Excessive Extracellular Matrix Research Articles

TWIST1+FAP+ fibroblasts in the pathogenesis of intestinal fibrosis in Crohn's disease.

Intestinal fibrosis, a severe complication of Crohn's disease (CD), is characterized by excessive extracellular matrix (ECM) deposition and induces intestinal strictures, but there are no effective anti-fibrosis drugs available for clinical application. We performed single-cell RNA sequencing (scRNA-seq) of fibrotic and non-fibrotic ileal tissues from CD patients with intestinal obstruction. Analysis revealed mesenchymal stromal cells (MSCs) as the major producers of ECM and the increased infiltration of its subset FAP+ fibroblasts in fibrotic sites, which was confirmed by immunofluorescence and flow cytometry. Single cell transcriptomic profiling of chronic Dextran Sulfate Sodium Salt (DSS) murine colitis model revealed Cd81+Pi16- fibroblasts exhibited transcriptomic and functional similarities to human FAP+ fibroblasts. Consistently, FAP+ fibroblasts were identified as the key subtype with the highest level of ECM production in fibrotic intestines. Furthermore, specific knockout or pharmacological inhibition of TWIST1, which was highly expressed by FAP+ fibroblasts, could significantly ameliorate fibrosis in mice. In addition, TWIST1 expression was induced by CXCL9+ macrophages enriched in fibrotic tissues via IL-1β and TGF-β signal. These findings suggest the inhibition of TWIST1 as a promising strategy for CD fibrosis treatment.

CTRP6 alleviates endometrial fibrosis by regulating Smad3 pathway in intrauterine adhesion†.

Intrauterine adhesion (IUA) is manifestations of endometrial fibrosis and excessive extracellular matrix deposition. C1q/tumor necrosis factor-related protein-6 (CTRP6) is a newly identified adiponectin paralog which has been reported to modulate the fibrosis process of several diseases; however, the endometrial fibrosis function of CTRP6 remains unknown. Our study aimed to assess the role of CTRP6 in endometrial fibrosis and further explore the underlying mechanism. Here, we found that the expression of CTRP6 was downregulated in the endometrial tissues of IUA. In vitro experiments demonstrated the reduced level of CTRP6 in facilitated transforming growth factor-β1 (TGF-β1)-induced human endometrial stromal cells (HESCs). In addition, CTRP6 inhibited the expression of α-smooth muscle actin (α-SMA) and collagen I in TGF-β1-treated HESCs. Mechanistically, CTRP6 activated the AMP-activated protein kinase (AMPK) and protein kinase B (AKT) pathway in HESCs, and AMPK inhibitor (AraA) or PI3K inhibitor (LY294002) pretreatment abolished the protective effect of CTRP6 on TGF-β1-induced fibrosis. CTRP6 markedly decreased TGF-β1-induced Smad3 phosphorylation and nuclear translocation, and AMPK or AKT inhibition reversed these effects. Notably, CTRP6-overexpressing treatment alleviated the fibrosis of endometrium in vivo. Therefore, CTRP6 ameliorates endometrial fibrosis, among which AMPK and AKT are essential for the anti-fibrotic effect of CTRP6 via the Smad3 pathway. Taken together, CTRP6 may be a potential therapeutic target for the treatment of intrauterine adhesion.

CTRP6 alleviates endometrial fibrosis by regulating Smad3 pathway in intrauterine adhesion†.

Intrauterine adhesion (IUA) is manifestations of endometrial fibrosis and excessive extracellular matrix deposition. C1q/tumor necrosis factor-related protein-6 (CTRP6) is a newly identified adiponectin paralog which has been reported to modulate the fibrosis process of several diseases; however, the endometrial fibrosis function of CTRP6 remains unknown. Our study aimed to assess the role of CTRP6 in endometrial fibrosis and further explore the underlying mechanism. Here, we found that the expression of CTRP6 was downregulated in the endometrial tissues of IUA. In vitro experiments demonstrated the reduced level of CTRP6 in facilitated transforming growth factor-β1 (TGF-β1)-induced human endometrial stromal cells (HESCs). In addition, CTRP6 inhibited the expression of α-smooth muscle actin (α-SMA) and collagen I in TGF-β1-treated HESCs. Mechanistically, CTRP6 activated the AMP-activated protein kinase (AMPK) and protein kinase B (AKT) pathway in HESCs, and AMPK inhibitor (AraA) or PI3K inhibitor (LY294002) pretreatment abolished the protective effect of CTRP6 on TGF-β1-induced fibrosis. CTRP6 markedly decreased TGF-β1-induced Smad3 phosphorylation and nuclear translocation, and AMPK or AKT inhibition reversed these effects. Notably, CTRP6-overexpressing treatment alleviated the fibrosis of endometrium in vivo. Therefore, CTRP6 ameliorates endometrial fibrosis, among which AMPK and AKT are essential for the anti-fibrotic effect of CTRP6 via the Smad3 pathway. Taken together, CTRP6 may be a potential therapeutic target for the treatment of intrauterine adhesion.

Tumor-associated macrophages restrict CD8+ T cell function through collagen deposition and metabolic reprogramming of the breast cancer microenvironment.

Tumor progression is accompanied by fibrosis, a condition of excessive extracellular matrix accumulation, which is associated with diminished antitumor immune infiltration. Here we demonstrate that tumor-associated macrophages (TAMs) respond to the stiffened fibrotic tumor microenvironment (TME) by initiating a collagen biosynthesis program directed by transforming growth factor-β. A collateral effect of this programming is an untenable metabolic milieu for productive CD8+ T cell antitumor responses, as collagen-synthesizing macrophages consume environmental arginine, synthesize proline and secrete ornithine that compromises CD8+ T cell function in female breast cancer. Thus, a stiff and fibrotic TME may impede antitumor immunity not only by direct physical exclusion of CD8+ T cells but also through secondary effects of a mechano-metabolic programming of TAMs, which creates an inhospitable metabolic milieu for CD8+ T cells to respond to anticancer immunotherapies.

Hydroxysafflor yellow A exerts anti-fibrotic and anti-angiogenic effects through miR-29a-3p/PDGFRB axis in liver fibrosis

BackgroundLiver fibrosis is a prevalent pathological process in chronic liver diseases characterized by excessive extracellular matrix (ECM) deposition and abnormal angiogenesis. Notably, hepatic stellate cells (HSCs) are the primary source of ECM. Activated HSCs not only secrete numerous pro-fibrotic cytokines but also are endowed with a pro-angiogenic phenotype to promote pathological angiogenesis. Therefore, targeted regulation of HSCs has emerged as a critical strategy for addressing liver fibrosis. Hydroxysafflor yellow A (HSYA) is a homology of medicine and food colourant with good pharmacological activity. However, the precise mechanisms of HSYA against liver fibrosis remain unclear. PurposeThe objective of this study was to elucidate the impact of HSYA on the modulation of tissue fibrosis and pathological angiogenesis, as well as the underlying mechanisms in vitro and in vivo. MethodsThe efficacy and mechanisms of HSYA on TGF-β1-induced HSCs and VEGFA-induced endothelial cells were investigated by MTT assay, EdU cell proliferation assay, cell scratch assay, Elisa assay, immunofluorescence assay, molecular docking, cell transfection assay, western blot analysis and RT-qPCR analysis. In CCl4-induced liver fibrosis mice model, H&E, Masson, and Sirius red staining were used to observe histopathology. Serum transaminase activity and liver biochemical indexes were tested by biochemical kit. Immunohistochemical, fluorescence in situ hybridization (FISH), western blot analysis and RT-qPCR analysis were implemented to determine the mechanism of HSYA in vivo. ResultsHerein, our findings confirmed that HSYA inhibited the proliferation, migration and activation of HSCs, as evidenced by a reduction in cell viability, relative migration rate, EdU staining intensity, and pro-fibrotic mRNAs and proteins expression in vitro. Mechanistically, HSYA played an anti-fibrotic and anti-angiogenic role by partially silencing PDGFRB in activated HSCs, thereby disrupting PDGFRB/MEK/ERK signal transduction and inhibiting HIF-1α, VEGFA and VEGFR2 protein expression. Importantly, PDGFRB was a target gene of miR-29a-3p. Treatment with HSYA reversed the down-regulation of miR-29a-3p and antagonized PDGFRB signaling pathway in TGF-β1+miR-29a-3p inhibitor-induced HSCs. Consistent with our in vitro study, HSYA exhibited a good hepatoprotective effect in CCl4-induced liver fibrosis mice by reducing serum ALT and AST levels, decreasing the contents of four fibrosis indicators (HA, PC-III, ColIV and LN) and hydroxyproline, and inhibiting the TGF-β1/TGFBR signaling pathway. In terms of mechanisms, HSYA alleviated pathological angiogenesis in fibrotic liver by deactivating PDGFRB signaling pathway and impairing the positive expression of CD31. Subsequently, FISH results further corroborated HSYA affected the activation of HSCs and angiogenesis, achieved by the concurrent upregulation of miR-29a-3p and downregulation of α-SMA and VEGFA. Additionally, treatment with HSYA also forged a link between HSCs and endothelial cells, as supported by inhibiting the aberrant proliferation of endothelial cells. ConclusionFundamentally, the current study has illustrated that HSYA ameliorates liver fibrosis by repressing HSCs-mediated pro-fibrotic and pro-angiogenic processes, which is contingent upon the regulatory effect of HSYA on the miR-29a-3p/PDGFRB axis. These findings provide compelling evidence bolstering the potential of HSYA as a therapeutic agent in liver fibrosis.

CTHRC1: An Emerging Hallmark of Pathogenic Fibroblasts in Lung Fibrosis.

Pulmonary fibrosis is a chronic, progressive, irreversible lung disease characterized by fibrotic scarring in the lung parenchyma. This condition involves the excessive accumulation of extracellular matrix (ECM) due to the aberrant activation of myofibroblasts in the alveolar environment. Transforming growth factor beta (TGF-β) signaling is a crucial driver of fibrogenesis because it promotes excessive ECM deposition, thereby leading to scar formation and lung damage. A primary target of TGF-β signaling in fibrosis is Collagen Triple Helix Repeat Containing 1 (CTHRC1), a secreted glycoprotein that plays a pivotal role in ECM deposition and wound repair. TGF-β transcriptionally regulates CTHRC1 in response to tissue injury and controls the wound healing response through functional activity. CTHRC1 may also play an essential role in re-establishing and maintaining tissue homeostasis after wound closure by modulating both the TGF-β and canonical Wnt signaling pathways. This dual function suggests that CTHRC1 regulates tissue remodeling and homeostasis. However, deregulated CTHRC1 expression in pathogenic fibroblasts has recently emerged as a hallmark of fibrosis in multiple organs and tissues. This review highlights recent studies suggesting that CTHRC1 can serve as a diagnostic and prognostic biomarker for fibrosis in idiopathic pulmonary fibrosis, systemic sclerosis, and post-COVID-19 lung fibrosis. Notably, CTHRC1 expression is responsive to antifibrotic drugs that target the TGF-β pathway, such as pirfenidone and bexotegrast, indicating its potential as a biomarker of treatment success. These findings suggest that CTHRC1 may present new opportunities for diagnosing and treating patients with lung fibrosis.

Aryl Hydrocarbon Receptor Activation Limits the Fatty Acid Synthesis and Subsequent "miR-193a-3p-HDAC3-FASN" Signals to Alleviate Intestinal Fibrosis.

Intestinal fibrosis is a common complication of Crohn's disease and characterized by excessive extracellular matrix (ECM) deposition. The aryl hydrocarbon receptor (AhR) detects micronutrients and microbial metabolites in diet and can attenuate intestinal fibrosis with unclear mechanisms. In this study, AhR activation was demonstrated to downregulate the transcription of collagen I and fibronectin in a Sp1- but not Sp3- or AP-1-dependent manner. A suppressed fatty acid synthesis was highlighted using untargeted metabolomics analyses, and synthetic products, palmitic acid (PA), were used as the intermediary agent. After a screening study, fatty acid synthase (FASN) was identified as the main targeted protein, and AhR activation regulated "HDAC3-acetylation" signals but not glycosylation to enhance FASN degradation. Furthermore, results of bioinformatics analysis and others showed that after being activated, AhR targeted miR-193a-3p to control HDAC3 transcription. Collectively, AhR activation inhibited ECM deposition and alleviated intestinal fibrosis by limiting fatty acid synthesis subsequent to the inhibition of "miR-193a-3p-HDAC3-FASN" signals.

Hypoxia in uterine fibroids: role in pathobiology and therapeutic opportunities.

Uterine fibroids are the most common tumors in females affecting up to 70% of women world-wide, yet targeted therapeutic options are limited. Oxidative stress has recently surfaced as a key driver of fibroid pathogenesis and provides insights into hypoxia-induced cell transformation, extracellular matrix pathophysiology, hypoxic cell signaling cascades, and uterine biology. Hypoxia drives fibroid tumorigenesis through (1) promoting myometrial stem cell proliferation, (2) causing DNA damage propelling transformation of stem cells to tumor initiating cells, and (3) driving excess extracellular matrix (ECM) production. Common fibroid-associated DNA mutations include MED12 mutations, HMGA2 overexpression, and Fumarate hydratase loss of function. Evidence suggests an interaction between hypoxia signaling and these mutations. Fibroid development and growth are promoted by hypoxia-triggered cell signaling via various pathways including HIF-1, TGFβ, and Wnt/β-catenin. Fibroid-associated hypoxia persists due to antioxidant imbalance, ECM accumulation, and growth beyond adequate vascular supply. Current clinically available fibroid treatments do not take advantage of hypoxia-targeting therapies. Growing pre-clinical and clinical studies identify ROS inhibitors, anti-HIF-1 agents, Wnt/β-catenin inhibition, and TGFβ cascade inhibitors as agents that may reduce fibroid development and growth through targeting hypoxia.

#2376 Tubular exosomes in renal ECM microenvironment promotes fibrosis progression by activating latent TGF-β1

Abstract Background and Aims Chronic kidney disease (CKD) is characterized with progressive fibrosis caused by excessive extracellular matrix (ECM) deposition. Tubular epithelial cells secreted increasing exosomes into extracellular space under pathological conditions with integrin as the prominent constituent membrane cargo. Due to the critical role of integrin in activation of TGF-β1 signalling, this study aims to investigate the performance of tubular-derived exosomes in the ECM microenvironment, exploring the potential mechanism of exosomal integrin mediated activation of latent TGF-β1. Method A fibrosis mouse model was established with unilateral ureteral obstruction (UUO) and an ITGβ6 overexpression cell line were established to observe the effect of ITGβ6+exosomes in vitro. Elastic or stress-relaxing hydrogels based on Polymethyl Methacrylate (PMMA) and acrylic acid-WGG(KA)3-heparin were constructed to mimic the in situ ECM under normal and fibrotic conditions with different stiffness (2kPa, 50kPa). Exosomes in hydrogels were tracked by single particle tracking to investigate their mobility and retention capability. Purified exosomal integrin β6 and inhibitor of exosome secretion, GW4869 was applied to clarify the role of exosomal integrin in latent TGF-β1 activation. Results In UUO model, immunofluorescence staining showed that latent TGF-β1 localized to ECM space and stored in the renal tubular interstitial which was activated with fibrotic progression. Additionally, we observed integrin β6 expression in tubule or interstitial increased with fibrotic progression. Isolated exosomes derived from cultured tubule cells under serum-free condition or renal tissues of UUO mice showed higher proportion of integrin β6+ subpopulation, as demonstrated via single exosome analysis by Nanoimaging (Oxford) . Interestingly, these exosomes diffused through hydrogels, while maintaining their original morphology and particle size. It turned out that stress-relaxing hydrogels with lower stiffness exhibited better retention of exosomes. Then, transwell experiment and single-particle tracking both revealed increased exosome diffusion rate in ECM with higher stiffness. Due to the critical role of integrin in the activation of latent TGFβ, we propose integrin β6+ exosome may exert such effect in ECM microenvironment. Impressively, purified ITGB6+ exosomes significantly increased TGF-β1 activation in TGF-β1 overexpressed HK-2 cells, while inhibiting exosome secretion via GW4869 treatment reversed such activation remarkably. Conclusion We demonstrated that tubular epithelial cell derived exosomal integrin β6 diffused in ECM space and promoted the activation of latent TGF-β1. Higher ECM stiffness correlates with accelerated movement of exosome which enhanced TGFβ1 activation. Exosomal integrin β6 may represent a novel manner of TGF-β1 activation with signal-spreading potential which may become a new therapeutic target for suppression of TGF-β1 activation in CKD treatment.

P25 Identification of early changes in extracellular microRNA biomarkers of scleroderma for point-of-care diagnosis

Abstract Introduction and aims Scleroderma is a rare autoimmune disease, characterized by excessive extracellular matrix deposition leading to chronic inflammation and fibrosis. As a progressive disease initially affecting the skin, it presents similarly to Raynaud phenomenon, which delays diagnosis and treatment. Clinical diagnostic tests lack sensitivity, therefore novel Methods of detection and sensitive biomarkers are required to improve diagnosis rates. MicroRNAs are short, stable, noncoding RNAs, often present in the extracellular space, and can be sampled from interstitial skin fluid (ISF) through the employment of hydrogel microneedles. We aim to use this technique to improve upon current clinical gold standards of diagnosing scleroderma, at earlier stages of the disease. Methods In this study, we performed next-generation small-RNA sequencing on ISF from healthy individuals, patients prior to a clinical diagnosis of scleroderma [termed very early detection of systemic sclerosis (VEDOSS)], and patients with scleroderma classified as low and high skin score, identifying a panel of microRNA biomarkers for scleroderma diagnosis. Swellable hydrogel microneedles were manufactured in house, applied to excised human tissue and preclinical mouse models to demonstrate the ability to access and quantify extracellular microRNAs in ISF. Results Patients who received a VEDOSS diagnosis have increased ISF abundance of miR-379-5p and miR-125a-5p and decreased miR-30e-3p compared with healthy patients. miR-379-5p, miR-363-3p and miR-221-3p are sensitive to the progression of scleroderma and could be used to track the disease with improved sensitivity to autoantibodies in blood or where clinical practices usually require a skin biopsy. Using preclinical models of skin disease, we could differentiate between conditions, with higher sensitivity than blood sampling using our microneedle technology. Conclusions By identifying extracellular mircoRNAs, we have shown the potential use of these sensitive biomarkers in a liquid biopsy that can be applied at point-of-care. Peptide nucleic acids complementary to our microRNA biomarkers, will be used to provide real-time on-the-chip readouts of these ­biomarkers.

The increase of long noncoding RNA Fendrr in hepatocytes contributes to liver fibrosis by promoting IL-6 production

Liver fibrosis/cirrhosis is a pathological state caused by excessive extracellular matrix deposition. Sustained activation of hepatic stellate cells (HSC) is the predominant cause of liver fibrosis, but the detailed mechanism is far from clear. In this study, we found that long noncoding RNA Fendrr is exclusively increased in hepatocytes in the murine model of CCl4- and bile duct ligation-induced liver fibrosis, as well as in the biopsies of liver cirrhosis patients. In vivo, ectopic expression of Fendrr aggravated the severity of CCl4-induced liver fibrosis in mice. In contrast, inhibiting Fendrr blockaded the activation of HSC and ameliorated CCl4-induced liver fibrosis. Our mechanistic study showed that Fendrr binds to STAT2 and enhances its enrichment in the nucleus, which then promote the expression of IL-6, and, ultimately, activates HSC in a paracrine manner. Accordingly, disrupting the interaction between Fendrr and STAT2 by ectopic expression of a STAT2 mutant attenuated the pro-fibrotic response inspired by Fendrr in the CCl4-induced liver fibrosis. Notably, the increase of Fendrr in patient fibrotic liver is positively correlated with the severity of fibrosis and the expression of IL-6. Meanwhile, hepatic IL-6 positively correlates with the extent of liver fibrosis and HSC activation as well, thus suggesting a causative role of Fendrr in HSC activation and liver fibrosis. In conclusion, these observations identify an important regulatory crosstalk between hepatocyte Fendrr and HSC activation in the progression of liver fibrosis, which might represent a potential strategy for therapeutic intervention.

Exploring unconventional targets in myofibroblast transdifferentiation outside classical TGF- signaling in renal fibrosis.

We propose that the key initiators of renal fibrosis are myofibroblasts which originate from four predominant sources-fibroblasts, pericytes, endothelial cells and macrophages. Increased accumulation of renal interstitial myofibroblasts correlates with an increase in collagen, fibrillar proteins, and fibrosis severity. The canonical TGF- pathway, signaling via Smad proteins, is the central molecular hub that initiates these cellular transformations. However, directly targeting these classical pathway molecules has proven challenging due their integral roles in metabolic process, and/or non-sustainable effects involving compensatory cross-talk with TGF-β. This review explores recently discovered alternative molecular targets that drive transdifferentiation into myofibroblasts. Discovering targets outside of the classical TGF-β/Smad pathway is crucial for advancing antifibrotic therapies, and strategically targeting the development of myofibroblasts offers a promising approach to control excessive extracellular matrix deposition and impede fibrosis progression.

Blocking the MIR155HG/miR-155 axis reduces CTGF-induced inflammatory cytokine production and α-SMA expression via upregulating AZGP1 in hypertrophic scar fibroblasts

Hypertrophic scarring (HS) is a pathological condition characterized by excessive fibrosis and inflammation, resulting in excessive extracellular matrix formation in the skin. MIR155HG, a long non-coding RNA, is abnormally upregulated in fibrotic tissues; however, its underlying mechanism is poorly understood. Using single-cell sequencing data, we analyzed connective tissue growth factor (CTGF) expression in various cell types in HS and normal skin tissues and MIR155HG expression in clinical samples. To investigate the mechanism of fibrosis, an in vitro model using CTGF-treated hypertrophic scar fibroblasts (HSFBs) was established and qRT-PCR, western blotting and ELISA assays were performed to investigate the expression of interleukin (IL)-1β, IL-6, and mesenchymal markers α-smooth muscle actin (α-SMA). CTGF stimulates MIR155HG level through phosphorylated STAT3 binding to the MIR155HG promoter. We analyzed the methylation of MIR155HG, assessed the levels of miR-155-5p/−3p in CTGF-treated HSFBs and identified differentially expressed genes among HS and NS samples using the Gene Expression Omnibus RNA sequencing data. The binding between miR-155-5p/−3p and AZGP1 was confirmed using a dual-luciferase assay and inflammatory cytokine production and α-SMA expression were investigated in rescue experiments. The findings revealed that CTGF elevated inflammatory cytokine production, α-SMA and MIR155HG expression in HSFBs. MIR155HG is upregulated in HS tissues due to low DNA methylation. Mechanistically, miR-155-5p/−3p was directly bound to MIR155HG 3’UTR. MIR155HG silencing inhibited cytokine production and α-SMA expression by repressing the generation of miR-155-5p/−3p in CTGF-treated HSFBs. Bioinformatics analysis and luciferase reporter assays revealed that miR-155-5p/−3p targets AZGP1. In addition, transfection with plasmids carrying AZGP1 cDNA significantly inhibited the signaling activity of miR-155-5p/−3 p-overexpressing HSFBs. Our findings highlight the importance of the MIR155HG/miR-155/AZGP1 axis in regulating cytokine production and α-SMA in HS.

Softness-Aided Mild Hyperthermia Boosts Stiff Nanomedicine by Regulating Tumor Mechanics.

Aberrant tumor mechanical microenvironment (TMME), featured with overactivated cancer-associated fibroblasts (CAFs) and excessive extracellular matrix (ECM), severely restricts penetration and accumulation of cancer nanomedicines, while mild-hyperthermia photothermal therapy (mild-PTT) has been developed to modulate TMME. However, photothermal agents also encounter the barriers established by TMME, manifesting in limited penetration and heterogeneous distribution across tumor tissues and ending with attenuated efficiency in TMME regulation. Herein, it is leveraged indocyanine green (ICG)-loaded soft nanogels with outstanding deformability, for efficient tumor penetration and uniform distribution, in combination with mild-PTT to achieve potent TMME regulation by inhibiting CAFs and degrading ECM. As a result, doxorubicin (DOX)-loaded stiff nanogels gain greater benefits in tumor penetration and antitumor efficacy than soft counterparts from softness-mediated mild-PTT. This study reveals the crucial role of nanomedicine mechanical properties in tumor distribution and provides a novel strategy for overcoming the barriers of solid tumors with soft deformable nanogels.

Exercise in Chronic Kidney Disease Patients Does Not Significantly Alter Fibrosis in Quadriceps Muscle

Background: Chronic kidney disease (CKD) is commonly associated with decreased physical activity and muscle dysfunction. Muscles in CKD become fibrotic, containing excess extracellular matrix (ECM), particularly collagen. Collagen is cross-linked in the ECM, and increased cross-linking is often associated with fibrosis, stiffness, and reduced ECM remodeling. However, it is not known how exercise in patients with CKD may impact the extent of collagen cross-linking or fibrosis. Additionally, muscle fibrosis may impair muscle adaptations to exercise including, fiber size, fiber type, or vasculature. We predicted that collagen content and cross-linking would decrease, and there would be more Type I fibers and larger fiber size in the patients that were in the exercise group. Methods: Participants were randomized to exercise (n=15) or no exercise (n=5) groups in the Exercise Study Testing Enhanced Energetics of Mitochondria (ESTEEM) clinical trial of non-dialysis patients with CKD. The exercise program consisted of 35 min sessions, 3 times a week for 12 weeks consisting of high-intensity interval training, strength training, and power walking. Vastus lateralis biopsies were obtained before and after the 12 weeks of exercise. A bundle of muscle tissue was powdered and separated into a pepsin soluble and insoluble fraction before applying a hydroxyproline assay for collagen content, with the insoluble fraction representing more cross-linked collagen. For histological analysis 10 μm thick cryosections were stained with Picrosirius Red, or immunofluorescent stains to investigate satellite cells, vasculature, fibro-adipogenic progenitors (FAPs), and muscle fiber types. Picrosirius red sections were imaged under polarized light to look at birefringence as a measure of collagen density. Sections that were stained for fiber type and vasculature were analyzed using semi-automatic muscle analysis using segmentation of histology (SMASH). Results: The overall amount of collagen and collagen cross-linking were not altered in either the exercise or no exercise groups. Picrosirius red stained sections did not reveal a change in ECM area or collagen packing density with exercise. Surprisingly minimum ferret diameter and percentage of type I (slow twitch) fibers decreased in most patients that underwent the exercise program. Type I and type IIa (fast oxidative) fiber size also significantly decreased, while there was no change in type IIx (fast glycolytic) fiber size. Conclusion: This study shows that exercise does not dramatically reduce ECM content or muscle fibrosis in patients with CKD. Surprisingly, the response to exercise was opposite of that anticipated in healthy subject with exercise in CKD producing smaller muscle fibers and a shift away from more oxidative type I fibers. This shows that exercise may have limited capacity to enhance function in CKD muscle, potentially due to irreversible fibrosis. Work supported by mitochondrial bioenergetics in persons with chronic kidney disease (NIH R03DK114502). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HSP47 complements cholesterol synthesis-independent HMGCoA-reductase pathway-mediated fibroblast bioenergetics

Fibrosis, characterized by excessive extracellular matrix (ECM) components deposition, is a common pathological process underlying numerous chronic diseases. Differentiating fibroblasts into myofibroblasts leads to the deposition of fibrous proteins like collagen in the ECM. Heat shock protein 47 (HSP47) is a molecular chaperone protein that assists in the folding of collagen. Recent studies show that inhibition of cholesterol synthesis-independent HMGCoA-reductase pathway enzyme geranylgeranyl pyrophosphate synthase 1 (GGPS1) decreases fibroblast differentiation and its bioenergetics. However, the role of HSP47 in this pathway is unknown, although recent observations show that HSP47 is decreased when GGPS1 is inhibited. Hence, we tested the hypothesis that HSP47 promotes cellular respiration in myofibroblasts. Seahorse assays were performed on different fibroblasts/myofibroblasts repeatedly and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in the presence and absence of HSP47 inhibitor (HY124817). Complex V of the electron transport chain was measured by immunoblotting ATP-5A. HY124817 tends to decrease the OCR, basal respiration rate, ATP-linked, and spare respiratory capacity. Co-incubation with HY124817 and digeranyl bisphosphonate (DGBP), an inhibitor of GGPS1, significantly (P = 0.043) inhibited further the ATP-linked OCR compared to the TGF group. Immunoblotting showed that Complex V did not change significantly across treatments. These results suggest that HSP47 is involved in fibroblast bioenergetics related to ATP-linked OCR in both differentiated myofibroblasts and normal fibroblasts. Inhibition of HSP47 complements the decrease in myofibroblast cellular respiration caused by the GGPS1 inhibition. This finding is novel in that HSP47 plays a role in fibroblast bioenergetics regulated by the cholesterol synthesis-independent HMGCoA-reductase pathway. Further research is necessary to identify what HSP47 targets and regulatory elements involved in this process. This would provide more insight into the mechanisms that underlie critical fibrosis conditions like myocardial fibrosis and potential therapeutics. This experience was supported by the Medical College of Wisconsin's Advancing Student Potential for Inclusion with Research Experiences (ASPIRE) program. The program is funded by a federal grant from the National Heart, Lung, and Blood Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiac Complications and COVID-19: A Review of Life-threatening Co-morbidities.

The novel 2019 coronavirus disease (COVID-19) was first reported in the last days of December 2019 in Wuhan, China. The presence of certain co-morbidities, including cardiovascular diseases (CVDs), are the basis for worse outcomes in patients with COVID-19. Relevant English-language literature was searched and retrieved from the Google Scholar search engine and PubMed database up to 2023 using COVID-19, SARS-CoV-2, Heart failure, Myocardial infarction, and Arrhythmia and Cardiac complication as keywords. Increased hemodynamic load, ischemia-related dysfunction, ventricular remodeling, excessive neurohumoral stimulation, abnormal myocyte calcium cycling, and excessive or insufficient extracellular matrix proliferation are associated with heart failure (HF) in COVID-19 patients. Inflammatory reaction due to the excessive release of inflammatory cytokines, leads to myocardial infarction (MI) in these patients. The virus can induce heart arrhythmia through cardiac complications, hypoxia, decreased heart hemodynamics, and remarkable inflammatory markers. Moreover, studies have linked cardiac complications in COVID-19 with poor outcomes, extended hospitalization time, and increased mortality rate. Patients with COVID-19 and CVDs are at higher mortality risk and they should be given high priority when receiving the treatment and intensive care during hospitalization.

Macrophage to Myofibroblast Transition Promotes Hypertension-Induced Cardiac Diastolic Dysfunction

Cardiac diastolic dysfunction is a pathological continuum of hypertension eventually leading to heart failure—which is responsible for ~13% of all deaths in the United States. A hallmark of this disease is cardiac fibrotic remodeling, in which excessive extracellular matrix components are deposited into the myocardium. Recent studies have shown the novel ability of macrophages to differentiate into a fibrotic myofibroblast-like phenotype—termed macrophage to myofibroblast transition (MMT). This phenomenon has been implicated in renal fibrosis but, has not been investigated in the heart during hypertension or diastolic dysfunction. Therefore, we hypothesize that MMT plays a causal role in cardiac fibrotic remodeling in hypertension, promoting diastolic dysfunction. As such, we seek to investigate the prevalence of myofibroblast-transitioned macrophages in the heart, as well as determine the gravity of MMT-mediated fibrotic cardiac remodeling. To understand the pathological progression of hypertension to diastolic dysfunction, we utilized the classic DOCA + NaCl model of hypertension in C57BL/6 mice. This investigation is multi-pronged, in that, we seek to gauge diastolic function in vivo, as well as investigate the microenvironment of the heart in primary in vitro experiments. To achieve this, we employ echocardiography and direct left ventricular pressure and volume measurements to determine the progression of hypertension to diastolic dysfunction in the adult mouse heart. For primary in vitro analyses, mouse hearts were isolated and digested, and leukocytes were isolated by Percoll gradient. Cells were stained with α-SMA (myofibroblast) and CD68 (monocyte/macrophage). Finally, the deposition of fibrotic collagen was assessed by both Masson’s trichrome and picrosirius red tissue stains. Mice were subjected to DOCA + salt to induce hypertension. Ten days after this treatment, the left ventricular pressure change as a function of time was measured in the left ventricle. This analysis revealed degraded heart function, realized by a significant decrease in -dp/dt as soon as ten days following the onset of hypertension. Following, another cohort of hypertensive WT mouse hearts was isolated for staining. The results of these assays revealed increased cardiac fibrosis in the hearts of hypertensive mice—likely contributing to the deficit in cardiac function. Finally, in addition to this, flow cytometric analyses of isolated cardiac leukocytes revealed that hypertensive mice had a significant increase in the α-SMA+/CD68+ population of cells in the hypertensive mouse heart, implicating MMT in the rapid pathological progression of hypertension to diastolic dysfunction. These results characterize the presence of MMT cells in the hypertensive mouse heart. Furthermore, the increase of myofibroblast transition is accompanied by decreased cardiac diastolic function and enhanced fibrotic remodeling in the heart—alluding to the role of MMT in the pathological progression of hypertension to diastolic dysfunction. Future efforts into this phenomenon involve the real-time progression of hypertension to diastolic dysfunction via echocardiography, as well as a final, definitive measurement of diastolic function by PV loop. Finally, we will employ an angiotensin II model of hypertension to ensure that this phenomenon is encapsulated in other forms of hypertension, rather than just the DOCA + Salt model. We thank our sources of funding:NIG R01-HL146713, AHA 23TPA1076467. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Targeting Interactions between Fibroblasts and Macrophages to Treat Cardiac Fibrosis.

Excessive extracellular matrix (ECM) deposition is a defining feature of cardiac fibrosis. Most notably, it is characterized by a significant change in the concentration and volume fraction of collagen I, a disproportionate deposition of collagen subtypes, and a disturbed ECM network arrangement, which directly affect the systolic and diastolic functions of the heart. Immune cells that reside within or infiltrate the myocardium, including macrophages, play important roles in fibroblast activation and consequent ECM remodeling. Through both direct and indirect connections to fibroblasts, monocyte-derived macrophages and resident cardiac macrophages play complex, bidirectional, regulatory roles in cardiac fibrosis. In this review, we discuss emerging interactions between fibroblasts and macrophages in physiology and pathologic conditions, providing insights for future research aimed at targeting macrophages to combat cardiac fibrosis.

Novel Inositol 1,4,5-Trisphosphate Receptor Inhibitor Antagonizes Hepatic Stellate Cell Activation: A Potential Drug to Treat Liver Fibrosis.

Liver fibrosis, characterized by excessive extracellular matrix (ECM) deposition, can progress to cirrhosis and increases the risk of liver cancer. Hepatic stellate cells (HSCs) play a pivotal role in fibrosis progression, transitioning from a quiescent to activated state upon liver injury, wherein they proliferate, migrate, and produce ECM. Calcium signaling, involving the inositol 1,4,5-trisphosphate receptor (IP3R), regulates HSC activation. This study investigated the efficacy of a novel IP3R inhibitor, desmethylxestospongin B (dmXeB), in preventing HSC activation. Freshly isolated rat HSCs were activated in vitro in the presence of varying dmXeB concentrations. The dmXeB effectively inhibited HSC proliferation, migration, and expression of fibrosis markers without toxicity to the primary rat hepatocytes or human liver organoids. Furthermore, dmXeB preserved the quiescent phenotype of HSCs marked by retained vitamin A storage. Mechanistically, dmXeB suppressed mitochondrial respiration in activated HSCs while enhancing glycolytic activity. Notably, methyl pyruvate, dimethyl α-ketoglutarate, and nucleoside supplementation all individually restored HSC proliferation despite dmXeB treatment. Overall, dmXeB demonstrates promising anti-fibrotic effects by inhibiting HSC activation via IP3R antagonism without adverse effects on other liver cells. These findings highlight dmXeB as a potential therapeutic agent for liver fibrosis treatment, offering a targeted approach to mitigate liver fibrosis progression and its associated complications.