Masahiro Zako

Chondroitin Sulfate Chains on Syndecan-1 and Syndecan-4 from Normal Murine Mammary Gland Epithelial Cells Are Structurally and Functionally Distinct and Cooperate with Heparan Sulfate Chains to Bind Growth Factors A NOVEL FUNCTION TO CONTROL BINDING OF MIDKINE, PLEIOTROPHIN, AND BASIC FIBROBLAST GROWTH FACTOR

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4. Functional analysis using a BIAcore system showed that basic fibroblast growth factor (bFGF) specifically bound only to the HS chains of both syndecans, whereas midkine (MK) and pleiotrophin (PTN) bound not only to the HS but also to the CS chains. Stronger binding of MK and PTN to the CS chains of syndecan-4 than those of syndecan-1 was revealed, supporting the structural and functional differences. Intriguingly, removal of the CS chains decreased the association and dissociation rate constants of MK, PTN, and bFGF for both syndecans, suggesting the simultaneous binding of these growth factors to both types of chains, producing a ternary complex that transfers the growth factors to the corresponding cell surface receptors more efficiently compared with the HS chains alone. The involvement of the core protein was also shown in the binding of MK and PTN to syndecan-1, suggesting the possibility of cooperation with the HS and/or CS chains in the binding of these growth factors and their delivery to the cell surface receptors.

The levator aponeurosis consists of two layers that include smooth muscle.

Purpose: To investigate the two-fold structure of the levator aponeurosis, which is partly composed of independent smooth muscles. Materials and Methods: Fifteen upper eyelids of 12 Asian postmortems, with age at death ranging from 72 to 91 years, were examined. In 9 eyelids, posterior lamella tissue of the upper eyelid was removed to observe the stratified structures of the levator aponeurosis. Six full-thickness eyelids were used to observe the attachment site or the continuity between the levator aponeurosis and its surrounding tissues. The eyelids were incised perpendicularly in the center of the eyelid; samples were stained with Masson trichrome and antismooth muscle actin antibody and examined microscopically. Results: Masson trichrome staining demonstrated the two-layered nature of the levator aponeurosis. The anterior layer was characterized by thick, robust fibrous tissue, and the posterior by thinner fibrous tissue. Although both layers contained muscle structures, the posterior layer contained more than the anterior. Immunostaining with antismooth muscle actin antibody revealed that the muscle in both layers was smooth muscle. The anterior layer continued to the orbital septum and the submuscular fibroadipose tissue; the posterior layers, located in front of Müller muscle and its tendon, attached to the anterior inferior one-third of the tarsus. Part of the anterior layer went through the orbicularis oculi muscle and attached to the subcuticular tissue. Conclusions: The levator aponeurosis is stratified, consisting of two layers than contain smooth muscle components in their proximal portions. It pulls mainly the preaponeurotic fat and anterior eyelid lamella. This partially regulates the tension of the eyelid and contributes to the ordered movement of the upper eyelid.

Transient Expression of PG-M/Versican, a Large Chondroitin Sulfate Proteoglycan in Developing Chicken Retina

Abstract: We previously showed the expression of PG‐M/versican in embryonic chicken retina. In this study, we characterized the alternatively spliced forms of PG‐M/versican and their developmental regulation to investigate the implication of PG‐M/versican in neurite outgrowth from retinal cells during development. On day 5, the immunolocalization of PG‐M was first observed at the inner surface of neural retina. On day 7, the pronounced staining was observed in the nerve fiber layer and inner plexiform layer where neural networks of ganglion cells were being formed. As the development proceeded, more intensive staining was observed in these layers. The staining peaked on day 14 and then decreased. Northern analysis and western blotting revealed the presence of a single‐sized transcript (13 kb) and the PG‐M/versican core protein (550 kDa) on day 14, but the absence of any transcripts or protein bands on day 20, indicating a transient expression of PG‐M+ (VO), the alternatively spliced form with the most abundant sites for the chondroitin sulfate attachment. Taken together, it is likely that PG‐M/versican is involved in neurite outgrowth from ganglion cells during retinal development, and antiadhesion activity of its chondroitin sulfate chains may be important for regulation.

Microscopic anatomy of Asian lower eyelids.

Purpose: To elucidate the microscopic anatomy of the Asian lower eyelid. Methods: Specimens (full-thickness sections of lower eyelids from 19 postmortem lower eyelids) from 11 Asians aged 73 to 96 years at death were fixed in 10% buffered formalin and microscopically examined. After pretreatment, sagittal sliced sections of the central part were stained with Masson trichrome. Results: The distinct junction of the orbital septum to the capsulopalpebral fascia (CPF) was confirmed in 7 eyelids in which orbital septum was clearly stained, with an average distance from the tarsus to the junction of 2.38 mm. The other 12 eyelids did not show a distinct junction, and the orbital septum was poorly defined anteriorly and indistinct posteriorly. There was a distinct layer between the orbicularis oculi muscle and the orbital septum. The inferior and the posterior attachments of the CPF to the tarsus were seen in all eyelids. Seventeen of the 19 eyelids had attachment of the CPF on the anterior aspect of the tarsus, from which an extension of the CPF through the pretarsal orbicularis oculi muscle was observed. All eyelids had anterior extension of the CPF through the preseptal orbicularis oculi muscle, which was overridden on the pretarsal orbicularis oculi muscle. Conclusions: The microscopic findings of Asian lower eyelids, especially fascial components, were mostly similar to those of non-Asian eyelids, but differences existed in higher or indistinct septum fusion, anterior and superior orbital fat projection, and the overriding of the preseptal orbicularis oculi muscle.

Direct insertion of the medial rectus capsulopalpebral fascia to the tarsus.

Purpose: To clarify the insertion of the medial rectus capsulopalpebral fascia to the tarsus in Asians. Methods: Specimens from 19 (11 right, 8 left) postmortem medial eyelids and orbits of 11 Asians (aged 45–96 years at death) were used. Samples had been fixed in 10% buffered formalin before their removal and microscopic examination. The tarsi were incised at 2 different heights in the upper and lower eyelids, as it was not disclosed which parts had the insertion of the medial rectus capsulopalpebral fascia. The first and second sections, parallel to the eyelid margin, were obtained, respectively, at 1 mm and 5 mm from the upper eyelid margin, and at 1 mm and 3 mm from the lower eyelid margin. Sections were stained with Masson trichrome. Results: Both upper and lower eyelids demonstrated similar findings. The first sections, which showed the medial rectus capsulopalpebral fascia and included many smooth muscle fibers, did not insert in the tarsi. However, the deep part of Horner muscle directly inserted, whereas the superficial part went in the dense fibrous tissue closely attaching on the tarsi. Then, some of the muscle branched out in the tarsi. The second sections showed that the medial rectus capsulopalpebral fascia had a direct insertion to the tarsi. Conclusions: The tarsi are supported medially by the medial rectus capsulopalpebral fascia and Horner muscle. The “medial eyelid retractors, ” comprising the medial rectus capsulopalpebral fascia and smooth muscles, were clearly defined, highlighting the relationship of the eyelid to the medial rectus muscle and offering a new pathogenesis and treatment for lateral tarsal shifts and lower medial ectropion.

Microscopic findings of lateral tarsal fixation in Asians.

Purpose: To identify microscopically lateral tarsal fixation in Asians. Methods: Specimens from 19 postmortem lateral eyelids and orbits of 11 Asians (11 right, 8 left; aged 45–96 years at death) were used. Samples damaged on sectioning and samples without tarsal plates were excluded. The samples were fixed in 10% buffered formalin and examined under a microscope. Two levels of tarsus were observed in the upper and lower eyelids, suggesting the possibility of different means of fixation. The first and second sections, which were incised parallel to the eyelid margin, were obtained at 1 mm and 5 mm from the upper eyelid margin, and at 1 mm and 3 mm from the lower eyelid margin. The sections were stained with Masson trichrome. Results: The first sections of all upper eyelids and those of the lower eyelids except one showed tarsal fixation by both the lateral rectus capsulopalpebral fascia (lr-CPF) and the tendon-ligament complex of the lateral canthal tendon (LCT), which in several cases received the muscle of Riolan. The second sections of all upper eyelids showed fixation by the lr-CPF and the ligamentous part of the LCT. The second sections of the lower eyelids were mostly similar to the second sections of upper eyelids, though some showed only ligamentous fixation. The lr-CPF in all cases included a small amount of smooth muscle fibers. Conclusions: The lateral aspect of the tarsus is supported by the lr-CPF and the LCT, which in some cases includes the muscle of Riolan.

Posterior layer advancement of the lower eyelid retractor in involutional entropion repair.

Purpose: To examine effectiveness of posterior layer advancement of the lower eyelid retractor in involutional entropion repair. Methods: Fifty lower eyelids (30 right and 20 left, average patient age 75.5 years) of 43 patients with involutional entropion underwent surgery. All cases were observed for at least 1 year postoperatively. During surgery, after detaching the anterior and posterior surfaces of the lower eyelid retractor, we positively advanced and fixed the posterior layer of the lower eyelid retractor to the tarsus. The anterior layer was used as reinforcement for the posterior layer. When lower eyelid retraction was intraoperatively observed, the suture was changed to fix to a more undercorrected position. Results: Of the 50 patients, only 1, who was the second case operated on, showed recurrent entropion 5 months postoperatively, but following a repeat operation using the same procedure no recurrence was seen in the next 2 years. Three eyelids showed a low degree of ectropion in the early postoperative period, but all improved within 1 month. No postoperative lower eyelid retraction was observed in any patient. Conclusions: Posterior layer advancement of the lower eyelid retractor is useful for entropion repair.

Incarceration of the Inferior Oblique Muscle Branch of the Oculomotor Nerve in Two Cases of Orbital Floor Trapdoor Fracture

BackgroundIncarceration of the inferior oblique muscle (IO) branch of the oculomotor nerve may occur in cases of orbital floor trapdoor fracture.CasesTwo orbital floor trapdoor fracture cases, with lesions located just outside of the inferior rectus muscle but without its incarceration, were examined pre- and postoperatively for visual acuity, intraocular details, the nine diagnostic ocular positions of gaze, binocular single vision field with the Hess chart, and by computed tomography (CT). One case was also examined by magnetic resonance imaging (MRI; T1-weighted images). A forced duction test was conducted intraoperatively.ObservationsEach case presented good visual acuity and neither globe showed any injury. Motility disturbance of the IO was shown in each case by binocular single vision field testing and the Hess chart. The possibility of the incarceration of the IO branch of the oculomotor nerve, which runs from the incarcerated lesion to the superior belly of the IO, in an orbital floor trapdoor fracture was shown on CT and MRI. Intraoperative forced duction testing revealed a restriction due to the incarceration of the connective tissue septa.ConclusionsAs inferred from the CT and MRI analyses conducted in this study, IO palsy may be one of the causes of ocular motility disturbance of the IO in an orbital floor trapdoor fracture, in addition to the ocular motility disturbance due to the connective tissue septa. Jpn J Ophthalmol 2005;49:246–252

Expression of a chondroitin sulfate proteoglycan, versican (PG-M), during development of rat cornea

Purpose: To understand the role of chondroitin sulfate proteoglycans during the development of rat cornea, expression of chondroitin sulfate and versican (PG-M) was studied. Methods: Chondroitin sulfate and keratan sulfate in rat cornea were analyzed by immunohistochemical techniques. Reverse transcription polymerase chain reaction (RT-PCR) for chondroitin sulfate proteoglycans was performed. Versican expression was studied by RT-PCR, immunohistochemical, and dot blot analyses. Expression of hyaluronan was evaluated histochemically using biotinylated hyaluronan binding protein. Results: Chondroitin sulfate was abundant in rat cornea at postnatal day 1 (P1) and became undetectable at P14. RT-PCR analysis showed that versican mRNA was highly expressed at P1 but was little expressed at P42. mRNAs for other chondroitin sulfate proteoglycans including biglycan, aggrecan, and decorin did not change much between P1 and P42. Expression for all versican splicing isoforms (V0–V3) was detectable from P1 through P14 but was undetectable after P21. mRNA for V0, the largest form with many chondroitin sulfate binding sites, decreased markedly in early stages from P1 to P14, whereas mRNA for V3, the shortest form with no chondroitin sulfate binding site, increased. mRNAs for middle-sized forms, V1 and V2, remained little changed during these periods. Immunohistchemical and dot blot analyses showed that versican is highly expressed at early stages of development and little expressed at adulthood. Similarly, hyaluronan, a versican-bound glycosaminoglycan, was highly expressed at early stages and little expressed at adulthood. Conclusions: Versican and hyaluronan, which can form a large molecular complex, may play an important role in the early phase of corneal development.

Correlation between optic nerve head structural parameters and glaucomatous visual field indices

We examined associations between optic nerve head structural parameters and glaucomatous visual field indices. The study population included patients with glaucomatous optic neuropathy who were evaluated at Aichi Medical University, Nagakute, Aichi, Japan, from October 2010 to January 2011. A total of 57 eyes from 33 patients were assessed. We measured visual field using a Humphrey field analyzer, peripapillary retinal nerve fiber layer thickness (RNFL-T), and Bruch’s membrane opening-minimum rim width (BMO-MRW) using spectral domain optical coherence tomography, and rim area with referring three-dimensional photography. Spearman’s rank correlation coefficients were calculated between the threshold of visual sensitivity or total deviation of visual field and the following five optic nerve head structural parameters: RNFL-T length, BMO-MRW length, rim area, and calculated RNFL-T and BMO-MRW volumes (each length multiplied by rim area). The Akaike information criterion was calculated to determine which structural parameter was the best predictor of each visual field index. Threshold of visual sensitivity had correlation coefficients of 0.23 with global sector of RNFL-T, 0.32 with BMO-MRW, 0.14 with rim area, 0.21 with RNFL-T volume, and 0.26 with BMO-MRW volume. The correlation coefficients for each parameter with total deviation of visual field were 0.22, 0.33, 0.28, 0.36, and 0.37, respectively. The Akaike information criterion of BMO-MRW showed the smallest values in analyses of both threshold of visual sensitivity and total deviation. The present results show that RNFL-T volume and BMO-MRW volume were more strongly correlated with total deviation than BMO-MRW, but BMO-MRW appeared to be the best predictor of the two glaucomatous visual field indices.