The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

The hyaluronan (HA)-rich extracellular matrix plays dynamic roles during tissue remodeling. Versican and serum-derived HA-associated protein (SHAP), corresponding to the heavy chains of inter-α-trypsin inhibitor, are major HA-binding molecules in remodeling processes, such as wound healing. Versican G1-domain fragment (VG1F) is generated by proteolysis and is present in either remodeling tissues or the mature dermis. However, the macrocomplex formation of VG1F has not been clarified. Therefore, we examined the VG1F-containing macrocomplex in pressure ulcers characterized by chronic refractory wounds. VG1F colocalized with SHAP-HA in specific regions of the granulation tissue but not with fibrillin-1. A unique VG1F-SHAP-HA complex was isolated from granulation tissues using gel filtration chromatography and subsequent cesium chloride-gradient ultracentrifugation under dissociating conditions. Consistent with this molecular composition, recombinant versican G1, but not versican G3, interacted with the two heavy chains of inter-α-trypsin inhibitor. The addition of recombinant VG1 in fibroblast cultures enhanced VG1F-SHAP-HA complex deposition in the pericellular extracellular matrix. Comparison with other VG1F-containing macrocomplexes, including dermal VG1F aggregates, versican-bound microfibrils, and intact versican, highlighted the tissue-specific organization of HA-rich extracellular matrix formation containing versican and SHAP. The VG1F-SHAP-HA complex was specifically detected in the edematous granulation tissues of human pressure ulcers and in inflamed stages in a mouse model of moist would healing, suggesting that the complex provides an HA-rich matrix suitable for inflammatory reactions.

Polypoid granulation tissue in pressure ulcers: Significance of describing individual ulcers.

Granulation tissue formation is required for the healing of deep pressure ulcers. The wound healing process is often delayed at the stage of granulation tissue formation. The pathogenesis of pressure ulcers showing granulation tissue may vary; however, no terminology has been defined to describe existing ulcers. Thus, we previously defined terminology for granulation tissue to describe individual ulcers. Based on these terms, we retrospectively evaluated the findings of deep pressure ulcers. In particular, we focused on polypoid granular tissue, a unique morphological feature. Polypoid granulation tissues were frequently observed in pressure ulcers over the sacrum compared with those over the foot. Chronological observation of a few cases indicated that external forces from specific directions during the healing process caused the development of polypoid granulation tissue. In addition, most pressure ulcers showing polypoid granulation tissue exhibited a trench-like appearance in individual wounds. Based on these observations, polypoid granulation tissue may generate from specific external forces, which lead to wound deformity. Morphological findings in an individual wound may be useful to predict the mechanical factors on existing pressure ulcers.

[A retrospective analysis of chemotherapy for gastric cancer in later-stage elderly patients].

AIM: Despite the significant advances in chemotherapy, the prognosis of unresectable or recurrent gastric cancer is still very poor. Given that older adults are likely to have a number of concomitant diseases and an impaired major organ function, cancer chemotherapy in elderly patients requires particular caution. We examined what factors are associated with the overall survival of gastric cancer patients undergoing chemotherapy. METHODS: A retrospective chart review of gastric cancer patients receiving oral fluoropyrimidines (N=130) was performed at Nagoya Memorial Hospital over 9 years. The overall survival was calculated from the beginning of chemotherapy until death or the most recent date of follow-up. The Kaplan-Meier method was used to plot survival curves, which were compared using the log-rank test. A multivariate analysis was performed using stepwise Cox proportional hazards models. A comprehensive geriatric assessment was conducted for the elderly patients. The chart review was approved by the ethics committee of Nagoya Memorial Hospital. RESULTS: The objective response rate and overall survival did not differ markedly between the patients < 75 years (N=64) and those ≥ 75 years of age (N=28). The addition of lentinan significantly prolonged the survival of the stage 4 gastric cancer patients. In a multivariate analysis of those ≥ 75 years of age, the only independent prognostic factor for the survival was the functional capacity, as measured by the TMIG Index of Competence. CONCLUSIONS: This comprehensive geriatric assessment was useful for predicting the longevity of patients with stage 4 gastric cancer ≥ 75 years of age.

Development of Pyriform Sinus Suctioning Programs for Aspiration Pneumonia Prevention During the Acute Stroke.

Aspiration due to dysphagia is a factor associated with pneumonia during acute stroke. In such cases, it is likely that secretions in the pyriform sinuses enter the laryngeal inlet. The present study was based on the idea that it is possible to reduce aspiration pneumonia by periodically suctioning and removing such secretions (pyriform sinus suctioning), a study was conducted in a single facility. The incidence of pneumonia as a dependent variable was compared between before (control) and after (intervention group) intervention with pyriform sinus suctioning as an independent variable. With a view of unifying the quality and frequency of intervention, two programs to: initially confirm the safety of such suctioning; subsequently enhance/evaluate knowledge and skills related to the procedure (educational); and specify conditions for the implementation and criteria for determining its appropriateness (practical), were developed. The study involved 33 (mean age: 74.6 ± 12.4) and 30 (80.0 ± 8.8) control and intervention group members, respectively, 25 (83.3%) of the latter were treated with pyriform sinus suctioning for 5 days after a stroke. Pneumonia developed in 7 (21.2%) and 2 (6.7%) of the former and latter, respectively. As individuals with a Japan Coma Scale (JCS) score of III or a midline shift on head CT tend to develop pharyngeal dysphagia, the patients were also divided into 2 groups to compare the incidence of pneumonia based on the risk level: low: Japan Coma Scale scores of I-II without a midline shift on head CT; and high: scores of II-III with it. In the latter, the incidence after intervention was markedly lower (p = 0.06, φ = 0.326), while the former did not show changes (p = 0.574, φ = 0.066), supporting the effectiveness of pyriform sinus suctioning to prevent aspiration pneumonia among patients with a low risk level.

A new concept: 'Relative position between the external force and the bony prominence' explains location-specific occurrence of superficial injury over an undermining lesion.

AIM: A pressure ulcer is localized injury to the skin and/or underlying tissue usually over a bony prominence, as a result of pressure, or pressure in combination with shear. Although the external forces and bony prominences differ depending on ulcer location, the way in which these anatomical differences affect pressure ulcer development is not well studied. METHODS: To clarify the location-dependent factors for pressure ulcer development, we focused on superficial injuries that develop over an undermining lesion, which we have termed them bilayer pressure ulcers. Because it is thought that a deep pressure ulcer is caused by ischemia at the deep lesion and a shallow pressure ulcer is caused by shear force to the superficial skin, a bilayer pressure ulcer can be considered a mixed phenotype, induced by both pressure and shear force. We retrospectively examined the frequency of bilayer pressure ulcers by location in a total of 568 pressure ulcers. RESULTS: The ratio of bilayer pressure ulcers to deep pressure ulcers staged III or more was significantly larger for pressure ulcers over the sacrum. CONCLUSION: A new concept, the relative position between the external force and bony prominence, could explain the frequency and developmental mechanism of bilayer pressure ulcers. The external forces, shape of the bony prominence, and mobility of the soft tissue may be responsible for this concept.

Bimatoprost, latanoprost, and tafluprost induce differential expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases.

BACKGROUND: Differences in the increase in matrix metalloproteinase (MMP) and decrease in tissue inhibitor of metalloproteinase (TIMP) activity may contribute to the different characteristics observed clinically on decreased intraocular pressure in patients with glaucoma or ocular hypertension. The purpose of this study was to investigate differences in the expression profiles of MMPs and TIMPs induced by the prostaglandin analogs bimatoprost, latanoprost, and tafluprost in human non-pigmented ciliary epithelial cells (HNPCECs). METHODS: HNPCECs were cultured for 24 h with 0, 10, 100, or 1000 µM of the free acid forms of bimatoprost, latanoprost, and tafluprost. We measured the expression levels of MMPs and TIMPs using real-time polymerase chain reaction, and compared the results. Enzyme activities of MMP-2 and -9 in conditioned media were measured by gelatin zymography. RESULTS: All prostaglandin analogs we examined dose-dependently increased expression levels of MMP-1, -2, -3, -9, and -17, whereas expression levels of TIMP-1 and -2 decreased with increasing concentrations of each analog. Each prostaglandin analog induced different levels of increases in MMPs and decreases in TIMPs. CONCLUSIONS: Unique expression profiles of MMPs and TIMPs induced by bimatoprost, latanoprost, and tafluprost, as shown in HNPCECs, may contribute to clinically different effects on intraocular pressure decreases in patients with glaucoma or ocular hypertension.

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia.

BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE). RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31 °C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

Homotypic versican G1 domain interactions enhance hyaluronan incorporation into fibrillin microfibrils.

Versican G1 domain-containing fragments (VG1Fs) have been identified in extracts from the dermis in which hyaluronan (HA)-versican-fibrillin complexes are found. However, the molecular assembly of VG1Fs in the HA-versican-microfibril macrocomplex has not yet been elucidated. Here, we clarify the role of VG1Fs in the extracellular macrocomplex, specifically in mediating the recruitment of HA to microfibrils. Sequential extraction studies suggested that the VG1Fs were not associated with dermal elements through HA binding properties alone. Overlay analyses of dermal tissue sections using the recombinant versican G1 domain, rVN, showed that rVN deposited onto the elastic fiber network. In solid-phase binding assays, rVN bound to isolated nondegraded microfibrils. rVN specifically bound to authentic versican core protein produced by dermal fibroblasts. Furthermore, rVN bound to VG1Fs extracted from the dermis and to nondenatured versican but not to fibrillin-1. Homotypic binding of rVN was also seen. Consistent with these binding properties, macroaggregates containing VG1Fs were detected in high molecular weight fractions of sieved dermal extracts and visualized by electron microscopy, which revealed localization to microfibrils at the microscopic level. Importantly, exogenous rVN enhanced HA recruitment both to isolated microfibrils and to microfibrils in tissue sections in a dose-dependent manner. From these data, we propose that cleaved VG1Fs can be recaptured by microfibrils through VG1F homotypical interactions to enhance HA recruitment to microfibrils.

Comparative analysis of hyaluronan's affinity for antivascular endothelial growth factor agents.

BACKGROUND: Differences in the efficacy of bevacizumab, an antivascular endothelial growth factor (VEGF) agent, against retinopathy with neovascularization when injected into the vitreous cavity of vitrectomized and nonvitrectomized eyes suggests the involvement of hyaluronan, a major component of the vitreous body. This study aimed to compare the affinities of hyaluronan for anti-VEGF agents in vitro. METHODS: We examined the affinities of hyaluronan for 3 anti-VEGF agents (bevacizumab, pegaptanib and ranibizumab). Tritium [(3)H]-labeled hyaluronan was incubated separately with each anti-VEGF agent. The ratio of bound and unbound hyaluronan measured using solid and liquid phase methods was calculated. RESULTS: Hyaluronan demonstrated a significantly greater affinity for bevacizumab than for pegaptanib or ranibizumab. CONCLUSIONS: The absence or presence of hyaluronan may be associated with the clinical efficacy of bevacizumab injected into the vitreous cavity due to the affinity of hyaluronan for bevacizumab.

Location-dependent depth and undermining formation of pressure ulcers.

AIM OF THE STUDY: We examined the location-specific properties of pressure ulcers, focusing on depth and undermining formation, which are often unfavorable factors for ulcer healing. METHODS: We conducted a retrospective observational study of 2 independent databases on pressure ulcers. Databases from a 200-bed hospital (database A) and a 300-bed hospital (database B) were collected during different time periods. Relationships between ulcer location, ulcer depth, and undermining formation were analyzed. All pressure ulcers were accurately diagnosed and classified according to their locations. RESULTS: A total of 282 pressure ulcers in 189 patients from database A and 232 pressure ulcers in 154 patients from database B were analyzed. It was found that pressure ulcers primarily developed over the sacrum. Ratio of stages III and IV pressure ulcers was high in pressure ulcers of the foot, ankle, and crus on the lower leg. Among the deep pressure ulcers, undermining formation was frequently observed on the greater trochanter, ilium, and sacrum. In contrast, pressure ulcers of the foot, ankle, and crus did not exhibit undermining formation. CONCLUSION: Our results revealed marked differences in pressure ulcer properties depending on their location. Factors affecting depth and undermining of pressure ulcers appear to be related to anatomical and physical properties of the bone and subcutaneous tissue.

Long-term remission of ocular and extraocular manifestations in Behçet's disease using infliximab.

OBJECTIVES: To investigate the long-term effect of infliximab on ocular and extraocular manifestations in patients with Behçet's disease. METHODS: Seven patients with active Behçet's disease and treated with infliximab at Aichi Medical University Hospital for more than 18 months were included in the study. We evaluated visual acuity, the average number of uveitis attacks involving the posterior segment, and general disease activity every 2 months. The Behçet's Disease Current Activity Form (BDCAF) was used for an overall index of disease activity. Anti-infliximab antibody levels were examined in the patients' sera. RESULTS: The follow-up period after initial introduction of infliximab ranged from 19 to 40 months (mean ± SD, 32 ± 8.7 months). The number of infliximab infusions ranged from 12 to 24 (19 ± 4.4). By the 2-month follow-up, the frequency of uveitis attacks involving the posterior segment and the BDCAF scores were significantly improved compared to the 2 months before introducing infliximab. Anti-infliximab antibodies were detected in the sera of all examined patients. CONCLUSIONS: Significant long-term improvement in both the frequency of uveitis attacks involving the posterior segment and overall disease activity was provided by the administration of infliximab to patients suffering from Behçet's disease, despite the presence of anti-infliximab antibodies.

Evidence for the heparin-binding ability of the ascidian Xlink domain and insight into the evolution of the Xlink domain in chordates.

The vertebrate Xlink domain is found in two types of genes: lecticans and their associated hyaluronan-and-proteoglycan-binding-link-proteins (HAPLNs), which are components of the extracellular matrix, and those represented by CD44 and stabilins, which are expressed on the surface of lymphocytes. In both types of genes, Xlink functions as a hyaluronan binding domain. We have already reported that protochordate ascidians possess only the latter type of gene. The present analysis of the expression of ascidian Xlink domain genes revealed that these genes function in blood cell migration and apoptosis. While the Xlink domain is found in various metazoans, including ascidians and nematodes, hyaluronan is believed to be specific for vertebrates. In comprehensive genome surveys for hyaluronan synthase (HAS), we found no HAS gene in ascidians. We also established that hyaluronan is absent from the ascidian body biochemically. Therefore, ascidians possess the Xlink domain, but they lack HA. We recovered one ascidian Xlink domain gene that encoded a heparin-binding protein, although it shows no affinity for hyaluronan. Based on these findings, we conclude that in invertebrates, the Xlink domain serves as heparin-binding protein domain and functions in blood cell migration and apoptosis. Its binding affinity for HA might have been acquired in the vertebrate lineage.

Increase in matrix metalloproteinase-2 level in the chicken retina after laser photocoagulation.

BACKGROUND AND OBJECTIVE: We investigated the levels of matrix metalloproteinase-2 (MMP-2), which has been implicated in various vitreoretinal diseases, in the retina after laser photocoagulation (LPC). MATERIALS AND METHODS: The time course of MMP-2 expression in 2-day-old chicken retinas before and 6 hours, 12 hours, 1 day, 2 days, 4 days, 8 days, 16 days, and 32 days after LPC was determined by real-time PCR and gelatin zymography. The basal level of MMP-2 in the retina and vitreous was also measured by gelatin zymography. MMP-2 localization in the retina was examined by immunohistochemistry. The localization of MMP-2 mRNA was determined by fluorescent in situ hybridization. The internal limiting membrane (ILM) was observed by scanning electron microscopy. RESULTS: MMP-2 mRNA expression in the retina peaked at day 4, but gelatin zymography showed that MMP-2 peaked 6 hours after LPC and the significant increase in the level of active MMP-2 lasted for more than 4 days. The concentration of MMP-2 in the vitreous was significantly higher than that in the retina. A distinct MMP-2 signal around the ILM was identified 6 hours after LPC, but MMP-2 mRNA was not detected there. Electron microscopy showed a damaged retinal surface after LPC. CONCLUSION AND OUTLOOK: The significant increase in retinal MMP-2 which lasted for more than 4 days after LPC may be induced by influx from the vitreous into the retina. This MMP-2 dynamics may contribute to pathological processes in the retina after LPC.

Competitive binding of heparin with hyaluronan to a specific motif in SPACR.

The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site-directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.

Characterization of a motif for specific binding to hyaluronan in chicken SPACR.

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.

A Rho-Associated Kinase Inhibitor Protects Permeability in a Cell Culture Model of Ocular Disease, and Reduces Aqueous Flare in Anterior Uveitis.

PURPOSE: Recent clinical and experimental studies have reported favorable results when using Rho-associated kinase (ROCK) inhibitors for ocular disease, and in cell culture. Disruption of the human, nonpigmented ciliary epithelial cells (HNPCECs) that comprise the blood-aqueous barrier (BAB) induces anterior uveitis; these cells therefore provide a useful cell model of ocular disease. In this study, we examined the effects of ROCK inhibitors in anterior uveitis and in HNPCECs. METHODS: Aqueous flare values and intraocular pressures (IOPs) were determined in patients with anterior uveitis, 2 weeks after administration of ripasudil hydrochloride hydrate, a commercial ROCK inhibitor used to treat glaucoma or ocular hypertension. We also investigated the effects of Y-27632, a second ROCK inhibitor, in HNPCECs following exposure to matrix metalloproteinases (MMPs) and human tumor necrosis factor-alpha (TNF-α). RESULTS: Patients with anterior uveitis, glaucoma, or ocular hypertension, referred to the Aichi Medical University from February to July 2015, were enrolled. Thirty eyes from 25 outpatients were studied. Aqueous flare values and IOPs were significantly decreased 2 weeks after ripasudil hydrochloride hydrate treatment, with no adverse events. In a cultured HNPCEC monolayer, permeability was markedly increased following exposure to MMPs-1, 3, 9, and TNF-α, with these effects attenuated by exposure to Y-27632. In cultured HNPCECs, Y-27632 provoked a marked alteration in cytoskeletal morphology without a significant change in expression levels of claudin-1 and occludin. CONCLUSION: ROCK inhibitors may confer favorable effects in anterior uveitis, possibly due to a reorganized BAB, although the relevant mechanisms remain unclear.

Expression profile of heat shock protein 108 during retinal development in the chick.

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.

Chemo-Immunotherapy Using Lentinan for the Treatment of Gastric Cancer with Liver Metastases.

Gastric cancer is the third leading cause of cancer-related mortality worldwide. Systemic chemotherapy is the main treatment option for advanced gastric cancer when the tumor is inoperable. Despite recent advances in chemotherapeutic agents, the prognosis of unresectable or recurrent gastric cancer remains extremely poor. In Japan, combination therapy including S-1 and cisplatin is the standard first-line treatment for advanced gastric cancer; however, the five-year survival rate remains very low. Lentinan, the backbone of beta-(1,3)-glucan with beta-(1,6) branches, an active ingredient purified from Shiitake mushrooms, has been approved as a biological response modifier for the treatment of gastric cancer. This agent has been used in combination with oral fluoropyrimidines to improve the overall survival of gastric cancer patients. A retrospective chart review on 138 metastatic gastric cancer patients receiving chemotherapy was performed in Nagoya Memorial Hospital from 1 September 2010 to 31 August 2015. 12 patients with liver metastases were treated by lentinan in combination with S-1-based chemotherapy. The rate of objective response was 42% (5/12) and the disease control rate was 83% (10/12) in response to chemo-immunotherapy using lentinan, with a median overall survival of 407 days (95% CI: 207-700 days).

Characteristic Formation of Hyaluronan-Cartilage Link Protein-Proteoglycan Complex in Salivary Gland Tumors.

Hyaluronan (HA) and its binding molecules, cartilage link protein (LP) and proteoglycan (PG), are structural components of the hydrated extracellular matrix. Because these molecules play important roles in the tumor microenvironment, we examined the distribution of HA, LP, versican, and aggrecan in salivary gland tumors using histochemical and immunohistochemical methods, including double staining. LP was present in pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) tissues, and aggrecan was absent in the malignant tumors that we investigated. LP colocalized with both HA and aggrecan in the chondromyxoid matrix of PA, suggesting the presence of a HA-LP-aggrecan complex. Furthermore, the HA-LP-versican complex could be observed in the pseudocystic space of the cribriform structures in ACC. The characteristic HA-LP-PG complex in PA and ACC might play a role in the behavior of tumors, and immunohistochemical analysis of these molecules could represent a diagnostic adjunct for salivary gland tumors.