Vegfc-expressing cells form heterotopic bone after musculoskeletal injury.

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels.

Comparative study on the expression of Pax3, Rad51 and VEGF-C in esophageal gastric junction adenocarcinoma and distal gastric adenocarcinoma.

The purpose of this study was to explore the differential expression of Pax3, Rad51 and VEGF-C in esophageal gastric junction adenocarcinoma and distal gastric adenocarcinoma and their relationship with cancer occurrence and development. 57 patients with gastric cancer were included and divided into esophageal gastric junction adenocarcinoma group (n=28) and distal gastric adenocarcinoma group (n=29). The positive expressions of Pax3, Rad51 and VEGF-C in the control group were lower than those in the esophageal gastric junction adenocarcinoma group and distal gastric adenocarcinoma group respectively (P<0.05). In esophageal gastric junction adenocarcinoma with low differentiation, positive expressions of Pax3, Rad51, and VEGF-C surpassed those in high/medium differentiation (P<0.05). Serosa-infiltrated cases exhibited higher Pax3 and Rad51 expressions compared to non-infiltrated cases (P<0.05). Rad51 and VEGF-C positivity were notably elevated in cases with lymph node metastasis compared to those without (P<0.05). Distal gastric adenocarcinoma displayed higher VEGF expression than middle/low differentiated adenocarcinomas. Rad51 expression was significantly higher in women than in men (P<0.05). The positive rates of Pax3, Rad51, and VEGF-C were markedly increased in esophageal gastric junction adenocarcinoma and distal gastric adenocarcinoma compared to normal gastric tissue, and these were associated with the degree of differentiation, depth of invasion, and lymph node metastasis in patients. Particularly, Rad51 exhibited a positive correlation with cancer cell differentiation, invasion depth, and lymph node metastasis in cancer tissue.

Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema.

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in the lymphedematous arm compared to the normal arm in patients. The role of APLN in LD was confirmed in APLN knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of nonintegrative RNA delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial.

DHCR7 promotes lymph node metastasis in cervical cancer through cholesterol reprogramming-mediated activation of the KANK4/PI3K/AKT axis and VEGF-C secretion.

Cervical cancer (CC) patients with lymph node metastasis (LNM) have a poor prognosis. However, the molecular mechanism of LNM in CC is unclear, and there is no effective clinical treatment. Here, we found that 7-dehydrocholesterol reductase (DHCR7), an enzyme that catalyzes the last step of cholesterol synthesis, was upregulated in CC and closely related to LNM. Gain-of-function and loss-of-function experiments proved that DHCR7 promoted the invasion ability of CC cells and lymphangiogenesis in vitro and induced LNM in vivo. The LNM-promoting effect of DHCR7 was partly mediated by upregulating KN motif and ankyrin repeat domains 4 (KANK4) expression and subsequently activating the PI3K/AKT signaling pathway. Alternatively, DHCR7 promoted the secretion of vascular endothelial growth factor-C (VEGF-C), and thereby lymphangiogenesis. Interestingly, cholesterol reprogramming was needed for the DHCR7-mediated promotion of activation of the KANK4/PI3K/AKT axis, VEGF-C secretion, and subsequent LNM. Importantly, treatment with the DHCR7 inhibitors AY9944 and tamoxifen (TAM) significantly inhibited LNM of CC, suggesting the clinical application potential of DHCR7 inhibitors in CC. Collectively, our results uncover a novel molecular mechanism of LNM in CC and identify DHCR7 as a new potential therapeutic target.

CCBE1 regulates the development and prevents the age-dependent regression of meningeal lymphatics.

Recent studies have described the importance of lymphatics in numerous organ-specific physiological and pathological processes. The role of meningeal lymphatics in various neurological and cerebrovascular diseases has been suggested. It has also been shown that these structures develop postnatally and are altered by aging and that the vascular endothelial growth factor C (VEGFC)/ vascular endothelial growth factor receptor 3 (VEGFR3) signaling plays an essential role in the development and maintenance of them. However, the molecular mechanisms governing the development and maintenance of meningeal lymphatics are still poorly characterized. Recent in vitro cell culture-based experiments, and in vivo studies in zebrafish and mouse skin suggest that collagen and calcium binding EGF domains 1 (CCBE1) is involved in the processing of VEGFC. However, the organ-specific role of CCBE1 in developmental lymphangiogenesis and maintenance of lymphatics remains unclear. Here, we aimed to investigate the organ-specific functions of CCBE1 in developmental lymphangiogenesis and maintenance of meningeal lymphatics during aging. We demonstrate that inducible deletion of CCBE1 leads to impaired postnatal development of the meningeal lymphatics and decreased macromolecule drainage to deep cervical lymph nodes. The structural integrity and density of meningeal lymphatics are gradually altered during aging. Furthermore, the meningeal lymphatic structures in adults showed regression after inducible CCBE1 deletion. Collectively, our results indicate the importance of CCBE1-dependent mechanisms not only in the development, but also in the prevention of the age-related regression of meningeal lymphatics. Therefore, targeting CCBE1 may be a good therapeutic strategy to prevent age-related degeneration of meningeal lymphatics.

Inhibition of PFKFB3 Expression Stimulates Macrophage-Mediated Lymphangiogenesis Post-Acute Myocardial Infarction.

BACKGROUND: The dilation of lymphatic vessels plays a critical role in maintaining heart function, while a lack thereof could contribute to heart failure (HF), and subsequently to an acute myocardial infarction (AMI). Macrophages participate in the induction of lymphangiogenesis by secreting vascular endothelial cell growth factor C (VEGF-C), although the precise mechanism remains unclear. METHODS: Intramyocardial injections of adeno-associated viruses (AAV9) to inhibit the expression of VEGFR3 (VEGFR3 shRNA) or promote the expression of VEGFR3 (VEGFR3 ORF) in the heart; Myh6-mCherry B6 D2-tg mice and flow cytometry were used to evaluate the number of myocellular debris in the mediastinal lymph nodes; fluorescence staining and qPCR were used to evaluate fluorescence analysis; seahorse experiment was used to evaluate the level of glycolysis of macrophages; Lyz2𝐶𝑟𝑒, VEGFCfl/fl, and PFKFB3fl/fl mice were used as a model to knock out the expression of VEGF-C and PFKFB3 in macrophages. RESULTS: The escalation of VEGFR3 in cardiac tissue can facilitate the drainage of myocardial debris to the mediastinal lymph nodes, thereby improving cardiac function and reducing fibrosis after reperfusion injury. Conversely, myeloid VEGF-C deficiency displayed an increase in macrophage counts and inflammation levels following reperfusion injury. The inhibition of the critical enzyme PFKFB3 in macrophage glycolysis can stimulate the manifestation of VEGF-C in macrophages. A deficiency in myeloid PFKFB3 is associated with induced lymphangiogenesis following reperfusion injury. CONCLUSIONS: Our initial investigations suggest that the suppression of PFKFB3 expression in macrophages could potentially stimulate the production of VEGF-C in these immune cells, which in turn may facilitate lymphangiogenesis and mitigate the inflammatory effects of I/R injury.

Local Adenoviral Delivery of Vascular Endothelial Growth Factor C Induces Lymphangiogenesis in the Conjunctiva in Rabbits.

INTRODUCTION: The purpose of this study was to determine if conjunctival lymphangiogenesis can be induced using adenoviral delivery of vascular endothelial growth factor C (VEGF-C). METHODS: Seventeen New Zealand white rabbits received a subconjunctival injection containing 3.5 × 107 plaque-forming units of an adenoviral vector containing the gene-encoding VEGF-C (Ad-VEGF-C). The contralateral eye was used for control experiment (the same volume of either saline or an empty vector). After 2 weeks, the animals were examined with trypan blue conjunctival lymphangiography, and the eyes were harvested for histology and immunohistochemistry (podoplanin and CD31). RESULTS: Trypan blue conjunctival lymphangiography revealed significantly more extensive conjunctival vessel network in the Ad-VEGF-C group compared with control: 1.35 ± 0.67 versus 0.28 ± 0.17 vessel length/analysed area (p = <0.0001). This finding was confirmed with immunohistochemistry, where a significant increase in the number of lymphatic vessels was found compared to control; 34 ± 9 per mm2 versus 13 ± 8 per mm2 (p = 0.0019). Furthermore, there was a significant increase in lymphatic cross-sectional area; 32,500 ± 7,900 µm2 per mm2 versus 17,600 ± 9,700 µm2 per mm2 (p = 0.0149). Quantification of blood vessels revealed no significant difference in blood vessel density between Ad-VEGF-C and control; 19 ± 9 per mm2 versus 14 ± 8 per mm2 (p = 0.1971). There was no significant difference in total blood vessel area; 13,200 ± 7,600 µm2 per mm2 versus 7,100 ± 3,000 µm2 per mm2 (p = 0.0715). Eyes treated with an adenoviral vector (VEGF-C or empty vector) responded with a reactive cellular response, predominantly lymphocytes, towards the vector. CONCLUSION: The study demonstrates the feasibility of inducing conjunctival lymphangiogenesis with a single subconjunctival injection of Ad-VEGF-C. Future studies will explore how this can be used with a therapeutic purpose.

Vascular endothelial growth factor (VEGF) -A, -C and VE-cadherin as potential biomarkers in early breast cancer patients.

BACKGROUND: Vascular endothelial growth factor (VEGF) -A and -C act as multifunctional molecules and growth factors, while VE-cadherin (cadherin 5, CDH5) is the endothelial junction protein. AIM: To assess the relationship between intratumoral VEGF -A, -C and CDH5 levels and clinical outcome, in primary, early-stage, breast cancer patients. PATIENTS AND METHODS: The study included 69 node-negative (N0) breast cancer patients, all of whom had not received any prior hormonal or chemotherapeutic systemic therapy that would affect the course of disease. The median follow-up period was 144 months. Intratumoral mRNA levels of VEGF -A, -C and CDH5 were determined by RT-qPCR. Prognostic performance was evaluated by Cox proportional hazards regression, Kaplan-Meier analysis, as well as by the multivariable approach based on the least absolute shrinkage and selection operator (LASSO) logit regression. Classification of patients into the low and high subgroups was performed using the outcome-oriented cut-off point categorization approach. RESULTS: Of the measured mRNAs, only CDH5 mRNA (t = -2.17; p = 0.04) and VEGF-C mRNA (t = -2.41; p = 0.03) showed significant differences between values in patient subgroups with distant metastasis and those without recurrences, respectively. These t-test results were in agreement with the Cox regression by which CDH5 mRNA reached the most pronounced hazard ratio (HR=2.07; p = 0.05), followed by VEGF-C mRNA (HR=1.59; p = 0.005). HR values above 1.0 indicate that high levels of either CDH5 or VEGF-C mRNAs associated with a higher risk of poor clinical outcome. Distant recurrence incidence was 26% for the CDH5high and 3% for the CDH5low subgroup (Kaplan-Meier analysis). Distant recurrence incidence was 23% for the VEGF-Chigh and 0% for VEGF-Clow subgroup. The independent prognostic value of VEGF-C mRNA was confirmed by LASSO regression. CONCLUSION: Intratumoral VEGF-A levels did not associate with disease outcome in primary, early-stage, breast cancer patients, whilst raised levels of either CDH5 or VEGF-C prognosticated a high risk of distant metastasis.

Visfatin upregulates VEGF-C expression and lymphangiogenesis in esophageal cancer by activating MEK1/2-ERK and NF-κB signaling.

Lymph node metastasis is a recognized prognostic factor in esophageal cancer. Adipokines, including visfatin, and the molecule vascular endothelial growth factor (VEGF)-C, are implicated in lymphangiogenesis, but whether any association exists between esophageal cancer, adipokines and VEGF-C is unknown. We examined the relevance of adipokines and VEGF-C in esophageal squamous cell carcinoma (ESCC) in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. We found significantly higher levels of visfatin and VEGF-C expression in esophageal cancer tissue than in normal tissue. Immunohistochemistry (IHC) staining identified that higher levels of visfatin and VEGF-C expression were correlated with advanced stage ESCC. Visfatin treatment of ESCC cell lines upregulated VEGF-C expression and VEGF-C-dependent lymphangiogenesis in lymphatic endothelial cells. Visfatin induced increases in VEGF-C expression by activating the mitogen-activated protein kinase kinases1/2-extracellular signal-regulated kinase (MEK1/2-ERK) and Nuclear Factor Kappa B (NF-κB) signaling cascades. Transfecting ESCC cells with MEK1/2-ERK and NF-κB inhibitors (PD98059, FR180204, PDTC, and TPCK) and siRNAs inhibited visfatin-induced increases in VEGF-C expression. It appears that visfatin and VEGF-C are promising therapeutic targets in the inhibition of lymphangiogenesis in esophageal cancer.

IL-1RA inhibits esophageal carcinogenesis and lymphangiogenesis via downregulating VEGF-C and MMP9.

Interleukin-1 receptor antagonist (IL-1RA) has been shown to play an important role in cancer progression. However, its pathogenic effects and molecular mechanism in the malignant progression of esophageal squamous cell carcinoma (ESCC) remain largely unknown. This study was designed to explore the function of IL-1RA in ESCC and determine the relationship between IL-1RA and lymph node metastasis in ESCC patients. The clinical relevance of IL-1RA in relation to the clinicopathological features and prognosis of 100 ESCC patients was analyzed. The function and underlying mechanisms of IL-1RA in the growth, invasion, and lymphatic metastasis in ESCC were explored both in vitro and in vivo. The therapeutic effect of anakinra, an IL-1 receptor antagonist, on ESCC was also evaluated in animal experiments. Downregulation of IL-1RA was observed in ESCC tissues and cells and was found to be strongly correlated with pathological stage (P = 0.034) and lymphatic metastasis (P = 0.038). Functional assays demonstrated that upregulation of IL-1RA reduced cell proliferation, migration, and lymphangiogenesis both in vitro and in vivo. Mechanistic studies revealed that overexpression of IL-1RA activated the epithelial-to-mesenchymal transition (EMT) in the ESCC cells through activation of MMP9 and regulation of the expression and secretion of VEGF-C through the PI3K/NF-κB pathway. Anakinra treatment resulted in significant inhibition of tumor growth, lymphangiogenesis, and metastasis. IL-1RA inhibits lymph node metastasis of ESCC by regulating the EMT through activation of matrix metalloproteinase 9(MMP9) and lymphangiogenesis, driven by VEGF-C and the NF-κB signaling pathway. Anakinra may be an effective drug for the inhibition of ESCC tumor formation and lymph node metastasis.

Molecular mechanisms of miR-1236 in the assessment of tumor lymphangiogenesis in human ovarian cancer patients.

BACKGROUND: Tumor lymphangiogenesis is a critical component in the progression of cancers and specific microRNAs have been reported to be implicated in this process. Recent studies revealed the involvement of miR-1236 in lymphangiogenic signaling by targeting vascular endothelial growth factor receptor 3 (VEGFR3). However, the prognostic importance of miR-1236 and its clinical relevance for lymphangiogenesis in ovarian cancer (OC) remains unclear. METHODS: The study included 52 ovarian tumors and 28 normal ovarian tissues. Quantitative real-time PCR was utilized to analyze the VEGFR3, VEGF-C, LYVE-1 and PROX1 mRNA expression as well as miR-1236. VEGFR3 protein expression was measured by immunohistochemistry staining. Immunohistochemistry for the podoplanin marker (D2-40) was performed to measure lymphatic vessel density (LVD). In addition, diagnostic evaluation based on the receiver-operating characteristic (ROC) curve was performed. The influence of miR-1236 on overall survival was evaluated by Kaplan-Meier method. RESULTS: Here, we show that miR-1236 expression was significantly decreased in ovarian tumors compared with control tissues (p < 0.001) and correlated with advanced clinical stage, lymph node metastasis, distant metastasis and patient survival (All P < 0.05). Moreover, in ovarian tumors, LVD as well as the gene expression of VEGFR3, VEGF-C and LYVE-1, but not PROX1, were found to be remarkably higher compared with control tissues. We also detected a more robust positive staining for VEGFR3 in OC tissues than in control tissues. Furthermore, our results demonstrated an inverse association of miR-1236 expression with LVD, VEGFR3, LYVE-1 and PROX1 expression in OC tissues. The ROC curve analysis indicated that miR-1236 expression has the potential to be used as a diagnostic and prognostic biomarker in OC. Survival analysis further verified a lowered overall survival rate in patients with low miR-1236 expression than in those with high expression. CONCLUSIONS: Our results provide evidence for the translational involvement of miR-1236 in the lymphangiogenesis of OC by regulating lymphangiogenesis-related factors and support the clinical importance of miR-1236 as a new diagnostic and prognostic biomarker for OC.

Eltrombopag Inhibits Metastasis in Breast Carcinoma by Targeting HuR Protein.

Eltrombopag is a small molecule TPO-R agonist that has been shown in our previous studies to inhibit tumor growth by targeting Human antigen R (HuR) protein. HuR protein not only regulates the mRNA stability of tumor growth-related genes, but it also regulates the mRNA stability of a variety of cancer metastasis-related genes, such as Snail, Cox-2, and Vegf-c. However, the role and mechanisms of eltrombopag in breast cancer metastasis have not been fully investigated. The purpose of this study was to investigate whether eltrombopag can inhibit breast cancer metastasis by targeting HuR. Our study first found that eltrombopag can destroy HuR-AU-rich element (ARE) complexes at the molecular level. Secondly, eltrombopag was found to suppress 4T1 cell migration and invasion and inhibit macrophage-mediated lymphangiogenesis at the cellular level. In addition, eltrombopag exerted inhibitory effects on lung and lymph node metastasis in animal tumor metastasis models. Finally, it was verified that eltrombopag inhibited the expressions of Snail, Cox-2, and Vegf-c in 4T1 cells and Vegf-c in RAW264.7 cells by targeting HuR. In conclusion, eltrombopag displayed antimetastatic activity in breast cancer in an HuR dependent manner, which may provide a novel application for eltrombopag, hinting at the multiple effects of HuR inhibitors in cancer therapy.

Exosomal miR-10527-5p Inhibits Migration, Invasion, Lymphangiogenesis and Lymphatic Metastasis by Affecting Wnt/ß-Catenin Signaling via Rab10 in Esophageal Squamous Cell Carcinoma.

Background: Cancer cell-derived exosomal microRNAs (miRNAs) play critical role in orchestrating intercellular communication between tumor cells and tumor microenvironmental factors, including lymphatic endothelial cells (LECs). Nevertheless, the functions and underlying mechanisms of exosomal miRNAs in lymphatic metastasis and lymphangiogenesis in esophageal squamous cell carcinoma (ESCC) remain unclear. Methods: Small RNA sequencing, Gene Expression Omnibus (GEO) analysis and qRT‒PCR were performed to identify the candidate exosomal miRNAs involved in ESCC metastasis. Receiver operating characteristic curve analysis was conducted to evaluate the diagnostic potential of exosomal miR-10527-5p in predicting lymph node metastasis (LNM) status. An in vitro coculture system was used to investigate the effects of exosomal miR-10527-5p on ESCC cells and human LECs (HLECs), followed by a popliteal LNM assay in vivo. The relationship between miR-10527-5p and Rab10 was identified by dual-luciferase reporter, fluorescence in situ hybridization and qRT‒PCR assays. Then, a series of rescue assays were performed to further investigate whether Rab10 is involved in exosomal miR-10527-5p mediated ESCC metastasis. Results: MiR-10527-5p was found to be notably reduced in both the plasma exosomes and tumor tissues of ESCC patients with LNM, and plasma exosomal miR-10527-5p had a high sensitivity and specificity for discrimination of LNM status. Moreover, exosome-shuttled miR-10527-5p suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) of ESCC cells as well as the migration and tube formation of HLECs via Wnt/ß-catenin signaling in vitro and in vivo. Further investigation revealed that Rab10 was a direct target of miR-10527-5p, and re-expression of Rab10 neutralized the inhibitory effects of exosomal miR-10527-5p. Conclusion: Our study demonstrated that exosomal miR-10527-5p had a strong capability to predict preoperative LNM status and anti-lymphangiogenic effect. Exosomal miR-10527-5p inhibited lymphangiogenesis and lymphatic metastasis of ESCC in a vascular endothelial growth factor-C (VEGF-C)-independent manner, showing potential as a therapeutic target for ESCC patients.

The Effect of Lymphangiogenesis in Transplant Arteriosclerosis.

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

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

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

VEGFC Gene Expression Is Associated with Tumor Progression and Disease-Free Survival in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (CSCC) is one of the most common cancers in the skin. CSCC belongs to the non-melanoma skin cancers, and its incidence is increasing every year around the world. The principal routes of tumor progression are related to angiogenesis and lymphangiogenesis. In this study, we assess the gene expression of the relevant biomarkers of both routes in 49 formalin-fixed paraffin-embedded (FFPE) CSCC samples in an attempt to determine a molecular profile that correlates with the tumor progression and disease-free survival (DFS). The results were enhanced by a posttranscriptional analysis using an immunofluorescence assay. Overexpression of the vascular endothelial growth factor C (VEGFC) gene was found in patients with tumor progression (p = 0.022) and in patients with perineural invasion (p = 0.030). An increased expression of protein VEGFC in samples with tumor progression supported these results (p = 0.050). In addition, DFS curves showed differences (p = 0.027) for tumors with absent-low VEGFC expression versus those with high levels of VEGFC expression. No significant influence on DFS was detected for the remaining analyzed genes. VEGFC expression was found to be a risk factor in the disease progression (HR = 2.675; 95% CI: 1.089-6.570; p = 0.032). Our main results suggest that VEGFC gene expression is closely related to tumor progression, DFS, and the presence of perineural invasion.

NEAT1 promotes the malignant development of bladder cancer by regulating the miR-101/VEGF-C pathway in vitro and in vivo.

BACKGROUND: NEAT1 has been shown to play an oncogenic role in many kinds of cancers. However, detailed roles of NEAT1 in bladder cancer are largely unknown. METHODS: In the present study, the expression of NEAT1, miR-101 and VEGF-C was detected in human bladder cancer samples. The relationship between NEAT1 and the prognosis of patients with bladder cancer was analysed. In vitro experiments explored the effects of NEAT1 on biological behaviours of bladder cancer T24 and 5637 cells. Bioinformatics prediction and luciferase assays were used to assay the regulatory mechanism of action of NEAT1 and miR-101. Loss and gain of the expression of miR-101 and VEGF-C were used to explore the effects of the NEAT1/miR-101/VEGF-C pathway on T24 and 5637 cells. The effect of NEAT1 on the growth of bladder cancer in vivo was explored using an orthotopic tumourigenesis model. RESULTS: NEAT1 and VEGF-C were significantly upregulated in bladder cancer samples, and miR-101 was significantly downregulated. NEAT1 upregulation was associated with poorer recurrence-free survival of patients with bladder cancer. Overexpression of NEAT1 promoted the proliferation, migration and invasion of bladder cancer cells. The results of the luciferase assay indicated that miR-101 was a target of NEAT1. The promoting effects of NEAT1 on bladder cancer cells were reversed by miR-101 upregulation, and inhibition of miR-101 enhanced the effects of NEAT1. Overexpression of VEGF-C had a clear synergistic effect with the action of NEAT1. Overexpression of NEAT1 increased tumour growth and induced the development of liver metastasis. CONCLUSIONS: In conclusion, our data indicated that NEAT1 was expressed at high levels in bladder cancer patients and correlated with unfavourable prognosis. NEAT1 promoted malignant development of bladder cancer in vitro and in vivo by regulating the miR-101/VEGF-C pathway.

Bioactive VEGF-C from E. coli.

Vascular endothelial growth factor-C (VEGF-C) stimulates lymphatic vessel growth in transgenic models, via viral gene delivery, and as a recombinant protein. Expressing eukaryotic proteins like VEGF-C in bacterial cells has limitations, as these cells lack specific posttranslational modifications and provisions for disulfide bond formation. However, given the cost and time savings associated with bacterial expression systems, there is considerable value in expressing VEGF-C using bacterial cells. We identified two approaches that result in biologically active Escherichia coli-derived VEGF-C. Expectedly, VEGF-C expressed from a truncated cDNA became bioactive after in vitro folding from inclusion bodies. Given that VEGF-C is one of the cysteine-richest growth factors in humans, it was unclear whether known methods to facilitate correct cysteine bond formation allow for the direct expression of bioactive VEGF-C in the cytoplasm. By fusing VEGF-C to maltose-binding protein and expressing these fusions in the redox-modified cytoplasm of the Origami (DE3) strain, we could recover biological activity for deletion mutants lacking the propeptides of VEGF-C. This is the first report of a bioactive VEGF growth factor obtained from E. coli cells circumventing in-vitro folding.

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

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

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

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