Endogenous Repair Mechanisms Research Articles

Introduction: Thematic Papers Issue on Peripheral Nerve Regeneration and Repair.

Injuries to the peripheral nerves result in loss of motor, sensory and autonomic functions in the denervated segments of the body, thus having strong impact in the quality of life of affected patients. Neurons are able to regenerate their injured axons in the peripheral nerves; however, the endogenous repair mechanisms usually do not allow for a satisfactory functional recovery, especially after severe nerve injuries. The interest on regeneration after peripheral nerve injuries has increased in the recent years due to the numerous advances derived from studies of neurobiology, cell therapy, and tissue engineering. This Thematic Papers Issue brings together a number of papers, authored by researchers in the field, which cover a wide spectrum of topics related to regeneration and repair of peripheral nerve injuries. The Issue proposal originated from the recent 4th International Symposium on Peripheral Nerve Regeneration (ISPNR2017) which was hosted by Xavier Navarro and the European Society for the Study of Peripheral Nerve Repair and Regeneration in Barcelona, Spain. Anat Rec, 301:1614-1617, 2018. © 2018 Wiley Periodicals, Inc.

Hitting the Mark: Optimizing the Use of Calcium Phosphate Injections for the Treatment of Bone Marrow Lesions of the Proximal Tibia and Distal Femur

Increased contact pressures of the osteoarthritic joint can lead to underlying osseous injury, with resultant marrow edema changes of the subchondral bone. These osteoarthritis-related bone marrow lesions can subsequently lead to persistent pain and further disability. Limited joint preservation treatment options exist to alleviate symptoms or potentially alter the natural history of the affected joint; however, recent success with injectable calcium phosphate has provided early pain relief and may provide a scaffold for endogenous repair mechanisms. In this Technical Note, a comprehensive surgical approach using injectable calcium phosphate to target bone marrow lesions of the proximal tibia and distal femur is presented. Critical technique considerations include the use of magnetic resonance and fluoroscopic imaging to target the area of the subchondral bone while refraining from overfilling and/or forced pressurization during delivery and the use of postinjection arthroscopy to prevent potential injurious sequelae.

Achievable Central Nervous System Concentrations of the Green Tea Catechin EGCG Induce Stress in Glioblastoma Cells in Vitro

The polyphenolic compounds present in green tea are preventative against cancer in several animal tumor models. However, direct cytotoxic effects on cancer cells have also been reported. In order to determine whether drinking of green tea has chemopreventive or cytotoxic effects on brain cancer cells, we investigated the effect of the major green tea polyphenol EGCG as a pure substance and as tea extract dietary supplement on primary human glioblastoma cell cultures at the CNS-achievable concentration of 100 nM reported in the literature. We compared this with the effect of the cytotoxic concentration of 500 μM determined to be specific for the investigated primary glioblastoma cultures. After treatment with 500 µM EGCG, strong induction of autophagy and apoptosis was observed. Under treatment with 100 nM EGCG, glioblastoma cells proliferated over the entire observation period of 6 days without any detectable signs of cell death. Only within the first 12 h of treatment was increased accumulation of autophagic vacuoles and increased reactive oxygen species production as a stress response demonstrated. Mild forms of stress, such as treatment with 100 nM EGCG, activate different endogenous repair mechanisms to protect cells. Our data imply that drinking of green tea may have chemopreventive effects, but no direct cytotoxic properties.

Cancer follows chromosome missegregation when all endogenous repair mechanisms fail

Almost all solid tumors consist of aneuploid cells with highly abnormal chromosome numbers. Such cancer cells could very well originate from chromosome missegregation which is a disturbingly common phenomenon, happening in 0.01 to 4 percent of cell divisions. Missegregated cells are aneuploid, typically lacking a chromosome or having one in surplus. Missegregated cells have mutation in the gene dose of the perhaps a thousand genes on a chromosome in one step. After missegregation cell division cannot be done right, as at least one daughter cell has a faulty chromosome number. At division in cells with surplus chromosomes the number will tend to increase due to mismatch in the division machinery. The organism has a number of repair mechanisms in place to prevent potential damage of accumulating aneuploidy. The first is the roll-back of the cell division itself, leading to tetraploidy or sometimes two nuclei in one cell; another is the prevention of further divisions. A very important one is induction of apoptosis, the cellular suicide. A special case is the elimination of the nucleus itself in the formation of red blood cells. Many aneuploid cells are probably eliminated by the immune system. A hypothetical mechanism would be the prevention of metastasis. Missegregation increases with age when the chromosomes lose their protective telomere ends at the Hayflick limit after about 50 divisions, and the unraveled chromosomes fuse and break. For cancer to develop all of these repair mechanisms must fail. The hypothesis offers a straightforward rationale for the multiple hit hypothesis of cancer development.

Meet the First Authors.

Meet the First Authors.

Abstract 473: IL-7 Increases Fusion of Rat Bone Marrow Mesenchymal Stem Cells With Cardiomyocytes In Vitro and Improve Cardiac Function In Vivo

Heart diseases are one of the main causes of morbidity and mortality all over the world particularly in the developing countries. Cardiac endogenous repair mechanisms have limited potential to regenerate myocardium following myocardial infarction. Mesenchymal stem cells (MSCs) hold significant promise as potential therapeutic candidates following cardiac tissue damage. Engrafted stem cells consistently experience programmed cell death and a large number of MSCs die in the ischemic area of the heart after transplantation. To ensure the survival of engrafted stem cells, it is beneficial to precondition these stem cells prior to their transplantation into the injured heart. In this study, we used interleukin-7 (IL-7), a hematopoietic growth factor having significant role in the cell survival, to transfect MSCs and observed its effect on cell-cell fusion in vitro and cardiac function in vivo . Cell fusion analyzed by co-culturing IL-7 transfected MSCs with neonatal rat cardiomyocytes (CMs) showed significantly (p<0.01) higher number of fused cells as compared to that in case of normal MSCs. Transplantation of IL-7 transfected MSCs into the rat model of myocardial infarction showed improvement in the cardiac function by significantly increasing the ejection fraction (EF) and fractional shortening (FS) (p<0.01), and significantly decreasing the diastolic and systolic left ventricular internal diameters (LVIDd and LVIDs) (p<0.001) and end systolic and diastolic volumes (ESV and EDV) (p<0.01 and p<0.001 respectively) as assessed by echocardiography after 4 weeks. IL-7 is able to enhance the fusogenic properties of MSCs and improve cardiac function. This Improvement in cardiac function may be attributed to the supportive action of IL-7 on cell proliferation and cell survival contributing to the regeneration of damaged myocardium.

T-DNA associated reciprocal translocation reveals differential survival of male and female gametes

In Arabidopsis thaliana, chromosomal rearrangements are commonly associated with compound T-DNA insertions due to endogenous double-stranded break and repair mechanisms. During the production of a double homozygous mutant for the CCCH zinc finger proteins, AtC3H6 and AtC3H68, we uncovered evidence for a reciprocal I-V chromosomal translocation in SALK_093620. Mapping the T-DNA borders in this insertion line confirmed an insertion site at the AtC3H6 locus on chromosome I and identified a second insertion in an intergenic region at the end of chromosome V. Our investigation of the progeny distribution and gamete transmission from reciprocal crosses indicated that male and female gametes are differentially affected by this predicted translocation. Male gametes with the haploid genotype AtC3H6(+);AtC3H68(+) had significantly higher transmission than female gametes with the same genotype. Our work highlights the importance of detailing T-DNA insertion events in transgenetic lines produced through Agrobacterium-mediated transformation, which can uncover novel chromosomal anomalies and imbalanced genome compositions.

Mesenchymal Stem Cell-Based Therapy for Cardiovascular Disease: Progress and Challenges.

Administration of mesenchymal stem cells (MSCs) to diseased hearts improves cardiac function and reduces scar size. These effects occur via the stimulation of endogenous repair mechanisms, including regulation of immune responses, tissue perfusion, inhibition of fibrosis, and proliferation of resident cardiac cells, although rare events of transdifferentiation into cardiomyocytes and vascular components are also described in animal models. While these improvements demonstrate the potential of stem cell therapy, the goal of full cardiac recovery has yet to be realized in either preclinical or clinical studies. To reach this goal, novel cell-based therapeutic approaches are needed. Ongoing studies include cell combinations, incorporation of MSCs into biomaterials, or pre-conditioning or genetic manipulation of MSCs to boost their release of paracrine factors, such as exosomes, growth factors, microRNAs, etc. All of these approaches can augment therapeutic efficacy. Further study of the optimal route of administration, the correct dose, the best cell population(s), and timing for treatment are parameters that still need to be addressed in order to achieve the goal of complete cardiac regeneration. Despite significant progress, many challenges remain.

In vivo Electrical Stimulation of Neural Stem Cells via Conductive Polymer Scaffold Improves Endogenous Repair Mechanisms of Stroke Recovery (P4.028)

In vivo Electrical Stimulation of Neural Stem Cells via Conductive Polymer Scaffold Improves Endogenous Repair Mechanisms of Stroke Recovery (P4.028)

F-102 Inflammation and immune modulation: tackling age-related stem cell dysfunction

In aging and diseased tissues, regeneration and regenerative therapies are limited by stem cell dysfunction and unfavorable tissue environments. Promising strategies to improve success include interventions that enhance stem cell function and that harness and boost endogenous tissue repair mechanisms. We study stem cells and tissue repair in barrier epithelia and the retina of Drosophila and mice to explore the causes and consequences of age-related regenerative dysfunction. These studies have led to the discovery of interventions targeting age-related inflammation, stem cell proliferation, stem cell metabolism, innate immune responses, and the commensal microbiota as strategies to enhance regeneration and extend lifespan. I will discuss these strategies and provide perspectives for the development of targeted interventions to improve tissue function in the elderly. I will highlight strategies to improve stem cell activity by targeting endogenous proliferation, differentiation and nutrient response pathways, and strategies to improve tissue repair by modulating innate immunity and host/commensal interactions. Combining such strategies is likely to significantly improve tissue homeostasis and regenerative therapies in the elderly, ultimately extending the healthy years of life.

Stroke Repair via Biomimicry of the Subventricular Zone

Stroke is among the leading causes of death and disability worldwide, 85% of which are ischemic. Current stroke therapies are limited by a narrow effective therapeutic time and fail to effectively complete the recovery of the damaged area. Magnetic resonance imaging of the subventricular zone (SVZ) following infarct/stroke has allowed visualization of new axonal connections and projections being formed, while new immature neurons migrate from the SVZ to the peri-infarct area. Such studies suggest that the SVZ is a primary source of regenerative cells for the repair and regeneration of stroke-damaged neurons and tissue. Therefore, the development of tissue engineered scaffolds that serve as a bioreplicative SVZ niche would support the survival of multiple cell types that reside in the SVZ. Essential to replication of the human SVZ microenvironment is the establishment of microvasculature that regulates both the healthy and stroke-injured blood brain barrier, which is dysregulated post-stroke. In order to reproduce this niche, understanding how cells interact in this environment is critical, in particular neural stem cells, endothelial cells, pericytes, ependymal cells, and microglia. Remodeling and repair of the matrix-rich SVZ niche by endogenous reparative mechanisms may then support functional recovery when enhanced by an artificial niche that supports the survival and proliferation of migrating vascular and neuronal cells. Critical considerations to mimic this area include an understanding of resident cell types, delivery method, and the use of biocompatible materials. Controlling stem cell survival, differentiation, and migration are key factors to consider when transplanting stem cells. Here, we discuss the role of the SVZ architecture and resident cells in the promotion and enhancement of endogenous repair mechanisms. We elucidate the interplay between the extracellular matrix composition and cell interactions prior to and following stroke. Lastly, we review current cell and neuronal niche biomimetic materials that allow for a tissue- engineered approach in order to promote structural and functional restoration of neural circuitry. By creating an artificial mimetic SVZ, tissue engineers can strive to facilitate tissue regeneration and functional recovery.

Microarray analysis for delineating the gene expression in biopsies of gastrocnemius muscle of patients with chronic critical limb ischaemia compared with non-ischaemic controls.

Microarray analysis has been carried out in this pilot study to compare delineated gene expression profiles in the biopsies of skeletal muscle taken from patients with chronic critical limb ischaemia (CLI) and non-ischaemic control subjects. Biopsy of gastrocnemius muscle was obtained from six patients with unreconstructed CLI referred for surgical major amputation. As control, biopsies of six patients undergoing elective knee arthroplasty without evidence of peripheral arterial occlusive disease were taken. The differences in gene expression associated with angiogenic processes in specimens obtained from ischaemic and non-ischaemic skeletal muscle were confirmed by quantitative real-time polymerase chain reaction (PCR) analysis. Compared with non-ischaemic skeletal muscle biopsy of chronic-ischaemic skeletal muscle contained 55 significantly up-regulated and 45 down-regulated genes, out of which 64 genes had a known genetic product. Tissue samples of ischaemic muscle were characterized by increased expression of cell survival factors (e. g. tissue factor pathway inhibitor 2) in combination with reduced expression of cell proliferation effectors (e. g. microfibrillar-associated protein 5 and transferrin receptor). The expression of growth factors (e. g. early growth response 3 and chemokine receptor chemokine C-X-C motif ligand 4) which play a central role in arterial and angiogenic processes and anti-angiogenetic factors (e. g. pentraxin 3) were increased in chronic ischaemic skeletal muscle. An increased expression of extracellular matrix proteins (e. g. cysteine-rich angiogenic inducer 61) was also observed. Gene expression profiles in biopsies of gastrocnemius muscle in patients with chronic critical limb ischaemia showed an increase in pro-survival factors, extracellular matrix protein deposition, and impaired proliferation, compared with non-ischaemic controls. Further studies are required to analyse the endogenous repair mechanism.

Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTP\u03c3 receptors promotes a beneficial inflammatory response following spinal cord injury

BackgroundTraumatic spinal cord injury (SCI) results in upregulation of chondroitin sulfate proteoglycans (CSPGs) by reactive glia that impedes repair and regeneration in the spinal cord. Degradation of CSPGs is known to be beneficial in promoting endogenous repair mechanisms including axonal sprouting/regeneration, oligodendrocyte replacement, and remyelination, and is associated with improvements in functional outcomes after SCI. Recent evidence suggests that CSPGs may regulate secondary injury mechanisms by modulating neuroinflammation after SCI. To date, the role of CSPGs in SCI neuroinflammation remains largely unexplored. The recent discovery of CSPG-specific receptors, leukocyte common antigen-related (LAR) and protein tyrosine phosphatase-sigma (PTPσ), allows unraveling the cellular and molecular mechanisms of CSPGs in SCI. In the present study, we have employed parallel in vivo and in vitro approaches to dissect the role of CSPGs and their receptors LAR and PTPσ in modulating the inflammatory processes in the acute and subacute phases of SCI.MethodsIn a clinically relevant model of compressive SCI in female Sprague Dawley rats, we targeted LAR and PTPσ by two intracellular functionally blocking peptides, termed ILP and ISP, respectively. We delivered ILP and ISP treatment intrathecally to the injured spinal cord in a sustainable manner by osmotic mini-pumps for various time-points post-SCI. We employed flow cytometry, Western blotting, and immunohistochemistry in rat SCI, as well as complementary in vitro studies in primary microglia cultures to address our questions.ResultsWe provide novel evidence that signifies a key immunomodulatory role for LAR and PTPσ receptors in SCI. We show that blocking LAR and PTPσ reduces the population of classically activated M1 microglia/macrophages, while promoting alternatively activated M2 microglia/macrophages and T regulatory cells. This shift was associated with a remarkable elevation in pro-regenerative immune mediators, interleukin-10 (IL-10), and Arginase-1. Our parallel in vitro studies in microglia identified that while CSPGs do not induce an M1 phenotype per se, they promote a pro-inflammatory phenotype. Interestingly, inhibiting LAR and PTPσ in M1 and M2 microglia positively modulates their inflammatory response in the presence of CSPGs, and harnesses their ability for phagocytosis and mobilization. Interestingly, our findings indicate that CSPGs regulate microglia, at least in part, through the activation of the Rho/ROCK pathway downstream of LAR and PTPσ.ConclusionsWe have unveiled a novel role for LAR and PTPσ in regulating neuroinflammation in traumatic SCI. Our findings provide new insights into the mechanisms by which manipulation of CSPG signaling can promote recovery from SCI. More importantly, this work introduces the potential of ILP/ISP as a viable strategy for modulating the immune response following SCI and other neuroinflammatory conditions of the central nervous system.

Endogenous factors and mechanisms of renoprotection and renal repair.

An imbalance between renal damaging molecules and nephroprotective factors contributes to the development and progression of kidney diseases. Molecules with renoprotective properties might serve as biomarkers, drug targets as well as therapeutic options themselves. For this review, we generated a set of renoprotective factors based on GeneRIF (Gene Reference Into Function) information available at NCBI's PubMed. The final set of manually curated renoprotective factors was investigated with respect to tissue-specific expression, subcellular location distribution and involvement in biological processes using information from gene ontology as well as information from protein-protein interaction databases. We furthermore investigated the factors in the context of clinical trials of renal disease and diabetes. One hundred and ninety-three factors could be retrieved from the set of GeneRIFs on nephroprotection and renal repair. A large number of factors were either secretory molecules or plasma membrane receptors. Next to the elevated expression in renal tissue, also higher expression in connective tissue and pancreas was observed. The proteins could be assigned to the broad functional categories of cell proliferation and signalling, inflammatory response, apoptosis, blood pressure regulation as well as cellular response to different kinds of insults such as hypoxia, heat or mechanical stimulus. Eight factors are studied in clinical trials with additional ones being targeted by compounds. We have generated a set of renoprotective factors based on the literature information, which was functionally annotated and evaluated with respect to tested compounds in kidney disease and diabetes clinical trials.

Cell-Based and Exosome Therapy in Diabetic Stroke.

SummaryStroke is a global health concern and it is imperative that therapeutic strategies with wide treatment time frames be developed to improve neurological outcome in patients. Patients with diabetes mellitus who suffer a stroke have worse neurological outcomes and long‐term functional recovery than nondiabetic stroke patients. Diabetes induced vascular damage and enhanced inflammatory milieu likely contributes to worse post stroke outcomes. Diabetic stroke patients have an aggravated pathological cascade, and treatments that benefit nondiabetic stroke patients do not necessarily translate to diabetic stroke patients. Therefore, there is a critical need to develop therapeutics for stroke specifically in the diabetic population. Stem cell based therapy for stroke is an emerging treatment option with wide therapeutic time window. Cell‐based therapies for stroke promote endogenous central nervous system repair and neurorestorative mechanisms such as angiogenesis, neurogenesis, vascular remodeling, white matter remodeling, and also modulate inflammatory and immune responses at the local and systemic level. Emerging evidence suggests that exosomes and their cargo microRNA mediate cell therapy derived neurorestorative effects. Exosomes are small vesicles containing protein and RNA characteristic of its parent cell. Exosomes are transported by biological fluids and facilitate communication between neighboring and remote cells. MicroRNAs, a class of naturally occurring, small noncoding RNA sequences, contained within exosomes can regulate recipient cell's signaling pathways and alter protein expression either acting alone or in concert with other microRNAs. In this perspective article, we summarize current knowledge and highlight the promising future of cell based and exosome therapy for stroke and specifically for diabetic stroke. stem cells translational medicine 2018;7:451–455

Neuroprotective strategies for retinal disease.

Diseases that affect the eye, including photoreceptor degeneration, diabetic retinopathy, and glaucoma, affect 11.8 million people in the US, resulting in vision loss and blindness. Loss of sight affects patient quality of life and puts an economic burden both on individuals and the greater healthcare system. Despite the urgent need for treatments, few effective options currently exist in the clinic. Here, we review research on promising neuroprotective strategies that promote neuronal survival with the potential to protect against vision loss and retinal cell death. Due to the large number of neuroprotective strategies, we restricted our review to approaches that we had direct experience with in the laboratory. We focus on drugs that target survival pathways, including bile acids like UDCA and TUDCA, steroid hormones like progesterone, therapies that target retinal dopamine, and neurotrophic factors. In addition, we review rehabilitative methods that increase endogenous repair mechanisms, including exercise and electrical stimulation therapies. For each approach, we provide background on the neuroprotective strategy, including history of use in other diseases; describe potential mechanisms of action; review the body of research performed in the retina thus far, both in animals and in humans; and discuss considerations when translating each treatment to the clinic and to the retina, including which therapies show the most promise for each retinal disease. Despite the high incidence of retinal diseases and the complexity of mechanisms involved, several promising neuroprotective treatments provide hope to prevent blindness. We discuss attractive candidates here with the goal of furthering retinal research in critical areas to rapidly translate neuroprotective strategies into the clinic.

Vive la radiorésistance!: converging research in radiobiology and biogerontology to enhance human radioresistance for deep space exploration and colonization

While many efforts have been made to pave the way toward human space colonization, little consideration has been given to the methods of protecting spacefarers against harsh cosmic and local radioactive environments and the high costs associated with protection from the deleterious physiological effects of exposure to high-Linear energy transfer (high-LET) radiation. Herein, we lay the foundations of a roadmap toward enhancing human radioresistance for the purposes of deep space colonization and exploration. We outline future research directions toward the goal of enhancing human radioresistance, including upregulation of endogenous repair and radioprotective mechanisms, possible leeways into gene therapy in order to enhance radioresistance via the translation of exogenous and engineered DNA repair and radioprotective mechanisms, the substitution of organic molecules with fortified isoforms, and methods of slowing metabolic activity while preserving cognitive function. We conclude by presenting the known associations between radioresistance and longevity, and articulating the position that enhancing human radioresistance is likely to extend the healthspan of human spacefarers as well.

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin.

Multiple sclerosis (MS) is a chronic autoimmune, inflammatory disease in the central nervous system (CNS) characterized by loss of oligodendrocytes, myelin axons, and neurons. Remyelination is an endogenous repair mechanism, which recovers the loss of myelin and is able to preserve functional axons. The hope is that the development of new treatments aiming at promoting remyelination will halt and potentially partially reverse the progressive neurological decline in MS. The development of such drugs requires adequate models. In this chapter, we will discuss the surgical procedure of injection of lysolecithin into ventral thoraco-lumbar spinal cord white matter of mice, which is particularly suitable for investigating remyelination using transgenic animals.

Exploring Stem Cells and Inflammation in Tendon Repair and Regeneration.

Tendon injuries are frequent and are responsible for substantial morbidity both in sports and in the workplace. Despite the endogenous mechanisms of tendon repair and regeneration, tendon healing upon injury is slow and often insufficient to restore complete biomechanics functionality.Inflammation has a pivotal role in tendon healing and failed healing responses contribute to the progression of tendinopathies. However, the molecular and cellular mechanisms involved are poorly understood requiring further insights.During inflammation, bioactive molecules such as cytokines secreted locally at the injury site, influence resident stem cells that contribute as modulatory agents over the niche towards homeostasis, holding great promise as therapeutic agents for tendon pathological conditions associated to unresolved inflammation and failed healing.This review overviews the role of cytokines and resident cells, focusing on the participation of tendon stem cell population in inflammation and tendon healing upon injury and their potential action in resolution of pathological conditions.

Functional Insulator Scanning of CpG Islands to Identify Regulatory Regions of Promoters Using CRISPR.

The ability to mutate a promoter in situ is potentially a very useful approach for gaining insights into endogenous gene regulation mechanisms. The advent of CRISPR/Cas systems has provided simple, efficient, and targeted genetic manipulation in eukaryotes, which can be applied to studying genome structure and function.The basic CRISPR toolkit comprises an endonuclease, Cas9, and a short DNA-targeting sequence, made up of a single guide RNA (sgRNA). The catalytic domains of Cas9 are rendered active upon dimerization of Cas9 with sgRNA, resulting in targeted double stranded DNA breaks. Among other applications, this method of DNA cleavage can be coupled to endogenous homology-directed repair (HDR) mechanisms for the generation of site-specific editing or knockin mutations, at both promoter regulatory and gene coding sequences.A well-characterized regulatory feature of promoter regions is the high abundance of CpGs. These CpG islands tend to be unmethylated, ensuring a euchromatic environment that promotes gene transcription. Here, we demonstrate CRISPR-mediated editing of two CpG islands located within the promoter region of the MDR1 gene (Multi Drug Resistance 1). Cas9 is used to generate double stranded breaks across multiple target sites, which are then repaired while inserting the beta globin (β-globin) insulator, 5'HS5. Thus, we are screening through promoter regulatory sequences with a chromatin barrier element to identify functional regions via "insulator scanning." Transcriptional and functional assessment of MDR1 expression provides evidence of genome engineering. Overall, this method allows the scanning of CpG islands to identify their promoter functions.