CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain via m6A-dependent stabilization of 5-HT3A mRNA in sensory neurons.

The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management.

Patch repair of deep wounds by mobilized fascia.

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism1-5. Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.

Histone methylation-mediated microRNA-32-5p down-regulation in sensory neurons regulates pain behaviors via targeting Cav3.2 channels.

microRNA (miRNA)­mediated gene regulation has been studied as a therapeutic approach, but its functional regulatory mechanism in neuropathic pain is not well understood. Here, we identify that miRNA-32-5p (miR-32-5p) is a functional RNA in regulating trigeminal-mediated neuropathic pain. High-throughput sequencing and qPCR analysis showed that miR-32-5p was the most down-regulated miRNA in the injured trigeminal ganglion (TG) of rats. Intra-TG injection of miR-32-5p agomir or overexpression of miR-32-5p by lentiviral delivery in neurons of the injured TG attenuated established trigeminal neuropathic pain. miR-32-5p overexpression did not affect acute physiological pain, while miR-32-5p down-regulation in intact rats was sufficient to cause pain-related behaviors. Nerve injury increased the methylated histone occupancy of binding sites for the transcription factor glucocorticoid receptor in the miR-32-5p promoter region. Inhibition of the enzymes that catalyze H3K9me2 and H3K27me3 restored the expression of miR-32-5p and markedly attenuated pain behaviors. Further, miR-32-5p­targeted Cav3.2 T-type Ca2+ channels and decreased miR-32-5p associated with neuropathic pain caused an increase in Cav3.2 protein expression and T-type channel currents. Conversely, miR-32-5p overexpression in injured TG suppressed the increased expression of Cav3.2 and reversed mechanical allodynia. Together, we conclude that histone methylation-mediated miR-32-5p down-regulation in TG neurons regulates trigeminal neuropathic pain by targeting Cav3.2 channels.

Fibroblasts as confederates of the immune system.

Fibroblastic stromal cells are as diverse, in origin and function, as the niches they fashion in the mammalian body. This cellular variety impacts the spectrum of responses elicited by the immune system. Fibroblast influence on the immune system keeps evolving our perspective on fibroblast roles and functions beyond just a passive structural part of organs. This review discusses the foundations of fibroblastic stromal-immune crosstalk, under the scope of stromal heterogeneity as a basis for tissue-specific tutoring of the immune system. Focusing on the skin as a relevant immunological organ, we detail the complex interactions between distinct fibroblast populations and immune cells that occur during homeostasis, injury repair, scarring, and disease. We further review the relevance of fibroblastic stromal cell heterogeneity and how this heterogeneity is central to regulate the immune system from its inception during embryonic development into adulthood.

The Multifaceted Functions of TRPV4 and Calcium Oscillations in Tissue Repair.

The transient receptor potential vanilloid 4 (TRPV4) specifically functions as a mechanosensitive ion channel and is responsible for conveying changes in physical stimuli such as mechanical stress, osmotic pressure, and temperature. TRPV4 enables the entry of cation ions, particularly calcium ions, into the cell. Activation of TRPV4 channels initiates calcium oscillations, which trigger intracellular signaling pathways involved in a plethora of cellular processes, including tissue repair. Widely expressed throughout the body, TRPV4 can be activated by a wide array of physicochemical stimuli, thus contributing to sensory and physiological functions in multiple organs. This review focuses on how TRPV4 senses environmental cues and thereby initiates and maintains calcium oscillations, critical for responses to organ injury, tissue repair, and fibrosis. We provide a summary of TRPV4-induced calcium oscillations in distinct organ systems, along with the upstream and downstream signaling pathways involved. In addition, we delineate current animal and disease models supporting TRPV4 research and shed light on potential therapeutic targets for modulating TRPV4-induced calcium oscillation to promote tissue repair while reducing tissue fibrosis.

Adiponectin receptor 1-mediated stimulation of Cav3.2 channels in trigeminal ganglion neurons induces nociceptive behaviors in mice.

BACKGROUND: Adipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons. RESULTS: Adiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCß1). Blocking PKCß1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCß1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2. CONCLUSION: Our study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCß1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.

TLR4-dependent shaping of the wound site by MSCs accelerates wound healing.

We here address the question whether the unique capacity of mesenchymal stem cells to re-establish tissue homeostasis depends on their potential to sense pathogen-associated molecular pattern and, in consequence, mount an adaptive response in the interest of tissue repair. After injection of MSCs primed with the bacterial wall component LPS into murine wounds, an unexpected acceleration of healing occurs, clearly exceeding that of non-primed MSCs. This correlates with a fundamental reprogramming of the transcriptome in LPS-treated MSCs as deduced from RNAseq analysis and its validation. A network of genes mediating the adaptive response through the Toll-like receptor 4 (TLR4) pathway responsible for neutrophil and macrophage recruitment and their activation profoundly contributes to enhanced wound healing. In fact, injection of LPS-primed MSCs silenced for TLR4 fails to accelerate wound healing. These unprecedented findings hold substantial promise to refine current MSC-based therapies for difficult-to-treat wounds.

MSCs rescue impaired wound healing in a murine LAD1 model by adaptive responses to low TGF-ß1 levels.

Mutations in the CD18 gene encoding the common ß-chain of ß2 integrins result in impaired wound healing in humans and mice suffering from leukocyte adhesion deficiency syndrome type 1 (LAD1). Transplantation of adipose tissue-derived mesenchymal stem cells (MSCs) restores normal healing of CD18-/- wounds by restoring the decreased TGF-ß1 concentrations. TGF-ß1 released from MSCs leads to enhanced myofibroblast differentiation, wound contraction, and vessel formation. We uncover that MSCs are equipped with a sensing mechanism for TGF-ß1 concentrations at wound sites. Low TGF-ß1 concentrations as occurring in CD18-/- wounds induce TGF-ß1 release from MSCs, whereas high TGF-ß1 concentrations suppress TGF-ß1 production. This regulation depends on TGF-ß receptor sensing and is relayed to microRNA-21 (miR-21), which subsequently suppresses the translation of Smad7, the negative regulator of TGF-ß1 signaling. Inactivation of TGF-ß receptor, or overexpression or silencing of miR-21 or Smad7, abrogates TGF-ß1 sensing, and thus prevents the adaptive MSC responses required for tissue repair.

Furnishing Wound Repair by the Subcutaneous Fascia.

Mammals rapidly heal wounds through fibrous connective tissue build up and tissue contraction. Recent findings from mouse attribute wound healing to physical mobilization of a fibroelastic connective tissue layer that resides beneath the skin, termed subcutaneous fascia or superficial fascia, into sites of injury. Fascial mobilization assembles diverse cell types and matrix components needed for rapid wound repair. These observations suggest that the factors directly affecting fascial mobility are responsible for chronic skin wounds and excessive skin scarring. In this review, we discuss the link between the fascia's unique tissue anatomy, composition, biomechanical, and rheologic properties to its ability to mobilize its tissue assemblage. Fascia is thus at the forefront of tissue pathology and a better understanding of how it is mobilized may crystallize our view of wound healing alterations during aging, diabetes, and fibrous disease and create novel therapeutic strategies for wound repair.

Melanocortin type 4 receptor-mediated inhibition of A-type K+ current enhances sensory neuronal excitability and mechanical pain sensitivity in rats.

α-Melanocyte-stimulating hormone (α-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that α-MSH suppresses the transient outward A-type K+ current (IA) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to α-MSH concentration-dependently decreased IA This α-MSH-induced IA decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated IA response. Blocking Gi/o-protein signaling with pertussis toxin or by dialysis of TG neurons with the Gßγ-blocking synthetic peptide QEHA abolished the α-MSH-mediated decrease in IA Further, α-MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38α abrogated the α-MSH-induced IA response. Additionally, α-MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by IA blockade. Taken together, our findings suggest that α-MSH suppresses IA by activating MC4R, which is coupled sequentially to the Gßγ complex of the Gi/o-protein and downstream class I PI3K-dependent p38α signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.

Newly Defined ATP-Binding Cassette Subfamily B Member 5 Positive Dermal Mesenchymal Stem Cells Promote Healing of Chronic Iron-Overload Wounds via Secretion of Interleukin-1 Receptor Antagonist.

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+ -derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5+ -derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5+ -derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057-1074.

Local and transient inhibition of p21 expression ameliorates age-related delayed wound healing.

Nonhealing chronic wounds in the constantly growing elderly population represent a major public health problem with high socioeconomic burden. Yet, the underlying mechanism of age-related impairment of wound healing remains elusive. Here, we show that the number of dermal cells expressing cyclin-dependent kinase inhibitor p21 was elevated upon skin injury, particularly in aged population, in both man and mouse. The nuclear expression of p21 in activated wound fibroblasts delayed the onset of the proliferation phase of wound healing in a p53-independent manner. Further, the local and transient inhibition of p21 expression by in vivo delivered p21-targeting siRNA ameliorated the delayed wound healing in aged mice. Our results suggest that the increased number of p21+ wound fibroblasts enforces the age-related compromised healing, and targeting p21 creates potential clinical avenues to promote wound healing in aged population.

Scars or Regeneration?-Dermal Fibroblasts as Drivers of Diverse Skin Wound Responses.

Scarring and regeneration are two physiologically opposite endpoints to skin injuries, with mammals, including humans, typically healing wounds with fibrotic scars. We aim to provide an updated review on fibroblast heterogeneity as determinants of the scarring-regeneration continuum. We discuss fibroblast-centric mechanisms that dictate scarring-regeneration continua with a focus on intercellular and cell-matrix adhesion. Improved understanding of fibroblast lineage-specific mechanisms and how they determine scar severity will ultimately allow for the development of antiscarring therapies and the promotion of tissue regeneration.

Cholecystokinin type B receptor-mediated inhibition of A-type K+ channels enhances sensory neuronal excitability through the phosphatidylinositol 3-kinase and c-Src-dependent JNK pathway.

BACKGROUND: Cholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K+ channels. RESULTS: CCK-8 reversibly and concentration-dependently decreased A-type K+ channel (IA) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K+ current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gαo. Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-Gß antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated IA response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated IA decrease, whereas c-Src inhibition attenuated the CCK-8-induced p-JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by IA blockade. CONCLUSION: Our findings indicate that CCK-8 attenuated IA through CCK-BR that is coupled to the Gßγ-dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.

Suppression of Neutrophil-Mediated Tissue Damage-A Novel Skill of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation. Stem Cells 2016;34:2393-2406.

Urotensin-II receptor stimulation of cardiac L-type Ca2+ channels requires the ßγ subunits of Gi/o-protein and phosphatidylinositol 3-kinase-dependent protein kinase C ß1 isoform.

Recent studies have demonstrated that urotensin-II (U-II) plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output. However, the mechanisms underlying these effects of U-II in cardiomyocytes still remain unknown. We show by electrophysiological studies that U-II dose-dependently potentiates L-type Ca(2+) currents (ICa,L) in adult rat ventricular myocytes. This effect was U-II receptor (U-IIR)-dependent and was associated with a depolarizing shift in the voltage dependence of inactivation. Intracellular application of guanosine-5'-O-(2-thiodiphosphate) and pertussis toxin pretreatment both abolished the stimulatory effects of U-II. Dialysis of cells with the QEHA peptide, but not scrambled peptide SKEE, blocked the U-II-induced response. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin as well as the class I PI3K antagonist CH132799 blocked the U-II-induced ICa,L response. Protein kinase C antagonists calphostin C and chelerythrine chloride as well as dialysis of cells with 1,2bis(2aminophenoxy)ethaneN,N,N',N'-tetraacetic acid abolished the U-II-induced responses, whereas PKCα inhibition or PKA blockade had no effect. Exposure of ventricular myocytes to U-II markedly increased membrane PKCß1 expression, whereas inhibition of PKCß1 pharmacologically or by shRNA targeting abolished the U-II-induced ICa,L response. Functionally, we observed a significant increase in the amplitude of sarcomere shortening induced by U-II; blockade of U-IIR as well as PKCß inhibition abolished this effect, whereas Bay K8644 mimicked the U-II response. Taken together, our results indicate that U-II potentiates ICa,L through the ßγ subunits of Gi/o-protein and downstream activation of the class I PI3K-dependent PKCß1 isoform. This occurred via the activation of U-IIR and contributes to the positive inotropic effect on cardiomyocytes.