COL1A1 mRNA Research Articles

The lncRNA XIST/miR-29b-3p/COL3A1 axis regulates central carbon metabolism in head and neck squamous cell carcinoma and is associated with poor tumor prognosis.

Recent evidence shows that COL3A1 promotes the progression of many types of cancer. The purpose of our study is to explore the correlation between COL3A1 and the prognosis of patients with head and neck squamous cell carcinoma (HNSCC) and its potential mechanism. We initially screened the differentially expressed gene COL3A1 in The Cancer Genome Atlas (TCGA) database, and the association between the expression level of COL3A1, prognosis, and the clinical parameters of HNSCC patients was verified. A nomogram was constructed according to the multivariate analysis results. Next, a heatmap of COL3A1 co-expressed genes was constructed in TCGA database. The TargetScan database is used to explore the microRNAs (miRNA) related to COL3A1. The starBase database was used to explore and predict the long non-coding RNAs (lncRNAs) that the candidate miRNAs might bind to. Finally, the potential mechanism of action was investigated using Gene Set Enrichment Analysis (GSEA). COL3A1 expression is elevated in HNSCC tumor tissues, and HNSCC patients with high COL3A1 expression have worse prognostic factors. COL3A1 was positively correlated with the central carbon metabolism-related proteins: epidermal growth factor receptor (EGFR), phosphoglycerate mutase 1 (PGAM1), hexokinase 3 (HK3), and phosphofructokinase, platelet (PFKP). The TargetScan database showed that the best candidate miRNA for binding to the three prime untranslated region (3'UTR) end of COL3A1 mRNA was hsa-miR-29b-3p, which was negatively correlated with COL3A1. The starBase database showed that the lncRNA X Inactive Specific Transcript (lncRNA XIST) was the best candidate upstream non-coding RNA for regulating hsa-miR-29b-3p. GSEA showed that COL3A1 may be involved in the poor prognosis of HNSCC by participating in carbon metabolism, glucose metabolism, oxidative stress, and the Wingless-Type MMTV Integration Site Family (Wnt) and vascular endothelial growth factor A-vascular endothelial growth factor receptor 2 (VEGFA-VEGFR2) pathways. Low COL3A1 expression can be employed as a new HNSCC predictive biomarker, and the prognosis of HNSCC patients with lower COL3A1 expression can be greatly improved. At the same time, we found that the lncRNA XIST/miR-29b-3p/COL3A1 axis may regulate the central carbon metabolism of HNSCC and is associated with poor prognosis. These findings point to a potential target for developing HNSCC anticancer therapies.

P067 Fractal analysis of extracellular matrix as a new histological method for observer-independent quantification of intestinal fibrosis in Crohn’s Disease

Abstract Background The prevention of intestinal fibrosis formation is still an unsolved problem in the management of Crohn’s Disease (CD) as no specific antifibrotic therapies are available yet. Appropriate analysis of fibrosis severity is essential to assess the therapeutic efficacy of potential anti-fibrotic drugs. However, up to now, no standardized method exists, that allows for automated observer-independent histological evaluation of intestinal fibrosis severity. The aim of this study was thus to develop an objective and observer-independent histological method for the quantification of intestinal fibrosis in surgical specimens of Crohn’s patients using structural analysis of the extracellular matrix (ECM). Methods To quantify the structural complexity of the total ECM, fractal dimension analysis was performed using the FracLac plugin in FIJI/ImageJ in fibrotic and control sections of surgical specimens from patients with CD (n = 28) stained with Masson's trichrome. To selectively assess the structure of the collagen matrix, polarized light microscopy was used on Picrosirius red-stained sections. A score to quantify collagen fiber alignment and the color of the polarized light was established. As a reference, sections were scored by a pathologist using an established histological fibrosis score (0 - 4), the proportionate collagen fraction was measured and COL1A1 and COL3A1 mRNA expression was assessed. Results Fractal dimension as a measure for the structural complexity correlated significantly with the histological fibrosis score (r = .68, p < .0001), and differences in fractal dimension differed significantly between sections with different fibrosis scores, indicating that during fibrosis progression, there is not only an accumulation of ECM but also structural changes within the ECM network. Using Picrosirius polarized light microscopy, we found that sections with low fibrosis scores showed a meshwork-like alignment of the collagen fibers while sections with high fibrosis scores showed a parallelization of long collagen fibers. Fiber alignment (r =.71, p < .0001) positively correlated with the histological fibrosis score. All measurements were significantly higher in sections from the fibrotic focus compared to the resection margin. Conclusion Fractal dimension analysis was suitable for fibrosis quantification in histological samples from surgical specimen of CD patients. Picrosirius polarized light microscopy underlined our findings on structural changes of the ECM network during fibrosis progression in CD. Automated structural analysis of the ECM network using fractal analysis has the potential to serve as an observer-independent histological tool for fibrosis quantification in surgical specimens of patients with CD.

CXCL9 Links Skin Inflammation and Fibrosis through CXCR3-Dependent Upregulation of Col1a1 in Fibroblasts

Morphea is characterized by initial inflammation followed by fibrosis of the skin and soft tissue. Despite its substantial morbidity, the pathogenesis of morphea is poorly studied. Previous work showed that CXCR3 ligands CXCL9 and CXCL10 are highly upregulated in the sera and lesional skin of patients with morphea. We found that an early inflammatory subcutaneous bleomycin mouse model of dermal fibrosis mirrors the clinical, histological, and immune dysregulation observed in human morphea. We used this model to examine the role of the CXCR3 chemokine axis in the pathogenesis of cutaneous fibrosis. Using the REX3 (Reporting the Expression of CXCR3 ligands) mice, we characterized which cells produce CXCR3 ligands over time. We found that fibroblasts contribute the bulk of CXCL9-RFP and CXCL10-BFP by percentage, whereas macrophages produce high amounts on a per-cell basis. To determine whether these chemokines are mechanistically involved in pathogenesis, we treated Cxcl9-, Cxcl10-, or Cxcr3-deficient mice with bleomycin and found that fibrosis is dependent on CXCL9 and CXCR3. Addition of recombinant CXCL9 but not CXCL10 to cultured mouse fibroblasts induced Col1a1 mRNA expression, indicating that the chemokine itself contributes to fibrosis. Taken together, our studies provide evidence that CXCL9 and its receptor CXCR3 are functionally required for inflammatory fibrosis.

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts.

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleledby siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFβ1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.

Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy.

The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis

Osteoarthritis-related cartilage extracellular matrix remodelling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n=5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analysed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9% to 14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33% to 64%]). This resulted in an altered ribosome translation modus (e.g CrPV IRES, FC 3, 95% CI [2.2 to 4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3 to 2.0]). Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Collagen type VII α1 chain: A promising prognostic and immune infiltration biomarker of pancreatic cancer.

Pancreatic cancer (PC) is a stubborn malignancy with high lethality and a low 5-year overall survival (OS) rate. Collagen type VII α1 chain (COL7A1), a major component of the extracellular matrix, serves important roles in numerous physiological processes and various illnesses. COL7A1 protein acts as an anchoring fibril between the external epithelial cells and the underlying stroma, and mutation of COL7A1 could cause recessive dystrophic epidermolysis bullosa. Raw data for PC were acquired from The Cancer Genome Atlas and the Gene Expression Omnibus database, and raw data for the normal pancreas were obtained from the Genotype-Tissue Expression database. COL7A1 mRNA expression in PC tissues was compared with that in either paired (GSE15471 dataset) or unpaired (all other data) normal pancreas tissues. The association between COL7A1 mRNA expression and clinicopathological factors was assessed using logistic regression analysis. Cox analysis and Kaplan-Meier analysis were used to evaluate the role of COL7A1 mRNA expression in prognosis and nomograms were constructed. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, Gene Set Enrichment Analysis (GSEA) and single-sample GSEA (ssGSEA) were performed to evaluate the relevant functions of COL7A1 and correlation with immune cell infiltration. Furthermore, reverse transcription-quantitative PCR was used to assess the mRNA expression levels of COL7A1 in PC. The present study demonstrated that COL7A1 mRNA expression was higher in PC tissues compared with in normal pancreas tissues. The Kaplan-Meier survival analysis indicated that patients with PC with high COL7A1 mRNA expression had shorter overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) times compared with patients with PC with low COL7A1 mRNA expression. Multivariate analysis demonstrated that COL7A1 mRNA expression was an independent risk factor for OS, DSS and PFI. Nomogram and calibration plots were constructed to predict the prognosis of patients with PC. GSEA demonstrated that high mRNA expression levels of COL7A1 were associated with multiple cancer-related pathways. ssGSEA analysis indicated that COL7A1 expression was positively associated with natural killer CD56bright cells and T helper (Th)2 cells, and negatively associated with Th17 cells and eosinophils. The results of the present study suggested that COL7A1 could be an independent biomarker and an influential moderator of immune infiltration in PC.

Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing

Maturation of the 3′ end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3′ untranslated regions (UTRs). The 3′UTR contains motifs that regulate mRNA metabolism; thus, changing the 3′UTR length via APA can significantly affect gene expression. Endochondral ossification is a central process in bone healing, but the impact of APA on gene expression during this process is unknown. Here, we report the widespread occurrence of APA, which impacts multiple pathways that are known to participate in bone healing. Importantly, the progression of endochondral ossification involves global 3′UTR shortening, which is coupled with an increased abundance of shortened transcripts relative to other transcripts; these results highlight the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of transcripts that undergo APA in the fracture callus revealed an intricate regulatory network in which APA enhances the expression of the collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA exerts this effect by shortening the 3′UTRs of the Col1a1 and Col1a2 mRNAs, thus removing the binding sites of miR-29a-3p, which would otherwise strongly promote the degradation of both transcripts. Taken together, our study is the first to characterize the crucial roles of APA in regulating the 3′UTR landscape and modulating gene expression during fracture healing.

Monocyte Chemoattractant Protein-1-Supplemented Plasma Enhances Adiponectin and Adipogenesis-Related Gene Expression.

Increasing adipogenesis has been explored to treat metabolic diseases and atherosclerosis through the release of adiponectin. The effects and mechanism of platelet-rich plasma treatment on fat graft survival and adipogenesis have not been clarified. Here, we aimed to study the effects of monocyte chemoattractant protein-1 (MCP-1)-supplemented plasma on adipogenesis-related gene expression and adiponectin levels. Stromal vascular fractions (SVFs) purified from the inguinal adipose tissue of obese and diabetic (Leprdb/db) mice were treated with plasma from control (Lepr+/+) mice supplemented with 10 or 50 ng of MCP-1. The expression of adiponectin and interleukin-33 (IL-33) mRNA in adipose tissue was increased in Leprdb/db mice, whereas control (Lepr+/+) plasma reduced expression of IL-33 mRNA as well as peroxisome proliferator-activated receptor gamma (PPARγ), pJNK, and pNF-κB protein, and increased the expression of IL-10 mRNA in SVFs of Leprdb/db mice. MCP-1-supplemented control plasma increased the expression of adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), dipeptidyl peptidase 4 (DPP4), IL-33, and PDGFα mRNA and the expression of adiponectin protein as well as PPARγ of SVFs and the expression of PPARγ mRNA in adipose tissue macrophages (ATMs). Injection of MCP-1-supplemented plasma into adipose tissue of Leprdb/db mice increased the expression of IL-33 and Col3a1 mRNA in SVFs and IL-33, FABP4, PDGFα, PPARγ and PPARγ2 of ATMs, protein expression of adiponectin and PPARγ of SVFs, and plasma adiponectin levels, as well as DPP4 activity. In conclusion, our results demonstrate that control plasma decreases adipogenesis and increases IL-10, and decreases IL-33, pJNK, and pNF-κB in adipose tissue. MCP-1-supplemented plasma enhances adipogenesis-related gene expression in SVFs and adiponectin levels, which may be mediated through an increase of IL-33 and PPARγ. Thus, our findings suggest that MCP-1-supplemented plasma represents a novel therapy to stimulate local adipogenesis and systemic adiponectin levels.

Vascular smooth muscle cell phenotype is modulated by ligands of the lymphotoxin β receptor and the tumor necrosis factor receptor

ObjectiveVascular smooth muscle cells (VSMCs) undergo a phenotypic-switching process during the generation of unstable atheroma plaques. In this investigation, the potential implication of the tumor necrosis factor superfamily (TNFSF) ligands, in the gene expression signature associated with VSMC plasticity was studied. Material and methodsHuman aortic (ha)VSMCs were obtained commercially and treated with the cytokine TNFSF14, also called LIGHT, the lymphotoxin alpha (LTα), the heterotrimer LTα1β2 or with vehicle for 72h. The effect of the different treatments on gene expression was analyzed by quantitative PCR and included the study of genes associated with myofibroblast-like cell function, osteochondrogenesis, pluripotency, lymphorganogenesis and macrophage-like cell function. ResultsHaVSMCs displayed a change in myofibroblast-like cell genes which consisted in reduced COL1A1 and TGFB1 mRNA levels when treated with LTα or LIGHT and with augmented MMP9 expression levels when treated with LTα. LTα and LIGHT treatments also diminished the expression of genes associated with osteochondrogenesis and pluripotency SOX9, CKIT, and KLF4. By contrary, all the above genes were no affected by the treatment with the trimer LTα1β2. In addition, haVSMC treatment with LTα, LTα1β2 and LIGHT altered lymphorganogenic cytokine gene expression which consisted of augmented CCL20 and CCL21 mRNA levels by LTα and a reduction in the gene expression of CCL21 and CXCL13 by LIGHT and LTα1β2 respectively. Neither, LTα or LIGHT or LTα1β2 treatments affected the expression of macrophage-like cell markers in haVSMC. ConclusionsAltogether, indicates that the TNFSF ligands through their interconnected network of signaling, are important in the preservation of VSMC identity against the acquisition of a genetic expression signature compatible with functional cellular plasticity.

Hyperin up-regulates miR-7031-5P to promote osteogenic differentiation of MC3T3-E1 cells.

To investigate the effects of Hyperin (Hyp) on osteogenic differentiation of MC3T3-E1 cells. Differentially expressed miRNA was screened by miRNA Microarray. miR-7031-5P overexpression and knockdown MC3T3-E1 cell models were constructed by transfecting miR-7031-5P mimics and inhibitor. Alizarin red staining (ARS) assay was used to observe the formation of mineralized nodules in MC3T3-E1 cells. ALP activity was detected by using ALP detection kit. Western blot assay was used to examine the changes in osteogenic differentiation-related proteins. The relationship between miR-7031-5P and Wnt7a was revealed by dual luciferase report experiments. We found that miR-7031-5P was up-regulated in MC3T3-E1 cells after Hyp treatment. The results indicated that compared with the untreated group, Hyp promoted the formation of mineralized nodules and the alkaline phosphatase (ALP) activity of MC3T3-E1 cells via overexpressing miR-7031-5P. Besides, elevated miR-7031-5P increased OPN, COL1A1, and Runx2 mRNA expression. More importantly, Wnt7a was identified as the downstream target gene of miR-7031-5P promoting osteogenic differentiation of MC3T3-E1 cells. Hyp up-regulated miR-7031-5P to promote osteogenic differentiation of MC3T3-E1 cells by targeting Wnt7a.

Effects of ‘NEOGEN V.BIOME LIPOSOME™’ on the Increased Expression of COL1A1 and HAS2 mRNA in Human Dermal Fibroblasts

Purpose: This study investigated functional cosmetic agent for anti-wrinkle effects of 'NEOGEN V.BIOME LIPOSOME™', which is a liposome in the form of a proprietary technology including a vitamin C active ingredient, on human dermal fibroblasts (HDFs).Methods: The cytotoxicity of 'NEOGEN V.BIOME LIPOSOME™' was measured by performing a water-soluble tetrazolium salt (WST-1) assay. The relative mRNA expression of collagen (COL1A1 ) and hyaluronan synthase (HAS2) was measured by quantitative real-time PCR (qRT-PCR) assay.Results: HDF cell viability of 'NEOGEN V.BIOME LIPOSOME™' was no cytotoxic effect at concentrations below 0.25%. The expression of COL1A1 and HAS2 mRNA was increased in a concentration-dependent manner compared with the negative control group.Conclusion: “NEOGEN V.BIOME LIPOSOME™“ helps to increase collagen and hyaluronan synthase production by promoting COL1A1 and HAS2 mRNA expression in HDFs; therefore, it is expected to function as an anti-wrinkle cosmetic material.

Caffeic Acid Phenethyl Ester Loaded PEG-PLGA Nanoparticles Enhance Wound Healing in Diabetic Rats.

Delayed wound healing is a serious complication of diabetes and a main reason for foot amputation. Caffeic acid phenethyl ester (CAPE) is a main active constituent of honeybee propolis with reported appealing pharmacological activities. In the current study, CAPE was loaded onto PEG-PLGA nanoparticles and showed a particle size of 198 ± 7.3 nm and polydispersity index of 0.43 ± 0.04. An in vivo study was performed to appraise the wound-healing activity of CAPE-loaded PEG-PLGA nanoparticles (CAPE-NPs) in diabetic rats. Wound closure was significantly accelerated in rats treated with CAPE-NPs. This was confirmed via histological examinations of skin tissues that indicated expedited healing and enhanced collagen deposition. This was accompanied by observed antioxidant activity as evidenced by the prevention of lipid peroxidation and the exhaustion of superoxide dismutase (SOD) and catalase (CAT) activities. In addition, CAPE-NPs showed superior anti-inflammatory activity as compared with the regular formula of CAPE, as they prevented the expression of interleukin-6 (IL-6) as well as tumor necrosis-alpha (TNF-α). The pro-collagen actions of CAPE-NPs were highlighted by the enhanced hyroxyproline content and up-regulation of Col 1A1 mRNA expression. Furthermore, the immunohistochemial assessment of skin tissues indicated that CAPE-NPs enhance proliferation and angiogenesis, as shown by the increased expression of transforming growth factor β1 (TGF-β1) and platelet-derived growth factor subunit B (PDGF-B). In conclusion, CAPE-loaded PEG-PLGA nanoparticles possess potent healing effects in diabetic wounds. This is mediated, at least partially, by its antioxidant, anti-inflammatory, and pro-collagen as well as angiogenic activities.

Acteoside Derived from Cistanche Improves Glucocorticoid-Induced Osteoporosis by Activating PI3K/AKT/mTOR Pathway

Objective Glucocorticoids are widely used in clinical practice; however, they can cause side effects, such as osteoporosis. Acteoside (ACT) from Cistanche has been used to combat a variety of diseases. The study was conducted to evaluate the efficacy of ACT in glucocorticoid-induced osteoporosis (GIOP) and its potential mechanism. Methods Dexamethasone (Dex) was injected intramuscularly to induce osteoporosis in a rat model, and ACT was given orally. ACT was supplemented in vivo in Dex-stimulated osteoblastic MC3T3-E1 cells. RT-qPCR was performed to assess the mRNA levels of bone formation (Runx2, CoL1A1), and bone resorption (OPG and RANKL). A commercial ELISA kit was applied to assess serum OC and CTX levels. Western blot was performed to assess protein levels in the PI3K/AKT/mTOR signaling pathway. CCK-8 assay and flow cytometry were performed to assess osteoblast viability and apoptosis. Results ACT reduced Dex-induced bone microstructure deterioration, increased serum levels of OC, and decreased the levels of CTX (P < 0.05). In the MC3T3-E1 cells, Dex inhibited cell viability and promoted apoptosis; however, this effect was greatly attenuated by ACT (P < 0.05). Concurrently, ACT reversed the reduction in Runx2, osterix, CoL1A1, and OPG mRNA levels, ALP activity, and the promotion of RANKL by Dex. Additionally, ACT attenuated Dex-induced inhibition of p-AKT/AKT, p-mTOR/mTOR, and p-PI3K/PI3K protein levels by Dex (P < 0.05), while the PI3K/AKT/mTOR pathway inhibitor LY294002 diminished the potential effect of ACT (P < 0.05). Conclusion ACT from Cistanche may exert osteoprotective effects by activating the PI3K/AKT/mTOR signaling pathway to alleviate Dex-induced osteoporosis.

Scleral Remolding-Related Gene Expression After Scleral Collagen Cross-Linking Using Ultraviolet A and Riboflavin in Myopic Guinea Pig Model

Purpose This study was conducted to evaluate scleral remolding-related gene expression after scleral collagen cross-linking (SCXL) using ultraviolet A (UVA) and riboflavin in lens-induced myopia (LIM) guinea pigs. Methods A total of 100 4-week-old pigmented guinea pigs were randomly divided into five groups (n = 20): SCXL + LIM, LIM, SCXL, Sham, and Control. Refraction, anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were measured using streak retinoscope and A-scan ultrasonography. SCXL was performed using 0.1% riboflavin solution and 365 nm UVA irradiation. Lens-induced myopia was achieved by wearing −10 D concave lenses. Quantitative real-time PCR (qPCR) and western blot were used to measure mRNA and protein levels, respectively. Results Myopia was successfully induced in the LIM group, while myopic refraction was higher and ACD and AL were shorter in SCXL + LIM compared with LIM, suppressing myopia progression. The scleral COL1A1 mRNA levels were significantly decreased and MMP2 and ACTA2 mRNA levels were significantly increased in LIM compared with other groups, while COL1A1 mRNA levels were increased and MMP2 and ACTA2 mRNA levels were decreased in SCXL + LIM compared with LIM. The scleral COL1A1 protein levels were significantly increased at 1 week and 4 weeks and MMP2 protein levels were significantly decreased at 1 week in SCXL compared with SCXL + LIM, LIM and Control. MMP2 protein levels were significantly decreased in SCXL + LIM and SCXL compared with LIM at 4 weeks. The differences in TGFB1, BMP2, CCN2, ITGA2, and ITGB1 mRNA levels and ACTA2 protein levels between the five groups were not significantly different. Conclusion SCXL using UVA and riboflavin could influence the expression of scleral remolding-related genes, including COL1A1, MMP2, TIMP2, and ACTA2, and thus contribute to improving collagen synthesis and reducing collagen degradation and might have an effect on slowing myopia progression.

Investigating the use of translational readthrough inducing drugs in keratocytes from a mouse model of brittle cornea syndrome

AbstractPurpose: Brittle Cornea Syndrome (BCS) is a rare recessive condition characterized by extreme thinning of the cornea. The collagen‐rich corneal stroma accounts for 90% of corneal thickness in humans, and is a major determinant of visual acuity. BCS results from homozygous loss‐of‐function mutations in ZNF469 (BCS1) or PRDM5 (BCS2). Our mouse model of BCS1, caused by a premature termination codon (PTC) mutation in Zfp469, showed that the stroma is thinner due to of decreased type 1 collagen expression1. To elucidate disease mechanisms and test potential therapies, we established a primary keratocyte disease‐in‐a‐dish model of BCS1. Translational readthrough inducing drugs (TRIDs) can restore full‐length proteins truncated by PTC and are a potential treatment for 20% of BCS patients with PTC mutations. Primary keratocytes from our BCS1 mouse model were treated with TRIDs to assess their efficacy in improving the BCS phenotype in vitro.Methods: Zfp469BCS1/BCS1 keratocytes were treated with TRIDs PTC124 (ataluren), amlexanox, gentamicin, geneticin or vehicle‐only, for 2 or 5 days. Following treatment, Zfp469 and Col1a1 mRNA levels were evaluated by RT‐qPCR. Changes to keratocyte cell‐derived matrices were assessed using immunostaining or the collagen dye CNA35.Results: Zfp469BCS1/BCS1 keratocytes express 40% more Zfp469 mRNA than wildtype cells; this was unchanged by treatment with amlexanox, geneticin or gentamicin. PTC124 decreased expression of Zfp469 by 50%. Expression of Col1a1, a key component of the stromal matrix, was significantly decreased by PTC124 treatment (&lt;65% relative to vehicle‐only Zfp469BCS1/BCS1 keratocytes). Collagen deposition, visualized using CNA35 dyes, was decreased by PTC124 treatment in BCS and wildtype keratocytes.Conclusions: BCS is a devastating condition. Extreme thinning of the corneal stroma, as a result of impaired collagen production by keratocytes, makes the cornea prone to rupture. This causes irreversible sight loss. There is currently no treatment for BCS. Our initial work suggests that TRIDs may not restore functional full‐length Zfp469 in primary keratocytes and are unlikely to be of therapeutic benefit in BCS.Reference1. Stanton CM et al. A mouse model of brittle cornea syndrome caused by mutation in Zfp469. Dis Model Mech. 2021;14(9):dmm049175.

302 Availability of mRNA Obtained from Peripheral Blood Mononuclear Cells for Mutational Analysis in Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa (DEB) is an inheritable blistering disease caused by mutations in COL7A1, which encodes type VII collagen. To address the issue of genotype-phenotype correlations in DEB, analyzing the consequences of COL7A1 mutations using mRNA is indispensable. Herein we established a novel method for testing the effect of mutations in DEB using COL7A1 mRNA extracted from peripheral blood mononuclear cells (PBMCs). We investigated the consequences of four COL7A1 mutations (c.6573 + 1G > C, c.6216 + 5G > T, c.7270C > T and c.2527C > T) in three Japanese individuals with recessive DEB.

ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation

Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

Bone Sialoprotein Is Critical for Alveolar Bone Healing in Mice

Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout (Ibsp–/–) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatalIbsp–/– and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro–computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp–/– mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp–/– versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.

Abstract 9449: Increased Neprilysin Expression Links Atrial Fibrotic Remodeling in Cardiovascular Surgery Patients

Introduction: Deposition of extracellular matrix (ECM) such as collagen leads to fibrosis, which plays essential roles in structural remodeling in atrial fibrillation (AF). Neprilysin (NEP) is an integral membrane-bound metallopeptidase with a wide spectrum of substrates and physiological functions. Hypothesis: We investigated the association of NEP and the atrial fibrosis-related genes in cardiovascular surgery patients including heart valve disease. Methods: Atrial mRNA expression including collagen and NEP were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) in 59 cases (sinus rhythm (SR) 24 patients, AF 35 patients) in which LA tissues were removed from the appendage during surgery. NEP mRNA expression was also analyzed in adipose tissues (AT) obtained from both epicardial and subcutaneous adipose tissues. The NEP protein expression was measured by western blotting analysis and immunohistochemistry staining, and serum level of NEP was measured by ELISA. Results: The immunohistochemical staining showed the expression of NEP in both AT and LA. Higher expression of NEP mRNA and protein in adipose tissues was observed, compared with LA. Serum NEP concentration did not significantly correlate with NEP mRNA and protein expression of AT and LA. The significant correlations were observed among ECM (COL1A1, COL3A1, fibronectin) and the five selected fibrosis-related genes (TGFβ1, CTGF, miR21, NOX2 and BAX) attributed to the fibrosis of myocardium in LA. NEP mRNA expression of LA also positively correlated with the ECM and the fibrosis-related gene expression level. The multiple regression analysis revealed TGFβ1, NOX2, and NEP as independent predictors of atrial COL1A1 mRNA expression level. In addition, it revealed NEP as an independent predictor of COL1A1 mRNA expression level, even adjusted by age, sex, and BMI. Serum NEP concentration did not correlate with the ECM and the fibrosis-gene expression level. Patients with AF had higher atrial expression of NEP protein, compared to those with SR. Conclusions: Increased atrial expression of NEP links atrial fibrotic remodeling and developing AF in cardiovascular surgery patients. However, serum NEP did not reflect atrial NEP expression in LA.