scholarly journals In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface.

1984 ◽  
Vol 98 (6) ◽  
pp. 2091-2106 ◽  
Author(s):  
I I Singer ◽  
D W Kawka ◽  
D M Kazazis ◽  
R A Clark
Keyword(s):  
Granulation Tissue ◽  
Wound Repair ◽  
Immunofluorescence Microscopy ◽  
Actin Microfilaments ◽  
Surface Adhesion ◽  
Microfilament Bundles ◽  
Binding Surface ◽  
Double Label ◽  
Fibroblast Adhesion

The fibronexus ( FNX ), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substrate-binding surface of fibroblasts in tissue culture (Singer, 1. 1., 1979, Cell, 16:675-685). To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds. At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy. These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy. We observed three kinds of FNXs : (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs , in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils. Goniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial. Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin. The finding that these FNXs appear to connect collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation.

scholarly journals Wound repair and regeneration potential of the fruits of Terminalia bellarica

2015 ◽  
Vol 4 (5) ◽  
pp. 253-258
Author(s):  
S. Kirubanandan ◽  
◽  
S. Renganathan ◽  
Keyword(s):  
Wound Healing ◽  
Matrix Metalloproteinase ◽  
Granulation Tissue ◽  
Wound Repair ◽  
Treated Group ◽  
Bioactive Molecules ◽  
Synergistic Activity ◽  
Regeneration Potential ◽  
Soxhlet Apparatus

The infection at the wound site is a severe kind of problem and it delays regeneration of epidermis and dermis in the wound and slows wound closure. Due to the secretion of microbial enzymes by wound pathogens, a variety of extracellular matrix proteins were degraded. Synthetic antimicrobial therapy used in the wound management and eradication of pathogens. However, it has many shortcomings such as anti-microbial resistance, cyto-toxicity against host tissue and absence of synergistic activity. In order to overcome these limitations, Pyto-pharmaceuticals extracted from herbal plants were applied to manage the wound infection and treatment. The objective of this work is to evaluate the wound repair and regeneration potential of the fruits of Terminalia bellarica which has a variety of pharmacological activities such as astringent, antiseptic and laxative. The dry fruits of Terminalia bellarica were grounded into powder form using the grinder. Extraction was performed by using Soxhlet apparatus with 95% (v/v) ethanol. The dried extract was dissolved in Dimethyl Sulfoxide (DMSO) and used to assay the antibacterial activity against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853. An ointment was prepared from the ethanol extract (10% w/w) and assessed for its in vivo wound healing potential on infected rat model by rate of healing, bacterial count, biochemical analysis, and expression of matrix metalloproteinase. In addition to that, the collagen content in the granulation tissue was estimated to comment on wound regeneration potential of the fruits of Terminalia bellarica. The treated group has shown significantly improved wound regeneration and well formed epidermis and dermis in the granulation tissue. Furthermore, Assessment of granulation tissue on every fourth day showed significant reduction in bacterial pathogens CFU with significant elevated level of collagen, hexosamine, uronic acid, in the treated group (P<0.05). The reduced level expression of matrix metalloproteinase expression observed in the treated group by gelatin zymography and the synthesis of substantial amount of collagen in the granulation tissue confirms our in vivo assessment. The results showed the antibacterial and wound healing activities of Terminalia bellarica fruits ointment, necessary for the management of infected open dermal wounds. The isolation of bioactive molecules from Terminalia bellarica fruits and its interaction various cells using cell culture studies would be future work.

scholarly journals Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor.

1985 ◽  
Vol 100 (4) ◽  
pp. 1219-1227 ◽  
Author(s):  
J M Davidson ◽  
M Klagsbrun ◽  
K E Hill ◽  
A Buckley ◽  
R Sullivan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Granulation Tissue ◽  
Dna Content ◽  
Wound Repair ◽  
Cultured Cells ◽  
Costal Cartilage ◽  
Relative Increase ◽  
Bovine Articular Cartilage

Cartilage-derived growth factor (CDGF), a cationic polypeptide of approximately 18,000 mol wt, was prepared from bovine articular cartilage; other sources were bovine and human scapular and costal cartilage. Previous studies have shown that CDGF stimulates the proliferation of cultured mouse fibroblasts as well as chondrocytes and endothelial cells from various sources. In this study, CDGF was shown to stimulate dose-dependently the accumulation of DNA and collagen by rat embryo fibroblasts and a population of fibroblasts derived from granulation tissue. CDGF also stimulated the proliferation of cultured bovine capillary endothelial cells dose-dependently. To evaluate the effects of CDGF in vivo, we implanted polyvinyl alcohol sponges subcutaneously in rats. 6 d postimplantation, sponges were injected with 300 micrograms of partially purified CDGF, a dose which takes into account the cell numbers in the sponges as compared with cell cultures. CDGF rapidly disappeared from the sponges and only approximately 10% of the initial dose was present at 4 h. Despite its transient presence, CDGF caused a relative increase in sponge DNA content of 2.6-fold at 48 h and 2.4-fold at 72 h. We repeated the sponge experiment by using 500-ng injections of CDGF purified to near homogeneity by heparin-Sepharose chromatography. Purified CDGF caused significant increases in sponge collagen, protein, and DNA content at 48 and 72 h after a single injection. The effects of CDGF were abolished by heat and unaffected by reduction of disulfide linkages. Morphologically, CDGF did not evoke an inflammatory response, and its effect on proliferating endothelial cells and fibroblasts was, therefore, probably direct. However, increases in DNA content of sponges could not be fully accounted for by increased DNA synthesis, which suggests that recruitment may be an important component of the in vivo response. Taken together, the effects of CDGF on cultured cells and granulation tissue suggest that the sustained presence of CDGF in vivo may greatly enhance its effects upon wound repair.

scholarly journals Dynamin-mediated Internalization of Caveolae

1998 ◽  
Vol 141 (1) ◽  
pp. 85-99 ◽  
Author(s):  
John R. Henley ◽  
Eugene W.A. Krueger ◽  
Barbara J. Oswald ◽  
Mark A. McNiven
Keyword(s):  
Mammalian Cells ◽  
Immunofluorescence Microscopy ◽  
In Vivo Studies ◽  
Coated Pits ◽  
Toxin B ◽  
Cholera Toxin B ◽  
Double Label ◽  
Biochemical Analyses ◽  
Redundant Functions

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.

scholarly journals Co-Transplantation of Skin-Derived Precursors and Collagen Sponge Facilitates Diabetic Wound Healing by Promoting Local Vascular Regeneration

2015 ◽  
Vol 37 (5) ◽  
pp. 1725-1737 ◽  
Author(s):  
Tingyu Ke ◽  
Mei Yang ◽  
Duo Mao ◽  
Meifeng Zhu ◽  
Yongzhe Che ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Repair ◽  
Expression Patterns ◽  
Collagen Sponge ◽  
Health Concern ◽  
Diabetic Wound ◽  
Paracrine Signalling ◽  
Vascular Regeneration ◽  
Diabetic Wound Healing

Background/Aims: Impaired diabetes wound healing can often lead to serious complications and remains a major health concern due to the lack of effective therapeutic approaches. Compromised angiogenesis, disrupted growth factor and cytokine activity are all attributable to diabetic wound healing impairment. The skin-derived precursors (SKPs) have been shown to differentiate into vascular and nerve cells, both of which are crucial components for wound repair. Given their easy accessibility and multipotency, the SKPs were proposed as an ideal therapeutic candidate for diabetic wound healing. Since the efficacy of cell therapy is limited by poor cell survival, collagen sponge was employed for better SKPs delivery. Methods: SKPs were isolated and transplanted directly to the wound areas of diabetic mice in the absence and presence of collagen sponge. The effects of SKPs and/or collagen sponge on diabetic wound healing were examined histologically as well as immunostaining of isolectin and α-SMA. Mechanisms via which the SKPs facilitate wound healing were then investigated by transplanting SKPs that have been pre-labelled with a fluorescence dye, Dil. Expression patterns of Dil and an SKP marker, nestin, was also examined. Results and Conclusion: Accelerated wound healing and enhanced local capillary regeneration could be observed 14 days after skin ablation from both SKPs and collagen sponge co-transplanted and collagen sponge only groups. Subsequent analyses further revealed superior pro-angiogenic effects from the SKP and collagen sponge co-delivered group, which are mainly attributable to in vivo transdifferentation and paracrine signalling of the SKPs.

Nanoscale Characterisation of Salivary Pellicle

2004 ◽  
Vol 841 ◽  
Author(s):  
Michelle E. Dickinson ◽  
Adrian B. Mann
Keyword(s):  
Dental Enamel ◽  
Tooth Surface ◽  
Surface Adhesion ◽  
Human Enamel ◽  
Oral Physiology ◽  
Salivary Pellicle ◽  
Initial Adsorption ◽  
Dietary Agents

ABSTRACTSalivary pellicle is an organic biofilm formed by the physisorption of proteins and carbohydrates onto the surface of dental enamel exposed to the oral environment. The pellicle has several key roles in oral physiology including lubrication and reduction of friction between teeth during mastication, as well as chemical protection of the enamel against acidic solutions. However, pellicle proteins are known to react with dietary compounds to cause extrinsic staining on the tooth surface.In this study, nanoindentation and AFM have been used in vitro to examine the acquired salivary pellicle formed in vivo on dental enamel. The mechanical properties, growth, structure and morphology of pellicle grown in vivo on human enamel surfaces have been analysed. In addition, the effects of dietary agents such as polyphenols on the pellicle's morphology and properties have been studied.It was found that initial adsorption of proteins on the enamel surface occurred within 30 seconds of exposure to the oral cavity, with full growth achieved within 2 hours. Differences in the properties of the pellicles such as surface adhesion, and time dependent effects due to polyphenol interaction were measured using nanoindentation. It was seen that the polyphenol interaction has a significant effect on these properties. These results suggest that the stained pellicle is mechanically stiffer, but also less viscous and more fluid like. This could explain why traditional tooth brushing techniques do not efficiently remove this layer.

scholarly journals Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.

1984 ◽  
Vol 98 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
W C Thompson ◽  
D J Asai ◽  
D H Carney
Keyword(s):  
Differential Staining ◽  
Hypotonic Medium ◽  
Cytoplasmic Microtubule ◽  
Alpha Tubulin ◽  
Apparent Correlation ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
Fibroblastic Cells ◽  
In Cells

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.

scholarly journals Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures.

1984 ◽  
Vol 99 (5) ◽  
pp. 1696-1705 ◽  
Author(s):  
P C Marchisio ◽  
D Cirillo ◽  
L Naldini ◽  
M V Primavera ◽  
A Teti ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Laying Hens ◽  
Cell Types ◽  
Medullary Bone ◽  
Transformed Cell ◽  
The Core ◽  
Low Calcium Diet

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.

scholarly journals Structures linking microfilament bundles to the membrane at focal contacts

1993 ◽  
Vol 122 (2) ◽  
pp. 485-496 ◽  
Author(s):  
SJ Samuelsson ◽  
PW Luther ◽  
DW Pumplin ◽  
RJ Bloch
Keyword(s):  
Single Bundle ◽  
Actin Microfilaments ◽  
Lateral Interactions ◽  
Membrane Interactions ◽  
Membrane Particles ◽  
Focal Contacts ◽  
Microfilament Bundles ◽  
Immunogold Cytochemistry ◽  
Beta 1 Integrin

We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.

Abstract 13373: Exercise Improves Angiogenic Function of Circulating Exosomes in Type 2 Diabetes: Role of Extracellular SOD

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kareem Abdelsaid ◽  
Sudhahar Varadarajan ◽  
Archita Das ◽  
Yutao Liu ◽  
Xuexiu Fang ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Wound Healing ◽  
Endothelial Dysfunction ◽  
Wound Repair ◽  
Cell Communication ◽  
Tube Formation ◽  
Skin Wound ◽  
Skin Wound Healing

Background: Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) have detrimental effects. Exercise not only improves endothelial dysfunction and angiogenesis in T2DM but also induces secretion of exosomes into circulation. Extracellular superoxide dismutase (ecSOD) is a major secretory Cu containing antioxidant enzyme that catalyzes dismutation of O 2 •- to H 2 O 2 and its full activity requires Cu transporter ATP7A. We reported that ecSOD-derived H 2 O 2 in endothelial cells (ECs) enhances angiogenesis while impaired ATP7A-ecSOD axis in diabetes induces endothelial dysfunction. Here we examined whether exercise-derived exosomes (Exe-Exo) may have pro-angiogenic effects via regulating ATP7A-ecSOD axis in T2DM. Results: Two weeks of voluntary wheel exercise of control C57Bl6 mice increased plasma exosome levels (6.2-fold) characterized by Nanosight, TEM and exosome markers (CD63, CD81, Tsg101). Treatment of HUVECs with equal number of exosomes revealed that angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold) were significantly enhanced by Exe-Exo compared to sedentary-derived exosomes (Sed-Exo). This was associated with increased ATP7A (2.9-fold) and ecSOD (1.4-fold) expression in Exe-Exo. Sed-Exo from high fat-induced T2DM mice significantly decreased EC migration (40%) and tube formation (10%) as well as ATP7A expression (28%) compared to Sed-Exo from control mice, which were restored by T2DM Exe-Exo, but not by T2DM/ecSOD KO Exe-Exo. Furthermore, exosomes overexpressing ecSOD (ecSOD-Exo) which mimic exercise increased angiogenesis and H2O2 levels in ECs, which were inhibited by overexpression of catalase. In vivo, skin wound healing model showed that direct application of T2DM Sed-Exo delayed while T2DM Exe-Exo enhanced wound healing of control mice. Furthermore, defective wound healing in T2DM mice or ecSOD KO mice were rescued by ecSOD-Exo application. Conclusion: Exercise training improves pro-angiogenic function of circulating exosomes in T2DM via increasing ATP7A-ecSOD axis, which may provide an effective therapy for promoting angiogenesis and wound repair in metabolic and cardiovascular diseases.

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

2007 ◽  
Vol 292 (2) ◽  
pp. L529-L536 ◽  
Author(s):  
Amiq Gazdhar ◽  
Patrick Fachinger ◽  
Coretta van Leer ◽  
Jaroslaw Pierog ◽  
Mathias Gugger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Gene Transfer ◽  
Pulmonary Fibrosis ◽  
Wound Repair ◽  
Alveolar Epithelial Cells ◽  
Epithelial Repair ◽  
Alveolar Epithelial ◽  
Hepatocyte Growth

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-β1 (TGF-β1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-β1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

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