Hikaru Sonoda

Circulating Exosomal microRNAs as Biomarkers of Colon Cancer

Purpose Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined. Experimental Design Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients. Results The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis. Conclusion Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease.

Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen

Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remains challenging. Here we describe a sensitive and rapid analytical technique for profiling circulating EVs directly from blood samples of patients with colorectal cancer. EVs are captured by two types of antibodies and are detected by photosensitizer-beads, which enables us to detect cancer-derived EVs without a purification step. We also show that circulating EVs can be used for detection of colorectal cancer using the antigen CD147, which is embedded in cancer-linked EVs. This work describes a new liquid biopsy technique to sensitively detect disease-specific circulating EVs and provides perspectives in translational medicine from the standpoint of diagnosis and therapy.

Distinctive localization and opposed roles of vasohibin-1 and vasohibin-2 in the regulation of angiogenesis

We recently isolated a novel angiogenesis inhibitor, vasohibin-1, and its homologue, vasohibin-2. In this study we characterize the role of these 2 molecules in the regulation of angiogenesis. In a mouse model of subcutaneous angiogenesis, the expression of endogenous vasohibin-1 was low in proliferating ECs at the sprouting front but high in nonproliferating endothelial cells (ECs) in the termination zone. In contrast, endogenous vasohibin-2 was preferentially expressed in mononuclear cells mobilized from bone marrow that infiltrated the sprouting front. When applied exogenously, vasohibin-1 inhibited angiogenesis at the sprouting front where endogenous vasohibin-1 was scarce but did not influence vascularity in the termination zone where endogenous vasohibin-1 was enriched. Exogenous vasohibin-2 prevented the termination of angiogenesis in the termination zone and increased vascularity in this region. Angiogenesis was persistent in the termination zone in the vasohibin-1 knockout mice, whereas angiogenesis was deficient at the sprouting front in the vasohibin-2 knockout mice. Supplementation of deficient proteins normalized the abnormal patterns of angiogenesis in the vasohibin knockout mice. These results indicate that vasohibin-1 is expressed in ECs in the termination zone to halt angiogenesis, whereas vasohibin-2 is expressed in infiltrating mononuclear cells in the sprouting front to promote angiogenesis.

Vasohibin-1 Expression in Endothelium of Tumor Blood Vessels Regulates Angiogenesis

In this study, we characterized the significance of the vascular endothelial growth factor-inducible angiogenesis inhibitor vasohibin-1 to tumors. In pathological sections of non-small cell lung carcinoma, vasohibin-1 was present in the endothelial cells of blood vessels of the tumor stroma, but not in the lymphatics. In cancer cells, the presence of vasohibin-1 was associated with hypoxia-inducible factor 1alpha/vascular endothelial growth factor and fibroblast growth factor-2 expression. We then examined the function of vasohibin-1 in the mouse by subcutaneously inoculating with Lewis lung carcinoma cells. Resultant tumors in vasohibin-1(-/-) mice contained more immature blood vessels and fewer apoptotic tumor cells than tumors in wild-type mice. In wild-type mice that had been inoculated with Lewis lung carcinoma cells, tail vein injection of adenovirus containing the human vasohibin-1 gene inhibited tumor growth and tumor angiogenesis. Moreover, the remaining tumor vessels in adenoviral human vasohibin-1 gene-treated mice were small, round, and mature, surrounded by mural cells. The addition of adenoviral human vasohibin-1 gene to cisplatin treatment improved cisplatins antitumor activity in mice. These results suggest that endogenous vasohibin-1 is not only involved in tumor angiogenesis, but when sufficient exogenous vasohibin-1 is supplied, it blocks sprouting angiogenesis by tumors, matures the remaining vessels, and enhances the antitumor effect of conventional chemotherapy.

Isolation and Characterization of Vasohibin-2 as a Homologue of VEGF-Inducible Endothelium-Derived Angiogenesis Inhibitor Vasohibin

Objective—We recently isolated vasohibin, a novel vascular endothelial growth factor (VEGF)-inducible endothelium-derived angiogenesis inhibitor. Our aim is to find DNA sequences homologous to vasohibin and determine their expression profile. Methods and Results—By the search of DNA sequences in the database, we found one homologous gene and designated it vasohibin-2. Overall amino acid sequence homology between the prototype vasohibin (vasohibin-1) and vasohibin-2 was >50%. Vasohibin-2 exhibited antiangiogenic activity. Vasohibin-2 expression in cultured endothelial cells was low and not inducible by the stimulation that induced vasohibin-1. However, the immunohistochemical analysis revealed that vasohibin-1 and -2 were diffusely expressed in endothelial cells in embryonic organs during mid-gestation. After that time point, vasohibin-1 and -2 became faint, but persisted to a certain extent in arterial endothelial cells from late gestation to neonate. Expression of vasohibin-1 and -2 could be augmented in vivo by local transfection with the VEGF gene in the embryonic brain or by cutaneous wounding in adult mice. Conclusion—These results suggest that vasohibin-2, in combination with vasohibin-1, forms a novel family of angiogenesis inhibitors.

Vasohibin-1, a Negative Feedback Regulator of Angiogenesis, Ameliorates Renal Alterations in a Mouse Model of Diabetic Nephropathy

OBJECTIVE The involvement of proangiogenic factors such as vascular endothelial growth factor as well as the therapeutic efficacy of angiogenesis inhibitors in early diabetic nephropathy has been reported. Vasohibin-1 (VASH-1) is a unique endogenous angiogenesis inhibitor that is induced in endothelial cells by proangiogenic factors. We investigated the therapeutic efficacy of VASH-1 in an early diabetic nephropathy model. RESEARCH DESIGN AND METHODS Streptozotocin- induced type 1 diabetic mice received intravenous injections of adenoviral vectors encoding VASH-1 (AdhVASH-1) or β-gal (AdLacZ) every other week and were killed after 28 days. RESULTS Treatment with AdhVASH-1 resulted in sustained increase in the protein levels of VASH-1 in the liver and sera, in the absence of any inflammatory alterations. AdhVASH-1 treatment significantly suppressed renal hypertrophy, glomerular hypertrophy, glomerular hyperfiltration, albuminuria, increase of the CD31+ glomerular endothelial area, F4/80+ monocyte/macrophage infiltration, the accumulation of type IV collagen, and mesangial matrix compared with AdLacZ-treated diabetic mice. Increase in the renal levels of transforming growth factor-β1, monocyte chemoattractant protein-1, and receptor for advanced glycation end products in diabetic animals was significantly suppressed by AdhVASH-1 (real-time PCR and immunoblot). VASH-1 significantly suppressed the increase of transforming growth factor-β, monocyte chemoattractant protein-1, and receptor for advanced glycation end products, induced by high ambient glucose in cultured mouse mesangial cells. Increased phosphorylation of VEGFR2 was suppressed in AdVASH-1–treated diabetic animals and in cultured glomerular endothelial cells. Endogenous mouse VASH-1 was localized to the mesangial and endothelial area in glomeruli of diabetic mice. CONCLUSIONS These results suggest the potential therapeutic efficacy of VASH-1 in treating early diabetic nephropathy potentially mediated via glomerular endothelial and mesangial cells.

Endogenous Angiogenesis Inhibitor Vasohibin1 Exhibits Broad-Spectrum Antilymphangiogenic Activity and Suppresses Lymph Node Metastasis

During cancer progression, the angiogenesis that occurs is involved in tumor growth and hematogenous-distant metastasis, whereas lymphangiogenesis is involved in regional lymph node metastasis. Angiogenesis is counterregulated by various endogenous inhibitors; however, little is known about endogenous inhibitors of lymphangiogenesis. We recently isolated vasohibin1 as an angiogenesis inhibitor intrinsic to the endothelium and further demonstrated its anticancer activity through angiogenesis inhibition. Here, we examined the effect of vasohibin1 on lymphangiogenesis. Vasohibin1 exhibited broad-spectrum antilymphangiogenic activity in the mouse cornea induced by factors including VEGF-A, VEGF-C, FGF2, and PDGF-BB. We then inoculated highly lymph node-metastatic cancer cells into mice and examined the effect of vasohibin1 on lymph node metastasis. Tail-vein injection of adenovirus containing the human vasohibin1 gene inhibited tumor lymphangiogenesis and regional lymph node metastasis. Moreover, local injection of recombinant vasohibin1 inhibited lymph node metastasis. These results suggest vasohibin1 to be the first known intrinsic factor having broad-spectrum antilymphangiogenic activity and indicate that it suppresses lymph node metastasis.

The Vasohibin Family A Negative Regulatory System of Angiogenesis Genetically Programmed in Endothelial Cells

Biological phenomena are under the precise control by the genome. For the regulation of angiogenesis, proangiogenic genes such as VEGFs and angiopoietins are highly conserved, act specifically on endothelial cells, and play a fundamental role. In this sense, nature should prepare specific antiangiogenic genes as well. However, this counterpart of genomic regulation of angiogenesis remains to be established. We recently isolated a novel endothelium-derived angiogenesis inhibitor and named it vasohibin. Vasohibin is dominantly expressed in endothelial cells, induced by the stimulation with VEGF or FGF-2, and selectively affects on endothelial cells and inhibits angiogenesis. Although the mechanism of how vasohibin inhibits angiogenesis remains to be elucidated, our discovery of vasohibin as an endothelium-derived VEGF-inducible angiogenesis inhibitor should shed light on the genomic basis of the negative regulation of angiogenesis.

Amelioration of renal alterations in obese type 2 diabetic mice by vasohibin-1, a negative feedback regulator of angiogenesis

The involvement of VEGF-A as well as the therapeutic efficacy of angiogenesis inhibitors in diabetic nephropathy have been reported. We recently reported the therapeutic effects of vasohibin-1 (VASH-1), an endogenous angiogenesis inhibitor, in a type 1 diabetic nephropathy model (Nasu T, Maeshima Y, Kinomura M, Hirokoshi-Kawahara K, Tanabe K, Sugiyama H, Sonoda H, Sato Y, Makino H. Diabetes 58: 2365-2375, 2009). In this study, we investigated the therapeutic efficacy of VASH-1 on renal alterations in obese mice with type 2 diabetes. Diabetic db/db mice received intravenous injections of adenoviral vectors encoding human VASH-1 (AdhVASH-1) and were euthanized 8 wk later. AdhVASH-1 treatment resulted in significant suppression of glomerular hypertrophy, glomerular hyperfiltration, albuminuria, increase in the CD31(+) glomerular endothelial area, F4/80(+) monocyte/macrophage infiltration, the accumulation of type IV collagen, and mesangial matrix. An increase in the renal levels of VEGF-A, VEGFR-2, transforming growth factor (TGF)-β1, and monocyte chemoattractant protein-1 in diabetic animals was significantly suppressed by AdhVASH-1 (immunoblotting). AdhVASH-1 treatment significantly recovered the loss and altered the distribution patterns of nephrin and zonula occludens (ZO)-1 and suppressed the increase in the number of fibroblast-specific protein-1 (FSP-1(+)) and desmin(+) podocytes in diabetic mice. In vitro, recombinant human VASH-1 (rhVASH-1) dose dependently suppressed the upregulation of VEGF induced by high ambient glucose (25 mM) in cultured mouse podocytes. In addition, rhVASH-1 significantly recovered the mRNA levels of nephrin and the protein levels of ZO-1 and P-cadherin and suppressed the increase in protein levels of desmin, FSP-1, Snail, and Slug in podocytes under high-glucose condition. Taken together, these results suggest the potential use of VASH-1 as a novel therapeutic agent in type 2 diabetic nephropathy mediated via antiangiogenic effects and maintenance of podocyte phenotype in association with antiproteinuric effects.

Isolation of a small vasohibin-binding protein (SVBP) and its role in vasohibin secretion

Upon stimulation with angiogenic factors, vascular endothelial cells (ECs) secrete a negative-feedback regulator of angiogenesis, vasohibin-1 (VASH1). Because VASH1 lacks a classical signal sequence, it is not clear how ECs secrete VASH1. We isolated a small vasohibin-binding protein (SVBP) composed of 66 amino acids. The level of Svbp mRNA was relatively high in the bone marrow, spleen and testes of mice. In cultured ECs, Vash1 mRNA was induced by VEGF, and Svbp mRNA was expressed constitutively. The interaction between VASH1 and SVBP was confirmed using the BIAcore system and immunoprecipitation analysis. Immunocytochemical analysis revealed that SVBP colocalized with VASH1 in ECs. In polarized epithelial cells, SVBP accumulated on the apical side, whereas VASH1 was present throughout the cells and partially colocalized with SVBP. Transfection of SVBP enhanced VASH1 secretion, whereas knockdown of endogenous SVBP markedly reduced VASH1 secretion. SVBP increased the solubility of VASH1 protein in detergent solution and inhibited the ubiquitylation of VASH1 protein. Moreover, co-transfection of SVBP significantly augmented the inhibitory effect of VASH1 on EC migration. These results indicate that SVBP acts as a secretory chaperone for VASH1 and contributes to the anti-angiogenic activity of VASH1.