COL1A1 Research Articles

Proteomic Composition of Acute Ischemic Stroke Thrombi Retrieved via Endovascular Thrombectomy Is Associated with Stroke Etiology.

The objective of this study is to investigate the protein components of acute ischemic stroke (AIS) thrombi using four-dimensional independent data acquisition (4D-DIA) proteomics and reveal the correlations between thrombotic protein components and AIS etiology. From April to September 2023, we enrolled a total of 30 patients who underwent endovascular thrombectomy at our institute and were diagnosed in accordance with large artery atherosclerosis (LAA; n = 15) or cardioembolism (CE; n = 15). Thromboembolic material was collected for 4D-DIA proteomic detection. We then analyzed it for differentially expressed proteins (DEPs; fold change [FC] ≥ 1.5 or ≤ 0.67), performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, and mapped protein-protein interactions (PPIs). In the 30 retrieved clots, 5115 proteins were expressed. Of these, we screened 246 DEPs between the LAA and CE groups, such as histone H4, collagen α1, and differentially expressed in neoplastic versus normal cells domain-containing protein 6A. GO analysis revealed that the DEPs' most important biological process was cellular process, the most important Cell Component was cell part, the molecular function was binding, and the most significantly enriched pathway was thiamine metabolism. PPI results revealed complicated interactions among these DEPs, of which superoxide dismutase, catalase, and γ-enolase might play important roles. This study outlines a promising molecular approach to differentiating the etiology of AIS between CE and LAA through the proteomics of retrieved thrombi, which might also inform future research into thrombotic biology.

Mechanistic role for mTORC1 signaling in profibrotic toxicity of low-dose cadmium

Cadmium (Cd) is a toxic environmental metal that occurs naturally in food and drinking water. Cd is of increasing concern to human health due to its association with age-related diseases and long biological half-life. Previous studies show that low-dose Cd exposure via drinking water induces mechanistic target of rapamycin complex 1 (mTORC1) signaling in mice; however, the role of mTORC1 pathway in Cd-induced pro-fibrotic responses has not been established. In the present study, we used human lung fibroblasts to examine whether inhibiting the mTORC1 pathway prevents lung fibrosis signaling induced by low-dose Cd exposure. Results show that rapamycin, a pharmacological inhibitor of mTORC1, inhibited Cd-dependent phosphorylation of ribosomal protein S6, a downstream marker of mTORC1 activation. Rapamycin also decreased Cd-dependent increases in pro-fibrotic markers, α-smooth muscle actin, collagen 1α1 and fibronectin. Cd activated mitochondrial spare respiratory capacity in association with increased cell proliferation. Rapamycin decreased these responses, showing that mTORC1 signaling supports mitochondrial energy supply for cell proliferation, an important step in fibroblast trans-differentiation into myofibroblasts. Collectively, these results establish a key mechanistic role for mTORC1 activation in environmental Cd-dependent lung fibrosis.

Revealing the structural microenvironment of high metastatic risk uveal melanomas following decellularisation

Uveal melanoma (UM) is a rare aggressive intraocular tumour that spreads most commonly to the liver in tumours with loss of one copy of chromosome 3 (HR-M3); current treatments for metastatic disease remain largely ineffective. Pre-clinical research is increasingly using three-dimensional models that better recapitulate the tumour microenvironment (TME). One aspect of the TME is the acellular extracellular matrix (ECM) that influences cell proliferation, migration and response to therapy. Although commercial matrices are used in culture, the composition and biochemical properties may not be representative of the tumour ECM in vivo. This study identifies UM metastatic risk specific ECM proteins by developing methodology for decellularisation of low- and high- metastatic risk tissue samples (LR-D3 vs. HR-M3). Proteomic analysis revealed a matrisome signature of 34 core ECM and ECM-associated proteins upregulated in HR-M3 UM. Combining additional UM secretome and whole cell iTRAQ proteomic datasets revealed enriched GO and KEGG pathways including ‘regulating ECM binding’ and ‘PI3K/Akt signalling’. Structural analyses of decellularised matrices revealed microarchitecture of differing fibre density and expression differences in collagen 4, collagen 6A1 and nidogen 1, between metastatic risk groups. This approach is a powerful tool for the generation of ECM matrices relevant to high metastatic risk UM.

Role of Lysyl Oxidase-Like Protein 3 in the Pathogenesis of Graves' Orbitopathy in Orbital Fibroblasts.

The lysyl oxidase (LOX) family has been implicated in the pathogenesis of diseases caused by inflammation and fibrosis. Therefore, we aimed to examine the role of lysyl oxidase-like protein 3 (LOXL3) in Graves' orbitopathy (GO) pathogenesis and its potential as a treatment target. Quantitative real-time polymerase chain reaction compared the transcript levels of the five LOX family subtypes in orbital tissue explants obtained from patients with GO (n = 18) and healthy controls (n = 15). The effects of LOXL3 inhibition on interleukin (IL)-1β-induced proinflammatory cytokines, transforming growth factor (TGF)-β-induced profibrotic proteins, intracellular signaling molecules, and adipogenic markers were evaluated using Western blotting. Adipogenic differentiation was identified using Oil Red O staining. LOX and LOXL3 transcript levels were high in GO tissues. Stimulation with IL-1β, TGF-β, and insulin-like growth factor-1 significantly increased LOXL3 messenger RNA expression in GO fibroblasts. Furthermore, silencing LOXL3 attenuated the IL-1β-induced production of proinflammatory cytokines (IL-6, IL-8, and intercellular adhesion molecule-1) and TGF-β-induced production of profibrotic proteins (fibronectin, collagen 1α, and alpha-smooth muscle actin). It also reduced the IL-1β or TGF-β-induced expression of phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells, protein kinase B, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. Additionally, LOXL3 silencing suppressed adipocyte differentiation and the expression of adipogenic transcription factors (leptin, AP-2, peroxisome proliferator-activated receptor gamma, and CCAAT/enhancer-binding protein). LOXL3 is crucial in GO pathogenesis. LOXL3 inhibition reduced inflammatory cytokine production, fibrotic protein expression, and fibroblast differentiation into adipocytes. This study highlights LOXL3 as a potential therapeutic target for GO.

Endoplasmic Reticulum Oxidative Stress Promotes Glutathione-Dependent Oxidation of Collagen-1A1 and Promotes Lung Fibroblast Activation.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in COL1A1 (collagen 1A1) S-glutathionylation (COL1A1-SSG) in lung tissues from subjects with IPF compared with control subjects in association with increases in ERO1A (endoplasmic reticulum [ER] oxidoreductin 1) and enhanced oxidation of ER-localized PRDX4 (peroxiredoxin 4), reflecting an increased oxidative environment of the ER. Human lung fibroblasts exposed to TGFB1 (transforming growth factor-β1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished the oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 concentrations, and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted the activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared with COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly and increased the expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling because of increased resistance to collagenase-mediated degradation and fibroblast activation.

MiR-424/322 attenuates cardiac remodeling by modulating the nuclear factor-activated T-cell 3/furin pathway

BackgroundCardiac remodeling is implicated in numerous physiologic and pathologic conditions, including scar formation, heart failure, and cardiac arrhythmias. Nuclear factor-activated T-cell cytoplasmic (NFATc) is a crucial transcription factor that regulates cardiac remodeling. MicroRNA (miR)-424/322 has pathophysiological roles in the cardiovascular and respiratory systems by modulating hypoxia and inflammatory pathways. The role of miR-424/322 in regulating cardiac remodeling is under investigation. We identified several cardiac hypertrophy and fibrosis-related molecules as putative targets of miR-424/322. We propose that miR-424/322 could have crucial roles in cardiac remodeling by modulating several key molecules for cardiac fibrosis and hypertrophy. MethodsHuman cardiac fibroblasts (HCFs) and a myogenic cell line H9c2 cells were used for in vitro experiments. A murine model of angiotensin II (AngII)-induced cardiac remodeling was used to assess the roles of miR-322 on cardiac hypertrophy and fibrosis in vivo. Immunoblotting, immunofluorescence, real-time polymerase chain reaction and cell proliferation, Sirius Red, and dual-luciferase reporter assays were used to decipher the molecular mechanism. ResultsWe found that miR-322 knockout mice were susceptible to AngII-induced cardiac fibrosis and hypertrophy in vivo. Administration of miR-424/322 inhibitors aggravated AngII-induced overexpression of NFATc3, furin, natriuretic peptides and collagen 1A1 in H9c2 cells and HCFs. miR-424/322 mimics reversed the AngII-induced fibrosis, hypertrophy, and proliferation by targeting NFATc3 and furin in vitro. miR-424/322 could be transactivated by NFATc3. Exogenous miR-322 ameliorated AngII-induced hypertrophy and cardiac fibrosis in vivo. ConclusionsThe NFATc3/miR-424/322/furin axis is crucial for developing cardiac remodeling, and exogenous miR-322 mimics could have therapeutic potential in cardiac remodeling.

A new paradigm of bone active risedronate-functionalized polycaprolactone/gelatin scaffold synergistically promotes osteogenic differentiation in rat bone marrow-derived mesenchymal stem cells for bone tissue engineering

Modern developments in bone tissue engineering focus on designing biomimetic materials that exhibit suitable mechanical and physiochemical properties. In this study, we present the development of a new biomaterial scaffold using a newly constructed polymer that contains bisphosphonate in combination with gelatin (GAT). To achieve scaffolding, a chemical grafting technique was used to develop risedronate (RS)-functionalized polycaprolactone (PCL-RS). The freeze-casting process was used to construct the porous PCL-RS/GAT scaffold after incorporating gelatin into the PCL-RS polymer solution. A scaffold with pores that are in perfect position and a porosity of 81.84% was successfully acquired. The biodegradability assessment, 48% of its early weight was lost after five weeks. The PCL-RS/GAT scaffold exhibited an elastic modulus of 32.01 MPa and a tensile strength of 3.9 MPa. The MTT analysis showed the PCL-RS/GAT favorable cytocompatibility with rat bone marrow-derived mesenchymal stem cells (BM-MSC). In addition, the cells cultured in the PCL-RS/GAT scaffold exhibited the greatest ALP activity and mineralization. The RT-PCR test results indicated that the PCL-RS/GAT scaffold had a high RUNX2, COL 1A1, and OCN gene expression, suggesting a strong osteoinductive capacity. The results indicate that the PCL-RS/GAT scaffold is a suitable biomimetic platform for tissue engineering in bone.

Impact of paracrine effects of different clonal hematopoiesis-driver mutations in human macrophages on cardiac cells

Abstract Background Age-acquired, non-malignant somatic mutations in hematopoietic stem cells can lead to expansion of blood cells, known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP-driving mutations in a spectrum of genes are correlated with an increased incidence of and poor prognosis in patients with cardiovascular disease. Recent reports suggest increased inflammation in carriers of frequent mutations in DNMT3A and TET2. Less frequent mutations in splicing factors, signaling molecules and epigenetic regulators were found to be associated with greater myeloid malignancy and cardiovascular mortality risks, but paracrine effects of these mutations on cardiac tissue is not known. Purpose We aim to assess the impact of less frequent CHIP-driver mutations on paracrine activities of macrophages on cardiac cells. Methods & Results In order to identify interesting CHIP-driver mutations, we analysed frequency and all-cause mortality of 56 potential CHIP driver genes in two cohorts of patients with heart failure and aortic stenosis (1168 patients). Besides the well-studied mutations in TET2 and DNMT3A, we identified nine mutations with a relative higher frequency and impact on prognosis, among them splicing factors SF3B1, SRSF2, ZRSR2, TP53-related genes PPM1D and PHF6, signaling mediators CALR and GNAS, and epigenetic regulators EZH2, KDM6A. To mimic CHIP-driving conditions, we assessed the effect of siRNA-mediated silencing of the so far understudied genes on the paracrine activity of primary human macrophages, which are differentiated from primary CD14+ sorted monocytes. We achieved >40% reductions in mRNA expression for each of the targeted genes (p<0.05). To gain first insights into a potential paracrine activity of individual mutations, we incubated cardiomyocytes with the conditioned medium collected from macrophages. Notably, supernatants from ZRSR2-, PPM1D- or CALR-silenced macrophages significantly reduced cardiomyocyte beating frequencies (53.1%, 64.9% and 63.7% of control, respectively; all p<0.05). Interestingly, only supernatants of ZRSR2-, CALR-silenced macrophages induced hypertrophy (all p<0.05). Next, we assessed the effects on primary human cardiac fibroblasts. Treatment with conditioned media did not affect collagen 1A1 expression and cell counts, but conditioned media of EZH2-, and CALR-silenced macrophages induced fibroblast senescence evaluated by senescence-associated β-galactosidase staining (158.3% and 154.1% of control, respectively; both p<0.05). The mechanisms driving the different effects are currently under investigation. Conclusion Loss of genes from a broad spectrum of clonal hematopoiesis drivers differentially affects the paracrine activity of human monocyte-derived macrophages. Silencing of ZRSR2 or CALR in macrophages had most profound effects on cardiomyocytes. Surprisingly, EZH2 and CALR-silenced macrophages augmented senescence of cardiac fibroblasts, a finding that further needs to be explored.

Loss of Y-chromosomal genes in macrophages induces paracrine phenotypic alterations in cardiac cells

Abstract Background At the intersection of gender medicine and ageing, mosaic loss of the Y chromosome (mLoY) in hematopoietic cells has emerged as a novel cardiovascular risk factor. As the most prevalent chromosomal aberration in men, mLoY is age-associated and characterised by interindividual varying frequencies of LoY cells in blood. It is associated with an increased risk for cardiovascular disease and mortality. A murine model of mLoY demonstrates myocardial fibrosis and reduced lifespan. However, the interactions between LoY hematopoietic and cardiac cells remain elusive. It is uncertain, whether the loss of single genes or combinations of multiple genes drive the observed effects, impeding the development of targeted therapies. Purpose We aimed to examine the impact of individual Y-chromosomal genes in macrophages concerning their paracrine effects on cardiac cells types. Methods and results To identify target genes, we analysed two single-nuclei RNA sequencing datasets of patient-derived PBMCs and THP-1 monocytic cells for their expression of Y-chromosomal genes. We identified nine genes that exhibited robust and concordant expression in both datasets. Seven were protein coding (RPS4Y, DDX3Y, EIF1AY, ZFY, UTY, USP9Y and KDM5D) and two were non-coding (TTTY14 and LINC00278). Using siRNA pools, we achieved >50% reductions in mRNA content in THP-1 M0 macrophages for all genes (p<0.05) and collected conditioned media (CM). As previous data suggested increased myocardial fibrosis in mLoY, we assessed the impact of CM on primary human cardiac fibroblasts. Notably, treatment with CM from ZFY, TTTY14 or LINC00278 silenced macrophages induced collagen 1A1 protein expression by 1.44-, 1.30- and 1.86-fold, respectively (all p<0.05), suggesting that loss of these genes might contribute to myocardial fibrosis. To explore potential effects on cardiomyocytes, neonatal rat cardiomyocytes were exposed to CM. We observed reduced beating frequencies upon treatment with CM from ZFY, TTTY14 or UTY silenced macrophages (relative fold-changes 0.45, 0.40, 0.53, respectively; all p<0.05). While suggestive of cardiomyocyte stress, we found no changes in the expression of the stress markers Nppa or Nppb, possibly indicating direct effects on ion transporters. Given the strong association of cardiovascular disease with endothelial cell dysfunction, we investigated the effects of CM on human umbilical vein endothelial cells (HUVEC). CM from ZFY and KDM5D silenced macrophages induced ICAM protein expression in HUVEC by 1.9- and 1.6-fold, respectively (both p<0.05). Conclusion Loss of individual Y-chromosomal genes in monocytic cells elicits distinct paracrine effects on various cardiac cell types. Particularly, silencing of ZFY in macrophages demonstrated profound effects on all investigated cell types, warranting further investigation. Additionally, two long non-coding RNAs, previously unexplored in cardiovascular disease, influenced paracrine activity of macrophages.

Rational design of bioengineered recombinant collagen-like protein enhances GelMA hydrogel for diabetic wound healing

Gelatin methacryloyl (GelMA) holds significant potential in tissue engineering; however, its clinical applications are often constrained by its lack of functional groups. To overcome this limitation, recombinant proteins with multiple biofunctional domains present a promising strategy for GelMA functionalization, enhancing its biological properties. In this study, we developed a rationally designed recombinant collagen-like protein (RC) engineered with multiple biofunctional domains, which demonstrated the ability to upregulate collagen 1α (COL-1α) expression in NIH-3 T3 cells. By utilizing EDC/NHS chemistry, the purified RC was conjugated to GelMA, resulting in GelMA-RC hydrogels that significantly improved cell viability and migration compared to unmodified GelMA. Subsequent in vivo studies showed that RC-modified GelMA exhibited superior wound healing efficacy, largely attributed to enhanced expression of cytokeratin-14 (CK-14) and COL-1α. These findings underscore the potential of RC-functionalized GelMA in promoting diabetic wound repair and suggest broader applicability for functionalizing other biomaterials.

Quantitative tissue analysis reveals AK2, COL1A1, & PLG protein signatures: Targeted therapeutics for meningioma.

Meningioma is the most prevalent primary intracranial brain tumor and accounts for one-third of all CNS tumors. Meningioma is known to be the most common yet life-threatening brain tumor with a higher recurrence rate. Globally, there is an increase in the healthcare burden due to meningioma and hence in its research. The present clinical approach includes surgical resection, chemotherapy, and radiation therapies to which the malignancy does not seem to respond efficiently. Targeted therapies and molecular markers provide elite patient treatment and care for individuals suffering from meningiomas as compared to conventional measures. Although there is proteomic data on meningioma the knowledge of potential biomarkers differentiating the grades is scarce. To identify the best set of biomarkers, validation of reported markers in large and independent sample cohorts in the future is necessary. A total of 12 samples, 3 each of control (which made pool 1) Meningioma grade I (which made 2 sets: pool 2 and pool 3), and Meningioma grade II (which made pool 4) were taken for LC-MS/MS. After this, the expression of three proteins was checked by immunocytochemistry, flow cytometry, and western blotting. Protein expression was analyzed using various techniques like mass spectrometry, immunocytochemistry, flow cytometry, and western blotting. Mass spectrometry is the most commonly used standard and reliable technique for identifying and quantifying protein expression. We got three highly upregulated proteins namely AK2, COL1A1, and PLG using this technique. The biomarker potential of these proteins was further checked by ICC, western blotting, and flow cytometry. Three important proteins were found to be upregulated namely AK2 (Adenylate Kinase 2), COL1A1 (Collagen 1A1), and PLG (Plasminogen). The order of increased protein expression was control < MG grade I < MG grade II according to mass spectrometry and western blotting. In immunocytochemistry, we found that COL1A1 expression increases significantly with grades in comparison to control. Similarly, AK2 and PLG also showed little increase but not as much as COL1A1. In flow cytometry, PLG showed higher upregulation in grades than control. While AK2 and COL1A1 showed little increase in expression in grades than control. All techniques, especially mass spectrometry and western blotting presented higher expression of these proteins in grades as compared to control. In the quest to find a suitable therapeutic marker, this study incorporates quantitative labeling and detection followed by flow cytometry, Immunocytochemistry, and western blotting for early diagnosis and treatment of meningioma. The article further explores the efficacy of some proteins namely AK2, COL1A1 & PLG to be the targeted molecules.

Bioactivity-Guided Isolation of Antistroke Compounds from Gymnadenia conopsea (L.) R. Br.

A bioactivity-guided separation strategy was used to identify novel antistroke compounds from Gymnadenia conopsea (L.) R. Br., a medicinal plant. As a result, 4 undescribed compounds (1-2, 13, and 17) and 13 known compounds, including 1 new natural product (3), were isolated from G. conopsea. The structures of these compounds were elucidated through comprehensive spectroscopic techniques, such as 1D/2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and quantum chemical calculations. An oxygen-glucose deprivation/reoxygenation (OGD/R)-injured rat pheochromocytoma (PC12) cell model was used to evaluate the antistroke effects of the isolates. Compounds 1-2, 10-11, 13-15, and 17 provided varying degrees of protection against OGD/R injury in the PC12 cells at concentrations of 12.5, 25, and 50 µM. Among the tested compounds, compound 17 demonstrated the most potent neuroprotective effect, which was equivalent to that of the positive control drug (edaravone). Then, transcriptomic and bioinformatics analyses were conducted to reveal the regulatory effect of compound 17 on gene expression. In addition, quantitative real-time PCR (qPCR) was performed to verify the results of the transcriptomic and bioinformatics analyses. These results suggest that the in vitro antistroke effect of compound 17 may be associated with the regulation of the Col27a1 gene. Thus, compound 17 is a promising candidate for the development of novel antistroke drugs derived from natural products, and this topic should be further studied.

Phosphodiesterase 4 is overexpressed in keloid epidermal scars and its inhibition reduces keratinocyte fibrotic alterations

BackgroundEpidermal remodeling and hypertrophy are hallmarks of skin fibrotic disorders, and keratinocyte to mesenchymal (EMT)-like transformations drive epidermis alteration in skin fibrosis such as keloids and hypertrophic scars (HTS). While phosphodiesterase 4 (PDE4) inhibitors have shown effectiveness in various fibrotic disorders, their role in skin fibrosis is not fully understood. This study aimed to explore the specific role of PDE4B in epidermal remodeling and hypertrophy seen in skin fibrosis.MethodsIn vitro experiments examined the effects of inhibiting PDE4A-D (with Roflumilast) or PDE4B (with siRNA) on TGFβ1-induced EMT differentiation and dedifferentiation in human 3D epidermis. In vivo studies investigated the impact of PDE4 inhibition on HOCl-induced skin fibrosis and epidermal hypertrophy in mice, employing both preventive and therapeutic approaches.ResultsThe study found increased levels of PDE4B (mRNA, protein) in keloids > HTS compared to healthy epidermis, as well as in TGFβ-stimulated 3D epidermis. Keloids and HTS epidermis exhibited elevated levels of collagen Iα1, fibronectin, αSMA, N-cadherin, and NOX4 mRNA, along with decreased levels of E-cadherin and ZO-1, confirming an EMT process. Inhibition of both PDE4A-D and PDE4B prevented TGFβ1-induced Smad3 and ERK1/2 phosphorylation and mesenchymal differentiation in vitro. PDE4A-D inhibition also promoted mesenchymal dedifferentiation and reduced TGFβ1-induced ROS and keratinocyte senescence by rescuing PPM1A, a Smad3 phosphatase. In vivo, PDE4 inhibition mitigated HOCl-induced epidermal hypertrophy in mice in both preventive and therapeutic settings.ConclusionsOverall, the study supports the potential of PDE4 inhibitors, particularly PDE4B, in treating skin fibrosis, including keloids and HTS, shedding light on their functional role in this condition.

Comprehensive ultrasonographic evaluation of normal and fibrotic kidneys in a mouse model with an ultra-high-frequency transducer.

This study aimed to establish baseline morphological and functional data for normal mouse kidneys via a clinical 33 MHz ultra-high-frequency (UHF) transducer, compare the data with the findings from fibrotic mice, and assess correlations between ultrasonography (US) parameters and fibrosis-related markers. This retrospective study aggregated data from three separate experiments (obstructive nephropathy, diabetic nephropathy, and acute-to-chronic kidney injury models). Morphological parameters (kidney size, parenchymal thickness [PT]) and functional (shear-wave speed [SWS], stiffness, resistive index [RI], and microvascular imaging-derived vascular index [VI]) were assessed and compared between normal and fibrotic mouse kidneys. Semi-quantitative histopathologic scores were calculated and molecular markers (epithelial cadherin), Collagen 1A1 [Col1A1], transforming growth factor-β, and α-smooth muscle actin [α-SMA]) were evaluated using western blots. Correlations with US parameters were explored. Clinical UHF US successfully imaged the kidneys of the experimental mice. A three-layer configuration was prevalent in the normal mouse kidney parenchyma (34/35) but was blurred in most fibrotic mouse kidneys (33/40). US parameters, including size (11.14 vs. 10.70 mm), PT (2.07 vs. 1.24 mm), RI (0.64 vs. 0.77), VI (22.55% vs. 11.47%, only for non-obstructive kidneys), SWS (1.67 vs. 2.06 m/s), and stiffness (8.23 vs. 12.92 kPa), showed significant differences between normal and fibrotic kidneys (P&lt;0.001). These parameters also demonstrated strong discriminative ability in receiver operating characteristic curve analysis (area under the curve, 0.76 to 0.95; P&lt;0.001). PT, VI, and RI were significantly correlated with histological fibrosis markers (ρ=-0.64 to -0.68 for PT and VI, ρ=0.71-0.76 for RI, P&lt;0.001). VI exhibited strong negative correlations with Col1A1 (ρ=-0.76, P=0.006) and α-SMA (ρ=-0.75, P=0.009). Clinical UHF US effectively distinguished normal and fibrotic mouse kidneys, indicating the potential of US parameters, notably VI, as noninvasive markers for tracking fibrosis initiation and progression in mouse kidney fibrosis models.

Evaluation Recovery of Ulcerative Colitis with a Lactobacillus Cocktail Derived from Traditional Dairy Products: In vivo Study.

This investigation investigates the anti-inflammatory and fibrinolytic effects of a cocktail of probiotics derived from traditional dairy products in a murine model of ulcerative colitis (UC). A mix of newly isolated probiotics containing L. plantarum, L. brevis, L. delbrueckii, and L. helveticus was characterized and orally administered to inbred eight-week-old C57BL/6 male mice (n = 6). Clinical symptoms, pathohistological changes, and inflammatory and fibrosis markers were analyzed in the existence and absence of probiotics in colitis mice. Dairy lactobacillus probiotics potently attenuated colitis symptoms by decreasing dextran sulfate sodium (DSS)-induced body weight loss, colon shortening, rectal bleeding, and rectal prolapse. Consistently, a cocktail of probiotics could significantly improve histopathological grading by suppressing crypt loss, mucosal damage, and inflammation scores in colitis tissues. Moreover, the mix of probiotics suppressed pro-inflammatory genes including interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), and increased anti-oxidant markers and activity such as superoxide dismutase and catalase in colon tissue. Furthermore, compared to the no-treated group, the administration of probiotics reduced fibrosis by decreasing collagen deposition in tissue sections and down-regulating levels of pro-fibrotic genes including alpha-actin-2 (Acta2), collagen (Col) 1a1, and Col 1a2 in colitis tissue homogenates. The results show the newly isolated cocktail of probiotics elicits a potent protective effect on UC symptoms in mice model. Further study on these probiotics is required to fully explore their effectiveness, strength, and safety considerations.

Phosphodiesterase 4 is overexpressed in human keloids and its inhibition reduces fibroblast activation and skin fibrosis

There is a pressing medical need for improved treatments in skin fibrosis including keloids and hypertrophic scars (HTS). This study aimed to characterize the role of phosphodiesterase 4 (PDE4), specifically PDE4B in fibrotic skin remodeling in vitro and in vivo.In vitro, effects of PDE4A-D (Roflumilast) or PDE4B (siRNA) inhibition on TGFβ1-induced myofibroblast differentiation and dedifferentiation were studied in normal (NHDF) and keloid (KF) human dermal fibroblasts. In vivo, the role of PDE4 on HOCl-induced skin fibrosis in mice was addressed in preventive and therapeutic protocols.PDE4B (mRNA, protein) was increased in Keloid > HTS compared to healthy skin and in TGFβ-stimulated NHDF and KF. In Keloid > HTS, collagen Iα1, αSMA, TGFβ1 and NOX4 mRNA were all elevated compared to healthy skin confirming skin fibrosis.In vitro, inhibition of PDE4A-D and PDE4B similarly prevented TGFβ1-induced Smad3 and ERK1/2 phosphorylation and myofibroblast differentiation, elevated NOX4 protein and proliferation in NHDF. PDE4A-D inhibition enabled myofibroblast dedifferentiation and curbed TGFβ1-induced reactive oxygen species and fibroblast senescence. In KF PDE4A-D inhibition restrained TGFβ1-induced Smad3 and ERK1/2 phosphorylation, myofibroblast differentiation and senescence. Mechanistically, PDE4A-D inhibition rescued from TGFβ1-induced loss in PPM1A, a Smad3 phosphatase. In vivo, PDE4 inhibition mitigated HOCl-induced skin fibrosis in mice in preventive and therapeutic protocols.The current study provides novel evidence evolving rationale for PDE4 inhibitors in skin fibrosis (including keloids and HTS) and delivered evidence for a functional role of PDE4B in this fibrotic condition.

Analysis of core functional components in Yinchenhao Decoction and their pathways for treating liver fibrosis

To analyze the core functional component groups (CFCG) in Yinchenhao Decoction (YCHD) and their possible pathways for treating hepatic fibrosis based on network pharmacology. PPI data were extracted from DisGeNET, Genecards, CMGRN and PTHGRN to construct a weighted network using Cytoscape 3.9.1. The data of the chemical components in YCHD were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the potential active components and targets were selected using PreADMET Web server and SwissTargetPrediction. A fusion model was constructed to obtain the functional effect space and evaluate the effective proteins to identify the CFCG followed by GO and KEGG pathway enrichment analyses for all the targets. In cultured human hepatic stellate cells (LX-2 cells), the cytotoxicity of different compounds in YCHD was tested using CCK-8 assay; the effects of these compounds on collagen α1 (Col1a1) mRNA expression and the pathways in 20 ng/mL TGF-β1-stimulated cells were analyzed using RT-qPCR and Western blotting. A total of 1005 pathogenic genes, 226 potential active components and 1529 potential targets in YCHD and 52 potential targets of CFCG were obtained. Benzyl acetate, vanillic acid, clorius, polydatin, lauric acid and ferulic acid were selected for CCK-8 verification, and they all showed minimal cytotoxicity below the concentration of 200 μmol/L. Clorius, polydatin, lauric acid and ferulic acid all effectively inhibited TGF-β1-induced LX-2 cell activation. At the concentration of 200 μmol/L, all these 4 components inhibited PI3K, p-PI3K, AKT, p-AKT, ERK, p-ERK, P38 MAPK and p-P38 MAPK expressions in TGF-β1-induced LX-2 cells. The therapeutic effect of YCHD on hepatic fibrosis is probably mediated by its core functional components including benzyl acetate, vanillic acid, clorius, polydatin, lauric acid and ferulic acid, which inhibit the PI3K-AKT and MAPK pathways in hepatic stellate cells.

α7-nAChR/P300/NLRP3-regulated pyroptosis mediated poor articular cartilage quality induced by prenatal nicotine exposure in female offspring rats

Nicotine is developmentally toxic. Prenatal nicotine exposure (PNE) affects the development of multiple fetal organs and causes susceptibility to a variety of diseases in offspring. In this study, we aimed to investigate the effect of PNE on cartilage development and osteoarthritis susceptibility in female offspring rats. Wistar rats were orally gavaged with nicotine on days 9–20 of pregnancy. The articular cartilage was obtained at gestational day (GD) 20 and postnatal week (PW) 24, respectively. Further, the effect of nicotine on chondrogenic differentiation was explored by the chondrogenic differentiation model in human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). The PNE group showed significantly shallower Safranin O staining and lower Collagen 2a1 content of articular cartilage in female offspring rats. Further, we found that PNE activated pyroptosis in the articular cartilage at GD20 and PW24. In vitro experiments revealed that nicotine inhibited chondrogenic differentiation and activated pyroptosis. After interfering with nod-like receptors3 (NLRP3) expression by SiRNA, it was found that pyroptosis mediated the chondrogenic differentiation inhibition of WJ-MSCs induced by nicotine. In addition, we found that α7-nAChR antagonist α-BTX reversed nicotine-induced NLRP3 and P300 high expression. And, P300 SiRNA reversed the increase of NLRP3 mRNA expression and histone acetylation level in its promoter region induced by nicotine. In conclusion, PNE caused chondrodysplasia and poor articular cartilage quality in female offspring rats. PNE increased the histone acetylation level of NLRP3 promoter region by α7-nAChR/P300, which resulting in the high expression of NLRP3. Further, NLRP3 mediated the inhibition of chondrogenic differentiation by activating pyroptosis.

A plasma peptidomic signature reveals extracellular matrix remodeling and predicts prognosis in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is plagued with high mortality and difficulty in identifying at-risk patients. The extracellular matrix undergoes significant remodeling during inflammatory liver injury and could potentially be used for mortality prediction. EDTA plasma samples were collected from patients with AH (n = 62); Model for End-Stage Liver Disease score defined AH severity as moderate (12-20; n = 28) and severe (>20; n = 34). The peptidome data were collected by high resolution, high mass accuracy UPLC-MS. Univariate and multivariate analyses identified differentially abundant peptides, which were used for Gene Ontology, parent protein matrisomal composition, and protease involvement. Machine-learning methods were used to develop mortality predictors. Analysis of plasma peptides from patients with AH and healthy controls identified over 1600 significant peptide features corresponding to 130 proteins. These were enriched for extracellular matrix fragments in AH samples, likely related to the turnover of hepatic-derived proteins. Analysis of moderate versus severe AH peptidomes was dominated by changes in peptides from collagen 1A1 and fibrinogen A proteins. The dominant proteases for the AH peptidome spectrum appear to be CAPN1 and MMP12. Causal graphical modeling identified 3 peptides directly linked to 90-day mortality in >90% of the learned graphs. These peptides improved the accuracy of mortality prediction over the Model for End-Stage Liver Disease score and were used to create a clinically applicable mortality prediction assay. A signature based on plasma peptidome is a novel, noninvasive method for prognosis stratification in patients with AH. Our results could also lead to new mechanistic and/or surrogate biomarkers to identify new AH mechanisms.

Alterations of Matrisome Gene Expression in Naturally Aged and Photoaged Human Skin In Vivo.

The main component of human skin is a collagen-rich extracellular matrix (ECM), known as the matrisome. The matrisome is essential for maintaining the structural integrity and mechanical properties of the skin. Recently, we reported notable decreases in matrisome proteins in natural aging and photoaging human skin. This study aims to investigate the mRNA expression of the core matrisome proteins in human skin, comparing young versus aged and sun-protected versus sun-exposed skin by quantitative real-time PCR and immunostaining. Our findings reveal a notable decrease in core matrisome transcription in aged skin. The mRNA expression of the core matrisome, such as collagen 1A1 (COL1A1), decorin, and dermatopontin, is significantly reduced in aged skin compared to its young skin. Yet, the majority of collagen mRNA expression levels of aged sun-exposed skin are similar to those found in young sun-exposed skin. This discrepancy is primarily attributable to a substantial decrease in collagen transcription in young sun-exposed skin, suggesting early molecular changes in matrisome transcription due to sun exposure, which preceded the emergence of clinical signs of photoaging. These findings shed light on the mRNA transcript profile of major matrisome proteins and their alterations in naturally aged and photoaged human skin, offering valuable insights into skin matrisome biology.