Takayuki Shiomi

Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis

Vascular endothelial growth factor (VEGF) is a strong angiogenic mitogen and plays important roles in angiogenesis under various pathophysiological conditions. The in vivo angiogenic activity of secreted VEGF may be regulated by extracellular inhibitors, because it is also produced in avascular tissues such as the cartilage. To seek the binding inhibitors against VEGF, we screened the chondrocyte cDNA library by a yeast two‐hybrid system by using VEGF165 as bait and identified connective tissue growth factor (CTGF) as a candidate. The complex formation of VEGF165 with CTGF was first established by immunoprecipitation from the cells overexpressing both binding partners. A competitive affinity‐binding assay also demonstrated that CTGF binds specifically to VEGF165 with two classes of binding sites (Kd = 26 ± 11 nM and 125 ± 38 nM). Binding assay using deletion mutants of CTGF indicated that the thrombospondin type‐1 repeat (TSP‐1) domain of CTGF binds to the exon 7‐coded region of VEGF165 and that the COOH‐terminal domain preserves the affinity to both VEGF165 and VEGF121. The interaction of VEGF165 with CTGF inhibited the binding of VEGF165 to the endothelial cells and the immobilized KDR/IgG Fc; that is, a recombinant protein for VEGF165 receptor. By in vitro tube formation assay of endothelial cells, full‐length CTGF and the deletion mutant possessing the TSP‐1 domain inhibited VEGF165‐induced angiogenesis significantly in the complex form. This antiangiogenic activity of CTGF was demonstrated further by in vivo angiogenesis assay by using Matrigel injection model in mice. These data demonstrate for the first time that VEGF165 binds to CTGF through a protein‐to‐protein interaction and suggest that the angiogenic activity of VEGF165 is regulated negatively by CTGF in the extracellular environment.

MT1-MMP and MMP-7 in invasion and metastasis of human cancers

Previous experimental and biochemical studies on matrix metalloproteinases (MMPs) have indicated that MMPs are implicated in cancer invasion and metastases. Studies on the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in various human cancer tissues have further demonstrated that activation of proMMP-2 mediated by a combination of TIMP-2 and MT1-MMP (the proMMP-2/TIMP-2/MT1-MMP system) correlates well with the progression of most of these cancers such as the breast carcinomas, thyroid papillary carcinomas, gastric adenocarcinomas, oral squamous cell carcinomas and gliomas, whereas MMP-7 plays an important role in the metastases of endometrial and gastrointestinal carcinomas. Although MMP-7 is a typical secreted MMP, a member of transmembrane 4 superfamily (TM4SF) captures proMMP-7 on the carcinoma cell membranes through interaction with its propeptide, leading to its pericellular activation. Thus, these results strongly suggest that proteolysis at the cell-extracellular matrix interfaces of cancer cells by the proMMP-2/TIMP-2/MT1-MMP and proMMP-7/TM4SF systems plays crucial roles in the progression of human cancers. In this article, we address the current views on the roles of these MMPs acting on the cell membranes in human cancer invasion and metastases.

Vascular Endothelial Growth Factor Isoforms and Their Receptors Are Expressed in Human Osteoarthritic Cartilage

To assess the possible involvement of vascular endothelial growth factor (VEGF) in the pathology of osteoarthritic (OA) cartilage, we examined the expression of VEGF isoforms and their receptors in the articular cartilage, and the effects of VEGF on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in OA chondrocytes. Reverse transcriptase-polymerase chain reaction analyses demonstrated that mRNAs for three VEGF isoforms (VEGF(121), VEGF(165), and VEGF(189)) are detectable in all of the OA and normal (NOR) cartilage samples. However, the mRNA expression of their receptors (VEGFR-1 = Flt-1, VEGFR-2 = KDR and neuropilin-1) was recognized only in the OA samples. The protein expression of VEGFR-1 and VEGFR-2 in OA chondrocytes was also demonstrated by immunohistochemistry of the OA cartilage tissue and cultured OA chondrocytes. In situ hybridization and immunohistochemistry indicated that VEGF is expressed in the chondrocytes in the superficial and transitional zones of OA cartilage. A linear correlation was obtained between VEGF immunoreactivity and Mankin scores in the cartilage (r = 0.906, P < 0.001). The production levels of VEGF determined by enzyme-linked immunosorbent assay were significantly 3.3-fold higher in OA than in NOR samples (P < 0.001). Among MMP-1, -2, -3, -7, -8, -9, and -13, TIMP-1 and -2 measured by their sandwich enzyme immunoassay systems, the production of MMP-1 and MMP-3 but not TIMP-1 or TIMP-2 was significantly enhanced by the treatment of cultured OA chondrocytes with VEGF (P < 0.05), whereas no such effect was obtained with cultured NOR chondrocytes. These results demonstrate that VEGF and its receptors are expressed in OA cartilage, and suggest the possibility that VEGF is implicated for the destruction of OA articular cartilage through the increased production of MMPs.

MMP-1 drives immunopathology in human tuberculosis and transgenic mice

Mycobacterium tuberculosis can cause lung tissue damage to spread, but the mechanisms driving this immunopathology are poorly understood. The breakdown of lung matrix involves MMPs, which have a unique ability to degrade fibrillar collagens at neutral pH. To determine whether MMPs play a role in the immunopathology of tuberculosis (TB), we profiled MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), in sputum and bronchoalveolar lavage fluid from patients with TB and symptomatic controls. MMP-1 concentrations were significantly increased in both HIV-negative and HIV-positive patients with TB, while TIMP concentrations were lower in HIV-negative TB patients. In primary human monocytes, M. tuberculosis infection selectively upregulated MMP1 gene expression and secretion, and Ro32-3555, a specific MMP inhibitor, suppressed M. tuberculosis-driven MMP-1 activity. Since the mouse MMP-1 ortholog is not expressed in the lung and mice infected with M. tuberculosis do not develop tissue destruction equivalent to humans, we infected transgenic mice expressing human MMP-1 with M. tuberculosis to investigate whether MMP-1 caused lung immunopathology. In the MMP-1 transgenic mice, M. tuberculosis infection increased MMP-1 expression, resulting in alveolar destruction in lung granulomas and significantly greater collagen breakdown. In summary, MMP-1 may drive tissue destruction in TB and represents a therapeutic target to limit immunopathology.

Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis

The avascularity of cardiac valves is abrogated in several valvular heart diseases (VHDs). This study investigated the molecular mechanisms underlying valvular avascularity and its correlation with VHD. Chondromodulin-I, an antiangiogenic factor isolated from cartilage, is abundantly expressed in cardiac valves. Gene targeting of chondromodulin-I resulted in enhanced Vegf-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography showed aortic valve thickening, calcification and turbulent flow, indicative of early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation and mobilization of endothelial cells and induced their apoptosis; these effects were partially inhibited by chondromodulin-I small interfering RNA. In human VHD, including cases associated with infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in areas of chondromodulin-I downregulation. These findings provide evidence that chondromodulin-I has a pivotal role in maintaining valvular normal function by preventing angiogenesis that may lead to VHD.

ADAM12 is selectively overexpressed in human glioblastomas and is associated with glioblastoma cell proliferation and shedding of heparin-binding epidermal growth factor

ADAMs (a disintegrin and metalloproteinases) are multifunctional molecules involved in cell-cell fusion, cell adhesion, membrane protein shedding, and proteolysis. In the present study, we examined the mRNA expression of 13 different ADAM species with putative metalloproteinase activity in human astrocytic tumors, nonneoplastic brain tissues, and other intracranial tumors by reverse transcriptase-polymerase chain reaction, and found that prototype membrane-anchored ADAM12 (ADAM12m) is predominantly expressed in glioblastomas. Real-time quantitative polymerase chain reaction indicated that the expression level of ADAM12m is remarkably at least 5.7-fold higher in glioblastomas (n = 16) than in nonneoplastic brain tissues (n = 6), low grade (n = 7) and anaplastic astrocytic tumors (n = 9) (P < 0.05 for each group), and intracranial neurinomas (n = 5) (P < 0.01). In situ hybridization showed that glioblastoma cells are responsible for the gene expression. By immunohistochemistry, ADAM12m was predominantly immunolocalized on the cell membranes of glioblastoma cells. Immunoblotting analysis demonstrated that ADAM12m is expressed as an activated N-glycosylated form of approximately 90 kd in glioblastoma tissues. There was a direct correlation between the mRNA expression levels of ADAM12m and proliferative activity (MIB1-positive cell index) of gliomas (r = 0.791, P < 0.0001; n = 32). Protein bands consistent with the soluble form of heparin-binding epidermal growth factor, a substrate of ADAM12m, were observed by immunoblotting in glioblastoma samples with the ADAM12m expression, and inhibited by treatment with ADAM inhibitor of the glioblastomas. These data demonstrate for the first time that among the 13 different ADAM species, ADAM12m is highly expressed in human glioblastomas, and suggest the possibility that ADAM12m plays a role in the prominent proliferation of the glioblastomas through shedding of heparin-binding epidermal growth factor.

Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non‐neoplastic diseases

Cellular functions within tissues are strictly regulated by the tissue microenvironment which comprises extracellular matrix and extracellular matrix‐deposited factors such as growth factors, cytokines and chemokines. These molecules are metabolized by matrix metalloproteinases (MMP), a disintegrin and metalloproteinases (ADAM) and ADAM with thrombospondin motifs (ADAMTS), which are members of the metzincin superfamily. They function in various pathological conditions of both neoplastic and non‐neoplastic diseases by digesting different substrates under the control of tissue inhibitors of metalloproteinases (TIMP) and reversion‐inducing, cysteine‐rich protein with Kazal motifs (RECK). In neoplastic diseases MMP play a central role in cancer cell invasion and metastases, and ADAM are also important to cancer cell proliferation and progression through the metabolism of growth factors and their receptors. Numerous papers have described the involvement of these metalloproteinases in non‐neoplastic diseases in nearly every organ. In contrast to the numerous review articles on their roles in cancer cell proliferation and progression, there are very few articles discussing non‐neoplastic diseases. This review therefore will focus on the properties of MMP, ADAM and ADAMTS and their implications for non‐neoplastic diseases of the cardiovascular system, respiratory system, central nervous system, digestive system, renal system, wound healing and infection, and joints and muscular system.

ADAM28 Is Overexpressed in Human Breast Carcinomas: Implications for Carcinoma Cell Proliferation through Cleavage of Insulin-like Growth Factor Binding Protein-3

A disintegrin and metalloproteinases (ADAMs) are involved in various biological events including cell adhesion, cell fusion, membrane protein shedding, and proteolysis. In the present study, our reverse transcription-PCR analysis showed that among the 12 different ADAM species with a putative metalloproteinase motif, prototype membrane-anchored ADAM28m and secreted-type ADAM28s are selectively expressed in human breast carcinoma tissues. By real-time quantitative PCR, their expression levels were significantly higher in carcinomas than in nonneoplastic breast tissues. In situ hybridization, immunohistochemistry, and immunoblotting analyses indicated that ADAM28 is predominantly expressed in an active form by carcinoma cells within carcinoma tissues. A direct correlation was observed between mRNA expression levels and proliferative activity of the carcinoma cells. Treatment of ADAM28-expressing breast carcinoma cells (MDA-MB231) with insulin-like growth factor-I (IGF-I) increased cell proliferation, cleavage of IGF binding protein (IGFBP)-3, as well as IGF-I cell signaling; these processes were all significantly inhibited by treatment with ADAM inhibitor or anti-ADAM28 antibody. Down-regulation of ADAM28 expression in MDA-MB231 cells with small interfering RNA significantly reduced cell proliferation, IGFBP-3 cleavage, and growth of xenografts in mice. In addition, cleavage of IGFBP-3 in breast carcinoma tissues was correlated with ADAM28 expression levels and inhibited by treatment with ADAM inhibitor or anti-ADAM28 antibody. These results show that ADAM28 is overexpressed in an activated form in human breast carcinoma cells and suggest that ADAM28 is involved in cell proliferation through enhanced bioavailability of IGF-I released from the IGF-I/IGFBP-3 complex by selective IGFBP-3 cleavage in human breast carcinomas.

Expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic cartilage.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, 4, 5, 8, 9 and 15, members of the ADAMTS gene family, have the ability to degrade a major cartilage proteoglycan, aggrecan, at the specific sites, and thus are called ‘aggrecanases’. The expression of these ADAMTS species was examined in human osteoarthritic articular cartilage on reverse transcription–polymerase chain reaction. The results demonstrated the predominant expression of ADAMTS4 in osteoarthritic cartilage, while ADAMTS5 was constitutively expressed in osteoarthritic and normal cartilage. ADAMTS9 was expressed mainly in normal cartilage, whereas no or negligible expression of ADAMTS1, 8 and 15 was observed in either osteoarthritic or normal cartilage. In situ hybridization for ADAMTS4 indicated that chondrocytes in osteoarthritic cartilage expressed the mRNA. Two monoclonal antibodies to ADAMTS4 were developed, and immunolocalized ADAMTS4 to chondrocytes in the proteoglycan‐depleted zones of osteoarthritic cartilage, showing a direct correlation with the Mankin scores. Immunoblotting indicated a major protein band of 58 kDa in the chondrocyte culture media and osteoarthritic cartilage tissue homogenates. These data demonstrate that among the six ADAMTS species, ADAMTS4 is mainly expressed in an active form in osteoarthritic cartilage, and suggest that ADAMTS4 may play an important role in the degradation of aggrecan in human osteoarthritic cartilage.

ADAM28 is overexpressed in human non-small cell lung carcinomas and correlates with cell proliferation and lymph node metastasis

ADAM (a disintegrin and metalloproteinases) are a recently discovered gene family of proteins with sequence similarity to the reprolysin family of snake venom metalloproteinases, and about one‐third of the family members have the catalytic site consensus sequence in their metalloproteinase domains. We screened the mRNA expression of 11 different ADAM species with putative metalloproteinase activity in human non‐small cell lung carcinomas by RT‐PCR, and found that prototype membrane‐anchored ADAM28 (ADAM28m) and secreted ADAM28 (ADAM28s) are predominantly expressed in the carcinoma tissues. Real‐time quantitative PCR demonstrated that the expression levels of ADAM28m and ADAM28s are significantly 16.8‐fold and 9.0‐fold higher in the carcinomas than in the non‐carcinoma tissues, respectively. In addition, the expression levels of ADAM28m and ADAM28s were significantly higher in the carcinomas with >30 mm in diameter than in those ≦30 mm. The expression levels were also significantly higher in the carcinomas with lymph node metastasis than in those without metastasis. MIB1‐positive cell index of the carcinomas had a direct correlation with the expression levels of ADAM28m and ADAM28s (r = 0.667, p < 0.001 and r = 0.535, p < 0.01, respectively). In situ hybridization and immunohistochemistry demonstrated that ADAM28 is expressed predominantly in the carcinoma cells. Immunoblot analysis showed the activated form of ADAM28 in the carcinoma tissues. These data demonstrate for the first time that ADAM28 is overexpressed and activated in human non‐small cell lung carcinomas, and suggest the possibility that ADAM28 plays a role in cell proliferation and progression of the human lung carcinomas.