Galectin-3 inhibition reduces fibrotic scarring and promotes functional recovery after spinal cord injury in mice.

BACKGROUND: In the context of spinal cord injury (SCI), infiltrating macrophages assume prominence as the primary inflammatory cells within the lesion core, where the fibrotic scar is predominantly orchestrated by platelet-derived growth factor receptor beta (PDGFRß+) fibroblasts. Galectin-3, a carbohydrate-binding protein of the lectin family, is notably expressed by infiltrating hematogenous macrophages and mediates cell-cell interactions. Although Galectin-3 has been shown to contribute to the endocytic internalization of PDGFRß in vitro, its specific role in driving fibrotic scar formation after SCI has not been determined. METHODS: We employed a crush mid-thoracic (T10) SCI mouse model. Galectin-3 inhibition after SCI was achieved through intrathecal injection of the Galectin-3 inhibitor TD139 or in situ injection of lentivirus carrying Galectin-3-shRNA (Lv-shLgals3). A fibrosis-induced mice model was established by in situ injection of platelet-derived growth factor D (PDGFD) or recombinant Galectin-3 (rGalectin-3) into the uninjured spinal cord. Galectin-3 internalization experiments were conducted in PDGFRß+ fibroblasts cocultured in conditioned medium in vitro. RESULTS: We identified the spatial and temporal correlation between macrophage-derived Galectin-3 and PDGFRß in fibroblasts from 3 to 56 days post-injury (dpi). Administration of TD139 via intrathecal injection or in situ injection of Lv-shLgals3 effectively mitigated fibrotic scar formation and extracellular matrix deposition within the injured spinal cord, leading to better neurological outcomes and function recovery after SCI. Furthermore, the fibrosis-inducing effects of exogenous PDGFD in the uninjured spinal cord could be blocked by TD139. In vitro experiments further demonstrated the ability of PDGFRß+ fibroblasts to internalize Galectin-3, with Galectin-3 inhibition resulting in reduced PDGFRß expression. CONCLUSIONS: Our finding underscores the pivotal role of macrophage-derived Galectin-3 in modulating the sustained internalized activation of PDGFRß within fibroblasts, providing a novel mechanistic insight into fibrotic scarring post-SCI.

Advanced Wound Healing and Scar Reduction Using an Innovative Anti-ROS Polysaccharide Hydrogel with Recombinant Human Collagen Type III.

Excessive fibrotic scar formation during skin defect repair poses a formidable challenge, impeding the simultaneous acceleration of wound healing and prevention of scar formation and hindering the restoration of skin integrity and functionality. Drawing inspiration from the structural, compositional, and biological attributes of skin, we developed a hydrogel containing modified recombinant human collagen type III and thiolated hyaluronic acid to address the challenges of regenerating skin appendages and improving the recovery of skin functions after injury by reducing fibrotic scarring. The hydrogel displayed favorable biocompatibility, antioxidant properties, angiogenic potential, and fibroblast migration stimulation in vitro. In a rat full-layer defect model, it reduced inflammation, promoted microvascular formation, and significantly enhanced the wound healing speed and effectiveness. Additionally, by upregulating fibrosis-associated genes, such as TGFB1, it facilitated collagen accumulation and a beneficial balance between type I and type III collagen, potentially expediting skin regeneration and functional recovery. In conclusion, the utilization of rhCol III-HS demonstrated considerable potential as a wound dressing, offering a highly effective strategy for the restoration and rejuvenation of complete skin defects.

Shh regulates M2 microglial polarization and fibrotic scar formation after ischemic stroke.

BACKGROUND: Fibrotic scar formation is a critical pathological change impacting tissue reconstruction and functional recovery after ischemic stroke. The regulatory mechanisms behind fibrotic scarring in the central nervous system (CNS) remain largely unknown. While macrophages are known to play a role in fibrotic scar formation in peripheral tissues, the involvement of microglia, the resident immune cells of the CNS, in CNS fibrosis requires further exploration. The Sonic Hedgehog (Shh) signaling pathway, pivotal in embryonic development and tissue regeneration, is also crucial in modulating fibrosis in peripheral tissues. However, the impact and regulatory mechanisms of Shh on fibrotic scar formation post-ischemic stroke have not been thoroughly investigated. METHODS: This study explores whether Shh can regulate fibrotic scar formation post-ischemic stroke and its underlying mechanisms through in vivo and in vitro manipulation of Shh expression. RESULTS: Our results showed that Shh expression was upregulated in the serum of acute ischemic stroke patients, as well as in the serum, CSF, and ischemic regions of MCAO/R mice. Moreover, the upregulation of Shh expression was positively correlated with fibrotic scar formation and M2 microglial polarization. Shh knockdown inhibited fibrotic scar formation and M2 microglial polarization while aggravating neurological deficits in MCAO/R mice. In vitro, adenoviral knockdown or Smoothened Agonist (SAG) activation of Shh expression in BV2 cells following OGD/R regulated their polarization and influenced the expression of TGFß1 and PDGFA, subsequently affecting fibroblast activation. CONCLUSION: These results suggest that Shh regulates M2 microglial polarization and fibrotic scar formation after cerebral ischemia.

Therapeutic application of nicotinamide: As a potential target for inhibiting fibrotic scar formation following spinal cord injury.

AIM: We aimed to confirm the inhibitory effect of nicotinamide on fibrotic scar formation following spinal cord injury in mice using functional metabolomics. METHODS: We proposed a novel functional metabolomics strategy to establish correlations between gene expression changes and metabolic phenotypes using integrated multi-omics analysis. Through the integration of quantitative metabolites analysis and assessments of differential gene expression, we identified nicotinamide as a functional metabolite capable of inhibiting fibrotic scar formation and confirmed the effect in vivo using a mouse model of spinal cord injury. Furthermore, to mimic fibrosis models in vitro, primary mouse embryonic fibroblasts and spinal cord fibroblasts were stimulated by TGFß, and the influence of nicotinamide on TGFß-induced fibrosis-associated genes and its underlying mechanism were examined. RESULTS: Administration of nicotinamide led to a reduction in fibrotic lesion area and promoted functional rehabilitation following spinal cord injury. Nicotinamide effectively downregulated the expression of fibrosis genes, including Col1α1, Vimentin, Col4α1, Col1α2, Fn1, and Acta2, by repressing the TGFß/SMADs pathway. CONCLUSION: Our functional metabolomics strategy identified nicotinamide as a metabolite with the potential to inhibit fibrotic scar formation following SCI by suppressing the TGFß/SMADs signaling. This finding provides new therapeutic strategies and new ideas for clinical treatment.

Piezo1 promotes peripheral nerve fibrotic scar formation through Schwann cell senescence.

After peripheral nerve injury (PNI), the long-term healing process at the injury site involves a progressive accumulation of collagen fibers and the development of localized scar tissue. Excessive formation of scar tissue within nerves hinders the process of nerve repair. In this study, we demonstrate that scar formation following nerve injury induces alterations in the local physical microenvironment, specifically an increase in nerve stiffness. Recent research has indicated heightened expression of Piezo1 in Schwann cells (SCs). Our findings also indicate Piezo1 expression in SCs and its association with suppressed proliferation and migration. Transcriptomic data suggests that activation of Piezo1 results in elevated expression of senescence-associated genes. GO enrichment analysis reveals upregulation of the TGF-ß pathway. Overall, our study highlights the potential for Piezo1-induced signaling to regulate SC senescence and its potential significance in the pathophysiology of fibrotic scar formation surrounding peripheral nerves.

Meningeal Damage and Interface Astroglial Scarring in the Rat Brain Exposed to a Laser-Induced Shock Wave(s).

In the past decade, signature clinical neuropathology of blast-induced traumatic brain injury has been under intense debate, but interface astroglial scarring (IAS) seems to be convincing. In this study, we examined whether IAS could be replicated in the rat brain exposed to a laser-induced shock wave(s) (LISW[s]), a tool that can produce a pure shock wave (primary mechanism) without dynamic pressure (tertiary mechanism). Under certain conditions, we observed astroglial scarring in the subpial glial plate (SGP), gray-white matter junctions (GM-WM), ventricular wall (VW), and regions surrounding cortical blood vessels, accurately reproducing clinical IAS. We also observed shock wave impulse-dependent meningeal damage (dural microhemorrhage) in vivo by transcranial near-infrared (NIR) reflectance imaging. Importantly, there were significant correlations between the degree of dural microhemorrhage and the extent of astroglial scarring more than 7 days post-exposure, suggesting an association of meningeal damage with astroglial scarring. The results demonstrated that the primary mechanism alone caused the IAS and meningeal damage, both of which are attributable to acoustic impedance mismatching at multi-layered tissue boundaries. The time course of glial fibrillary acidic protein (GFAP) immunoreactivity depended not only on the LISW conditions but also on the regions. In the SGP, significant increases in GFAP immunoreactivity were observed at 3 days post-exposure, whereas in the GM-WM and VW, GFAP immunoreactivity was not significantly increased before 28 days post-exposure, suggesting different pathological mechanisms. With the high-impulse single exposure or the multiple exposure (low impulse), fibrotic reaction or fibrotic scar formation was observed, in addition to astroglial scarring, in the cortical surface region. Although there are some limitations, this seems to be the first report on the shock-wave-induced IAS rodent model. The model may be useful to explore potential therapeutic approaches for IAS.

Inhibition of CK2 Diminishes Fibrotic Scar Formation and Improves Outcomes After Ischemic Stroke via Reducing BRD4 Phosphorylation.

Fibrotic scars play important roles in tissue reconstruction and functional recovery in the late stage of nervous system injury. However, the mechanisms underlying fibrotic scar formation and regulation remain unclear. Casein kinase II (CK2) is a protein kinase that regulates a variety of cellular functions through the phosphorylation of proteins, including bromodomain-containing protein 4 (BRD4). CK2 and BRD4 participate in fibrosis formation in a variety of tissues. However, whether CK2 affects fibrotic scar formation remains unclear, as do the mechanisms of signal regulation after cerebral ischemic injury. In this study, we assessed whether CK2 could modulate fibrotic scar formation after cerebral ischemic injury through BRD4. Primary meningeal fibroblasts were isolated from neonatal rats and treated with transforming growth factor-ß1 (TGF-ß1), SB431542 (a TGF-ß1 receptor kinase inhibitor) or TBB (a highly potent CK2 inhibitor). Adult SD rats were intraperitoneally injected with TBB to inhibit CK2 after MCAO/R. We found that CK2 expression was increased in vitro in the TGF-ß1-induced fibrosis model and in vivo in the MCAO/R injury model. The TGF-ß1 receptor kinase inhibitor SB431542 decreased CK2 expression in fibroblasts. The CK2 inhibitor TBB reduced the increases in proliferation, migration and activation of fibroblasts caused by TGF-ß1 in vitro, and it inhibited fibrotic scar formation, ameliorated histopathological damage, protected Nissl bodies, decreased infarct volume and alleviated neurological deficits after MCAO/R injury in vivo. Furthermore, CK2 inhibition decreased BRD4 phosphorylation both in vitro and in vivo. The findings of the present study suggested that CK2 may control BRD4 phosphorylation to regulate fibrotic scar formation, to affecting outcomes after ischemic stroke.

M2a macrophages regulate fibrosis and affect the outcome after stroke via PU.1/mTOR pathway in fibroblasts.

The moderate formation of the fibrotic scar plays an important role in functional recovery after stroke. M2a macrophages have been identified as an important source of early fibrosis after cerebral ischemia. However, the underlying mechanisms by which macrophages interact with fibroblasts in this context remain largely unknown. Therefore, our study aimed to further investigate the potential mechanisms underlying the effects of macrophages on fibroblasts following ischemic stroke. In vitro and in vivo, recombinant rat interleukin 4 (IL4) was used to induce macrophages to polarize into M2a macrophages. In vitro, primary Sprague-Dawley newborn rat meningeal-derived fibroblasts were treated with PU.1 knockdown, the PU.1 inhibitor DB1976 or the mTOR inhibitor rapamycin, which were then co-cultured with M2a macrophage conditioned medium (MCM). In vivo, Sprague-Dawley adult rats were infected with negative control adenoviruses or PU.1-shRNA adenoviruses. Ten days after infection, an injury model of middle cerebral artery occlusion/reperfusion (MCAO/R) was constructed. Subsequently, IL4 was injected intracerebroventricularly to induce M2a macrophages polarization. In vitro, M2a MCM upregulated PU.1 expression and promoted the differentiation, proliferation, migration and extracellular matrix generation of fibroblasts, which could be reversed by treatment with the PU.1 inhibitor DB1976 or PU.1 knockdown. In vivo, PU.1 expression in fibroblasts was increased within ischemic core following MCAO/R, and this upregulation was further enhanced by exposure to IL4. Treatment with IL4 promoted fibrosis, increased angiogenesis, reduced apoptosis and infarct volume, as well as mitigated neurological deficits after MCAO/R, and these effects could be reversed by PU.1 knockdown. Furthermore, both in vivo and in vitro studies showed that IL4 treatment increased the levels of phosphorylated Akt and mTOR proteins, which were markedly decreased by PU.1 knockdown. Additionally, the use of an mTOR inhibitor rapamycin obviously suppressed the migration and differentiation of fibroblasts, and Col1 synthesis. In conclusion, our findings suggest for the first time that M2a macrophages, at least in part, regulate fibrosis and affect the outcome after cerebral ischemic stroke via the PU.1/mTOR signaling pathway in fibroblasts.

Evaluating the Efficacy of a Thermoresponsive Hydrogel for Delivering Anti-Collagen Antibodies to Reduce Posttraumatic Scarring in Orthopedic Tissues.

Excessive posttraumatic scarring in orthopedic tissues, such as joint capsules, ligaments, tendons, muscles, and peripheral nerves, presents a significant medical problem, resulting in pain, restricted joint mobility, and impaired musculoskeletal function. Current treatments for excessive scarring are often ineffective and require the surgical removal of fibrotic tissue, which can aggravate the problem. The primary component of orthopedic scars is collagen I-rich fibrils. Our research team has developed a monoclonal anti-collagen antibody (ACA) that alleviates posttraumatic scarring by inhibiting collagen fibril formation. We previously established the safety and efficacy of ACA in a rabbit-based arthrofibrosis model. In this study, we evaluate the utility of a well-characterized thermoresponsive hydrogel (THG) as a delivery vehicle for ACA to injury sites. Crucial components of the hydrogel included N-isopropylacrylamide, poly(ethylene glycol) diacrylate, and hyaluronic acid. Our investigation focused on in vitro ACA release kinetics, stability, and activity. Additionally, we examined the antigen-binding characteristics of ACA post-release from the THG in an in vivo context. Our preliminary findings suggest that the THG construct exhibits promise as a delivery platform for antibody-based therapeutics to reduce excessive scarring in orthopedic tissues.

Building a pathway to recovery: Targeting ECM remodeling in CNS injuries.

Extracellular matrix (ECM) is a complex and dynamic network of proteoglycans, proteins, and other macromolecules that surrounds cells in tissues. The ECM provides structural support to cells and plays a critical role in regulating various cellular functions. ECM remodeling is a dynamic process involving the breakdown and reconstruction of the ECM. This process occurs naturally during tissue growth, wound healing, and tissue repair. However, in the context of central nervous system (CNS) injuries, dysregulated ECM remodeling can lead to the formation of fibrotic and glial scars. CNS injuries encompass various traumatic events, including concussions and fractures. Following CNS trauma, the formation of glial and fibrotic scars becomes prominent. Glial scars primarily consist of reactive astrocytes, while fibrotic scars are characterized by an abundance of ECM proteins. ECM remodeling plays a pivotal and tightly regulated role in the development of these scars after spinal cord and brain injuries. Various factors like ECM components, ECM remodeling enzymes, cell surface receptors of ECM molecules, and downstream pathways of ECM molecules are responsible for the remodeling of the ECM. The aim of this review article is to explore the changes in ECM during normal physiological conditions and following CNS injuries. Additionally, we discuss various approaches that target various factors responsible for ECM remodeling, with a focus on promoting axon regeneration and functional recovery after CNS injuries. By targeting ECM remodeling, it may be possible to enhance axonal regeneration and facilitate functional recovery after CNS injuries.

M2 macrophages mediate fibrotic scar formation in the early stages after cerebral ischemia in rats.

In the central nervous system, the formation of fibrotic scar after injury inhibits axon regeneration and promotes repair. However, the mechanism underlying fibrotic scar formation and regulation remains poorly understood. M2 macrophages regulate fibrotic scar formation after injury to the heart, lung, kidney, and central nervous system. However, it remains to be clarified whether and how M2 macrophages regulate fibrotic scar formation after cerebral ischemia injury. In this study, we found that, in a rat model of cerebral ischemia induced by middle cerebral artery occlusion/reperfusion, fibrosis and macrophage infiltration were apparent in the ischemic core in the early stage of injury (within 14 days of injury). The number of infiltrated macrophages was positively correlated with fibronectin expression. Depletion of circulating monocyte-derived macrophages attenuated fibrotic scar formation. Interleukin 4 (IL4) expression was strongly enhanced in the ischemic cerebral tissues, and IL4-induced M2 macrophage polarization promoted fibrotic scar formation in the ischemic core. In addition, macrophage-conditioned medium directly promoted fibroblast proliferation and the production of extracellular matrix proteins in vitro. Further pharmacological and genetic analyses showed that sonic hedgehog secreted by M2 macrophages promoted fibrogenesis in vitro and in vivo, and that this process was mediated by secretion of the key fibrosis-associated regulatory proteins transforming growth factor beta 1 and matrix metalloproteinase 9. Furthermore, IL4-afforded functional restoration on angiogenesis, cell apoptosis, and infarct volume in the ischemic core of cerebral ischemia rats were markedly impaired by treatment with an sonic hedgehog signaling inhibitor, paralleling the extent of fibrosis. Taken together, our findings show that IL4/sonic hedgehog/transforming growth factor beta 1 signaling targeting macrophages regulates the formation of fibrotic scar and is a potential therapeutic target for ischemic stroke.

Fingolimod (FTY720) Hinders Interferon-γ-Mediated Fibrotic Scar Formation and Facilitates Neurological Recovery After Spinal Cord Injury.

Following spinal cord injury (SCI), fibrotic scar inhibits axon regeneration and impairs neurological function recovery. It has been reported that T cell-derived interferon (IFN)-γ plays a pivotal role in promoting fibrotic scarring in neurodegenerative disease. However, the role of IFN-γ in fibrotic scar formation after SCI has not been declared. In this study, a spinal cord crush injury mouse was established. Western blot and immunofluorescence showed that IFN-γ was surrounded by fibroblasts at 3, 7, 14, and 28 days post-injury. Moreover, IFN-γ is mainly secreted by T cells after SCI. Further, in situ injection of IFN-γ into the normal spinal cord resulted in fibrotic scar formation and inflammation response at 7 days post-injection. After SCI, the intraperitoneal injection of fingolimod (FTY720), a sphingosine-1-phosphate receptor 1 (S1PR1) modulator and W146, an S1PR1 antagonist, significantly reduced T cell infiltration, attenuating fibrotic scarring via inhibiting IFN-γ/IFN-γR pathway, while in situ injection of IFN-γ diminished the effect of FTY720 on reducing fibrotic scarring. FTY720 treatment inhibited inflammation, decreased lesion size, and promoted neuroprotection and neurological recovery after SCI. These findings demonstrate that the inhibition of T cell-derived IFN-γ by FTY720 suppressed fibrotic scarring and contributed to neurological recovery after SCI.

Delayed inhibition of collagen deposition by targeting bone morphogenetic protein 1 promotes recovery after spinal cord injury.

Fibrotic scars appear after spinal cord injury (SCI) and are mainly composed of fibroblasts and excess extracellular matrix (ECM), including different types of collagen. The temporal and spatial distribution and role of excess collagens and ECM after SCI are not yet fully understood. Here, we identified that the procollagen type I C-terminal propeptide (PICP), a marker of collagen type I deposition, and bone morphogenetic protein 1 (BMP1), a secreted procollagen c-proteinase (PCP) for type I collagen maturation, were significantly elevatedin cerebrospinal fluid of patients with SCI compared with healthy controls, and were associated with spinal cord compression and neurological symptoms. We revealed the deposition of type I collagen in the area damaged by SCI in mice and confirmed that BMP1 was the only expressed PCP and induced collagen deposition. Furthermore, transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) can activate the expression of BMP1. However, inhibition of BMP1 at the acute phase eliminated fibrotic scars in the damaged area and inhibited activation and enrichment of astrocytes, which made the damage difficult to repair and increased hematoma. Unexpectedly, knockdown of Bmp1 by adeno-associated virus or the inhibition of BMP1 biological function by specific inhibitors and monoclonal antibodies at different time points after injury led to distinct therapeutic effects. Only delayed inhibition of BMP1 improved axonal regeneration and myelin repair at the subacute stage post-injury, and led to the recovery of motor function, suggesting that scarring had a dual effect. Early inhibition of the scarring was not conducive to limiting inflammation, while excessive scar formation inhibited the growth of axons. After SCI, the collagen deposition indicators increased in both human cerebrospinal fluid and mouse spinal cord. Therefore, suppression of BMP1 during the subacute phase improves nerve function after SCI and is a potential target for scar reduction.

The role of monocytes in optic nerve injury.

Monocytes, including monocyte-derived macrophages and resident microglia, mediate many phases of optic nerve injury pathogenesis. Resident microglia respond first, followed by infiltrating macrophages which regulate neuronal inflammation, cell proliferation and differentiation, scar formation and tissue remodeling following optic nerve injury. However, microglia and macrophages have distinct functions which can be either beneficial or detrimental to the optic nerve depending on the spatial context and temporal sequence of their activity. These divergent effects are attributed to pro- and anti-inflammatory cytokines expressed by monocytes, crosstalk between monocyte and glial cells and even microglia-macrophage communication. In this review, we describe the dynamics and functions of microglia and macrophages in neuronal inflammation and regeneration following optic nerve injury, and their possible role as therapeutic targets for axonal regeneration.

Multiple Genetic Loci Associated with Pug Dog Thoracolumbar Myelopathy.

Pug dogs with thoracolumbar myelopathy (PDM) present with a specific clinical phenotype that includes progressive pelvic limb ataxia and paresis, commonly accompanied by incontinence. Vertebral column malformations and lesions, excessive scar tissue of the meninges, and central nervous system inflammation have been described. PDM has a late onset and affects more male than female dogs. The breed-specific presentation of the disorder suggests that genetic risk factors are involved in the disease development. To perform a genome-wide search for PDM-associated loci, we applied a Bayesian model adapted for mapping complex traits (BayesR) and a cross-population extended haplotype homozygosity test (XP-EHH) in 51 affected and 38 control pugs. Nineteen associated loci (harboring 67 genes in total, including 34 potential candidate genes) and three candidate regions under selection (with four genes within or next to the signal) were identified. The multiple candidate genes identified have implicated functions in bone homeostasis, fibrotic scar tissue, inflammatory responses, or the formation, regulation, and differentiation of cartilage, suggesting the potential relevance of these processes to the pathogenesis of PDM.

Astrocytic Cebpd Regulates Pentraxin 3 Expression to Promote Fibrotic Scar Formation After Spinal Cord Injury.

Astroglial-fibrotic scars resulted from spinal cord injury affect motor and sensory function, leading to paralysis. In particular, the fibrotic scar is a main barrier that disrupts neuronal regeneration after spinal cord injury. However, the association between astrocytes and fibrotic scar formation is not yet understood. We have previously demonstrated that the transcriptional factor Cebpd contributes to astrogliosis, which promotes glial scar formation after spinal cord injury. Herein, we show that fibrotic scar formation was decreased in the epicenter region in Cebpd-/- mice after contusive spinal cord injury and astrocytic Cebpd promoted fibroblast migration through secretion of Ptx3. Furthermore, the expression of Mmp3 was increased under recombinant protein Ptx3 treatment in fibroblasts by observing microarray data, resulting in fibroblast migration. In addition, regulation of Mmp3 occurs through the NFκB signaling pathway by using an irreversible inhibitor of IκBα phosphorylation in pretreated fibroblasts. Of note, we used the synthetic peptide RI37, which blocks fibroblast migration and decreases fibroblast Mmp3 expression in IL-1ß-treated astrocyte conditioned media. Collectively, our data suggest that fibroblast migration can be affected by astrocytic Cebpd through the Ptx3/NFκB/Mmp3 axis pathway and that the RI37 peptide may act as a therapeutic medicine to inhibit fibrotic scar formation after spinal cord injury.

Hematogenous Macrophages Contribute to Fibrotic Scar Formation After Optic Nerve Crush.

Although glial scar formation has been extensively studied after optic nerve injury, the existence and characteristics of traumatic optic nerve fibrotic scar formation have not been previously characterized. Recent evidence suggests infiltrating macrophages are involved in pathological processes after optic nerve crush (ONC), but their role in fibrotic scar formation is unknown. Using wild-type and transgenic mouse models with optic nerve crush injury, we show that macrophages infiltrate and associate with fibroblasts in the traumatic optic nerve lesion fibrotic scar. We dissected the role of hematogenous and resident macrophages, labeled with Dil liposomes intravenously administered, and observed that hematogenous macrophages (Dil+ cells) specifically accumulate in the center of traumatic fibrotic scar while Iba-1+ cells reside predominantly at the margins of optic nerve fibrotic scar. Depletion of hematogenous macrophages results in reduced fibroblast density and decreased extracellular matrix deposition within the fibrotic scar area following ONC. However, retinal ganglion cell degeneration and function loss after optic nerve crush remain unaffected after hematogenous macrophage depletion. We present new and previously not characterized evidence that hematogenous macrophages are selectively recruited into the fibrotic core of the optic nerve crush site and critical for this fibrotic scar formation.

Inhibition of BRD4 decreases fibrous scarring after ischemic stroke in rats by inhibiting the phosphorylation of Smad2/3.

AIMS: Fibrous scarring may play a much more important role in preventing secondary expansion of tissue damage and hindering repair and regeneration than glial scarring after central nervous system (CNS) injury. However, relatively little is known about how fibrous scars form and how fibrous scar formation is regulated after CNS injury. Bromodomain-containing protein 4 (BRD4) is involved in fibrosis in many tissues, and transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signaling is one of the critical pathways of fibrosis. However, it is unclear whether and how BRD4 affects fibrous scar formation after ischemicbraininjury. In the present study, whether BRD4 can regulate the formation of fibrous scars after ischemic stroke via TGF-ß1/Smad2/3 signaling was assessed. MATERIALS AND METHODS: Primary meningeal fibroblasts isolated from neonatal SD rats were treated with TGF-ß1, SB431542 (a TGF-ß1 receptor inhibitor) and JQ1 (a small-molecule BET inhibitor that can also inhibit BRD4). BRD4 was knocked down in adult Sprague-Dawley (SD) rats by using adenovirus before middle cerebral artery occlusion/reperfusion (MCAO/R) injury. The proliferation and migration of meningeal fibroblasts in vitro were evaluated with the Cell Counting Kit-8 (CCK-8) assay and scratch test, respectively. Neurological function was assessed with Longa scores, modified Bederson Scores and modified neurological severity scores (mNSSs). The infarct volume was assessed with TTC staining. The protein expression of synaptophysin (SY), BRD4, Smad2/3, p-Smad2/3, α-smooth muscle actin (α-SMA), collagen-1 (COL1) and fibronectin (FN) in vivo and in vitro was examined with immunocytochemistry, immunofluorescence, and Western blotting. KEY FINDINGS: BRD4 expression was upregulated in a TGF-ß1-induced meningeal fibroblast fibrosis model and was downregulated by the TGF-ß1 receptor inhibitor SB431542 in vitro. JQ1, a small-molecule BET inhibitor, inhibited BRD4 and decreased TGF-ß1-induced meningeal fibroblast proliferation, migration and activation. Furthermore, MCAO/R injury induced fibrosis and upregulated BRD4 expression in the cerebral infarct center. BRD4 knockdown by adenovirus inhibited fibrous scarring, promoted synaptic survival, decreased the infarct volume, and improved neurological function after MCAO/R injury. Moreover, inhibition of BRD4, either by JQ1 in vitro or adenovirus in vivo, decreased the phosphorylation of Smad2/3. CONCLUSIONS: This study is the first to indicate that inhibition of BRD4 delays fibrous scarring after ischemic stroke through mechanisms involving the phosphorylation of Smad2/3.

Imatinib inhibits pericyte-fibroblast transition and inflammation and promotes axon regeneration by blocking the PDGF-BB/PDGFRß pathway in spinal cord injury.

BACKGROUND: Fibrotic scar formation and inflammation are characteristic pathologies of spinal cord injury (SCI) in the injured core, which has been widely regarded as the main barrier to axonal regeneration resulting in permanent functional recovery failure. Pericytes were shown to be the main source of fibroblasts that form fibrotic scar. However, the mechanism of pericyte-fibroblast transition after SCI remains elusive. METHODS: Fibrotic scarring and microvessels were assessed using immunofluorescence staining after establishing a crush SCI model. To study the process of pericyte-fibroblast transition, we analyzed pericyte marker and fibroblast marker expression using immunofluorescence. The distribution and cellular origin of platelet-derived growth factor (PDGF)-BB were examined with immunofluorescence. Pericyte-fibroblast transition was detected with immunohistochemistry and Western blot assays after PDGF-BB knockdown and blocking PDGF-BB/PDGFRß signaling in vitro. Intrathecal injection of imatinib was used to selectively inhibit PDGF-BB/PDGFRß signaling. The Basso mouse scale score and footprint analysis were performed to assess functional recovery. Subsequently, axonal regeneration, fibrotic scarring, fibroblast population, proliferation and apoptosis of PDGFRß+ cells, microvessel leakage, and the inflammatory response were assessed with immunofluorescence. RESULTS: PDGFRß+ pericytes detached from the blood vessel wall and transitioned into fibroblasts to form fibrotic scar after SCI. PDGF-BB was mainly distributed in the periphery of the injured core, and microvascular endothelial cells were one of the sources of PDGF-BB in the acute phase. Microvascular endothelial cells induced pericyte-fibroblast transition through the PDGF-BB/PDGFRß signaling pathway in vitro. Pharmacologically blocking the PDGF-BB/PDGFRß pathway promoted motor function recovery and axonal regeneration and inhibited fibrotic scar formation. After fibrotic scar formation, blocking the PDGFRß receptor inhibited proliferation and promoted apoptosis of PDGFRß+ cells. Imatinib did not alter pericyte coverage on microvessels, while microvessel leakage and inflammation were significantly decreased after imatinib treatment. CONCLUSIONS: We reveal that the crosstalk between microvascular endothelial cells and pericytes promotes pericyte-fibroblast transition through the PDGF-BB/PDGFRß signaling pathway. Our finding suggests that blocking the PDGF-BB/PDGFRß signaling pathway with imatinib contributes to functional recovery, fibrotic scarring, and inflammatory attenuation after SCI and provides a potential target for the treatment of SCI.

Fibrotic Scar in CNS Injuries: From the Cellular Origins of Fibroblasts to the Molecular Processes of Fibrotic Scar Formation.

Central nervous system (CNS) trauma activates a persistent repair response that leads to fibrotic scar formation within the lesion. This scarring is similar to other organ fibrosis in many ways; however, the unique features of the CNS differentiate it from other organs. In this review, we discuss fibrotic scar formation in CNS trauma, including the cellular origins of fibroblasts, the mechanism of fibrotic scar formation following an injury, as well as the implication of the fibrotic scar in CNS tissue remodeling and regeneration. While discussing the shared features of CNS fibrotic scar and fibrosis outside the CNS, we highlight their differences and discuss therapeutic targets that may enhance regeneration in the CNS.