Vasohibin-1 Research Articles

AB262. Expression of vasohibin-1 in prostate cancer and its progress research

BackgroundTo search the expression of vasohibin-1 (VASH-1) and CD34 in prostate cancer tissues. Then explore the relationship between their express with the clinical pathological characteristics of patients.MethodsUsing immunohistochemical staining method, we calculate the average optical density (AOD) by Image Pro Plus 6.0, and the expression of VASH-1 was evaluated by the AOD. The staining of CD34 was evaluated by microvessel density (MVD), the MVD was measured by Weidner judging standard.ResultsVASH-1 mainly expressed in the cytoplasm of vascular endothelial cells and the prostate cancer cells, and CD34 was expressed in the cytoplasm of vascular endothelial cells. In the all 50 cases of prostate cancer tissues, the average AOD of VASH-1 was 0.053±0.039, and the average of MVD was 51.14±24.91. The expression of VASH-1 was positively correlated with the expression of CD34 (r=0.58, P<0.01). The expression of VASH-1 and CD34 were both significant correlated with Gleason Score, pT stage and the distant metastasis (P<0.05, their expression were significant improved by the increase of Gleason Score and pT stage), but there was no significant correlation with the age and PSA level (P>0.05).ConclusionsIn prostate cancer, the expression of VASH-1 was positively correlated with the level of MVD. And the expression of VASH-1 was increased, which indicated that the prognosis was poor.

AB188. Vasohibin-1 and microvessel density predict prognosis in clear cell renal cell carcinoma

ObjectiveTo investigate the expression of vasohibin-1(VASH-1) and microvessel density (MVD) marked with CD34 in clear cell renal cell carcinoma (ccRCC) and analyze their correlations with prognosis.MethodsFrom January 2006 to December 2008, the clinical and pathologic data of 79 patients with ccRCC were retrospectively analyzed. Expressions of VASH-1 and CD34 in their tissue samples were detected by immunohistochemical (IHC) staining. For VASH-1 assessment, the staining intensity of positive tumor cells were evaluated semi-quantitatively and measured by Average Optical Density (AOD) for further analysis. The microvessel density (MVD) was counted using Weidner’s method. Statistical analysis was performed using SPSS 19.0.ResultsThe AOD of VASH1 in the 79 cancer tissues was 0.0435±0.0178. The MVD value marked by CD34 in the 79 cancer tissues was (55±34) profiles/HPF. The expression of MVD and VASH1 significantly varied with tumor stages and necrosis presence (P<0.05). Assessed by Spearman’s correlation, the expression of VASH1 was negatively associated with MVD (r=−0.641, P<0.05). The overall survival (OS) and recurrence-free survival (RFS) in ccRCC patients with high VASH1 expression were significantly worse than those low VASH1 expression (P<0.05).The multivariate analysis showed that age, tumor stage and necrosis presence were significantly correlated to OS and RFS in ccRCC patients.ConclusionsVASH1 is potentially valuable in prognostic evaluation of ccRCC.

Vasohibin-1 expression inhibits advancement of ovarian cancer producing various angiogenic factors.

Vasohibin‐1 (VASH1) is a negative feedback regulator of angiogenesis, the first to be discovered, and was identified in vascular endothelial growth factor (VEGF)‐stimulated vascular endothelial cells. Vasohibin‐1 inhibits abnormal vascularization induced by various angiogenic factors including fibroblast growth factor and platelet‐derived growth factor (PDGF), in addition to VEGF. By focusing on this characteristic of VASH1, we investigated the antitumor effects of VASH1 expression on ovarian cancer cells that produce different angiogenic factors. By using a high VEGF‐producing ovarian cancer cell line, SHIN‐3, and a high PDGF‐producing ovarian cancer cell line, KOC‐2S, the cells were transfected with either a VEGF antagonist, soluble VEGF receptor‐1 (sVEGFR‐1, or sFlt‐1), or VASH1 genes to establish their respective cellular expression. The characteristics of these transfectants were compared with controls. We previously reported that the expression of sFlt‐1 inhibited tumor vascularization and growth of high VEGF‐producing ovarian cancer cells, reduced peritoneal dissemination and ascites development, and prolonged the survival time of the host. However, in the current study, the expression of sFlt‐1 had no such effect on the high PDGF‐producing ovarian cancer cells used here, whereas VASH1 expression inhibited tumor vascularization and growth, not only in high VEGF‐producing cells, but also in high PDGF‐producing cells, reduced their peritoneal dissemination and ascites, and prolonged the survival time of the host. These results suggest that VASH1 is an effective treatment for ovarian cancer cells that produce different angiogenic factors.

MP90-09 A NEW STRATEGY FOR THE TREATMENT OF PROSTATE CANCER BY TARGETING VASOHIBIN-2

INTRODUCTION AND OBJECTIVES: Recently, we isolated a novel angiogenic molecule, vasohibin-2 (VASH2) which is an intrinsic factor specifically expressed in activated vascular endothelial cells (ECs). Previous studies found that the expression of VASH2 was restricted to ECs and tumor cells. Although VASH2 promoting the angiogenesis in several cancers have been investigated, it has not been fully characterized yet that the VASH2 reflects the activity of tumor and the predictor of progression in prostate cancer (Pca). The aim of this study was to address the role of VASH2 expression and the treatment by targeting VASH2 in Pca. METHODS: We retrospectively analyzed the clinical records of patients with localized Pca performed radical prostatectomy and immunohistochemically analyzed the expression of VASH2. Furthermore, we analyzed the expression of VASH2 in human umbilical vein ECs (HUVEC) and Pca cell lines. The cell viability of these cells treated with anti-VASH2 antibody (V2-Ab) was determined by using the WST-1 assay. DU145 was injected subcutaneously in BALB/c nude mice and then V2-Ab was injected intraperitoneally in mice. RESULTS: The average follow-up period was 5.6 years. The expression of VASH2 was observed to ECs of microvessels in the tumor stroma and Pca cells. The density of VASH2 positive vessels were significantly associated with Gleason score and pathological T stage (p<0.05). High VASH2 density was significantly associated with preoperative PSA (p1⁄40.012), T stage (p1⁄40.026), and the intensity of VASH2 positive tumor cells (p1⁄40.048). The PSA recurrence-free survival rate was significantly worse in high VASH2 group than in low VASH2 group (p1⁄40.019). We showed that the protein level of VASH2 in HUVEC and DU145 was higher than that of the Pca cell lines (LNCap, C4-2). WST-1 assay indicated that the treatment with V2-Ab caused a decreased cell proliferation of HUVEC (67%, p<0.001) and DU145 (80%, p1⁄40.017) at high dose. We examined time course changes in DU145 tumor growth in mice. The average tumor size in the V2-Ab treatment group and in the control group on day 27 was 85mm and 389mm, respectively (p1⁄40.007). CONCLUSIONS: The present study clearly demonstrates VASH2 have the cell apoptosis mediated by V2-Ab. The results suggest that VASH2 could become a new therapeutic target in patients with CRPC.

Protective role of gambogic acid in experimental pulmonary fibrosis in vitro and in vivo

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive disorder with poor prognosis. The treatment options for IPF are very limited. Gambogic acid (GA) has anticancer effect and anti-proliferative activity which is extracted from a dried yellow resin of the Garcinia hanburyi Hook.f. [Clusiaceae (Guttiferae)] in Southeast Asia. However, the anti-fibrotic activities of GA have not been previously investigated. MethodsIn this study, the effects of GA on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells and endothelial–mesenchymal transition (EndoMT) in human pulmonary microvascular endothelial cells (HPMECs), on the proliferation of human lung fibroblasts (HLF-1) were investigated in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis was investigated in vivo. ResultsIn TGF-β1 stimulated A549 cells, treatment with GA resulted in a reduction of EMT with a decrease in vimentin and p-Smad3 and an increase in E-cadherin instead. In TGF-β1 stimulated HPMECs, treatment with GA resulted in a reduction of EndoMT with a decrease in vimentin, and an increase in VE-cadherin instead. In the hypoxic HPMECs, treatment with GA reduced Vasohibin-2 (VASH-2), whereas increased VASH-1. In TGF-β1 stimulated HLF-1, treatment with GA reduced HLF-1 proliferation with a decrease in platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF-2) expressions. In vivo, treatment with GA for 2 weeks resulted in an amelioration of the BLM-induced pulmonary fibrosis in rats with a lower VASH-2. Instead, it was observed a higher VASH-1 expression at early stage of fibrosis at 1mg/kg, with reductions of the pathological score, collagen deposition, α-SMA, PDGF and FGF-2 expressions at fibrotic stage at 0.5mg/kg and 1mg/kg. ConclusionIn summary, GA reversed EMT and EndoMT, as well as HLF-1 proliferation in vitro and prevented pulmonary fibrosis in vivo by modulating VASH-2/VASH-1 and suppressing the TGF-β1/Smad3 pathway.

Ponatinib attenuates experimental pulmonary arterial hypertension by modulating Wnt signaling and vasohibin-2/vasohibin-1

AimsAn abnormal ratio of vasohibin-2/vasohibin-1 may be involved in the abnormal angiogenesis and vascular remodeling during pulmonary arterial hypertension (PAH). Main methodsTo evaluate the pharmacological actions of Ponatinib (AP) in experimental model of PAH, the effects of AP on TGF-β1-mediated endothelial-mesenchymal transition (EndoMT) in human pulmonary microvascular endothelial cells (HPMEC), and the hypoxic human pulmonary artery smooth muscle cells (HPASMC) proliferation and HPMEC in vitro, and on bleomycin (BLM)-induced PAH in vivo were investigated. Key findingsAP treatment resulted in a reduction of EndoMT in HPMECs with a decrease of vimentin, whereas an increase of VE-cadherin, reduction of fibroblast growth factor (FGF-2), vascular endothelial growth factor (VEGF) and vasohibin-2 (VASH-2), whereas an increase of vasohibin-1 (VASH-1) in the hypoxic HPMEC, a reduction of the HPASMC proliferation with decreases of wnt5a, β-catenin and cyclin D1 expression. AP ameliorated BLM-induced PAH in rats with reductions of FGF-2, VEGF, von Willebrand factor (vWF) and VASH-2 expression, whereas an increase of VASH-1 expression. AP ameliorated BLM-induced PAH in rats with reductions of the pathological score and the collagen deposition. In addition, AP ameliorated hemodynamics and right ventricular hypertrophy. SignificancesOur results identified a therapeutic potential of AP in PAH therapy might be modulated VASH-2/VASH-1 and the Wnt signaling.

Expression of pro-angiogenic genes in mesenchymal stem cells derived from dermis of patients with psoriasis.

Recent experimental studies revealed that angiogenesis and lymphangiogenesis are closely related to psoriasis. Our microarray analysis suggested that the pro-angiogenic genes platelet endothelial cell adhesion molecule-1 (PECAM1), facio-genital dysplasia-5 (FGD5), prostaglandin-endoperoxide synthase-1 (PTGS1), melanoma cell adhesion molecule (MCAM), vasohibin-2 (VASH2), and stabilin-1 (STAB1) are differentially expressed in dermal mesenchymal stem cells in psoriasis. The aim of this study was to investigate the mRNA and protein expression of PECAM1, FGD5, PTGS1, MCAM, VASH2, and STAB1 for angiogenesis and the possible mechanisms in psoriasis. We studied 12 patients with plaque psoriasis and 14 healthy controls matched for age and sex. Dermal mesenchymal stem cells were expanded, passaged, and identified by cellular morphology, immunophenotyping, and multipotential differentiation. The mRNA and protein expression of the above-mentioned six genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. The significantly decreased expression of PECAM1, PTGS1, FGD5, and MCAM at both mRNA and protein level (except VASH2 and STAB1) were demonstrated in mesenchymal stem cells from psoriatic skin lesions compared with non-lesional from healthy controls. We provide the first report that pro-angiogenic genes PECAM1, PTGS1, FGD5, and MCAM rather than VASH2 and STAB1 may be play a vital role in pathological dermal angiogenesis disorders of psoriasis. Therefore, anti-angiogenesis is attractive and offers future potential for application in patients with psoriasis.

The angiogenesis regulator vasohibin-1 inhibits ovarian cancer growth and peritoneal dissemination and prolongs host survival.

Vasohibin-1 (VASH1) is expressed in vascular endothelial cells stimulated by several angiogenic growth factors and displays autocrine activity to regulate angiogenesis via a negative feedback mechanism. In this study, we investigated the effect of VASH1 on ovarian cancer progression using VASH1-expressing ovarian cancer cells in vitro and in vivo. The growth ability of ovarian cancer cells engineered to express the VASH1 gene remained unchanged in vitro. However, we showed that VASH1 secretion by tumor cells inhibited the growth of human umbilical vein endothelial cells. Further, animal experiments showed that VASH1 expression inhibited tumor angiogenesis and growth. In a murine model of peritoneal dissemination of ovarian cancer cells, VASH1 inhibited peritoneal dissemination and ascites, resulting in significantly prolonged survival in mice. This indicates that VASH1 exerts an antitumor effect on ovarian cancer by inhibiting angiogenesis in the tumor environment. These findings suggest that a novel therapy based on VASH1 could be a useful therapeutic strategy for ovarian cancer.

Vasohibin-1 suppresses colon cancer.

Vasohibin-1 (VASH1) is an endogenous angiogenesis inhibitor.However, the clinical relevance of VASH1 in colon cancer and its regulations on cancer angiogenesis and cancer cell biological characteristics are still unknown. Here we showed that stromal VASH1 levels were negatively correlated with tumor size, advanced clinical stage and distant metastases in colon cancer patients. Overexpression of VASH1 in colon cancer cells induced apoptosis and senescence, inhibiting cancer cell growth and colony formation in vitro and tumor growth in vivo. In addition, knockdown of VASH1 in cancer cells promoted cell growth, adhesion and migration in vitro, and enhanced tumorigenesis and metastasis in vivo.

Expression of Vasohibin-1 in Human Carotid Atherosclerotic Plaque.

In patients with carotid plaque, intraplaque hemorrhage arising from ruptured neovascular vessels within the neointima is an important cause of stroke. The expression of Vasohibin-1 (VASH1), a negative feedback regulator of angiogenesis, occurs in the microvessel endothelial cells of various solid tumors and the arterial wall. However, the roles of VASH1 in the pathogenesis of atherosclerotic diseases remain unclear. The present study aimed to clarify the relevance of the VASH1 expression and plaque instability in human carotid plaques. We used quantitative real-time PCR and immunostaining to examine 12 atheromatous plaque specimens obtained via carotid endarterectomy. The distal areas of specimens lacking macroscopic atherosclerotic lesions served as controls. Compared with that observed in the controls, the VASH1 gene expression increased significantly in the atheromatous plaque (p=0.018). Moreover, the VASH1 mRNA levels correlated positively with those of VEGFA, CD31 and VCAM1 (r=0.788, p=0.004; r=0.99, p ＜ 0.001; r=0.94, p ＜ 0.001, respectively). Finally, the immunohistochemical analyses revealed the VASH1 expression in the neointimal microvessel endothelial cells of carotid plaque. The VASH1 expression levels in atheroma reflect both enhanced neovascularization and the inflammatory burden. Therefore, the VASH1 level may be a novel biomarker for evaluating plaque instability in patients with carotid arteriosclerosis and predicting ischemic stroke.

Novel Link between Inhibition of Angiogenesis and Tolerance to Vascular Stress.

The functional integrity of the vascular endothelium is an essential component required for the maintenance of vascular health, thus counteracting the onset of vascular diseases, including atherosclerosis and vascular complications of diabetes. In light of this important role, the vascular endothelium is expected to have a self-defense system. One candidate factor of such a system is vasohibin-1 (VASH1), a protein that is preferentially expressed in vascular endothelial cells (ECs). The unique features of VASH1 are its anti-angiogenic activity and ability to promote the stress tolerance and survival of ECs. This review summarizes current knowledge regarding VASH1 in terms of its roles in maintaining vascular integrity and protecting the vasculature against various forms of stress.

MiR200-upregulated Vasohibin 2 promotes the malignant transformation of tumors by inducing epithelial-mesenchymal transition in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) typically relies on tumor transformation and angiogenesis for its malignant behavior, including growth and metastasis. Previously, we reported that Vasohibin2 (VASH2) is preferentially expressed in hepatocellular carcinoma (HCC) tumor tissues and promotes angiogenesis. Here, we further investigated the role of VASH2 in HCC tumor progression.ResultsBioinformatics analyses and luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200a/b/c. We then used HepG2 and Hep3B cells, two representative hepatic cancer cell lines, to examine the role of VASH2 in tumors. VASH2 knockdown in HepG2 cells inhibited epithelial-mesenchymal transition (EMT), but VASH2 overexpression in Hep3B cells promoted EMT. Western blot analyses showed that VASH2 promoted EMT through the ZEB1/2 pathway.ConclusionVASH2 promoted invasion, reduced apoptosis and increased the proportion of stem cells in vitro and in vivo. These results indicated that VASH2 expression in HCC cells promotes the malignant transformation of tumors by inducing EMT.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0062-x) contains supplementary material, which is available to authorized users.

Abstract 4732: Vasohibine-1 negative and EZH2 positive may predict the prognosis of cholangiocarcinoma

Abstract Background: Vasohibin-1 (VASH1) is isolated as an endogenous angiogenesis inhibitor produced by the vascular endothelium, and the status of VASH1 expression has been documented in various cancer prognosis. Some reports shows that VASH1 was negatively regulated by enhancer of zeste homolog 2 (EZH2) epigenetically. The aim of this study was to assess the relationship between EZH2 and VASH1, and the prognostic value of this two molecules in intrahepatic cholangiocarcinoma (ICC) patients. Method: We examine 47 cases of intrahepatic cholangiocarcinoma to which the R0 liver resection had been performed by our department between 1993 years and 2010. Immunohistochemical staining of VASH1 and EZH2 was performed using paraffin-embedded block, and we examined the relation among the expression of VASH1 and a clinicopathological factor (sex, age, tumor diameter, tumor number, differentiation level, microscopic vascular invasion, and metastasis in lymph node), EZH2 expression, recurrence-free survival (RFS) and overall survival (OS). Result: VASH1 and EZH2 positivity was observed in 24 (51.1%) and 24 (51.1%) patients respectively, and VASH1 positivity was inversely correlated with EZH2 high expression (R=0.3870, p=0.0018). VASH1 positivity was not significantly correlate with RFS or OS. On the other hands, VASH1 negative and EZH2 positive was observed in 17 (36.2%) patients, and was a significant disadvantage to both RFS (relative risk (RR) 3.39, 95% confidence interval (CI) 1.28-8.67, p=0.0157) and OS (RR 3.69, 95% CI 1.20-11.2, 0.0237) in univariate Cox hazard analysis. In multivariate analysis, VASH1 negative and EZH2 positive group was an independent risk factor for both RFS (RR 4.19, 95% CI 1.33-13.13, p=0.0150), and OS (RR 8.11, 95% CI 2.01-42.1, p=0.0030). Conclusion: The expression of VASH1 was inversely correlate with EZH2 expression. VASH1 negative and EZH2 positive was an independent risk factor for OS and DFS in ICC patients. Citation Format: Shigeki Nakagawa, Yasuo Sakamoto, Ryuma Tokunaga, Takaaki Higashi, Keita Sakamoto, Akira Chikamoto, Takatoshi Ishiko, Toru Beppu, Hideo Baba. Vasohibine-1 negative and EZH2 positive may predict the prognosis of cholangiocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4732. doi:10.1158/1538-7445.AM2014-4732

Exacerbation of diabetic renal alterations in mice lacking vasohibin-1.

Vasohibin-1 (VASH1) is a unique endogenous inhibitor of angiogenesis that is induced in endothelial cells by pro-angiogenic factors. We previously reported renoprotective effect of adenoviral delivery of VASH1 in diabetic nephropathy model, and herein investigated the potential protective role of endogenous VASH1 by using VASH1-deficient mice. Streptozotocin-induced type 1 diabetic VASH1 heterozygous knockout mice (VASH1+/−) or wild-type diabetic mice were sacrificed 16 weeks after inducing diabetes. In the diabetic VASH1+/− mice, albuminuria were significantly exacerbated compared with the diabetic wild-type littermates, in association with the dysregulated distribution of glomerular slit diaphragm related proteins, nephrin and ZO-1, glomerular basement membrane thickning and reduction of slit diaphragm density. Glomerular monocyte/macrophage infiltration and glomerular nuclear translocation of phosphorylated NF-κB p65 were significantly exacerbated in the diabetic VASH1+/− mice compared with the diabetic wild-type littermates, accompanied by the augmentation of VEGF-A, M1 macrophage-derived MCP-1 and phosphorylation of IκBα, and the decrease of angiopoietin-1/2 ratio and M2 macrophage-derived Arginase-1. The glomerular CD31+ endothelial area was also increased in the diabetic VASH1+/− mice compared with the diabetic-wild type littermates. Furthermore, the renal and glomerular hypertrophy, glomerular accumulation of mesangial matrix and type IV collagen and activation of renal TGF-β1/Smad3 signaling, a key mediator of renal fibrosis, were exacerbated in the diabetic VASH1+/− mice compared with the diabetic wild-type littermates. In conditionally immortalized mouse podocytes cultured under high glucose condition, transfection of VASH1 small interfering RNA (siRNA) resulted in the reduction of nephrin, angiopoietin-1 and ZO-1, and the augmentation of VEGF-A compared with control siRNA. These results suggest that endogenous VASH1 may regulate the development of diabetic renal alterations, partly via direct effects on podocytes, and thus, a strategy to recover VASH1 might potentially lead to the development of a novel therapeutic approach for diabetic nephropathy.

Role of the vasohibin family in the regulation of fetoplacental vascularization and syncytiotrophoblast formation.

Vasohibin-1 (VASH1) and vasohibin-2 (VASH2), the 2 members of the vasohibin family, have been identified as novel regulators of angiogenesis. VASH1 ceases angiogenesis, whereas VASH2 stimulates sprouting. Here we characterized their functional role in the placenta. Immunohistochemical analysis of human placental tissue clarified their distinctive localization; VASH1 in endothelial cells and VASH2 in trophoblasts. We then used a mouse model to explore their function. Wild-type, Vash1(−/−), and Vash2(−/−) mice on a C57BL6 background were used in their first pregnancy. As expected, the fetal vascular area was increased in the Vash1(−/−) mice, whereas it was decreased in the Vash2(−/−) mice relative to wild-type. In addition, we noticed that the Vash2(−/−) mice at 18.5dpc displayed thinner villi of the labyrinth and larger maternal lacunae. Careful observation by an electron microscopy revealed that the syncytiotrophoblast formation was defective in the Vash2(−/−) mice. To test the possible involvement of VASH2 in the syncytiotrophoblast formation, we examined the fusion of BeWo cells, a human trophoblastoid choriocarcinoma cell line. The forskolin treatment induced the fusion of BeWo cells, and the knockdown of VASH2 expression significantly inhibited this cell fusion. Conversely, the overexpression of VASH2 by the infection with adenovirus vector encoding human VASH2 gene significantly increased the fusion of BeWo cells. Glial cell missing-1 and endogenous retrovirus envelope glycoprotein Syncytin 1 and Syncytin 2 are known to be involved in the fusion of trophoblasts. However, VASH2 did not alter their expression in BeWo cells. These results indicate that VASH1 and VASH2 showed distinctive localization and opposing function on the fetoplacental vascularization. Moreover, our study shows for the first time that VASH2 expressed in trophoblasts is involved in the regulation of cell fusion for syncytiotrophoblast formation.

Vasohibin‑2 promotes proliferation in human breast cancer cells via upregulation of fibroblast growth factor‑2 and growth/differentiation factor‑15 expression.

Vasohibin-2 (VASH2) is an angiogenic factor, and has been previously reported to be a cancer-related gene, with cytoplasmic and karyotypic forms. In the current study VASH2 expression in human breast cancer tissue and adjacent non-cancerous tissue was investigated with immunohistochemistry. MCF-7 and BT474 human breast cancer cells were transfected with lentiviral constructs to generate in vitro VASH2 overexpression and knockdown models. In addition, BALB/cA nude mice were inoculated subcutaneously with transfected cells to generate in vivo models of VASH2 overexpression and knockdown. The effect of VASH2 on cell proliferation was investigated using a bromodeoxyuridine assay in vitro and immunohistochemistry of Ki67 in xenograft tumors. Growth factors were investigated using a human growth factor array, and certain factors were further confirmed by an immunoblot. The results indicated that the expression level of cytoplasmic VASH2 was higher in breast cancer tissues with a Ki67 (a proliferation marker) level of ≥14%, compared with tissues with a Ki67 level of <14%. VASH2 induced proliferation in vitro and in vivo. Four growth factors activated by VASH2 were identified as follows: Fibroblast growth factor 2 (FGF2), growth/differentiation factor-15 (GDF15), insulin-like growth factor-binding protein (IGFBP)3 and IGFBP6. FGF2 and GDF15 may contribute to VASH2-induced proliferation. The current study identified a novel role for VASH2 in human breast cancer, and this knowledge suggests that VASH2 may be a novel target in breast cancer treatment.

Urinary and plasma levels of vasohibin-1 can predict renal functional deterioration in patients with renal disorders.

Vasohibin-1 (VASH-1) is a negative feedback regulator of angiogenesis, and a small vasohibin-binding protein (SVBP) serves as its secretory chaperone and contributes to its antiangiogenic effects. In the present study, we aimed to define the clinical significance of VASH-1 and SVBP in patients with chronic kidney disease (CKD). We recruited 67 Japanese hospitalized patients with renal disorders with (n = 45) or without (n = 22) renal biopsy samples and 10 Japanese healthy controls. We evaluated the correlations between the plasma and urinary levels of VASH-1/VASH-1-SVBP complex/SVBP and the clinicopathological parameters. The plasma levels of VASH-1 were inversely correlated with age and systolic and diastolic blood pressure and positively correlated with crescent formation. Increased plasma and urinary levels of VASH-1 and VASH-1-SVBP complex were significantly correlated with worse renal outcomes. These results demonstrate an association between elevated urinary and plasma levels of VASH-1 and progressive decline of the renal function, thus suggesting a potential role for VASH-1 in predicting a worse renal prognosis in patients with renal disease, including CKD.

Vasohibin-1 deficiency enhances renal fibrosis and inflammation after unilateral ureteral obstruction.

Tubulointerstitial injuries are known to predict the deterioration of renal function in chronic kidney disease (CKD). We recently reported the protective role of Vasohibin‐1(VASH‐1), a negative feedback regulator of angiogenesis, in diabetic nephropathy, but its impact on tubulointerstitial injuries remains to be elucidated. In the present study, we evaluated the role of endogenous VASH‐1 in regulating the tubulointerstitial alterations induced by unilateral ureteral obstruction (UUO), and assessed its role on fibrogenesis and the activation of Smad3 signaling in renal fibroblasts. UUO was induced in female Vasohibin‐1 heterozygous knockout mice (VASH‐1+/−) or wild‐type (WT) (VASH‐1+/+) littermates. Mice were sacrificed on Day 7 after left ureter ligation, and the kidney tissue was obtained. Interstitial fibrosis, the accumulation of type I and type III collagen and monocytes/macrophages infiltration in the obstructed kidneys (OBK) were significantly exacerbated in VASH‐1+/− mice compared with WT mice (Day 7). The increases in the renal levels of TGF‐β1, pSmad3, NF‐κB pp65, CCL2 mRNA, and the number of interstitial fibroblast‐specific protein‐1 (FSP‐1)+ fibroblasts in the OBK were significantly aggravated in VASH‐1+/− mice. In addition, treatment with VASH‐1 siRNA enhanced the TGF‐β1‐induced phosphorylation of Smad3, the transcriptional activation of the Smad3 pathway and the production of type I/type III collagen in fibroblasts, in vitro. Taken together, our findings demonstrate a protective role for endogenous VASH‐1 on tubulointerstitial alterations via its regulation of inflammation and fibrosis and also show the direct anti‐fibrotic effects of VASH‐1 on renal fibroblasts through its modulation of TGF‐β1 signaling.

Vasohibin-2 modulates tumor onset in the gastrointestinal tract by normalizing tumor angiogenesis.

BackgroundVasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of Apc Min/+ mice and Apc Min/+ /Vash2 -/- mice.MethodsApc Min/+ mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. Apc Min/+ mice were mated with Vash2 -/- mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2 -/- gene by PCR. Intestinal tumors from Apc Min/+ mice and Apc Min/+ /Vash2 -/- mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed.ResultsWe found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in Apc Min/+ mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2-/- mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2-/- mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine.ConclusionWe propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.

Vasohibin 2 Decreases the Cisplatin Sensitivity of Hepatocarcinoma Cell Line by Downregulating p53

Hepatocellular carcinoma (HCC) is a prevalent problem worldwide. Chemotherapy, especially cisplatin (CDDP)-based systemic chemotherapy, is the best option for advanced liver cancer. However, CDDP resistance is becoming common and hindering the clinical application of CDDP. Meanwhile, no consensus has been reached regarding the chemotherapeutic use of vasohibin 2 (VASH2), which promotes the angiogenesis and proliferation of cancer cells. In this work, a tissue microarray was used to observe VASH2 and its possible role in cancer treatment. Results showed that VASH2 was highly expressed in HCC tissues and was significantly correlated with cancer differentiation. To further investigate the efficacy and mechanism of the combination of VASH2 with anti-cancer drugs in liver cancer cells, we stably built VASH2 overexpression and knockdown cell lines. We found that VASH2 can influence the CDDP sensitivity and that the cell overexpression of VASH2 had a higher cell viability and lower apoptosis rate after CDDP exposure. We also observed that VASH2 overexpression downregulated wild-type p53, as well as suppressed the expression of the pro-apoptotic protein BCL2-associated X protein (Bax) and cleaved caspase-3 (CC-3) after treatment by CDDP. Conversely, the knockdown of VASH2 significantly inhibited these effects. In an in vivo chemosensitivity study, nude mice were subcutaneously injected with tumor cells and received CDDP treatment through intraperitoneal administration every 3 days. We found that VASH2 knockdown markedly limited the tumor growth and enhanced the CDDP toxicity and apoptosis of tumor cells. Western blot analysis revealed that tumor cells with downregulated VASH2 had a higher expression of wild-type p53, Bax, and CC-3 than control cells. Overall, our results indicated the novel roles of VASH2 in the chemoresistance of hepatocarcinoma cells to CDDP and suggested that VASH2 may be a promising anticancer target.