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Mitsutaka Kadota,
Misako Sato,
Beverly Duncan, Akira Ooshima,
Howard H Yang,
Natacha Diaz-Meyer,
Sheryl Gere,
Shun-Ichiro Kageyama,
Junya Fukuoka,
Takuya Nagata,
Kazuhiro Tsukada,
Barbara K Dunn,
Lalage M Wakefield,
Maxwell P Lee
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ABSTRACT: To identify genetic events that characterize cancer progression, we conducted a comprehensive genetic evaluation of 161 primary breast tumors. Similar to the "mountain-and-hill" view of mutations, gene amplification also shows high- and low-frequency alterations in breast cancers. The frequently amplified genes include the well-known oncogenes ERBB2, FGFR1, MYC, CCND1, and PIK3CA, whereas other known oncogenes that are amplified, although less frequently, include CCND2, EGFR, FGFR2, and NOTCH3. More importantly, by honing in on minimally amplified regions containing three or fewer genes, we identified six new amplified genes: POLD3, IRAK4, IRX2, TBL1XR1, ASPH, and BRD4. We found that both the IRX2 and TBL1XR1 proteins showed higher expression in the malignant cell lines MCF10CA1h and MCF10CA1a than in their precursor, MCF10A, a normal immortalized mammary epithelial cell line. To study oncogenic roles of TBL1XR1, we performed knockdown experiments using a short hairpin RNA approach and found that depletion of TBL1XR1 in MCF10CA1h cells resulted in reduction of cell migration and invasion as well as suppression of tumorigenesis in mouse xenografts. Intriguingly, our mutation analysis showed the presence of activation mutations in the PIK3CA gene in a subset of tumors that also had DNA copy number increases in the PIK3CA locus, suggesting an additive effect of coexisting activating amino acid substitution and dosage increase from amplification. Our gene amplification and somatic mutation analysis of breast primary tumors provides a coherent picture of genetic events, both corroborating and novel, offering insight into the genetic underpinnings of breast cancer progression.
Cancer Research 09/2009; 69(18):7357-65. · 7.86 Impact Factor
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ABSTRACT: Purpose. Keratinization of the ocular surface epithelium is associated with various disorders impairing vision. We immunohistochemically determined whether the ocular surface epithelia express involucrin, and whether its expression pattern may differ in benign vs. malignant disorders. Expression of cytokeratins was also examined to provide further information relative to the epithelial differentiation. Methods. We evaluated 17 specimens; 6 specimens of the normal ocular surface epithelia, 3 specimens from cases of conjunctival intraepithelial neoplasia (CIN), 6 of conjunctival squamous cell carcinoma (SCC) and 2 of conjunctivae from cases of superior limbic keratoconjunctivitis (SLK). Results. Corneal epithelium exhibited intracellular immunoreactivity for involucrin. Four of the 6 specimens of bulbar conjunctival epithelium showed involucrin immunoreactivity in the perimembranous region, whereas the fornical conjunctiva was negative. Cornified envelope in SLK specimens was positive for involucrin. The CIN showed its immunoreactivity in the perimembranous region in all levels of the hyperproliferative epithelium without keratinization, i.e., similar to the bulbar conjunctiva. The neoplastic cells of well-differentiated SCC showed involucrin in the perimembranous region, and those of moderately- to poorly-differentiated SCC have involucrin in their cytoplasm. The expression pattern of cytokeratins was unrelated to grade of malignancy in ocular SCC. Conclusion. The epithelia of normal subjects and of CIN expresses involucrin without keratinization. In contrary, the keratinized SLK epithelium markedly expresses involucrin in the cornified envelope. The subcellular immunolocalization of involucrin in the ocular SCC may help in evaluating the differentiation, i.e., malignancy, of neoplastic cells.
07/2009; 21(5):877-885.
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ABSTRACT: PURPOSE. We investigated the effects of OPC-15161 on the growth of cultured human Tenon's capsule fibroblasts (TCFs), as well as on the production of type I procollagen, fibronectin, and laminin. These effects were examined in the presence or absence of TGF-ß1. METHODS. Cell proliferation was assayed by counting cell number and assay of DNA synthesis. Cytotoxicity was determined by the MTT method. Matrix components were assayed by enzyme immunoassay of material in the medium and in the cell lysate with or without OPC-15161. Total protein content was determined. Cellular ultrastructure was also evaluated. RESULTS. Treatment with OPC-15161 (up to 100.0 µg ml -1) significantly reduced the proliferation and DNA synthesis of TCFs. No significant decrease in MTT values was observed in confluent TCF cultures with OPC-15161 (up to 100.0 µg ml -1) . TGF-ß1 enhanced the TCF production of procollagen I and fibronectin. OPC-15161 significantly decreased the procollagen I content in both the medium, in the cell lysate of TGF-ß1-stimulated cells, and fibronectin content in the lysate. OPC-15161 did not affect the laminin or total protein content, either with or without TGF-ß1. No ultrastructural evidence of cytotoxicity was observed. CONCLUSIONS. OPC-15161 inhibited the proliferation of TCFs, and reduced their production of procollagen I and fibronectin in the presence of TGF-ß1 without evidence of cytotoxicity. OPC-15161 may be useful in inhibiting the excessive fibrosis produced in the wound in response to filtering surgery.
07/2009; 17(9):933-940.
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ABSTRACT: Purpose. We previously reported that extracellular matrix (ECM) accumulation in human capsular opacification included collagen types I, III, IV, V, and VI. To further characterize the ECM in capsular opacification we performed immunohistochemistry to localize collagen types XII and XIV (fibril-associated collagens with interrupted triple helices, or FACITs) in specimens of human capsular opacification and in cultures of bovine lens epithelial cells (LECs). Methods. Cryosections and paraffin sections of human capsular opacification specimens or uninjured lens capsules, as well as cultured bovine LECs, were processed for immunohistochemistry using antibodies against collagen types I to VI, XII, and XIV. A rat crystalline lens was punctured through the central cornea and the eye was processed for immunohistochemistry for FACITs after healing intervals. Results. In the absence of injury human LECs were unstained for FACITs, but as early as 10 days after operation, LECs in healing capsules were immunoreactive. Collagen types I, III, IV, V, and VI were also detected. ECM deposited in confluent LEC cultures stained for FACITs. Normal rat LECs were not stained for FACITs, but ECM accumulated in injured lens stained for them. Conclusions. LECs up-regulate FACITs during post-opera-tive healing. FACITs, as well as other collagen types, are deposited in ECM in healing injured rat lens, in human capsular opacification and in LEC cultures. ECM components may regulate LEC behavior during postoperative healing.
07/2009; 23(6):463-468.
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ABSTRACT: Introduction Smad-3, a key cytoplasmic mediator of transforming growth factor-β (TGF-β) signalling, mediates many of its inflammatory and fibrotic effects in vivo (Roberts et al. 2001). Smad-3 null mice are protected against cutaneous injury induced by ionizing irradiation (Flanders et al. 2002). Here, we report on our continuing studies on radioprotection as well as protection against tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in Smad-3 null mice.Methods For radioprotection studies, the flank skin of Smad-3+/+ wild-type (WT) and Smad-3–/– knockout (KO) mice was exposed to 30 Gy of localized -irradiation and analysed for histology and gene expression at various times post irradiation. In the UUO model, the right proximal ureter of WT and KO mice was ligated, and 1–2 weeks later kidneys were analysed for inflammation, fibrosis and gene expression.Results Six weeks after exposure to irradiation, skin from KO mice shows less epidermal acanthosis and influx of mast cells, macrophages and neutrophils than skin of WT mice. Paradoxically, at 6–8 h post irradiation, KO skin shows a significantly greater number of neutrophils. Irradiated KO skin also exhibits less immunoreactive TGF-β, fewer myofibroblasts and less scarring than does WT. Smad-3 null dermal fibroblasts do not respond to the chemotactic effects of TGF-β and show less induction of fibrogenic cytokines when treated with irradiation plus TGF-β compared to WT cells. Following UUO, normal kidney architecture is preserved in KO mice, while kidneys from WT mice are enlarged with an influx of mononuclear cells and increased expression of collagen and TGF-β1. Additionally, renal tubules in obstructed kidneys of KO mice remain positive for E-cadherin without expression of -smooth muscle actin, while the opposite expression pattern is seen in obstructed kidneys of WT mice. TGF-β treatment of primary cultures of WT renal tubular epithelial cells results in a phenotypic change from a cobblestone pattern to a spindle-shaped fibroblastic appearance, while KO cells treated with TGF-β maintain their original appearance.Conclusion Smad-3 plays an important role in mediating pathogenic inflammation and fibrosis in several model systems and is also essential for TGF-β1-induced epithelial–mesenchymal transition in renal tubular epithelial cells. Inhibitors of the Smad-3 pathway may have clinical applications in the treatment of a number of fibrotic conditions.
International Journal of Experimental Pathology 06/2008; 85(1):A13 - A13. · 2.57 Impact Factor
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Hiroki Suemoto,
Yasuteru Muragaki,
Katsuhiro Nishioka,
Misako Sato, Akira Ooshima,
Shunji Itoh,
Ikuji Hatamura,
Michitaka Ozaki,
Attila Braun,
Erika Gustafsson,
Reinhard Fässler
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ABSTRACT: Mutations in the TRPS1 gene lead to the tricho-rhino-phalangeal syndrome, which is characterized by skeletal defects and abnormal hair development. The TRPS1 gene encodes an atypical member of the GATA-type family of transcription factors. Here we show that mice with a disrupted Trps1 gene develop a chondrodysplasia characterized by diminished chondrocyte proliferation and decreased apoptosis in growth plates. Our analyses revealed that Trps1 is a repressor of Stat3 expression, which in turn controls chondrocyte proliferation and survival by regulating the expression of cyclin D1 and Bcl2. Our conclusion is supported (i) by siRNA-mediated depletion of Stat3 in Trps1-deficient chondrocytes, which normalized the expression of cyclin D1 and Bcl2, (ii) by overexpression of Trps1 in ATDC5 chondrocytes, which diminished Stat3 levels and increased proliferation and apoptosis, and (iii) by mutational analysis of the GATA-binding sites in the Stat3 gene, which revealed that their integrity is critical for the direct association with Trps1 and for Trps1-mediated repression of Stat3. Altogether our findings identify Trps1 as a novel regulator of chondrocytes proliferation and survival through the control of Stat3 expression.
Developmental Biology 01/2008; 312(2):572-81. · 4.07 Impact Factor
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Binwu Tang,
Naomi Yoo,
Mary Vu,
Mizuko Mamura,
Jeong-Seok Nam, Akira Ooshima,
Zhijun Du,
Pierre-Yves Desprez,
Miriam R Anver,
Aleksandra M Michalowska,
Joanna Shih,
W Tony Parks,
Lalage M Wakefield
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ABSTRACT: The transforming growth factor-beta (TGF-beta) pathway has tumor-suppressor activity in many epithelial tissues. Because TGF-beta is a potent inhibitor of epithelial cell proliferation, it has been widely assumed that this property underlies the tumor-suppressor effect. Here, we have used a xenograft model of breast cancer to show that endogenous TGF-beta has the potential to suppress tumorigenesis through a novel mechanism, involving effects at two distinct levels in the hierarchy of cellular progeny that make up the epithelial component of the tumor. First, TGF-beta reduces the size of the putative cancer stem or early progenitor cell population, and second it promotes differentiation of a more committed, but highly proliferative, progenitor cell population to an intrinsically less proliferative state. We further show that reduced expression of the type II TGF-beta receptor correlates with loss of luminal differentiation in a clinical breast cancer cohort, suggesting that this mechanism may be clinically relevant. At a molecular level, the induction of differentiation by TGF-beta involves down-regulation of Id1, and forced overexpression of Id1 can promote tumorigenesis despite persistence of the antiproliferative effect of TGF-beta. These data suggest new roles for the TGF-beta pathway in regulating tumor cell dynamics that are independent of direct effects on proliferation.
Cancer Research 10/2007; 67(18):8643-52. · 7.86 Impact Factor
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ABSTRACT: The Fas-Fas ligand interaction is the most important pathway in starting apoptosis. In addition, several recent reports have emerged documenting non-apoptotic roles for Fas. However, a non-apoptotic role of Fas in dermal fibroblasts remains unknown. The present study investigated whether Fas stimulation not only promotes apoptosis but also stimulates elements of the inflammatory response such as angiogenesis and macrophage infiltration. Fas stimulation was performed by treating cultured human dermal fibroblasts with an agonistic anti-Fas monoclonal antibody (mAb). Anti-Fas mAb-treated fibroblasts showed a significantly greater increase of caspase-3 and caspase-8 activity compared with control fibroblasts. Addition of the anti-Fas mAb induced DNA fragmentation, as confirmed by the DNA ladder assay. Terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining showed that treatment with the anti-Fas mAb induced an increase of apoptotic fibroblasts in a time-dependent manner. At both mRNA and protein levels, anti-Fas mAb-treated fibroblasts showed significantly higher expression of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein (MCP)-1 compared with control fibroblasts. A pan-caspase inhibitor (Z-VAD-FMK) significantly inhibited VEGF and MCP-1 expression. After transplantation of fibroblasts into mice with severe combined immunodeficiency, the nodules derived from anti-Fas mAb-treated fibroblasts showed more abundant neovascularization, increased macrophage infiltration, and more apoptotic cells in comparison with nodules derived from control fibroblasts. The results of both in vitro and in vivo studies confirmed significantly higher angiogenic activity and macrophage chemotactic activity of anti-Fas mAb-treated fibroblasts compared with control fibroblasts.
The Journal of Dermatology 03/2007; 34(2):99-109. · 1.49 Impact Factor
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ABSTRACT: To investigate the possible involvement of collagen in the characteristic structure and function of human corpora lutea (CL), type V collagen expression was determined in the CL tissues during the menstrual cycle and early pregnancy.
In vitro experiment.
Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama, Japan.
Regulatory cycling women and pregnant women with ovarian tumor and ectopic pregnancy who underwent adnexectomy.
Composition of the various types of collagen in human CL was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Expression of type V collagen.
The ratios of type III to type I collagen and the ratios of type V to type I collagen in the CL tissues were significantly increased in early pregnancy compared with those in the menstrual cycle.
These results suggest that alterations in composition of collagen might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.
Fertility and sterility 02/2007; 87(1):178-81. · 3.97 Impact Factor
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Shizuya Saika,
Kazuo Ikeda,
Osamu Yamanaka,
Kathleen C Flanders,
Yuka Okada,
Takeshi Miyamoto,
Ai Kitano, Akira Ooshima,
Yuji Nakajima,
Yoshitaka Ohnishi,
Winston W-Y Kao
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ABSTRACT: Animal cornea is an avascular transparent tissue that is suitable for research on wound healing-related scarring and neovascularization. Here we show that loss of tumor necrosis factor alpha (TNFalpha) potentiates the undesirable, pathogenic response of wound healing in an alkali-burned cornea in mice. Excessive invasion of macrophages and subsequent formation of a vascularized scar tissue were much more marked in TNFalpha-null knockout (KO) mice than in wild-type mice. Such an unfavorable outcome in KO mice was abolished by Smad7 gene introduction, indicating the involvement of transforming growth factor beta or activin/Smad signaling. Bone marrow transplantation from wild-type mice normalized healing of the KO mice, suggesting the involvement of bone marrow-derived inflammatory cells in this phenomenon. Co-culture experiments showed that loss of TNFalpha in macrophages, but not in fibroblasts, augmented the fibroblast activation as determined by detection of alpha-smooth muscle actin, the hallmark of myofibroblast generation, mRNA expression of collagen Ialpha2 and connective tissue growth factor, and detection of collagen protein. TNFalpha in macrophages may be required to suppress undesirable excessive inflammation and scarring, both of which are promoted by transforming growth factor beta, and for restoration of tissue architecture in a healing alkali-burned cornea in mice.
American Journal Of Pathology 07/2006; 168(6):1848-60. · 4.89 Impact Factor
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ABSTRACT: We examined the role of interleukin-7 (IL-7) in modulation of production of extracellular matrix (ECM), immunolocalization of Smads, and cell migration and expressions of transforming growth factor-beta (TGF-beta) in cultured human subconjunctival fibroblasts. IL-7 is capable of inducing Smad7, an inhibitory Smad that interferes with TGF-beta/Smad signal.
The effects of IL-7 on ECM production, immunolocalization of Smads, type I collagen, fibronectin, alpha -smooth muscle actin (alpha -SMA), and cell migration were examined in human subconjunctival fibroblast culture with or without TGF-beta1. ECM production, such as type I collagen and fibronectin, was measured by immunoassay or real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cell migration was examined using an in vitro wound model in monolayer cultures. We also examined the effects of IL-7, PKC inhibitor, and STAT inhibitor on the expressions of TGF-beta1 and type I collagen alpha1 chain (col1A1) m-RNA by using real-time RT-PCR.
IL-7 reduced the ECM production much more markedly in the cells treated with TGF-1beta than in the control fibroblasts. TGF-beta1 strongly showed immunolocalization of phospho-Smad2, and IL-7 also showed immunolocalization of Smad7 in the nuclei. The immunoreactivities of alpha -SMA and fibronectin were weaker in the presence of IL-7 than in the control cells. IL-7 also delayed defect closure in the monolayer cell sheets, and the delay was recovered by exogenous type I collagen or fibronectin. Each of IL-7, BIS I, or AGS 490 reduced the mRNA expressions of TGF-beta1 and col1A1.
These findings indicate that IL-7 is involved in ECM production in the subconjunctival fibroblasts activated by exogenous TGF-beta1, suggesting that administration of IL-7 can be a novel therapeutic strategy in preventing undesirable bleb scar formation during healing after filtration surgery.
Current Eye Research 07/2006; 31(6):491-9. · 1.28 Impact Factor
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Masahide Mizobuchi,
Hiroaki Ogata,
Ikuji Hatamura,
Fumihiko Koiwa,
Fumie Saji,
Kazuhiro Shiizaki,
Shigeo Negi,
Eriko Kinugasa, Akira Ooshima,
Shozo Koshikawa,
Tadao Akizawa
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ABSTRACT: Cardiovascular disease is the most frequent cause of death in patients with end-stage kidney disease (ESKD). Vascular calcification is a confirmed risk factor for cardiovascular events in the general population and has a high occurrence in patients with ESKD. Despite the high prevalence of vascular calcification in ESKD, the pathogenesis of the disorder is still obscure. The present study examined the expressions of bone-associated factors in calcified arteries in subtotally nephrectomized rats with severe secondary hyperparathyroidism (SHPT).
Seven-week-old male Sprague-Dawley rats were divided into five groups as follows: sham-operated rats that received a normal diet [0.8% of phosphorus (P), 1.1% of calcium (Ca)] (Sham), sham-operated rats that received a high-phosphorus and low-calcium (HPLCa) diet (1.2% P, 0.4% Ca) (Sham+HPLCa), 5/6 nephrectomized rats that received a normal diet as the uraemic control group (Nx), and 5/6 nephrectomized rats that received a HPLCa diet to induce the development of SHPT (Nx+HPLCa), and 5/6 nephrectomized and parathyroidectomized rats that received a HPLCa diet (Nx+PTx+HPLCa). The feeding period of each group was 10 weeks. The rats were then sacrificed and their serum was examined. The upper part of the abdominal aorta was used to investigate the expression of mRNAs of core-binding factor alpha-1 (Cbfa1) and sodium-dependent phosphate cotransporter (Pit-1) by real-time reverse transcriptase polymerase chain reaction (real-time PCR) analysis. The lower part was examined for calcification by von Kossa staining.
Serum P level and Ca x P products increased significantly in the Nx+HPLCa group compared with those of any other groups. Severe hyperparathyroidism was also observed in the Nx+HPLCa group. Vascular calcification (medial layer) was observed in the Nx+HPLCa group only. There was a significant increase in Cbfa1 and Pit-1 mRNA expression levels in the aorta of the Nx+HPLCa group compared with that of any other groups.
These results suggest that medial layer vascular calcification in uraemic rats with severe hyperphosphataemia and SHPT may be caused in part by Cbfa1 and Pit-1.
Nephrology Dialysis Transplantation 05/2006; 21(4):911-6. · 3.40 Impact Factor
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ABSTRACT: In a cirrhotic liver, the regenerative ability and specific functions are impaired; a hepatic resection increases the possibility of postoperative liver failure. Hepatocyte growth factor (HGF) stimulates liver regeneration, accelerates restoration of hepatic function, and improves fibrosis. A truncated type II transforming growth factor-beta receptor (TbetaTR), which specifically inhibits TGF-beta signaling as a dominant-negative receptor, appears to prevent the progression of liver fibrosis. We demonstrated the therapeutic efficacy of adenovirus-mediated HGF and TbetaTR gene transduction after partial hepatectomy for liver cirrhosis.
Rats were treated with dimethylnitrosamine for 3 weeks, and they all had severe cirrhosis. After partial hepatectomy (10%), we injected adenovirus expressing bacterial beta-galactosidase (AdLacZ), adenovirus expressing a truncated type II TGF-beta receptor (AdTbetaTR), adenovirus expressing hepatocyte growth factor (AdHGF), or AdTbetaTR + AdHGF into the portal vein, which was followed by an additional 2-week dimethylnitrosamine treatment.
On histologic examination, fibrotic tissue had decreased in the livers of the AdTbetaTR + AdHGF-treated rats compared with rats that were treated by AdLacZ, AdTbetaTR alone, and AdHGF alone. Liver function, which included serum levels of alanine aminotransferase, improved significantly in AdTbetaTR + AdHGF-treated rats compared with all other groups. The number of hepatocytes that were positive for proliferating-cell nuclear antigen was greater (P < .05) in AdHGF alone and AdTbetaTR + AdHGF-treated rat livers than in AdLacZ- and AdTbetaTR-treated rats. All AdTbetaTR + AdHGF-treated rats survived >60 days, and AdTbetaTR + AdHGF treatment markedly improved the survival rate after a partial hepatectomy.
Our results suggest that the combination of HGF and TbetaTR gene therapy may increase the possibility of hepatectomy in a cirrhotic liver by improving fibrosis, hepatic function, and hepatocyte regeneration.
Surgery 05/2006; 139(4):563-73. · 3.10 Impact Factor
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ABSTRACT: To investigate the characteristic structure and function of human corpora lutea (CL), various types of collagen expression were determined in the CL tissues during the menstrual cycle and early pregnancy.
In vitro experiment.
Department of obstetrics and gynecology at a medical university.
Regulatory cycling women and pregnant women with ovarian tumor and ectopic pregnancy who underwent adnexectomy.
Immunohistochemistry for human type I, III, and IV collagen with specific monoclonal antibodies was used for analysis.
Expression of type I, III, and IV collagen.
Immunohistochemical staining for type I and III collagen revealed intense staining of the CL stroma during early pregnancy, as compared with those in the menstrual cycle. Moreover, pericellular intense immunostaining for type IV collagen was observed around the luteal cells, especially luteal granulosa cells, of early pregnancy.
These results suggest that alterations in distribution of collagen might play an important role in determining the physiology and structure of the CL during the menstrual cycle and early pregnancy.
Fertility and sterility 05/2006; 85 Suppl 1:1093-6. · 3.97 Impact Factor
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ABSTRACT: Smad7 is a molecule that blocks the Smad2/3 signal. Herein, we examined the effects of Smad7 gene introduction on post-injury conjunctival wound healing in mice. Its effects on the cultured human subconjunctival fibroblasts (SCFs) were also investigated.
A circumferential incision was made in the equatorial conjunctiva by using scissors in the right eye of fully anesthetized adult C57BL/6 mice (n=72). Smad7 cDNA-expressing adenoviral vector was topically applied. The control eye received nonfunctioning adenoviral vector. After 2, 5, 7, and 30 days the eyes were processed for histological or immunohistochemical examination to evaluate wound healing of conjunctiva. The expressions of type-I collagen and growth factors were evaluated by real time-reverse transcriptase-polymerase chain reaction. The effects of Smad7 gene introduction on the cultured human SCFs were also studied.
Marked Smad7 protein expression was detected in the vector-treated conjunctival epithelium and fibroblasts that coincided with green fluorescein protein expression, whereas faint endogenous Smad7 expression was observed in the control tissue. In vivo Smad7 gene introduction blocked Smad2/3 nuclear translocation with suppression of alpha-smooth muscle actin (alphaSMA) and vascular endothelial growth factor (VEGF) in fibroblasts and invasion of macrophages. Smad7 gene transfer suppressed mRNA expressions of connective tissue growth factor (CTGF), VEGF, and monocyte chemoattractant protein-1 in vivo and those of type-I collagen, alphaSMA, and CTGF in vitro.
Smad7 gene transfer modulates injury-induced wound healing of conjunctival tissue in mice, suggesting that this strategy may be effective in preventing excessive scarring following filtration surgery. The mechanism might include suppression of activation of fibroblasts and reduction of macrophage invasion.
Molecular vision 02/2006; 12:841-51. · 2.20 Impact Factor
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ABSTRACT: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model.
Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in lens epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model.
Two days post-burn of the ocular surface, lens epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type lens cells. The majority of myofibroblast-type lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the lens epithelium.
Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the lens epithelium in this cataract model.
Molecular vision 02/2006; 12:681-91. · 2.20 Impact Factor
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ABSTRACT: We have examined the effect of adenovirus-mediated expression of bone morphogenic protein-7 (BMP-7) and inhibitors of differentiation 2 and 3 (Id2 and Id3) on injury-induced epithelial-to-mesenchymal transition (EMT) of lens epithelium in mice. Id2 and Id3 are known to be upregulated by BMP-7 and to antagonize Smad2/3 signaling. The Cre-LoxP system adenoviral gene transfer was used. Three microliters of adenoviral solution (2 x 10(7) PFU/mul) were injected into the right lens of adult male C57BL/6 mice (n = 144) at the time of capsular injury induced using a hypodermic needle under both general and topical anesthesia. A mixture of Cre-adenovirus (Cre-Ad) and vector encoding mBMP-7, mId2, or mId3 was administered in a test group. Control lenses were treated with Cre-Ad alone. After healing intervals of 5 or 10 days, the animals were killed and then we performed histological processes or RNA extraction from the lens. RT-PCR, real-time RT-PCR, and immunohistochemistry showed expression of each introduced gene in the lens. Exogenous BMP-7 upregulated expression of Id2 and Id3 in injured lenses, and gene introduction of Id2 or Id3 also upregulated BMP-7 expression. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of the lens epithelial cells as evaluated by histology and expression patterns of alpha-smooth muscle actin and collagens in association with reduction of Smad2 COOH-terminal phosphorylation. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of lens epithelial cells and subsequent sealing of the capsular break with fibrous tissue in mice.
AJP Cell Physiology 02/2006; 290(1):C282-9. · 3.54 Impact Factor
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ABSTRACT: Keloids are tumor-like lesions that result from excessive scar formation during healing of wounds. Histologically, keloids show an increased blood vessel density compared with normal dermis or normal scars. However, the angiogenic activity of keloid fibroblasts remains unknown. In this study, we investigated angiogenic activity of keloid fibroblasts. Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF) were investigated as elements of the angiogenic factors. Expressions of TGF-beta1 and VEGF in conditioned medium were measured with enzyme-linked immunosorbent assay (EIA) and Northern blot analysis. Participation of TGF-beta1 in the production of VEGF was also investigated with addition of TGF-beta1 and a neutralizing anti-TGF-beta1 antibody. A modified Boyden chamber assay was performed to assess the chemotactic activity of vascular endothelial cells. Angiogenic activity in vivo was evaluated by neovascularization of nodules formed by implantation of fibroblasts into severe combined immunodeficiency (SCID) mice. EIA showed that the concentrations of TGF-beta1 and VEGF in conditioned medium were increased 2.5- and 6-fold, respectively, after the culture of keloid fibroblasts compared with normal fibroblasts. Northern blot analysis revealed that the expression of TGF-beta1 and VEGF mRNA was upregulated 3.6- and 6-fold, respectively, in keloid fibroblasts compared with normal fibroblasts. Addition of TGF-beta1 to keloid fibroblast cultures increased VEGF production by 3.5-fold, while there was a 6-fold in culture of normal fibroblasts. A neutralizing anti-TGF-beta1 antibody reduced VEGF secretion to control levels, suggesting that TGF-beta1 mediated the upregulation of VEGF expression. A modified Boyden chamber assay demonstrated that the chemotactic activity of vascular endothelial cells was more strongly (sevenfold) induced by keloid fibroblast-conditioned medium than by normal fibroblast-conditioned medium. Anti-VEGF antibody inhibited chemotaxis to basal levels. When SCID mice underwent implantation of fibroblasts into the back, the nodules formed by keloid fibroblasts were three times larger than those formed by normal fibroblasts. Although abundant neovascularization was observed in keloid fibroblast nodules, neovascularization was scarce in normal fibroblast nodules. Both in vitro and in vivo studies confirmed the significantly higher angiogenic activity of keloid fibroblasts compared with normal fibroblasts, and TGF-beta1 and VEGF were clearly shown to be involved. These results suggest that angiogenesis in keloids is promoted by endogenous TGF-beta1 and VEGF.
Archives for Dermatological Research 11/2005; 297(4):161-9. · 2.28 Impact Factor
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Shizuya Saika,
Osamu Yamanaka,
Kazuo Ikeda,
Shokei Kim-Mitsuyama,
Kathleen C Flanders,
Jiyun Yoo,
Anita B Roberts,
Iku Nishikawa-Ishida,
Yoshitaka Ohnishi,
Yasuteru Muragaki, Akira Ooshima
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ABSTRACT: Proliferative vitreoretinopathy (PVR) is one of the major causes of the failure of retinal detachment surgery. Its pathogenesis includes a fibrotic reaction by the retinal pigment epithelium and other retina-derived non-neural cells, leading to fixation of the detached retina. We examined the role of p38 mitogen-activated protein kinase (MAPK) in transforming growth factor (TGF)-beta2-dependent enhancement of the fibrogenic reaction in a human retinal pigment epithelial cell line, ARPE-19, and also evaluated the therapeutic efficacy of inhibiting p38MAPK by adenoviral gene transfer of dominant-negative (DN) p38MAPK in a mouse model of PVR. Exogenous TGF-beta2 activates p38MAPK in ARPE-19 cells. It also suppresses cell proliferation, but this was unaffected by addition of the p38MAPK inhibitor, SB202190. SB202190 interfered with TGF-beta2-dependent cell migration and production of collagen type I and fibronectin, but had no effect on basal levels of these activities. While SB202190 did not affect phosphorylation of the C-terminus of Smads2/3, it did suppress the transcriptional activity of Smads3/4 as indicated by a reporter gene, CAGA12-Luc. Gene transfer of DN-p38MAPK attenuated the post-retinal detachment fibrotic reaction of the retinal pigment epithelium in vivo in mice, supporting its effectiveness in preventing/treating PVR.
Laboratory Investigation 08/2005; 85(7):838-50. · 3.64 Impact Factor
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Shizuya Saika,
Takeshi Miyamoto,
Osamu Yamanaka,
Tadashi Kato,
Yoshitaka Ohnishi,
Kathleen C Flanders,
Kazuo Ikeda,
Yuji Nakajima,
Winston W-Y Kao,
Misako Sato,
Yasuteru Muragaki, Akira Ooshima
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ABSTRACT: We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.
American Journal Of Pathology 06/2005; 166(5):1393-403. · 4.89 Impact Factor
