Masanari Obika

ADAMTS1 Is a Unique Hypoxic Early Response Gene Expressed by Endothelial Cells

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is a member of the matrix metalloproteinase family. We have previously reported that ADAMTS1 was strongly expressed in myocardial infarction. In this study, we investigated whether hypoxia induced ADAMTS1 and investigated its regulatory mechanism. In hypoxia, the expression level of ADAMTS1 mRNA and protein rapidly increased in endothelial cells, but not in other cell types. Interestingly, the induction of ADAMTS1 by hypoxia was transient, whereas vascular endothelial growth factor induction by hypoxia in human umbilical vein endothelial cells (HUVEC) increased in a time-dependent manner. CoCl2, a transition metal that mimics hypoxia, induced ADAMTS1 in HUVEC. The phosphatidylinositol 3-kinase inhibitor LY294002 dose-dependently inhibited the increase of ADAMTS1 mRNA expression in hypoxia. We characterized the promoter region of ADAMTS1, and the secreted luciferase assay system demonstrated that hypoxia induced luciferase secretion in the culture medium 4.6-fold in HUVEC. In the promoter region of ADAMTS1, we found at least three putative hypoxia-inducible factor (HIF) binding sites, and the chromatin immunoprecipitation assay revealed HIF-1 binding to HIF binding sites in the promoter region of ADAMTS1 under hypoxia. Recombinant ADAMTS1 protein promoted the migration of HUVEC under hypoxic conditions. In summary, we found that ADAMTS1 is transiently induced by hypoxia in endothelial cells, and its transcription is mediated by HIF-1 binding. Our data indicate that ADAMTS1 is a novel acute hypoxia-inducible gene.

Tumor-specific expression of the RGD-α3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice

Angiogenesis plays an essential role in tumor growth. This study investigated expression of the noncollagenous domain of α3(IV) collagen (α3(IV)NC1) transduced into tumors and its inhibition of tumor growth. We hypothesized that if a human telomerase reverse transcriptase (hTERT) promoter‐driven RGD motif containing α3(IV)NC1 (hTERT/ RGD‐α3(IV)NC1) were expressed in telomerase‐expressing tumor cells, it would inhibit tumor growth by its anti‐angiogenic property. Adenoviral transduction of hTERT/RGD‐α3(IV)NC1 expressed RGD‐α3(IV)NC1 in hTERT‐positive tumor cell lines. However, hTERT/RGD‐α3(IV)NC1 did not express RGD‐α3(IV)NC1 in hTERT‐negative cells such as keratinocytes and fibroblasts. The secreted RGD‐α3(IV)NC1 in the conditioned medium from tumor cells inhibited cell proliferation as well as tube formation in cultured endothelial cells, but had no effect on other types of cells. In an in vivo model, adenoviral hTERT/RGD‐α3(IV)NC1 gene therapy showed limited expression of RGD‐α3(IV)NC1 in tumors and resulted in a significant decrease of vessel density in tumors. The growth of subcutaneous (s.c.) tumors in nude mice was significantly suppressed by treatment with hTERT/ RGD‐α3(IV)NC1. In addition, long‐term inhibition of tumor growth was achieved by intermittent administration of hTERT/RGD‐α3(IV)NC1. In conclusion, our findings demonstrate that tumor‐specific anti‐angiogenic gene therapy utilizing RGD‐α3(IV)NC1 under the hTERT promoter inhibited angiogenesis in tumors, resulting in an antitumor effect.—Miyoshi, T., Hirohata, S., Ogawa, H., Doi, M., Obika, M., Yonezawa, T., Sado, Y., Kusachi, S., Kyo, S., Kondo, S., Shiratori, Y., Hudson, B. G., Ninomiya, Y. Tumor‐specific expression of the RGD‐α3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice. FASEB J. 20, 1264–1275 (2006)

Tumor growth inhibitory effect of ADAMTS1 is accompanied by the inhibition of tumor angiogenesis.

Angiogenesis plays an important role in tumor progression. Several reports have demonstrated that a disintegrin and metalloproteinase with thrombospondin motifs1 (ADAMTS1) inhibited angiogenesis via multiple mechanisms. The aim of this study was to investigate the effect of ADAMTS1 on endothelial cells in vitro and on tumor growth with regard to angiogenesis in vivo. We examined the effects of the transfection of ADAMTS1 using two constructs, full‐length ADAMTS1 (full ADAMTS1) and catalytic domain‐deleted ADAMTS1 (delta ADAMTS1). Transfection of both the full ADAMTS1 and delta ADAMTS1 gene constructs demonstrated the secretion of tagged‐ADAMTS1 protein into the conditioned medium, so we examined the effects of ADAMTS1‐containing conditioned medium on endothelial cells. Both types of conditioned media inhibited endothelial tube formation, and this effect was completely abolished after immunoprecipitation of the secreted protein from the medium. Both types of conditioned media also inhibited endothelial cell migration and proliferation. We then examined the impact of ADAMTS1 on endothelial cell apoptosis. Both conditioned media increased the number of Annexin V‐positive endothelial cells and caspase‐3 activity and this effect was attenuated when z‐vad was added. These results indicated that ADAMTS1 induced endothelial cell apoptosis. We next examined the effects of ADAMTS1 gene transfer into tumor‐bearing mice. Both full ADAMTS1 and delta ADAMTS1 significantly inhibited the subcutaneous tumor growth. Collectively, our results demonstrated that ADAMTS1 gene transfer inhibited angiogenesis in vitro and in vivo, likely as a result of the induction of endothelial cell apoptosis by ADAMTS1 that occurs independent of the protease activity.

ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy.

ADAMTS-4 and Biglycan are Expressed at High Levels and Co-Localize to Podosomes During Endothelial Cell Tubulogenesis In Vitro

Proteolysis of the extracellular matrix influences vascular growth. We examined the expression of ADAMTS-1, -4, and -5 metalloproteinases and their proteoglycan substrates versican, decorin, and biglycan as human umbilical vein endothelial cells (HUVECs) formed tubes within type I collagen gels in vitro. Tubulogenic and control HUVEC cultures expressed low levels of ADAMTS-1 and -5 mRNAs, but ADAMTS-4 mRNA was relatively abundant and was significantly elevated (as was ADAMTS-4 protein) in tubulogenic cultures versus controls. Immunocytochemistry revealed ADAMTS-4 in f-actin- and cortactin-positive podosome-like puncta in single cells and mature tubes. Tubulogenic and control cultures expressed low levels of versican and decorin mRNAs; however, peak levels of biglycan mRNA were 400- and 16,000-fold that of versican and decorin, respectively. Biglycan mRNA was highest at 3 hr, declined steadily through day 7 and, at 12 hr and beyond, was significantly lower in tubulogenic cultures than in controls. Western blots of extracellular matrix from tubulogenic cultures contained bands corresponding to biglycan and its cleavage products. By immunocytochemistry, biglycan was found in the pericellular matrix surrounding endothelial tubes and in cell-associated puncta that co-localized with ADAMTS-4 and cortactin. Collectively, our results suggest that ADAMTS-4 and its substrate biglycan are involved in tubulogenesis by endothelial cells.

Circulating white blood cell count correlates with left ventricular indices independently of the extent of risk area for myocardial infarction after successful reperfusion.

Objective — To test the hypothesis that the circulating white blood cell (WBC) and neutrophil counts are related to left ventricular (LV) indices in patients with the same risk area for acute myocardial infarction (AMI), we examined 100 consecutive AMI patients who had the culprit lesion at segment 6 according to the American Heart Association classification and who underwent successful direct coronary angioplasty. Methods and results — The LV ejection fraction (LVEF), end-systolic volume (LVESVI) and end-diastolic volume index (LVEDVI) were obtained by left ventriculography performed 4 weeks after AMI onset. Univariate analysis disclosed that the counts of WBC and neutrophils on admission, and the maximal WBC count correlated negatively with LVEF (r = –0.46, p < 0.001; r = –0.54, p < 0.001 and r = –0.40, p < 0.001, respectively) and positively with LVESVI (r = 0.43, p < 0.001; r = 0.55, p < 0.001, and r = 0.30, p < 0.01, respectively). The counts of WBC and neutrophils on admission also correlated with LVEDVI (r = 0.28, p < 0.01 and r = 0.41, p < 0.001, respectively). Multivariate analysis with other clinical and angiographic factors revealed that the counts of WBC and neutrophils on admission correlated with LVEF (partial correlation coefficient, r = –0.37, p < 0.001 and r = –0.52, p < 0.001, respectively), with LVESVI (r = 0.34, p < 0.01 and r = 0.56, p < 0.001, respectively) and with LVEDVI (r = 0.28, p < 0.01 and r = 0.44, p < 0.001, respectively). The maximal WBC count also correlated with LVEF and LVESVI (r = –0.40, p < 0.001 and r = 0.21, p < 0.05, respectively). Conclusion — The present study revealed that the circulating WBC count correlated with function and volume of the successfully reperfused LV after AMI in patients with the same risk area for AMI, indicating that the WBC count needs to be taken into consideration as an independent factor affecting the LV indices.