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Hidetoshi Ito

Publications and source records attributed to Hidetoshi Ito.

10 recordsLinked to original sources
PubMed2006

Testing the utility of rationally engineered recombinant collagen-like proteins for applications in tissue engineering.

Collagens are attractive proteins as materials for tissue engineering. Over the last decade, significant progress has been made in developing technologies for large-scale production of native-like human recombinant collagens. Yet, the rational design of customized collagen-like proteins for smart biomaterials to enhance the quality of engineered tissues has not been explored. We mapped the D4 domain of human collagen II as most critical for supporting migration of chondrocytes and used this information to genetically engineer a collagen-like protein consisting of tandem repeats of the D4 domain (mD4 collagen). This novel collagen has been utilized to fabricate a scaffold for support of chondrocytes. We determined superior qualities of cartilaginous constructs created by chondrocytes cultured in scaffolds containing the mD4 collagen in comparison to those formed by chondrocytes cultured in bare scaffolds or those coated with wild-type collagen II. Our results are a first attempt to rationally engineer collagen-like proteins with characteristics tailored for specific needs of cartilage engineering and provide a basis for rational engineering of similar proteins for a variety of biomedical applications.

Cartilage↗
PubMed2006

Therapeutic strategies for secondary hyperparathyroidism in dialysis patients.

Secondary hyperparathyroidism (SHPT) leads not only to bone disorders, but also to cardiovascular complications in long-term dialysis patients. Conventional treatment with calcium (Ca) supplement, phosphate (P) binders and active vitamin D analogs lead in part to amelioration of SHPT, but are simultaneously associated with unacceptable side-effects, including hypercalcemia, hyperphosphatemia, and increased Ca x P products, which are the risk factors for cardiovascular disease in dialysis patients. Conventional treatment has been unable to facilitate the attainment of optimal management of SHPT proposed in the K/DOQI guidelines. Cinacalcet HCl (cinacalcet), a novel calcimimetic compound, restores the sensitivity of the Ca-sensing receptor in parathyroid cells, and decreases serum parathyroid hormone (PTH) without introducing hypercalcemia or hyperphosphatemia. Cinacalcet treatment enables a significant number of patients to achieve the K/DOQI guideline. Based on experimental data, calcimimetics could ameliorate cardiovascular calcification and remodeling in uremic rats with SHPT. Clinical trials have shown that cinacalcet significantly reduced the risks of parathyroidectomy, fracture and cardiovascular hospitalization among long-term dialysis patients with SHPT. Parathyroid intervention therapy (parathyroidectomy and percutaneous direct injection) is also a useful alternative. In the present article, we review novel therapeutic strategies for SHPT.

Calcium↗
PubMed2005

Guilty by association: some collagen II mutants alter the formation of ECM as a result of atypical interaction with fibronectin.

Among the structural components of extracellular matrices (ECM) fibrillar collagens play a critical role, and single amino acid substitutions in these proteins lead to pathological changes in tissues in which they are expressed. Employing a biologically relevant experimental model consisting of cells expressing R75C, R519C, R789C, and G853E procollagen II mutants, we found that the R789C mutation causing a decrease in the thermostability of collagen not only alters individual collagen molecules and collagen fibrils, but also has a negative impact on fibronectin. We propose that thermolabile collagen molecules are able to bind to fibronectin, thereby altering intracellular and extracellular processes in which fibronectin takes part, and we postulate that such an atypical interaction could change the architecture of the ECM of affected tissues in patients harboring mutations in genes encoding fibrillar collagens.

Cell Line, Tumor↗
PubMed2004

Single amino acid substitutions in procollagen VII affect early stages of assembly of anchoring fibrils.

Procollagen VII is a homotrimer of 350-kDa pro-alpha1(VII) chains, each consisting of a central collagenous domain flanked by the noncollagenous N-terminal NC1 domain and the C-terminal NC2 domain. After secretion from cells, procollagen VII molecules form anti-parallel dimers with a C-terminal 60-nm overlap. Characteristic alignment of procollagen VII monomers forming a dimer depends on site-specific binding between the NC2 domain and the triple-helical region adjacent to Cys-2634 of the interacting procollagen VII molecules. Formation of the intermolecular disulfide bonds between Cys-2634 and either Cys-2802 or Cys-2804 is promoted by the cleavage of the NC2 domain by procollagen C-proteinase. By employing recombinant procollagen VII variants harboring G2575R, R2622Q, or G2623C substitutions previously disclosed in patients with dystrophic epidermolysis bullosa, we studied how these amino acid substitutions affect intermolecular interactions. Binding assays utilizing an optical biosensor demonstrated that the G2575R substitution increased affinity between mutant molecules. In contrast, homotypic binding between the R2622Q or G2623C molecules was not detected. In addition, kinetics of heterotypic binding of all analyzed mutants to wild type collagen VII were different from those for binding between wild type molecules. Moreover, solid-state binding assays demonstrated that R2622Q and G2623C substitutions prevent formation of stable assemblies of procollagen C-proteinase-processed mutants. These results indicate that single amino acid substitutions in procollagen VII alter its self-assembly and provide a basis for understanding the pathomechanisms leading from mutations in the COL7A1 gene to fragility of the dermal-epidermal junction seen in patients with dystrophic forms of epidermolysis bullosa.

Amino Acid Substitution↗
PubMed2004

Thermostability gradient in the collagen triple helix reveals its multi-domain structure.

A triple-helical conformation and stability at physiological temperature are critical for the mechanical and biological functions of the fibril-forming collagens. Here, we characterized the role of consecutive domains of collagen II in stabilizing the triple helix. Analysis of melting temperatures of genetically engineered collagen-like proteins consisting of tandem repeats of the D1, D2, D3 or D4 collagen II periods revealed the presence of a gradient of thermostability along the collagen molecule with thermolabile N-terminal domains and thermostable C-terminal domains. These results imply a multi-domain character of the collagen triple helix. Assays of thermostabilities of the Arg75Cys and Arg789Cys collagen II mutants suggest that, in contrast to the thermostable domains, the thermolabile domains are able to accommodate amino acid substitutions without altering the thermostability of the entire collagen molecule.

Blotting, Western↗
PubMed2004

Inhibition of cyclooxygenase-2 suppresses invasiveness of oral squamous cell carcinoma cell lines via down-regulation of matrix metalloproteinase-2 and CD44.

Expression of cyclooxygenase-2 (COX-2) in tumors is known to be associated with enhanced angiogenesis, suppression of host immunity, and tumor invasion. In the present study, human oral squamous cell carcinoma (OSCC) cell lines NA and HSC-4 were used to evaluate the effects of NS-398, a selective inhibitor of COX-2, and COX-2 antisense oligonucleotide (COX-2 AS) on the invasion activity of OSCC cells. Matrigel invasion assay revealed that the invasiveness of NA and HSC-4 was suppressed by treatment with either NS-398 or COX-2 AS. These reagents down-regulated the secretion of matrix metalloproteinase-2 (MMP-2) to culture supernatant as well as the expression of MMP-2 mRNA and protein. Membrane-type 1 matrix metalloproteinase (MT1-MMP), an activator of proMMP-2, was also down-regulated by treatment with these reagents. Furthermore, expression of CD44 on the surface of these cells was reduced by treatment with either NS-398 or COX-2 AS. In addition, MMP-2 antisense oligonucleotides reduced the expression of CD44 on the surface of both OSCC cell lines. These findings suggest that NS-398 and COX-2 AS suppress the invasiveness of OSCC cells via down-regulation of MMP-2 and CD44. Genetic or pharmacological inhibition of COX-2 may therefore be a beneficial strategy in the treatment of OSCC patients.

Carcinoma, Squamous Cell↗
PubMed2004

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils.

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.

Binding Sites↗
PubMed2004

[Pathogenesis of secondary hyperparathyroidism and renal bone disease].

Patients with chronic renal disease (CKD)almost always develop secondary hyperparathyroidism (SHPT) due to hypocalcemia, phosphate retention, and abnormalities in vitamin D (VD) metabolism. Concomitant decreases in VD receptor and calcium sensing receptor in the parathyroid glands render them more resistant to the action of VD and calcium, and accelerate parathyroid cell growth. Several types of bone diseases are known to occur in CKD patients. Excessive secretion of parathyroid hormone (PTH) due to SHPT causes high-turnover bone disease, called osteitis fibrosa. Among low-turnover bone disease (LTBD), osteomalacia which is characterized by calcification defect is often complicated with VD deficiency and/or aluminum accumulation. Recently, frequency of adynamic bone disease caused by PTH suppression, another type of LTBD, is increasing probably due to calcium salts as phosphate binder with or without VD treatment.

Aluminum↗
PubMedDate not supplied

Comparison of the expression profile of metastasis-associated genes between primary and circulating cancer cells in oral squamous cell carcinoma.

Tumor metastasis is a complex process involving several distinct steps such as escape from a primary tumor, dissemination through the circulation, lodgment in small vessels at distinct sites, penetration of the vessel wall and growth in the new site as a secondary tumor. To compare the expression profile of metastasis-associated genes between circulating cancer cells in peripheral blood and cells in the primary lesion of oral squamous cell carcinoma (OSCC), we employed a combination analysis of laser captured microdissection (LCM) and immunomagnetic separation (IMS) techniques for capturing primary and circulating cancer cells, respectively. Total RNAs were then extracted from each cell and mRNA expression of CK19, matrix metalloproteinases (MMP-1, -2, -7, -9) and CD44, including its variant forms (CD44s, v6, v9), were analyzed by RT-PCR. Although CD44 including its variant forms were expressed in 20%(CD44s) to 30%(v6, v9) of the primary lesion, 40%(v6) to 90%(CD44s) of blood samples were CD44-positive. Furthermore, MMPs were expressed in 30%(MMP-1, -2) to 60%(MMP-7) of primary samples, whereas most blood samples were negative for the expression of MMPs. These results suggested that circulating cancer cells might express different characteristics after being released from the primary lesion.

Aged↗