Hiroki Miyashita

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.

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.

HEX Acts as a Negative Regulator of Angiogenesis by Modulating the Expression of Angiogenesis-Related Gene in Endothelial Cells In Vitro

Objective—The hematopoietically expressed homeobox (HEX) is transiently expressed in endothelial cells (ECs) during vascular formation in embryo. Here, we investigated whether HEX played any role in angiogenesis-related properties of ECs in vitro. Methods and Results—We transiently overexpressed HEX in human umbilical vein ECs (HUVECs). To our surprise, HEX completely abrogated the response of HUVECs to vascular endothelial growth factor (VEGF) with regard to proliferation, migration, and invasion and abolished network formation by HUVECs on Matrigel. cDNA microarray analysis and quantitative real-time reverse transcription–polymerase chain reaction combined with Western blotting revealed that HEX significantly repressed the expression of VEGF receptor-1, VEGF receptor-2, neuropilin-1, tyrosine kinase with Ig and EGF homology domains (TIE)-1, TIE-2, and the integrin &agr;v subunit, whereas it augmented the expression of endoglin in HUVECs. We established murine embryonic stem cells that were stably transfected with HEX sense cDNA or antisense cDNA, and we examined the in vitro differentiation to ECs. Although the expression of VEGF receptor-2 was decreased in sense transfectants, the number of cells expressing VE-cadherin, a specific marker of ECs, was not altered. Conclusions—Our present results suggest that HEX may not affect the differentiation of ECs but acts as a negative regulator of angiogenesis.

Vascular Endothelial Zinc Finger 1 Is Involved in the Regulation of Angiogenesis: Possible Contribution of Stathmin/OP18 as a Downstream Target Gene

Objective—Vascular endothelial zinc finger 1 (Vezf1) is a recently identified zinc finger transcription factor that is expressed in endothelial cells (ECs) during vascular development in mouse embryo. Here, we present that Vezf1 was expressed in ECs at the site of postnatal angiogenesis. We therefore examined whether Vezf1 was involved in the regulation of angiogenesis. Methods and Results—The specific downregulation of Vezf1 by antisense oligodeoxynucleotide (AS-ODN) significantly inhibited the proliferation, migration, and network formation of cultured ECs as well as angiogenesis in vivo. Vezf1 AS-ODN downregulated the expression of stathmin/oncoprotein18 (OP18), a microtubule-destabilizing protein, in ECs, whereas transient transfection of Vezf1 cDNA increased the expression of stathmin/OP18 in ECs. To explore the relationship between Vezf1 and stathmin/OP18, we specifically downregulated stathmin/OP18. We found that stathmin/OP18 AS-ODN inhibited the proliferation, migration, and network formation of ECs as Vezf1 AS-ODN did. Moreover, Vezf1 AS-ODN decreased G2/M population of ECs and increased apoptosis, which reproduced the characteristic feature of stathmin/OP18 inhibition. Conclusion—These results suggest that Vezf1 is involved in the regulation of angiogenesis, at least in part, through the expression of stathmin/OP18 in ECs.

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.

Puromycin insensitive leucyl-specific aminopeptidase (PILSAP) is involved in the activation of endothelial integrins

We previously reported that mouse orthologue of puromycin insensitive leucyl‐specific aminopeptidase (mPILSAP) played an important role in angiogenesis by regulating the proliferation and migration of endothelial cells (ECs) (Miyashita et al., 2002 . Blood 99:3241–3249). Here, we examined the mechanism as to how mPILSAP regulates the migration of ECs. Cell adhesion through integrins plays a crucial role in cell migration, and ECs use at least type‐1 collagen receptor integrin α2β1, fibronectin receptor α5β1, and vitronectin receptors αvβ3 and αvβ5. mPILSAP antisense oligodeoxynucleotide (AS‐ODN) or leucinethiol (LT), a leucyl‐aminopeptidase inhibitor, did not affect the attachment but did significantly inhibit the spreading of cells of the murine endothelial cell line MSS31 when they were plated on vitronectin‐, fibronectin‐, or type‐1 collagen, although they did not affect the expression of integrin α2, α5, αv, β1, β3, and β5 subunits on the cell surface. AS‐ODN and LT also inhibited the tyrosine phosphorylation of FAK when cells were plated on vitronectin, fibronectin, or type‐1 collagen. This inhibition of cell spreading and of tyrosine phosphorylation of FAK could be negated by Mg2+. These results suggest that mPILSAP is involved in the activation of endothelial integrins.

Angiogenesis Inhibitor Vasohibin-1 Enhances Stress Resistance of Endothelial Cells via Induction of SOD2 and SIRT1

Vasohibin-1 (VASH1) is isolated as an endothelial cell (EC)-produced angiogenesis inhibitor. We questioned whether VASH1 plays any role besides angiogenesis inhibition, knocked-down or overexpressed VASH1 in ECs, and examined the changes of EC property. Knock-down of VASH1 induced premature senescence of ECs, and those ECs were easily killed by cellular stresses. In contrast, overexpression of VASH1 made ECs resistant to premature senescence and cell death caused by cellular stresses. The synthesis of VASH1 was regulated by HuR-mediated post-transcriptional regulation. We sought to define the underlying mechanism. VASH1 increased the expression of (superoxide dismutase 2) SOD2, an enzyme known to quench reactive oxygen species (ROS). Simultaneously, VASH1 augmented the synthesis of sirtuin 1 (SIRT1), an anti-aging protein, which improved stress tolerance. Paraquat generates ROS and causes organ damage when administered in vivo. More VASH1 (+/−) mice died due to acute lung injury caused by paraquat. Intratracheal administration of an adenovirus vector encoding human VASH1 augmented SOD2 and SIRT1 expression in the lungs and prevented acute lung injury caused by paraquat. Thus, VASH1 is a critical factor that improves the stress tolerance of ECs via the induction of SOD2 and SIRT1.

Enhanced cancer metastasis in mice deficient in vasohibin-1 gene.

Vasohibin-1 (VASH1) is isolated as an endogenous angiogenesis inhibitor produced by the vascular endothelium. We previously reported that tumor growth and tumor angiogenesis were augmented in VASH1 (−/−) mice. Here we examined whether VASH1 plays any role in cancer metastasis. When Lewis lung carcinoma (LLC) cells were inoculated in the footpad to observe spontaneous metastasis, a significant increase in lung metastasis together with inguinal lymph node metastasis was evident in the VASH1 (−/−) mice. Histological analyses revealed that vessels of the footpad tumor in VASH1 (−/−) mice were more immature, having fewer mural cells. However, when LLC cells were injected into a tail vein, the extent of lung metastasis was unchanged between wild-type mice and VASH1 (−/−) mice. When VASH1 in endothelial cells in culture was knocked-down by siRNA, we observed a decrease in the content of ZO-1, a component of tight junctions, which decrease resulted in increased transmigration of cancer cells across the endothelial cell monolayer. These results indicate that endogenous VASH1 tightens the endothelial barrier and makes tumor vessels resistant to cancer metastasis.