Extracellular Matrix Deposition Research Articles

The role of apoptosis, immunogenic cell death, and macrophage polarization in carbon ion radiotherapy for keloids: Targeting the TGF-β1/SMADs signaling pathway

Keloids, characterized by excessive extracellular matrix (ECM) deposition and aberrant fibrous tissue proliferation, present significant therapeutic challenges due to their recalcitrant and recurrent nature. This study explores the efficacy of Carbon Ion Radiotherapy (CIRT) as a novel therapeutic approach for keloids, focusing on its impact on fibroblast proliferation, apoptosis induction, immunogenic cell death (ICD), macrophage polarization, and the TGF-β/SMAD signaling pathway. Utilizing a murine model of keloid formed by subcutaneous injection of zeocin in C57BL/6 mice, we demonstrated that CIRT effectively reduces collagenous fiber synthesis and collagen production in keloid tissues. Further, CIRT was shown to inhibit keloid fibroblast proliferation and to induce apoptosis, as evidenced by increased expression of apoptosis-related proteins and confirmed through flow cytometry and TUNEL assay. Notably, CIRT induced mitochondrial stress, leading to enhanced immunogenicity of cell death, characterized by increased expression of ICD markers and secretion of interferon-γ. Additionally, CIRT promoted a shift from M2 to M1 macrophage polarization, potentially reducing TGF-β release and mitigating ECM deposition. Our findings suggest that CIRT mediates its therapeutic effects through the inhibition of the TGF-β/SMAD signaling pathway, thereby attenuating ECM formation and offering a promising avenue for keloid treatment. This study underscores the potential of CIRT as an innovative strategy for managing keloids, highlighting its multifaceted impact on key cellular processes involved in keloid pathogenesis.

Culture Conditions Differentially Regulate the Inflammatory Niche and Cellular Phenotype of Tracheo-Bronchial Basal Stem Cells.

Human bronchial epithelial cells (HBECs) derived from the tracheo-bronchial regions of human airways provide an excellent in vitro model for studying pathological mechanisms and evaluating therapeutics in human airway cells. This cell population comprises a mixed population of basal cells (BCs), the predominant stem cell in airways capable of both self-renewal and functional differentiation. Despite their potential for regenerative medicine, BCs exhibit significant phenotypic variability in culture. To investigate how culture conditions influence BC phenotype and function, we expanded three independent BC isolates in three media, airway epithelial cell growth medium (AECGM), dual-SMAD inhibitor (DSI)-enriched AECGM, and Pneumacult Ex plus (PEx+). Extensive RNA sequencing, immune assays and electrical measurements revealed that PEx+ media significantly drove cell proliferation and a broad pro-inflammatory phenotype in BCs. In contrast, BCs expanded in AECGM, displayed increased expression of structural and extracellular matrix components at high passage. Whereas culture in AECGM increased expression of some cytokines at high passage, DSI suppressed inflammation altogether thus implicating TGF-β in BC inflammatory processes. Differentiation capacity declined with time in culture irrespective of expansion media except for PLUNC expressing secretory cells that were elevated at high passage in AECGM and PEx+ suggestive of an immune modulatory role of PLUNC in BCs. These findings underscore the profound impact of media conditions on inflammatory niche and function of in vitro expanded BCs. The broad pro-inflammatory phenotype driven by PEx+ media, in particular, should be considered in the development of cell-based models for airway diseases and therapeutic application.

Rapid detection of guttae area using aniline blue staining in Fuchs endothelial corneal dystrophy mouse model.

Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial degeneration resulting in impaired visual acuity. Excessive deposition of extracellular matrix (guttae) on Descemet's membrane (DM) is the hallmark of FECD. We sought to detect the guttae area rapidly using aniline blue (AB) staining in FECD mouse model. FECD mouse model was established via ultraviolet A (UVA) exposure. Masson's trichrome staining was utilized to stain the corneal sections. AB staining was utilized to stain both whole cornea tissues and stripped Descemet's membrane-endothelium complex (DMEC) flat mounts, while immunofluorescence staining of collagen I was employed to stain guttae areas. In Masson's trichrome staining, corneal collagen fibrils were stained blue with AB. The DMEC flat mounts were stained into relative dark blue areas and relative light blue areas using 2% AB staining. The areas of dark blue could almost overlap with collagen I-positive areas, and have an acellular centre and a moderately distinct boundary line with the surrounding corneal endothelial cells. In conclusion, AB staining is a rapid and effective method for the evaluation of the guttae areas in the FECD mouse model.

Profiling of collagen and extracellular matrix deposition from cell culture using in vitro ExtraCellular matrix mass spectrometry imaging (ivECM-MSI)

While numerous approaches have been reported towards understanding single cell regulation, there is limited understanding of single cell production of extracellular matrix phenotypes. Collagens are major proteins of the extracellular microenvironment extensively used in basic cell culture, tissue engineering, and biomedical applications. However, identifying compositional regulation of collagen remains challenging. Here, we report the development of In vitro ExtraCellular Matrix Mass Spectrometry Imaging (ivECM-MSI) as a tool to rapidly and simultaneously define collagen subtypes from coatings and basic cell culture applications. The tool uses the mass spectrometry imaging platform with reference libraries to produce visual and numerical data types. The method is highly integrated with basic in vitro strategies as it may be used with conventional cell chambers on minimal numbers of cells and with minimal changes to biological experiments. Applications tested include semi-quantitation of collagen composition in culture coatings, time course collagen deposition, deposition altered by gene knockout, and changes induced by drug treatment. This approach provides new access to proteomic information on how cell types respond to and change the extracellular microenvironment and provides a holistic understanding of both the cell and extracellular response.

Extracellular matrix production and oxygen diffusion regulate chemotherapeutic response in osteosarcoma spheroids.

Osteosarcoma (OS) survival rates and outcome have not improved in 50 years since the advent of modern chemotherapeutics. Thus, there is a critical need for an improved understanding of the tumor microenvironment to identify better therapies. Extracellular matrix (ECM) deposition and hypoxia are known to abrogate the efficacy of various chemical and cell-based therapeutics. Here, we aim to mechanistically investigate the combinatorial effects of hypoxia and matrix deposition with the use of OS spheroids. We use two murine OS cell lines with differential metastatic potential to form spheroids. We form spheroids of two sizes, use ascorbate-2-phosphate supplementation to enhance ECM deposition, and study cell response under standard (21% O2) and physiologic (5% O2) oxygen tensions. Finally, we examine chemotherapeutic responses to doxorubicin treatment. ECM production and oxygen tension are key determinants of spheroid size through cell organization based on nutrient and oxygen distribution. Interestingly, highly metastatic OS is more susceptible to chemotherapeutics compared to less metastatic OS when matrix production increases. Together, these data suggest that dynamic interactions between ECM production and oxygen diffusion may result in distinct chemotherapeutic responses despite inherent tumor aggressiveness. This work establishes OS spheroids as a valuable tool for early OS tumor formation investigation and holds potential for novel therapeutic target and prognostic indicator discovery.

Abstract P355: Multiomic Analyses Reveal Sex-specific Mechanisms Governing Cardiac Fibrosis.

Cardiac fibrosis—the deposition of excess extracellular matrix in the heart—occurs during pathological cardiac remodelling and precedes cardiac dysfunction and heart failure. Experimental and clinical data show that both cardiac fibrosis and heart failure exhibit sex-specific differences, particularly in the absence of ischemic injury. However, no study to date has systematically examined this, or the hormonal mechanisms driving these differences, using high-resolution single-cell and spatial omics. To address this gap, we applied single-cell and spatial omics to map the quality and distribution of non-ischemic cardiac fibrosis in human and mouse hearts. Further, to query the impact of sex hormones on cardiac fibrosis development, we analysed hearts of castrated (CAST) and ovariectomized (OVX) mice, which were ectopically infused with angiotensin-II (AngII). Using single cell transcriptomic data of hypertensive mouse hearts, we identified a fibroblast population driving cardiac fibrosis with a unique gene signature. We confirmed the presence of these cells in multiple mouse models and aged donor human hearts. Spatial mapping of fibrosis showed a sex-specific distribution of fibrosis, specifically in the development of perivascular fibrosis in the heart. Hypertensive male mice exhibited extensive perivascular fibrosis, and coronary artery adventitial hypertrophy, which was absent in females. However, OVX resulted in the development of perivascular fibrosis in hypertensive mice suggesting a cardioprotective role of estrogen. Confirming these observations, spatial transcriptomic analyses showed distinct fibrotic signatures driving perivascular and interstitial fibrosis, and transcriptomic differences between females and males. Our findings provide fundamental new insights towards the importance of biological sex in driving cardiac fibrosis, pointing to new mechanisms that may be manipulated to ameliorate development of heart failure.

The Effects of Mesenchymal Stem Cells-Derived Exosomes on Metabolic Reprogramming in Scar Formation and Wound Healing.

Pathological scarring results from aberrant cutaneous wound healing due to the overactivation of biological behaviors of human skin fibroblasts, characterized by local inordinate inflammation, excessive extracellular matrix and collagen deposition. Yet, its underlying pathogenesis opinions vary, which could be caused by increased local mechanical tension, enhanced and continuous inflammation, gene mutation, as well as cellular metabolic disorder, etc. Metabolic reprogramming is the process by which the metabolic pattern of cells undergoes a systematic adjustment and transformation to adapt to the changes of the external environment and meet the needs of their growth and differentiation. Therefore, the abnormality of metabolic reprogramming in cells within wounds and scars attaches great importance to scar formation. Mesenchymal stem cells-derived exosomes (MSC-Exo) are the extracellular vesicles that play an important role in tissue repair, cancer treatment as well as immune and metabolic regulation. However, there is not a systematic work to detail the relevant studies. Herein, we gave a comprehensive summary of the existing research on three main metabolisms, including glycometabolism, lipid metabolism and amino acid metabolism, and MSC-Exo regulating metabolic reprogramming in wound healing and scar formation for further research reference.

FGF21 Ameliorates Fibroblasts Activation and Systemic Sclerosis by Inhibiting CK2α/GLI2 Signaling Axis

Systemic sclerosis is a typical fibrotic disease of unknown etiology that is characterized by abnormal fibroblast activation and excessive deposition of extracellular matrix. Unfortunately, effective therapeutic approaches are lacking. FGF21 plays a key role in mediating a variety of biological activities. However, its specific function in systemic sclerosis is unclear. In this study, we found that the expression of FGF21 was significantly downregulated in fibrotic skin tissue and in TGF-β-stimulated fibroblasts. Furthermore, our studies demonstrated that treatment with recombinant FGF21 in the skin significantly alleviated bleomycin-induced and TBRI-activated fibrosis and inhibited the activation of fibroblasts, whereas skin fibrosis was exacerbated by deletion of FGF21. Mechanistically, FGF21 inhibits the activity of CK2α and promotes the degradation of GLI2. In conclusion, these results indicate that FGF21 attenuates skin fibrosis through the CK2α/GLI2 signaling pathway and therefore may be a potential therapeutic target for systemic sclerosis.

Hepatic stellate cell single cell atlas reveals a highly similar activation process across liver disease aetiologies

Background & AimsThe progression of chronic liver disease (CLD) is characterized by excessive extracellular matrix deposition, disrupting hepatic architecture and function. Upon liver injury, hepatic stellate cells (HSCs) differentiate towards myofibroblasts and become inflammatory, proliferative and fibrogenic. To date it is still unclear whether HSC activation is driven by similar mechanisms in different aetiologies. MethodsHSCs from multiple publicly available single-cell RNA-sequencing datasets were annotated and merged into a single-cell HSC activation atlas. Spheroid co-cultures of primary mouse hepatocytes/HSCs (n=5) and ELISAs on patient plasma samples (n=80) were performed to validate the mechanistic insight obtained from the HSC atlas. ResultsWe established an HSC activation atlas in which HSCs are clearly divided into 3 distinct transcriptomic profiles: quiescent HSCs, initiatory HSCs and myofibroblasts. These transcriptomic profiles are present in each of the investigated mouse liver injury models as well as in human chronic liver diseases, indicating that HSC activation is a conserved process. This activation process is driven by a core set of transcription factors independent of liver injury or species. Furthermore, we reveal novel ligands associated with activation of HSCs in multiple liver injury models and validate the profibrotic effect of parathyroid hormone. Finally, we identify COLEC10 as a conserved marker for quiescent HSCs and a biomarker of liver fibrosis in patients with different CLDs (p<0.0001). ConclusionsWe reveal unexpected similarities in the regulatory mechanisms of HSCs across diverse liver injury settings and species. The HSC activation atlas has the potential to provide novel insights into liver fibrosis and steer novel treatment options. Impact and implicationsThis study establishes a single-cell atlas of hepatic stellate cells across various liver injuries, highlighting a conserved activation process between different injuries and across species. The discovery of novel activating ligands and the biomarker COLEC10 in human plasma enhances diagnostic and therapeutic strategies. Additionally, the conserved activation process supports the use of any mouse model for mechanistic studies and testing of new anti-fibrotic compounds, streamlining preclinical research efforts.

Pancreatic stellate cells and the interleukin family: Linking fibrosis and immunity to pancreatic ductal adenocarcinoma (Review).

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive form of cancer with a low survival rate. A successful treatment strategy should not be limited to targeting cancer cells alone, but should adopt a more comprehensive approach, taking into account other influential factors. These include the extracellular matrix (ECM) and immune microenvironment, both of which are integral components of the tumor microenvironment. The present review describes the roles of pancreatic stellate cells, differentiated cancer‑associated fibroblasts and the interleukin family, either independently or in combination, in the progression of precursor lesions in pancreatic intraepithelial neoplasia and PDAC. These elements contribute to ECM deposition and immunosuppression in PDAC. Therapeutic strategies that integrate interleukin and/or stromal blockade for PDAC immunomodulation and fibrogenesis have yielded inconsistent results. A deeper comprehension of the intricate interplay between fibrosis, and immune responses could pave the way for more effective treatment targets, by elucidating the mechanisms and causes of ECM fibrosis during PDAC progression.

Integrin-β1 aggravates paraquat-induced pulmonary fibrosis by activation of FAK/ ERK1/2 pathway depending on fibrotic ECM

BackgroundIrreversible pulmonary fibrosis induced by paraquat is the most prevalent cause of death in patients with paraquat poisoning. Pulmonary fibrosis is characterized by abnormal deposition of extracellular matrix (ECM). Currently, the role of fibrotic ECM microenvironment in paraquat-induced pulmonary fibrosis has not been established. MethodsRat pulmonary fibrosis model was induced by paraquat, ATN-161 (an integrin-β1 antagonist) was given to investigate their effect on Rat survival and pulmonary fibrosis. Lungs were decellularized to generate normal and fibrotic acellular ECM scaffolds using Triton and SDS. Fibroblasts were cocultured with ECM scaffolds to established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. Then we explored the effect of fibrotic ECM microenvironment systematically promoting on integrin-β1/FAK/ERK1/2 pathway and established 3D culture systems to investigate the relationship between fibrotic ECM and the differentiation of fibroblasts. ResultsAntagonism of integrin-β1 could alleviate paraquat-induced pulmonary fibrosis and ameliorate survival status of rats. Compared to normal ECM, fibrotic extracellular microenvironment promoted the differentiation of fibroblasts to myofibroblasts. Antagonism of integrin-β1 could also ameliorate the promotion of fibrotic extracellular microenvironment on differentiation of fibroblasts to myofibroblasts. Fibrotic ECM microenvironment promotes fibroblasts transforming into myofibroblasts through integrin-β1/FAK/ERK1/2 signaling pathway. Moreover, this phenomenon holds independent on exogenous integrin-β1. ConclusionsActivation of integrin-β1/FAK/ERK1/2 pathway aggravates paraquat-induced pulmonary fibrosis depend on fibrotic ECM and integrin-β1 may be a prospective therapeutic target for paraquat-induced pulmonary fibrosis in the future.

Injectable decellularized extracellular matrix hydrogel with cell-adaptable supramolecular network enhances cartilage regeneration by regulating inflammation and facilitating chondrogenesis

Osteoarthritis, characterized by progressive loss of cartilage, affects millions of people worldwide and clinically lacks effective treatments. Herein, we present an injectable, biodegradable supramolecular decellularized extracellular matrix (dECM) hydrogel, which can be injected into the joints and provide biomimetic microenvironments to drive cartilage regeneration. Benefiting from the cell-adaptable dynamic network and cartilage-specific compositions, the supramolecular dECM hydrogel not only enhances cell proliferation and migration, but also facilitates chondrogenesis. Furthermore, compared with the conventional dECM hydrogels, the supramolecular dECM hydrogel is instrumental in regulating inflammation, which consequently leads to a marked suppression of genes linked to cell death and cartilage degradation. In vivo analysis confirms that stem cells, when delivered via the supramolecular dECM hydrogel, remain viable for up to three months within the restored cartilage. The regenerated tissue exhibits hyaline-cartilage structure, good mechanical properties, and cartilage-specific extracellular matrix deposition that align closely with those of native cartilage. We envision that this supramolecular dECM hydrogel could serve as a valuable platform for cartilage regeneration and could strongly facilitate the clinical translation of dECM-based materials for tissue regeneration.

RNA-seq transcriptomic profiling of TGF-β2-exposed human trabecular meshwork explants: Advancing insights beyond conventional cell culture models

Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is closely linked to increased intraocular pressure (IOP), with the trabecular meshwork (TM) playing a critical role in its regulation. The TM, located at the iridocorneal angle, acts as a sieve, filtering the aqueous humor from the eye into the collecting ducts, thus maintaining proper IOP levels. The transforming growth factor-beta 2 (TGF-β2) signaling pathway has been implicated in the pathophysiology of primary open-angle glaucoma POAG particularly, in the dysfunction of the TM. This study utilizes human TM explants to closely mimic in vivo conditions, thereby minimizing transcriptional changes that could arise from cell culture enabling an exploration of the transcriptomic impacts of TGF-β2. Through bulk RNA sequencing and immunohistological analysis, we identified distinct gene expression patterns and morphological changes induced by TGF-β2 exposure (5 ng/ml for 48 h). Bulk RNA sequencing identified significant upregulation in genes linked to extracellular matrix (ECM) regulation and fibrotic signaling. Immunohistological analysis further elucidated the morphological alterations, including cytoskeletal rearrangements and ECM deposition, providing a visual confirmation of the transcriptomic data. Notably, the enrichment analysis unveils TGF-β2’s influence on both bone morphogenic protein (BMP) and Wnt signaling pathways, suggesting a complex interplay of molecular mechanisms contributing to TM dysfunction in glaucoma. This characterization of the transcriptomic modifications on an explant model of TM obtained under the effect of this profibrotic cytokine involved in glaucoma is crucial in order to develop and test new molecules that can block their signaling pathways.

MR Molecular Image Guided Treatment of Pancreatic Cancer with Targeted ECO/miR-200c Nanoparticles in Immunocompetent Mouse Tumor Models.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplasia due to increased deposition of extracellular matrix (ECM) proteins. This work investigates the efficacy of targeted ECO/miR-200c nanoparticles (ELNP) on ECM remodeling in PDAC and tumor proliferation with MR molecular imaging (MRMI) with MT218 in immunocompetent mouse models. The miR-200c mediated regulation of EMT markers was measured in PDAC cells in vitro. Wild-type mice bearing mutated KRAS-driven KPC subcutaneous or orthotopic tumors were dosed weekly with RGD-ELNP/miR-200c at 1mg-RNA/kg for a total of 4 doses. We utilized MT218-MRMI to non-invasively monitor the alteration of tumor ECM EDN-FN levels by miR-200c and tumor response to the treatment. The changes were also validated by posthumous histopathology. Transfection of PDAC cells with ELNP/miR-200c downregulated the expression of FN1 and EDB-FN and some mesenchymal markers, inhibiting 3D spheroid formation and migration of KPC PDAC cells. RGD-ELNP/miR-200c treatment resulted in significant signal reduction in the MT218 enhanced MRMI images of both subcutaneous and orthotopic KPC tumors compared to those prior to treatment and treated with a non-specific control. MT218-MRMI results were suggestive of EDB-FN downregulation in tumors, which was later confirmed by immunohistochemistry. Tumor growth in subcutaneous tumors was significantly attenuated with RGD-ELNP/miR-200c and was an observed trend in orthotopic tumors. Substantial necrosis and remodeling were observed in both models treated with RGD-ELNP/miR-200c based on H&E staining. These results demonstrate the feasibility of RGD-ELNP/miR-200c in modulating PDAC ECM and restraining tumor growth and the utility of MT218-MRMI for non-invasively monitoring miR-200c efficacy.

Chlorquinaldol Alleviates Lung Fibrosis in Mice by Inhibiting Fibroblast Activation through Targeting Methionine Synthase Reductase.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited treatment options. Thus, it is essential to investigate potential druggable targets to improve IPF treatment outcomes. By screening a curated library of 201 small molecules, we have identified chlorquinaldol, a known antimicrobial drug, as a potential antifibrotic agent. Functional analyses have demonstrated that chlorquinaldol effectively inhibits the transition of fibroblasts to myofibroblasts in vitro and mitigates bleomycin-induced pulmonary fibrosis in mice. Using a mass spectrometry-based drug affinity responsive target stability strategy, we revealed that chlorquinaldol inhibited fibroblast activation by directly targeting methionine synthase reductase (MTRR). Decreased MTRR expression was associated with IPF patients, and its reduced expression in vitro promoted extracellular matrix deposition. Mechanistically, chlorquinaldol bound to the valine residue (Val-467) in MTRR, activating the MTRR-mediated methionine cycle. This led to increased production of methionine and s-adenosylmethionine, counteracting the fibrotic effect. In conclusion, our findings suggest that chlorquinaldol may serve as a novel antifibrotic medication, with MTRR-mediated methionine metabolism playing a critical role in IPF development. Therefore, targeting MTRR holds promise as a therapeutic strategy for pulmonary fibrosis.

The novel peptide PEP-Z-2 potentially treats renal fibrosis in vivo and in vitro by regulating TGF-β/Smad/AKT/MAPK signaling

Renal fibrosis is a process in which excessive deposition of extracellular matrix leads to an increase in tissue hardness and gradual destruction of the renal parenchyma. Chronic kidney disease (CKD) commonly progresses to end-stage renal disease (ESRD), ultimately leading to renal failure. This disease has high incidence and mortality rates, but to date, effective treatment options are lacking. PEP-Z-2 is a collagen peptide isolated from redlip croaker scales and may have potential fibroprotective activity. In this study, PEP-Z-2 was found to alleviate unilateral ureteral obstruction (UUO)- and folic acid (FA)-induced kidney injury in a mouse model, reduce collagen deposition in tissues, normalize renal function, reduce the expression of fibrosis markers, reduce reactive oxygen species (ROS) production, and restore the balance of the oxidant/antioxidant system. In vitro experiments also demonstrated that PEP-Z-2 inhibits the TGF-β-induced differentiation of fibroblasts and renal tubular epithelial cells into myofibroblasts and reduces the production of extracellular matrix (ECM) proteins such as fibronectin, Col I, and α-SMA, demonstrating notable therapeutic effects on renal fibrosis. This effect is achieved by regulating the TGF-β/Smad/AKT/MAPK pathway. Our research suggested that PEP-Z-2 is a potential therapeutic drug for renal fibrosis, and peptides from aquatic organisms may constitute a new class of candidate drugs for the treatment of renal fibrosis and even other types of organ fibrosis.

White adipose tissue remodeling in Little Brown Myotis (Myotis lucifugus) with white-nose syndrome.

White-nose syndrome (WNS) is a fungal wildlife disease of bats that has caused precipitous declines in certain Nearctic bat species. A key driver of mortality is premature exhaustion of fat reserves, primarily white adipose tissue (WAT), that bats rely on to meet their metabolic needs during winter. However, the pathophysiological and metabolic effects of WNS have remained ill-defined. To elucidate metabolic mechanisms associated with WNS mortality, we infected a WNS susceptible species, the Little Brown Myotis (Myotis lucifugus), with Pseudogymnoascus destructans (Pd) and collected WAT biopsies for histology and targeted lipidomics. These results were compared to the WNS-resistant Big Brown Bat (Eptesicus fuscus). A similar distribution in broad lipid class was observed in both species, with total WAT primarily consisting of triacylglycerides. Baseline differences in WAT chemical composition between species showed that higher glycerophospholipids (GPs) levels in E. fuscus were dominated by unsaturated or monounsaturated moieties and n-6 (18:2, 20:2, 20:3, 20:4) fatty acids. Conversely, higher GP levels in M. lucifugus WAT were primarily compounds containing n-3 (20:5 and 22:5) fatty acids. Following Pd-infection, we found that perturbation to WAT reserves occurs in M. lucifugus, but not in the resistant E. fuscus. A total of 66 GPs (primarily glycerophosphocholines and glycerophosphoethanolamines) were higher in Pd-infected M. lucifugus, indicating perturbation to the WAT structural component. In addition to changes in lipid chemistry, smaller adipocyte sizes and increased extracellular matrix deposition was observed in Pd-infected M. lucifugus. This is the first study to describe WAT GP composition of bats with different susceptibilities to WNS and highlights that recovery from WNS may require repair from adipose remodeling in addition to replenishing depot fat during spring emergence.

β-arrestin2 promotes angiogenesis of liver sinusoidal endothelial cells through the VEGF/VEGFR2 pathway to aggravate cirrhosis

Excessive extracellular matrix deposition and increased intrahepatic angiogenesis are prominent features of cirrhosis. β-arrestin2 is thought to be involved in the pathological processes of various fibrotic diseases. This study aimed to investigate the role and possible mechanism of β-arrestin2 in the angiogenesis of cirrhosis. Firstly, β-arrestin2 expression in liver tissues of cirrhotic patients was detected, and the correlation between β-arrestin2 and α-SMA, CD-31, PDGF, and VEGF indexes was analyzed. Then, after liver cirrhosis induced by CCL4 in Arrb2-KO mice (β-arrestin2 coding gene), liver histopathological changes were observed, and the expressions of α-SMA, CD-31, PDGF, VEGF, and VEGFR2 were detected. Finally, VEGF-A was used to treat human liver sinusoidal endothelial cells (LSECs) to simulate pathological conditions. After transfection with si-ARRB2, the cell activity, MDA and GSH-PX activities, cell invasion, angiogenesis, and the expressions of α-SMA, CD-31, and VEGF/VEGFR2 pathway were detected. Results showed that β-arrestin2 expression in the liver increased significantly during cirrhosis and was positively correlated with angiogenesis. In vivo, Arrb2-KO significantly inhibited fibrosis and angiogenesis in cirrhotic mice, and decreased the expressions of α-SMA, CD31, PDGF, VEGF, and VEGFR2. Studies using LSECs in vitro showed that after intervention of ARRB2, the activity of LSECs and the number of invasions and tubule formations were significantly reduced. Similarly, after transfection with si-ARRB2, the expressions of α-SMA, CD31, PDGF, VEGF, and VEGFR2 in LSECs were significantly decreased. Collectively, β-arrestin2 aggravated cirrhosis by promoting the angiogenesis of LSECs. Blocking β-arrestin2 may be an important target against angiogenesis and fibrosis in cirrhosis.

Liquid Crystalline Hydroxyapatite Nanorods Orchestrate Hierarchical Bone-Like Mineralization.

Bone matrix exhibits exceptional mechanical properties due to its unique nanocomposite structure of type I collagen fibrils and hydroxyapatite (HAp) nanoparticles in hierarchical liquid crystalline (LC) order. However, the regeneration mechanism of this LC structure is elusive. This study investigates the role of the LC structure of HAp nanorods in guiding aligned mineralization and its underlying molecular mechanism. A unidirectionally oriented LC phase of HAp nanorods is developed through engineering-assisted self-assembling. This is used to study the growth direction of long-range aligned extracellular matrix (ECM) and calcium deposit formation during the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. It is found that 2 key regulatory genes, COL1A1 and COL4A6, lead to the formation of aligned ECM. Activation of the PI3K-Akt pathway enhances osteogenesis and promotes ordered calcium deposits. This study provides evidence for elucidating the mechanism of LC-induced ordered calcium deposition at hierarchical levels spanning from the molecular to macro-scale, as well as the switch from ordered to disordered mineralization. These findings illuminate bone regeneration, contribute to the development of biomimetic artificial bone with long-range ordered structures, and suggest a basis for therapeutic targeting of microstructure-affected bone disorders and the broader field of cell-ECM interactions.

EGR1 interacts with p-SMAD at the endothelin-1 gene promoter to regulate gene expression in TGFβ1-stimulated IMR-90 fibroblasts

Pulmonary fibrosis is a severe and progressive lung disease characterized by lung tissue scarring. Transforming growth factor beta 1 (TGFβ1) is crucial in causing pulmonary fibrosis by promoting the activation of fibroblasts and their differentiation into myofibroblasts, which are responsible for excessive extracellular matrix deposition. This study aimed to identify genes activated by TGFβ1 that promote fibrosis and to understand the regulatory pathway controlling myofibroblast. Endothelin-1 (ET-1) was identified as the top-ranking gene in the fibrosis-related gene set using quantitative PCR array analysis. TGFβ1 upregulated EGR1 expression through the ERK1/2 and JNK1/2 MAPK pathways. EGR1 and p-SMAD2 proteins interacted with the ET-1 gene promoter region to regulate TGFβ1-induced ET-1 expression in IMR-90 pulmonary fibroblasts. Mice lacking the Egr1 gene showed reduced ET-1 levels in a model of pulmonary fibrosis induced by intratracheal administration of bleomycin. These findings suggest that targeting EGR1 is a promising approach for treating pulmonary fibrosis, especially idiopathic pulmonary fibrosis, by affecting ET-1 expression and profibrotic reactions.