COL1A1 Research Articles

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.

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.

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.

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.

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.

Expression, characterization, and application of human-like recombinant gelatin

Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. With irreplaceable biological functions in the field of biomedical science and tissue engineering, it has been widely applied. The amino acid sequence of recombinant human-like gelatin was constructed through a newly designed hexamer composed of six protein monomer sequences in series, with the minimum repeating unit being the characteristic Gly-X-Y sequence found in type III human collagen α1 chain. The nucleotide sequence was subsequently inserted into the genome of Pichia pastoris to enable soluble secretion expression of recombinant gelatin. At the shake flask fermentation level, the yield of recombinant gelatin is up to 0.057 g/L, and its purity can rise up to 95% through affinity purification. It was confirmed in the molecular weight determination and amino acid analysis that the amino acid composition of the obtained recombinant gelatin is identical to that of the theoretically designed. Furthermore, scanning electron microscopy revealed that the freeze-dried recombinant gelatin hydrogel exhibited a porous structure. After culturing cells continuously within these gelatin microspheres for two days followed by fluorescence staining and observation through confocal laser scanning microscopy, it was observed that cells clustered together within the gelatin matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant gelatin as a biomedical material.

Examining the Synergic Effect of Exosomes Derived from Mouse Mesenchymal Stem Cells and Low-frequency Electromagnetic Field on Chondrogenic Differentiation

Background: Cartilage has intrinsically limited healing power, and regeneration of cartilage damages has remained a challenge. Secreted products of mesenchymal stem cells have shown a new therapeutic strategies for cartilage injuries. Also it has been observed that low frequency electromagnetic field plays a key role in biological processes. Objective: This research was performed to investigate the synergic effect of mesenchymal stem cell-derived exosomes and low frequency electromagnetic field on chondrogenic differentiation. Methods: In this in vitro study, mesenchymal stem cell-derived exosomes were identified using AFM, SEM, TEM microscopy, and DLS method. Cells were treated in chondrogenic medium by exosomes, low frequency electromagnetic field, and the synergy of both. The cell survival was examined using MTT and Annexin methods, and cartilage differentiation was confirmed by Alcian blue staining. The expression of Sox-9, Acan, Col 2a1 and Col 10a1 genes was examined via Real- Time PCR technique on day 14 post-treatment. Results: The results confirmed the presence of exosomes with an approximate size of less than 100 nm. The results of Alcian blue revealed greater expression of glycosaminoglycans in the synergic treatment group compared to the other groups. Real-time PCR showed a significant increase in the expression of Sox-9, Acan, and Col 2a1 genes, as well as a significant reduction of Col 10a1 gene expression in the synergic treatment group compared to other groups. Conclusion: This study indicated that the synergic effect of exosome and low-frequency electromagnetic fields would lead to enhanced chondrogenic differentiation, which can be further explored in future clinical studies

Downregulation of LOX Overexpression Promotes Retinal Ganglion Cells Survival in an Acute Ocular Hypertension Model

Purpose To investigate the effect of reducing Lysyl oxidase (LOX) overexpression on retinal ganglion cells (RGCs) apoptosis in an acute ocular hypertension (AOH) rat model. Methods AOH rat model was performed by anterior chamber perfusion and either received an intravitreal injection with β-aminopropionitrile (BAPN) or normal saline. After 2wk, Quantification of survival RGCs in the retina was performed using Retrograde FluoroGold labeling. The mRNA expression levels of LOX, LOXL1-4, collagen 1a1 (Col1a1), collagen 3a1 (Col3a1), collagen4a1 (Col4a1), elastin (Eln), fibronectin1 (Fbn1), fibronectin4 (Fbn4) were determined by RT-qPCR. LOX expression was determined by Western blot (WB) analysis and immunohistochemistry. The RNA expression of LOX, Eln and Col1a1 in RGCs retrograde-labeled with 1,1’-dioctadecyl-3,3,3’,3’ tetra-methylindocarbocyanine perchlorate(DiI)that selected through FACS sorting were determined by RT-qPCR analysis. Changes of the retinal function were detected by Electroretinogram (ERG) analysis. Results Results showed that significant LOX overexpression and loss of RGCs related to IOP exposure in AOH retinas. PCR analysis indicated significant increased mRNA level of Col1a1, Col3al and Eln in AOH retinas. Significant increase mRNA expression of LOX, Col1a1 and Eln in the RGCs were observed in AOH group compared with CON group. AOH rats injected with BAPN showed a significant decrease in LOX expression, reduced the loss of RGCs and retinal function damage. Conclusions The results demonstrated that changes of LOX and specific ECM components in retina were correlated with AOH. Findings from this study indicated that preventing LOX over-expression may be protective against RGCs loss and retinal function damage in AOH animal model.