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Kazuo Tsubota

Publications and source records attributed to Kazuo Tsubota.

At least 19 recordsLinked to original sources
PubMed2007

Functional analysis of an established mouse vascular endothelial cell line.

BACKGROUND: In vitrostudies using cell lines are useful for the understanding of cellular mechanisms. The purpose of our study is to develop a new immortalized aortic vascular endothelial cell (EC) line that retains endothelial characteristics and can facilitate the study of ECs. METHODS: A mouse aortic vascular EC line (MAEC) was established from p53-deficient mouse aorta and cultured for over 100 passages. The expression of endothelial markers was assessed, and the function of this cell line was analyzed by tube formation and binding assays. RESULTS: MAEC retained many endothelial properties such as cobblestone appearance, contact-inhibited growth, active uptake of acetylated low-density lipoprotein, existence of Weibel-Palade bodies and several EC markers. MAECs exhibited tube formation activity both in vitro and in vivo. Furthermore, crucially, tumor necrosis factor alpha, an inflammatory cytokine, promoted lymphocyte adhesion to MAECs, suggesting that MAECs may facilitate the study of atherosclerosis and local inflammatory reactions in vitro. CONCLUSION: We describe the morphological and cell biological characteristics of MAEC, providing strong evidence that it retained endothelial properties. This novel cell line can be a useful tool for studying the biology of ECs.

Animals↗
PubMed2007

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium.

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.

3T3 Cells↗
PubMed2007

Role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization.

PURPOSE: Recently, it was revealed that the inhibition of nonproteolytic activation of prorenin led to significant suppression of ocular inflammation in endotoxin-induced uveitis. The purpose of the present study was to investigate whether nonproteolytically activated prorenin plays a role in ischemia-induced retinal neovascularization. METHODS: C57BL/6 neonatal mice were reared in an 80% concentration of oxygen from postnatal (P) day 7 to P12, followed by room-air breathing to P17 to induce ischemia-initiated retinal neovascularization. Tissue localization of activated prorenin and prorenin receptor was examined by immunohistochemistry. Animals received intraperitoneal injections of handle-region peptide (HRP), a decoy peptide corresponding to the handle region of prorenin, which inhibits prorenin receptor-mediated upregulation of the renin-angiotensin system (RAS). A concanavalin A lectin perfusion-labeling technique was used to evaluate the areas of physiologic and pathologic retinal new vessels and the number of leukocytes adhering to the vasculature. Retinal mRNA expression and protein levels of intercellular adhesion molecule (ICAM)-1, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by RT-PCR and ELISA. RESULTS: Retinal vessels in ischemic retinopathy eyes were positive for activated prorenin and prorenin receptor. Pathologic, but not physiologic, retinal neovascularization was significantly attenuated in HRP-treated mice compared with vehicle- or control peptide-treated animals. The number of adherent leukocytes was also significantly reduced. Retinal mRNA expression and protein levels of ICAM-1, VEGF, VEGFR-1, and VEGFR-2 in ischemic retinopathy were also significantly suppressed by the application of HRP. CONCLUSIONS: The present findings suggest that nonproteolytic activation of prorenin selectively promotes pathologic, but not physiologic, retinal neovascularization through the inflammatory processes related to pathologic neovascularization.

Animals↗
PubMed2006

SOCS3 is required to temporally fine-tune photoreceptor cell differentiation.

Suppressor of cytokine signaling 3 (SOCS3) is an intracellular, ligand-induced negative feedback modulator of STAT3 activation that acts during inflammation. Here, we demonstrate that SOCS3 expression is important for normal retinal development in the perinatal period. STAT3 is highly activated in the late-embryonic retina, then downregulated at postnatal day 0 (P0), presumably by the depletion of upstream ligands. We found that SOCS3 was required after P0 to shut down the residual STAT3 activation; this loss of activated STAT3 leads to Rhodopsin expression and rod photoreceptor cell differentiation. SOCS3 deficiency failed to terminate STAT3 activation, thereby delaying expression of Rhodopsin and its upstream transcription factor, crx. Development subsequently continued, but its course was temporally erratic, probably because of faulty compensation. Interestingly, SOCS3 protein expression was first detected postnatally, after STAT3 activation was mostly downregulated. It initially appeared in some of the presumptive photoreceptor cells and gradually spread. SOCS3 mRNA level was constant from the late-embryonic to early-postnatal period. Post-transcriptional inhibition of SOCS3 protein expression maintains a high STAT3 activation during late embryogenesis, and after P0, releasing from the inhibition promptly terminates STAT3 activation. Thus, SOCS3 can act as a temporal fine-tuner of STAT3 activation during photoreceptor cell differentiation.

Animals↗
PubMed2006

Functional visual acuity in Stevens-Johnson syndrome.

PURPOSE: To evaluate the correlation of functional visual acuity (FVA) measurement with ocular surface findings in patients with Stevens-Johnson syndrome (SJS). DESIGN: Prospective comparative study. METHODS: Sixty-nine eyes of 38 patients with chronic SJS assessed at the Tokyo Dental College, Tokyo Medical Center, and the Kyoto Prefectural University of Medicine, Department of Ophthalmology, Kyoto, Japan, were studied. Twenty eyes of 10 normal subjects and 40 eyes of 20 patients with Sjögren syndrome (SS) were also studied. Conventional Landolt visual acuity (VA) and FVA examinations and slit-lamp examinations were performed. FVA was measured continuously by the FVA measurement system during a 30-second blink-free period in one eye. The visual maintenance ratio (VMR) was calculated as follows: VMR = [(2.7 - FVA)/(2.7 - baseline VA)], where logarithm of minimal angle of resolution values of FVA were entered into the formula and 2.7 represented the lowest visual acuity in this series. Slit-lamp examinations, Schirmer test, and fluorescein vital stainings were also performed in all subjects. RESULTS: VMR was markedly lower in patients with SJS compared with patients with SS and controls. FVA values showed a relation with the presence of corneal opacity and vascularization. CONCLUSIONS: The FVA measurement system is not only a useful tool in the evaluation of dynamic VA changes, but also reflects the ocular surface clinical findings in SJS.

Adult↗
PubMed2006

Angiotensin II type 1 receptor-mediated inflammation is required for choroidal neovascularization.

BACKGROUND: Choroidal neovascularization (CNV) is a critical pathogenesis in age-related macular degeneration, the most common cause of blindness in the developed countries. The aim of the current study was to determine the involvement of the renin-angiotensin system (RAS) with the development of CNV, using human surgical samples and the murine model of laser-induced CNV. METHODS AND RESULTS: In the human and murine CNV tissues, the vascular endothelium expressed angiotensin II type 1 receptor (AT1-R), AT2-R, and angiotensin II. The CNV volume was significantly suppressed by treatment with an AT1-R blocker telmisartan, but not with an AT2-R blocker. AT1-R signaling blockade with telmisartan inhibited various inflammatory mechanisms including macrophage infiltration and upregulation of VEGF, intercellular adhesion molecule-1 (ICAM-1), MCP-1, and IL-6 in the retinal pigment epithelium-choroid complex. A PPAR-gamma antagonist partially but significantly reversed the suppressive effect of telmisartan on in vivo induction of CNV and in vitro upregulation of ICAM-1 and MCP-1 in endothelial cells and IL-6 in macrophages, showing the dual contribution of PPAR-gamma-agonistic and AT1-R-antagonistic actions in the telmisartan treatment. CONCLUSIONS: AT1-R-mediated inflammation plays a pivotal role in the development of CNV, indicating the possibility of AT1-R blockade as a novel therapeutic strategy to inhibit CNV.

Angiogenesis Inducing Agents↗
PubMed2006

Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea.

We report the presence of neural crest-derived corneal precursors (COPs) that initiate spheres by clonal expansion from a single cell. COPs expressed the stem cell markers nestin, Notch1, Musashi-1, and ABCG2 and showed the side population cell phenotype. COPs were multipotent with the ability to differentiate into adipocytes, chondrocytes, as well as neural cells, as shown by the expression of beta-III-tubulin, glial fibrillary acidic protein, and neurofilament-M. COP spheres prepared from E/nestin-enhanced green fluorescent protein (EGFP) mice showed induction of EGFP expression that was not originally observed in the cornea, indicating activation of the neural-specific nestin second intronic enhancer in culture. COPs were Sca-1(+), CD34(+), CD45(-), and c-kit(-). Numerous GFP(+) cells were observed in the corneas of mice transplanted with whole bone marrow of transgenic mice ubiquitously expressing GFP; however, no GFP(+) COP spheres were initiated from these mice. On the other hand, COP spheres from transgenic mice encoding P0-Cre/Floxed-EGFP as well as Wnt1-Cre/Floxed-EGFP were GFP(+), indicating the neural crest origin of COPs, which was confirmed by the expression of the embryonic neural crest markers Twist, Snail, Slug, and Sox9. Taken together, these data indicate the existence of neural crest-derived, multipotent stem cells in the adult cornea.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: a model of age-related macular degeneration.

Oxidative stress has long been linked to the pathogenesis of neurodegenerative diseases; however, whether it is a cause or merely a consequence of the degenerative process is still unknown. We show that mice deficient in Cu, Zn-superoxide dismutase (SOD1) have features typical of age-related macular degeneration in humans. Investigations of senescent Sod1(-/-) mice of different ages showed that the older animals had drusen, thickened Bruch's membrane, and choroidal neovascularization. The number of drusen increased with age, and exposure of young Sod1(-/-) mice to excess light induced drusen. The retinal pigment epithelial cells of Sod1(-/-) mice showed oxidative damage, and their beta-catenin-mediated cellular integrity was disrupted, suggesting that oxidative stress may affect the junctional proteins necessary for the barrier integrity of the retinal pigment epithelium. These observations strongly suggest that oxidative stress may play a causative role in age-related retinal degeneration, and our findings provide evidence for the free radical theory of aging. In addition, these results demonstrate that the Sod1(-/-) mouse is a valuable animal model to study human age-related macular degeneration.

Animals↗
PubMed2006

Polymorphism of beta-adrenergic receptors and susceptibility to open-angle glaucoma.

PURPOSE: The human trabecular meshwork and ciliary body, which express beta-adrenergic receptors (ADRB1 and ADRB2), control aqueous humor dynamics. We investigated associations of ADRB polymorphisms with open-angle glaucoma (OAG), because ADRB gene polymorphisms alter receptor function. METHODS: We studied 240 Japanese controls and 505 Japanese OAG patients including 211 with primary open-angle glaucoma (POAG), and 294 with normal-tension glaucoma (NTG). Associations of four polymorphisms (Ser49Gly and Arg389Gly in the ADRB1 gene; Arg16Gly and Gln27Glu in the ADRB2 gene) were compared between patients and controls. Age, intraocular pressure (IOP), and visual field defects at diagnosis were examined for associations with polymorphisms. RESULTS: The Arg389Gly polymorphism in the ADRB1 gene showed significantly different allele and genotype frequencies in patients with NTG than in controls (p = 0.004 and 0.006, respectively). Other polymorphisms did not show a significant frequency difference. In POAG patients, carriers of Gly16 in the ADRB2 gene were significantly younger at diagnosis than noncarriers (p<0.001). The IOP at diagnosis was significantly higher in OAG patients carrying 27Glu in the ADRB2 gene than in patients without this allele (p<0.001). Clinical characteristics of OAG patients did not differ significantly in relation to other polymorphisms. CONCLUSIONS: Certain polymorphisms of the ADRB1 and ADRB2 genes influence the pathophysiology of OAG in Japanese patients.

Aged↗
PubMed2006

Proteomic analysis of soluble factors secreted by limbal fibroblasts.

PURPOSE: To identify soluble factors selectively secreted by limbal fibroblasts as possible regulators of limbal basal epithelium. METHODS: Limbal, corneal, and conjunctival fibroblasts were first expanded in vitro in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, and then maintained in serum-free medium for two weeks. Proteomic analysis of culture supernatants was done to compare differences in secreted matricellular proteins. Real time PCR and western blots were done to confirm the expression of secreted protein acid and rich in cysteine (SPARC), a protein found in abundance in extracellular proteins secreted by limbal fibroblasts. Immunohistochemistry of SPARC was done in human limbal tissue to show the spatial distribution of the protein. An adhesion assay was designed to demonstrate the effects of SPARC on an SV40 immortalized human corneal epithelial cell line (HCEC). RESULTS: Proteomic analysis revealed several proteins selectively secreted by limbal fibroblasts. The particular spots were identified as SPARC, vimentin, serine protease, collagen alpha 2 precursor, tissue inhibitor of metalloproteinase 2 (TIMP-2), and 5,10-methlenetetrahdrofolate reductase (FADH2). The expression of SPARC was confirmed by western blot analysis, and mRNA expression was significantly higher in limbal fibroblasts compared to central corneal fibroblasts when analyzed by real time PCR. Immunohistochemistry revealed higher distribution of SPARC in the subepithelial stroma of the limbus compared to the central cornea. The addition of 10 microg/ml murine SPARC in HCEC significantly reduced cell spreading at three h. CONCLUSIONS: The matricellular protein SPARC is preferentially secreted by limbal fibroblasts, and may modulate intercellular adhesion of basal limbal epithelial cells.

Animals↗
PubMed2006

Long-standing bullous keratopathy is associated with peripheral conjunctivalization and limbal deficiency.

OBJECTIVE: To investigate whether peripheral corneal neovascularization in bullous keratopathy (BK) is due to conjunctivalization, a sign of limbal stem cell deficiency. DESIGN: Observational case-control study. PARTICIPANTS: Sixteen BK patients. METHODS: Patients were divided into 2 groups: BK without peripheral neovascularization [NV(-) group; 5 patients, 5 eyes] and BK with neovascularization [NV(+) group; 11 patients, 13 eyes]. Evidence of conjunctivalization was evaluated by periodic acid-Schiff staining of impression cytology samples from the peripheral vascularized cornea. The 2 groups' durations of disease also were compared. Penetrating keratoplasty (PK) was performed in all 16 cases, and the 2 groups' durations of reepithelialization after PK were compared. MAIN OUTCOME MEASURES: Presence of goblet cells using impression cytology, duration of BK, and duration of postoperative reepithelialization. RESULTS: Goblet cells were found on the peripheral corneal surface in all eyes in the NV(+) group. However, all eyes in the NV(-) group were negative for goblet cells (P<0.0001). Duration of disease was 14.4+/-5.4 months in the NV(-) group and 66.2+/-65.5 months in the NV(+) group (P = 0.030). Duration of postoperative epithelialization was 6.2+/-2.2 days in the NV(-) group and 28.8+/-36.5 days in the NV(+) group (P = 0.046). CONCLUSION: Conjunctivalization of the peripheral cornea and delayed postoperative epithelialization in BK patients with NV suggest the presence of limbal stem cell deficiency in such patients. Patients with long-standing disease were found to be more prone to neovascularization. For this reason, early surgery may lead to a better surgical outcome.

Aged↗
PubMed2006

Laboratory findings in tear fluid analysis.

The tear film, composed of the lipid, aqueous and mucin layers, has many functions including defending the ocular surface. The tear film covering the ocular surface presents a mechanical and antimicrobial barrier and ensures an optical refractive surface. The lipid component originates from the meibomian glands of the tarsus and forms the superficial layer of the tear film. The aqueous component contains electrolytes, water, and a large variety of proteins, peptides and glycoproteins, and is primarily secreted by the lacrimal gland. Mucins are glycoproteins expressed by epithelial tissues of mucous surfaces. They protect tissues by functioning as antioxidants, providing lubrication, and inhibiting bacterial adherence. Quantitatively and qualitatively, its composition must be maintained within the fairly narrow limits to maintain a healthy and functional visual system. Abnormalities of the tear film, affecting the constituents or the volume, can rapidly result in serious dysfunction of the eyelids and conjunctiva and ultimately affect the transparency of the cornea. Many ocular surface tests have been developed for the clinical diagnosis of dry eye syndromes. This paper provides an overview on laboratory methods for the analysis of the tear film. Understanding the components of the tear film will aid in the treatment of dry eye syndromes and the ocular surface diseases.

Animals↗
PubMed2006

Dacryoadenitis associated with Acanthamoeba keratitis.

OBJECTIVE: To report the incidence of dacryoadenitis as a symptom associated with Acanthamoeba keratitis. METHODS: We investigated all cases of Acanthamoeba keratitis (20 patients and 21 eyes) diagnosed and treated at Tokyo Dental College, Ichikawa General Hospital, Ichikawa, Japan, between May 1, 1994, and November 30, 2005. We recorded the incidence of dacryoadenitis diagnosed using clinical signs of lacrimal gland swelling and pain on pressure, computed tomography, magnetic resonance imaging, and histopathologic analysis. RESULTS: Eight eyes (38%) of 8 patients had dacryoadenitis simultaneously with Acanthamoeba keratitis. Dacryoadenitis was diagnosed using histopathologic analysis and computed tomography in 1 patient, histopathologic analysis and magnetic resonance imaging in 1, magnetic resonance imaging in 2, and clinical signs alone in 4. Histopathologic examination in 2 patients revealed moderate infiltration of lymphocytes and plasma cells in the lacrimal gland compatible with dacryoadenitis. No Acanthamoeba organisms were found in the lacrimal gland. The standard protocol for Acanthamoeba keratitis was performed without particular treatment of dacryoadenitis in all patients. Lacrimal gland swelling improved after a mean of 10 weeks (range, 4-17 weeks) in conjunction with symptoms of keratitis; however, 1 patient (patient 1) required levator muscle surgery and blepharoplasty for residual ptosis. CONCLUSION: Dacryoadenitis is a clinical finding associated with Acanthamoeba keratitis.

Acanthamoeba Keratitis↗
PubMed2006

Collagen-immobilized poly(vinyl alcohol) as an artificial cornea scaffold that supports a stratified corneal epithelium.

The cornea is a transparent tissue of the eye, which is responsible for the refraction of incoming light. Both biological corneal equivalents and synthetic keratoprostheses have been developed to replace donor tissue as a means to restore vision. However, both designs have drawbacks in terms of stability and biocompatibility. Clinically available synthetic devices do not support an intact epithelium, which poses a risk of microbial infection or protrusion of the prosthesis. In the present study, type I collagen was immobilized onto poly(vinyl alcohol) (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. Human and rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-COL. The epithelial sheet expressed keratin 3/12 differentiation markers, the tight junction protein occludin, and had characteristic microvilli structures on transmission electron microscopy. Functionally, the stratified epithelium contained normal glycogen levels, and an apical tight-junction network was observed to exclude the diffusion of horseradish peroxidase. Furthermore, the epithelium-PVA-COL composite was suturable in the rabbit cornea, suggesting the possibility of using PVA-COL as a biocompatible material for keratoprosthesis.

Animals↗
PubMed2006

Clinical evaluation of the Smart Plug in the treatment of dry eyes.

PURPOSE: To evaluate the efficacy of a thermo sensitive punctum plug, the Smart Plug, in the treatment of dry eyes. DESIGN: Observational case-series study. METHODS: Eighteen eyes of 10 dry eye patients who previously failed conventional plug insertion were enrolled. Schirmer 1 test, vital staining scores, and tear clearance tests were performed before plug insertion, at 2 weeks, 1 month, and 3 months after insertion. RESULTS: Although Schirmer test values were not significantly different before and after plug insertion, the tear clearance rate considerably decreased (Before insertion: 14.8+/-11.4; After insertion: 8.2+/-6.6) (P<.05). Rose-Bengal and fluorescein scores improved significantly after plug insertion (Rose-Bengal: Before insertion: 6.4+/-2.0; After insertion: 3.3+/-1.6 points, Fluorescein: Before insertion: 4.8+/-2.3; After insertion: 2.1+/-1.3) (P<.05). No complications were observed. CONCLUSIONS: Smart Plug proved to be a safe and efficient option in the treatment of dry eye.

Dry Eye Syndromes↗
PubMed2006

Subjective visual sensation during vitrectomy under retrobulbar anesthesia.

PURPOSE: To report a precise illustrated image of patient's view during vitrectomy. DESIGN: Interventional case report. METHODS: A 56-year-old male with cystoid macular edema (CME) associated with epiretinal membrane underwent vitrectomy. The patient seemed to find gratification in being operated on because he was able to see what was going on. He was a professional artist in computer graphics, and he drew three different pictures depending on the surgical procedures. RESULTS: These pictures depicted the moving of pasty, whirling fluid during core vitrectomy, numerous black or gray spots like snowflakes by intravitrealy injected crystals of triamcinolone acetonide, and a sharp tapered instrument coming into the center and membrane-like material being peeled off. CONCLUSIONS: Patients may see a precise shape in a constant size, scale, and detailed movement as well, focused on the retina without described optics. Further investigation will be required to determine this mechanism.

Anesthesia, Local↗
PubMed2006

The impact of the onset time of atopic keratoconjunctivitis on the tear function and ocular surface findings.

PURPOSE: To investigate the tear and ocular surface findings between controls, children, and adults with atopic keratoconjunctivitis (AKC). DESIGN: Prospective comparative study. METHODS: Twenty eyes of 10 childhood-onset, 10 eyes of five adult-onset, AKC adult patients, and 12 eyes of six children with infantile-onset AKC, 14 eyes of seven normal adults and seven normal children were recruited. Corneal aesthesiometry, Schirmer test, tear film break-up time (BUT), vital staining, and conjunctival impression cytology were performed. RESULTS: The Schirmer and Rose Bengal scores in childhood-onset adult AKC patients were considerably worse than adult-onset adult AKC patients, pediatric subjects, and the controls (P < .05). The same significant relation was observed in impression cytology parameters. CONCLUSIONS: Ocular surface disease in childhood-onset adult AKC patients was characterized by greater ocular surface epithelial damage. Prolonged inflammation may be important in the evolution and progression of ocular surface disease in patients with longstanding active AKC.

Adult↗