Upregulation Of TIMP-1 Research Articles

The host cells suppress the proliferation of pseudorabies virus by regulating the PI3K/Akt/mTOR pathway.

PRV, known for its neurotropic properties, is capable of inducing severe neuronal damage. Our study discovered that following PRV infection, the expression of MMP2 was upregulated, leading to the degradation of ZO-1. Furthermore, upon PRV infection in the host, the promoter of TIMP1 is significantly activated, resulting in a significant increase in TIMP1 protein levels. This upregulation of TIMP1 inhibits the proliferation of PRV through the PI3K/Akt signaling pathway. This study elucidated the mechanism through which PRV, including the PRV XJ delgE/gI/TK strains, compromises the blood-brain barrier and identifies the antiviral response characterized by the activation of the PI3K/Akt signaling pathway within infected host cells. These findings provide potential therapeutic targets for the clinical management and treatment of PRV.

Molecular changes underlying pulmonary emphysema and chronic bronchitis in Chronic Obstructive Pulmonary Disease: An updated review.

The aim of this review is to update and synthesize the molecular mechanisms that lead to the heterogeneous effect on tissue remodeling observed in the two most important clinical phenotypes of chronic obstructive pulmonary disease (COPD), pulmonary emphysema (PE) and chronic bronchitis (CB). Clinical and experimental evidence suggests that this heterogeneous response to promote PE, CB, or both, is related to differentiated genetic, epigenetic, and molecular conditions. Specifically, a tendency toward PE could be related to a variant in the DSP gene, SIRT1 downregulation, macrophage polarization to M1, as well as the involvement of the noncanonical Wnt5A signaling pathway, among other alterations. Additionally, in advanced stages of COPD, PE development is potentiated by dysregulations in autophagy, which promotes senescence and subsequently cell apoptosis, through exacerbated inflammasome activation and release of caspases. On the other hand, CB or the pro-fibrotic phenotype could be potentiated by the downregulated activity of HDAC2, the activation of the TGF-β/Smad or Wnt/β-catenin signaling pathways, macrophage polarization to M2, upregulation of TIMP-1, and/or the presence of the epithelial-mesenchymal transition (EMT) mechanism. Interestingly, the upregulated activity of MMPs, especially MMP-9, is widely involved in the development of both phenotypes. Furthermore, MMP-9 and MMP-12 enhance the severity, perpetuation, and exacerbation of COPD, as well as the development of autoimmunity in this disease.

Deciphering the role of ferroptosis in rheumatoid arthritis: Synovial transcriptome analysis and immune infiltration correlation

The pathogenesis of rheumatoid arthritis (RA) remains elusive. The initiation of joint degeneration is characterized by the loss of self-tolerance in peripheral joints. Ferroptosis, a form of regulated cell death, holds significant importance in the pathophysiology of inflammatory arthritis, primarily due to iron accumulation and the subsequent lipid peroxidation.The present study investigated the association between synovial lesions and ferroptosis-related genes using previously published data from rheumatoid patients. Transcriptome differential gene analysis was employed to identify ferroptosis-related differentially expressed genes (FRDEGs). To validate FRDEGs and screen hub genes, we used weighted gene co-expression network analysis (WGCNA) and receiver operating characteristic (ROC) curves. Subsequently, immune infiltration analysis and single cell analysis were conducted to investigate the relationship between various synovial tissues cells and FRDEGs. The findings were further confirmed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunohistochemical staining, and immunofluorescence techniques.Upon intersecting DEGs with ferroptosis-related genes, we identified a total of 104 FRDEGs. Through the construction of a protein-protein interaction (PPI) network, we pinpointed the top 20 most highly concentrated genes as hub genes. Subsequent analyses using ROC curve and WGCNA validated eight FRDEGs: TIMP1, JUN, EGFR, SREBF1, ADIPOQ, SCD, AR, and FABP4. Immuno-infiltration analyses revealed significant infiltration of immune cell in RA synovial tissues and their correlations with the FRDEGs. Notably, TIMP1 demonstrated a positive correlation with various immune cell populations. Single-cell sequencing date of RA synovial tissue revealed predominant expression of TIMP1 is in fibroblasts. RT–qPCR, immunohistochemistry, and immunofluorescence analyses confirmed significant upregulation of TIMP1 at both mRNA and protein levels in RA synovial tissues and fibroblast-like synoviocytes (FLS).The findings provide novel insights into pathophysiology of peripheral immune tolerance deficiency in RA. The dysregulation of TIMP1, a gene associated with ferroptosis, was significantly observed in RA patients, suggesting its potential as a promising biomarker and therapeutic target.

P13 Keratinocyte heterogeneity and dynamics in human skin wound healing

Abstract Introduction and aims Human chronic skin wounds have been extensively characterized, but ethical limitations of collecting and studying wounds from healthy individuals means there is a sparsity of data regarding re-epithelialization in normal wound healing. Here, we characterize ex vivo models of wound closure in human skin to better understand the keratinocyte dynamics of normal healing. Methods We conducted single-cell RNA sequencing (scRNA-Seq) from ex vivo skin explants 2 days postwounding (n = 4) to interrogate heterogeneity in keratinocytes relative to the wound edge. Immunostaining was also conducted on sections from skin explants at different timepoints during wound closure. Scratch assays were conducted using isolated human keratinocytes. Specific zones of keratinocytes were separated relative to the original scratch, 6 h postscratch, and used for real-time polymerase chain reaction analyses. Results We found that partial re-epithelialization and full healing of 2-mm wounds occurred 2–3 days and 4 days postwounding, respectively. Analyses of the scRNA-Seq data showed an increase in S100A7 expression in suprabasal keratinocyte subclusters in wounded skin compared with unwounded skin. Using immunostaining we also showed that S100A7 is expressed at the leading edge of the regenerating epidermis. In an in vitro scratch wound model, the keratinocytes nearest to the wound edge also show an upregulation of S100A7, EGFR, EPHA4 and TIMP1, as well as a downregulation in PCNA, implying a decrease in cell proliferation in leading keratinocytes. Conclusions S100A7 was previously reported to be increased at the wound edge in human keratinocytes, highlighting that transcriptional changes in our ex vivo and in vitro human wound healing models reflect those occurring in vivo. The decrease in PCNA in keratinocytes in the leading edge also recapitulates Results previously shown in mouse models of wound healing. These data on normal re-epithelialization will be compared with published data on human chronic wounds and other skin diseases.

Comprehensive analysis of basement membrane-related gene based on single-cell and bulk RNA sequencing data to predict prognosis and evaluate immune characteristics in colorectal cancer.

Basement membrane-related genes (BMs) participate in regulating cell polarity, invasion, metastasis, and survival across different tumor types. Nevertheless, the specific functions of BMs in colorectal cancer (CRC) remain uncertain. To investigate the clinical relevance of BMs in CRC, we retrieved both gene expression and clinical data from The Cancer Genome Atlas (TCGA) datasets for subsequent analysis. The Kaplan-Meier (K-M) survival curve was employed to evaluate prognosis in high- and low-risk groups. Furthermore, additional analyses, including nomogram construction, functional enrichment, examination of the tumor immune microenvironment, prediction of small-molecule drugs, and more, were conducted to delve into the significance of BM-related signatures in CRC. Single-cell data from seven CRC patients were obtained from the TISCH2 database, and expression validation and cell source exploration of BM-related signatures were performed. Lastly, the expression and function of TIMP1, a key gene in BMs that may play a role in the progression of CRC, was validated in vitro through a series of basic experiments. We constructed a seven BMs-based model to categorize CRC patients into high-risk and low-risk groups. K-M survival analysis indicated a poorer prognosis for high-risk CRC patients. Cox regression analysis further identified the risk score as an independent prognostic factor for CRC patients. The nomogram model exhibited superior discrimination and calibration abilities of CRC patients. Based on the results from GO/KEGG and GSEA, genes in the high-risk subgroup were implicated in immune-related pathways and exhibited a positive correlation with immune checkpoints. In single-cell data, we found that TIMP1 is highly expressed in many cells, especially in malignant tumor cells. We also observed up-regulation of TIMP1 in CRC cell lines, promoting cancer invasion and migration in vitro. Our study has discovered a novel prognostic index derived from BM-related genes in CRC patients. Specifically, the new model enables patient stratification, improving the selection of individuals likely to benefit from immunotherapy.

Exploration and validation of signature genes and immune associations in septic cardiomyopathy

ABSTRACT An early and accurate diagnosis of septic cardiomyopathy is vital for improving the overall prognosis of sepsis. In our research, we aimed to identify signature genes and their immune connections in septic cardiomyopathy. By analyzing the mouse myocardial transcriptome from sepsis induced by cecum ligation and puncture (CLP), we identified four distinct k-means clusters. Further analysis of human myocardial datasets using Weighted Gene Co-expression Network Analysis (WGCNA) revealed a strong correlation between the MEturquoise module and septic cardiomyopathy (cor = 0.79, p < .001). Through the application of Cytoscape plug-in MCODE and comprehensive analysis, we pinpointed two signature genes, THBS1 and TIMP1. These genes demonstrated significant involvement in immune cell infiltration, as detected by CIBERSORT, and displayed promising prognostic potential as validated by external datasets. Our experimental validation confirmed the up-regulation of both THBS1 and TIMP1 in septic murine hearts, underscoring their positive association with septic cardiomyopathy.

TGF-β2 Induces Epithelial-Mesenchymal Transitions in 2D Planer and 3D Spheroids of the Human Corneal Stroma Fibroblasts in Different Manners.

To examine the epithelial-mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen (COL) 1, 4 and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 3, 9 and 14, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-β2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of COL1, COL4, FN, αSMA, TIMP1, TIMP, and most ER stress-related genes and the downregulation of COL6 and MMP3. In the case of 3D spheroids, TGF-β2 induced the downsizing and stiffening of 3D spheroids and the upregulation of COL6, MMP14, and most ER stress-related genes. These findings suggest that TGF-β2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids.

Characterizing the impact of SGLT2 inhibition on hepatic inflammation and cellular response in a type 2 diabetic mouse model

Type 2 diabetes (T2D) is associated with several comorbidities including nonalcoholic fatty liver disease (NAFLD). NAFLD is characterized by the presence of steatosis with or without hepatic fibrosis and is associated with insulin resistance, inflammation, and hyperlipidemia. Recently, sodium-glucose co-transport 2 inhibitors (SGLT2i) such as Empagliflozin (Empa), have emerged as a treatment option for patients with T2D by blocking renal glucose reabsorption and improving glycemic control. Whether these inhibitors may attenuate T2D-associated NAFLD is still under investigation. To explore this possibility, we challenged TallyHo (TH) mice with a 60% high-milk fat diet (HMFD) with or without Empa (10mg/kg BW) for 12 weeks. Like humans, TH mice possess several quantitative trace loci associated with diabetic/obese phenotypes. In this study, we found that Empa treatment decreased hepatic steatosis in both male and female TH mice. However, the 12-week dietary challenge was not sufficient to induce fibrosis and inflammation (Kurtz et al. 2022). Thus, we have extended the trial to 24-weeks with the hypothesis that Empa promotes anti-inflammatory responses through macrophage infiltration and cellular signaling. Protein expression of the cGAS-STING pathway (STING, pTBK1, TBK1, pIRF3, IRF3), inflammasome pathway (AIM2, ASC/TMS1) and apoptosis signaling pathway (Caspase-3) showed no significant changes between diet or treatment. Additionally, activity of p50 and p65 of NF-κB (measured through nuclear expression) was not changed between treatment groups.While Empa does not appear to alter cellular-mediated inflammatory pathways, we did note changes in cytokine expression. Using a large-scale screen to probe for more than 60 inflammatory markers, we observed elevated levels of three pro-inflammatory cytokines (IL-3, CCL2, M-CSF; p&lt;0.05) in livers from Empa-treated mice while there was downregulation of p40/p70 (p&lt;0.01). The screen of plasma samples identified elevated levels of MIP-1γ (p&lt;0.01) and identified decreased levels of three cytokines (IL-9, TIMP1, TPO; p&lt;0.05) in Empa-treated mice. To further understand the role of macrophage infiltration as a response to increased inflammation, immunofluorescence on liver section with F4/80, a macrophage marker, was performed. There were no significant differences in the density or localization of Kupffer cells between treatments and diets for both sexes. Thus, it is hypothesized that Empa promotes the polarization of macrophages from M1 (pro-inflammatory) to M2 (anti-inflammatory) to promote tissue regeneration and repair. Staining of M1 and M2 macrophage markers are currently underway. Additionally, it has been determined that the upregulation of TIMP1 leads to matrix deposition. Efforts are currently underway to quantify portal inflammation using QuPath. Collectively, the data shows that Empa treatment for 24-weeks does not alter cellular signaling, and potentially alters macrophage signaling to combat NAFLD. NIDDK This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Thymoquinone Suppresses Angiogenesis in DEN-Induced Hepatocellular Carcinoma by Targeting miR-1-3p.

Hepatocellular carcinoma (HCC) is characterized by its high vascularity and metastasis. Thymoquinone (TQ), the main bio-active constituent of Nigella sativa, has shown anticancer and hepatoprotective effects. TQ's anticancer effect is mediated through miRNA regulation. miR-1-3p plays a significant role in various cancers but its role in HCC invasiveness remains poorly understood. Bio-informatics analysis predicted that the 3'-UTR of TIMP3 is a target for miR-1-3p; Rats were equally divided into four groups: Group 1, the negative control; Group 2 received TQ; Group 3 received DEN; and Group 4 received DEN after pretreatment with TQ. The expression of TIMP3, MMP2, MMP9, and VEGF in rats' liver was determined immunohistochemically. RT-qPCR was used to measure the miR-1-3p level in rats' liver, and TIMP3, MMP2, MMP9, and VEGF in the HepG2 cells after being transfected with miR-1-3p mimic or inhibitor; In rats pretreated with TQ, a decreased expression of MMP2, MMP9 and VEGF, and increased expression levels of TIMP3 and miR-1-3p were detected. Treating the HepG2 cells with miR-1-3p mimic led to the upregulation of TIMP3 and downregulation of MMP2, MMP9, and VEGF, and showed a significant delay in wound healing; These results suggested that the anti-angiogenic effect of TQ in HCC may be mediated through the regulation of miR-1-3p.

Plumbagin downregulates UHRF1, p-Akt, MMP-2 and suppresses survival, growth and migration of cervical cancer CaSki cells

Plumbagin is a natural compound known to impede growth of cancerous cells. However, anti-cervical cancer effects of plumbagin and its underlying molecular mechanism still remains elusive. In this study, plumbagin reduced the viability of CaSki cells in a concentration dependent manner and suppressed their colony formation potential. It led to G2/M phase arrest with downregulation of E2F1 and upregulation of p21. Plumbagin reduced mitochondrial membrane potential and concomitantly increased the percentage of apoptotic cells as revealed by annexin V-propidium iodide staining. Real Time PCR and western blotting confirmed that plumbagin induced apoptosis by reducing the expression of pAkt, procaspase 9 and full-length PARP. Furthermore, scratch assay showed that plumbagin suppressed migratory potential of CaSki cells which could be due to the reduced expression and activity of MMP-2 and upregulation of TIMP2. Interestingly, plumbagin also downregulated UHRF1 expression. Transient silencing of UHRF1 like plumbagin, induced G2/M phase arrest, enhanced apoptosis and suppressed metastasis of CaSki cells suggesting the role of UHRF1 in mediating anti-cancer activities of plumbagin. Plumbagin at IC20 (1 μM) interacted synergistically with cisplatin and reduced its IC50 value by 13.23 fold with improved effectivity as revealed by augmented apoptosis in CaSki cells.

Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-β2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-β2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

TARGETING THE HISTONE DEMETHYLASE JMJD2B/KDM4B AS A NOVEL THERAPEUTIC APPROACH IN IL-13-MEDIATED FIBROSIS IN AIRWAY FIBROBLASTS OF SEVERE ASTHMATICS

TYPE: Abstract TOPIC: Allergy and Airway PURPOSE: Asthma is a heterogeneous disorder characterized by chronic inflammation and remodeling of the airways. Asthma responses are driven mainly by type 2 immune responses, where IL-13 plays a key role in asthma pathogenesis. We previously identified JMJD2B as an IL-13-regulated epigenetic modifier in asthmatic airway fibroblasts. Therefore, this study aimed to target JMJD2B to potentially alleviate IL-13-mediated fibrosis in asthma. METHODS: Bronchial fibroblasts isolated from asthmatic and healthy individuals were stimulated with IL-13 and treated with JIB-04, a pan-selective inhibitor of histone demethylase(s). Gene and protein expression of extracellular matrix markers were assessed using qRT-PCR, western blotting and matrix metalloproteinases (MMPs) activity assay. Chromatin immunoprecipitation (ChIP) assay was used to determine the bindig of JMJD2B to promoter region of Tissue inhibitors of matrix metalloproteinases (TIMPs). RESULTS: Here we show that JIB-04 inhibited JMJD2B activity by reducing the demethylation of its downstream target, H3K36me3, in asthmatic fibroblasts. Inhibition of JMJD2B significantly affected the viability of the bronchial fibroblasts at 48 hours. JMJD2B inhibition was further associated with the downregulation of extracellular matrix (ECM) proteins such as MMP-2, MMP-9, collagen-1, and fibronectin, and upregulation of TIMP-2, at both the gene and protein levels. This was accompanied by inhibition of IL-13 mediated MMPs activity. JIB-04 further prevented the association of JMJD2B on TIMP-2 promoter region. CONCLUSIONS: Our results demonstrate the potential of JMJD2B inhibition in attenuating IL-13-mediated fibrosis in asthma. CLINICAL IMPLICATIONS: Therapeutic targeting of JMJD2B using JIB-04 is a promising candidate to alleviate IL-13-mediated responses in chronic disorders such as asthma. DISCLOSURE: Nothing to declare. KEYWORD: Asthma

Sciatic Nerve Intrafascicular Injection Induces Neuropathy by Activating the Matrix Modulators MMP-9 and TIMP-1.

Background: Peripheral nerve block (PNB) under echo guidance may not prevent intrafascicular anesthetic injection-induced nerve injury. This study investigated whether unintended needle piercing alone, or the intrafascicular nerve injectant could induce neuropathy. Methods: 120 adult male Sprague-Dawley rats were divided into four groups: 1) group S, only the left sciatic nerve was exposed; 2) group InF-P, the left sciatic nerve was exposed and pierced with a 30 G needle; 3) group InF-S, left sciatic nerve was exposed and injected with saline (0.9% NaCl 30 µL); 4) group InF-R, left sciatic nerve was exposed and injected with 0.5% (5 mg/mL, 30 µL) ropivacaine. Behaviors of thermal and mechanical stimuli responses from hindpaws, sciatic nerve vascular permeability and tight junction protein expression, and macrophage infiltration were assessed. Pro-inflammatory cytokine expression and TIMP-1 and MMP-9 activation at the injection site and the swollen, and distal sites of the sciatic nerve were measured by cytokine array, western blotting, and immunofluorescence of POh14 and POD3. Results: Intrafascicular saline and ropivacaine into the sciatic nerve, but not needle piercing alone, significantly induced mechanical allodynia that lasted for seven days. In addition, the prior groups increased vascular permeability and macrophage infiltration, especially in the swollen site of the sciatic nerve. Thermal hypersensitivity was induced and lasted for only 3 days after intrafascicular saline injection. Obvious upregulation of TIMP-1 and MMP-9 on POh6 and POh14 occurred regardless of intrafascicular injection or needle piercing. Compared to the needle piercing group, the ratio of MMP-9/TIMP-1 was significantly higher in the intrafascicular injectant groups at the injected and swollen sites of the sciatic nerve. Although no gross changes in the expressions of tight junction proteins (TJPs) claudin-5 and ZO-1, the TJPs turned to apparent fragmentation and fenestration-like degenerative change in swollen endothelial cells and thickened microvessels. Conclusion: Intrafascicular nerve injection is a distinct mechanism that induces neuropathy. It is likely that the InF nerve injection-induced neuropathy was largely due to dramatic, but transient, increases in enzymatic activities of MMP-9 and activating TIMP-1 in the operated nerves. The changes in enzymatic activities then contributed to certain levels of extracellular matrix degradation, which leads to increases in endoneurial vascular permeability.

Abstract 384: Regulation Of The Smooth Muscle Cell Phenotypic Switch In Resolution Of Vascular Injury

Background: The modulation of vascular smooth muscle cells (VSMC) phenotypes is a highly important process in the formation of neointima and in resolution of inflammation after vascular injury. Underlying genetic mechanisms that control the phenotypic switch of VSMC in disease are not completely understood. On this basis, the phenotypic regulation of VSMC in restenosis development was examined in the mouse carotid artery injury model. Methods and Results: Wire-induced injury, an established mouse-model to study restenosis, was applied to induce vascular injury in C57BL/6 mice. Time-resolved single-cell RNA-sequencing (scRNAseq) and massive-analysis-of-cDNA-ends (MACE)-RNAseq of neointima obtained by laser capture microdissection were performed to detect genetic changes in neointima development. Single-cell RNA-sequencing of C57BL/6 mice at different time points after wire-induced injury revealed an expanding, highly proliferative activated VSMC cluster, which exhibited low expression of SMC marker genes. Analysis for differentially regulated genes (DEG) and pseudotime clustering identified a unique marker gene signature, including high expression of TIMP Metallopeptidase Inhibitor 1 (Timp1), which was identified as main ordering gene of the modulation processes′ pseudo temporal states. MACEseq confirmed upregulation of Timp1 selectively in neointima forming VSMCs. Integration with atherosclerosis scRNAseq data indicated differences in gene signatures of activated VSMC populations. Transcription factor analysis of modulated VSMC identified BACH1, a factor involved in reactive oxidative stress response and proliferation of SMCs, as potential regulatory target for re-differentiation of VSMCs after vascular injury. Conclusion: ScRNAseq identified a unique cell cluster responsible for neointima formation. Targeting the transcription factors driving the cluster (like BACH1) can be exploited to combat neointima development.

MOLECULAR BASIS OF THE DEVELOPMENT OF INSULIN RESISTANCE IN OBESE ADOLESCENT AND ADULT MEN

The aim of this work was to study the association between the expression of glucose metabolism related genes and insulin resistance, which expression is changed in obese adolescents and adult men with and without insulin resistance, for better understanding the molecular basis of the development of obesity complications and evaluation of possible contribution of these genes in development of insulin resistance. Methods. The expression level of genes related to glucose metabolism and their regulations was studied by real-time qPCR in adipose tissue and blood cells using SYBRGreen Mix and specific for each mRNA forward and reverse primers. Total RNA was extracted using TRIZOL reagent. For reverse transcription of mRNAs we used Thermo Scientific Verso cDNA Synthesis Kit (Germany). The values of mRNA expressions were normalized to the level of ACTB mRNA and represented as percent of control (100 %). Results. It was shown that in obese patients with insulin resistance the expression level of IRS1 (insulin receptor substrate 1), HK2 (hexokinase 2), PFKFB2 (6-phosphofructokinase/fructose-2,6-bisphosphatase 2) and PFKFB3 as well as circadian factors CLOCK and ARNTL genes in subcutaneous adipose tissue is significantly decreased as compared to obese men with normal sensitivity to insulin. At the same time, the development of insulin resistance in obese patients leads to up-regulation of PFKFB4, PER1, HSPA6, ALDH1A3, COL5A1, TIMP1, TIMP2, SPARC, and VCAN gene expressions in subcutaneous adipose tissue. The expression level of IGF1 (insulin-like growth factor 1) and IGFBP5 (IGF binding protein 5) as well as ENO1 (enolase 1) and ENO2 is down-regulated in the blood of obese adolescent with insulin resistance, but IGFBP2 and IGFBP7 gene expressions are significantly increased in these patients. Conclusions. The results of this investigation provide evidence that the development of insulin resistance in obese patients is associated with gene specific changes in the expression of many very important regulatory genes, which are endoplasmic reticulum stress responsible.

Extravesicular TIMP-1 is a non-invasive independent prognostic marker and potential therapeutic target in colorectal liver metastases

Molecular reprogramming of stromal microarchitecture by tumour-derived extracellular vesicles (EVs) is proposed to favour pre-metastatic niche formation. We elucidated the role of extravesicular tissue inhibitor of matrix metalloproteinase-1 (TIMP1EV) in pro-invasive extracellular matrix (ECM) remodelling of the liver microenvironment to aid tumour progression in colorectal cancer (CRC). Immunohistochemistry analysis revealed a high expression of stromal TIMP1 in the invasion front that was associated with poor progression-free survival in patients with colorectal liver metastases. Molecular analysis identified TIMP1EV enrichment in CRC-EVs as a major factor in the induction of TIMP1 upregulation in recipient fibroblasts. Mechanistically, we proved that EV-mediated TIMP1 upregulation in recipient fibroblasts induced ECM remodelling. This effect was recapitulated by human serum-derived EVs providing strong evidence that CRC release active EVs into the blood circulation of patients for the horizontal transfer of malignant traits to recipient cells. Moreover, EV-associated TIMP1 binds to HSP90AA, a heat-shock protein, and the inhibition of HSP90AA on human-derived serum EVs attenuates TIMP1EV-mediated ECM remodelling, rendering EV-associated TIMP1 a potential therapeutic target. Eventually, in accordance with REMARK guidelines, we demonstrated in three independent cohorts that EV-bound TIMP1 is a robust circulating biomarker for a non-invasive, preoperative risk stratification in patients with colorectal liver metastases.

Identification of early response to hypertonic dextrose prolotherapy markers in knee osteoarthritis patients by an inflammation-related cytokine array.

Osteoarthritis (OA) is one of the most common forms of arthritis, and hypertonic dextrose prolotherapy has long been used clinically to treat knee OA. The aim of this study was to investigate the inflammation-related protein-expression profile characterizing the efficacy of the hypertonic dextrose prolotherapy in knee OA as prognostic markers. OA patients over the age of 65 were recruited for Western Ontario McMaster University Osteoarthritis (WOMAC) index, knee X-ray evaluation and knee joint synovial fluid analysis before and after hypertonic dextrose prolotherapy. The expressions of inflammation-related factors were measured using a novel cytokine antibody array methodology. The cytokine levels were quantified by quantitative protein expression and analyzed by ELISA using the patients' knee-joint synovial fluid. The WOMAC Index and minimum joint space width before receiving the intra-articular injection and at 2-week intervals were compared. Twelve patients who received OA intervention were enrolled and finally a clinical evaluation of 12 knee joints and knee synovial fluid samples were analyzed. In this study, after receiving hypertonic dextrose prolotherapy, the OA patients clearly demonstrated a significant improvement in WOMAC index and increasing tendency in the medial minimum joint space width after intervention. Meanwhile, we observed a significantly associated tendency between hypertonic dextrose treatment of knee OA and the upregulation of MMP2, TIMP-1, EGF, CXCL9 and IL-22. The findings provide knee OA patients receiving hypertonic dextrose prolotherapy, which is accompained by the improvemeny of knee symptoms and associated tendency of upregulation of MMP2, EGF, CXCL 9 and IL-22.

The Short-Chain Fatty Acid Butyrate Attenuates Pulmonary Vascular Remodeling and Inflammation in Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a progressive cardiovascular disorder in which local vascular inflammation leads to increased pulmonary vascular remodeling and ultimately to right heart failure. The HDAC inhibitor butyrate, a product of microbial fermentation, is protective in inflammatory intestinal diseases, but little is known regarding its effect on extraintestinal diseases, such as PH. In this study, we tested the hypothesis that butyrate is protective in a Sprague–Dawley (SD) rat model of hypoxic PH. Treatment with butyrate (220 mg/kg intake) prevented hypoxia-induced right ventricular hypertrophy (RVH), hypoxia-induced increases in right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, and permeability. A reversal effect of butyrate (2200 mg/kg intake) was observed on elevated RVH. Butyrate treatment also increased the acetylation of histone H3, 25–34 kDa, and 34–50 kDa proteins in the total lung lysates of butyrate-treated animals. In addition, butyrate decreased hypoxia-induced accumulation of alveolar (mostly CD68+) and interstitial (CD68+ and CD163+) lung macrophages. Analysis of cytokine profiles in lung tissue lysates showed a hypoxia-induced upregulation of TIMP-1, CINC-1, and Fractalkine and downregulation of soluble ICAM (sICAM). The expression of Fractalkine and VEGFα, but not CINC-1, TIMP-1, and sICAM was downregulated by butyrate. In rat microvascular endothelial cells (RMVEC), butyrate (1 mM, 2 and 24 h) exhibited a protective effect against TNFα- and LPS-induced barrier disruption. Butyrate (1 mM, 24 h) also upregulated tight junctional proteins (occludin, cingulin, claudin-1) and increased the acetylation of histone H3 but not α-tubulin. These findings provide evidence of the protective effect of butyrate on hypoxic PH and suggest its potential use as a complementary treatment for PH and other cardiovascular diseases.

IGF-1 Facilitates Cartilage Reconstruction by Regulating PI3K/AKT, MAPK, and NF-kB Signaling in Rabbit Osteoarthritis.

PurposeThe pathogenesis of osteoarthritis (OA) is characterized by joint degeneration. The pro-inflammatory cytokine interleukin (IL)-1β plays a vital role in the pathogenesis of OA by stimulation of specific signaling pathways like NF-κB, PI3K/Akt, and MAPKs pathways. The catabolic role of growth factors in the OA may be inhibited cytokine-activated pathogen. The purpose of this study was to investigate the potential effects of insulin-like growth factor-1 (IGF-1) on IL-1β-induced apoptosis in rabbit chondrocytes in vitro and in an in vivo rabbit knee OA model.MethodsIn the present study, the OA developed in chondrocyte with the treatment of IL-1β and articular cartilage ruptures by removal of cartilage from the rabbit knee femoral condyle. After IGF-1 treatment, immunohistochemistry and qRT-PCR were identified OA expression with changes in MMPs (matrix metalloproteinases). The production of ROS (intracellular reactive oxygen species) in the OA was detected by flow cytometry. Further, the disease progression was microscopically investigated and pathophysiological changes were analyzed using histology. The NF-κB, PI3K/Akt and P38 (MAPK) specific pathways that are associated with disease progression were also checked using the Western blot technique.ResultsThe expression of MMPs and various apoptotic markers are down-regulated following administration of IGF-1 in a dose-dependent fashion while significantly up-regulation of TIMP-1. The results showed that higher levels of ROS were observed upon treatment of chondrocytes and chondral OA with IL-1β. Collectively, our results indicated that IGF-1 protected NF-κB pathway by suppression of PI3K/Akt and MAPKs specific pathways. Furthermore, the macroscopic and pathological investigation showed that it has a chondroprotective effect by the formation of hyaline cartilage.ConclusionOur results indicate a protective effect of IGF-1 against OA pathogenesis by inhibition of NF-κB signaling via regulation of the MAPK and PI3K/Akt signaling pathways and prevention of apoptosis by suppression of ROS production.

Molecular toxicological alterations in the mouse hearts induced by sub-chronic thiazolidinedione drugs administration.

Thiazolidinediones are well-known anti-diabetic drugs. However, they are not widely used due to their cardiotoxic effects. Therefore, in this study, we aimed to determine the molecular toxicological alterations induced in the mouse hearts after thiazolidinedione administration. Balb/c mice received doses clinically equivalent to those given to humans of the most commonly used thiazolidinediones, pioglitazone, and rosiglitazone for 30days. After that, RNA samples were isolated from the hearts. The mRNA expression of cytochrome (cyp) p450 genes that synthesize the cardiotoxic 20-hydroxyeicosatetraenoic acid (20-HETE) in addition to 92 cardiotoxicity biomarker genes were analyzed using quantitative polymerase chain reaction array technique. The analysis demonstrated that thiazolidinediones caused a significant upregulation (p<0.5) of the mRNA expression of cyp1a1, cyp4a12, itpr1, ccl7, ccr1, and b2m genes. In addition, thiazolidinediones caused a significant (p<0.05) downregulation of the mRNA expression of adra2a, bsn, col15a1, fosl1, Il6, bpifa1, plau, and reg3b genes. The most affected gene was itpr1 gene, which was upregulated by pioglitazone and rosiglitazone by sevenfold and 3.5-fold, respectively. In addition, pioglitazone caused significant upregulation of (p<0.05) hamp, ppbp, psma2, sik1, timp1, and ucp1 genes, which were not affected significantly (p>0.05) by rosiglitazone administration. In conclusion, this study showed that thiazolidinediones induce toxicological molecular alterations in the mouse hearts, such as the induction of cyp450s that synthesize 20-HETE, chemokine activation, inflammatory responses, blood clotting, and oxidative stress. These findings may help us understand the mechanism of cardiotoxicity involved in thiazolidinedione administration.