofibroblast growth, migration and collagen synthesis. Further studies are underway to determine the role of VEGF-D in fibrous tissue formation during cardiac repair following MI.