Adult Tissue Repair Research Articles

A computational model reveals an early transient decrease in fiber cross-linking that unlocks adult regeneration

The decline in regeneration efficiency after birth in mammals is a significant roadblock for regenerative medicine in tissue repair. We previously developed a computational agent based-model (ABM) that recapitulates mechanical interactions between cells and the extracellular-matrix (ECM), to investigate key drivers of tissue repair in adults. Time calibration alongside a parameter sensitivity analysis of the model suggested that an early and transient decrease in ECM cross-linking guides tissue repair toward regeneration. Consistent with the computational model, transient inhibition or stimulation of fiber cross-linking for the first six days after subcutaneous adipose tissue (AT) resection in adult mice led to regenerative or scar healing, respectively. Therefore, this work positions the computational model as a predictive tool for tissue regeneration that with further development will behave as a digital twin of our in vivo model. In addition, it opens new therapeutic approaches targeting ECM cross-linking to induce tissue regeneration in adult mammals.

Approaches to Study Wound-Induced Hair Neogenesis (WIHN).

Embryonic wound repair proceeds with complete regeneration of the tissue without any scar formation, whereas tissue repair in adults usually results in scars and the tissue does not completely regain its preinjured state. Wound-induced hair neogenesis (WIHN) in adult rodents results in de novo hair follicle formation in the center of large wounds, mimicking regeneration processes seen in fetal tissue. The investigation of WIHN therefore provides a unique quantitative framework for scrutinizing the mechanistic underpinnings of regenerative repair, which can have clinical implications in the context of scarless healing. In this chapter, we present a detailed protocol for inducing wounds that lead to hair neogenesis in laboratory mice and facilitating the identification and characterization of distinct stages in neogenic hair follicle development. Additionally, we present a whole-mount alkaline phosphatase assay to distinguish de novo hair follicles. These protocols can facilitate studies toward obtaining a comprehensive understanding of WIHN and shedding light on the intricate molecular and cellular processes involved in mammalian regenerative repair.

Fibroblast growth factor 10

Fibroblast growth factor 10 (FGF10) is a major morphoregulatory factor that plays essential signaling roles during vertebrate multiorgan development and homeostasis. FGF10 is predominantly expressed in mesenchymal cells and signals though FGFR2b in adjacent epithelia to regulate branching morphogenesis, stem cell fate, tissue differentiation and proliferation, in addition to autocrine roles. Genetic loss of function analyses have revealed critical requirements for FGF10 signaling during limb, lung, digestive system, ectodermal, nervous system, craniofacial and cardiac development. Heterozygous FGF10 mutations have been identified in human genetic syndromes associated with craniofacial anomalies, including lacrimal and salivary gland aplasia. Elevated Fgf10 expression is associated with poor prognosis in a range of cancers. In addition to developmental and disease roles, FGF10 regulates homeostasis and repair of diverse adult tissues and has been identified as a target for regenerative medicine.

Vinculin is essential for sustaining normal levels of endogenous forces at cell-cell contacts

Transmission of cell-generated (i.e., endogenous) tension at cell-cell contacts is crucial for tissue shape changes during morphogenesis and adult tissue repair in tissues such as epithelia. E-cadherin-based adhesions at cell-cell contacts are the primary means by which endogenous tension is transmitted between cells. The E-cadherin-β-catenin-α-catenin complex mechanically couples to the actin cytoskeleton (and thereby the cell’s contractile machinery) both directly and indirectly. However, the key adhesion constituents required for substantial endogenous force transmission at these adhesions in cell-cell contacts are unclear. Due to the role of α-catenin as a mechanotransducer that recruits vinculin at cell-cell contacts, we expected α-catenin to be essential for sustaining normal levels of force transmission. Instead, using the traction force imbalance method to determine the inter-cellular force at a single cell-cell contact between cell pairs, we found that it is vinculin that is essential for sustaining normal levels of endogenous force transmission, with absence of vinculin decreasing the inter-cellular tension by over 50%. Our results constrain the potential mechanical pathways of force transmission at cell-cell contacts and suggest that vinculin can transmit forces at E-cadherin adhesions independent of α-catenin, possibly through β-catenin. Furthermore, we tested the ability of lateral cell-cell contacts to withstand external stretch and found that both vinculin and α-catenin are essential to maintain cell-cell contact stability under external forces.

Novel Frizzled-specific antibody-based Wnt mimetics and Wnt superagonists selectively activate WNT/β-catenin signaling in target tissues

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/β-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion invivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and invivo research, and serve as bases for future therapeutic development.

Canonical fibroblast growth factors in viral infection.

Fibroblast growth factors (FGFs) are a family of proteins that play a crucial role in the development and maintenance of various tissues in the body. There are three function-al groups of FGFs: canonical FGFs (cFGFs), intracellularly retained FGFs, and metabolic (also called endocrine) FGFs. cFGFs are secreted and act in an autocrine/paracrine fashion to regulate differentiation during foetal development, as well as tissue repair in adults. Recent studies have also begun to unravel the role of cFGFs during viral infections, suggesting that FGF-2 and other canonical FGFs may have an important virus-specific role, also by the regulation of the immune response. Because dysregulation in the FGF pathways is pivotal in cancer development, FGFs are the target of many anticancer drugs. These drugs may be repurposed to treat viral infection, since dysregulation of FGF signalling has been implicated in the pathogenesis of viral infections, such as hepatitis C. Overall, the role of cFGFs during viral infection is an underrepresented area of current research. This review focuses on overviewing the effects of canonical FGFs during infection by different viruses. Many studies highlight that the effects of FGFs during viral infection may be complex and context-dependent. While there is evidence to suggest that FGFs may have a beneficial impact on the immune response and tissue repair during viral infection, further studies are needed to fully understand the mechanisms underlying these effects and to determine in what cases FGFs could be targeted as a therapeutic approach for viral infection.

Magnetic Nanomaterials: Advanced Tools for Cartilage Extracellular Matrix Regeneration

After decades of basic, translational, and clinical research, cartilage regeneration therapies are still anticipated to impact the clinical landscape consistently. However, biological joint resurfacing and the regeneration of functional, integrated cartilage capable of wholly and permanently restoring joint function is still unavailable. Albeit structurally relatively simple, the cartilage extracellular matrix has a complex function in joint organ biomechanics, developmental processes, and adult life tissue turnover, repair, disease, and aging. The intricated cross-talk extracellular matrix-cellular component and its bidirectional role in maintaining tissue homeostasis are only beginning to be elucidated. Magnetic responsive nanomaterials (MNMs) have proven to be versatile tools for drug delivery, scaffold fabrication, alignment, and orienting cell distribution and cell fate. Due to their responsiveness to an applied magnetic field, MNMs can be remotely actuated for scaffold cell positioning, scaffold fabrication, or the delivery of touchless mechanical stimulation. In addition, MNMs possess excellent biocompatibility and are degradable through cellular iron storage metabolic pathways. Current directions for using MNMs to augment tissue engineering that recommends them as essential tools for fabricating bio-functional cartilage grafts are presented and discussed.

Driving adult tissue repair via re-engagement of a pathway required for fetal healing

Fetal cutaneous wound closure and repair differ from that in adulthood. In this work, we identify an oxidant stress sensor protein, nonselenocysteine-containing phospholipid hydroperoxide glutathione peroxidase (NPGPx), that is abundantly expressed in normal fetal epidermis (and required for fetal wound closure), though not in adult epidermis, but is variably re-induced upon adult tissue wounding. NPGPx is a direct target of the miR-29 family. Following injury, abundance of miR-29 is lowered, permitting a prompt increase in NPGPx transcripts and protein expression in adult wound-edge tissue. NPGPx expression was required to mediate increased keratinocyte migration induced by miR-29 inhibition invitro and invivo. Increased NPGPx expression induced increased SOX2 expression and β-catenin nuclear localization in keratinocytes. Augmenting physiologic NPGPx expression via experimentally induced miR-29 suppression, using cutaneous tissue nanotransfection or targeted lipid nanoparticle delivery of anti-sense oligonucleotides, proved to be sufficient to overcome the deleterious effects of diabetes on this specific pathway to enhance tissue repair.

The crosstalk between long non-coding RNAs and the hedgehog signaling pathway in cancer.

Hedgehog (Hh) is a conserved signaling pathway that is involved in embryo development as well as adult tissue maintenance and repair in invertebrates and vertebrates. Abnormal activation of this pathway in various types of malignant drug- and apoptosis-resistant tumors has made it a therapeutic target against tumorigenesis. Thus, understanding the molecular mechanisms that promote the activation or inhibition of this pathway is critical. Long non-coding RNAs (lncRNAs), a subclass of non-coding RNAs with a length of > 200 nt, affect the expression of Hh signaling components via a variety of transcriptional and post-transcriptional processes. This review focuses on the crosstalk between lncRNAs and the Hh pathway in carcinogenesis, outlines the broad role of Hh-related lncRNAs in tumor progression, and illustrates their clinical diagnostic, prognostic, and therapeutic potential in tumors.

The Contribution of Innervation to Tissue Repair and Regeneration.

Animals such as amphibians have an incredible capacity for regeneration with some being able to regrow their tail or appendages. Although some mammalian tissues like the skin and bones can repair following injury, there are only a few examples of true multilineage regeneration, including the distal portion of the digit tip. In both amphibians and mammals, however, to achieve successful repair or regeneration, it is now appreciated that intact nerve innervation is a necessity. Here, we review the current state of literature and discuss recent advances that identify axon-derived signals, Schwann cells, and nerve-derived mesenchymal cells as direct and indirect supporters of adult tissue homeostasis and repair. We posit that understanding how nerves positively influence repair and regeneration could lead to targeted regenerative medicine strategies to enhance tissue repair in humans.

Mechanisms of Feedback Regulation of Vitamin A Metabolism.

Vitamin A is an essential nutrient required throughout life. Through its various metabolites, vitamin A sustains fetal development, immunity, vision, and the maintenance, regulation, and repair of adult tissues. Abnormal tissue levels of the vitamin A metabolite, retinoic acid, can result in detrimental effects which can include congenital defects, immune deficiencies, proliferative defects, and toxicity. For this reason, intricate feedback mechanisms have evolved to allow tissues to generate appropriate levels of active retinoid metabolites despite variations in the level and format, or in the absorption and conversion efficiency of dietary vitamin A precursors. Here, we review basic mechanisms that govern vitamin A signaling and metabolism, and we focus on retinoic acid-controlled feedback mechanisms that contribute to vitamin A homeostasis. Several approaches to investigate mechanistic details of the vitamin A homeostatic regulation using genomic, gene editing, and chromatin capture technologies are also discussed.

Analysis of Age-Related Circular RNA Expression Profiles in Mesenchymal Stem Cells of Rat Bone Marrow.

As multicellular organisms age, they undergo a reduction in tissue and organ function. Researchers have put forward a theory that stem cell aging is the main factor responsible for decreased tissue and organ function. The adult stem cells guarantee the maintenance and repair of adult tissues and organs. Among adult stem cells, mesenchymal stem cells (MSCs) are emerging as hopeful candidates for cell-based therapy of numerous diseases. In recent years, high-throughput sequencing technologies have evolved to identify circular RNAs (circRNAs) associated with an increasing number of diseases, such as cancer and age-related diseases. It has been reported that circRNAs can compete with microRNAs (miRNAs) to affect the stability or translation of target RNAs and further regulate gene expression at the transcriptional level. However, the role of circRNAs expressed in MSCs in aging mechanisms has not yet been deciphered. The aim of this study was to explore and analyze the expression profiles of age-related circRNAs in MSCs. In this study, bone marrow MSCs were extracted from aged and young rats and analyzed using high-throughput sequencing and bioinformatics. The reliability of high-throughput RNA sequencing was verified by quantitative real-time polymerase chain reaction. The most important circRNA functions and pathways were further selected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) analysis. Age-related circRNAs were found in the circrNA–miRNA–mRNA interaction network. The results of high-throughput sequencing showed that 4,229 circRNAs were involved in age-related senescence of MSCs. Compared with the young group, there were 29 differentially expressed circRNAs in the aged group, of which four were upregulated and 25 were downregulated. GO analysis covered three domains: biological process (BP), cellular component (CC), and molecular function (MF). The terms assigned to the BP domain were cellular metabolic processes and cellular macromolecule metabolic processes. The identified CC terms were intracellular and intracellular part, and the identified MF terms were binding and protein binding. The top five KEGG pathways were mitophagy–animal–Rattus norvegicus, prostate cancer–Rattus norvegicus, pathways in cancer–Rattus norvegicus, lysosome–Rattus norvegicus, and autophagy–animal–Rattus norvegicus. Altogether, circRNAs may play a major role in age-related MSC senescence. This study provides new mechanistic insights into MSC senescence, possibly leading to novel therapeutic strategies for age-related diseases.

Protein arginine methyltransferase 1 regulates cell proliferation and differentiation in adult mouse adult intestine

BackgroundAdult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma thyroid hormone (T3) levels are high. Furthermore, deficiency in T3 signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a T3 receptor coactivator, is highly expressed during intestinal maturation in mouse.MethodsWe have analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium.ResultsWe show that PRMT1 is expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we have demonstrated that the expression of PRMT1 in the intestinal epithelium is critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium results in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus are also increased. Furthermore, there are increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remains unchanged.ConclusionsOur finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in T3-signaling and the importance of T3 for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to T3-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.

WNT signaling in atrial fibrillation.

The Wnt signaling pathway regulates physiological processes such as cell proliferation and differentiation, cell fate decisions, and stem cell maintenance and, thus, plays essential roles in embryonic development, but also in adult tissue homeostasis and repair. The Wnt signaling pathway has been associated with heart development and repair and has been shown to be crucially involved in proliferation and differentiation of progenitor cells into cardiomyocytes. The investigation of the role of the Wnt signaling pathway and the regulation of its expression/activity in atrial fibrillation has only just begun. The present minireview (I) provides original data regarding the expression of Wnt signaling components in atrial tissue of patients with atrial fibrillation or sinus rhythm and (II) summarizes the current state of knowledge of the regulation of Wnt signaling components' expression/activity and the contribution of the various levels of the Wnt signal transduction pathway to the processes of the development, maintenance, and progression of atrial fibrillation.

Pharmacologically Targeting the WNT/β-Catenin Signaling Cascade: Avoiding the Sword of Damocles.

WNT/β-catenin signaling plays fundamental roles in numerous developmental processes and in adult tissue homeostasis and repair after injury, by controlling cellular self-renewal, activation, division, differentiation, movement, genetic stability, and apoptosis. As such, it comes as no surprise that dysregulation of WNT/β-catenin signaling is associated with various diseases, including cancer, fibrosis, neurodegeneration, etc. Although multiple agents that specifically target the WNT/β-catenin signaling pathway have been studied preclinically and a number have entered clinical trials, none has been approved by the FDA to date. In this chapter, we provide our insights as to the reason(s) it has been so difficult to safely pharmacologically target the WNT/β-catenin signaling pathway and discuss the significant efforts undertaken towards this goal.

Deregulated immune cell recruitment orchestrated by FOXM1 impairs human diabetic wound healing

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of “ideal” adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.

Secretome of Senescent Adipose-Derived Mesenchymal Stem Cells Negatively Regulates Angiogenesis.

Nowadays, paracrine regulation is considered as a major tool of mesenchymal stem cell (MSC) involvement in tissue repair and renewal in adults. Aging results in alteration of tissue homeostasis including neovascularization. In this study, we examined the influence of replicative senescence on the angiogenic potential of adipose-derived MSCs (ASCs). Angiogenic activity of conditioned medium (CM) from senescent and “young” ASCs was evaluated in chorioallantoic membrane (CAM) assay in ovo using Japanese quail embryos. Also, the formation of capillary-like tubes by human umbilical vein endothelial cells (HUVECs) in 3D basement membrane matrix “Matrigel” and HUVEC migration capacity were analyzed. Multiplex, dot-blot and gene expression analysis were performed to characterize transcription and production of about 100 angiogenesis-associated proteins. The results point to decreased angiogenic potential of senescent ASC secretome in ovo. A number of angiogenesis-associated proteins demonstrated elevation in CM after long-term cultivation. Meanwhile, VEGF (key positive regulator of angiogenesis) did not change transcription level and concentration in CM. Increasing both pro- (FGF-2, uPA, IL-6, IL-8 etc.) and antiangiogenic (IL-4, IP-10, PF4, Activin A, DPPIV etc.) factors was observed. Some proangiogenic genes were downregulated (IGF1, MMP1, TGFB3, PDGFRB, PGF). Senescence-associated secretory phenotype (SASP) modifications after long-term cultivation lead to attenuation of angiogenic potential of ASC.

Modulation of retinoid signaling: therapeutic opportunities in organ fibrosis and repair.

The vitamin A metabolite, retinoic acid, is an important signaling molecule during embryonic development serving critical roles in morphogenesis, organ patterning and skeletal and neural development. Retinoic acid is also important in postnatal life in the maintenance of tissue homeostasis, while retinoid-based therapies have long been used in the treatment of a variety of cancers and skin disorders. As the number of people living with chronic disorders continues to increase, there is great interest in extending the use of retinoid therapies in promoting the maintenance and repair of adult tissues. However, there are still many conflicting results as we struggle to understand the role of retinoic acid in the multitude of processes that contribute to tissue injury and repair. This review will assess our current knowledge of the role retinoic acid signaling in the development of fibroblasts, and their transformation to myofibroblasts, and of the potential use of retinoid therapies in the treatment of organ fibrosis.

Cultured cell-derived extracellular matrices to enhance the osteogenic differentiation and angiogenic properties of human mesenchymal stem/stromal cells.

Cell-derived extracellular matrix (ECM) consists of a complex assembly of fibrillary proteins, matrix macromolecules, and associated growth factors that mimic the composition and organization of native ECM micro-environment. Therefore, cultured cell-derived ECM has been used as a scaffold for tissue engineering settings to create a biomimetic micro-environment, providing physical, chemical, and mechanical cues to cells, and support cell adhesion, proliferation, migration, and differentiation. Here, we present a new strategy to produce different combinations of decellularized cultured cell-derived ECM (dECM) obtained from different cultured cell types, namely, mesenchymal stem/stromal cells (MSCs) and human umbilical vein endothelial cells (HUVECs), as well as the coculture of MSC:HUVEC and investigate the effects of its various compositions on cell metabolic activity, osteogenic differentiation, and angiogenic properties of human bone marrow (BM)-derived MSCs, vital features for adult bone tissue regeneration and repair. Our findings demonstrate that dECM presented higher cell metabolic activity compared with tissue culture polystyrene. More importantly, we show that MSC:HUVEC ECM enhanced the osteogenic and angiogenic potential of BM MSCs, as assessed by in vitro assays. Interestingly, MSC:HUVEC (1:3) ECM demonstrated the best angiogenic response of MSCs in the conditions tested. To the best of our knowledge, this is the first study that demonstrates that dECM derived from a coculture of MSC:HUVEC impacts the osteogenic and angiogenic capabilities of BM MSCs, suggesting the potential use of MSC:HUVEC ECM as a therapeutic product to improve clinical outcomes in bone regeneration.

Inhibiting Hedgehog: An Update on Pharmacological Compounds and Targeting Strategies

Important steps in embryonic development are governed by the Hedgehog (Hh) signaling pathway, an evolutionary conserved signal transduction cascade. However, Hh activity not only is crucial during embryo formation but also is involved in adult tissue repair and in several malignancies. Particularly due to its link to cancer, small molecule Hh pathway inhibitors have been developed and the first compounds have been approved for use in Hh-driven basal cell carcinoma. Almost all advanced Hh inhibitors target the critical signaling component Smoothened (SMO), but preclinical research has identified additional compounds that can block the Hh pathway along its entire signaling cascade, which, in light of emerging drug resistance occurring with SMO inhibitors, is of high importance. Herein we give an overview on currently known Hh pathway inhibitors, delineating their respective strengths and weaknesses and describing potential drug targeting strategies to interfere with Hh signaling in different cancer settings.