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Background: The increase in the collagen I (COL I)/COL III ratio enhances vessel wall stiffness and renders vessels less resistant to blood flow and pressure changes. Activated microglia enhance inflammation-induced fibrosis. Hypotheses: The COL I/COL III ratio in human and mouse brain arteriovenous malformations (bAVMs) is associated with bAVM hemorrhage, and the depletion of microglia decreases the COL I/COL III ratio and hemorrhage. Method: COL I, COL III, and hemorrhages were analyzed in 12 human bAVMs and 6 control brains, and mouse bAVMs induced in three mouse lines with activin receptor-like kinase 1 (n = 7) or endoglin (n = 7) deleted in the endothelial cells or brain focally (n = 5). The controls for the mouse study were no-gene-deleted litter mates. Mouse bAVMs were used to test the relationships between the Col I/Col III ratio and hemorrhage and whether the transient depletion of microglia reduces the Col I/Col III ratio and hemorrhage. Results: The COL I/COL III ratio was higher in the human and mouse bAVMs than in controls. The microhemorrhage in mouse bAVMs was positively correlated with the Col I/Col III ratio. Transient depletion of microglia reduced the Col I/Col III ratio and microhemorrhage. Conclusions: The COL I/COL III ratio in the bAVMs was associated with bAVM hemorrhage. The depletion of microglia reduced the bAVM Col I/Col III ratio and hemorrhage.

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I. [ABSTRACT FROM AUTHOR]

This research examines the anti‐aging potential of the flavonoid derivative of isoquercitrin known as enzymatically modified isoquercitrin (EMIQ). Initial HPLC analyses showed that EMIQ used in the study contained 1–12 glucosides and 10.7% pentahydroxyflavonoids, promising potent antioxidant properties. In subsequent in‐vitro studies with UVA‐exposed human dermal fibroblasts (HDFa), EMIQ demonstrated protective properties by reducing collagen damage. It modulated both the TGFβ/Smad pathway and the MMP1 pathway, contributing to collagen preservation. This protective effect was further confirmed using the T‐Skin™ model, a reconstructed full‐thickness human skin model, which illustrated that EMIQ could defend the physiological structures of both the epidermis and dermis against UV radiation. A 28‐day clinical trial with 30 volunteers aged 31–55 years highlighted EMIQ's effectiveness. Participants using EMIQ‐containing Essence displayed reduced facial trans‐epidermal water loss and skin roughness, alongside improved skin elasticity. This study emphasizes EMIQ's potential as an anti‐photoaging ingredient in cosmetics, warranting further research. The findings pave the way for developing innovative skincare products addressing photoaging effects. [ABSTRACT FROM AUTHOR]

Objective(s): Slit guidance ligand 3 (SLIT3) has been identified as a potential therapeutic regulator against fibroblast activity and fibrillary collagen production in an autocrine manner. However, this research aims to investigate the potential role of SLIT3 in cardiac fibrosis and fibroblast differentiation and its underlying mechanism.Materials and Methods: C57BL/6 mice (male, 8-10 weeks, n=47) were subcutaneously infused with Ang II (2.0 mg/kg/day) for 4 weeks. One to two-day-old Sprague-Dawley (SD) rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg) and ketamine (50 mg/kg) and the cardiac fibroblast was isolated aseptically. The mRNA and protein expression were analyzed using RT-qPCR and Western blotting.Results: The SLIT3 expression level was increased in Ang II-induced mice models and cardiac fibroblasts. SLIT3 significantly increased migrated cells and α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts. Ang II-induced increases in mRNA expression of collagen I (COL1A1), and collagen III (COL3A1) was attenuated by SLIT3 inhibition. SLIT3 knockdown attenuated the Ang II-induced increase in mRNA expression of ACTA2 (α-SMA), Fibronectin, and CTGF. SLIT3 suppression potentially reduced DHE expression and decreased malondialdehyde (MDA) content, and the superoxide dismutase (SOD) and catalase (CAT) levels were significantly increased in cardiac fibroblasts. Additionally, SLIT3 inhibition markedly decreased RhoA and ROCK1 protein expression, whereas ROCK inhibitor Y-27632 (10 μM) markedly attenuated the migration of cardiac fibroblasts stimulated by Ang II and SLIT3.Conclusion: The results speculate that SLIT3 could significantly regulate cardiac fibrosis and fibroblast differentiation via the RhoA/ROCK1 signaling pathway.

The ovary generally undergoes tissue remodeling during larval to pupal transition, which includes membrane degeneration and ovariole growth. At the same time, the hormones produced by insects significantly change during metamorphosis. However, the regulatory mechanism for ovarian development and hormones is not fully understood in insects. Herein, we found that matrix metalloproteinase 2 (MMP2) was highly expressed in the ovarian capsules and ovarioles, and the development was abnormal after knocking out MMP2 in Bombyx mori. The process of abnormal degradation of collagen I due to MMP2 deletion, which resulted in abnormal development of ovarioles and eggs, was analyzed in detail. The proteomics of ovaries in the MMP2‐knock out and wild type strains showed a critically significant difference in the expression of a protein, insulin‐like peptide (ILP). Additional analysis revealed significant alteration of ILP during ovarian development, and abnormal expression of ILP significantly affected ovarian development in vivo and MMP2 expression in vitro and in vivo. These results showed that MMP2 regulation of ovarian tissue remodeling is closely related to ILP expression. Our study provides new insights into the regulatory mechanism of MMP2 and ovarian development in B. mori. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis, arising from prolonged liver injury, entails the activation of hepatic stellate cells (HSCs) into myofibroblast-like cells expressing alpha-smooth muscle actin (α-SMA), thereby driving extracellular matrix deposition and fibrosis progression. Strategies targeting activated HSC reversal and hepatocyte regeneration show promise for fibrosis management. Previous studies suggest that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) can suppress HSC activation, but ensuring EV purity is essential for clinical use. This study investigated the effects of MSC-derived EVs cultured in chemically defined conditions on liver spheroids and activated HSCs. Umbilical cord- and bone marrow-derived MSCs were expanded in chemically defined media, and EVs were isolated using filtration and differential ultracentrifugation. The impact of MSC-EVs was evaluated on liver spheroids generated in Sphericalplate 5D™ and on human HSCs, both activated by transforming growth factor beta 1 (TGF-β1). MSC-EVs effectively reduced the expression of profibrotic markers in liver spheroids and activated HSCs induced by TGF-β1 stimulation. These results highlight the potential of MSC-EVs collected under chemically defined conditions to mitigate the activated phenotype of HSCs and liver spheroids, suggesting MSC-EVs as a promising treatment for hepatic fibrosis. [ABSTRACT FROM AUTHOR]

Objective To explore the phenotypic regulation mechanism of silence information regulator 1 (SIRT1) agonist SRT1720 on hypertrophic scar fibroblasts. Methods From January to June 2022, Department of Burns and Cutaneous Surgery, The First Affiliated Hospital of the Air Force Medical University，according to random number table method, hypertrophic scar fibroblasts (HSF) were divided into PBS control group and SRT1720-treated group. The two groups were respectively treated with equal amounts of PBS and SRT1720 with a final concentration of 2 μmol/L for 24 h. The mRNA expressions of collagen type I (Collagen I) and α-smooth muscle actin (α-SMA) in HSF were detected by a real-time fluorescence quantitative reverse transcription PCR (RT-PCR). The protein expressions of Collagen I and α-SMA in HSF were detected by Western blotting. The intracellular expression and localization of Collagen I and α-SMA of each group in HSF were detected using immunofluorescence method. Statistical analysis was performed using GraphPad Prim 8.0, the data was analyzed by independent sample t test, and statistically significant differences were considered at P<0.05. Results The mRNA expressions of Collagen I and α-SMA in HSF in the SRT1720-treated group was (0.34±0.06) and (0.43±0.08), respectively, which were significantly lower than that in the negative control group of (1.00±0.12) and (1.00±0.13), t=8.57 and 6.79, P<0.01. The protein expression of Collagen I and α-SMA in HSF in the SRT1720-treated group was (0.70±0.05) and (0.64±0.09), which were significantly lower than that in the negative control group of (1.14±0.04) and (1.10±0.05), t=12.61 and 7.37, P<0.01). Immunofluorescence staining showed that Collagen I and α-SMA were mainly localized in the cytoplasm of HSF, and the expression of Collagen I and α-SMA in the SRT1720-treated group was reduced compared with that in the negative control group. Conclusion SRT1720 could significantly inhibit the expression of Collagen I and α-SMA in scar fibroblasts by activating SIRT1, thereby inhibiting extracellular matrix (ECM) synthesis and abnormal transdifferentiation of fibroblasts. [ABSTRACT FROM AUTHOR]

Tremella polysaccharide is the main active substance in Tremella, possessing various functions such as anti- oxidant and anti-aging effects. The widely occurring chemical contaminant in the environment, benzo[a]pyrene (BaP), can be absorbed through the skin and metabolized, thereby posing a risk of carcinogenicity. In this experiment, the antioxidant activity of crude Tremella polysaccharide was measured in vitro, and then cellular assays were conducted to determine the changes in mRNA expression of collagen (Collagen I, Collagen III), Elastin and matrix metalloproteinases-1 (MMP-1) in fibroblasts under the combined effect of BaP and Tremella polysaccharide, along with the protein content of Collagen I. The anti-aging effect of polysaccharides was also determined. It was found that Tremella polysaccharide had good in vitro antioxidant activity with the highest superoxide anion radical scavenging rate of 70.28%, DPPH scavenging rate of 50.10% and hydroxyl radical scavenging rate of 32.69%. Tremella polysaccharide only at 0.1 µg/mL could effectively inhibit the stimulation of related gene expression by BaP at 20 µmol/L (P<0.05). The expression of Collagen I, Collagen III and Elastin genes increased in a polysaccharide concentration-dependent manner. Under BaP exposure, although collagen gene expression increased to 1.26-fold, the heavily expressed MMP-1 caused the degradation of collagen, therefore, under the effect of Tremella polysaccharide, the content of Collagen-1 decreased to 3.06 µg/L (which was lower than the value of the blank control: 3.48 µg/L). The results indicated that Tremella polysaccharide could effectively inhibit the BaP-induced damage to cells and promote the expression of cell genes (on which the concentration had an important influence). [ABSTRACT FROM AUTHOR]

Introduction and Hypothesis: The objective was to investigate the correlation between endogenous vaginal microecological alterations and female pelvic organ prolapse (POP). Methods: Patients who underwent vaginal hysterectomy were retrospectively analyzed as the POP group (n = 30) and the non-POP group (n = 30). The vaginal microbial metabolites and enzyme levels were tested using the dry chemoenzymatic method. The mRNA and protein expression were tested using real-time quantitative PCR and immunohistochemistry. SPSS version 25.0 and GraphPad Prism 8.0 were performed for statistical analysis. Results: Compared with the non-POP group, the vaginal pH, H2O2 positivity and leukocyte esterase positivity were higher in patients with POP (all p < 0.05). Further analysis showed that patients with pelvic organ prolapse quantification (POP-Q) stage IV had higher rates of vaginal pH, H2O2 positivity and leukocyte esterase positivity than those with POP-Q stage III. Additionally, the mRNA expression of decorin (DCN), transforming growth factor beta 1 (TGF-β1), and matrix metalloproteinase-3 (MMP-3) in uterosacral ligament tissues were higher, whereas collagen I and III were lower. Similarly, the positive expression of MMP-3 in uterosacral ligament tissue was significantly upregulated in the POP group compared with the non-POP group (p = 0.035), whereas collagen I (p = 0.004) and collagen III (p = 0.019) in uterosacral ligament tissue were significantly downregulated in the POP group. Correlation analysis revealed that there was a significant correlation between vaginal microecology and collagen metabolism. In addition, MMP-3 correlated negatively with collagen I and collagen III (p = 0.002, r = −0.533; p = 0.002, r = −0.534 respectively), whereas collagen I correlated positively with collagen III (p = 0.001, r = 0.578). Conclusions: Vaginal microecological dysbiosis affects the occurrence of female POP, which could be considered a novel therapeutic option. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of silencing connective tissue growth factor (CTGF) gene on growth, apoptosis, and expression of proliferating cell nuclear antigen (PCNA), CTGF, integrin alpha 5 (Itgα5) and collagen Ⅰ (ColⅠ) in rat hepatic stellate cell line T6 (HSC_T6). Methods The CTGF shRNA lentiviral vectors with high infectivity were transfected into HSC_T6 cells, and the cells transfected with empty virus or not transfected served as control cells. Fluorescence inverted microscopy was used to observe the cell growth and the expression of green fluorescent protein (GFP) in the transfected HSC_T6 cells. CCK-8 assay and flow cytometry were employed respectively to detect the cell viability and apoptosis. The expression changes in PCNA, CTGF, Itgα5 and ColⅠ were measured by real-time PCR, Western blotting and immunofluorescence assay. Results Microscopic observation displayed that transfection with CTGF shRNA lentiviral vectors resulted in high expression of GFP in HSC_T6 cells. CCK-8 assay showed that silencing CTGF induced inhibited growth in the transfected cells, with significant difference since 72 h after transfection (P < 0.05). Flow cytometry indicated that transfection of CTGF shRNA lentivirus led to the apoptosis of HSC_T6 cells. The results of real-time PCR, Western blotting and immunofluorescence assay confirmed that CTGF shRNA lentivirus effectively down-regulated the expression of PCNA, CTGF, Itgα5 and ColⅠ, with notable differences when compared the expression in control cells (P < 0.05). Conclusion Silencing CTGF can down-regulate the expression of CTGF, Itgα5 and ColⅠ, inhibit the growth and induce the apoptosis of HSC_T6 cells, which might be closely associated with its interference for the CTGF/Itgα5 signaling pathway.

Background: Vulvovaginal atrophy is part of the genitourinary syndrome of menopause caused by hypoestrogenic changes. This pathophysiological mechanism alters the concentration of collagen and elastin, which modifies the vaginal mucosa and impairs the function of the pelvic floor muscles. Quercetin is one of the flavonols found in plants, fruits, and vegetables. Quercetin helps to improve the syndrome through a variety of actions and estrogen-like effects. This study aimed to analyze the impact of quercetin on collagen I, collagen III, and elastin in a vaginal menopausal rat model. Method: This study's research design was an in vivo randomized control group post-test. The research was conducted at the animal laboratory, Faculty of Medicine, Universitas Airlangga. Rattus norvegicus, used in the study, were divided into 5 groups: normal rat, menopausal model without treatment, menopausal model given 17-β estradiol valerate 0.18 mg/kg, quercetin 12.5 mg/kg, and quercetin 50 mg/kg. The effectiveness of therapy was assessed from the immunohistochemical expression of collagen I, collagen III, and elastin in vaginal tissue. Results: The standard group in this study had the highest average expression levels of collagen I and elastin. The group of menopausal models without treatment in the study had the highest average expression of collagen III. Collagen I expression (p < 0.001), collagen III expression (p < 0.001), and elastin expression (p < 0.001) all showed significant differences. The menopausal rat model without treatment and the standard group showed the most differences in the expression of collagen I, collagen III, and elastin. Conclusions: The expression of collagen I, collagen III, and elastin in the vagina of the menopausal model was affected by the administration of quercetin at a level of 50 mg/kg. Thus, quercetin can be an alternative herbal treatment option to improve vulvovaginal atrophy in menopausal conditions.

Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.

Aim: The aim of the research was to analyse the regulatory effect of astragaloside (AST) on the immune microenvironment of diabetic non-healing wound (DNHW), and to analyse the clinical efficacy and mechanism of wound repair in multiple layers. Material and methods: Ninety adult male Wistar rats, which were kept healthy (SPF) under natural infection, were randomly divided into three groups, namely, blank, control and observation groups, with 30 rats in each group. After adaptive feeding for 7 days, the diabetes model was established. After the model was formed, the wounds were uniformly prepared, and then the blank group only was shaved. Both the control group and the observation group were treated with moist exposure therapy. The control group was covered with physiological saline gauze, while the observation group was covered with AST gauze. The healing status of the wounds in both groups was observed and recorded on the 1st, 7th, and 14th days after formation. And the levels of a-smooth muscle actin (α-SMA) and collagen I (COL-1) in the wound tissue were measured. Results: On the 1st day after wound formation, the wound healing area, α-SMA, and COL-1 levels in the three groups were consistent (p > 0.05). On the 7th and 14th days after wound formation, the wound healing area in the three groups increased compared within the group, but only the control and observation groups had significantly higher wound healing area than on the 1st day after wound formation (p < 0.05). In addition, the blank group had lower levels of α-SMA and COL-1, while the control and observation groups had higher levels of α-SMA and COL-1 (p < 0.05). In the comparison between groups, the wound healing area, α-SMA, and COL-1 levels in the control and observation groups were higher than those in the blank group, while the wound healing area, αa-SMA, and COL-1 levels in the observation group were higher than those in the control group (p < 0.05). Conclusions: AST can regulate the immune microenvironment of DNHW, improve α-SMA and COL-1, and accelerate the wound healing of DNHW. [ABSTRACT FROM AUTHOR]

We studied the effect of exosomes derived from mesenchymal stem cells on the synthesis of collagen I and α-smooth muscle actin (α-SMA) by rat fibroblast culture. Exosomes were isolated from the verified culture of mesenchymal stem cells and also verified. Fibrosis was modeled using a fibroblast culture supplemented with recombinant TGF-β1 (5 ng/ml) and immunocytochemical analysis of the expression of collagen I and α-SMA markers was carried out. After 6-day incubation, the expression of the studied markers increased in comparison with the control. Addition of exosomes to the fibroblast culture reduced the production of collagen and SMA, which allows considering exosomes as a promising drug for the treatment of pathologies associated with fibrosis. [ABSTRACT FROM AUTHOR]

Hepatic fibrosis, arising from prolonged liver injury, entails the activation of hepatic stellate cells (HSCs) into myofibroblast-like cells expressing alpha-smooth muscle actin (α-SMA), thereby driving extracellular matrix deposition and fibrosis progression. Strategies targeting activated HSC reversal and hepatocyte regeneration show promise for fibrosis management. Previous studies suggest that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) can suppress HSC activation, but ensuring EV purity is essential for clinical use. This study investigated the effects of MSC-derived EVs cultured in chemically defined conditions on liver spheroids and activated HSCs. Umbilical cord- and bone marrow-derived MSCs were expanded in chemically defined media, and EVs were isolated using filtration and differential ultracentrifugation. The impact of MSC-EVs was evaluated on liver spheroids generated in Sphericalplate 5D™ and on human HSCs, both activated by transforming growth factor beta 1 (TGF-β1). MSC-EVs effectively reduced the expression of profibrotic markers in liver spheroids and activated HSCs induced by TGF-β1 stimulation. These results highlight the potential of MSC-EVs collected under chemically defined conditions to mitigate the activated phenotype of HSCs and liver spheroids, suggesting MSC-EVs as a promising treatment for hepatic fibrosis.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-β1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-β1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-β1.

Mechanical loading is critical for collagen network maintenance and remodelling in adult skeletal tissues, but the role of loading in collagen network formation during development is poorly understood. We test the hypothesis that mechanical loading is necessary for the onset and maturation of spatial localization and structure of collagens in prenatal cartilage and bone, using in vivo and in vitro mouse models of altered loading. The majority of collagens studied was aberrant in structure or localization, or both, when skeletal muscle was absent in vivo. Using in vitro bioreactor culture system, we demonstrate that mechanical loading directly modulates the spatial localization and structure of collagens II and X. Furthermore, we show that mechanical loading in vitro rescues aspects of the development of collagens II and X from the effects of fetal immobility. In conclusion, our findings show that mechanical loading is a critical determinant of collagen network establishment during prenatal skeletal development.

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis largely due to the intense fibrotic desmoplastic reaction, characterized by high levels of extracellular matrix (ECM) collagen I that constitutes a niche for the cancer stem cells (CSCs). The role of the ECM composition in determining metabolic plasticity is still unknown. As ECM collagen I content increased, the CSCs switched from glucose to mostly glutamine metabolism. While all the bioenergetic modulators (BMs) decreased cell viability and increased cell death in all extracellular matrix types, a distinct, collagen I-dependent profile was observed in CSCs, in which the CSCs switched from glucose to mostly glutamine metabolism. Furthermore, all BMs synergistically potentiated the cytotoxicity of paclitaxel albumin nanoparticles (NAB-PTX) in both cell lines. Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 10 percent largely due to the intense fibrotic desmoplastic reaction, characterized by high levels of extracellular matrix (ECM) collagen I that constitutes a niche for a subset of cancer cells, the cancer stem cells (CSCs). Cancer cells undergo a complex metabolic adaptation characterized by changes in metabolic pathways and biosynthetic processes. The use of the 3D organotypic model in this study allowed us to manipulate the ECM constituents and mimic the progression of PDAC from an early tumor to an ever more advanced tumor stage. To understand the role of desmoplasia on the metabolism of PDAC parenchymal (CPC) and CSC populations, we studied their basic metabolic parameters in organotypic cultures of increasing collagen content to mimic in vivo conditions. We further measured the ability of the bioenergetic modulators (BMs), 2-deoxyglucose, dichloroacetate and phenformin, to modify their metabolic dependence and the therapeutic activity of paclitaxel albumin nanoparticles (NAB-PTX). While all the BMs decreased cell viability and increased cell death in all ECM types, a distinct, collagen I-dependent profile was observed in CSCs. As ECM collagen I content increased (e.g., more aggressive conditions), the CSCs switched from glucose to mostly glutamine metabolism. All three BMs synergistically potentiated the cytotoxicity of NAB-PTX in both cell lines, which, in CSCs, was collagen I-dependent and the strongest when treated with phenformin + NAB-PTX. Metabolic disruption in PDAC can be useful both as monotherapy or combined with conventional drugs to more efficiently block tumor growth. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) contributes to impaired wound healing. This study was aimed to evaluate the effect of stromal vascular fraction (SVF) gel extracted from rats on diabetic ulcers wound healing and peripheral nerve repair. 60 Sprague Dawley (SD) rats were divided into 6 groups, including control, model, SVF-gel low dose (SVF-gel-L), SVF-gel high dose (SVF-gel-H), ST2825, and SVF-gel-H + CL075 groups. Wound closure rate was recorded. The histopathological changes and deposition change of collagen fibre were identified. The content of TNF-α, IL-1β, VEGF, and bFGF were detected. Immunohistochemical, immunofluorescence and western blot were employed to determine the protein expression. We identified SVF-gel could promoted wound healing, restored normal cutaneous structures of the wound, promoted collagen deposition, while diminished fibrosis and inflammation. In addition, SVF-gel promoted angiogenesis and peripheral nerve recovery, diminished the expression of TLRs/MyD88/NF-κB signaling pathway. However, the protective effect of SVF-gel could be revised by CL075 co-treatment. Furthermore, ST2825 also promoted wound healing, but its effect was lower than that with SVF-gel-H treatment. SVF gel promotes the healing of diabetic skin ulcer tissue and regeneration of damaged peripheral nerve, diminished inflammatory factor infiltration. The mechanism maybe related to suppress the activation of TLRs/MyD88/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Fibrosis is an important phenomenon as it can occur early in the pathogenesis of asthma; it may be associated with disease severity and resistance to therapy. There is a strong evidence that infection caused by human rhinovirus (HRV) contributes to remodelling process, but there is lack of studies clearly explaining this pathway. Synthetic peroxisome proliferator‐activated receptor (PPAR) γ presents immunomodulatory and anti‐inflammatory features. In this study, we examined immunomodulatory properties of ciglitazone – PPAR‐γ agonist, in development and modulation of airway remodelling. Epithelial cells (NHBE) and two lines of fibroblasts (WI‐38, HFL1) were stimulated with ciglitazone and rhinovirus. The expression of genes related to airway remodelling process were analysed in the cells; moreover NF‐κB, c‐Myc and STAT3 were silenced in order to estimate potential pathways involved. Ciglitazone decreased mRNA expression of MMP‐9 and TGF‐β. It also modified the expression of α‐SMA and collagen after rhinovirus infection. Transcription factors knockdown altered the levels of expression. The results suggest possible anti‐fibrotic activity of PPAR‐γ agonist in human airway cells. Ciglitazone has been shown to be dependent on NF‐κB‐ and STAT3‐related pathways, thus, the PPAR‐γ agonist may have therapeutic potential for the treatment of airway remodelling in asthma. [ABSTRACT FROM AUTHOR]

The development of uremic cardiomyopathy (UC) and cardiac fibrosis in chronic kidney disease (CKD) was comparatively analyzed in model experiments on SPF males of Sprague–Dawley and Wistar rats. CKD was induced by left subtotal nephrectomy (NE) followed after 2 weeks by right radical NE. Sham-operated (Sham) animals served as controls. Four weeks after radical NE, blood pressure (BP) was measured, and the expression of pro-fibrotic factors (Fli1, pro-collagen I, collagen I, collagen IV) in left ventricular myocardial tissue was assessed by real-time PCR and immunoblotting. After NE, Sprague–Dawley rats exhibited a significant increase in systolic BP, as well as left ventricular hypertrophy. In Wistar rats, the difference between BP in NE and Sham animals was smaller, while the left ventricular mass to body weight ratio did not change. Experimental CKD in Sprague–Dawley rats was accompanied by a 1.5–2.5-fold suppression of Fli1 gene expression and a decrease in Fli1 protein content in cardiac muscle tissue, while no significant differences were observed in Wistar rats. Pro-collagen I and collagen I levels in the heart of rats of both strains changed at neither transcription nor translation levels. Such a difference in the development of pathological processes indicates an ineffectiveness of the applied type of NE to induce UC and investigate pro-fibrotic processes in Wistar vs. Sprague–Dawley rats. [ABSTRACT FROM AUTHOR]

Previous research testifies that c‐Myc may promote keloid fibroblast proliferation and collagen accumulation. Ubiquitin‐specific peptidase 37 (USP37)‐mediated deubiquitination and stabilisation of c‐Myc are vital for lung cancer proliferation, while the potential role of USP37 in keloid fibroblasts is not investigated. Elevated USP37, c‐Myc, and Collagen I content were detected in keloid tissue with RT‐PCR or ELISA assay. USP37 over‐expression plasmids or USP37 short hairpin RNAs (shRNAs) were transfected into keloid fibroblasts with Lipofectamine 3000 to decipher the role of USP37 in keloid fibroblasts. USP37 overexpression could promote the proliferation of keloid fibroblasts with increased c‐Myc and Collagen I expression. On the other hand, USP37 shRNAs inhibited the proliferation of keloid fibroblasts with diminished c‐Myc and Collagen I expression. It was worth noting that C‐Myc overexpression promoted the proliferation of keloid fibroblasts inhibited by USP37 shRNAs with increasing Collagen I expression. All of these results demonstrate that USP37 could regulate c‐Myc to promote the proliferation and collagen deposit of keloid fibroblasts, and USP37 could be targeted in future keloid therapy. [ABSTRACT FROM AUTHOR]

The interactions between cell and cellular matrix confers plasticity to each body tissue, influencing the cellular migratory capacity. Macrophages rely on motility to promote their physiological function. These phagocytes are determinant for the control of invasive infections, and their immunological role largely depends on their ability to migrate and adhere to tissue. Therefore, they interact with the components of the extracellular matrix through their adhesion receptors, conferring morphological modifications that change their shape during migration. Nevertheless, the need to use in vitro cell growth models with the conditioning of three-dimensional synthetic matrices to mimic the dynamics of cell-matrix interaction has been increasingly studied. This becomes more important to effectively understand the changes occurring in phagocyte morphology in the context of infection progression, such as in Chagas disease. This disease is caused by the intracellular pathogen Trypanosoma cruzi, capable of infecting macrophages, determinant cells in the anti-trypanosomatid immunity. In the present study, we sought to understand how an in vitro extracellular matrix model interferes with T. cruzi infection in macrophages. Using different time intervals and parasite ratios, we evaluated the cell morphology and parasite replication rate in the presence of 3D collagen I matrix. Nevertheless, microscopy techniques such as scanning electron microscopy were crucial to trace macrophage-matrix interactions. In the present work, we demonstrated for the first time that the macrophage-matrix interaction favors T. cruzi in vitro replication and the release of anti-inflammatory cytokines during macrophage infection, in addition to drastically altering the morphology of the macrophages and promoting the formation of migratory macrophages. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-1 (MMP-1) is a zinc-containing endopeptidase that degrades dermal collagen and other extracellular matrix molecules. It is recognized as one of the most important indicators of cellular senescence and age-related skin changes. Here, we introduced a novel MMP-1 peptide nucleic acid (PNA) derivative—PNA-20 carboxyethyl fluorene (CEF)—which can interact with and consequently silence the MMP-1 gene sequence. The investigation on the efficacy of PNA-20 CEF in MMP-1 silencing in human dermal fibroblasts revealed significantly decreased expression of MMP-1 at both gene and protein levels. Treatment with PNA-20 CEF showed significantly increased expression of collagen I protein, indicating its potential role in preventing the degradation of collagen I and consequently combating the skin aging process. Its topical application on 3D human skin tissue showed successful absorption into the epidermis and the upper dermis. Furthermore, the additional 4-week single-arm prospective study on 21 Asian women revealed improvements in facial wrinkles, skin moisture, elasticity, and density after the use of the topical PNA-20 CEF cosmeceutical formulation. Additional in-vitro and ex-vivo studies are needed for a comprehensive understanding of the skin anti-aging effects of MMP-1 PNA. [ABSTRACT FROM AUTHOR]

• Genetic analysis by RNAi reveals that collagen I is under tonic repression in fibroblasts. • Bradykinin receptor 2 (BDKRB2), a G-protein couple receptor repress collagen I expression and is a modulator of ECM homeostasis. • BDKRB2 effect is independent of bradykinin and canonical TGFβ-SMAD signalling and is conveyed through ERK1/2 inhibition. • BDKRB2 regulation of collagen I appears relevant in various fibrotic conditions such as Keloids and Idiopathic Pulmonary Fibrosis. Imbalance of collagen I expression results in severe pathologies. Apart from activation by the TGFβ-receptor/Smad pathway, control of collagen I expression remains poorly understood. Here, we used human dermal fibroblasts expressing a mCherry fluorescent protein driven by endogenous COL1A1 promoter to functionally screen the kinome and phosphatome. We identify 8 negative regulators, revealing that collagen is under tonic repression. The cell surface receptor BDKRB2 represses collagen I and other pro-fibrotic genes. Interestingly, it also promotes other basal membrane ECM genes. This function is independent of the natural ligand, bradykinin, and of SMAD2/3 factors, instead requiring constant ERK1/2 repression. TGFβ stimulation induces rapid BDKRB2 transcriptional downregulation. Human fibrotic fibroblasts have reduced BDKRB2 levels and enhancing its expression in keloid fibroblasts represses COL1A1. We propose that tonic signalling by BDKRB2 prevents collagen overproduction in skin fibroblasts. [ABSTRACT FROM AUTHOR]

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and CIS genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder. [ABSTRACT FROM AUTHOR]

Background: The increase in the collagen I (COL I)/COL III ratio enhances vessel wall stiffness and renders vessels less resistant to blood flow and pressure changes. Activated microglia enhance inflammation-induced fibrosis. Hypotheses: The COL I/COL III ratio in human and mouse brain arteriovenous malformations (bAVMs) is associated with bAVM hemorrhage, and the depletion of microglia decreases the COL I/COL III ratio and hemorrhage. Method: COL I, COL III, and hemorrhages were analyzed in 12 human bAVMs and 6 control brains, and mouse bAVMs induced in three mouse lines with activin receptor-like kinase 1 (n = 7) or endoglin (n = 7) deleted in the endothelial cells or brain focally (n = 5). The controls for the mouse study were no-gene-deleted litter mates. Mouse bAVMs were used to test the relationships between the Col I/Col III ratio and hemorrhage and whether the transient depletion of microglia reduces the Col I/Col III ratio and hemorrhage. Results: The COL I/COL III ratio was higher in the human and mouse bAVMs than in controls. The microhemorrhage in mouse bAVMs was positively correlated with the Col I/Col III ratio. Transient depletion of microglia reduced the Col I/Col III ratio and microhemorrhage. Conclusions: The COL I/COL III ratio in the bAVMs was associated with bAVM hemorrhage. The depletion of microglia reduced the bAVM Col I/Col III ratio and hemorrhage.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, lack of attached gingiva and thin and fragile gums leading to gingival recession. Connective tissue abnormalities of pEDS typically include easy bruising, pretibial plaques, distal joint hypermobility, hoarse voice, and less commonly manifestations such as organ or vessel rupture. pEDS is caused by heterozygous missense mutations in C1R and C1S genes of the classical complement C1 complex. Previously we showed that pEDS pathogenic variants trigger intracellular activation of C1r and/or C1s, leading to extracellular presence of activated C1s. However, the molecular link relating activated C1r and C1s proteases to the dysregulated connective tissue homeostasis in pEDS is unknown. Using cell- and molecular-biological assays, we identified activated C1s (aC1s) as an enzyme which degrades collagen I in cell culture and in in vitro assays. Matrix collagen turnover in cell culture was assessed using labelled hybridizing peptides, which revealed fast and comprehensive collagen protein remodeling in patient fibroblasts. Furthermore, collagen I was completely degraded by aC1s when assays were performed at 40°C, indicating that even moderate elevated temperature has a tremendous impact on collagen I integrity. This high turnover is expected to interfere with the formation of a stable ECM and result in tissues with loose compaction a hallmark of the EDS phenotype. Our results indicate that pathogenesis in pEDS is not solely mediated by activation of the complement cascade but by inadequate C1s-mediated degradation of matrix proteins, confirming pEDS as a primary connective tissue disorder.

Skin thickness is closely related to the appearance of human skin, such as sagging and wrinkling, which primarily depends on the level of collagen I synthesized by fibroblasts in the dermal layer. To explore the underlying genetics of the development of skin thickness, we used the indigenous Chinese Chenghua pigs, considered to have superior skin thickness, as model animals. We first performed whole transcriptome sequencing analysis to identify significant skin morphological differences between Chenghua pigs and Large White pigs and obtained some differentially expressed coding RNAs (454 mRNAs) and noncoding RNAs (612 circRNAs, 188 miRNAs, and 19 lncRNAs); moreover, some competing endogenous RNA (ceRNA) networks were constructed. Interestingly, we then identified a circRNA, namely circ0044633, which plays an important role in promoting fibroblast proliferation along with myofibroblast transition and collagen I synthesis by sponging miR-23b and regulating CADM3 and MAP4K4 expression via activation of the downstream AKT and ERK pathways in vitro. Furthermore, overexpression of circ004463 increased the mouse skin thickness and collagen I content in vivo. These results revealed a whole transcript profile of skin tissue and identified an important circ0044633-miR-23b-CADM3/MAP4K4 axis related to fibroblast proliferation and collagen I synthesis during the development of skin thickness. [Display omitted] [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is a common arrhythmia. Angiotensin‐receptor blocker (ARB) is related to AF treatment. This study explored the mechanism of ARB in AF. AF rat models were established by Ach‐CaCl2 mixed solution injection. Rats were treated with ARB by gavage and injected with pcDNA3.1‐based frizzled homolog 8 (FZD8) overexpression plasmids (oe‐FZD8) through the tail vein. The 12‐lead electrocardiogram was recorded by biological signal acquisition and processing system and AF duration was recorded, and atrial effective refractory period (AERP) was monitored by electrophysiology. Atrial fibrosis degree, FZD8 messenger RNA and protein levels, collagen I, collagen III, transforming growth factor β1 (TGF‐β1), fibronectin, α smooth muscle actin (α‐SMA), WBT‐5B, and p‐JNK1/2 levels, interleukin 1 β (IL‐1β) and interleukin 6 (IL‐6) levels were detected by Masson staining, reverse transcription quantitative polymerase chain reaction, western blot assay, immunohistochemistry, and enzyme‐linked immunosorbent assay. ACh‐CaCl2‐induced AF rats showed a large area of fused necrosis, abnormal collagen fibre proliferation, high atrial fibrosis degree, and increased atrial fibrosis area in atrial interstitium, elevated collagen I, collagen III, TGF‐β1, fibronectin, α‐SMA, IL‐1β, and IL‐6 levels, whereas these trends were averted by ARB treatment. FZD8 was highly expressed in AF rat myocardium. ARB repressed FZD8 expression, prolonged AERP and reduced AF incidence. FZD8 overexpression annulled the effects of ARB on improving AF rat myocardial fibrosis. ARB inactivated the WNT‐5A pathway by suppressing FZD8. ARB inactivated the WNT‐5A pathway by silencing FZD8, therefore, alleviating AF rat atrial fibrosis. [ABSTRACT FROM AUTHOR]

C‐MYC‐mediated keloid fibroblasts proliferation and collagen deposit may contribute to the development of keloids. F‐box and leucine‐rich repeat protein 6 (FBXL6) is reported to be involved in tumour progression, while the role of FBXL6 in keloid fibroblasts is not deciphered. Normal control skins, hypertrophic scars and keloid tissues were collected and prepared for FBXL6 detection. FBXL6 short hairpin RNAs (shRNAs) or FBXL6 over‐expression plasmids were transfected into keloid fibroblasts, and then c‐MYC plasmids were further transfected. Cell viability was assayed with a Cell‐Counting Kit‐8 kit. The relative expression of FBXL6, Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I was detected with real‐time PCR and Western blot. Elevated FBXL6 expression could be observed in keloid tissues and hypertrophic scars. FBXL6 shRNAs transfection could inhibit the viability of keloid fibroblasts with diminished c‐MYC expression and down‐regulated Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I expression. At the same time, overexpressed FBXL6 could promote the proliferation of keloid fibroblasts. Overexpression of c‐MYC could promote the proliferation of keloid fibroblasts reduced by FBXL6 shRNAs with up‐regulated Cyclin A1 and Collagen I expression. FBXL6 could promote the growth of keloid fibroblasts by inducing c‐MYC expression, which could be targeted in keloids treatment. [ABSTRACT FROM AUTHOR]

Metformin, a biguanide, exerts different functions through various signaling pathways. In order to investigate the function and mechanism of metformin in burn wounds, we established burn rat models, subcutaneously injected metformin to treat the wounds, and observed the morphologies and the expression of collagen I, collagen III, fibronectin, and pro-inflammatory markers. In vitro experiments were performed to investigate the effects of metformin on the proliferation, migration, and collagen I synthesis of the mouse embryonic fibroblast (NIH 3T3) cell line and on the proliferation, apoptosis, and immune response of the mouse mononuclear macrophage (RAW 264.7) cell line. Finally, we studied the regulatory effects of metformin on a co-culture of RAW 264.7/NIH 3T3 cells. We found that 100 mM of metformin reduced dermal thickness, collagen I deposition, and mRNA expression of IL1β and CCL2 in rat burn wounds. In vitro experiments revealed that metformin inhibited the proliferation of NIH 3T3 and RAW 264.7 cells. Metformin attenuated NIH 3T3 cell migration via the AMPK/mTOR pathway and attenuated collagen I synthesis through the TGFβ1/Smad3 pathway. Metformin inhibited the apoptosis of RAW 264.7 cells induced by 10 μg/mL LPS. Metformin downregulated the mRNA expression of IL1β and CCL2 in RAW 264.7 cells under 1 μg/mL LPS induction by inhibiting NF-κB p65 phosphorylation. In a RAW 264.7/NIH 3T3 co-culture, metformin attenuated collagen I synthesis in NIH 3T3 cells by inhibiting RAW 264.7 paracrine secretion of TGF-β1. This provides new evidence related to the development of metformin for potentially improving burn wound healing. [ABSTRACT FROM AUTHOR]

Background: The key focal adhesion protein β1 integrin plays an essential role in early skeletal development. However, roles of β1 integrin expression in osteocytes during the regulation of bone homeostasis and mechanotransduction are incompletely understood. Materials and methods: To study the in vivo function of osteocyte β1 integrin in bone, we utilized the 10-kb Dmp1 (Dentin matrix acidic phosphoprotein 1)-Cre to generate mice with β1 integrin deletion in this cell type. Micro-computerized tomography, bone histomorphometry and immunohistochemistry were performed to determine the effects of osteocyte β1 integrin loss on bone mass accrual and biomechanical properties. In vivo tibial loading model was applied to study the possible involvement of osteocyte β1 integrin in bone mechanotransduction. Results: Loss of β1 integrin expression in osteocytes resulted in a severe low bone mass and impaired biomechanical properties in load-bearing long bones and spines, but not in non-weight-bearing calvariae, in mice. The loss of β1 integrin led to enlarged size of lacunar-canalicular system, abnormal cell morphology, and disorientated nuclei in osteocytes. Furthermore, β1 integrin loss caused shortening and disorientated collagen I fibers in long bones. Osteocyte β1 integrin loss did not impact the osteoclast activities, but significantly reduced the osteoblast bone formation rate and, in the meantime, enhanced the adipogenic differentiation of the bone marrow stromal cells in the bone microenvironment. In addition, tibial loading failed to accelerate the anabolic bone formation and improve collagen I fiber integrity in mutant mice. Conclusions: Our studies demonstrate an essential role of osteocyte β1 integrin in regulating bone homeostasis and mechanotransduction.The transnational potential of this article: This study reveals the regulatory roles of osteocyte β1 integrin in vivo for the maintenance of bone mass accrual, biomechanical properties, extracellular matrix integrity as well as bone mechanobiology, which defines β1 integrin a potential therapeutic target for skeletal diseases, such as osteoporosis.

Abstract Background Mesenchymal stem cells (MSCs) have emerged as a promising cell-based therapy for acute kidney injury (AKI). However, the optimal route of MSC transplantation remains controversial, and there have been no comparisons of the therapeutic benefits of MSC administration through different delivery routes. Methods In this study, we encapsulated MSCs into a collagen matrix to help achieve local MSC retention in the kidney and assessed the survival of MSCs in vitro and in vivo. After transplanting collagen matrix-encapsulated-MSCs (Col-MSCs) under the renal capsule or into the parenchyma using the same cell dose and suspension volume in an ischemia/reperfusion injury model, we evaluated the treatment efficacy of two local transplantation routes at different stages of AKI. Results We found that Col-MSCs could be retained in the kidney for at least 14 days. Both local MSC therapies could reduce tubular injury, promote the proliferation of renal tubular epithelial cells on Day 3 and alleviate renal fibrosis on Day 14 and 28. MSC transplantation via the subcapsular route exerts better therapeutic effects for renal functional and structural recovery after AKI than MSC administration via the parenchymal route. Conclusions Subcapsular MSC transplantation may be an ideal route of MSC delivery for AKI treatment, and collagen I can provide a superior microenvironment for cell–cell and cell–matrix interactions to stabilize the retention rate of MSCs in the kidney.

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes. We previously demonstrated that contact of megakaryocytes to Collagen I fibers initiated the formation of linear podosomes required for proplatelets extension and release of mature platelets. MKs linear podosomes have the particularity of displaying mechanical pulling activity but, unlike other linear podosomes, they lack the ability of digesting the extracellular matrix (ECM), as we recently demonstrated. The Cdc42 small GTPase is required for actomyosin-dependent maturation of the demarcation membrane system (DMS), a membrane reservoir for PPT and platelets components. Cdc42 is a known protein of the podosomes core, and is instrumental to accurate platelets release into the sinusoids. Indeed, FRET analysis showed that Cdc42 activity was very high and central to DMS formation. Unexpectedly, even though we found the protein in linear podosomes, almost undetectable Cdc42 activity was detected in those structures. This observation suggests that Cdc42 could also act as scaffold to assemble proteins required for PPT formation/elongation along Collagen I fibers in MKs. Eventually, we demonstrated that linear podosomes appear as points of contact between Collagen I fibers and DMS membranes, to mechanically extend PPT along Collagen bundles, independently of Cdc42 activity. • Cdc42 links the demarcation membrane system to the linear podosomes in megakaryocytes. • Low CDC42 activity is required in linear podosomes. • Demarcation membrane system is the main site of Cdc42 activity in megakaryocytes. [ABSTRACT FROM AUTHOR]

The lung is one of the most common sites for cancer metastasis. Collagens in the lung provide a permissive microenvironment that supports the colonization and outgrowth of disseminated tumor cells. Therefore, down-regulating the production of collagens may contribute to the inhibition of lung metastasis. It has been suggested that miR-29 exhibits effective anti-fibrotic activity by negatively regulating the expression of collagens. Indeed, our clinical lung tumor data shows that miR-29a-3p expression negatively correlates with collagen I expression in lung tumors and positively correlates with patients’ outcomes. However, suitable carriers need to be selected to deliver this therapeutic miRNA to the lungs. In this study, we found that the chemotherapy drug cisplatin facilitated miR-29a-3p accumulation in the exosomes of lung tumor cells, and this type of exosomes exhibited a specific lung-targeting effect and promising collagen down-regulation. To scale up the preparation and simplify the delivery system, we designed a lung-targeting liposomal nanovesicle (by adjusting the molar ratio of DOTAP/cholesterol–miRNAs to 4:1) to carry miR-29a-3p and mimic the exosomes. This liposomal nanovesicle delivery system significantly down-regulated collagen I secretion by lung fibroblasts in vivo, thus alleviating the establishment of a pro-metastatic environment for circulating lung tumor cells.

Introduction: The purpose of this study was to determine whether miR-29a regulates cell survival and apoptosis and the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MMP-2, and collagen I in scleral fibroblasts. Methods: We transfected scleral fibroblasts with the miR-29a mimic and inhibitor. The effects of miR-29a on cell proliferation and apoptosis were determined using the CCK-8 assay and flow cytometry, respectively. Quantitative polymerase chain reaction (qPCR) was used to determine whether miR-29a regulates the mRNA levels of PTEN, MMP-2, and collagen I. The protein expression of PTEN, MMP-2, and collagen I was also assessed by western blot analysis. Results: The results of CCK-8 showed that, at 0, 24, 48, and 72 h after transfection, the relative optical density values in the mimic group were 0.233 ± 0.005, 0.380 ± 0.008, 0.650 ± 0.040, and 0.906 ± 0.032, and in the inhibitor group were 0.272 ± 0.011, 0.393 ± 0.029, 0.597 ± 0.059, and 0.950 ± 0.101, respectively. The flow cytometry results showed that the apoptosis rates of each group were as follows: the mimic group (0.043 ± 0.007), the NC group (0.040 ± 0.006), the inhibitor group (0.032 ± 0.003), the inhibitor NC group (0.027 ± 0.010), the lipofectamine group (0.027 ± 0.005), and the blank group (0.031 ± 0.009). The qPCR results indicated that in the mimic group, PTEN (0.795 ± 0.182, p = 0.2783), MMP-2 (0.621 ± 0.105, p = 0.0033), and COL1A1 (0.271 ± 0.100, p = 0.0002) expression decreased, whereas in the inhibitor group, PTEN (1.211 ± 0.100, p = 0.2614), MMP-2 (1.161 ± 0.053, p = 0.1190), and COL1A1 (1.7040 ± 0.093, p = 0.0003) increased. Western blot analysis showed that in the mimic group, the expression of PTEN (0.392 ± 0.039, p < 0.0001), MMP-2 (0.577 ± 0.017, p < 0.0001), and COL1A1 (0.072 ± 0.006, p < 0.0001) protein decreased, whereas in the inhibitor group, PTEN (1.043 ± 0.042, p = 0.9413), MMP-2 (1.397 ± 0.075, p = 0.0002), and COL1A1 (1.935 ± 0.081, p < 0.0001) expression increased. Conclusion: MiR-29a inhibits the expression of PTEN, MMP-2, and collagen I on scleral fibroblasts, which may provide a basis studies in sclera. [ABSTRACT FROM AUTHOR]

The use of collagen in cell cultures promotes cell proliferation and differentiation, and it has been commercialized. In this study, we separated and purified collagen from adipose tissue discarded during liposuction and prepared collagen-coated dishes. After collagen was identified from human adipose tissue, type identification and quantification were performed using SDS-PAGE and FPLC. Collagen type I was used to coat culture dishes. Human skin fibroblasts and human adipose tissue-derived stem cells were seeded at a density of 2.5×105 cells/mL on prepared dishes at a collagen concentration of 3 mg/mL and cultured for 7 days. Cell viability was then measured and analyzed. The WST-1 assay was used to evaluate the results. The amount of collagen in 300 g of adipose tissue was 25.5 mg for type I, 41.4 mg for type III, 10.6 mg for type IV, 6.5 mg for type V, and 15 mg for type VI. The highest rates were observed for adipose stem cells cultured on human adipose tissue-derived collagen-coated dishes. In cell cultures, cell affinity was higher when cells and the substrate used were of the same origin, and affinity was stronger when the tissue of origin was the same., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Hepatic fibrosis progresses concomitantly with a variety of biomechanical alternations, especially increased liver stiffness. These biomechanical alterations have long been considered as pathological consequences. Recently, growing evidence proposes that these alternations result in the fibrotic biomechanical microenvironment, which drives the activation of hepatic stellate cells (HSCs). Here, an inorganic ascorbic acid-oxidase (AAO) mimicking nanozyme loaded with liquiritigenin (LQ) is developed to trigger remodeling of the fibrotic biomechanical microenvironment. The AAO mimicking nanozyme is able to consume intracellular ascorbic acid, thereby impeding collagen I deposition by reducing its availability. Simultaneously, LQ inhibits the transcription of lysyl oxidase like 2 (LOXL2), thus impeding collagen I crosslinking. Through its synergistic activities, the prepared nanosystem efficiently restores the fibrotic biomechanical microenvironment to a near-normal physiological condition, promoting the quiescence of HSCs and regression of fibrosis. This strategy of remodeling the fibrotic biomechanical microenvironment, akin to "pulling the rug out from under", effectively treats hepatic fibrosis in mice, thereby highlighting the importance of tissue biomechanics and providing a potential approach to improve hepatic fibrosis treatment., (© 2024 Wiley‐VCH GmbH.)

Periodontium regeneration and repair is a controversial and difficult point in the treatment of periodontosis. The proliferation, differentiation, migration and adhesion of periodontal ligament cells and the dynamic relationship between periodontal ligament cells and their extracellular matrix proteins are the basis of periodontium morphological reconstruction, functional maintenance and tissue repair. This article reviews the mechanism of estrogen-regulated periodontal membrane fine repair and periodontal tissue reconstruction to provide the basis for follow-up research on the treatment of periodontitis and the promotion of periodontal tissue repair and reconstruction by exogenous estrogen-mediated periodontal membranes. Under the regulation of certain concentrations of estrogen, the proliferation and differentiation ability of periodontal ligament stem cells (PDLSCs) and bone mesenchymal stem cells (BMSCs) to other periodontal ligament cells were enhanced. At the same time, PDLSCs, BMSCs, human periodontal ligament fibroblasts (HPLFSs), osteoblasts and cementoblasts synthesized and secreted collagen I (COLI), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCN) into the extracellular matrix. They interact with fibronectin (FN) and cementum attachment protein (CAP) in the extracellular matrix to form a variety of chain complexes and regulate each other, thus promoting the growth, migration, adhesion and fibrosis of periodontal ligament cells, repairing the collagen fiber skeleton of the periodontal ligament and adhering the two ends to the new cementum and the inherent alveolar bone.

Introduction: Pancreatic adenocarcinoma (PAAD) is an aggressive malignancy, with a major mortality resulting from the rapid progression of metastasis. Unfortunately, no effective treatment strategy has been developed for PAAD metastasis to date. Thus, unraveling the mechanisms involved in PAAD metastatic phenotype may facilitate the treatment for PAAD patients. Objectives: PIK3CB is an oncogene implicated in cancer development and progression but less is known about whether PIK3CB participates in PAAD metastasis. Therefore, the objective of this study is to explore the mechanism(s) of PIK3CB in PAAD metastasis. Methods: In our study, we examined the PIK3CB expression pattern using bioinformatic analysis and clinical material derived from patients with PAAD. Subsequently, a series of biochemical experiments were conducted to investigate the role of PIK3CB as potential mechanism(s) underlying PAAD metastasis in vivo using nude mice and in vitro using cell lines. Results: We observed that PIK3CB was involved in PAAD progression. Notably, we identified that PIK3CB was involved in PAAD metastasis. Downregulation of PIK3CB significantly reduced PAAD metastatic potential in vivo. Furthermore, a series of bioinformatic analyses showed that PIK3CB was involved in cell adhesion in PAAD. Notably, PIK3CB depletion inhibited invasion potential specifically via suppressing cell adhesion to collagen I in PAAD cells. Conclusion: Collectively, our findings indicate that PIK3CB is involved in PAAD metastasis through cell-matrix adhesion. We proposed that PIK3CB is a potential therapeutic target for PAAD therapy.

Wound healing is a complex biological process in which many molecules, including microRNA molecules, play an essential role in its regulation. It is well-established that reducing miR-155 expression can accelerate wound healing. This study investigated the effect of using nanoparticles loaded with vitamin C on miR-133, collagen I, and III expressions. In this study, first, nanoparticles of albumin protein were produced and then loaded with vitamin C. 3T3 mouse fibroblast cells were affected by these nanoparticles, and cell behavior was investigated to evaluate the toxicity and appropriate doses. In addition, the expression of collagen I and III genes was studied. The results showed that nanoparticles containing vitamin C in 20 µg/ml concentration had a positive effect on collagen I and III expressions compared to the control group. Moreover, we observed a decrease in the expression of miR-133 in comparison to the control group. Therefore, according to the results of this study, it can be argued that nanoparticles containing vitamin C can significantly decrease the expression of the miR-133 gene and lead to collagen I and III gene overexpression in fibroblasts cells, which is directly effective in wound healing.

Preeclampsia is a common obstetric disorder affecting 2-8% of pregnancy worldwide. Fibrosis is an important histological change occurring in preeclamptic placenta, and might depend on the excess deposition of collagen I. However, the role of fibrotic placenta and collagen I in the pathogenesis of preeclampsia remains unclear. Therefore, we analyzed the collagen deposition and the expression of Collagen I in human placenta by Masson staining, Sirius red staining and western blotting. Further, the role of collagen I in preeclampsia pathogenesis was studied in C57BL/6 mice. HTR-8/SVneo cells were used to investigate the mechanisms underlying the effects of collagen I in trophoblasts by transcriptome sequencing and pharmacological agonists. Human preeclamptic placenta exhibited a significantly higher degree of fibrosis in stem villi and terminal villi than normal placenta, and was characterized by collagen I deposition. In vivo, a single injection of collagen I on gestational day 0.5 led to an increase in systolic pressure of pregnant mice from gestational days 4.5-17.5, to a decrease in weight and number of embryos, and to enhanced placental collagen I expression and degree of fibrosis compared with control mice. In vitro, collagen I attenuated the proliferation and invasion of HTR-8SV/neo cells. This effect could be reversed by treatment with agonists of ERK and β-catenin. Moreover, transcriptome sequencing demonstrated that signaling pathways related to cell proliferation and invasion were significantly downregulated in HTR-8SV/neo cells. Thus, we propose that collagen I induced preeclampsia-like symptoms by suppressing the proliferation and invasion of trophoblasts through inhibition of the ERK phosphorylation and WNT/β-catenin signaling pathways. Our findings could pave the way to the discovery of small-molecule inhibitors for preeclampsia treatment and future studies with larger sample size are required. [ABSTRACT FROM AUTHOR]

Stimuli-responsive nanomaterials have the prospective to enable the fabrication of new extracellular matrix-like substrates with unique structures and cell-instructive capabilities. The development of biocompatible collagen substrates with on-demand ordered architectures is an open challenge since it is well-known that the directionality of the collagen fibres affects important cell behaviour, such as proliferation, differentiation, and ultimately, tissue regeneration. Here, an easy and cheap approach to fabricate anisotropic collagen-based substrates exhibiting cells-instructing ability was proposed. Paramagnetic iron oxide nanoparticles (IOPs) coated with polyethylene glycol were synthetized by a coprecipitation and solvothermic method and mixed with a collagen precursors solution. The suspension was then immersed within a static and low-intensity magnetic field to trigger the IOPs self-assembly. Guided by the external stimulus, IOPs assembled along the magnetic field lines into long filamentous structures within the collagen matrix. The solidification of the pre-cursors solution in the presence of filamentous IOPs’ structures promotes the collagen organization into ordered fashions. The obtained collagen substrate demonstrated good cytocompatibility and cells’ instructive properties. [ABSTRACT FROM AUTHOR]

Astrocyte scar formation after spinal cord injury (SCI) efficiently limits the accurate damage but physically restricts the following axon regeneration. Lately, fine tuning scar formation is becoming a novel strategy to develop SCI treatment, yet how to leverage these opposite effects remains challenging. Here, utilizing an improved drug administration approach, we show that in a mouse model of spinal cord injury, continual deletion of microglia, especially upon scar formation, by pexidartinib decreases the amount of microglia-derived collagen I and reforms the astrocyte scar. The astrocytes become less compacted in the scar, which permits axon regeneration and extension. Although continual microglia deletion did not significantly improve the locomotive performance of the SCI mice, it did ameliorate their weight loss, possibly by improving their relevant health conditions. We thus identified a novel approach to regulate astrocyte scars for improved axon regeneration, which is indicative of the clinical treatment of SCI patients. [ABSTRACT FROM AUTHOR]

Astrocyte scar formation after spinal cord injury (SCI) efficiently limits the accurate damage but physically restricts the following axon regeneration. Lately, fine tuning scar formation is becoming a novel strategy to develop SCI treatment, yet how to leverage these opposite effects remains challenging. Here, utilizing an improved drug administration approach, we show that in a mouse model of spinal cord injury, continual deletion of microglia, especially upon scar formation, by pexidartinib decreases the amount of microglia-derived collagen I and reforms the astrocyte scar. The astrocytes become less compacted in the scar, which permits axon regeneration and extension. Although continual microglia deletion did not significantly improve the locomotive performance of the SCI mice, it did ameliorate their weight loss, possibly by improving their relevant health conditions. We thus identified a novel approach to regulate astrocyte scars for improved axon regeneration, which is indicative of the clinical treatment of SCI patients.

The extracellular matrix surrounding the tumor undergoes changes in its organization during the metastasis process. The present study aims to quantify total collagen, collagen I (Col I) and collagen III (Col III), analyze the alignment of collagen fibers and assess the basement membrane integrity in samples from patients with metastatic and non-metastatic prostate cancer. Tissue samples from 60 patients were classified into groups based on prognostic parameters: better prognosis (n = 20), worse prognosis without metastasis (n = 23) and metastatic (n = 17). Picrosirius red with further analysis under polarizing microscope was used to quantify (with validation using immunohistochemistry) and analyze collagen alignment, and Periodic Acid Schiff staining was used to analyze the basement membrane integrity. The Col I/Col III ratio was found to be higher in the metastatic group than in the groups with better prognosis (p = 0.012) and worse prognosis without metastasis (p = 0.018). Basement membrane integrity constitution in malignant tumor tissue differed from that of adjacent non-tumor tissue (p < 0.001). Moreover, the worsening in the tumor tissue integrity was positively correlated with worse prognostic parameters. All in all, absence of Col III and basement membrane integrity might be indicators of poor prognosis in prostate cancer. [Display omitted] • Type III collagen (Col III) had lower deposition in metastatic prostate cancer (PCa) group. • Disruptions in basement membrane integrity were associated with PCa invasive parameters. • Col III and basement membrane are good predictive markers for a worse PCa prognosis. [ABSTRACT FROM AUTHOR]

The interactions between cell and cellular matrix confers plasticity to each body tissue, influencing the cellular migratory capacity. Macrophages rely on motility to promote their physiological function. These phagocytes are determinant for the control of invasive infections, and their immunological role largely depends on their ability to migrate and adhere to tissue. Therefore, they interact with the components of the extracellular matrix through their adhesion receptors, conferring morphological modifications that change their shape during migration. Nevertheless, the need to use in vitro cell growth models with the conditioning of three-dimensional synthetic matrices to mimic the dynamics of cell-matrix interaction has been increasingly studied. This becomes more important to effectively understand the changes occurring in phagocyte morphology in the context of infection progression, such as in Chagas disease. This disease is caused by the intracellular pathogen Trypanosoma cruzi, capable of infecting macrophages, determinant cells in the anti-trypanosomatid immunity. In the present study, we sought to understand how an in vitro extracellular matrix model interferes with T. cruzi infection in macrophages. Using different time intervals and parasite ratios, we evaluated the cell morphology and parasite replication rate in the presence of 3D collagen I matrix. Nevertheless, microscopy techniques such as scanning electron microscopy were crucial to trace macrophage-matrix interactions. In the present work, we demonstrated for the first time that the macrophage-matrix interaction favors T. cruzi in vitro replication and the release of anti-inflammatory cytokines during macrophage infection, in addition to drastically altering the morphology of the macrophages and promoting the formation of migratory macrophages.