Inhibiting VEGFC - mediated hepatocyte - macrophage regulatory axis contributes to protective effects of naringin against high - fat diet - induced hepatic fibrosis.

Transgenic Induction of Vascular Endothelial Growth Factor-C Is Strongly Angiogenic in Mouse Embryos but Leads to Persistent Lymphatic Hyperplasia in Adult Tissues

RETRACTED: Paracrine signaling by VEGF-C promotes non-small cell lung cancer cell metastasis via recruitment of tumor-associated macrophages

Radiotherapy is an important treatment regimen for prostate cancer. We have shown that the lymphangiogenic growth factor, Vascular Endothelial Growth Factor C (VEGF-C), induces docetaxel resistance and autophagy in prostate cancer (Muders et al., Cancer Res 2009 and Stanton et al., Cancer Res 2013). In this study we have evaluated the role of VEGF-C and VEGF-C induced autophagy in radioresistance of prostate cancer using cell culture experiments and human tissue samples. The human prostate carcinoma cell lines PC-3, LNCaP and DU145 were used in these cell culture experiments. Clonogenic survival of prostate cancer cells, after RNA interference (RNAi) of VEGF-C, autophagy related gene 5 (ATG5), and after addition of recombinant human VEGF-C following irradiation therapy, was evaluated. Autophagic flux was tested after ionizing irradiation using a Light-Chain-3 II immunoblot. To confirm our in vitro findings, human tissues of patients who underwent radical prostatectomy and adjuvant radiotherapy at Mayo Clinic were evaluated for VEGF-C expression. The quantity and intensity of VEGF-C staining was evaluated by two pathologists independently and correlated with biochemical relapse free survival (BRFS). The follow-up time is up to 23 years after radiotherapy. Multivariate Cox analysis was performed. VEGF-C levels correlated with a significantly higher radioresistance in colony formation assays and human tissue samples by multivariate analysis. The risk of biochemical recurrence increased 2.8 fold (95% confidence interval: 1.109 to 7.237; p = 0.03) when VEGF-C was highly expressed in prostatectomy patients with adjuvant radiotherapy. In line with our studies on autophagy in VEGF-C mediated docetaxel resistance of prostate cancer, autophagic flux was reduced after VEGF-C depletion during radiation. Interestingly, RNAi for ATG5 showed no effect on radiosensitivity which suggests that the decrease in autophagic flux after VEGF-C depletion is not responsible for the increased radiosensitivity. We conclude that VEGF-C is an important mediator of radiotherapy resistance in prostate cancer. In contrast to our published results on chemotherapy resistance, VEGF-C induced autophagy appears to be less important for resistance against ionizing radiation. Other VEGF-C dependent mechanisms for therapy resistance are under investigation. Citation Format: Steffi Haberlau, Pia Hönscheid, Rafael E. Jimenez, Gustavo B. Baretton, Donald J. Tindall, Mechthild Krause, Kaustubh Datta, Michael H. Muders. Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2015-3304

Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis

Quantification of vascular endothelial growth factor-C (VEGF-C) by a novel ELISA.

RNA-mediated gene silencing of VEGF-C inhibits the growth of human pancreatic cancer in vivo

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

Vascular endothelial growth factor-C (VEGF-C) has been found to be significantly associated with lymphangiogenesis and regional lymph node metastasis in various human tumors. The present work was aimed to explore the role of VEGF-C in malignant progression of human bladder cancer T24 cell line. First, the expression of VEGF-C in T24 cells was detected by western blotting. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was employed to measure the cellular proliferation after treatment with various concentrations of recombinant human VEGF-C (rhVEGF-C). Then, lentivirus vector-based RNA interference (RNAi) was used to inhibit VEGF-C expression of T24 cells. The alterations of T24 cells regarding proliferation, invasiveness, and the apoptosis induced by mitomycin C (MMC) were evaluated. The results showed that the proliferation rate of T24 cells rose from 27.3% to 65.0%, with increasing rhVEGF-C concentration. T24 cells stably transfected with VEGF-C small interference RNA showed 85% reduction in VEGF-C mRNA expression (p < 0.05). The VEGF-C protein level was significantly downregulated (p < 0.05) and the growth and invasiveness were also inhibited (p < 0.05) compared with the control group. Further, the inhibition of VEGF-C expression markedly enhanced the apoptosis of T24 cells induced by MMC (p < 0.05). These were associated with the decreased ratio of Bcl-2/Bax, activation of Caspase-3, decreased expression of MMP-9, as well as the downregulation of phosphorylated p38 MAPK and Akt. The present study suggests that VEGF-C can enhance the proliferation and invasiveness of bladder cancer T24 cells, which is due to suppression of apoptosis and facilitation of migration, accompanied with upregulation of p38 MAPK and Akt phosphorylation. RNAi targeting VEGF-C could effectively suppress malignant progression and enhance chemosensitivity of T24 cells. Thus, inhibition of VEGF-C expression is a potential and promising therapeutic strategy for bladder cancer.

Vascular endothelial growth factor C (VEGF-C) is a key mediator of lymphangiogenesis, acting via its receptors VEGF-R2 and VEGF-R3. High expression of VEGF-C in tumors correlates with increased lymphatic vessel density, lymphatic vessel invasion, sentinel lymph node metastasis and poor prognosis. Recently, we found that in a chemically induced skin carcinoma model, increased VEGF-C drainage from the tumor enhanced lymphangiogenesis in the sentinel lymph node and facilitated metastatic spread of cancer cells via the lymphatics. Hence, interference with the VEGF-C/VEGF-R3 axis holds promise to block metastatic spread, as recently shown by use of a neutralizing anti-VEGF-R3 antibody and a soluble VEGF-R3 (VEGF-C/D trap). By antibody phage-display, we have developed a human monoclonal antibody fragment (single-chain Fragment variable, scFv) that binds with high specificity and affinity to the fully processed mature form of human VEGF-C. The scFv binds to an epitope on VEGF-C that is important for receptor binding, since binding of the scFv to VEGF-C dose-dependently inhibits the binding of VEGF-C to VEGF-R2 and VEGF-R3 as shown by BIAcore and ELISA analyses. Interestingly, the variable heavy domain (VH) of the anti-VEGF-C scFv, which contains a mutation typical for camelid heavy chain-only antibodies, is sufficient for binding VEGF-C. This reduced the size of the potentially VEGF-C-blocking antibody fragment to only 14.6 kDa. Anti-VEGF-C VH-based immunoproteins hold promise to block the lymphangiogenic activity of VEGF-C, which would present a significant advance in inhibiting lymphatic-based metastatic spread of certain cancer types.

Opposing Roles of JNK and p38 in Lymphangiogenesis in Melanoma

CCAAT/enhancer-binding protein delta (C/EBP-δ), a transcription factor, is elevated in carcinoma compared to normal tissue. This study reports a novel function of C/EBP-δ in lymphangiogenesis and tumor metastasis. Genetic deletion of C/EBP-δ in mice resulted in a significant reduction of lymphangiogenesis and pulmonary metastases, with a dramatic reduction of VEGF-C and its cognate receptor VEGFR3 in lymphatic endothelial cells (LECs). In contrast, no difference of VEGF-C in tumor tissues and bone marrow was observed between null and wild type mice. Consistently, forced expression of C/EBP-δ increased VEGF-C and VEGFR3 expression in cultured LECs. These findings suggest a specific and important role of C/EBP-δ in regulating VEGFR3 signaling in LECs. Furthermore, expression of C/EBP-δ in cultured LECs significantly increased cell motility, and knockdown of C/EBP-δ inhibited cell motility and lymphatic vascular network formation in vitro. Forced expression of VEGF-C, but not recombinant VEGF-C, rescued knockdown of C/EBP-δ-induced cell apoptosis, indicative of autonomous VEGF-C autocrine signaling essential for LEC survival. Moreover, hypoxia induces C/EBP-δ expression, and C/EBP-δ regulates HIF-1α expression. Blocking HIF-1α activity totally blocked CEBP-δ induced VEGF-C and VEGFR3 expression in LECs. Together, these findings reveal a new function of CEBP-δ in lymphangiogenesis via regulating VEGFR3 signaling in LECs.

For many types of human cancer, the expression of vascular endothelial growth factor-C (VEGF-C) correlates with enhanced tumor-associated lymphatic vessel density, metastasis formation and poor prognosis. In experimental animals, VEGF-C produced by primary tumors can induce lymphangiogenesis within and/or at the periphery of the tumor, and promotes metastasis formation. Tumor-induced lymphangiogenesis is therefore thought to expedite entry of tumor cells into the lymphatic vasculature and their trafficking to regional lymph nodes, thereby fostering metastatic dissemination. Tumour-produced VEGF-C can also drain to the regional lymph nodes and induce lymphangiogenesis there. Whether this activity promotes metastasis formation remains unclear. To address this issue we manipulated VEGF-C activity and VEGFR-3 activation in the lymph nodes draining syngeneic rat breast cancers using intra-dermal delivery of either recombinant VEGF-C or VEGFR-3 blocking antibodies to induce or suppress lymph node lymphangiogenesis, respectively. Recombinant VEGF-C induced lymph node lymphangiogenesis, but was not sufficient to promote metastasis formation by poorly metastatic NM-081 breast tumours. Conversely, inhibition of lymph node lymphangiogeneis induced by highly metastatic MT-450 breast tumours suppressed the outgrowth of lymph node metastases, but not the initial colonization of the lymph nodes. Lung metastasis was also not affected. We conclude that tumor-derived VEGF-C draining to regional lymph nodes promotes the outgrowth of lymph node metastases. VEGF-C may induce lung metastasis independently of its effects on lymph node metastasis.

Vascular endothelial growth factor C (VEGF-C) recently has been described to be a relatively specific growth factor for the lymphatic vascular system. Here we report that ectopic application of recombinant VEGF-C also has potent angiogenic effects in vivo. VEGF-C is sufficiently potent to stimulate neovascularization from limbal vessels in the mouse cornea. Similar to VEGF, the angiogenic response of corneas induced by VEGF-C is intensive, with a high density of new capillaries. However, the outgrowth of microvessels stimulated by VEGF-C was significantly longer than that induced by VEGF. In the developing embryo, VEGF-C was able to induce branch sprouts from the established blood vessels. VEGF-C also induced an elongated, spindle-like cell shape change and actin reorganization in both VEGF receptor (VEGFR)-2 and VEGFR-3-overexpressing endothelial cells, but not in VEGFR-1-expressing cells. Further, both VEGFR-2 and VEGFR-3 could mediate proliferative and chemotactic responses in endothelial cells on VEGF-C stimulation. Thus, VEGF-C may regulate physiological angiogenesis and participate in the development and progression of angiogenic diseases in addition to lymphangiogenesis.

Obesity, aging, and metabolic diseases contribute to non-alcoholic fatty liver disease (NAFLD) that can progress to non-alcoholic steatohepatitis (NASH) due to ongoing inflammation and pathological angiogenesis. At present, NASH is a leading cause of cirrhosis, hepatocarcinoma cancer (HCC) and liver transplant within the United States. Pathological angiogenesis of liver sinusoidal endothelial cells (LSECs) is one of the hallmarks of liver injury that disrupt normal LSEC physiology. Therefore, strategies which target factors involved in LSEC dysregulation may improve the progression of hepatic diseases. Examination of a human tissue microarray identified increased levels of vascular endothelial growth factor-C (VEGF-C) in NASH livers compared with healthy individuals. Additionally, single nuclei RNA sequencing and immunofluorescent staining revealed that the receptors for VEGF-C, VEGFR2 and VEGFR3, are primarily expressed by the LSECs. Using murine models of NASH, we further investigated the role of VEGF-C during disease progression. Chronic over expression of VEGF-C by adeno-associated viral (AAV) infection in mice fed a western diet with carbon tetrachloride injections increased NASH progression compared with control AAV infected NASH mice. Interestingly, overexpression of VEGF-C C156S, a variant of VEGF-C which exclusively binds to VEGFR3, resulted in significantly increased hepatic steatosis, but only minimal fibrosis. To determine whether blockade of VEGF-C signaling would delay disease progression, we administered lenvatinib, a tyrosine kinase inhibitor which primarily blocks VEGFR2 and VEGFR3 at low doses, to NASH mice. We discovered that low dose lenvatinib resulted in significantly decreased amount of fibrosis, steatosis, and tumor formation. Analysis of vehicle treated and lenvatinib treated livers by single nuclei RNA sequencing uncovered significant changes in endothelial cell genes associated with extracellular interactions and inflammation, as well as hepatocyte genes involved in lipid synthesis and metabolism. Taken together, these findings indicate that chronic VEGF-C production in NASH plays a role in promoting liver fibrosis and steatosis as well as HCC development, and that blockade of the downstream receptors for VEGF-C, VEGFR2 and VEGFR3, may be a promising therapeutic strategy to mitigate disease severity. Citation Format: Seock-Won Youn, Jason W.-L. Eng, Bhairavi Swaminathan, Pamela Teneqexhi, Rahul Vadakath, Jan K. Kitajewski. Chronic VEGF-C signaling exacerbates the progression of non-alcoholic steatohepatitis and hepatocarcinoma through endothelial VEGFR2 and VEGFR3. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4605.

To investigate mechanisms underlying lymphatic node metastasis in pancreatic cancer, we examined roles of vascular endothelial growth factor-C (VEGF-C) in tumor lymphangiogenesis. We measured VEGF-C secretion by pancreatic cancer cell lines using enzyme-linked immunosorbent assay and examined effects of different cell lines on lymphatic endothelial cells (LECs) in vitro. We identified VEGF-C high-secretion (MIA PaCa-2) and low-secretion cell lines (BxPC-3). The trend of enhancement of LEC proliferation by recombinant human VEGF-C (rVEGF-C) was not statistically significant. Numbers of migrating cells were increased by rVEGF-C treatment in a dose-dependent manner. The MIA PaCa-2 cell culture supernatant caused greater LEC migration than the BxPC-3 supernatant. The VEGF-C effects were significantly inhibited by rVEGF receptor 3 (rVEGF R3)/Fc chimera. In LEC/fibroblast coculture on collagen gel, LEC capillary formation was significantly enhanced by coculture with MIA PaCa-2 cells compared with BxPC-3 cells. Enhanced capillary formation with MIA PaCa-2 cells was inhibited by rVEGF R3/Fc chimera, implying VEGF-C involvement in progression of LEC sprouting in a tumor microenvironment. Because VEGF-C secreted by pancreatic cancer cells plays an important role in LEC migration in pancreatic cancer lymphangiogenesis, it is possible that rVEGF R3/Fc chimera might have a role in controlling lymph node metastasis.