Quercetin attenuates lactate production and extracellular matrix secretion in keratoconus.
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ABSTRACT: Keratoconus(KC) is an ecstatic corneal disease leading to corneal-thinning and the formation of a cone-like cornea. Elevated lactate levels, increased oxidative stress, and myofibroblast formation have all been previously reported. In the current study, we assess the role of Quercetin on collagen secretion and myofibroblast formation in KC in vitro. Human corneal fibroblasts(HCFs) and human keratoconus cells(HKCs) were treated with a stable Vitamin C derivative and cultured for 4 weeks, stimulating formation of a self-assembled extracellular matrix. All samples were analyzed using Western blots and targeted tandem mass spectrometry. Our data showed that Quercetin significantly down regulates myofibroblast differentiation and fibrotic markers, such as α-smooth muscle actin (α-SMA) and Collagen III (Col III), in both HCFs and HKCs. Collagen III secretion was reduced 80% in both HCFs and HKCs following Quercetin treatment. Furthermore, Quercetin reduced lactate production by HKCs to normal HCF levels. Quercetin down regulated TGF-βR2 and TGF-β2 expression in HKCs suggesting a significant link to the TGF-β pathway. These results assert that Quercetin is a key regulator of fibrotic markers and ECM assembly by modulating cellular metabolism and TGF-β signaling. Our study suggests that Quercetin is a potential therapeutic for treatment of corneal dystrophies, such as KC.
Project description:ObjectiveOsteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage damage. In order to find a safer and more effective drug to treat OA, we investigated the role of quercetin-3-O-β-D-glucuronide (Q3GA) in OA.MethodsWe used qRT-PCR and western blots to detect the effects of Q3GA on extracellular matrix (ECM) and inflammation related genes and proteins in interleukin-1β (IL-1β) induced chondrocytes. We determined the effect of Q3GA on the NF-κB pathway using western blots and immunofluorescence. Moreover, the effect of Q3GA on the Nrf2 pathway was evaluated through molecular docking, western blots, and immunofluorescence experiments and further validated by transfection with Nrf2 siRNA. Subsequently, we established a rat model of OA and injected Q3GA into the joint cavity for treatment. After 5 weeks of Q3GA administration, samples were obtained for micro-computed tomography scanning and histopathological staining to determine the effects of Q3GA on OA rats.ResultsWe found that Q3GA reduced the degradation of ECM and the expression of inflammatory related proteins and genes in primary chondrocytes of rats induced by IL-1β, as well as the expression of nitric oxide (NO) and reactive oxygen species (ROS). It inhibited the activation of the NF-κB pathway by increasing the expression of Nrf2 in the nucleus. In addition, Q3GA inhibited cartilage degradation in OA rats and promoted cartilage repair.ConclusionQ3GA attenuates OA by inhibiting ECM degradation and inflammation via the Nrf2/NF-κB axis.The translational potential of this articleThe results of our study demonstrate the promising potential of Q3GA as a candidate drug for the treatment of OA and reveal its key mechanisms.
Project description:Keratoconus (KC) is a common corneal ectatic disease that affects 1:500-1:2000 people worldwide and is associated with a progressive thinning of the corneal stroma that may lead to severe astigmatism and visual deficits. Riboflavin-mediated collagen crosslinking currently remains the only approved treatment to halt progressive corneal thinning associated with KC by improving the biomechanical properties of the stroma. Treatments designed to increase collagen deposition by resident corneal stromal keratocytes remain elusive. In this study, we evaluated the effects of arginine supplementation on steady-state levels of arginine and arginine-related metabolites (e.g., ornithine, proline, hydroxyproline, spermidine, and putrescine) and collagen protein expression by primary human corneal fibroblasts isolated from KC and non-KC (healthy) corneas and cultured in an established 3D in vitro model. We identified lower cytoplasmic arginine and spermidine levels in KC-derived constructs compared to healthy controls, which corresponded with overall higher gene expression of arginase. Arginine supplementation led to a robust increase in cytoplasmic arginine, ornithine, and spermidine levels in controls only and a significant increase in collagen type I secretion in KC-derived constructs. Further studies evaluating safety and efficacy of arginine supplementation are required to elucidate the potential therapeutic applications of modulating collagen deposition in the context of KC.
Project description:Drawing inspiration for biomaterials from biological systems has led to many biomedical innovations. One notable bioinspired device, Velcro, consists of two substrates with interlocking ability. Generating reversibly interlocking biomaterials is an area of investigation, as such devices can allow for modular tissue engineering, reversibly interlocking biomaterial interfaces, or friction-based coupling devices. Here, a biaxially interlocking interface generated using electrostatic flocking is reported. Two electrostatically flocked substrates are mechanically and reversibly interlocked with the ability to resist shearing and compression forces. An initial high-throughput screen of polyamide flock fibers with varying diameters and fiber lengths is conducted to elucidate the roles of different fiber parameters on scaffold mechanical properties. After determining the most desirable parameters via weight scoring, polylactic acid (PLA) fibers are used to emulate the ideal scaffold for in vitro use. PLA flocked scaffolds are populated with osteoblasts and interlocked. Interlocked flocked scaffolds improved cell survivorship under mechanical compression and sustained cell viability and proliferation. Additionally, the compression and shearing resistance of cell-seeded interlocking interfaces increased with increasing extracellular matrix deposition. The introduction of extracellular matrix-reinforced interlocking interfaces may serve as binders for modular tissue engineering, act as scaffolds for engineering tissue interfaces, or enable friction-based couplers for biomedical applications.
Project description:The hepatitis C viral (HCV) genome is translated through an internal ribosome entry site (IRES) as a single polyprotein precursor that is subsequently cleaved into individual mature viral proteins. Nonstructural protein 5A (NS5A) is one of these proteins that has been implicated in regulation of viral genome replication, translation from the viral IRES and viral packaging. We sought to identify cellular proteins that interact with NS5A and determine whether these interactions may play a role in viral production. Mass spectrometric analysis of coimmunoprecipitated NS5A complexes from cell extracts identified heat shock proteins (HSPs) 40 and 70. We confirmed an NS5A/HSP interaction by confocal microscopy demonstrating colocalization of NS5A with HSP40 and with HSP70. Western analysis of coimmunoprecipitated NS5A complexes further confirmed interaction of HSP40 and HSP70 with NS5A. A transient transfection, luciferase-based, tissue culture IRES assay demonstrated NS5A augmentation of HCV IRES-mediated translation, and small interfering RNA (siRNA)-mediated knockdown of HSP70 reduced this augmentation. Treatment with an inhibitor of HSP synthesis, Quercetin, markedly reduced baseline IRES activity and its augmentation by NS5A. HSP70 knockdown also modestly reduced viral protein accumulation, whereas HSP40 and HSP70 knockdown both reduced infectious viral particle production in an HCV cell culture system using the J6/JFH virus fused to the Renilla luciferase reporter. Treatment with Quercetin reduced infectious particle production at nontoxic concentrations. The marked inhibition of virus production by Quercetin may partially be related to reduction of HSP40 and HSP70 and their potential involvement in IRES translation, as well as viral morphogenesis or secretion.Quercetin may allow for dissection of the viral life cycle and has potential therapeutic use to reduce virus production with low associated toxicity.
Project description:Objective: To elucidate the age and sex-specific impact of the IFP secretome on aged chondrocytes, and uncover the exact mechanism driving these effects. To explore mediators of the IFP on chondrocytes, we performed mass-spectrometry proteomics of CM from young versus aged male IFP and mass spectrometry on aged male chondrocytes treated with CM of the same experimental groups. Network analysis interrogated age-dependent changes in communication between IFP-derived secreted proteins and chondrocytes. Results: Col2 and aggrecan expression were significantly increased in chondrocytes co-cultured with young IFP tissue compared to aged IFP tissue, and young CM compared to aged CM. There was no significant increase in SOX9 under any of the different conditions. Similar trends were seen in female experiments. Multi-omics results combined with network analysis suggested IFPs secrete succinate co-A ligase related factors that regulate Hox-dependent spliceosome gene expression in chondrocytes in an age-dependent manner. Conclusions: This study suggests that a young IFP secretome enhances ECM production by aged chondrocytes, in part through enhanced mitochondrial activity. However, this beneficial effect decreases with age.
Project description:Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.
Project description:Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.
Project description:AimTo identify changes in the expression of genes coding for extracellular matrix (ECM) proteins in patients with non-inflammatory corneal disorder keratoconus (KC), patients with corneal scarring, and normal controls.Materials and methodsTotal RNA extracted from corneal tissue of 13 KC patients, 2 patients with corneal scaring and 4 normal controls was analyzed using Human Extracellular Matrix & Adhesion Molecules Profiler PCR Array. Statistically significant changes in gene expression were identified using the Data Analysis software.ResultsComparison of KC and control corneas with thresholds of 1.5 or greater fold change and a p-value of 0.05 or lower, revealed 21 differentially expressed genes, 16 genes were downregulated and 5 were upregulated. Among transcripts downregulated in KC patients we identified THBS1, ADAMTS1, SPP1, several collagens and integrins. We found TGFBI (BIGH3) gene was the most significantly upregulated transcript.ConclusionDevelopment of keratoconus results in deregulation of gene expression of extracellular matrix and adhesion molecules.Clinical significanceDownregulation of collagens and upregulation of TGFBI repeatedly identified in KC patients may be used as clinical markers of the disease.
Project description:As the main loading-bearing tissue of eye, sclera exerts an important role in the pathophysiology of glaucoma. Intraocular pressure (IOP) generates mechanical strain on sclera. Recent studies have demonstrated that sclera, especially the peripapillary sclera, undergoes complicated remodelling under the mechanical strain. However, the mechanisms of the hypertensive scleral remodelling in human eyes remained uncertain. In this study, peripapillary human scleral fibroblasts (ppHSFs) were applied cyclic mechanical strain by Flexcell-5000™ tension system. We found that CXC- ligands and CXCR2 were differentially expressed after strain. Increased cell proliferation and inhibited cell motility were observed when CXCR2 was upregulated under the strain, whereas cell proliferation and motility did not have a significant change when CXCR2 was knocked down. CXCR2 could facilitate cell proliferation ability, modulate the mRNA and protein expressions of type I collagen and matrix metalloproteinase 2 via JAK1/2-STAT3 signalling pathway. In addition, CXCR2 might inhibit cell migration via FAK/MLC2 pathway. Taken together, CXCR2 regulated protein production and affected cell behaviours of ppHSFs. It might be a potential therapeutic target for the hypertensive scleral remodelling.
Project description:Epithelial-Mesenchymal Transition (EMT) is essential for tissue patterning and organization. It involves both regulation of cell motility and alterations in the composition and organization of the extracellular matrix (ECM); a complex environment of proteoglycans and fibrous proteins that promotes tissue homeostasis, regulates signaling in response to chemical and biomechanical stimuli and is often dysregulated in diseases such as cancer and fibrosis. Here, we demonstrate that Basonuclin-2 (BNC2), a mesenchymal-expressed gene that has been widely associated with cancer and developmental defects by genome-wide association study (GWAS), is a novel regulator of ECM composition and degradation. We find that at endogenous levels, BNC2 controls the expression of specific collagens, matrix metalloproteases and other matrisomal components in breast cancer cells, and in fibroblasts that are primarily responsible for the deposition and processing of the ECM within the tumour microenvironment. In so doing, BNC2 modulates the motile and invasive properties of cancers which likely explains the association of high BNC2 expression with increasing cancer grade and poor patient prognosis.