Balance of pro‐ versus anti‐angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF₁₆₅ has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti‐angiogenic isoform of VEGF‐A, generated from distal splice site selection in the terminal exon of VEGF (VEGF₁₆₅b) and shown to be down‐regulated in epithelial malignancies. The expression of both the pro‐ (VEGF_xxx) and the anti‐angiogenic (VEGF_xxxb) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGF_xxxb and VEGF_xxx were both expressed in GN, specific up‐regulation of the VEGF_xxx isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up‐regulation of the angiogenic isoforms relative to VEGF_xxxb compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF₁₆₅ is up‐regulated in NB and that there is a difference in the balance of isoform expression from anti‐angiogenic VEGF₁₆₅b to angiogenic VEGF₁₆₅. Treatment with recombinant human VEGF₁₆₅b significantly reduced the growth rate of established xenografts of SK‐N‐BE(2)‐C cells (4.24 ± 1.01 fold increase in volume) compared with those treated with saline (9.76 ± 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF₁₆₅b‐treated tumours (19.4 ± 1.9 vessels/mm³) in contrast to the saline‐treated tumours (45.5 ± 8.6 vessels/mm³). VEGF₁₆₅b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK‐N‐BE(2)‐C cells after 48 h. In conclusion, VEGF₁₆₅b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF₁₆₅b in NB and other paediatric malignancies. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunomodulatory glc/man‐directed Dolichos lablab lectin (DLL) evokes anti‐tumour response in vivo by counteracting angiogenic gene expressions

Neovascularization and jeopardized immunity has been critically emphasized for the establishment of malignant progression. Lectins are the diverse class of carbohydrate interacting proteins, having great potential as immunopotentiating and anti‐cancer agents. The present investigation sought to demonstrate the anti‐proliferative activity of Dolichos lablab lectin (DLL) encompassing immunomodulatory attributes. DLL specific to glucose and mannose carbohydrate moieties has been purified to homogeneity from the common dietary legume D. lablab. Results elucidated that DLL agglutinated blood cells non‐specifically and displayed striking mitogenicity to human and murine lymphocytes in vitro with interleukin (IL)‐2 production. The DLL‐conditioned medium exerted cytotoxicity towards malignant cells and neoangiogenesis in vitro. Similarly, in‐vivo anti‐tumour investigation of DLL elucidated the regressed proliferation of ascitic and solid tumour cells, which was paralleled with blockade of tumour neovasculature. DLL‐treated mice showed an up‐regulated immunoregulatory cytokine IL‐2 in contrast to severely declined levels in control mice. Mechanistic validation revealed that DLL has abrogated the microvessel formation by weakening the proangiogenic signals, specifically nuclear factor kappa B (NF‐κB), hypoxia inducible factor 1α (HIF‐1 α), matrix metalloproteinase (MMP)‐2 and 9 and vascular endothelial growth factor (VEGF) in malignant cells leading to tumour regression. In summary, it is evident that the dietary lectin DLL potentially dampens the malignant establishment by mitigating neoangiogenesis and immune shutdown. For the first time, to our knowledge, this study illustrates the critical role of DLL as an immunostimulatory and anti‐angiogenic molecule in cancer therapeutics.     

VEGF‐C,VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours

Ramani P, Nash R, Radevsky L, Patel A, Luckett M & Rogers C
(2012) Histopathology
VEGF‐C, VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours Aims: More than 50% of neuroblastomas (NBs) present with haematogenous and/or lymphatic metastasis; however, little is known about the clinicopathological significance in NBs of the key lymphangiogenesis growth factors vascular endothelial growth factor (VEGF)‐C and VEGF‐D and the receptor VEGFR‐3. Methods and results: Ninety‐three NBs and nine ganglioneuromas (GNs) were immunostained for VEGF‐C, VEGF‐D and VEGFR‐3. VEGF‐C and VEGF‐D were present in 76% and 82% of the NBs, respectively. There was no significant difference in VEGF‐C expression between NBs and GNs. VEGF‐D expression was significantly higher in NBs compared with GNs and in MYCN‐amplified NBs. VEGFR‐3 tumoral cell expression (VEGFR‐3c), present in 48% of the NBs, was significantly higher in NBs from children ≥18 months at presentation and those belonging to a high‐risk group. VEGFR‐3 lymphovascular density was increased significantly in NBs compared with GNs and in NBs associated with adverse clinicopathological and biological factors. Lymphovascular invasion, assessed in VEGFR‐3‐stained vessels, was present in ～50% of NBs. Cox regression analyses demonstrated that VEGFR‐3c expression was associated with a significantly shorter event‐free survival and that its effect was independent of the important pathological variable, mitosis–karyorrhexis index. Conclusions: VEGF‐D and VEGFR‐3 up‐regulation support tumour progression in NB and VEGFR‐3c may provide a useful prognostic marker in NBs.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

LMO2 promotes angiogenesis probably by up-regulation of bFGF in endothelial cells: an implication of its pathophysiological role in infantile haemangioma

Sun Z‐J, Cai Y, Chen G, Wang R, Jia J, Chen X‐M, Zheng L‐W & Zhao Y‐F
(2010) Histopathology 57 , 622–632
LMO2 promotes angiogenesis probably by up‐regulation of bFGF in endothelial cells: an implication of its pathophysiological role in infantile haemangioma Aims: Infantile haemangiomas (IHs) are common benign vascular tumours distinctive for their perinatal presentation, rapid growth during the first year of life and subsequent slow involution. Recent research has indicated that endothelial cells of haemangiomas express LIM‐only protein 2 (LMO2). The aim of this study was to investigate the role of LMO2 in the pathogenesis of IHs was investigated. Methods and results: Immunoreactivity of LMO2 was assessed in specimens of 19 IH. Stable transfection of LMO2 into human endothelial cell lines (EAhy926) was performed to evaluate the role of LMO2 in terms of the change in cell proliferation, cell cycle and cell migration as well as the expression level of angiogenic factors. Immunoreactivity for LMO2 was detected in all IH specimens, specifically in the nucleus of the endothelial cells. The intensity of LMO2 immunostaining decreased significantly from proliferative to involuting stages. Furthermore, the overexpression of LMO2 enhanced the proliferation and migration of the endothelial cells and promoted G0/G1–S‐phase transition in vitro, together with an up‐regulation of bFGF expression. Conclusions: LMO2 probably promotes angiogenesis by up‐regulation of bFGF expression and thereby consequently influences progression of IH.     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness

Dhakal H P, Naume B, Synnestvedt M, Borgen E, Kaaresen R, Schlichting E, Wiedswang G, Bassarova A, Holm R, Giercksky K‐E & Nesland J M
(2012) Histopathology  61, 350–364 Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness Aims: Vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR‐1) and VEGF receptor 2 (VEGFR‐2) play a role in breast cancer growth and angiogenesis. We examined the expression and relationship with clinical outcome and other prognostic factors. Methods and results: Tumour sections from 468 breast cancer patients were immunostained for VEGF, VEGFR‐1, and VEGFR‐2, and their relationships with tumour vascularity, disseminated tumour cells (DTCs) in bone marrow and other clinicopathological parameters were evaluated. VEGF, VEGFR‐1 and VEGFR‐2 immunoreactivities were observed in invasive breast carcinoma cells. VEGF expression was significantly associated with VEGFR‐1 and VEGFR‐2 expression (P < 0.001). High‐level cytoplasmic expression of VEGFR‐1 was associated with significantly reduced distant disease‐free survival (DDFS) (P = 0.017, log‐rank) and breast cancer‐specific survival (BCSS) (P = 0.005, log‐rank) for all patients, and for node‐negative patients without systemic treatment (DDFS, P = 0.03, log‐rank; BCSS, P = 0.009, log‐rank). VEGFR‐1 expression was significantly associated with histopathological markers of aggressiveness (P < 0.05). Significantly reduced survival was observed in DTC‐positive patients as compared with DTC‐negative patients in the combined moderate/high VEGFR‐1 group (P < 0.001 for DDFS and BCSS), and the same was true for DDFS in the moderate VEGFR‐2 group (P = 0.006). Conclusions: High‐level expression of VEGFR‐1 indicates reduced survival. Higher‐level expression of VEGFR‐1 or VEGFR‐2 in primary breast carcinomas combined with the presence of DTC selects a prognostically unfavourable patient group.     

Peritumoral brain edema in angiomatous supratentorial meningiomas: an investigation of the vascular endothelial growth factor A pathway

The aim of this work was to study the vascular endothelial growth factor A (VEGF‐A) pathway and peritumoral brain edema (PTBE) through comparison of non‐angiomatous and angiomatous meningiomas. Meningiomas are common intracranial tumors, which often have PTBE. VEGF‐A is an integral part of PTBE formation and angiogenesis, and the capillary‐rich angiomatous meningiomas are known for their PTBE. The VEGF‐A receptor VEGFR‐2 is responsible for the angiogenic effect of VEGF‐A on endothelial cells, which is enhanced by the co‐receptor neuropilin‐1. Forty non‐angiomatous, 22 angiomatous meningiomas, and 10 control tissue samples were collected for the study. Magnetic resonance images were available for 40 non‐angiomatous and 10 angiomatous meningiomas. Tissue sections were immunostained for CD34, MIB‐1, estrogen‐ and progesterone receptors. ELISA, chemiluminescence, and RT‐qPCR were used for VEGF‐A, VEGFR‐2, and neuropilin‐1 protein and mRNA quantification. Angiomatous meningiomas had larger PTBE (695 vs 218 cm³, p = 0.0045) and longer capillary length (3614 vs 605 mm/mm³, p < 0.0001). VEGF‐A mRNA, neuropilin‐1 mRNA, and VEGFR‐2 protein levels were higher in angiomatous meningiomas independently of the capillary length (p < 0.05). Neuropilin‐1 protein levels were lower in angiomatous meningiomas (p < 0.0001). The VEGF‐A pathway and tumor capillary length may be essential for PTBE‐formation in meningiomas. Further investigations of this pathway could lead to earlier therapy and targeted pharmacological treatment options.     

Expression and Cellular Distribution of Vascular Endothelial Growth Factor‐C System in Cortical Tubers of the Tuberous Sclerosis Complex

Cortical tubers are malformations of cortical development in patients with tuberous sclerosis complex (TSC), and highly associated with pediatric intractable epilepsy. Recent evidence has shown that signaling mediated through vascular endothelial growth factor‐C (VEGF‐C) and its receptors, VEGFR‐2 and VEGFR‐3, has direct effects on both neurons and glial cells. To understand the potential role of VEGF‐C system in the pathogenesis of cortical tubers, we investigated the expression patterns of VEGF‐C signaling in cortical tubers compared with age‐matched normal control cortex (CTX). We found that VEGF‐C, VEGFR‐2 and VEGFR‐3 were clearly upregulated in tubers at both the mRNA and protein levels, compared with CTX. The in situ hybridization and immunostaining results demonstrated that VEGF‐C, VEGFR‐2 and VEGFR‐3 were highly expressed in dysplastic neurons (DNs), giant cells (GCs) and reactive astrocytes within tubers. Most DNs/GCs expressing VEGF‐C and its receptors co‐labeled with neuronal rather than astrocytic markers, suggesting a neuronal lineage. In addition, protein levels of Akt‐1, p‐Bad and ERK1/2, the important downstream factors of the VEGF‐C pathway, were significantly increased in cortical tubers, indicating involvement of VEGF‐C–dependent prosurvival signaling in cortical tubers. Taken together, our results suggest a putative role for the VEGF‐C signaling pathway in the pathogenesis of cortical tubers.     

Advanced glycation end product Nε‐carboxymethyllysine induces endothelial cell injury: the involvement of SHP‐1‐regulated VEGFR‐2 dephosphorylation

N^ε‐carboxymethyllysine (CML), a major advanced glycation end product, plays a crucial role in diabetes‐induced vascular injury. The roles of protein tyrosine phosphatases and vascular endothelial growth factor (VEGF) receptors in CML‐related endothelial cell injury are still unclear. Human umbilical vein endothelial cells (HUVECs) are a commonly used human EC type. Here, we tested the hypothesis that NADPH oxidase/reactive oxygen species (ROS)‐mediated SH2 domain‐containing tyrosine phosphatase‐1 (SHP‐1) activation by CML inhibits the VEGF receptor‐2 (VEGFR‐2, KDR/Flk‐1) activation, resulting in HUVEC injury. CML significantly inhibited cell proliferation and induced apoptosis and reduced VEGFR‐2 activation in parallel with the increased SHP‐1 protein expression and activity in HUVECs. Adding recombinant VEGF increased forward biological effects, which were attenuated by CML. The effects of CML on HUVECs were abolished by SHP‐1 siRNA transfection. Exposure of HUVECs to CML also remarkably escalated the integration of SHP‐1 with VEGFR‐2. Consistently, SHP‐1 siRNA transfection and pharmacological inhibitors could block this interaction and elevating [³H]thymidine incorporation. CML also markedly activated the NADPH oxidase and ROS production. The CML‐increased SHP‐1 activity in HUVECs was effectively attenuated by antioxidants. Moreover, the immunohistochemical staining of SHP‐1 and CML was increased, but phospho‐VEGFR‐2 staining was decreased in the aortic endothelium of streptozotocin‐induced and high‐fat diet‐induced diabetic mice. We conclude that a pathway of tyrosine phosphatase SHP‐1‐regulated VEGFR‐2 dephosphorylation through NADPH oxidase‐derived ROS is involved in the CML‐triggered endothelial cell dysfunction/injury. These findings suggest new insights into the development of therapeutic approaches to reduce diabetic vascular complications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Ephrin‐A3 and Ephrin‐A4 Contribute to Microglia‐Induced Angiogenesis in Brain Endothelial Cells

The association of microglia with brain vasculature during development and the reduced brain vascular complexity in microglia‐deficient mice suggest the role of microglia in cerebrovascular angiogenesis. However, the underlying molecular mechanism remains unclear. Here, using an in vitro angiogenesis model, we found the culture supernatant of BV2 microglial cells significantly enhanced capillary‐like tube formation and migration of brain microvascular endothelial cells (BMECs). The expression of angiogenic factors, ephrin‐A3 and ephrin‐A4, were specifically upregulated in BMECs exposed to BV2‐derived culture supernatant. Knockdown of ephrin‐A3 and ephrin‐A4 in BMECs by siRNA significantly attenuated the enhanced angiogenesis and migration of BMECs induced by BV2 supernatant. Our further results indicated that the ability of BV2 supernatant to promote endothelial angiogenesis was caused by the soluble tumor necrosis factor α (TNF‐α) released from BV2 microglial cells. Moreover, the upregulations of ephrin‐A3 and ephrin‐A4 in BMECs in response to BV2 supernatant were effectively abolished by neutralization antibody against TNF‐α and TNF receptor 1, respectively. The present study provides evidence that microglia upregulates endothelial ephrin‐A3 and ephrin‐A4 to facilitate in vitro angiogenesis of brain endothelial cells, which is mediated by microglia‐released TNF‐α. Anat Rec, 297:1908–1918, 2014. © 2014 Wiley Periodicals, Inc.     

Expression of oestrogen receptor α and β is higher in skeletal muscle of highly endurance‐trained than of moderately active men

Aim: Two known oestrogen receptors (ERs), ERα and the recently cloned ERβ, are expressed in the human skeletal muscle of both males and females. The effects of oestrogen and the role of ERs in skeletal muscle tissue are not well known. Oestrogen receptors and some of their target genes are involved in angiogenic processes. It was hypothesized that ERs are expressed at a higher level in a group with higher oxidative capacity, and that such an enhanced expression would parallel expression of the angiogenic factor – vascular endothelial growth factor (VEGF). Method: Muscle biopsies were taken from vastus lateralis in 10 highly endurance‐trained males and 10 moderately active males and analysed for the expression of ERs and VEGF. Results: The major findings in the present study were the higher mRNA levels of ERα, ERβ and VEGF in the highly endurance‐trained than in the moderately active group. Conclusion: These data suggest that the greater mRNA expression of ERα and ERβ and the oestrogen‐associated angiogenic factor VEGF support the hypothesis of an involvement of ERs in the adaptation of skeletal muscle to endurance training.     

Impact of VEGF‐dependent tumour micro‐environment on EDB fibronectin expression by subcutaneous human tumour xenografts in nude mice

Fibronectin (FN) is an extracellular matrix cell‐adhesive glycoprotein. The alternative spliced isoform EDB‐FN (extra domain B containing FN) is highly expressed in tumour blood vessels and stroma and represents a candidate for tumour targeting. To investigate the impact of different angiogenic micro‐environments on EDB‐FN expression, we used a tumour model in which human endometrial adenocarcinoma Tet‐FGF2 cells overexpressing fibroblast growth factor‐2 (FGF2) driven by the tetracycline‐responsive promoter were further transfected with a VEGF antisense cDNA, generating AS‐VEGF/Tet‐FGF2 cells. In this model, the expression of FGF2 plus VEGF results in fast‐growing, highly vascularized Tet‐FGF2 tumours. Down‐regulation of FGF2 production by tetracycline administration and/or of VEGF production by AS‐VEGF transduction inhibited tumour growth and vascularization, with profound changes in tumour micro‐environment. Quantitative RT‐PCR analysis using human EDB‐FN primers shows that subcutaneous grafting in immunodeficient mice is per se sufficient to cause a dramatic up‐regulation of EDB‐FN expression by these cells, as well as by human oesophageal cancer KYSE 30 cells and renal carcinoma Caki‐1 cells. However, in vivo down‐regulation of VEGF expression, as occurs in AS‐VEGF/Tet‐FGF2 tumours, and to a lesser extent of FGF2 expression, as occurs in tetracycline‐treated Tet‐FGF2 tumour‐bearing animals, causes significant inhibition of EDB‐FN production in tumour grafts, as shown by immunohistochemistry and quantitative RT‐PCR analysis. Accordingly, treatment of Tet‐FGF2 tumour‐bearing animals with the neutralizing anti‐murine VEGF receptor‐2 antibody DC101, or of Caki‐1 tumour‐bearing animals with the anti‐VEGF antibody bevacizumab, inhibited EDB‐FN expression in tumour grafts. EDB‐FN down‐regulation was paralleled by a decrease in vascularity, thus confirming EDB‐FN as a marker of tumour angiogenesis. These data demonstrate that the angiogenic micro‐environment, and in particular the VEGF/VEGFR‐2 system, plays a key role in modulating EDB‐FN expression by tumour cells in vivo. This may have implications for the design of therapeutic strategies targeting EDB‐FN in combination with anti‐angiogenic and/or cytotoxic drugs. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunohistochemical expression of vascular endothelial growth factor A (VEGF), and its receptors (VEGFR1, 2) in normal and pathologic conditions of the human thymus.

Vascular endothelial growth factor A (VEGF-A) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. In the tumor microenvironment, an upregulation of both VEGF and its receptors occurs, leading to a high concentration of occupied receptors on tumor vascular endothelium. Also, VEGF is involved in the development of the normal vascular network of the thymus. Little is known about VEGF expression in normal and malignant thymic tissue. Our purpose was to study the pattern and localization of VEGF expression in benign conditions of the thymus and thymoma to determine a possible correlation with VEGF receptors VEGFR1, VEGFR2 and microvascular density. All cases were positive for VEGF and VEGFR1, 2 in the epithelial cells, in a cytoplasmic, granular pattern. In the normal thymus, there were positive epithelial cells with subcapsular distribution and Hassall's corpuscle epithelial cells. In acute thymic involution, the positive fields were correlated with dilation and stasis of blood vessels and lymphocyte depletion. Rare positive cells were found in other types of involution; the myasthenic thymus showed an intense and diffuse reaction in lymphoid follicles of the medulla. A strong reaction for VEGF was observed in type B3 thymomas in neoplastic epithelial cells, normal endothelial cells, plasma within the blood vessels and focally in the stroma adjacent to the tumor. Receptors for VEGF were positive in neoplastic epithelial cells and endothelium. We hypothesized that VEGF acts as an immunoregulatory factor in the normal thymus and as proangiogenic and autocrine factor in thymomas.     

Genetic ablation of the alpha 6‐integrin subunit in Tie1Cre mice enhances tumour angiogenesis

Laminins are expressed highly in blood vessel basement membranes and have been implicated in angiogenesis. α6β1‐ and α6β4‐integrins are major receptors for laminins in endothelial cells, but the precise role of endothelial α6‐integrin in tumour angiogenesis is not clear. We show that blood vessels in human invasive ductal carcinoma of the breast have decreased expression of the α6‐integrin‐subunit when compared with normal breast tissue. These data suggest that a decrease in α6‐integrin‐subunit expression in endothelial cells is associated with tumour angiogenesis. To test whether the loss of the endothelial α6‐integrin subunit affects tumour growth and angiogenesis, we generated α6fl/fl‐Tie1Cre+ mice and showed that endothelial deletion of α6‐integrin is sufficient to enhance tumour size and tumour angiogenesis in both murine B16F0 melanoma and Lewis cell lung carcinoma. Mechanistically, endothelial α6‐integrin deficiency elevated significantly VEGF‐mediated angiogenesis both in vivo and ex vivo. In particular, α6‐integrin‐deficient endothelial cells displayed increased levels of VEGF‐receptor 2 (VEGFR2) and VEGF‐mediated downstream ERK1/2 activation. By developing the first endothelial‐specific α6‐knockout mice, we show that the expression of the α6‐integrin subunit in endothelial cells acts as a negative regulator of angiogenesis both in vivo and ex vivo. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Smad4 Inhibits VEGF‐A and VEGF‐C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer

Smad4 is a critical factor in the TGF‐β pathway and is involved in tumor progression and metastasis, but the role of Smad4 in colon cancer cells is unclear. The aim of this study is to explore the effect and the underlying mechanism of Smad4 on the growth, migration and apoptosis of colon cancer cells as well as vascular endothelial growth factor (VEGF)‐A and VEGF‐C secreted by these cells. In this study, we showed that Smad4, VEGF‐A, and VEGF‐C are independent prognostic factors of colon cancer, and Smad4 expression was negatively correlated with VEGF‐A and ‐C in samples. We found that Smad4 mRNA and protein levels in colon cancer cells, particularly in HCT‐116 cells, were significantly lower than those in the human intestinal epithelial cell line (HIEC). Smad4 overexpression promoted tumor cell apoptosis, inhibited VEGF‐A and ‐C expression in vitro and in vivo, but had no effect on cell proliferation and migration. Tail vein injection of the virus inhibited xenograft growth in nude mice. Importantly, we also demonstrated that Smad4 could increase the phosphorylation level of Smad3, but not Smad2, which may be one of the mechanisms underlying these effects of Smad4 in colon cancer. Therefore, Smad4 may be a new target for the treatment of colon cancer. Anat Rec, 300:1560–1569, 2017. © 2017 Wiley Periodicals, Inc.     

Role of FGF10 on tumorigenesis by MS‐K

Murine MS‐K and NFSA cell lines formed tumor after inoculation into mouse and both cell lines expressed high level of vascular endothelial growth factor‐A (vegf‐A) and produced same level of VEGF‐A. However, poor blood vessel formation, and necrosis was significantly observed in NFSA‐tumor, contrary to well‐developed blood vessel formation in MS‐K tumor. The microarray analysis showed high expression of fibroblast growth factor‐10 (fgf‐10) in MS‐K than NFSA. In this report, the role of fgf‐10 on tumor growth was studied. MS‐K enhanced more proliferation of endothelial cells by direct co‐culture than NFSA, and rFGF10 supported the proliferation of HUVEC in combination with VEGF‐A. fgf‐10‐knocked down MS‐K, MS‐K (fgf‐10‐KD), proliferated slower in vitro and the tumorigenicity of them was also slower than control. The blood vessel formation in these MS‐K (fgf‐10‐KD) clones was reduced compared with the MS‐K (normal). qPCR analysis showed the suppression of vegf‐A, vegf‐C and fgfr‐1‐expression in the MS‐K (fgf‐10‐KD) clones. Taken together, these results indicated that FGF10, which was produced from tumor cells, was essential for the proliferation of tumor cell itself and also supports proliferation of endothelial cells. Thus, FGF10 plays an important role for tumor growth by both paracrine and autocrine manner.     

Corneal Dystrophy‐Causing SLC4A11 Mutants: Suitability for Folding‐Correction Therapy

SLC4A11 mutations cause some cases of the corneal endothelial dystrophies, congenital hereditary endothelial corneal dystrophy type 2 (CHED2), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). SLC4A11 protein was recently identified as facilitating water flux across membranes. SLC4A11 point mutations usually cause SLC4A11 misfolding and retention in the endoplasmic reticulum (ER). We set about to test the feasibility of rescuing misfolded SLC4A11 protein to the plasma membrane as a therapeutic approach. Using a transfected HEK293 cell model, we measured functional activity present in cells expressing SLC4A11 variants in combinations representing the state found in CHED2 carriers, affected CHED2, FECD individuals, and unaffected individuals. These cells manifest respectively about 60%, 5%, and 25% of the water flux activity, relative to the unaffected (WT alone). ER‐retained CHED2 mutant SLC4A11 protein could be rescued to the plasma membrane, where it conferred 25%–30% of WT water flux level. Further, some ER‐retained CHED2 mutants expressed at 30°C supported increased water flux compared with 37°C cultures. Caspase activation and cell vitality assays revealed that expression of SLC4A11 mutants in HEK293 cells does not induce cell death. We conclude that therapeutics able to increase cell surface localization of ER‐retained SLC4A11 mutants hold promise to treat CHED2 and FECD patients.