RESUMEN

Minimally invasive transcatheter interventional therapy utilizing cardiac occluders represents the primary approach for addressing congenital heart defects and left atrial appendage (LAA) thrombosis. However, incomplete endothelialization and delayed tissue healing after occluder implantation collectively compromise clinical efficacy. In this study, we have customized a recombinant humanized collagen type I (rhCol I) and developed an rhCol I-based extracellular matrix (ECM)-mimetic coating. The innovative coating integrates metal-phenolic networks with anticoagulation and anti-inflammatory functions as a weak cross-linker, combining them with specifically engineered rhCol I that exhibits high cell adhesion activity and elicits a low inflammatory response. The amalgamation, driven by multiple forces, effectively serves to functionalize implantable materials, thereby responding positively to the microenvironment following occluder implantation. Experimental findings substantiate the coating's ability to sustain a prolonged anticoagulant effect, enhance the functionality of endothelial cells and cardiomyocyte, and modulate inflammatory responses by polarizing inflammatory cells into an anti-inflammatory phenotype. Notably, occluder implantation in a canine model confirms that the coating expedites reendothelialization process and promotes tissue healing. Collectively, this tailored ECM-mimetic coating presents a promising surface modification strategy for improving the clinical efficacy of cardiac occluders.

Asunto(s)

Materiales Biocompatibles Revestidos , Matriz Extracelular , Cicatrización de Heridas , Animales , Matriz Extracelular/metabolismo , Perros , Humanos , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Colágeno Tipo I/metabolismo , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Células Endoteliales de la Vena Umbilical Humana , Repitelización/efectos de los fármacos , Adhesión Celular/efectos de los fármacos

RESUMEN

G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9Cre/+ mice with G9afl/fl mice to generate G9afl/fl; Sox9Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development.

Asunto(s)

Diferenciación Celular , Proliferación Celular , N-Metiltransferasa de Histona-Lisina , Ratones Noqueados , Tendones , Animales , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Tendones/metabolismo , Tendones/embriología , Ratones , Tenocitos/metabolismo , Histonas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética

RESUMEN

BACKGROUND: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial. METHODS: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation. RESULTS: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor. CONCLUSIONS: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.

Asunto(s)

Factor de Crecimiento del Tejido Conjuntivo , Células Estrelladas Hepáticas , Lisofosfolípidos , Transducción de Señal , Esfingosina , Factores de Transcripción , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Humanos , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Línea Celular , Cirrosis Hepática/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Lisoesfingolípidos/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Vía de Señalización Hippo

RESUMEN

BACKGROUND: Elevated extracellular matrix (ECM) accumulation is a major contributing factor to the pathogenesis of fibrotic diseases. Recent studies have indicated that N6-methyladenosine (m6A) RNA modification plays a pivotal role in modulating RNA stability and contribute to the initiation of various pathological conditions. Howbeit, the precise mechanism by which m6A influences ECM deposition remains unclear. METHODS: In this study, we used hypertrophic scars (HTSs) as a paradigm to investigate ECM-related diseases. We focused on the role of ALKBH5-mediated m6A demethylation within the pathological progression of HTSs and examined its correlation with clinical stages. The effects of ALKBH5 ablation on ECM components were studied both in vivo and in vitro. Downstream targets of ALKBH5, along with their underlying mechanisms, were identified using integrated high-throughput analysis, RNA-binding protein immunoprecipitation and RNA pull-down assays. Furthermore, the therapeutic potential of exogenous ALKBH5 overexpression was evaluated in fibrotic scar models. RESULTS: ALKBH5 was decreased in fibroblasts derived from HTS lesions and was negatively correlated with their clinical stages. Importantly, ablation of ALKBH5 promoted the expression of COL3A1, COL1A1, and ELN, leading to pathological deposition and reconstruction of the ECM both in vivo and in vitro. From a therapeutic perspective, the exogenous overexpression of ALKBH5 significantly inhibited abnormal collagen deposition in fibrotic scar models. As determined by integrated high-throughput analysis, key ECM components including COL3A1, COL1A1, and ELN are direct downstream targets of ALKBH5. By means of its mechanism, ALKBH5 inhibits the expression of COL3A1, COL1A1, and ELN by removing m6A from mRNAs, thereby decreasing their stability in a YTHDF1-dependent manner. CONCLUSIONS: Our study identified ALKBH5 as an endogenous suppressor of pathological ECM deposition, contributing to the development of a reprogrammed m6A-targeted therapy for HTSs.

Asunto(s)

Desmetilasa de ARN, Homólogo 5 de AlkB , Matriz Extracelular , Fibrosis , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Matriz Extracelular/metabolismo , Fibrosis/metabolismo , Humanos , Ratones , Animales , Desmetilación , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/genética , Masculino , Cadena alfa 1 del Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo

RESUMEN

BACKGROUND: The AGEs levels in tissues of diabetics and elderly tend to be higher than in normal individuals. This study aims to determine the effects of AGEs on Achilles tendon repair. MATERIALS AND METHODS: Thirty-six male eight-week-old Sprague Dawley rats were selected in this study. The rats were randomly divided into two experimental groups and a control group after the transection of the Achilles tendon. During the tendon repair, the experimental groups were injected around the Achilles tendon with 350mmol/L (low dose group) and 1000mmol/L (high dose group) D-ribose 0.2 ml respectively to increase the AGEs level, while in the control group were given the same amount of PBS. The injections were given twice a week for six weeks. Collagen-I, TNF-α, and IL-6 expression in the healed Achilles tendon was assessed. Additionally, macroscopic, pathological, and biomechanical evaluations of Achilles tendon repair were conducted. RESULTS: The repaired Achilles tendons in the high dose group showed severe swelling and distinctive adhesions. The histological score went up with the increase of the AGEs in the Achilles tendon (p<0.001). TNF- α and IL-6 in the Achilles tendon increased (p<0.001, p<0.001), and the production of collagen-I decreased with the accumulation of AGEs in the repaired Achilles tendon (p<0.001). The tensile strength of Achilles tendon in the high dose group was impaired significantly. CONCLUSION: In current study, the compromised tendon repair model induced by AGEs was successfully established in rat. The study demonstrated that AGEs significantly impair Achilles tendon repair.

Asunto(s)

Tendón Calcáneo , Productos Finales de Glicación Avanzada , Ratas Sprague-Dawley , Traumatismos de los Tendones , Cicatrización de Heridas , Animales , Masculino , Tendón Calcáneo/lesiones , Tendón Calcáneo/patología , Tendón Calcáneo/metabolismo , Tendón Calcáneo/cirugía , Tendón Calcáneo/efectos de los fármacos , Productos Finales de Glicación Avanzada/metabolismo , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/fisiopatología , Ratas , Cicatrización de Heridas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Colágeno Tipo I/metabolismo , Interleucina-6/metabolismo , Modelos Animales de Enfermedad

RESUMEN

OBJECTIVE: To analyze the core functional component groups (CFCG) in Yinchenhao Decoction (YCHD) and their possible pathways for treating hepatic fibrosis based on network pharmacology. METHODS: 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-ß1-stimulated cells were analyzed using RT-qPCR and Western blotting. RESULTS: 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-ß1-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-ß1-induced LX-2 cells. CONCLUSION: 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.

Asunto(s)

Medicamentos Herbarios Chinos , Células Estrelladas Hepáticas , Cirrosis Hepática , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Medicina Tradicional China/métodos , Factor de Crecimiento Transformador beta1/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Ácidos Cumáricos/farmacología , Línea Celular , Transducción de Señal/efectos de los fármacos , Farmacología en Red , Cadena alfa 1 del Colágeno Tipo I

RESUMEN

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and limited treatment options. This study evaluates the prognostic value of stromal markers in TNBC, focusing on the tumor-stroma ratio (TSR) and overall stroma ratio (OSR) in whole slide images (WSI), as well as the expression of type-I collagen, type-III collagen, and fibrillin-1 on tissue microarrays (TMAs), using both visual assessment and digital image analysis (DIA). A total of 101 female TNBC patients, primarily treated with surgery between 2005 and 2016, were included. We found that high visual OSR correlates with worse overall survival (OS), advanced pN categories, lower stromal tumor-infiltrating lymphocyte count (sTIL), lower mitotic index, and patient age (p < 0.05). TSR showed significant connections to the pN category and mitotic index (p < 0.01). High expression levels of type-I collagen (>45%), type-III collagen (>30%), and fibrillin-1 (>20%) were linked to significantly worse OS (p = 0.004, p = 0.013, and p = 0.005, respectively) and progression-free survival (PFS) (p = 0.028, p = 0.025, and p = 0.002, respectively), validated at the mRNA level. Our results highlight the importance of stromal characteristics in promoting tumor progression and metastasis and that targeting extracellular matrix (ECM) components may offer novel therapeutic strategies. Furthermore, DIA can be more accurate and objective in evaluating TSR, OSR, and immunodetected stromal markers than traditional visual examination.

Asunto(s)

Biomarcadores de Tumor , Neoplasias de la Mama Triple Negativas , Humanos , Femenino , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/mortalidad , Pronóstico , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Anciano , Adulto , Células del Estroma/metabolismo , Células del Estroma/patología , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Fibrilina-1/metabolismo , Fibrilina-1/genética , Procesamiento de Imagen Asistido por Computador/métodos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Anciano de 80 o más Años

RESUMEN

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-ß1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

Asunto(s)

Colágeno Tipo I , Fibrina , Células Madre , Tendones , Andamios del Tejido , Factor de Crecimiento Transformador beta1 , Humanos , Tendones/citología , Tendones/metabolismo , Andamios del Tejido/química , Células Madre/metabolismo , Células Madre/citología , Fibrina/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tendinopatía/metabolismo , Tendinopatía/patología , Células Cultivadas , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Persona de Mediana Edad , Masculino , Supervivencia Celular/efectos de los fármacos , Ingeniería de Tejidos/métodos , Proteínas de la Membrana

RESUMEN

Extended exposure to UVB (280-315 nm) radiation results in oxidative damage and inflammation of the skin. Previous research has demonstrated that pilose antler extracts have strong anti-inflammatory properties and possess antioxidant effects. This study aimed to elucidate the mechanism of pilose antler protein in repairing photodamage caused by UVB radiation in HaCaT cells and ICR mice. Pilose antler protein (PAP) was found to increase the expression of type I collagen and hyaluronic acid in HaCaT cells under UVB irradiation while also inhibiting reactive oxygen species (ROS) production and oxidative stress in vitro. In vivo, the topical application of pilose antler protein effectively attenuated UVB-induced skin damage in ICR mice by reducing interleukin-1ß (IL-ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and inhibiting skin inflammation while alleviating UVB-induced oxidative stress. It was shown that pilose antler protein repaired UVB-induced photodamage through the MAPK and TGF-ß/Smad pathways.

Asunto(s)

Cuernos de Venado , Células HaCaT , Ratones Endogámicos ICR , Estrés Oxidativo , Especies Reactivas de Oxígeno , Piel , Rayos Ultravioleta , Rayos Ultravioleta/efectos adversos , Animales , Humanos , Cuernos de Venado/química , Ratones , Estrés Oxidativo/efectos de los fármacos , Piel/efectos de los fármacos , Piel/efectos de la radiación , Piel/patología , Piel/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Colágeno Tipo I/metabolismo , Ciervos , Ácido Hialurónico/farmacología , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Osteogenesis imperfecta (OI) is a disorder of low bone mass and increased fracture risk due to a range of genetic variants that prominently include mutations in genes encoding type I collagen. While it is well known that OI reflects defects in the activity of bone-forming osteoblasts, it is currently unclear whether OI also reflects defects in the many other cell types comprising bone, including defects in skeletal vascular endothelium or the skeletal stem cell populations that give rise to osteoblasts and whether correcting these broader defects could have therapeutic utility. Here, we find that numbers of skeletal stem cells (SSCs) and skeletal arterial endothelial cells (AECs) are augmented in Col1a2oim/oim mice, a well-studied animal model of moderate to severe OI, suggesting that disruption of a vascular SSC niche is a feature of OI pathogenesis. Moreover, crossing Col1a2oim/oim mice to mice lacking a negative regulator of skeletal angiogenesis and bone formation, Schnurri 3 (SHN3), not only corrected the SSC and AEC phenotypes but moreover robustly corrected the bone mass and spontaneous fracture phenotypes. As this finding suggested a strong therapeutic utility of SHN3 inhibition for the treatment of OI, a bone-targeting AAV was used to mediate Shn3 knockdown, rescuing the Col1a2oim/oim phenotype and providing therapeutic proof-of-concept for targeting SHN3 for the treatment of OI. Overall, this work both provides proof-of-concept for inhibition of the SHN3 pathway and more broadly addressing defects in the stem/osteoprogenitor niche as is a strategy to treat OI.

Asunto(s)

Modelos Animales de Enfermedad , Osteogénesis Imperfecta , Nicho de Células Madre , Animales , Ratones , Huesos/patología , Huesos/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Ratones Endogámicos C57BL , Osteogénesis/efectos de los fármacos , Osteogénesis Imperfecta/patología , Osteogénesis Imperfecta/genética , Células Madre/metabolismo , Células Madre/patología , Masculino , Femenino

RESUMEN

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.

Asunto(s)

Envejecimiento de la Piel , Piel , Humanos , Envejecimiento de la Piel/genética , Envejecimiento de la Piel/efectos de la radiación , Piel/metabolismo , Piel/efectos de la radiación , Adulto , Anciano , Persona de Mediana Edad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/genética , Regulación de la Expresión Génica/efectos de la radiación , Masculino , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Adulto Joven , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I/genética , Luz Solar

RESUMEN

AIMS: Tacrolimus is an effective immunosuppressant commonly used post-transplantation and in certain autoimmune diseases. However, its long-term administration is associated with renal fibrosis through transforming growth factor-beta/suppressor of mother against decapentaplegic (TGF-ß/Smad) signaling that could be partly attributed to endothelial dysfunction alongside decreased nitric oxide (NO) release. Our study aimed to investigate the prospective renal anti-fibrotic effect of enhanced NO production by nebivolol against tacrolimus-stimulated TGF-ß1/Smad3 signaling. MATERIALS AND METHODS: To illustrate the proposed mechanism of nebivolol, Nω-nitro-L-arginine methyl ester (L-NAME); nitric oxide synthase inhibitor; was co-administered with nebivolol. Rats were treated for 30 days as control, tacrolimus, tacrolimus/nebivolol, tacrolimus/L-NAME, and tacrolimus/nebivolol/L-NAME groups. KEY FINDINGS: Our results revealed that renal NO content was reduced in tacrolimus-treated rats, while treatment with tacrolimus/nebivolol enhanced NO content via up-regulated endothelial nitric oxide synthase (eNOS), but down-regulated inducible nitric oxide synthase (iNOS) expression. That participated in the inhibition of TGF-ß1/Smad3 signaling induced by tacrolimus, where the addition of L-NAME abolished the defensive effects of nebivolol. Subsequently, the deposition of collagen I and alpha-smooth muscle actin (α-SMA) was retarded by nebivolol, emphasized by reduced Masson's trichrome staining. In accordance, there was a strong negative correlation between eNOS and both TGF-ß1 and collagen I protein expression. The protective effects of nebivolol were further confirmed by the improvement in kidney function biomarkers and histological features. SIGNIFICANCE: It can be suggested that treatment with nebivolol along with tacrolimus could effectively suppress renal TGF-ß1/Smad3 fibrotic signaling via the enhancement of endothelial NO production, thus curbing renal fibrosis development.

Asunto(s)

Colágeno Tipo I , Riñón , Nebivolol , Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , Transducción de Señal , Proteína smad3 , Tacrolimus , Factor de Crecimiento Transformador beta1 , Animales , Nebivolol/farmacología , Proteína smad3/metabolismo , Óxido Nítrico/metabolismo , Tacrolimus/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Ratas , Óxido Nítrico Sintasa de Tipo III/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Riñón/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Colágeno Tipo I/metabolismo , Inmunosupresores/farmacología , Fibrosis , NG-Nitroarginina Metil Éster/farmacología , Ratas Wistar , Ratas Sprague-Dawley

RESUMEN

Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle (n = 8), or the anti-MBG antibody (n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC50 = 0.28 µmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.

Asunto(s)

Bufanólidos , Fibrosis , Ratas Sprague-Dawley , Insuficiencia Renal Crónica , Vasodilatación , Animales , Masculino , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismo , Vasodilatación/efectos de los fármacos , Ratas , Bufanólidos/farmacología , Aorta/efectos de los fármacos , Aorta/metabolismo , Anticuerpos/farmacología , Nefrectomía , Nitroprusiato/farmacología , Proteína Proto-Oncogénica c-fli-1/metabolismo , Colágeno Tipo I/metabolismo , Endotelina-1/metabolismo

RESUMEN

The present study investigated the effect of adding allopathic doxorubicin (DOX 0.3 µg/mL), the vehicle of ultradiluted/dynamized doxorubicin (0.2 % ethanol), different dynamizations of ultradiluted/dynamized doxorubicin (DOX 6CH, DOX 12CH and DOX 30CH), both in the absence or presence of chemical stress induced by doxorubicin at 0.3 µg/mL on follicular survival and activation, antioxidant capacity of the medium, Catalase activity (CAT), production of reactive protein thiol, maintenance of type I and III collagen fibers and accumulation of lipofuscin in porcine ovarian tissue cultured in vitro for 48 hours. To do this, part of the ovarian tissue fragments was fixed for the uncultured control and the rest were cultured in: MEM (cultured control), DOX 0.3 µg/mL, Ethanol, DOX 6CH, DOX 12CH, DOX 30CH, DOX (0.3 µg/mL) + DOX 6CH, DOX (0.3 µg/mL) + DOX 12CH, DOX (0.3 µg/mL) + DOX 30CH treatments. The results showed that, in general, ultradiluted/dynamized doxorubicin (DOX 6CH, DOX 12CH and DOX 30CH) mitigated the toxic effect of allopathic doxorubicin (0.3 µg/mL) on the morphology of preantral follicles, the content of type I and III collagen fibers, and the production of lipofuscin in the tissue. However, only DOX (0.3 µg/mL) + DOX 6CH attenuated the oxidative stress induced by DOX (0.3 µg/mL), maintaining adequate CAT activity that was similar to the uncultured control. Additionally, when the three isolated ultradiluted/dynamized doxorubicin were considered, only DOX 12CH increased the reduced thiol levels compared to the uncultured control and MEM. In conclusion, supplementing the culture medium with ultradiluted/dynamized DOX (DOX 6CH, DOX 12CH and DOX 30CH) attenuated the toxicity induced by allopathic doxorubicin during the in vitro culture of pig preantral follicles enclosed in ovarian tissue.

Asunto(s)

Antibióticos Antineoplásicos , Doxorrubicina , Folículo Ovárico , Animales , Doxorrubicina/toxicidad , Femenino , Porcinos , Antibióticos Antineoplásicos/toxicidad , Folículo Ovárico/efectos de los fármacos , Catalasa/metabolismo , Técnicas de Cultivo de Tejidos , Lipofuscina/metabolismo , Estrés Oxidativo/efectos de los fármacos , Antioxidantes/farmacología , Colágeno Tipo I/metabolismo , Ovario/efectos de los fármacos , Compuestos de Sulfhidrilo/metabolismo , Colágeno Tipo III/metabolismo

RESUMEN

BACKGROUND: Abdominal aortic aneurysm (AAA) is a complex vascular disorder characterized by the progressive dilation of the abdominal aorta, with a high risk of rupture and mortality. Understanding the cellular interactions and molecular mechanisms underlying AAA development is critical for identifying potential therapeutic targets. METHODS: This study utilized datasets GSE197748, GSE164678 and GSE183464 from the GEO database, encompassing bulk and single-cell RNA sequencing data from AAA and control samples. We performed principal component analysis, differential expression analysis, and functional enrichment analysis to identify key pathways involved in AAA. Cell-cell interactions were investigated using CellPhoneDB, focusing on fibroblasts, vascular smooth muscle cells (VSMCs), and macrophages. We further validated our findings using a mouse model of AAA induced by porcine pancreatic enzyme infusion, followed by gene expression analysis and co-immunoprecipitation experiments. RESULTS: Our analysis revealed significant alterations in gene expression profiles between AAA and control samples, with a pronounced immune response and cell adhesion pathways being implicated. Single-cell RNA sequencing data highlighted an increased proportion of pro-inflammatory macrophages, along with changes in the composition of fibroblasts and VSMCs in AAA. CellPhoneDB analysis identified critical ligand-receptor interactions, notably collagen type I alpha 1 chain (COL1A1)/COL1A2-CD18 and thrombospondin 1 (THBS1)-CD3, suggesting complex communication networks between fibroblasts and VSMCs. In vivo experiments confirmed the upregulation of these genes in AAA mice and demonstrated the functional interaction between COL1A1/COL1A2 and CD18. CONCLUSION: The interaction between fibroblasts and VSMCs, mediated by specific ligand-receptor pairs such as COL1A1/COL1A2-CD18 and THBS1-CD3, plays a pivotal role in AAA pathogenesis.

Asunto(s)

Aneurisma de la Aorta Abdominal , Músculo Liso Vascular , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/metabolismo , Animales , Ratones , Análisis de la Célula Individual/métodos , Humanos , Análisis de Secuencia de ARN/métodos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Macrófagos/metabolismo , Progresión de la Enfermedad , Fibroblastos/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Modelos Animales de Enfermedad , Masculino , Ratones Endogámicos C57BL , Perfilación de la Expresión Génica/métodos , Comunicación Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo

RESUMEN

Wound healing is a complex process orchestrated by interactions between a variety of cell types, including keratinocytes, fibroblasts, endothelial cells, inflammatory cells, and bioactive factors such as extracellular matrix (ECM) components, growth factors, and cytokines. Chronic wounds exhibit delayed proliferative phase initiation, reduced angiogenesis, impaired ECM synthesis, and persistent inflammatory response. Chronic wounds are one of the main challenges to the healthcare system worldwide, with a high cost for medical services. Hence, investigation of new approaches to accelerate wound healing is essential. Phytomedicines are considered as potential agents for improving the wound healing by accelerating epithelization, collagen synthesis, and angiogenesis. These natural compounds have various advantages including availability, ease of application, and high effectiveness in wound managment. This study aimed to investigate the biological effects of saffron or Crocus sativus L. (C. sativus) petal extract on cell survival, migration, and angiogenesis using MTT, scratch and in vitro tube formation assays. Moreover, the expression of collagen type I alpha 1 (COL1A1) and vascular endothelial growth factor (VEGF) were evaluated in human dermal fibroblasts (HDF)s and human umbilical vein endothelial cells (HUVEC)s, respectively. The effect of the C. sativus extract on the skin of diabetic mice was also monitored. The results showed that C. sativus petal extract promoted the viability and migration of HDFs and HUVECs. Moreover, C. sativus petal extract enhanced the formation of tube-like structures by HUVECs cultured on the Matrigel basement membrane matrix, indicating its potential to stimulate angiogenesis. Gene expression studies have shown the the C. sativus extract increases wound healing by upregulation of COL1A1 and VEGF, which are crucial factors involved in collagen deposition, epithelialization, and angiogenesis. Histological analysis revealed that C. sativus petal extract enhanced vascularity and increased the number of fibroblasts and collagen synthesis, ultimately accelerating wound closure compared to wounds treated with eucerin and commercial ointment in diabetic mice. Therefore, C. sativus petal extract has potential as a herbal treatment to improve the healing of diabetic wounds.

Asunto(s)

Crocus , Fibroblastos , Células Endoteliales de la Vena Umbilical Humana , Extractos Vegetales , Factor A de Crecimiento Endotelial Vascular , Cicatrización de Heridas , Cicatrización de Heridas/efectos de los fármacos , Crocus/química , Animales , Humanos , Extractos Vegetales/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Ratones , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Diabetes Mellitus Experimental/tratamiento farmacológico , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cadena alfa 1 del Colágeno Tipo I , Flores/química , Neovascularización Fisiológica/efectos de los fármacos , Masculino

RESUMEN

The clinical treatment of bone defects includes allogeneic bone transplantation and autologous bone transplantation. However, they all have their own limitations, and the scope of application is limited. In recent years, bone tissue engineering scaffolds based on a variety of materials have been well developed and achieved good bone regeneration ability. However, most scaffold materials always face problems such as high biotoxicity, leading to inflammation and poor bioactivity, which limits the bone regeneration effect and prolongs the bone regeneration time. In our work, we prepared hydroxyapatite, erythropoietin (EPO), and osteogenic growth peptide (OGP) codoped type-I collagen (Col I) polypeptide nanofiber membranes (NFMs) by electrostatic spinning. In cell experiments, the composite NFMs had low cytotoxicity and promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells. Quantitative real-time polymerase chain reaction and alkaline phosphatase staining confirmed the high expression of osteogenic genes, and alizarin red S staining directly confirmed the appearance of calcium nodules. In animal experiments, the loaded hydroxyapatite formed multiple independent mineralization centers in the defect center. Under the promotion of Col I, EPO, and OGP, the bone continued to grow along the mineralization centers as well as inward the defect edge, and the bone defect completely regenerated in about two months. The hematological and histological analyses proved the safety of the experiments. This kind of design to promote bone regeneration by simulating bone composition, introducing mineralization center and signal molecules, can shorten repair time, improve repair effect, and has good practical prospects in the future.

Asunto(s)

Regeneración Ósea , Colágeno Tipo I , Durapatita , Células Madre Mesenquimatosas , Nanofibras , Osteogénesis , Nanofibras/química , Animales , Regeneración Ósea/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/química , Colágeno Tipo I/farmacología , Colágeno Tipo I/genética , Osteogénesis/efectos de los fármacos , Durapatita/química , Durapatita/farmacología , Ratas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Diferenciación Celular/efectos de los fármacos , Andamios del Tejido/química , Ingeniería de Tejidos/métodos , Péptidos/farmacología , Péptidos/química , Eritropoyetina/química , Ratas Sprague-Dawley , Péptidos y Proteínas de Señalización Intercelular/farmacología , Masculino , Membranas Artificiales , Histonas

RESUMEN

Scleral hypoxia is considered a trigger in scleral remodeling-induced myopia. Identifying differentially expressed molecules within the sclera is essential for understanding the mechanism of myopia. We developed a scleral fibroblast hypoxia model and conducted RNA sequencing and bioinformatic analysis. RNA interference technology was then applied to knock down targeted genes with upregulated expression, followed by an analysis of COLLAGEN I protein level. Microarray data analysis showed that the expression of Adamts1 and Adamts5 were upregulated in fibroblasts under hypoxia (t-test, p < 0.05). Western blot analysis confirmed increased protein levels of ADAMTS1 and ADAMTS5, and a concurrent decrease in COLLAGEN I in hypoxic fibroblasts. The knockdown of either Adamts1 or Adamts5 in scleral fibroblasts under hypoxia resulted in an upregulation of COLLAGEN I. Moreover, a form-deprivation myopia (FDM) mouse model was established for validation. The sclera tissue from FDM mice exhibited increased levels of ADAMTS1 and ADAMTS5 protein and a decrease in COLLAGEN I, compared to controls. The study suggests that Adamts1 and Adamts5 may be involved in scleral remodeling induced by hypoxia and the development of myopia.

Asunto(s)

Proteína ADAMTS1 , Proteína ADAMTS5 , Western Blotting , Modelos Animales de Enfermedad , Fibroblastos , Ratones Endogámicos C57BL , Miopía , Esclerótica , Animales , Proteína ADAMTS1/metabolismo , Proteína ADAMTS1/genética , Esclerótica/metabolismo , Esclerótica/patología , Ratones , Miopía/metabolismo , Miopía/genética , Miopía/patología , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Fibroblastos/metabolismo , Fibroblastos/patología , Células Cultivadas , Hipoxia/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Masculino , Regulación de la Expresión Génica

RESUMEN

Osteogenesis imperfecta (OI), a rare genetic connective tissue disorder, primarily arises from pathogenic variants affecting the production or structure of collagen type I. In addition to skeletal fragility, individuals with OI may face an increased risk of developing ophthalmic diseases. This association is believed to stem from the widespread presence of collagen type I throughout various parts of the eye. However, the precise consequences of abnormal collagen type I on different ocular tissues remain unknown. Of particular significance is the sclera, where collagen type I is abundant and crucial for maintaining the structural integrity of the eye. Recent research on healthy individuals has uncovered a unique organizational pattern of collagen fibers within the sclera, characterized by fiber arrangement in both circular and radial layers around the optic nerve head. While the precise function of this organizational pattern remains unclear, it is hypothesized to play a role in providing mechanical support to the optic nerve. The objective of this study is to investigate the impact of abnormal collagen type I on the sclera by assessing the fiber organization near the optic nerve head in individuals with OI and comparing them to healthy individuals. Collagen fiber orientation of the sclera was measured using polarization-sensitive optical coherence tomography (PS-OCT), an extension of the conventional OCT that is sensitive to materials that exhibit birefringence (axial changes in light refraction). Birefringence was quantified and used as imaging contrast to extract collagen fiber orientation as well as the thickness of the radially oriented scleral layer. Three individuals with OI, exhibiting different degrees of disease severity, were assessed and analyzed, along with seventeen healthy individuals. Mean values obtained from individuals with OI were descriptively compared to those of the healthy participant group. PS-OCT revealed a similar orientation pattern of scleral collagen fibers around the optic nerve head between OI individuals and healthy individuals. However, two OI participants exhibited reduced mean birefringence of the radially oriented scleral layer compared to the healthy participant group (OI participant 1 oculus dexter et sinister (ODS): 0.34°/µm, OI participant 2: ODS 0.26°/µm, OI participant 3: OD: 0.29°/µm, OS: 0.28°/µm, healthy participants: ODS 0.38 ± 0.05°/µm). The radially oriented scleral layer was thinner in all OI participants although within ±2 standard deviations of the mean observed in healthy individuals (OI participant 1 OD: 101 µm, OS 104 µm, OI participant 2: OD 97 µm, OS 98 µm, OI participant 3: OD: 94 µm, OS 120 µm, healthy participants: OD 122.8 ± 13.6 µm, OS 120.8 ± 15.1 µm). These findings imply abnormalities in collagen organization or composition, underscoring the necessity for additional research to comprehend the ocular phenotype in OI.

Asunto(s)

Colágeno Tipo I , Osteogénesis Imperfecta , Esclerótica , Tomografía de Coherencia Óptica , Humanos , Osteogénesis Imperfecta/patología , Tomografía de Coherencia Óptica/métodos , Esclerótica/metabolismo , Esclerótica/patología , Adulto , Masculino , Femenino , Colágeno Tipo I/metabolismo , Adulto Joven , Disco Óptico/patología , Persona de Mediana Edad , Adolescente , Colágeno/metabolismo

RESUMEN

BACKGROUND: Transforming growth factor-beta1 (TGF-ß1)-mediated renal fibrosis is a critical pathological process of chronic kidney disease worsening to end-stage renal disease. Recent studies have shown that long noncoding RNA H19 (lncRNA H19) is widely involved in the formation and progression of fibrosis in multiple organs. However, its molecular events in renal fibrosis remain to be elucidated. METHODS: Rats were treated with adenine intragastrically and HK-2 cells were induced by TGF-ß1 to construct renal fibrosis models in vivo and in vitro, respectively. Renal histopathological examination was performed using HE and Masson staining. Gene expression levels of interleukin-1beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), TGF-ß1, fibronectin (Fn), alpha-smooth muscle actin (α-SMA), H19, let-7b-5p, TGF-ß receptor 1 (TGF-ßR1), and type I collagen (COL1A1) were detected by qRT-PCR. Immunohistochemistry, immunofluorescence, and western blot analysis were used to evaluate the expression of renal fibrosis biomarkers. Dual-luciferase reporter assay was used to verify the presence of binding sites between H19 and let-7b-5p, and between let-7b-5p and TGF-ßR1 and COL1A1. RESULTS: H19 was overexpressed in both in vivo and in vitro renal fibrosis models. H19 knockdown significantly reversed TGF-ß1-induced upregulation of fibronectin, COL1A1, and α-SMA and downregulation of E-cadherin in HK-2 cells, accompanied by an increase in let-7b-5p. Let-7b-5p was bound to H19 in HK-2 cells, and its overexpression inhibited TGF-ß1-induced HK-2 cell fibrosis. Further experiments determined that let-7b-5p directly targets TGF-ßR1 and COL1A1 in HK-2 cells. In addition, inhibition of let-7b-5p reversed the reduction in HK-2 cell fibrosis induced by H19 knockdown. Finally, knockdown of H19 alleviated renal fibrosis in vivo and was associated with regulation of the let-7b-5p/TGF-ßR1/COL1A1 axis. CONCLUSION: Our results indicate that knockdown of H19 inhibits renal tubular epithelial fibrosis by negatively regulating the let-7b-5p/TGF-ßR1/COL1A1 axis, which may provide new mechanistic insights into CRF progression.

Asunto(s)

Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo I , Fibrosis , MicroARNs , ARN Largo no Codificante , Ratas Sprague-Dawley , Receptor Tipo I de Factor de Crecimiento Transformador beta , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , MicroARNs/metabolismo , MicroARNs/genética , Humanos , Ratas , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Masculino , Colágeno Tipo I/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Línea Celular , Riñón/patología , Riñón/metabolismo , Transducción de Señal