The cellular localization of prosystemin: a functional role for phloem parenchyma in systemic wound signaling

The systemin precursor, prosystemin, has been previously shown to be sequestered in vascular bundles of tomato (Lycopersicon esculentum Mill.) plants, but its subcellular compartmentalization and association with a specific cell type has not been established. We present in situ hybridization and immunocytochemical evidence at the light, confocal, and transmission electron microscopy levels that wound-induced and methyl jasmonate-induced prosystemin mRNA and protein are exclusively found in vascular phloem parenchyma cells of minor veins and midribs of leaves, and in the bicollateral phloem bundles of petioles and stems of tomato. Prosystemin protein was also found constitutively in parenchyma cells of various floral organs, including sepals, petals and anthers. At the subcellular level, prosystemin was found compartmentalized in the cytosol and the nucleus of vascular parenchyma cells. The cumulative data indicate that vascular phloem parenchyma cells are the sites for the synthesis and processing of prosystemin as a first line of defense signaling in response to herbivore and pathogen attacks.Abbreviations IgG immunoglobulin - TEM transmission electron microscope     

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin. Here we show that FGFR1-IIIb is expressed in normal and wounded mouse skin. Reduced expression of this type of receptor was found in wounds of healing-impaired genetically diabetic mice, suggesting that downregulation of FGFR1-IIIb is associated with wound healing defects. To address this possibility, we deleted the IIIb exon of FGFR1 in mice. The lack of FGFR-IIIb did not alter the expression of either FGFR1-IIIc, other FGF receptor genes or of FGFR1-IIIb ligands in normal and wounded skin. Histological analysis of the skin of FGFR1-IIIb knockout animals did not reveal any obvious abnormalities. Furthermore, full-thickness excisional skin wounds in these mice healed normally and no defects could be observed at the macroscopic or histological level. Finally, several genes that encode key players in wound repair were normally expressed in these animals. These data demonstrate that FGFR1-IIIb is dispensable for skin development and wound repair.     

Permanent restoration of human skin treated with cultured epithelium grafting--wound healing by stem cell based tissue engineering--

The technique of epidermal cell culture developed by Green and colleagues made a breakthrough in the treatment of massive wounds in vivo with grown cells in vitro. In the past two decades, progress of culture methods and clinical practice have been made and now it is possible to treat extensive skin defect with large amounts of cultured epithelium. Since 1985, we have been successfully used cultured epidermis as autografts for the permanent coverage of full-thickness burn wounds or excised burn scars, giant nevi, tattoos and so on. Furthermore, cultured epidermis has been available as allografts to promote the healing of chronic skin ulcers or deep dermal burn. In this paper we describe our clinical experience of cultured epithelium grafting for the treatment of wounds and predict new trial of wound management and regeneration based on tissue engineering concept.     

Oxygen modulates the growth of skin fibroblasts

Elevated oxygen tensions are inhibitory to the growth of skin fibroblasts. Skin fibroblasts grow better at oxygen tensions below 137 mm Hg regardless of seeding density. A wide range of oxygen tensions, including those in the physiological range, strongly modulate the growth of human skin fibroblasts. There were no significant differences between the responses of fetal and postnatal cell lines to changes in ambient oxygen tension. In all cases, higher oxygen tensions significantly impeded cell growth. Seeding cells at 10(4) cells/cm(2) afforded some protection from the deleterious effects of hyperoxia. Oxygen tensions exceeding the amount present in ambient room air also impeded cell growth at this higher seeding density, but the effect did not become significant until the oxygen partial pressure reached 241 mm Hg. At lower oxygen tensions, cells seeded at 10(3) cells/cm(2) grew more rapidly than did cells seeded at 10(4) cells/cm(2). These findings may have implications for the treatment of poorly healing wounds with hyperbaric oxygen.     

Developmental stages and fine structure of surface callus formed after debarking of living lime trees (Tilia sp.)

Wounding of trees by debarking during the vegetative period sometimes results in the formation of callus tissue which develops over the entire wound surface or on parts of it. This light and transmission electron microscopy study of living lime trees found that the formation of such a surface callus is subdivided into three stages. During the first stage, numerous cell divisions take place in regions where differentiating xylem remains at the wound surface after debarking. This young callus tissue consists of isodiametric parenchymatous cells. Cambium cells, sometimes also remaining at the wound surface, collapse and do not contribute to callus formation. During the second stage, cells in the callus undergo differentiation by forming a wound periderm with phellem, phellogen and phelloderm. In the third stage, a cambial zone develops between the wound periderm and the xylem tissue laid down prior to wounding. This process is initiated by anticlinal and periclinal divisions of a few callus cells only. Later this process extends tangentially to form a continuous belt of wound cambium. Subsequently, this cambium produces both wound xylem and wound phloem and thus contributes to further thickening.     

Inhibition of regeneration in the planarian Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological study of wound closure

At a concentration of 0.2% (21 m M) in culture water, magnesium chloride impaired muscle contraction and completely inhibited head regeneration in specimens of Dugesia polychroa cut prepharyngeally. The wound stayed open for nine days, with neoblasts accumulating beneath the wound without any signs of differentiation. Extremely delayed wound closure occurred by spreading epithelial cells, and was completed after 30 days in the magnesium chloride solution. Histological examination confirmed the absence of any regenerated head structures. Interestingly, the inhibitory effect was removed when such headless fragments were cut once more and kept in normal culture water: complete head regeneration then occurred at a normal rate. Among several possible explanations for the failure to regenerate, the following hypothesis is an attractive alternative: direct contact between parenchyma and epithelial cells during the period following injury seems to be an essential stimulus for the start of cell differentiation within the blastema, and the lack of such contact as a result of the drug action prevents normal regeneration. When the wound has eventually closed, a continuous basement membrane separates epithelium from parenchyma. Thus a direct contact between these tissues is never established. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

Live imaging early immune cell ontogeny and function in zebrafish Danio rerio

The success of a robust vertebrate inflammatory response is in part because of the migratory potential of its haematopoietic components. Once these cells converge at an inflammatory site, they interact with each other as well as non‐immune tissues and infectious agents to help manage both the scale and the duration of any ensuing response. Exactly how these blood cells, that constitute the innate and adaptive immune systems, contribute to such immune responses remain largely unknown. Traditionally, assessing these contributions relied upon histological analysis of fixed tissue sections complemented with in vitro dynamic data. Although informative, translating results from these studies into the multicellular whole‐animal setting remain difficult. Recently, non‐invasive live imaging of the immune system in animal models is providing significant insights into how immune cells function within their intact natural environment. Although the majority of these studies have been conducted within mice, another vertebrate, the zebrafish Danio rerio is being recognized as an ideal platform for non‐invasive live imaging applications. The optical transparency, rapid development, genetic tractability and highly conserved innate and adaptive immune systems of this well‐established developmental model have been exploited in a number of recent studies evaluating the immunocompetence of fluorescently tagged blood cells. In addition, similar live imaging studies are helping to dissect the ontogeny of blood‐cell development by tracking various haematopoietic precursor cells to assess their contribution to different blood lineages. This review will examine some recent advances that have helped D. rerio emerge as a live imaging platform as well as its potential to offer valuable insights into the genetics behind diseases associated with immune cell dysfunction.     

Anti-inflammatory and wound healing potential of cashew apple juice (Anacardium occidentale L.) in mice

Cashew apple is a tropical pseudofruit consumed as juice due to its excellent nutritional and sensory properties. In spite of being well known for its important antioxidant properties, the cashew apple has not been thoroughly investigated for its therapeutic potential. Thereby, this study evaluated the antioxidant capacity, anti-inflammatory, and wound-healing activities of cashew apple juice. Juices from ripe and immature cashew apples were analyzed for antioxidant, anti-inflammatory, and wound-healing properties. Those were evaluated in murine models of xylene-induced ear edema and wound excision. Swiss mice were treated with cashew juice by gavage. Edema thickness was measured and skin lesions were analyzed by planimetry and histology. Both antioxidant content and total antioxidant activity were higher in ripe cashew apple juice (RCAJ) than in unripe cashew apple juice (UNCAJ). The UNCAJ presented the main anti-inflammatory activity by a significant inhibition of ear edema (66.5%) when compared to RCAJ (10%). Moreover, UNCAJ also showed the best result for wound contraction (86.31%) compared to RCAJ (67.54%). Despite of higher antioxidant capacity, RCAJ did not promote better anti-inflammatory, and healing responses, which may be explained by the fact that treatment increased antioxidants level leading to a redox “imbalance” turning down the inflammatory response modulation exerted by reactive oxygen species (ROS). The results suggest that UNCAJ presents a greater therapeutic activity due to a synergistic effect of its phytochemical components, which improve the immunological mechanisms as well as an optimal balance between ROS and antioxidants leading to a better wound healing process.     

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.