TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

Elastin and collagen fibres in cutaneous wound healing.

Skin forms the outer barrier of the body. Upon injury, successful wound healing in normal skin restores tissue damage and counteracts the loss of extracellular matrix (ECM) proteins and cells. Collagens and elastin are the most abundant structural proteins of the ECM. In homeostasis, collagen type I is the prevalent form, but it is replaced by type III collagen upon wounding, and only later remodelled. In turn, unsuccessful healing results in scars, which tend to be inflexible and inelastic as compared to normal elastic dermis. Scar inelasticity may be due to the absence of mature elastin fibre formation and cross-linking. In this review, the available information on the process of formation of new collagen and elastic fibres during wound healing is analysed. The distinct roles of elastin and collagen proteins during healing are revisited and future research directions proposed which may help improve clinical management of open wounds and scars.

Pericardial delta like non-canonical NOTCH ligand 1 (Dlk1) augments fibrosis in the heart through epithelial to mesenchymal transition.

BACKGROUND: Heart failure due to myocardial infarction (MI) involves fibrosis driven by epicardium-derived cells (EPDCs) and cardiac fibroblasts, but strategies to inhibit and provide cardio-protection remains poor. The imprinted gene, non-canonical NOTCH ligand 1 (Dlk1), has previously been shown to mediate fibrosis in the skin, lung and liver, but very little is known on its effect in the heart. METHODS: Herein, human pericardial fluid/plasma and tissue biopsies were assessed for DLK1, whereas the spatiotemporal expression of Dlk1 was determined in mouse hearts. The Dlk1 heart phenotype in normal and MI hearts was assessed in transgenic mice either lacking or overexpressing Dlk1. Finally, in/ex vivo cell studies provided knowledge on the molecular mechanism. RESULTS: Dlk1 was demonstrated in non-myocytes of the developing human myocardium but exhibited a restricted pericardial expression in adulthood. Soluble DLK1 was twofold higher in pericardial fluid (median 45.7 [34.7 (IQR)) µg/L] from cardiovascular patients (n = 127) than in plasma (median 26.1 µg/L [11.1 (IQR)]. The spatial and temporal expression pattern of Dlk1 was recapitulated in mouse and rat hearts. Similar to humans lacking Dlk1, adult Dlk1-/- mice exhibited a relatively mild developmental, although consistent cardiac phenotype with some abnormalities in heart size, shape, thorax orientation and non-myocyte number, but were functionally normal. However, after MI, scar size was substantially reduced in Dlk1-/- hearts as compared with Dlk1+/+ littermates. In line, high levels of Dlk1 in transgenic mice Dlk1fl/fl xWT1GFPCre and Dlk1fl/fl xαMHCCre/+Tam increased scar size following MI. Further mechanistic and cellular insight demonstrated that pericardial Dlk1 mediates cardiac fibrosis through epithelial to mesenchymal transition (EMT) of the EPDC lineage by maintaining Integrin ß8 (Itgb8), a major activator of transforming growth factor ß and EMT. CONCLUSIONS: Our results suggest that pericardial Dlk1 embraces a, so far, unnoticed role in the heart augmenting cardiac fibrosis through EMT. Monitoring DLK1 levels as well as targeting pericardial DLK1 may thus offer new venues for cardio-protection.

Lupus dermal fibroblasts are proinflammatory and exhibit a profibrotic phenotype in scarring skin disease.

Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in patients with cutaneous lupus erythematosus (CLE) remain poorly understood. Dermal fibroblasts in patients with systemic lupus erythematosus (SLE) experience increased cytokine signaling in vivo, but the effect of inflammatory mediators on fibroblast responses in nonscarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from nonlesional skin of 22 patients with SLE and CLE and 34 individuals acting as healthy controls. Notably, inflammatory cytokine responses were exaggerated in SLE fibroblasts compared with those from individuals acting as healthy controls. In lesional CLE biopsies, these same inflammatory profiles were reflected in single-cell RNA-Seq of SFRP2+ and inflammatory fibroblast subsets, and TGF-ß was identified as a critical upstream regulator for inflammatory fibroblasts in scarring discoid lupus lesions. In vitro cytokine stimulation of nonlesional fibroblasts from patients who scar from CLE identified an upregulation of collagens, particularly in response to TGF-ß, whereas inflammatory pathways were more prominent in nonscarring patients. Our study revealed that SLE fibroblasts are poised to hyperrespond to inflammation, with differential responses among patients with scarring versus nonscarring disease, providing a potential skin-specific target for mitigating damage.

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.

Fibroblast mediated dynamics in diffusively uncoupled myocytes: a simulation study using 2-cell motifs.

In healthy hearts myocytes are typically coupled to nearest neighbours through gap junctions. Under pathological conditions such as fibrosis, or in scar tissue, or across ablation lines myocytes can uncouple from their neighbours. Electrical conduction may still occur via fibroblasts that not only couple proximal myocytes but can also couple otherwise unconnected regions. We hypothesise that such coupling can alter conduction between myocytes via introduction of delays or by initiation of premature stimuli that can potentially result in reentry or conduction blocks. To test this hypothesis we have developed several 2-cell motifs and investigated the effect of fibroblast mediated electrical coupling between uncoupled myocytes. We have identified various regimes of myocyte behaviour that depend on the strength of gap-junctional conductance, connection topology, and parameters of the myocyte and fibroblast models. These motifs are useful in developing a mechanistic understanding of long-distance coupling on myocyte dynamics and enable the characterisation of interaction between different features such as myocyte and fibroblast properties, coupling strengths and pacing period. They are computationally inexpensive and allow for incorporation of spatial effects such as conduction velocity. They provide a framework for constructing scar tissue boundaries and enable linking of cellular level interactions with scar induced arrhythmia.

Single-cell, single-nucleus, and spatial transcriptomics characterization of the immunological landscape in the healthy and PSC human liver.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which there is an unmet need to understand the cellular composition of the affected liver and how it underlies disease pathogenesis. We aimed to generate a comprehensive atlas of the PSC liver using multi-omic modalities and protein-based functional validation. METHODS: We employed single-cell and single-nucleus RNA sequencing (47,156 cells and 23,000 nuclei) and spatial transcriptomics (one sample by 10x Visium and five samples with Nanostring GeoMx DSP) to profile the cellular ecosystem in 10 PSC livers. Transcriptomic profiles were compared to 24 neurologically deceased donor livers (107,542 cells) and spatial transcriptomics controls, as well as 18,240 cells and 20,202 nuclei from three PBC livers. Flow cytometry was performed to validate PSC-specific differences in immune cell phenotype and function. RESULTS: PSC explants with parenchymal cirrhosis and prominent periductal fibrosis contained a population of cholangiocyte-like hepatocytes that were surrounded by diverse immune cell populations. PSC-associated biliary, mesenchymal, and endothelial populations expressed chemokine and cytokine transcripts involved in immune cell recruitment. Additionally, expanded CD4+ T cells and recruited myeloid populations in the PSC liver expressed the corresponding receptors to these chemokines and cytokines, suggesting potential recruitment. Tissue-resident macrophages, by contrast, were reduced in number and exhibited a dysfunctional and downregulated inflammatory response to lipopolysaccharide and interferon-γ stimulation. CONCLUSIONS: We present a comprehensive atlas of the PSC liver and demonstrate an exhaustion-like phenotype of myeloid cells and markers of chronic cytokine expression in late-stage PSC lesions. This atlas expands our understanding of the cellular complexity of PSC and has potential to guide the development of novel treatments. IMPACT AND IMPLICATIONS: Primary sclerosing cholangitis (PSC) is a rare liver disease characterized by chronic inflammation and irreparable damage to the bile ducts, which eventually results in liver failure. Due to a limited understanding of the underlying pathogenesis of disease, treatment options are limited. To address this, we sequenced healthy and diseased livers to compare the activity, interactions, and localization of immune and non-immune cells. This revealed that hepatocytes lining PSC scar regions co-express cholangiocyte markers, whereas immune cells infiltrate the scar lesions. Of these cells, macrophages, which typically contribute to tissue repair, were enriched in immunoregulatory genes and demonstrated a lack of responsiveness to stimulation. These cells may be involved in maintaining hepatic inflammation and could be a target for novel therapies.

The TGFß1/SMADs/Snail1 signaling axis mediates pericyte-derived fibrous scar formation after spinal cord injury.

AIMS: The deposition of fibrous scars after spinal cord injury (SCI) affects axon regeneration and the recovery of sensorimotor function. It has been reported that microvascular pericytes in the neurovascular unit are the main source of myofibroblasts after SCI, but the specific molecular targets that regulate pericyte participation in the formation of fibrous scars remain to be clarified. METHODS: In this study, a rat model of spinal cord dorsal hemisection injury was used. After SCI, epigallocatechin gallate (EGCG) was intraperitoneally injected to block the TGFß1 signaling pathway or LV-Snail1-shRNA was immediately injected near the core of the injury using a microsyringe to silence Snail1 expression. Western blotting and RT-qPCR were used to analyze protein expression and transcription levels in tissues. Nissl staining and immunofluorescence analysis were used to analyze neuronal cell viability, scar tissue, and axon regeneration after SCI. Finally, the recovery of hind limb function after SCI was evaluated. RESULTS: The results showed that targeted inhibition of Snail1 could block TGFß1-induced pericyte-myofibroblast differentiation in vitro. In vivo experiments showed that timely blockade of Snail1 could reduce fibrous scar deposition after SCI, promote axon regeneration, improve neuronal survival, and facilitate the recovery of lower limb motor function. CONCLUSION: In summary, Snail1 promotes the deposition of fibrous scars and inhibits axonal regeneration after SCI by inducing the differentiation of pericytes into myofibroblasts. Snail1 may be a promising therapeutic target for SCI.

Biological effect of laser-assisted scar healing (LASH) on standardized human three-dimensional wound healing skin models using fractional non-ablative 1540 nm Er:Glass or 1550 nm diode lasers.

PURPOSE: In postoperative wound healing after surgical operations or ablative laser treatments, recent studies suggest the timely use of non-ablative fractional laser treatments with the aim to improve wound healing and prevent pathological scar formation. However, the underlying molecular mechanisms are poorly understood. The aim of this study was to investigate the effects of laser-assisted scar healing (LASH) at the molecular level and to combine it with already established wound healing-promoting local treatments. METHODS: We irradiated full-thickness 3D skin models with a fractional ablative Er:YAG laser to set standardized lesions to the epidermal and upper dermal layer. Subsequently, LASH was induced by irradiating the models with either a fractional non-ablative 1540 nm Er:Glass or 1550 nm diode laser. In addition, we tested the combination of non-ablative fractional laser treatment and topical aftercare with a dexpanthenol-containing ointment (DCO). RESULTS: Histological analysis revealed that models irradiated with the 1540 nm Er:Glass or 1550 nm diode laser exhibited accelerated but not complete wound closure after 16 h. In contrast, additional topical posttreatment with DCO resulted in complete wound closure. At gene expression level, both non-ablative laser systems showed similar effects on epidermal differentiation and mild anti-inflammatory properties. The additional posttreatment with DCO enhanced the wound-healing effects of LASH, especially the upregulation of epidermal differentiation markers and anti-inflammatory cytokines at the gene expression level. CONCLUSION: This in vitro study deciphers the biological effects of LASH with a fractional non-ablative 1540 nm Er:Glass or a 1550 nm diode laser in 3D skin models. These data help to better understand the biological properties of the LASH technique and is important to optimize its application.

Automated spatially targeted optical micro proteomics identifies fibroblast- and macrophage-specific regulation of myocardial infarction scar maturation in rats.

Myocardial infarction (MI) results from occlusion of blood supply to the heart muscle causing death of cardiac muscle cells. Following myocardial infarction (MI), extracellular matrix deposition and scar formation mechanically stabilize the injured heart as damaged myocytes undergo necrosis and removal. Fibroblasts and macrophages are key drivers of post-MI scar formation, maturation, and ongoing long-term remodelling; however, their individual contributions are difficult to assess from bulk analyses of infarct scar. Here, we employ state-of-the-art automated spatially targeted optical micro proteomics (autoSTOMP) to photochemically tag and isolate proteomes associated with subpopulations of fibroblasts (SMA+) and macrophages (CD68+) in the context of the native, MI tissue environment. Over a time course of 6-weeks post-MI, we captured dynamic changes in the whole-infarct proteome and determined that some of these protein composition signatures were differentially localized near SMA+ fibroblasts or CD68+ macrophages within the scar region. These results link specific cell populations to within-infarct protein remodelling and illustrate the distinct metabolic and structural processes underlying the observed physiology of each cell type.

Single-Cell Mapping of Large and Small Arteries During Hypertensive Aging.

Vascular aging is directly related to several major diseases including clinical primary hypertension. Conversely, elevated blood pressure itself accelerates vascular senescence. However, the interaction between vascular aging and hypertension has not been characterized during hypertensive aging. To depict the interconnectedness of complex mechanisms between hypertension and aging, we performed single-cell RNA sequencing of aorta, femoral and mesentery arteries, respectively, from male Wistar Kyoto rats and male spontaneously hypertensive rats aging 16 or 72 weeks. We integrated 12 data sets to map the blood vessels of senile hypertension from 3 perspective: vascular aging, hypertension, and vascular type. We found that aging and hypertension independently exerted a significant impact on the alteration of cellular composition and artery remodeling, even greater when superimposed. Consistently, smooth muscle cells (SMCs) underwent phenotypic switching from contractile toward synthetic, apoptotic, and senescent SMCs with aging/hypertension. Furthermore, we identified 3 subclusters of Spp1high, encoding protein osteopontin (OPN), synthetic SMCs, Spp1high matrix activated fibroblasts, and Spp1high scar-associated macrophage involved in hypertensive aging. Spp1high scar-associated macrophage enriched for reactive oxygen species metabolic process and cell migration-associated function. Cell-cell communication analysis revealed Spp1-Cd44 receptor pairing was markedly aggravated in the hypertensive aging condition. Importantly, the concentration of serum OPN significantly potentiated in aged hypertensive patients compared with the normal group. Thus, we provide a comprehensive cell atlas to systematically resolve the cellular diversity and dynamic cellular communication changes of the vessel wall during hypertensive aging, identifying a protein marker OPN as a potential regulator of vascular remodeling during hypertensive aging.

The expression pattern of cytokeratin 6a in epithelial cells of different origin in dermo-epidermal skin substitutes in vivo.

Keratinocytes are the predominant cell type of skin epidermis. Through the programmed process of differentiation, they form a cornified envelope that provides a physical protective barrier against harmful external environment. Keratins are major structural proteins of keratinocytes that together with actin filaments and microtubules form the cytoskeleton of these cells. In this study, we examined the expression pattern and distribution of cytokeratin 6a (CK6a) in healthy human skin samples of different body locations, in fetal and scar skin samples, as well as in dermo-epidermal skin substitutes (DESSs). We observed that CK6a expression is significantly upregulated in fetal skin and scar tissue as well as in skin grafts after short-term transplantation. Importantly, the abundance of CK6a corresponds directly to the expression pattern of wound healing marker CK16. We postulate that CK6a is a useful marker to accurately evaluate the homeostatic state of DESSs.

Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis.

OBJECTIVES: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. METHODS: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). RESULTS: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. CONCLUSION: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. LEVEL OF EVIDENCE: NA Laryngoscope, 134:374-381, 2024.

Integrin αV mediated activation of myofibroblast via mechanoparacrine of transforming growth factor ß1 in promoting fibrous scar formation after myocardial infarction.

BACKGROUND: Myocardial infarction (MI) dramatically changes the mechanical stress, which is intensified by the fibrotic remodeling. Integrins, especially the αV subunit, mediate mechanical signal and mechanoparacrine of transforming growth factor ß1 (TGF-ß1) in various organ fibrosis by activating CFs into myofibroblasts (MFBs). We investigated a possible role of integrin αV mediated mechanoparacrine of TGF-ß1 in MFBs activation for fibrous reparation in mice with MI. METHODS: Heart samples from MI, sham, or MI plus cilengitide (14 mg/kg, specific integrin αV inhibitor) treated mice, underwent functional and morphological assessments by echocardiography, and histochemistry on 7, 14 and 28 days post-surgery. The mechanical and ultrastructural changes of the fibrous scar were further evaluated by atomic mechanics microscope (AFM), immunofluorescence, second harmonic generation (SHG) imaging, polarized light and scanning electron microscope, respectively. Hydroxyproline assay was used for total collagen content, and western blot for protein expression profile examination. Fibroblast bioactivities, including cell shape, number, Smad2/3 signal and expression of extracellular matrix (ECM) related proteins, were further evaluated by microscopic observation and immunofluorescence in polyacrylamide (PA) hydrogel with adjustable stiffness, which was re-explored in fibroblast cultured on stiff matrix after silencing of integrin αV. The content of total and free TGF-ß1 was tested by enzyme-linked immunosorbent assay (ELISA) in both infarcted tissue and cell samples. RESULT: Increased stiffness with heterogeneity synchronized with integrin αV and alpha smooth muscle actin (α-SMA) positive MFBs accumulation in those less mature fibrous areas. Cilengitide abruptly reduced collagen content and disrupted collagen alignment, which also decreased TGF-ß1 bioavailability, Smad2/3 phosphorylation, and α-SMA expression in the fibrous area. Accordingly, fibroblast on stiff but not soft matrix exhibited obvious MFB phenotype, as evidenced by enlarged cell, hyperproliferation, well-developed α-SMA fibers, and elevated ECM related proteins, while silencing of integrin αV almost abolished this switch via attenuating paracrine of TGF-ß1 and nuclear translocation of Smad2/3. CONCLUSION: This study illustrated that increased tissue stiffness activates CFs into MFBs by integrin αV mediated mechanoparacrine of TGF-ß1, especially in immature scar area, which ultimately promotes fibrous scar maturation.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Anti-Angiogenic and Anti-Scarring Dual Effect of Galectin-3 Inhibition in Mouse Models of Corneal Wound Healing.

Corneal scarring is the third leading cause of global blindness. Neovascularization of ocular tissues is a major predisposing factor in scar development. Although corneal transplantation is effective in restoring vision, some patients are at high risk for graft rejection due to the presence of blood vessels in the injured cornea. Current treatment options for controlling corneal scarring are limited, and outcomes are typically poor. In this study, topical application of a small-molecule inhibitor of galectin-3, GB1265, in mouse models of corneal wound healing, led to the reduction of the following in injured corneas: i) corneal angiogenesis; ii) corneal fibrosis; iii) infiltration of immune cells; and iv) expression of the proinflammatory cytokine IL-1ß. Four independent techniques (RNA sequencing, NanoString, real-time quantitative RT-PCR, and Western blot analysis) determined that decreased corneal opacity in the galectin-3 inhibitor-treated corneas was associated with decreases in the numbers of genes and signaling pathways known to promote fibrosis. These findings allowed for a high level of confidence in the conclusion that galectin-3 inhibition by the small-molecule inhibitor GB1265 has dual anti-angiogenic and anti-scarring effects. Targeting galectin-3 by GB1265 is, thus, attractive for the development of innovative therapies for a myriad of ocular and nonocular diseases characterized by pathologic angiogenesis and fibrosis.

CD36/Lyn kinase interactions within macrophages promotes pulmonary fibrosis in response to oxidized phospholipid.

Recent data from human studies and animal models have established roles for type II alveolar epithelial cell (AEC2) injury/apoptosis and monocyte/macrophage accumulation and activation in progressive lung fibrosis. Although the link between these processes is not well defined, we have previously shown that CD36-mediated uptake of apoptotic AEC2s by lung macrophages is sufficient to drive fibrosis. Importantly, apoptotic AEC2s are rich in oxidized phospholipids (oxPL), and amongst its multiple functions, CD36 serves as a scavenger receptor for oxPL. Recent studies have established a role for oxPLs in alveolar scarring, and we hypothesized that uptake and accrual of oxPL by CD36 would cause a macrophage phenotypic change that promotes fibrosis. To test this hypothesis, we treated wild-type and CD36-null mice with the oxPL derivative oxidized phosphocholine (POVPC) and found that CD36-null mice were protected from oxPL-induced scarring. Compared to WT mice, fewer macrophages accumulated in the lungs of CD36-null animals, and the macrophages exhibited a decreased accumulation of intracellular oxidized lipid. Importantly, the attenuated accrual of oxPL in CD36-null macrophages was associated with diminished expression of the profibrotic mediator, TGFß. Finally, the pathway linking oxPL uptake and TGFß expression was found to require CD36-mediated activation of Lyn kinase. Together, these observations elucidate a causal pathway that connects AEC2 injury with lung macrophage activation via CD36-mediated uptake of oxPL and suggest several potential therapeutic targets.

[Study of senescence protein p66Shc on myocardial tissue repair in adult mice].

Our previous study has shown that p66Shc plays an important role in the process of myocardial regeneration in newborn mice, and p66Shc deficiency leads to weakened myocardial regeneration in newborn mice. This study aims to explore the role of p66Shc protein in myocardial injury repair after myocardial infarction in adult mice, in order to provide a new target for the treatment of myocardial injury after myocardial infarction. Mouse myocardial infarction models of adult wild-type (WT) and p66Shc knockout (KO) were constructed by anterior descending branch ligation. The survival rate and heart-to-body weight ratio of two models were compared and analyzed. Masson's staining was used to identify scar area of injured myocardial tissue, and myocyte area was determined by wheat germ agglutinin (WGA) staining. TUNEL staining was used to detect the cardiomyocyte apoptosis. The protein expression of brain natriuretic peptide (BNP), a common marker of myocardial hypertrophy, was detected by Western blotting. The results showed that there was no significant difference in survival rate, myocardial scar area, myocyte apoptosis, and heart weight to body weight ratio between the WT and p66ShcKO mice after myocardial infarction surgery. Whereas the protein expression level of BNP in the p66ShcKO mice was significantly down-regulated compared with that in the WT mice. These results suggest that, unlike in neonatal mice, the deletion of p66Shc has no significant effect on myocardial injury repair after myocardial infarction in adult mice.

Targeting Lysyl Oxidase as a Potential Therapeutic Approach to Reducing Fibrotic Scars Post-operatively: Its Biological Role in Post-Surgical Scar Development.

Abdominal and pelvic surgery, or any surgical injury of the peritoneum, often leads to chronic abdominal adhesions that may lead to bowel obstruction, infertility, and pain. Current therapeutic strategies are usually ineffective, and the pathological mechanisms of the disease are unclear. Excess collagen cross-linking is a key mediator for extra-cellular matrix deposition and fibrogenesis. Lysyl oxidase is a key enzyme that catalyzes the formation of stabilizing cross-links in collagen. Dysregulation of Lysyl oxidase (Lox) expressing upregulates collagen cross-linking, leading ECM deposition. Tissue hypoxia during surgery induces molecular mechanisms and active transcription factors to promote the expression of several genes related to inflammation, oxidative stress, and fibrosis, such as transforming growth factor beta, and Lox. Studies have shown that targeting Lox improves clinical outcomes and fibrotic parameters in liver, lung, and myocardial fibrosis, therefore, Lox may be a potential drug target in the prevention of postsurgical adhesion.

Autophagy protects against vocal fold injury-induced fibrosis.

Vocal fold scar formation due to vocal fold injury (VFI) is a common cause of surgery or trauma-induced voice disorders. Severe scar formation can lead to reduced voice quality or even be life-threatening. Here, we investigated the role of autophagy in VFI, focusing on fibrosis as a consequence of autophagy in inducing VFI. A VFI model was constructed in rats by dissecting the lamina propria tissue from the thyroarytenoid muscle. Real-time PCR and Western blot were used to analyze expressions of autophagy markers, including Beclin1 and Atg7, in VFI. Tgfb1 and Col1a1 were assessed to determine the correlation of fibrosis with VFI progression and autophagy levels. Rat vocal fold fibroblasts were also treated with TGF-ß1 or rapamycin, which activates and suppresses autophagy respectively, to explore how autophagy regulates fibrosis in VFI. Initially, we observed that autophagy was downregulated in vocal fold mucosa after VFI in rats. This was particularly evident by the time-dependent downregulation of Beclin1 and Atg7 following VFI. Concurrently, levels of Tgfb1 and Col1a1 also surged, hinting at elevated fibrosis levels. Furthermore, our experiments with TGF-ß1 stimulation revealed that it inhibited autophagy in rat vocal fold fibroblasts. Interestingly, when we introduced rapamycin, this effect was reversed. Our data suggest that autophagy is a suppressor of VFI by alleviating fibrosis, making targeting autophagy a potential therapeutic route in VFI.NEW & NOTEWORTHY The study has demonstrated that autophagy is a suppressor of VFI by alleviating fibrosis, making autophagy a potential therapeutic target in VFI.