Mixture Of Collagen Research Articles

Agonist-Induced Thromboxane A2 Generation Requires Signaling Through Apoptosis Signal-Regulating Kinase 1 (ASK1): Genetic Ablation of Ask1 Severely Affects In Vivo Thrombosis

Abstract 187Oxidative stress is a major contributing factor for cardiovascular diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family, which responds to a diverse array of cellular stresses including oxidative stress. Although its downstream signaling effectors, the JNK and p38 MAP kinases, have been shown to be present and activated in platelets, the presence of ASK1 is not known. Here we show that ASK1 is highly expressed in human platelets. During activation of platelets by a variety of physiological agonists, ASK1 is rapidly activated by phosphorylation of threonine (T845) in its activation loop. In order to determine the physiological significance of activation of ASK1 we used Ask1 knockout mouse. Genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2×10−9). While all WT mice stopped bleeding with an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s, with 5 out of 9 mice that did not stop bleeding for up to 10 min. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine as determined by increased survival and a lack of exclusion of Evans blue dye from the lungs. To determine the cause of such a severe defect in thrombosis, we evaluated ex vivo platelet functions. We found that low doses of agonist-induced platelet aggregation were significantly reduced in Ask1 null mice compared to WT mice. Defective aggregation was found to be due in part to impaired activation of fibrinogen receptor as indicated by reduced fibrinogen binding. When the ability of Ask1 platelets to release platelet granular contents was analyzed, both a- and d-granule secretion were significantly inhibited as assessed by P-selectin exposure and 14C-serotonin release, respectively. Interestingly, aspirin treatment had no effect on the amount of 14C-serotonin release from Ask1 null platelets, but reduced the amount in WT platelets, bringing it to the same level as that of Ask1 null platelets, suggesting that agonist-induced thromboxane generation was impaired in the Ask1 null platelets. This was further confirmed by the complete lack of thrombin-induced TxA2 production in Ask1 null platelets as compared to WT. Since cPLA2 is a key enzyme in the pathway of TxA2 generation, we analyzed the effect of the absence of Ask1 on cPLA2 activation. We found a complete absence of cPLA2 activation-induced by thrombin in Ask1 null platelets while a robust activation of cPLA2 occurred in WT. Additionally, agonist-induced activation of the p38 MAP kinase, known to be involved in cPLA2 activation, was absent in Ask1 null platelets. Furthermore, the TxA2 mimetic U46619-induced platelet aggregation was also greatly reduced in Ask1 null platelets, suggesting that signaling through the TxA2 receptor is also impaired in Ask1 null platelets. These in vitro and in vivo results strongly suggest that ASK1 plays a major role in the regulation of hemostasis and thrombosis by regulating a number of signaling events that are critical for proper platelet function. Disclosures:No relevant conflicts of interest to declare.

Hypoxia-Inducible Factor Regulates Expression of Surfactant Protein in Alveolar Type II Cells In Vitro

Alveolar type II (ATII) cells cultured at an air-liquid (A/L) interface maintain differentiation, but they lose these properties when they are submerged. Others showed that an oxygen tension gradient develops in the culture medium as ATII cells consume oxygen. Therefore, we wondered whether hypoxia inducible factor (HIF) signaling could explain differences in the phenotypes of ATII cells cultured under A/L interface or submerged conditions. ATII cells were isolated from male Sprague-Dawley rats and cultured on inserts coated with a mixture of rat-tail collagen and Matrigel, in medium including 5% rat serum and 10 ng/ml keratinocyte growth factor, with their apical surfaces either exposed to air or submerged. The A/L interface condition maintained the expression of surfactant proteins, whereas that expression was down-regulated under the submerged condition, and the effect was rapid and reversible. Under submerged conditions, there was an increase in HIF1α and HIF2α in nuclear extracts, mRNA levels of HIF inducible genes, vascular endothelial growth factor, glucose transporter-1 (GLUT1), and the protein level of pyruvate dehydrogenase kinase isozyme-1. The expression of surfactant proteins was suppressed and GLUT1 mRNA levels were induced when cells were cultured with 1 mM dimethyloxalyl glycine. The expression of surfactant proteins was restored under submerged conditions with supplemented 60% oxygen. HIF signaling and oxygen tension at the surface of cells appears to be important in regulating the phenotype of rat ATII cells.

Role of polyphenolic compounds on biological activity of collagenous materials

Collagen, a unique connective tissue protein, is extensively used as biocompatible biomaterial in wound healing [1], cosmetics [2] and tissue engineering [3]. Due to its high sensitivity to enzymatic degradation, cross-linking of collagenous materials must be a compulsory step in their fabrication. Chemical cross-linking agents are able to introduce new covalent bonds in collagen structure, but are cytotoxic. The ability of natural plant polyphenolic compounds to stabilize collagen structure while preserving its cytocompatibility it is now established [4]. This study aimed to investigate the interaction of small molecules from plants with collagen and their effect on its biological properties. Three mixtures of collagen with a polyphenolic extract of Urtica dioica L., a plant-derived flavonoid (quercetin) and a flavin from milk (riboflavin), respectively, were conditioned as porous materials by freeze-drying technique. A collagen-glutaraldehyde mixture was used as control. Free amino groups present in the mixtures were spectrophotometrically assayed. An in vitro experimental model using bacterial collagenase was used to mimic the enzymatic attack on the collagenous materials implanted in vivo. The swelling capacity and in vitro cytocompatibility tested according to ISO 10993–5 on fibroblasts from NCTC cell line were evaluated. Results showed a good correlation between the free amino groups and the biodegradability of each mixture. The values of swelling capacity were at least 70-fold higher than the initial weight. Fibroblast viability and morphology showed a high cytocompatibility of the polyphenol-collagen mixtures. In conclusion, all these tests indicated an improved applicability of these mixtures for tissue wound healing.

Experimental Model of Large Pulmonary Embolism Employing Controlled Release of Subacute Caval Thrombus in Swine

Purpose To develop a catheter-based model of large pulmonary embolism (PE) in swine based on in situ venous thrombus formation. Materials and Methods Ten Yorkshire swine underwent transjugular implantation of a retrievable inferior vena cava (IVC) filter. A thrombin and collagen mixture was injected into a confined space created by two balloons inflated proximal and distal to the IVC filter. Animals were left to survive for 7 days ± 3 to allow thrombus to organize in situ. The caval thrombus was released on transcatheter retrieval of the IVC filter and embolized into the main and branch pulmonary arteries. The severity of PE was scored based on digital subtraction angiography with the Miller index. At necropsy, thrombi were recovered and analyzed histopathologically. Results Large PE was induced in all animals (Miller index score of 15 ± 5). Two animals developed saddle embolus with bilateral pulmonary artery occlusion, and five developed proximal occlusion of the left or right pulmonary artery. Nevertheless, no animal exhibited significant hemodynamic compromise. Large tubular thrombi were explanted in the size range of 5–10 cm long and 0.5–1 cm wide. Histologic analysis indicated an organized thrombus with infiltration of white blood cells and fibrin deposition. Conclusions Large caval thrombi can be formed in vivo and released at a predetermined time to induce large PE in a large animal model. This may help in the development and testing of new therapeutic approaches for PE.

Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model

Cytotherapeutic approaches have been investigated to overcome the limitations of existing procedures for periodontal regeneration. In this study, cell sheet transplantation was performed using three kinds of mesenchymal tissue (periodontal ligament, alveolar periosteum, and bone marrow)-derived cells to compare the differences between cell sources in a canine severe defect model (one-wall intrabony defect). Periodontal ligament cells (PDLCs), iliac bone marrow mesenchymal stromal cells (BMMSCs), and alveolar periosteal cells (APCs) were obtained from each dog; a total of four dogs were used. Three-layered cell sheets of each cell source supported with woven polyglycolic acid were autologously transplanted to the denuded root surface. One-wall intrabony defects were filled with a mixture of β-tricalcium phosphate (β-TCP) and collagen. Eight weeks after the transplantation, periodontal regeneration was significantly observed with both newly formed cementum and well-oriented PDL fibers more in the PDLC group than in the other groups. In addition, nerve filament was observed in the regenerated PDL tissue only in the PDLC group. The amount of alveolar bone regeneration was highest in the PDLC group, although it did not reach statistical significance among the groups. These results indicate that PDLC sheets combined with β-TCP/collagen scaffold serve as a promising tool for periodontal regeneration.

Glucosamine inhibits epithelial-to-mesenchymal transition and migration of retinal pigment epithelium cells in culture and morphologic changes in a mouse model of proliferative vitreoretinopathy

To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)-β signalling and several processes involved in proliferative vitreoretinopathy (PVR). We evaluated the surface levels of TGF-β receptor and its binding of TGF-β in ARPE-19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Glucosamine reduced the surface levels of TGF-β receptor and the ability of ARPE-19 cells to bind TGF-β. In ARPE-19 cells, TGF-β1 plus epidermal growth factor (EGF) or TGF-β2 increased the expression of alpha-smooth muscle actin (α-SMA) and decreased the expression of zona occludens protein (ZO-1). Transforming growth factor-(β2) also caused the release of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet-derived growth factor and CTGF stimulated cell migration, and TGF-β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF-β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial-mesenchymal transition and PVR.

Pocket related complications in 163 patients receiving anticoagulation or dual antiplatelet therapy: D-Stat Hemostat™ versus standard of care

BackgroundPocket hematoma is a common complication after cardiac rhythm device implantation (CRDI) in patients receiving anticoagulation or dual antiplatelet therapy (DAPT). We sought to assess the safety and effectiveness of D-Stat Flowable Hemostat™ ([D-Stat]—a mixture of thrombin and collagen) in these patients. MethodsWe conducted a prospective case control study and enrolled all patients admitted for CRDI receiving anticoagulation or DAPT. Participants received either D-Stat into the pectoral pocket as an adjunct to standard of care versus the standard of care (UC) alone. The primary endpoint was a composite of hematoma needing evacuation, and pocket infection. ResultsWe included a total of 163 patients (mean age 72±8years). 119 (73%) were male. 82 patients (50%) received D-stat and 81 (50%) usual care; 82 patients (50%) were treated with anticoagulation, 62 patients (38%) received DAPT, and 19 patients (12%) both. The primary endpoint occurred in 12/82 patients from the D-Stat group and 3/81 patients of the control group (14.6% versus 3.7%; p=0.03). The use of D-Stat tended for a higher incidence of pocket hematoma requiring evacuation (8.5% versus 2.4%; p=0.12), and an increased rate of pocket infections (6.1% versus 1.2%; p=0.21). Postoperative Immunoglobulin E levels (86±147IU/ml (D-Stat) versus 120±231IU/ml (UC)) and increase of C-reactive protein (D-Stat: ∆19mg/l versus UC: 14mg/l) were similar in both groups (p=0.26 and p=0.4, respectively). ConclusionsAdministration of D-Stat does not decrease the frequency of clinically relevant pocket hematomas. The observed rate of pocket infections in the D-Stat group is a matter of concern.

A cost-effective and simple culture method for primary hepatocytes

Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

Effects of Shenghua decoction on hemorheology, thrombosis and microcirculation

To investigate the effects of Shenghua decoction on hemorheology, thrombosis and microcirculation, and explore its approach and mechanism. The main hemorheological indexes and endothelial function were detected in acute stress blood stasis rats. The thrombus wet weight and thrombus dry weight were measured in the rat model of venous thrombosis, and the inhibitory rates in the formation of venous thrombosis were calculated. The number of paralysis or dead after 1-15 min was calculated in mice, induced by tail intravenous injection of a mixture of collagen and Epinephrine. Blood flow and the across netting were also determined on capillary vessel of uterus microcirculation. Shenghua decoction reduce blood viscidity of the rats, reduce the thrombosis of the murine, and promote the microcirculation of the uterus in rats. Shenghua decoction has the ability of blood-activating and stasis-eliminating. It also has an anti-thrombotic effect and can promote microcirculation.

Mesenchymal Stem Cell Responses to Bone-Mimetic Electrospun Matrices Composed of Polycaprolactone, Collagen I and Nanoparticulate Hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.

Engineering bioartificial tracheal tissue using hybrid fibroblast-mesenchymal stem cell cultures in collagen hydrogels☆

We aimed at providing the first in vitro and in vivo proof-of-concept for a novel tracheal tissue engineering technology. We hypothesized that bioartificial trachea (BT) could be generated from fibroblast and collagen hydrogels, mechanically supported by osteogenically-induced mesenchymal stem cells (MSC) in ring-shaped 3D-hydrogel cultures, and applied in an experimental model of rat trachea injury. Tube-shaped tissue was constructed from mixtures of rat fibroblasts and collagen in custom-made casting molds. The tissue was characterized histologically and mechanically. Ring-shaped tissue was constructed from mixtures of rat MSCs and collagen and fused to the tissue-engineered tubes to function as reinforcement. Stiffness of the biological reinforcement was enhanced by induction of osteogeneic differentiation in MSCs. Osteogenic differentiation was evaluated by assessment of osteocalcin (OC) secretion, quantification of calcium (Ca) deposit, and mechanical testing. Finally, BT was implanted to bridge a surgically-induced tracheal defect. A three-layer tubular tissue structure composed of an interconnected network of fibroblasts was constructed. Tissue collapse was prevented by the placement of MSC-containing ring-shaped tissue reinforcement around the tubular constructs. Osteogenic induction resulted in high OC secretion, high Ca deposit, and enhanced construct stiffness. Ultimately, when BT was implanted, recipient rats were able to breathe spontaneously.

Ossification Vesicles with Calcium Phosphate in the Eyes of the InsectCopium teucrii(Hemiptera: Tingidae)

Arthropod eyes are built of repeating units named ommatidia. Each single ommatidium unit contains a cluster of photoreceptor cells surrounded by support cells and pigment cells. The insect Copium eye ommatidia include additional calcium-phosphate deposits, not described in insects to date, which can be examined today using a combined set of modern microscopy and spectroscopy techniques. Teucrium gnaphalodes L'Her plants, growing in central Spain, develop galls induced by Copium insects. A survey of C. teucrii adult specimens resulted in surprising environmental scanning electron microscopy (ESEM) images, showing that their bright red eyes contain a calcium-phosphate mineralization. A complete survey of Copium eye specimens was performed by ESEM using energy-dispersive spectroscopy, backscattered electron detector and cathodoluminescence (CL) probes, field emission scanning electron microscopy, micro-Raman spectroscopy, and confocal laser scanning microscopy in order to learn ommatidia features, such as chemical composition, molecular structure, cell membrane, and internal ommatidium eye fluids and calcium-phosphate distribution deposits. The CL panchromatic images distinguish between the calcium-phosphate ommatidium and calcium-phosphate setae, which are more apatite rich. They show Raman bands attributable to bone tissue apatite biomaterials, such as bone, collagen, lipids, and blood, i.e., peptides, amide-S, amide-II, amide-III, and cytochrome P-450scc. The chemical composition of both galls and leaves of T. gnaphalodes was determined by gas chromatography – mass spectrometry (GC-MS) of their extracts. The spectrometric and microscopic images reveal that the calcium-phosphate mineralization is formed and constrained to Copium ommatidia, which are both matrix vesicles generating mixtures of apatite collagen and operational compound eyes of the insect.

A secreted variant of cartilage oligomeric matrix protein carrying a chondrodysplasia‐causing mutation (p.H587R) disrupts collagen fibrillogenesis

Mutations in human cartilage oligomeric matrix protein (COMP) cause multiple epiphyseal dysplasia or pseudoachondroplasia. Electron microscopic analyses of patient biopsy tissue have shown that, in most cases, mutated COMP is retained in granular or lamellar inclusions in the endoplasmic reticulum of chondrocytes. However, some mutations that do not interfere with protein trafficking, resulting in normal secretion of the mutated protein, have been identified. These mutations are likely to cause the chondrodysplasia phenotype, via events that occur after secretion. The aim of the present study was to identify such extracellular mechanisms associated with the pathogenesis of chondrodysplasias. A mutated but secreted COMP variant, p.H587R, as well as wild-type COMP were recombinantly expressed and purified from cell culture supernatants. Since recent studies have shown that COMP can facilitate collagen fibrillogenesis in vitro, the effect of the p.H587R mutation on this process was determined by analyzing the kinetics of fibrillogenesis in vitro and determining the structure of the collagen fibrils formed by immunogold electron microscopy. Mutated p.H587R COMP accelerated fibril formation by type I collagen in vitro to a slightly greater extent than that with wild-type COMP. However, p.H587R COMP induced aggregation and disorganization of fibril intermediates and end products. Mixtures of cartilage collagens or of type XI collagen alone produced similar results. The addition of p.H587R COMP to preformed fibrils induced aggregation and fusion of the fibrils, whereas wild-type COMP had little effect. The mutant COMP variant p.H587R generally interferes with normal collagen organization during fibrillogenesis. This constitutes a novel pathogenetic mechanism of COMP-associated chondrodysplasias.

Apoptosis Signal-Regulating Kinase 1 Regulates Platelet Function through Integrin aIIbb3 Outside-In Signaling

AbstractAbstract 328Atherosclerosis tends to develop in areas of the circulation where the blood flow is either low or turbulent, such as regions where the arteries bifurcate. Studies have shown that shear stress stimulates endothelial cells and modulates platelet function. However, the intracellular signaling events that coordinate these effects are not completely understood. The mitogen-activated protein kinase (MAPK) cascades are crucial in regulating cellular stress responses. It is known that a number of diseases, including cardiovascular diseases, are intimately related to stress related mechanisms mediated by MAPK cascades. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family which responses to diverse array of stresses. Here, we show for the first time that ASK1, a 155 kDa protein, is present in human and murine platelets and its role as a novel regulator of platelet function. In human platelets, when stimulated with thrombin, ASK1 is rapidly activated within 30 sec, as indicated by phosphorylation of Thr 845 on its activation loop. Interestingly, we found that ASK1 is rapidly inactivated after 3 min by dephosphorylation of phospho-Thr 845. Concomitantly, ASK1 is phosphorylated on Ser 967, a known inhibitory phosphorylation site. The activation of ASK1 coincided well with its downstream effector, p38 Map kinase. These results suggested that ASK1 may play a role in platelet function. We therefore tested the hemostatic functions in congenic Ask-1 knockout mice and compared it to C57/BL6 wild type (Wt) mice. We found a significant delay in tail bleeding time (P=0.2×10−9) in Ask1 null mice. While all Wt mice stopped bleeding with an average bleeding time of 100 sec, the Ask1 null mice had an average bleeding time of 576 sec, with 5 out of 9 mice that did not stop bleeding. A 10% FeCl3-induced carotid artery injury, a well-established in vivo thrombosis model, showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine. This finding was further supported by the histological examination of the lung tissue of mice surviving after 3 min of injection, which showed very few thrombi in Ask1 null mice compared to Wt mice. We next asked if the platelet function is affected in Ask1 null mice. We found that low dose of PAR-4 peptide-induced aggregation is significantly less in Ask1 null mice compared to Wt mice. When analyzed for the ability of Ask1 null platelets to retract clots, we found that while clot retraction was completed within 2h in Wt platelets, Ask1 null platelets failed to retract clots even after 18h. When assessed for phosphorylation of the integrin b3 subunit as an indicator of outside-in signaling, we found that Wt platelet exposure to immobilized fibrinogen showed a robust phosphorylation of b3 subunit. Interestingly, Ask1 null platelets failed to show any induction of b3 phosphorylation. These in vitro and in vivo results strongly suggest that ASK1 plays a significant role in the regulation of platelet function and hemostasis. Disclosures:No relevant conflicts of interest to declare.

The effect of interleukin-13 (IL-13) and interferon-γ (IFN-γ) on expression of surfactant proteins in adult human alveolar type II cells in vitro

BackgroundSurfactant proteins are produced predominantly by alveolar type II (ATII) cells, and the expression of these proteins can be altered by cytokines and growth factors. Th1/Th2 cytokine imbalance is suggested to be important in the pathogenesis of several adult lung diseases. Recently, we developed a culture system for maintaining differentiated adult human ATII cells. Therefore, we sought to determine the effects of IL-13 and IFN-γ on the expression of surfactant proteins in adult human ATII cells in vitro. Additional studies were done with rat ATII cells.MethodsAdult human ATII cells were isolated from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research. The cells were cultured on a mixture of Matrigel and rat-tail collagen for 8 d with differentiation factors and human recombinant IL-13 or IFN-γ.ResultsIL-13 reduced the mRNA and protein levels of surfactant protein (SP)-C, whereas IFN-γ increased the mRNA level of SP-C and proSP-C protein but not mature SP-C. Neither cytokine changed the mRNA level of SP-B but IFN-γ slightly decreased mature SP-B. IFN-γ reduced the level of the active form of cathepsin H. IL-13 also reduced the mRNA and protein levels of SP-D, whereas IFN-γ increased both mRNA and protein levels of SP-D. IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A.ConclusionsWe demonstrated that IL-13 and IFN-γ altered the expression of surfactant proteins in human adult ATII cells in vitro. IL-13 decreased SP-C and SP-D in human ATII cells, whereas IFN-γ had the opposite effect. The protein levels of mature SP-B were decreased by IFN-γ treatment, likely due to the reduction in active form cathpesin H. Similarly, the active form of cathepsin H was relatively insufficient to fully process proSP-C as IFN-γ increased the mRNA levels for SP-C and proSP-C protein, but there was no increase in mature SP-C. These observations suggest that in disease states with an overexpression of IL-13, there would be some deficiency in mature SP-C and SP-D. In disease states with an excess of IFN-γ or therapy with IFN-γ, these data suggest that there might be incomplete processing of SP-B and SP-C.

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Solitary neurofibroma of the gingiva with prominent differentiation of Meissner bodies: a case report

BackgroundOral neurofibromas are peripheral nerve sheath tumors, similar to schwannomas. Histological variations in oral neurofibromas are relatively uncommon.Case presentationHere, we present a case of unique variation in the observed characteristics of a neurofibroma, with no relation to neurofibromatosis type-1 or von Recklinghausen disease of the skin. The neurofibroma was observed in the right mandibular gingiva of a 32-year-old Japanese woman. Histologically, it differed from conventional neurofibromas in that the tumor was composed of a mixture of fine fibrillary collagen in sheets and/or cords of neoplastic Schwann cells containing numerous clusters of Meissner bodies. Histologically, these bodies were in contact with neoplastic Schwann cells. The Meissner bodies were immunopositive for S-100 protein, neuron-specific enolase, and vimentin, but were negative for calretinin. CD34-positive spindle cells were observed around the Meissner bodies. No recurrence or signs of other tumors have been observed in the patient for 5 years after tumor resection.ConclusionTo the best of our knowledge, no formal descriptions of sporadic, solitary neurofibromas containing numerous Meissner bodies occurring in the oral cavity are available in literature. We believe that an uncommon proliferation of Meissner bodies, as seen in the present case, may result from aberrant differentiation of neoplastic Schwann cells.

Proteomic evaluation on antiplatelet activity of piperlongumine derived from Piper longum

Piperlongumine (PL) abundantly present in Piper longum fruits strongly inhibited platelet cell aggregation. Proteomic studies using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and LC-MS/MS analysis showed that PL inhibited collagen-induced aggregation in rabbit platelet cells induced by collagen. Addition of PL protected aggregation of rabbit platelet cells from collagen induction and mediated proteomic changes as 33 proteins decreased and 24 proteins increased. Coagulation factor and fibrinogen were down-regulated and they are involved in aggregation of the platelet cells. Antithrombogenic effect of PL was determined using mice with an i.v. injection of a mixture of collagen and epinephrine. Protection rate of PL on thrombogenic effect of the mixtures was up to 47.9% and it was 8.6% higher than that of aspirin. Taken together, these results strongly suggest that PL is considered as a potent medicine for treating unexpected platelet cell aggregation.

Anatomic Study of the Nostril Sill

To define and investigate the normal nostril sill anatomy and classify the different forms.Photographs of the nose of 95 Korean adults and 26 preserved cadavers were studied. Classification of the different forms and cadaveric dissections and histologic studies were performed. Morphologically the noses were studied and classified into 3 types, and histologically the thickness of the soft tissue and muscle layers were measured at the nostril sill.The nostril sill was defined as the soft tissue bulge between 4 borders; medially the foot plate of the columella, laterally the ala, cephalically the vestibule, and caudally the upper lip, and classified into 3 forms. Type I: full nostril sill (55.7%), type II: point nostril sill (31.9%), and type III: flat nostril sill (12.1%). On histologic analysis, in type I, the sill consisted of fibro fatty tissue composed of a mixture of dermal collagen and gland which were thicker at the area of the sill. Also the muscle fibers beneath the sill were more protuberant in this area. The thickness of the soft tissue and muscle layers were significantly thicker at the sill area than the upper lip and vestibule. In types II and III, dermal thickening was not observed and the soft tissue thickness remained unchanged from the upper lip to the nasal vestibule. The muscle fibers showed no protuberance under the sill.This study provides references which will allow more accurate and proper correction and reconstruction of the nose and nostril sill.

Mechanical Interactions of Mouse Mammary Gland Cells with Collagen in a Three-Dimensional Construct

An effort to understand the development of breast cancer motivates the study of mammary gland cells and their interactions with the extracellular matrix. A mixture of mammary gland epithelial cells (normal murine mammary gland), collagen, and fluorescent beads was loaded into microchannels and observed via four-dimensional imaging. Collagen concentrations of 1.3, 2, and 3 mg/mL were used. The displacements of the beads were used to calculate strains in the 3D matrix. To ensure physiologically relevant materials properties for analysis, the collagen was characterized using independent tensile testing with strain rates in the range of those measured in the cell-gel constructs. 3D elastic theory for an isotropic material was employed to calculate the stress. The technique presented adds to the field of measuring cell-generated stresses by providing the capability of measuring 3D stresses locally around a single cell and using physiologically relevant materials properties for analysis. The highest strains were observed in the most compliant matrix. Additionally, the stresses fluctuated over time due to the cells' interaction with the collagen matrix.