RESUMO
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.
Assuntos
Crocus , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Extratos Vegetais , Fator A de Crescimento do Endotélio Vascular , Cicatrização , Cicatrização/efeitos dos fármacos , Crocus/química , Animais , Humanos , Extratos Vegetais/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Camundongos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Diabetes Mellitus Experimental/tratamento farmacológico , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cadeia alfa 1 do Colágeno Tipo I , Flores/química , Neovascularização Fisiológica/efeitos dos fármacos , MasculinoRESUMO
Ehlers-Danlos syndromes (EDS) represent a group of rare genetic disorders affecting connective tissues. Globally, approximately 1.5 million individuals suffer from EDS, with 10,000 reported cases in Canada alone. Understanding the histological properties of collagen in EDS has been challenging, but advanced techniques like atomic force microscopy (AFM) have opened up new possibilities for label-free skin imaging. This approach, which explores Type I collagen fibrils at the nanoscale, could potentially enhance EDS diagnosis and our knowledge of collagen type I-related connective tissue disorders. In the current study, we have employed AFM to examine ex-vivo skin biopsies from four individuals: one with classical EDS (cEDS), one with hypermobile EDS (hEDS), one with hEDS and Scleroderma (hEDS-Scleroderma), and one healthy control. Picrosirius red (PS) staining was used to highlight collagen differences in the samples. For each case, 14 images and 1400 force curves were obtained, with seven images and 700 force curves representing healthy collagen (PS-induced red staining) and the rest showcasing disrupted collagen (yellow staining). The results showed that PS staining was uniform throughout the control section, while cEDS and hEDS displayed localized areas of yellow staining. In the case of hEDS-Scleroderma, the yellow staining was widespread throughout the section. AFM images revealed irregular collagen fibrils in the disrupted, yellow-stained areas, contrasting with aligned and well-registered collagen fibrils in healthy, red-stained regions. Additionally, the study assessed the ability of non-AFM specialists to differentiate between healthy and disrupted collagen in AFM images, yielding substantial agreement among raters according to Fleiss's and Cohen's kappa scores (0.96 and 0.79±0.1, respectively). Biomechanical analysis revealed that normal healthy collagen exhibited a predominant population at 2.5 GPa. In contrast, EDS-affected collagen displayed subpopulations with lower compressive elastic modulus, indicating weaker collagen fibrils in EDS patients. Although these findings pertain to a limited number of cases, they offer valuable insights into the nanoscale collagen structure and biomechanics in individuals with EDS. Over time, these insights could be developed into specific biomarkers for the condition, improving diagnosis and treatment for EDS and related connective tissue disorders.
Assuntos
Síndrome de Ehlers-Danlos , Microscopia de Força Atômica , Síndrome de Ehlers-Danlos/patologia , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/genética , Humanos , Pele/patologia , Pele/metabolismo , Nanoestruturas/química , Feminino , Masculino , Colágeno/metabolismo , Adulto , Colágeno Tipo I/metabolismo , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Classifying diverse skin types is crucial for promoting skin health. However, efficiently identifying and analyzing relevant biomarkers from a vast array of available genetic data is challenging. Therefore, this study aimed to develop a precise and efficient platform for analyzing specific skin biomarkers using quantitative real-time PCR (qRT-PCR) with the minimal invasive skin sampling method (MISSM). MATERIALS AND METHODS: MISSM was used for RNA extraction from skin samples, followed by qRT-PCR analysis to quantify the expression of 20 biomarkers associated with skin characteristics (four biomarkers each for five skin characteristics). Noninvasive measurements from 299 Korean participants were utilized to correlate biomarker expression with skin parameters. Statistical analyses were conducted between biomarker expression levels and noninvasive skin measurements to select the relatively best-performing biomarker for each skin characteristic. RESULTS: Collagen type 1 alpha 1 (COL1A1) and moesin (MSN) were identified as skin aging biomarkers. Krüppel-like factor 4 (KLF4) and serine peptidase inhibitor Kazal type 5 (SPINK5) were identified as skin dryness biomarkers, whereas melan-A (MLANA) was selected as a biomarker for understanding pigmentation dynamics. Myelin protein zero like 3 (MPZL3) and high mobility group box 2 (HMGB2) were identified as markers of oily skin and skin sensitivity, respectively. Statistically significant correlations were found between the biomarker expression levels and noninvasive skin characteristic measurements. CONCLUSION: This study successfully developed a platform for the precise evaluation of individual skin characteristics using MISSM and qRT-PCR biomarker analysis. By selecting biomarkers that correlate with noninvasive measurements of skin characteristics, we demonstrated the platform's efficacy in assessing diverse skin conditions.
Assuntos
Biomarcadores , Fator 4 Semelhante a Kruppel , Reação em Cadeia da Polimerase em Tempo Real , Envelhecimento da Pele , Pele , Humanos , Biomarcadores/metabolismo , Biomarcadores/análise , Feminino , Masculino , Reação em Cadeia da Polimerase em Tempo Real/métodos , Pele/metabolismo , Adulto , Pessoa de Meia-Idade , Envelhecimento da Pele/genética , Envelhecimento da Pele/fisiologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Idoso , Adulto JovemRESUMO
BACKGROUND: Lung fibroblasts play a central role in maintaining lung homeostasis and facilitating repair through the synthesis and organization of the extracellular matrix (ECM). This study investigated the cross-talk between interleukin-1 alpha (IL-1α) and transforming growth factor-ß (TGF-ß) signaling, two key regulators in tissue repair and fibrosis, in the context of lung fibroblast repair in the healthy lung. RESULTS: Stimulation of lung fibroblasts with TGF-ß1 and TGF-ß2 induced collagen-I and fibronectin protein expression (p < 0.05), a response inhibited with co-treatment with IL-1α (p < 0.05). Additionally, TGF-ß1 and TGF-ß2 induced myofibroblast differentiation, and collagen-I gel contraction, which were both suppressed by IL-1α (p < 0.05). In contrast, interleukin (IL)-6, IL-8 and thymic stromal lymphopoietin induced by IL-1α, were unaffected by TGF-ß1 or TGF-ß2. Mechanistically, IL-1α administration led to the suppression of TGF-ß1 and TGF-ß2 signaling, through downregulation of mRNA and protein for TGF-ß receptor II and the downstream adaptor protein TRAF6, but not through miR-146a that is known to be induced by IL-1α. DISCUSSION: IL-1α acts as a master regulator, modulating TGF-ß1 and TGF-ß2-induced ECM production, remodeling, and myofibroblast differentiation in human lung fibroblasts, playing a vital role in balancing tissue repair versus fibrosis. Further research is required to understand the dysregulated cross-talk between IL-1α and TGF-ß signaling in chronic lung diseases and the exploration of therapeutic opportunities. METHODS: Primary human lung fibroblasts (PHLF) were treated with media control, or 1 ng/ml IL-1α with or without 50 ng/ml TGF-ß1 or TGF-ß2 for 1, 6 and 72 h. Cell lysates were assessed for the expression of ECM proteins and signaling molecules by western blot, miRNA by qPCR, mRNA by RNA sequencing and cell supernatants for cytokine production by ELISA. PHLFs were also seeded in non-tethered collagen-I gels to measure contraction, and myofibroblast differentiation using confocal microscopy.
Assuntos
Matriz Extracelular , Fibroblastos , Interleucina-1alfa , Pulmão , Transdução de Sinais , Fator de Crescimento Transformador beta1 , Humanos , Interleucina-1alfa/metabolismo , Interleucina-1alfa/genética , Matriz Extracelular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Pulmão/metabolismo , Pulmão/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/citologia , Diferenciação Celular , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fibronectinas/metabolismo , Fibronectinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta2RESUMO
PURPOSE: Prostate cancer (PCa) is a common malignancy in men, with an escalating mortality rate attributed to Recurrence and metastasis. Recent studies have illuminated collagen's critical regulatory role within the tumor microenvironment, significantly influencing tumor progression. Accordingly, this investigation is dedicated to examining the relationship between genes linked to collagen and the prognosis of PCa, with the objective of uncovering any possible associations between them. METHODS: Gene expression data for individuals with prostate cancer were obtained from the TCGA repository. Collagen-related genes were identified, leading to the development of a risk score model associated with biochemical recurrence-free survival (BRFS). A prognostic nomogram integrating the risk score with essential clinical factors was crafted and evaluated for efficacy. The influence of key collagen-related genes on cellular behavior was confirmed through various assays, including CCK8, invasion, migration, cell cloning, and wound healing. Immunohistochemical detection was used to evaluate PLOD3 expression in prostate cancer tissue samples. RESULTS: Our study identified four key collagen-associated genes (PLOD3, COL1A1, MMP11, FMOD) as significant. Survival analysis revealed that low-risk groups, based on the risk scoring model, had significantly improved prognoses. The risk score was strongly associated with prostate cancer prognosis. Researchers then created a nomogram, which demonstrated robust predictive efficacy and substantial clinical applicability.Remarkably, the suppression of PLOD3 expression notably impeded the proliferation, invasion, migration, and colony formation capabilities of PCa cells. CONCLUSION: The risk score, derived from four collagen-associated genes, could potentially act as a precise prognostic indicator for BRFS of patients. Simultaneously, our research has identified potential therapeutic targets related to collagen. Notably, PLOD3 was differentially expressed in cancer and para-cancer tissues in clinical specimens and it also was validated through in vitro studies and shown to suppress PCa tumorigenesis following its silencing.
Assuntos
Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo I , Nomogramas , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/mortalidade , Prognóstico , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Metaloproteinase 11 da Matriz/genética , Metaloproteinase 11 da Matriz/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Colágeno/metabolismo , Colágeno/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Microambiente Tumoral/genética , Idoso , Proliferação de Células/genética , Movimento Celular/genéticaRESUMO
Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC-MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.
Assuntos
Colágeno Tipo III , Colágeno Tipo I , Proteômica , Animais , Humanos , Cromatografia Líquida/métodos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/análise , Colágeno Tipo III/metabolismo , Colágeno Tipo III/análise , Formaldeído , Inclusão em Parafina/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Fixação de Tecidos/métodosRESUMO
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.
Assuntos
Aneurisma da Aorta Abdominal , Músculo Liso Vascular , Análise de Sequência de RNA , Análise de Célula Única , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/metabolismo , Animais , Camundongos , Análise de Célula Única/métodos , Humanos , Análise de Sequência de RNA/métodos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Macrófagos/metabolismo , Progressão da Doença , Fibroblastos/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Perfilação da Expressão Gênica/métodos , Comunicação Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismoRESUMO
BACKGROUND: Skin injuries have long been recognized as a prevalent type of physical injury. As a result, numerous research studies have been performed to discover an effective mechanism for wound healing. Therefore, tissue engineering of skin has developed as a potential solution for traditional methods of treating skin injuries. METHODS AND MATERIALS: Alginate/Chitosan hydrogel was mixed with 1, 10, 100, and 150 µM Obestatin, and evaluated the morphology, cumulative release, hemocompatibility and cytocompatibility, water absorption, cell viability, weight loss, and antibacterial characteristics of three-dimensional (3D) alginate (Alg) and chitosan (Cs) hydrogels during the process of wound curing. Various concentrations of Obestatin (Obes) were utilized for this purpose. Finally, the hydrogels that were made were tested on a full-thickness dermal wound in a Wistar rat model. The curative effects were determined by analyzing RNA expression and examining tissue stained with Masson's trichrome (MT) and hematoxylin-eosin (H&E). RESULTS: The biodegradability of this hydrogel was verified using weight loss testing, which demonstrated a reduction of around 90% after a period of 3 days. Furthermore, the MTT assay demonstrated that hydrogels have a beneficial effect on cell proliferation without inducing any harmful effects. Furthermore, the hydrogels produced demonstrated higher wound closure in vivo compared to the wounds treated with gauze (negative control group). Among the hydrogel groups, the chitosan/alginate/obestatin 100 µM group exhibited the apical percentage of wound closure, gene expression, and secondary epithelialization, but in 150 µM concentrations, we saw a lower rate of cell growth and proliferation and increase in hemolysis. In addition, RT-PCR analysis demonstrated that a concentration of 100 µM obestatin resulted in an upregulation in the expression of mRNA for vascular endothelial growth factor (VEGF), collagen type I & type III, and transforming growth factor-beta (TGF-ß). CONCLUSION: The present study suggests that 3D Alg/Cs hydrogels with a concentration of 100 µM obestatin have the potential for clinical application in the treatment of skin injuries.
Assuntos
Alginatos , Quitosana , Grelina , Hidrogéis , Pele , Fator de Crescimento Transformador beta1 , Fator A de Crescimento do Endotélio Vascular , Cicatrização , Animais , Humanos , Masculino , Ratos , Alginatos/farmacologia , Alginatos/química , Quitosana/química , Quitosana/farmacologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Grelina/farmacologia , Grelina/administração & dosagem , Hidrogéis/farmacologia , Ratos Wistar , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/lesões , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização/efeitos dos fármacosRESUMO
PURPOSE: Laser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells. METHODS: HCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0-2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining. RESULTS: The laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation. CONCLUSION: High-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.
Assuntos
Diferenciação Celular , Cemento Dentário , Lasers Semicondutores , Osteogênese , Humanos , Lasers Semicondutores/uso terapêutico , Diferenciação Celular/efeitos da radiação , Osteogênese/efeitos da radiação , Cemento Dentário/efeitos da radiação , Cemento Dentário/citologia , Fosfatase Alcalina/metabolismo , Células Cultivadas , Terapia com Luz de Baixa Intensidade/métodos , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismoRESUMO
Background: The revolution of orthopedic implant manufacturing is being driven by 3D printing of titanium implants for large bony defects such as those caused by diabetic Charcot arthropathy. Unlike traditional subtractive manufacturing of orthopedic implants, 3D printing fuses titanium powder layer-by-layer, creating a unique surface roughness that could potentially enhance osseointegration. However, the metabolic impairments caused by diabetes, including negative alterations of bone metabolism, can lead to nonunion and decreased osseointegration with traditionally manufactured orthopedic implants. This study aimed to characterize the response of both healthy and diabetic primary human osteoblasts cultured on a medical-grade 3D-printed titanium surface under high and low glucose conditions. Methods: Bone samples were obtained from six patients, three with Type 2 Diabetes Mellitus and three without. Primary osteoblasts were isolated and cultured on 3D-printed titanium discs in high (4.5 g/L D-glucose) and low glucose (1 g/L D-Glucose) media. Cellular morphology, matrix deposition, and mineralization were assessed using scanning electron microscopy and alizarin red staining. Alkaline phosphatase activity and L-lactate concentration was measured in vitro to assess functional osteoblastic activity and cellular metabolism. Osteogenic gene expression of BGLAP, COL1A1, and BMP7 was analyzed using reverse-transcription quantitative polymerase chain reaction. Results: Diabetic osteoblasts were nonresponsive to variations in glucose levels compared to their healthy counterparts. Alkaline phosphatase activity, L-lactate production, mineral deposition, and osteogenic gene expression remained unchanged in diabetic osteoblasts under both glucose conditions. In contrast, healthy osteoblasts exhibited enhanced functional responsiveness in a high glucose environment and showed a significant increase in osteogenic gene expression of BGLAP, COL1A1, and BMP7 (p<.05). Conclusion: Our findings suggest that diabetic osteoblasts exhibit impaired responsiveness to variations in glucose concentrations, emphasizing potential osteoblast dysfunction in diabetes. This could have implications for post-surgery glucose management strategies in patients with diabetes. Despite the potential benefits of 3D printing for orthopedic implants, particularly for diabetic Charcot collapse, our results call for further research to optimize these interventions for improved patient outcomes.
Assuntos
Diabetes Mellitus Tipo 2 , Glucose , Osteoblastos , Impressão Tridimensional , Titânio , Humanos , Titânio/farmacologia , Osteoblastos/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Células Cultivadas , Masculino , Fenótipo , Propriedades de Superfície , Feminino , Pessoa de Meia-Idade , Proteína Morfogenética Óssea 7/metabolismo , Osteogênese/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I/genética , IdosoRESUMO
Left atrial strain (LAS) measured by two-dimensional speckle tracking echocardiography (2DSTE) is considered to be a marker of LA structural remodeling, but it remains unsettled. We investigated the potential usefulness and clinical relevance of LAS to detect atrial remodeling including fibrosis by analyzing gene expression in cardiovascular surgery patients. Preoperative 2DSTE was performed in 131 patients (92 patients with sinus rhythm [SR] patients including paroxysmal AF [PAF], 39 atrial fibrillation [AF]) undergoing cardiovascular surgery. Atrial samples were obtained from the left atrial appendages, and mRNA expression level was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (24 PAF, 35 AF). Mean value of left atrial reservoir strain (mLASr) correlated with left atrial volume index (LAVI), and left atrial conduit strain (mLAScd). mLASr also correlated with left atrial contractile strain (mLASct) in SR patients including PAF. mLASr was significantly lower, and LAVI was higher, in the AF group, compared with SR patients including PAF. The expression of COL1A1 mRNA encoding collagen type I α1 significantly increased in AF patients (p = 0.031). mLASr negatively correlated with COL1A1 expression level, and multivariate regression analysis showed that mLASr was an independent predictor of atrial COL1A1 expression level, even after adjusting for age, sex, and BMI. But, neither mLAScd / mLASct nor LAVI (bp) correlated with COL1A1 gene expression. The expression level of COL1A1 mRNA strongly correlated with ECM-related genes (COL3A1, FN1). It also correlated ECM degradation-related genes (MMP2, TIMP1, and TIMP2), pro-fibrogenic cytokines (TGFB1 encoding TGFß1, END1, PDGFD, CTGF), oxidant stress-related genes (NOX2, NOX4), ACE, inflammation-related genes (NLRP, IL1B, MCP-1), and apoptosis (BAX). Among the fibrosis-related genes examined, univariable regression analysis showed that log (COL1A1) was associated with log (TGFB1) (adjusted R2 = 0.685, p<0.001), log (NOX4) (adjusted R2 = 0.622, p<0.001), log (NOX2) (adjusted R2 = 0.611, p<0.001), suggesting that TGFB1 and NOX4 was the potent independent determinants of COL1A1 expression level. mLASr negatively correlated with the ECM-related genes, and fibrosis-related gene expression level including TGFB1, NOX2, and NLRP3 in PAF patients. PAF patients with low mLASr had higher expression of the fibrosis-related gene expression, compared with those with high mLASr. These results suggest that LASr correlates with atrial COL1A1 gene expression associated with fibrosis-related gene expression. Patients with low LASr exhibit increased atrial fibrosis-related gene expression, even those with PAF, highlighting the utility of LAS as a marker for LA fibrosis in cardiovascular surgery patients.
Assuntos
Fibrilação Atrial , Remodelamento Atrial , Fibrose , Átrios do Coração , Humanos , Masculino , Feminino , Remodelamento Atrial/genética , Idoso , Pessoa de Meia-Idade , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/cirurgia , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Ecocardiografia , Cadeia alfa 1 do Colágeno Tipo I , Biomarcadores/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Função do Átrio EsquerdoRESUMO
One hundred eighty pairs of tissues of esophageal squamous cell carcinoma (ESCC) were tested by the transcriptome sequencing in order to explore etiology factors. The chi-square test and correlation analysis demonstrated that the relative expression levels of keratin 17 (KRT17) and collagen type I α1 chain (COL1A1) were significantly higher in EC with diabetes. Expression of KRT17 was correlated with blood glucose (r = 0.204, p = 0.001) and tumor size (r = -0.177, p = 0.038) in patients. COL1A1 correlated with age (r = -0.170, p = 0.029) and blood glucose levels (r = 0.190, p = 0.015). Experimental results of qRT-PCR: KRT17 and COL1A1 genes were highly expressed in ESCC (p < 0.05). When the two genes were used as a combination test, the positive detection rate of EC was 90.6%, and the ROC curve had greater power. The KRT17 and COL1A1 genes had the potential to be biomarkers for the diagnosis of ESCC.
Assuntos
Biomarcadores Tumorais , Cadeia alfa 1 do Colágeno Tipo I , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Queratina-17 , Humanos , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/metabolismo , Masculino , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , Queratina-17/genética , Queratina-17/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Idoso , Regulação Neoplásica da Expressão GênicaRESUMO
Activated hepatic stellate cells differentiate into myofibroblasts, which synthesize and secrete extracellular matrix (ECM) leading to liver fibrosis. It was previously demonstrated that bulleyaconitine A (BLA), an alkaloid from Aconitum bulleyanum, inhibits proliferation and promotes apoptosis of human hepatic Lieming Xu-2 (LX-2) cells. In this study, we analyzed the effect of BLA on the production of ECM and related proteins by LX-2 cells activated with acetaldehyde (AA). The cells were randomized into the control group, AA group (cells activated with 400 µM AA), and BLA+AA group (cells cultured in the presence of 400 µM AA and 18.75 µg/ml BLA). In the BLA+AA group, the contents of collagens I and III and the expression of α-smooth muscle actin and transforming growth factor-ß1 (TGF-ß1) were statistically significantly higher than in the control, but lower than in the AA group. Expression of MMP-1 in the BLA+AA group was also significantly higher than in the AA group, but lower than in the control. Expression of TIMP-1 in the BLA+AA group was significantly higher than in the control, but lower than in the AA group. Thus, BLA suppressed activation and proliferation of LX-2 cells by inhibiting TGF-ß1 signaling pathway and decreasing the content of collagens I and III by reducing the MMP-1/TIMP-1 ratio.
Assuntos
Acetaldeído , Aconitina , Actinas , Colágeno Tipo I , Matriz Extracelular , Células Estreladas do Fígado , Inibidor Tecidual de Metaloproteinase-1 , Fator de Crescimento Transformador beta1 , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Humanos , Acetaldeído/farmacologia , Acetaldeído/análogos & derivados , Aconitina/farmacologia , Aconitina/análogos & derivados , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/genética , Actinas/metabolismo , Actinas/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/genética , Linhagem Celular , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Proliferação de Células/efeitos dos fármacos , Aconitum/química , Cirrose Hepática/metabolismo , Cirrose Hepática/patologiaRESUMO
Purpose: Angiogenesis is a tightly controlled process that initiates the formation of new vessels and its dysfunction can lead to life-threatening diseases. Apoptotic extracellular vesicles (ApoEVs) have emerged as a proangiogenic agent with high safety and isolation efficiency profile, and ApoEVs from supernumerary tooth-derived pulp stem cells (SNTSC-ApoEVs) have their unique advantages with an easily accessible parental cell source and non-invasive cell harvesting. However, the detailed characteristics of SNTSC-ApoEVs are largely unknown. This study aimed to investigate the proangiogenic capacity and function molecule of SNTSC-ApoEVs. Methods: SNTSC-ApoEVs were isolated and characterized. In vitro effects of SNTSC-ApoEVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, wound healing, transwell, and tube formation assays. The mRNA and protein levels of proangiogenic genes were quantified by qRT-PCR, Western blot, and immunofluorescence analysis. A Matrigel plug model was established in 6-week-old male nu/nu mice for one week, and the in vivo impact of SNTSC-ApoEVs on micro-vessel formation was assessed by histological analysis. Proteomic analysis and RNA sequencing were performed to explore the active ingredients and underlying mechanisms. Results: SNTSC-ApoEVs enhanced the proliferation, migration, and angiogenesis of HUVECs in vitro. In the Matrigel plug model in vivo, SNTSC-ApoEVs promoted CD31-positive luminal structure formation. Apart from expressing general ApoEV markers, SNTSC-ApoEVs were enriched with multiple proteins related to extracellular matrix-cell interactions. Mechanistically, SNTSC-ApoEVs transferred COL1A1 to HUVECs and promoted endothelial functions by activating the PI3K/Akt/VEGF cascade. Conclusion: SNTSC-ApoEVs can promote angiogenesis by transferring the functional molecule COL1A1 and activating the PI3K/Akt/VEGF pathway, making SNTSC-ApoEVs a promising strategy for the treatment of angiogenesis-related diseases.
Assuntos
Apoptose , Colágeno Tipo I , Polpa Dentária , Vesículas Extracelulares , Células Endoteliais da Veia Umbilical Humana , Neovascularização Fisiológica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Dente Supranumerário , Fator A de Crescimento do Endotélio Vascular , Vesículas Extracelulares/química , Humanos , Polpa Dentária/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Neovascularização Fisiológica/fisiologia , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Camundongos , Colágeno Tipo I/metabolismo , Proliferação de Células , Células-Tronco/citologia , Células-Tronco/metabolismo , Transdução de Sinais , Camundongos Nus , Movimento Celular , AngiogêneseRESUMO
Spinal cord injury (SCI) leads to fibrotic scar formation at the lesion site, yet the heterogeneity of fibrotic scar remains elusive. Here we show the heterogeneity in distribution, origin, and function of fibroblasts within fibrotic scars after SCI in mice and female monkeys. Utilizing lineage tracing and single-cell RNA sequencing (scRNA-seq), we found that perivascular fibroblasts (PFs), and meningeal fibroblasts (MFs), rather than pericytes/vascular smooth cells (vSMCs), primarily contribute to fibrotic scar in both transection and crush SCI. Crabp2 + /Emb+ fibroblasts (CE-F) derived from meninges primarily localize in the central region of fibrotic scars, demonstrating enhanced cholesterol synthesis and secretion of type I collagen and fibronectin. In contrast, perivascular/pial Lama1 + /Lama2+ fibroblasts (LA-F) are predominantly found at the periphery of the lesion, expressing laminin and type IV collagen and functionally involved in angiogenesis and lipid transport. These findings may provide a comprehensive understanding for remodeling heterogeneous fibrotic scars after SCI.
Assuntos
Cicatriz , Fibroblastos , Fibrose , Laminina , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Cicatriz/patologia , Cicatriz/metabolismo , Camundongos , Feminino , Laminina/metabolismo , Meninges/patologia , Meninges/metabolismo , Fibronectinas/metabolismo , Modelos Animais de Doenças , Colágeno Tipo I/metabolismo , Camundongos Endogâmicos C57BL , Pericitos/metabolismo , Pericitos/patologia , Colágeno Tipo IV/metabolismo , Colesterol/metabolismoRESUMO
Due to the limitations of the current skin wound treatments, it is highly valuable to have a wound healing formulation that mimics the extracellular matrix (ECM) and mechanical properties of natural skin tissue. Here, a novel biomimetic hydrogel formulation has been developed based on a mixture of Agarose-Collagen Type I (AC) combined with skin ECM-related components: Dermatan sulfate (DS), Hyaluronic acid (HA), and Elastin (EL) for its application in skin tissue engineering (TE). Different formulations were designed by combining AC hydrogels with DS, HA, and EL. Cell viability, hemocompatibility, physicochemical, mechanical, and wound healing properties were investigated. Finally, a bilayered hydrogel loaded with fibroblasts and mesenchymal stromal cells was developed using the Ag-Col I-DS-HA-EL (ACDHE) formulation. The ACDHE hydrogel displayed the best in vitro results and acceptable physicochemical properties. Also, it behaved mechanically close to human native skin and exhibited good cytocompatibility. Environmental scanning electron microscopy (ESEM) analysis revealed a porous microstructure that allows the maintenance of cell growth and ECM-like structure production. These findings demonstrate the potential of the ACDHE hydrogel formulation for applications such as an injectable hydrogel or a bioink to create cell-laden structures for skin TE.
Assuntos
Materiais Biomiméticos , Hidrogéis , Engenharia Tecidual , Hidrogéis/química , Humanos , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Engenharia Tecidual/métodos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Cicatrização/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Dermatan Sulfato/química , Dermatan Sulfato/farmacologia , Fibroblastos/efeitos dos fármacos , Elastina/química , Matriz Extracelular/metabolismo , Biomimética/métodos , Sefarose/química , Derme/efeitos dos fármacos , Derme/metabolismo , Derme/citologia , AnimaisRESUMO
Liver fibrosis, a consequence of chronic liver damage or inflammation, is characterized by the excessive buildup of extracellular matrix components. This progressive condition significantly raises the risk of severe liver diseases like cirrhosis and hepatocellular carcinoma. The lack of approved therapeutics underscores the urgent need for novel anti-fibrotic drugs. Hepatic stellate cells (HSCs), key players in fibrogenesis, are promising targets for drug discovery. This study investigated the anti-fibrotic potential of Citrus hystrix DC. (KL) and its bioactive compound, ß-citronellol (ß-CIT), in a human HSC cell line (LX-2). Cells exposed to TGF-ß1 to induce fibrogenesis were co-treated with crude KL extract and ß-CIT. Gene expression was analyzed by real-time qRT-PCR to assess fibrosis-associated genes (ACTA2, COL1A1, TIMP1, SMAD2). The release of matrix metalloproteinase 9 (MMP-9) was measured by ELISA. Proteomic analysis and molecular docking identified potential signaling proteins and modeled protein-ligand interactions. The results showed that both crude KL extract and ß-CIT suppressed HSC activation genes and MMP-9 levels. The MAPK signaling pathway emerged as a potential target of ß-CIT. This study demonstrates the ability of KL extract and ß-CIT to inhibit HSC activation during TGF-ß1-induced fibrogenesis, suggesting a promising role of ß-CIT in anti-hepatic fibrosis therapies.
Assuntos
Monoterpenos Acíclicos , Células Estreladas do Fígado , Cirrose Hepática , Fator de Crescimento Transformador beta1 , Humanos , Actinas , Antifibróticos/farmacologia , Linhagem Celular , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Simulação de Acoplamento Molecular , Proteína Smad2/metabolismo , Proteína Smad2/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta1/farmacologia , Monoterpenos Acíclicos/farmacologiaRESUMO
BACKGROUND: Tianhe Zhuifeng Gao (TZG) is an authorized Chinese patent drug with satisfying clinical efficacy, especially for RA patients with cold-dampness syndrome. However, its underlying pharmacological mechanisms remain unclear. METHOD: Anti-arthritic effects of TZG were evaluated using an adjuvant-induced arthritis (AIA) rat model. Transcriptional regulatory network analysis based on synovial tissues obtained from AIA rats, combining with our previous analysis based on whole blood samples from RA patients with cold-dampness syndrome and co-immunoprecipitation were performed to identify involved dominant pathways, which were experimentally verified using AIA-wind-cold-dampness stimulation modified (AIA-M) animal model. RESULTS: TZG treatment dramatically attenuated joint injury and inflammatory response in AIA rats, and PSMC2-RUNX2-COL1A1 axis, which was closely associated with bone/cartilage damage, was inferred to be one of therapeutic targets of TZG against RA. Experimentally, TZG displayed obvious pharmacological effects for alleviating the joint inflammation and destruction through reinstating the body weight, reducing the arthritis score, the limbs diameters, the levels of RF and CRP, and the inflammatory cytokines, recovering the thymus and spleen indexes, diminishing bone and cartilage destruction, as well elevating the pain thresholds of AIA-M rats. In addition, TZG markedly reversed the abnormal energy metabolism in AIA-M rats through enhancing articular temperature, daily water consumption, and regulating expression levels of energy metabolism parameters and hormones. Moreover, TZG also significantly modulated the abnormal expression levels of PSMC2, RUNX2 and COL1A1 proteins in the ankle tissues of AIA-M rats. CONCLUSION: TZG may exert the bone protective effects in RA therapy via regulating bone and cartilage damage-associated PSMC2-RUNX2-COL1A1 axis.
Assuntos
Artrite Experimental , Artrite Reumatoide , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo I , Medicamentos de Ervas Chinesas , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Experimental/tratamento farmacológico , Ratos , Humanos , Masculino , Medicamentos de Ervas Chinesas/uso terapêutico , Medicamentos de Ervas Chinesas/farmacologia , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cartilagem/metabolismo , Cartilagem/patologia , Cartilagem/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Chondrogenesis is a complex cellular process that involves the transformation of mesenchymal stem cells (MSCs) into chondrocytes, the specialised cells that form cartilage. In recent years, three-dimensional (3D) culture systems have emerged as a promising approach to studying cell behaviour and development in a more physiologically relevant environment compared to traditional two-dimensional (2D) cell culture. The use of these systems provided insights into the molecular mechanisms that regulate chondrogenesis and has the potential to revolutionise the development of new therapies for cartilage repair and regeneration. This study demonstrates the successful application of Raman microspectroscopy (RMS) as a label-free, non-destructive, and sensitive method to monitor the chondrogenic differentiation of bone marrow-derived rat mesenchymal stem cells (rMSCs) in a collagen type I hydrogel, and explores the potential benefits of 3D hydrogels compared to conventional 2D cell culture environments. rMSCs were cultured on 3D substrates for 3 weeks and their differentiation was monitored by measuring the spectral signatures of their subcellular compartments. Additionally, the evolution of high-density micromass cultures was investigated to provide a comprehensive understanding of the process and complex interactions between cells and their surrounding extracellular matrix. For comparison, rMSCs were induced into chondrogenesis in identical medium conditions for 21 days in monolayer culture. Raman spectra showed that rMSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway at a significantly higher rate than the 2D culture cells. 3D cultures expressed stronger and more homogeneous chondrogenesis-associated peaks such as collagens, glycosaminoglycans (GAGs), and aggrecan while manifesting changes in proteins and lipidic content. These results suggest that 3D type I collagen hydrogel substrates are promising for in vitro chondrogenesis studies, and that RMS is a valuable tool for monitoring chondrogenesis in 3D environments.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Análise Espectral Raman , Análise Espectral Raman/métodos , Animais , Ratos , Células-Tronco Mesenquimais/citologia , Condrogênese , Hidrogéis/química , Células Cultivadas , Técnicas de Cultura de Células/métodos , Colágeno Tipo I/metabolismo , Colágeno Tipo I/química , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
BACKGROUND: Microneedles are tiny needles, typically ranging from tens to hundreds of micrometers in length, used in various medical procedures and treatments. The tested medical device named "CELLADEEP Patch" a dissolvable microneedle therapy system (MTS), made of hyaluronic acid and collagen. And the iontophoresis technique is also applied in the system. The study aimed to evaluate the effectiveness of the "CELLADEEP Patch" in skin improvement. METHODS: Ex vivo human-derived skin tissue models were used in this study and they were divided into three different groups, namely, the Untreated Group, the Negative Control Group, and the Test Group respectively. The Untreated Group received no treatment measures, the Negative Control Group was exposed to ultraviolet B radiation (UVB) irradiation, and the Test Group was exposed to UVB irradiation and treated with "CELLADEEP Patch". Skin moisture content, transdermal water loss, and skin elasticity were evaluated by three clinical devices. Additionally, histological staining and related mRNA expression levels were also analyzed. RESULTS: The results of skin moisture content, transdermal water loss, and skin elasticity evaluation consistently illustrated that the application of "CELLADEEP Patch" led to remarkable skin improvement. And the analysis of histological staining images also confirmed the effectiveness of the "CELLADEEP Patch", especially for increasing collagen density. Moreover, the upregulation of Collagen type 1 a (COL1A1) and hyaluronan synthase 3 mRNA expression and the decrease of Matrix metalloproteinase 1 (MMP-1) and Interleukin-1 beta (IL-1ß) mRNA expression reflected its wrinkle improvement, moisturizing and anti-inflammation function. CONCLUSION: "CELLADEPP Patch", the MTS combined with the iontophoresis technique, exhibits its effectiveness in moisturizing, skin elasticity improvement, and anti-inflammatory function when applied to ex vivo human-derived skin tissue models in experiments. The study has contributed to the understanding of the "CELLADEPP Patch" and laid the foundation for subsequent animal experiments and clinical trials.