Reduction in ACE2 expression in peripheral blood mononuclear cells during COVID-19 - implications for post COVID-19 conditions.

BACKGROUND: Severe COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC). METHODS: The IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10. RESULTS: Forty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals. CONCLUSIONS: This work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.

Naringin from Ganshuang granule inhibits inflammatory to relieve liver fibrosis through TGF-ß-Smad signaling pathway.

OBJECTIVE: The present study aims to investigate the specific protective effects and underlying mechanisms of Ganshuang granule (GSG) on dimethylnitrosamine (DMN)-induced hepatic fibrosis in rat models. METHODS: Hepatic fibrosis was experimentally evoked in rats by DMN administration, and varying dosages of GSG were employed as an intervention. Hepatocellular damage was assessed by measuring serum levels of aminotransferase and bilirubin, accompanied by histopathological examinations of hepatic tissue. The hepatic concentrations of platelet-derived growth factor (PDGF) and transforming growth factor-ß1 (TGF-ß1) were quantitated via enzyme-linked immunosorbent assay (ELISA). The expression of α-smooth muscle actin (α-SMA) within hepatic tissue was evaluated using immunohistochemical techniques. The levels of hepatic interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and a spectrum of interleukins (IL-2, IL-4, IL-6, IL-10) were quantified by quantitative real-time PCR (qRT-PCR). Additionally, hepatic stellate cells (HSCs) were cultured in vitro and exposed to TNF-α in the presence of naringin, a principal component of GSG. The gene expression levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metallopeptidase-1 (MMP-1) in these cells were also quantified by qRT-PCR. Proliferative activity of HSCs was evaluated by the Cell Counting Kit-8 assay. Finally, alterations in Smad protein expression were analyzed through Western blotting. RESULTS: Administration of GSG in rats with fibrosis resulted in reduced levels of serum aminotransferases and bilirubin, along with alleviation of histopathological liver injury. Furthermore, the fibrosis rats treated with GSG exhibited significant downregulation of hepatic TGF-ß1, PDGF, and TNF-α levels. Additionally, GSG treatment led to increased mRNA levels of IFN-γ, IL-2, and IL-4, as well as decreased expression of α-SMA in the liver. Furthermore, treatment with naringin, a pivotal extract of GSG, resulted in elevated expression of MMP-1 and decreased levels of TIMP-1 in TNF-α-stimulated HSCs when compared to the control group. Additionally, naringin administration led to a reduction in Smad expression within the HSCs. CONCLUSION: GSG has the potential to mitigate fibrosis induced by DMN in rat models through the regulation of inflammatory and fibrosis factors. Notably, naringin, the primary extract of GSG, may exert a pivotal role in modulating the TGF-ß-Smad signaling pathway.

Crocin's role in modulating MMP2/TIMP1 and mitigating hypoxia-induced pulmonary hypertension in mice.

To explore the molecular pathogenesis of pulmonary arterial hypertension (PAH) and identify potential therapeutic targets, we performed transcriptome sequencing of lung tissue from mice with hypoxia-induced pulmonary hypertension. Our Gene Ontology analysis revealed that "extracellular matrix organization" ranked high in the biological process category, and matrix metallopeptidases (MMPs) and other proteases also played important roles in it. Moreover, compared with those in the normoxia group, we confirmed that MMPs expression was upregulated in the hypoxia group, while the hub gene Timp1 was downregulated. Crocin, a natural MMP inhibitor, was found to reduce inflammation, decrease MMPs levels, increase Timp1 expression levels, and attenuate hypoxia-induced pulmonary hypertension in mice. In addition, analysis of the cell distribution of MMPs and Timp1 in the human lung cell atlas using single-cell RNAseq datasets revealed that MMPs and Timp1 are mainly expressed in a population of fibroblasts. Moreover, in vitro experiments revealed that crocin significantly inhibited myofibroblast proliferation, migration, and extracellular matrix deposition. Furthermore, we demonstrated that crocin inhibited TGF-ß1-induced fibroblast activation and regulated the pulmonary arterial fibroblast MMP2/TIMP1 balance by inhibiting the TGF-ß1/Smad3 signaling pathway. In summary, our results indicate that crocin attenuates hypoxia-induced pulmonary hypertension in mice by inhibiting TGF-ß1-induced myofibroblast activation.

Integrating bulk and single-cell sequencing reveals metastasis-associated CAFs in head and neck squamous cell carcinoma.

AIMS: Cancer-associated fibroblasts (CAFs) have been shown to promote the metastasis of head and neck squamous cell carcinoma (HNSCC), but the underlying mechanisms remain unclear. The purpose of this study is to identify gene in CAFs that are associated with metastasis and to preliminarily validate its impact on the metastasis of HNSCC. MATERIALS AND METHODS: Scissor analysis was utilized to process single-cell and bulk RNA sequencing datasets, identifying genes associated with the metastasis of HNSCC through differential gene expression analysis. A risk model was constructed using LASSO regression analysis. Quantitative real time-PCR and Western blot were employed to measure mRNA and protein expressions, respectively. Multiplex immunohistochemistry (mIHC) was used to assess protein expression in CAFs. siRNA was utilized to achieve gene knockdown. CCK-8 and Transwell assays were employed to evaluate the biological characteristics of HNSCC cells. KEY FINDINGS: Compare to the nonmetastatic primary CAFs (nmCAFs), tissue inhibitors of metalloproteinase-1 (TIMP1) was founded to be overexpressed in the cells and tissues of metastatic primary CAFs (mCAFs). Knocking down TIMP1 in CAFs can signifucantly inhibit the proliferation, invasion, and migration of HNSCC cells. SIGNIFICANCE: CAFs facilitate HNSCC cell metastasis by upregulating TIMP1, highlighting TIMP1 as a potential therapeutic target in HNSCC metastasis management.

A Novel Strategy for the Treatment of Aneurysms: Inhibition of MMP-9 Activity through the Delivery of TIMP-1 Encoding Synthetic mRNA into Arteries.

Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms.

TIMP-1 is an activator of MHC-I expression in myeloid dendritic cells with implications for tumor immunogenicity.

Immune checkpoint therapies (ICT) for advanced solid tumors mark a new milestone in cancer therapy. Yet their efficacy is often limited by poor immunogenicity, attributed to inadequate priming and generation of antitumor T cells by dendritic cells (DCs). Identifying biomarkers to enhance DC functions in such tumors is thus crucial. Tissue Inhibitor of Metalloproteinases-1 (TIMP-1), recognized for its influence on immune cells, has an underexplored relationship with DCs. Our research reveals a correlation between high TIMP1 levels in metastatic melanoma and increased CD8 + T cell infiltration and survival. Network studies indicate a functional connection with HLA genes. Spatial transcriptomic analysis of a national melanoma cohort revealed that TIMP1 expression in immune compartments associates with an HLA-A/MHC-I peptide loading signature in lymph nodes. Primary human and bone-marrow-derived DCs secrete TIMP-1, which notably increases MHC-I expression in classical type 1 dendritic cells (cDC1), especially under melanoma antigen exposure. TIMP-1 affects the immunoproteasome/TAP complex, as seen by upregulated PSMB8 and TAP-1 levels of myeloid DCs. This study uncovers the role of TIMP-1 in DC-mediated immunogenicity with insights into CD8 + T cell activation, providing a foundation for mechanistic exploration and highlighting its potential as a new target for combinatorial immunotherapy to enhance ICT effectiveness.

Time-course and muscle-specific gene expression of matrix metalloproteinases and inflammatory cytokines in response to acute treadmill exercise in rats.

BACKGROUND: The extracellular matrix (ECM) of skeletal muscle plays a pivotal role in tissue repair and growth, and its remodeling tightly regulated by matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammatory cytokines. This study aimed to investigate changes in the mRNA expression of MMPs (Mmp-2 and Mmp-14), TIMPs (Timp-1 and Timp-2), and inflammatory cytokines (Il-1ß, Tnf-α, and Tgfß1) in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats following acute treadmill exercise. Additionally, muscle morphology was examined using hematoxylin and eosin (H&E) staining. METHODS AND RESULTS: Male rats were subjected to acute treadmill exercise at 25 m/min for 60 min with a %0 slope. The mRNA expression of ECM components and muscle morphology in the SOL and EDL were assessed in both sedentary and exercise groups at various time points (immediately (0) and 1, 3, 6, 12, and 24 h post-exercise). Our results revealed a muscle-specific response, with early upregulation of the mRNA expression of Mmp-2, Mmp-14, Timp-1, Timp-2, Il-1ß, and Tnf-α observed in the SOL compared to the EDL. A decrease in Tgfß1 mRNA expression was evident in the SOL at all post-exercise time points. Conversely, Tgfß1 mRNA expression increased at 0 and 3 h post-exercise in the EDL. Histological analysis also revealed earlier cell infiltration in the SOL than in the EDL following acute exercise. CONCLUSIONS: Our results highlight how acute exercise modulates ECM components and muscle structure differently in the SOL and EDL muscles, leading to distinct muscle-specific responses.

Investigation of diabetes-related molecular changes in embryo-endometrium crosstalk.

The primary aim of this study was to explore the impact of diabetes on matrix metalloproteases and tissue inhibitors, crucial factors for successful implantation, and to elucidate the molecular mechanisms that undergo changes in the endometrium and the embryo during diabetic pregnancies. In this investigation, we established a streptozotocin-induced diabetic pregnant rat model. Microarray analysis followed by RT-PCR was utilized to identify gene regions exhibiting expression alterations. Subsequently, we assessed the effects of MMPs and tissue inhibitors using ELISA and immunohistochemistry techniques, in addition to analyzing changes at the genetic level. Diabetes led to the upregulation of MMP3, MMP9, and MMP20 on the 6.5th day of pregnancy, while causing the downregulation of MMP3, MMP9, and MMP11 on the 8.5th day of pregnancy. TIMP1 expression was downregulated on the 8.5th day compared to the control group. No statistically significant differences were observed between the groups regarding other TIMP expressions. KEGG pathway analysis revealed that diabetes induced alterations in the expression of genes associated with certain microRNAs, as well as signaling pathways such as cAMP, calcium, BMP, p53, MAPK, PI3K-Akt, Jak-STAT, Hippo, Wnt, and TNF. Additionally, gene ontology analysis unveiled changes in membrane structures, extracellular matrix, signaling pathways, ion binding, protein binding, cell adhesion molecule binding, and receptor-ligand activity. This study serves as a valuable guide for investigating the mechanisms responsible for complications in diabetic pregnancies. By revealing the early-stage effects of diabetes, it offers insight into the development of new diagnostic and treatment approaches, ultimately contributing to improved patient care.

HLA-B and TIMP1 as hub genes of the ventricular remodeling caused by hypertension.

RATIONALE: Myocardial fibrosis is an important pathological change that occurs during ventricular remodeling in patients with hypertension and is an important pathophysiological basis of cardiovascular disease. However, the molecular mechanism underlying this ventricular remodeling is unclear. METHODS: Bioinformatics analysis identified HLA-B and TIMP1 as hub genes in the process of myocardial fibrosis. Expression and correlation analyses of significant hub genes with ventricular remodeling were performed. Weighted gene co-expression network analysis (WGCNA) was performed to verify the role of HLA-B. ceRNA network was constructed to identify the candidate molecule drugs. Receiver operating characteristic (ROC) curves were analyzed. RESULTS: RT-qPCR was performed to verify the roles of HLA-B and TIMP1 in seven control individuals with hypertension and seven patients with hypertension and ventricular remodeling. The WGCNA showed that HLA-B was in the brown module and the correlation coefficient between HLA-B and ventricular remodeling was 0.67. Based on univariate logistic proportional regression analysis, HLA-B influences ventricular remodeling (P<0.05). RT-qPCR showed that the relative expression levels of HLA-B and TIMP1 were significantly higher in HLVR samples compared with their expression in the control group. CONCLUSIONS: HLA-B and TIMP1 might provide novel research targets for the diagnosis and treatment of HLVR.

Ability of NAD and Sirt1 to epigenetically suppress albuminuria.

The time for diabetic nephropathy (DN) to progress from mild to severe is long. Thus, methods to continuously repress DN are required to exert long-lasting effects mediated through epigenetic regulation. In this study, we demonstrated the ability of nicotinamide adenine dinucleotide (NAD) and its metabolites to reduce albuminuria through Sirt1- or Nampt-dependent epigenetic regulation. We previously reported that proximal tubular Sirt1 was lowered before glomerular Sirt1. Repressed glomerular Sirt1 was found to epigenetically elevate Claudin-1. In addition, we reported that proximal tubular Nampt deficiency epigenetically augmented TIMP-1 levels in Sirt6-mediated pathways, leading to type-IV collagen deposition and diabetic fibrosis. Altogether, we propose that the Sirt1/Claudin-1 axis may be crucial in the onset of albuminuria at the early stages of DN and that the Nampt/Sirt6/TIMP-1 axis promotes diabetic fibrosis in the middle to late stages of DN. Finally, administration of NMN, an NAD precursor, epigenetically potentiates the regression of the onset of DN to maintain Sirt1 and repress Claudin-1 in podocytes, suggesting the potential use of NAD metabolites as epigenetic medications for DN.

Predictive Model to Identify the Long Time Survivor in Patients with Glioblastoma: A Cohort Study Integrating Machine Learning Algorithms.

We aimed to develop and validate a predictive model for identifying long-term survivors (LTS) among glioblastoma (GB) patients, defined as those with an overall survival (OS) of more than 3 years. A total of 293 GB patients from CGGA and 169 from TCGA database were assigned to training and validation cohort, respectively. The differences in expression of immune checkpoint genes (ICGs) and immune infiltration landscape were compared between LTS and short time survivor (STS) (OS<1.5 years). The differentially expressed genes (DEGs) and weighted gene co-expression network analysis (WGCNA) were used to identify the genes differentially expressed between LTS and STS. Three different machine learning algorithms were employed to select the predictive genes from the overlapping region of DEGs and WGCNA to construct the nomogram. The comparison between LTS and STS revealed that STS exhibited an immune-resistant status, with higher expression of ICGs (P<0.05) and greater infiltration of immune suppression cells compared to LTS (P<0.05). Four genes, namely, OSMR, FMOD, CXCL14, and TIMP1, were identified and incorporated into the nomogram, which possessed good potential in predicting LTS probability among GB patients both in the training (C-index, 0.791; 0.772-0.817) and validation cohort (C-index, 0.770; 0.751-0.806). STS was found to be more likely to exhibit an immune-cold phenotype. The identified predictive genes were used to construct the nomogram with potential to identify LTS among GB patients.

Identification and validation of anoikis-related genes to clarify the prognosis and immune mechanisms of patients with low-grade glioma.

BACKGROUND: Low-grade glioma (LGG) has an extremely poor prognosis, and the mechanism leading to malignant development has not been determined. The aim of our study was to clarify the function and mechanism of anoikis and TIMP1 in the malignant progression of LGG. METHODS: We screened 7 anoikis-related genes from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to construct a prognostic-predicting model. The study assessed the clinical prognosis, pathological characteristics, and immune cell infiltration in both high- and low-risk groups. Additionally, the potential modulatory effects of TIMP1 on proliferation, migration, and anoikis in LGG were investigated both in vivo and in vitro. RESULTS: In this study, we identified seven critical genes, namely, PTGS2, CCND1, TIMP1, PDK4, LGALS3, CDKN1A, and CDKN2A. Kaplan‒Meier (K‒M) curves demonstrated a significant correlation between clinical features and overall survival (OS), and single-cell analysis and mutation examination emphasized the heterogeneity and pivotal role of hub gene expression imbalances in LGG development. Immune cell infiltration and microenvironment analysis further elucidated the relationships between key genes and immune cells. In addition, TIMP1 promoted the malignant progression of LGG in both in vitro and in vivo models. CONCLUSIONS: This study confirmed that TIMP1 promoted the malignant progression of LGG by inhibiting anoikis, providing insights into LGG pathogenesis and potential therapeutic targets.

Network pharmacology and experimental validation to reveal the pharmacological mechanisms of Sini decoction against renal fibrosis.

OBJECTIVE: To investigate the mechanism by which Sini decoction (, SND) improves renal fibrosis (Rf) in rats based on transforming growth factor ß1/Smad (TGF-ß1/Smad) signaling pathway. METHODS: Network pharmacology was applied to obtain potentially involved signaling pathways in SND's improving effects on Rf. The targets of SND drug components and the targets of Rf were obtained by searching databases, such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCSMP) and GeenCard. The intersection targets of two searches were obtained and underwent signaling pathway analysis using a Venn diagram. Then experimental pharmacology was utilized to prove and investigate the effects of SND on target proteins in the TGF-ß1/Smad signaling pathway. The Rf rat model was established by unilateral ureteral occlusion (UUO). The expression levels of transforming growth factor, matrix metalloproteinase-9 (MMP-9), matrix metal protease-2 (MMP-2), connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1) were determined by Masson staining of rat renal tissue, and immunohistochemical methods. The expression levels of Smad3, Smad2, and Smad7 in renal tissue were determined by Western blotting (WB). The mechanism of the improving effects of SND on Rf was investigated based on TGF-ß1/Smad signaling pathway. RESULTS: A total of 12 drug components of Fuzi (Radix Aconiti Lateralis Preparata), 5 drug components of Ganjiang (Rhizoma Zingiber), and 9 drug components of Gancao (Radix Glycy et Rhizoma) were obtained from the database search, and 207 shared targets were found. A total of 1063 Rf targets were found in the database search. According to the Venn diagram, in total, 96 intersection targets were found in two database searches. The metabolic pathways involved included TGF-ß signaling pathway, phosphatidylinositol-3-kinase/serine-threonine protein kinase signaling (PI3K/Akt) pathway, and hypoxia-inducible factor-1 (HIF-1) signaling pathway. Masson staining analysis showed that compared with the model group, the renal interstitial collagen deposition levels in the SSN and SND groups were significantly lower (P < 0.05). Immunohistochemical analysis, compared with the control group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the model group were significantly decreased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-ß1 were significantly increased (P < 0.01). Compared with the model group, the positive cell area expression levels of MMP-9/TIMP-1 and MMP-2/TIMP-1 in the kidney tissue of the SSN and SND groups were significantly increased (P < 0.05, P < 0.01), and the positive cell area expression levels of CTGF and TGF-ß1 in the kidney tissue were significantly decreased (P < 0.05, P < 0.01). WB results showed that the SSN group and the SND group could reduce the expression of Smad2 and Smad3 (P < 0.05) and increase the expression of Smad7 (P < 0.05).

GnT-V-mediated aberrant N-glycosylation of TIMP-1 promotes diabetic retinopathy progression.

BACKGROUND: Vascular endothelial growth factor (VEGF) signaling pathway plays an important role in the progression of diabetic retinopathy (DR). The glycosylation modification process of many key functional proteins in DR patients is abnormal. However, the potential involvement of abnormal N-glycoproteins in DR progression remains unclear. METHODS: Glycoproteomic profiling of the vitreous humor was performed. The level of protein and N-glycoprotein was confirmed by Western blot and Lectin blot, respectively. The cell viability and migration efficiency were detected by CCK-8 and Transwell assay. Flow cytometry was conducted to analyze the level of cell apoptosis and reactive oxygen specie. Malondialdehyde, superoxide dismutase activity and VEGF content were detected by Enzyme linked immunosorbent assays. The interaction of metalloproteinase 1 (TIMP-1) with N-acetylglucosamine transferase V (GnT-V) was detected by GST pull-down. Hematoxylin and eosin staining and choroidal and retinal flat mount stained with fluorescein isothiocyanate-Dextran assay were used for functional research in vivo. RESULTS: We found that N-glycosylation was up-regulated in DR rats and high glucose (HG)-induced human retinal pigment epithelium cell line ARPE-19. HG-induced inhibited the viability of ARPE-19 cells and promoted cell apoptosis and oxidative stress (OS), but these effects were reversed with kifunensine treatment, GnT-V knockdown and TIMP-1 mutation. Additionally, GnT-V binds to TIMP-1 to promote N-glycosylation of TIMP-1. Over-expression of GnT-V inhibited the viability of ARPE-19 cells and promoted cell apoptosis, OS and VEGF release, which these effects were reversed with TIMP-1 mutation. Interestingly, over-expression of GnT-V promoted retinal microvascular endothelial cells (RMECs) angiogenesis but was revered with TIMP-1 mutation, which was terminally boosted by VEGF-A treatment. Finally, knockdown of GnT-V relieved DR progression. CONCLUSION: The findings indicate that GnT-V can promote RMECs angiogenesis and ARPE-19 cells injury through activation VEGF signaling pathway by increasing TIMP-1 N-glycosylation level, which provides a new theoretical basis for the prevention of DR.

Aberrant TIMP-1 production in tumor-associated fibroblasts drives the selective benefits of nintedanib in lung adenocarcinoma.

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors.

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.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The role of sinusoidal endothelial cells and TIMP1 in the regulation of fibrosis in a novel human liver 3D NASH model.

BACKGROUND: The prevalence of NAFLD is rapidly increasing. NAFLD can progress to NASH, fibrosis, cirrhosis, and HCC, which will soon become the main causes of liver transplantation. To date, no effective drug for NASH has been approved by the Food and Drug Administration. This is partly due to the lack of reliable human in vitro models. Here, we present a novel human liver spheroid model that can be used to study the mechanisms underlying liver fibrosis formation and degradation. METHODS AND RESULTS: Such spheroids, which contain hepatocytes, stellate cells, KC, and LSECs, spontaneously develop fibrosis that is exacerbated by treatment with free fatty acids. Conditioned medium from activated LSECs caused similar activation of fibrosis in spheroids containing primary human hepatocyte and NPCs, indicating the action of soluble mediators from the LSECs. Spheroids containing LSECs treated with free fatty acids produced tissue inhibitor of metalloproteinases inhibitor 1, a matrix metalloproteinases inhibitor important for fibrosis progression. Tissue inhibitor of metalloproteinases inhibitor 1 knockdown using siRNA led to a reduction in collagen and procollagen accumulation, which could be partially rescued using a potent matrix metalloproteinases inhibitor. Interestingly, tissue inhibitor of metalloproteinases inhibitor 1 was found to be expressed at higher levels, specifically in a subtype of endothelial cells in the pericentral region of human fibrotic livers, than in control livers. CONCLUSION: Potential anti-NASH drugs and compounds were evaluated for their efficacy in reducing collagen accumulation, and we found differences in specificity between spheroids with and without LSECs. This new human NASH model may reveal novel mechanisms for the regulation of liver fibrosis and provide a more appropriate model for screening drugs against NASH.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Further structural optimization and SAR study of sungsanpin derivatives as cell-invasion inhibitors.

Metastasis is one of the major causes of death in patients with cancer, and cell invasion plays a fundamental part in this process. Because of the absence of efficacious treatments, caring for these patients is challenging. Recently, we optimized the structure of the naturally occurring lasso peptide sungsanpin. We identified two peptides, octapeptide S3 and cyclic peptide S4, which inhibited invasion into A549 cells effectively. We undertook an alanine scan of S3 to explore the structure-activity relationship. The linear octapeptide S3-4 and cyclic peptide S4-1 exhibited improved inhibition of invasion into A549 cells. We modified S3-4 to obtain S3-4K, which displayed much higher inhibitory activity against invasion into A549 cells than S3-4. Of all peptides tested, S4-1 upregulated significantly mRNA of tissue inhibitor matrix metalloproteinase TIMP-1 and TIMP-2.

Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. Astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. Here, we performed an unbiased proteomic analysis and identified a fibronectin-derived peptide called Anastellin (Ana) that was unique to the Timp1KO astrocyte secretome. Ana was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Ana is known to act upon the sphingosine-1-phosphate receptor 1, and we determined that Ana also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-experimental autoimmune encephalomyelitis ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 (Timp1KO) had no effect. Analysis of Timp1 and fibronectin (FN1) transcripts from primary human astrocytes from healthy and multiple sclerosis (MS) donors revealed lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Last, analyses of proteomic databases of MS samples identified Ana peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high disease activity. A role for Ana in MS as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and innate remyelination potential in the MS brain.