Biomedical subjects

Y Maeshima

Publications and source records attributed to Y Maeshima.

At least 19 recordsLinked to original sources

PubMed2001

Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis.

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.

Alkylation↗

PubMed2001

Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin.

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.

Angiogenesis Inhibitors↗

PubMed2001

Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation.

Type IV collagen is a major component of basement membranes and it provides structural and functional support to various cell types. Type IV collagen exists in a highly complex suprastructure form and recent studies implicate that protomer (the trimeric building unit of type IV collagen) assembly is mediated by the NC1 domain present in the C-terminus of each collagen alpha-chain polypeptide. Here we show that type IV collagen contributes to the maintenance of the epithelial phenotype of proximal tubular epithelial cells, whereas type I collagen promotes epithelial-to-mesenchymal transdifferentiation (EMT). In addition, the recombinant human alpha1NC1 domain inhibits assembly of type IV collagen NC1 hexamers and potentially disrupts the deposition of type IV collagen, facilitating EMT in vitro. Inhibition of type IV collagen assembly by the alpha1NC1 domain up-regulates the production of transforming growth factor-beta1 in proximal tubular epithelial cells, an inducer of EMT. These results strongly suggest that basement membrane architecture is pivotal for the maintenance of epithelial phenotype and that changes in basement membrane architecture potentially lead to up-regulation of transforming growth factor-beta1, which contributes to EMT during renal fibrosis.

Animals↗

PubMed2001

Methylprednisolone accelerates the resolution of glomerulonephritis by sensitizing mesangial cells to apoptosis.

Glucocorticoid has long been used to treat patients with glomerulonephritis because it ameliorates mesangial cell proliferation and proteinuria, in part by suppressing nuclear factor-kappa B (NF-kappaB) activation, which regulates the transcription of various pro-inflammatory genes. Recent evidence shows that NF-kappaB activation increases the resistance to TNF-alpha-induced apoptosis in mesangial cells. We examined glomerular cell proliferation and apoptosis along with NF-kappaB activation in the Thy-1.1 nephritis model. We also evaluated TNF-alpha-induced apoptosis in cultured mesangial cells. Methylprednisolone treatment ameliorated mesangial hypercellularity in Thy-1.1 nephritis by decreasing proliferating cells and increasing apoptosis in the glomeruli. These effects were associated with suppressed NF-kappaB activation. This in vitro study revealed that treatment with methylprednisolone and TNF-alpha induced cultured mesangial cell apoptosis. These results suggest that methylprednisolone may accelerate the resolution phase of Thy-1.1 nephritis in part by sensitizing mesangial cells to apoptosis.

Animals↗

PubMed2000

Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties.

Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin.

Angiogenesis Inhibitors↗

PubMed2000

Distinct antitumor properties of a type IV collagen domain derived from basement membrane.

Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.

Angiogenesis Inhibitors↗

PubMed2000

Anti-angiogenic cues from vascular basement membrane collagen.

Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes, which expose cryptic protein modules for interaction with the vascular endothelium. We speculate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important antiangiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of the alpha1 chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neovascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Additionally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the alpha1beta1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.

Animals↗

PubMed2000

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth.

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.

Angiogenesis Inhibitors↗

PubMed1998

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.

Animals↗

PubMed1998

Regulation of survival and death of mesangial cells by extracellular matrix.

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.

Apoptosis↗

PubMed1998

Expression of interleukin-8 in human glomerulonephritis.

Interleukin-8 (IL-8) is a cytokine, which possesses both chemotactic and activating properties for neutrophils, lymphocytes and basophils. Various evidence has indicated IL-8 to be implicated in the pathophysiology of immune-mediated renal diseases. We thus examined the expression of IL-8 in renal diseases. We detected the expression of IL-8 both in mRNA and the protein levels in renal biopsy specimens obtained from patients with IgA nephropathy and lupus nephritis. A significant correlation was found between the expression of IL-8 mRNA and the number of neutrophils in the glomerulus. We also found a negative correlation between the expression of IL-8 mRNA and creatinine clearance. Our study thus suggested IL-8 to be involved in the pathophysiology of proliferative glomerulonephritis.

Adolescent↗

PubMed1997

Inhibition of rheumatoid synovial fibroblast proliferation by antisense oligonucleotides targeting proliferating cell nuclear antigen messenger RNA.

OBJECTIVE: To evaluate the feasibility of antisense oligonucleotides as therapeutic agents to inhibit synovial cell growth in rheumatoid arthritis (RA). METHODS: Fibroblast-like cells established from RA synovium were stimulated with interleukin-1beta (IL-1beta) and treated with antisense or sense oligonucleotides targeting proliferating cell nuclear antigen (PCNA) messenger RNA (mRNA). Proliferation of these cells was determined by 3H-thymidine incorporation. Effects of antisense oligonucleotides on the expression of mRNA and protein were evaluated by reverse transcriptase-polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Antisense oligonucleotides targeting PCNA inhibited IL-1-stimulated fibroblast proliferation, whereas sense oligonucleotides had no effect. Both mRNA and protein levels of PCNA were suppressed in the cells treated with antisense oligonucleotides, indicating that the antiproliferative effect was occurring through an antisense mechanism. CONCLUSION: These results suggest that antisense strategies designed to suppress PCNA expression have potential use as therapeutic agents for RA.

Arthritis, Rheumatoid↗

PubMed1996

Antisense oligonucleotides to proliferating cell nuclear antigen and Ki-67 inhibit human mesangial cell proliferation.

Proliferating cell nuclear antigen (PCNA) and Ki-67 are cell cycle-associated nuclear proteins and are used as markers for proliferating cells. This study attempted to inhibit glomerular mesangial cell (MC) proliferation, which is the hallmark of many forms of glomerular disease, by inhibiting these nuclear proteins with antisense oligodeoxynucleotides. The antisense and sense phosphorothioate oligodeoxynucleotides complementary to PCNA and Ki-67 mRNA, including the initiation codon, were synthesized. Human MC were cultured in growth medium in the presence of sense or antisense oligodeoxynucleotides, and the effects of these oligodeoxynucleotides on mesangial cell proliferation were evaluated by direct cell count. Both PCNA and Ki-67 antisense oligodeoxynucleotides significantly inhibited mesangial cell proliferation as compared with sense oligodeoxynucleotides. Antisense oligodeoxynucleotides (10 microM) for PCNA and Ki-67 inhibited mesangial cell growth by greater than 50%. The effect of antisense oligodeoxynucleotides on target protein expression was examined by immunocytochemistry using specific monoclonal antibodies. Reverse transcription-polymerase chain reaction also was performed to evaluate the effect of antisense oligodeoxynucleotides on PCNA and Ki-67 mRNA expression. Studies of target protein and mRNA expression revealed that the inhibitory effects of the antisense oligonucleotides were mediated through decreases in the expression of both mRNA and protein. Sense oligodeoxynucleotides produced little effect. These results indicate that antisense oligodeoxynucleotides targeting PCNA and Ki-67 mRNA reduce the expression of these gene products and inhibit mesangial cell proliferation. Moreover, these results suggest the feasibility of antisense strategies designed to inhibit PCNA and Ki-67 expression for the inhibition of mesangial cell proliferation in vivo.

Cell Division↗

PubMed1996

Phenotypic modulation of the mesangium reflected by contractile proteins in diabetes.

The phenotypic change of the mesangial cell is considered to play a pivotal role in the accumulation of extracellular matrix in diabetic nephropathy. This investigation was undertaken to evaluate the expression of the various isoforms of contractile proteins in the streptozocin (STZ)-induced diabetic rat kidney and in renal biopsy specimens from patients with diabetic nephropathy. Specific antibodies to myosin heavy chain isoforms (SM1, SM2, SMemb), caldesmon, and alpha-smooth muscle actin and cDNAs for SMemb were used. Increased expression of SMemb at the mRNA and protein levels was demonstrated at 1 week after STZ administration in the rat. Both levels were increased at 4 weeks. Mesangial staining of caldesmon was observed at 4 weeks and that of alpha-smooth muscle actin at 24 weeks. Immunohistochemical mesangial staining of the contractile proteins was pronounced in patients with diabetic nephropathy in contrast to the trace mesangial staining in normal control subjects. These results indicate that the phenotypic change in mesangial cells occurs in the early stages of diabetes and that several stages in phenotypic changes may exist. Expression of the contractile protein isoforms, especially SMemb, should serve as a new marker for the subsequent glomerular hypertrophy and sclerosis.

Actins↗

PubMed1996

A patient with sarcoidosis associated with recurrent urolithiasis and tubular injury caused by calcium deposition.

A 38-year-old woman was hospitalized in January 1994 with renal dysfunction and hypercalcemia. Before admission, she was diagnosed as having urolithiasis, and had been treated twice with extracorporeal shock wave lithotripsy (ESWL). Ophthalmologically, she exhibited iritis and secondary glaucoma. Hypercalcemia, an extremely low titer of parathyroid hormone (PTH), and elevation of angiotensin-converting enzyme (ACE) and lysozyme activity were noted. These findings suggested sarcoidosis, although the chest X-ray showed only fibrotic changes. Hypercalcemia was suspected of having been caused secondarily by sarcoidosis. Since her laboratory data also showed renal dysfunction and abnormal urinalysis, a renal biopsy was performed. The histological findings indicated a tubular and interstitial disorder without glomerular abnormality; calcium deposition, which was detected by X-ray energy dispersive analysis, was observed in the tubular cytoplasm. Administration of prednisolone alleviated the renal dysfunction and decreased the elevation of ACE activity and lysozyme level of the blood. Sarcoidosis is sometimes associated with hypercalcemia, but rarely with renal dysfunction. These findings suggested that sarcoidosis may be associated with renal dysfunction due to tubular injury caused by calcium deposition in the tubules, and that glucocorticoid therapy was effective for these disorders.

Adult↗

PubMed1975

A new antibiotic, fumaramidmycin I. Production, biological properties and characterization of producer strain.

A new antibiotic, fumaramidmycin, has been isolated from a streptomycete NR-7GG1 which was characterized and named Streptomyces kurssanovii. The strain produced the antibiotic only when grown on agar plates but not in the submerged culture broth, where the contact with the vegetative mycelia appears to cause the inactivation of the antibiotic. The antibiotic shows an antimicrobial activity against both Gram-positive and Gram-negative bacteria.

Animals↗

PubMedDate not supplied

Antisense oligonucleotides.

Antisense technology was developed to inhibit gene expression by utilizing an oligonucleotide complementary to the mRNA which encodes the target gene. There are a few possible mechanisms for the inhibitory effects of antisense oligonucleotides. Among them, degradation of mRNA by RNase H is considered to be the major mechanism of action for antisense oligonucleotides. This technique was originally used to elucidate the function of a target gene, but may also have therapeutic applications, provided it is designed carefully and properly.

Animals↗

PubMedDate not supplied

Caldesmon isoform associated with phenotypic modulation of mesangial cells.

Caldesmon (CaD) is a major calmodulin- and actin-binding protein distributed in smooth muscle cells (SMC) and nonmuscle cells. There are at least two high-molecular-weight CaD (h-CaD) isoforms and four low-molecular-weight CaD (l-CaD) isoforms produced by alternative splicing. Isoformal interconversion is associated with phenotypic modulations of vascular SMC. We investigated the CaD isoform in human and rat glomerular mesangial cells (MC) to characterize the phenotypic changes of MC involved in glomerular diseases. A Western blot analysis and reverse-transcription analysis using exon-specific primers revealed that one l-CaD isoform lacking exons 1, 3b and 4 was predominantly expressed in human cultured MC. The expression of this isoform was markedly enhanced in anti-Thy1.1 nephritis rats and streptozotocin-induced diabetic rats, while little expression was observed in the normal glomerulus. Isoformal interconversion did not occur during the phenotypic changes of MC. These data suggested that the activated MC resembled dedifferentiated SMC in terms of the CaD expression pattern, and that CaD is a useful marker of the phenotypic modulations of MC.

Animals↗