A survey of integral α-helical membrane proteins

Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1) to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily, (2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane protein families

Analysis of Allogenicity of Mesenchymal Stem Cells in Engraftment and Wound Healing in Mice

Studies have shown that allogeneic (allo-) bone marrow derived mesenchymal stem cells (BM-MSCs) may enhance tissue repair/regeneration. However, recent studies suggest that immune rejection may occur to allo-MSCs leading to reduced engraftment. In this study, we compared allo-BM-MSCs with syngeneic BM-MSCs or allo-fibroblasts in engraftment and effect in wound healing. Equal numbers of GFP-expressing allo-BM-MSCs, syngeneic BM-MSCs or allo-fibroblasts were implanted into excisional wounds in GFP-negative mice. Quantification of GFP-expressing cells in wounds at 7, 14 and 28 days indicated similar amounts of allogeneic or syngeneic BM-MSCs but significantly reduced amounts of allo-fibroblasts. With healing progression, decreasing amounts of allogeneic and syngeneic BM-MSCs were found in the wound; however, the reduction was more evident (2 fold) in allo-fibroblasts. Similar effects in enhancing wound closure were found in allogeneic and syngeneic BM-MSCs but not in allo-fibroblasts. Histological analysis showed that allo-fibroblasts were largely confined to the injection sites while allo-BM-MSCs had migrated into the entire wound. Quantification of inflammatory cells in wounds showed that allo-fibroblast- but not allo-BM-MSC-treated wounds had significantly increased CD45+ leukocytes, CD3+ lymphocytes and CD8+ T cells. Our study suggests that allogeneic BM-MSCs exhibit ignorable immunogenicity and are equally efficient as syngeneic BM-MSCs in engraftment and in enhancing wound healing

Dopamine Regulates Angiogenesis in Normal Dermal Wound Tissues

Cutaneous wound healing is a normal physiological process and comprises different phases. Among these phases, angiogenesis or new blood vessel formation in wound tissue plays an important role. Skin is richly supplied by sympathetic nerves and evidences indicate the significant role of the sympathetic nervous system in cutaneous wound healing. Dopamine (DA) is an important catecholamine neurotransmitter released by the sympathetic nerve endings and recent studies have demonstrated the potent anti-angiogenic action of DA, which is mediated through its D2 DA receptors. We therefore postulate that this endogenous catecholamine neurotransmitter may have a role in the neovascularization of dermal wound tissues and subsequently in the process of wound healing. In the present study, the therapeutic efficacy of D2 DA receptor antagonist has been investigated for faster wound healing in a murine model of full thickness dermal wound. Our results indicate that treatment with specific D2 DA receptor antagonist significantly expedites the process of full thickness normal dermal wound healing in mice by inducing angiogenesis in wound tissues. The underlined mechanisms have been attributed to the up-regulation of homeobox transcription factor HoxD3 and its target α5β1 integrin, which play a pivotal role in wound angiogenesis. Since D2 DA receptor antagonists are already in clinical use for other disorders, these results have significant translational value from the bench to the bedside for efficient wound management along with other conventional treatment modalities

Dopamine Regulates Mobilization of Mesenchymal Stem Cells during Wound Angiogenesis

Angiogenesis is an important step in the complex biological and molecular events leading to successful healing of dermal wounds. Among the different cellular effectors of wound angiogenesis, the role of mesenchymal stem cells (MSCs) is of current interest due to their transdifferentiation and proangiogenic potentials. Skin is richly innervated by sympathetic nerves which secrete dopamine (DA) and we have recently shown that concentration of DA present in synaptic cleft can significantly inhibit wound tissue neovascularization. As recent reports indicate that MSCs by mobilizing into wound bed play an important role in promoting wound angiogenesis, we therefore investigated the effect of DA on the migration of MSCs in wound tissues. DA acted through its D2 receptors present in the MSCs to inhibit their mobilization to the wound beds by suppressing Akt phosphorylation and actin polymerization. In contrast, this inhibitory effect of DA was reversed after treatment with specific DA D2 receptor antagonist. Increased mobilization of MSCs was demonstrated in the wound site following blockade of DA D2 receptor mediated actions, and this in turn was associated with significantly more angiogenesis in wound tissues. This study is of translational value and indicates use of DA D2 receptor antagonists to stimulate mobilization of these stem cells for faster regeneration of damaged tissues

Long-Term Alterations of Cytokines and Growth Factors Expression in Irradiated Tissues and Relation with Histological Severity Scoring

PURPOSE: Beside its efficacy in cancer treatment, radiotherapy induces degeneration of healthy tissues within the irradiated area. The aim of this study was to analyze the variations of proinflammatory (IL-1α, IL-2, IL-6, TNF-α, IFN-γ), profibrotic (TGF-β1), proangiogneic (VEGF) and stem cell mobilizing (GM-CSF) cytokines and growth factors in an animal model of radiation-induced tissue degeneration. MATERIALS AND METHODS: 24 rats were irradiated unilaterally on the hindlimb at a monodose of 30 Gy. Six weeks (n=8), 6 months (n=8) and 1 year (n=8) after irradiation the mediators expression in skin and muscle were analyzed using Western blot and the Bio-Plex® protein array (BPA) technology. Additional histological severity for fibrosis, inflammation, vascularity and cellularity alterations scoring was defined from histology and immnunohistochemistry analyses. RESULTS: A significant increase of histological severity scoring was found in irradiated tissue. Skin tissues were more radio-sensitive than muscle. A high level of TGF-β1 expression was found throughout the study and a significant relation was evidenced between TGF-β1 expression and fibrosis scoring. Irradiated tissue showed a chronic inflammation (IL-2 and TNF-α significantly increased). Moreover a persistent expression of GM-CSF and VEGF was found in all irradiated tissues. The vascular score was related to TGF-β1 expression and the cellular alterations score was significantly related with the level of IL-2, VEGF and GM-CSF. CONCLUSION: The results achieved in the present study underline the complexity and multiplicity of radio-induced alterations of cytokine network. It offers many perspectives of development, for the comprehension of the mechanisms of late injuries or for the histological and molecular evaluation of the mode of action and the efficacy of rehabilitation techniques

Topical Insulin Accelerates Wound Healing in Diabetes by Enhancing the AKT and ERK Pathways: A Double-Blind Placebo-Controlled Clinical Trial

Background: Wound healing is impaired in diabetes mellitus, but the mechanisms involved in this process are virtually unknown. Proteins belonging to the insulin signaling pathway respond to insulin in the skin of rats. Objective: The purpose of this study was to investigate the regulation of the insulin signaling pathway in wound healing and skin repair of normal and diabetic rats, and, in parallel, the effect of a topical insulin cream on wound healing and on the activation of this pathway. Research Design and Methods: We investigated insulin signaling by immunoblotting during wound healing of control and diabetic animals with or without topical insulin. Diabetic patients with ulcers were randomized to receive topical insulin or placebo in a prospective, double-blind and placebo-controlled, randomized clinical trial (NCT 01295177) of wound healing. Results and Conclusions: Expression of IR, IRS-1, IRS-2, SHC, ERK, and AKT are increased in the tissue of healing wounds compared to intact skin, suggesting that the insulin signaling pathway may have an important role in this process. These pathways were attenuated in the wounded skin of diabetic rats, in parallel with an increase in the time of complete wound healing. Upon topical application of insulin cream, the wound healing time of diabetic animals was normalized, followed by a reversal of defective insulin signal transduction. In addition, the treatment also increased expression of other proteins, such as eNOS (also in bone marrow), VEGF, and SDF-1 alpha in wounded skin. In diabetic patients, topical insulin cream markedly improved wound healing, representing an attractive and cost-free method for treating this devastating complication of diabetes.Sao Paulo Research Foundation (FAPESP)Sao Paulo Research Foundation (FAPESP)National Institute of Science and Technology (INCT)National Institute of Science and Technology (INCT)National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer

Functional tissue engineering of ligament healing

Ligaments and tendons are dense connective tissues that are important in transmitting forces and facilitate joint articulation in the musculoskeletal system. Their injury frequency is high especially for those that are functional important, like the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) of the knee as well as the glenohumeral ligaments and the rotator cuff tendons of the shoulder. Because the healing responses are different in these ligaments and tendons after injury, the consequences and treatments are tissue- and site-specific. In this review, we will elaborate on the injuries of the knee ligaments as well as using functional tissue engineering (FTE) approaches to improve their healing. Specifically, the ACL of knee has limited capability to heal, and results of non-surgical management of its midsubstance rupture have been poor. Consequently, surgical reconstruction of the ACL is regularly performed to gain knee stability. However, the long-term results are not satisfactory besides the numerous complications accompanied with the surgeries. With the rapid development of FTE, there is a renewed interest in revisiting ACL healing. Approaches such as using growth factors, stem cells and scaffolds have been widely investigated. In this article, the biology of normal and healing ligaments is first reviewed, followed by a discussion on the issues related to the treatment of ACL injuries. Afterwards, current promising FTE methods are presented for the treatment of ligament injuries, including the use of growth factors, gene delivery, and cell therapy with a particular emphasis on the use of ECM bioscaffolds. The challenging areas are listed in the future direction that suggests where collection of energy could be placed in order to restore the injured ligaments and tendons structurally and functionally

Phospholipase D signaling: orchestration by PIP2 and small GTPases

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction. The synthesis of PIP2 by phosphoinositide 5-kinase (PIP5K) isoforms is tightly regulated by small GTPases and, interestingly, by PA as well, and the concerted formation of PIP2 and PA has been shown to mediate receptor-regulated cellular events. This review highlights the regulation of PLD by membrane receptors, and describes how the close encounter of PLD and PIP5K isoforms with small GTPases permits the execution of specific cellular functions