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.

Ointment vehicles regulate the wound-healing process by modifying the hyaluronan-rich matrix.

Topical ointment consists of an active ingredient and vehicle, and the vehicle largely comprises the volume of the ointment. During the treatment of chronic wounds, such as pressure ulcers, the vehicle has been considered inactive, only serving as a carrier of the main pharmaceutical. However, recent reports have indicated that the vehicle has distinct clinical effects that depend on its physicochemical properties. Therefore, an understanding of the action mechanism of the ointment vehicle in wound tissue is necessary. In this study, we established a mouse model to analyze tissue reactions induced by the following ointment vehicles, an oil-in-water emulsion (EM) vehicle; a macrogol ointment (MO), which is a water-soluble, hydrophilic vehicle; and a MOs containing sucrose (MS). EM-treated wounds exhibited an inflammatory reaction characterized by tissue edema and thick granulation tissue; however, MO- and MS-treated wounds did not exhibit this reaction. Moreover, EM-treated wounds exhibited infiltration of inflammatory cells unlike MO-treated wounds. In contrast, the formation of collagenous tissue was dominantly observed in MO-treated wounds. Because the vehicle regulates the water environment of the wound, the water-holding extracellular matrix molecules, including hyaluronan (HA) and proteoglycan, were examined using immunohistochemical and biochemical methods. The versican G1 fragment, serum-derived HA-associated protein (SHAP) and HA (the VG1F-SHAP-HA) complex characteristically found in inflammatory conditions of pressure ulcers was found in EM-treated wounds. To histologically analyze the mechanism of action of the vehicle, we evaluated the ointment vehicle-wound tissue interface in an en bloc manner. Formation of the HA-containing complex was observed locally between the vehicle and wound surface. On the basis of these data, ointment vehicles regulate the wound-healing process through the formation of HA-rich extracellular matrices at the ointment-wound interface. This study provides a better understanding of the treatment of deep-pressure ulcers with focus on ointment vehicles.

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.

Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells.

Addicsin (Arl6ip5) is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1) and participates in the regulation of intracellular glutathione (GSH) content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ), a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABAA) receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER) to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na+/K+ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABAA receptor antagonist, picrotoxin, but not by a competitive GABAA receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.

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.

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.

Generation of a transgenic mouse line for conditional expression of human IL-6.

IL-6 is a cytokine that is involved in various physiological and pathological conditions, and approaches using gain-of-function transgenic animals have contributed in elucidating IL-6 function. However, studies of the multiple functions of IL-6 in vivo are very time consuming because they require the generation of transgenic mice that harbor the gene encoding IL-6 under the control of specific promoters to mimic different pathologies. Here, we report the establishment of a conditional human IL-6 transgenic mouse, LGL-IL6, which conditionally expresses human IL-6 by taking advantage of the well-characterized Cre recombinase drivers.

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.

Type V collagen fibrils in mouse metanephroi.

Type V collagen (Col V) molecule, a minor component of kidney connective tissues, was found in adult cornea, and has been considered as a regulatory fibril-forming collagen that emerges into type I collagen to trigger the initiation of Col I fiber assembly. Col V was also found in injured, wound healing tissues or placenta, and was considered as a dysfunctional extracellular matrix (ECM). Reconstituted Col V fibril was characterized as an ECM to detach cells in vitro, and our previous study showed that the reconstituted Col V fibril facilitated the migration of glomerular endothelial cells and induced ECM remodeling, whereas Col V molecules stabilized cells. These facts suggest that not only the structure but also the function of Col V fibril are different from Col V molecule. Recently, Col V molecule has been reported existing in various developing tissues such as bone and lung, but Col V fibril has not been reported yet. In this study, we firstly explored the existence of Col V fibril in metanephroi, and found it distributed in the immature kidney tissues whereas disappeared when the tissues reached mature. It is likely that Col V fibril may form a prototype of pericellular microenvironment and the transient existence of Col V fibril may play a role as the pioneering ECM during metanephric tissue morphogenesis.

Iodoform gauze removes necrotic tissue from pressure ulcer wounds by fibrinolytic activity.

Iodoform gauze is used in clinical practice for treatment of infected wounds. However, effectiveness and action mechanism of iodoform gauze for removal of necrotic tissue are unknown. We therefore employed case control and biochemical studies in order to clarify the pharmacological activity of iodoform gauze. A clinical study demonstrated that treatment with iodoform gauze removed necrotic tissue more effectively than treatment with conventional ointments. More than 60% of iodoform gauze-treated wounds were completely debrided within 2 weeks. Consistent with the clinical observation, biochemical analyses revealed clear differences in wound fluid proteins after treatment with iodoform gauze or conventional gauze. The amount of macroaggregates of type I collagen from wounds were remarkably decreased in iodoform gauze. Moreover, iodoform gauze and iodoform itself released non-aggregative type I collagen from necrotic debris in vitro. Taken together, we conclude that iodoform gauze efficiently removes necrotic tissue by its lytic activity for collagen fibers.

The anti-inflammatory effects of matrix metalloproteinase-3 on irreversible pulpitis of mature erupted teeth.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation and the modulation of cell behavior. These proteinases have also been implicated in tissue repair and regeneration. Our previous studies have demonstrated that MMP-3 elicits stimulatory effects on the proliferation and the migration of endothelial cells as well as anti-apoptotic effects on these cells in vitro. In addition, we found that MMP-3 enhanced the regeneration of lost pulp tissue in a rat incisor pulp injury model. However, continuously erupting rodent incisors exhibit significantly different pulp organization compared with mature erupted teeth. Therefore, we have further extended these studies using a canine irreversible pulpitis model to investigate the effects of MMP-3. In this study, the crowns of the canine mature premolars were removed and the pulp tissues were amputated. The amputated pulp tissues remained exposed for 24 or 72 hours to induce mild or severe irreversible pulpitis, respectively, followed by sealing of the cavities. In both models, the whole pulp tissues became necrotic by day 14. In this mild pulpitis model, the regeneration of pulp tissue with vasculature and nerves was observed until 14 days after sealing with MMP-3, followed by extracellular matrix formation in the regenerated pulp tissues until day 28. The treatment with MMP-3 resulted in a decrease in the number of macrophage and antigen-presenting cells and a significant inhibition of IL-6 expression on day 3. The inhibition of MMP-3 activity abolished these anti-inflammatory effects. Immunofluorescence staining demonstrated that MMP-3 was involved in the modification of serum-derived hyaluronan-associated proteins and hyaluronan (SHAP-HA) complexes possibly through the degradation of versican. These results demonstrate that MMP-3 can act as an anti-inflammatory agent and suggest that MMP-3 might represent a useful therapy for the treatment of mild irreversible pulpitis.

Albumin production activity of primary rat hepatocytes is improved on type V collagen.

Primary rat hepatocytes were cultured on type V collagen. Hepatocyte spreading on type V collagen was inferior compared with that on type I collagen. However, the albumin production rates of hepatocytes cultured on type V collagen were approximately twice as high as those of hepatocytes cultured on type I collagen.

The role of type V collagen fibril as an ECM that induces the motility of glomerular endothelial cells.

Although type V collagen (Col V) is present in developing and mature connective tissues of glomeruli, its primary function has not been elucidated yet. The purpose of this study was to elucidate the role of Col V fibrils in glomerular cells. We isolated primary cells from porcine kidney and cultured them on Col V fibrils reconstructed from purified Col V molecules extracted from porcine cornea. Time-lapse observation showed that Col V fibrils induce dynamic movement of glomerular endothelial cells (GEC) by stimulating them to extend long filopodial protrusions and wide lamellipodia. Col V signaling mediated through beta1 integrin activated phosphorylation of paxillin at tyrosine 118 (paxillin-pY118) and of focal adhesion kinase at tyrosine 861 (FAKpY861) at the cell periphery; a second Col V signal was mediated through neuroglycan 2 and activated FAKpY397. FAKpY861 was present in loose attachment points between Col V fibrils and GEC, allowing the cells to migrate easily. Activation of FAKpY397 induced incomplete focal adhesion at the centers of cells and caused cell movement. Therefore both signaling pathways facilitated cell motility, which was inhibited by the addition of antibodies to beta1 integrin, NG2, and Col V. We suggest that Col V fibrils activate 'outside-in' signaling in GEC and induce their dynamic motility.