TIMP-1 Expression Research Articles

Effects of MMP2 and its inhibitor TIMP2 on DNA damage, apoptosis and senescence of human lens epithelial cells induced by oxidative stress.

Oxidative stress-induced lens epithelial cells (LECs) death plays a pivotal role in pathogenesis of age-related cataract (ARC), causing significant visual impairment. Apoptosis of porcine granulosa cells mediated by MMP2 is linked to DNA damage. The current study aimed to investigate the potential mechanism of MMP2 in DNA damage, apoptosis and senescence of lens epithelial cells caused by oxidative stress. HLE-B3 cells were treated with different doses of H2O2 for 24h, and CCK-8 was used to detect cell viability. Furthermore, western blotting was used to detect the expressions of MMP2, Bcl2, Bax, cleaved caspase3, γ-H2AX, p16, p21, and TIMP2. DCFH-DA staining was used to assess ROS levels. Moreover, EdU staining was used to detect cell proliferation, and flow cytometry was used to detect cell apoptosis. Then, 15A3 immunofluorescence staining and γ-H2AX staining were used to detect DNA damage. In addition, SA-β-gal staining was used to observe cell senescence. The present findings suggest that oxidative stress triggers damage to LECs viability and elevates the expression of MMP2. Furthermore, MMP2 interference attenuates H2O2-induced active damage, apoptosis, DNA damage, and cellular senescence in LECs. Additionally, TIMP2 expression is down-regulated in H2O2-induced LECs, which suppresses the expression of MMP2 induced by H2O2. These findings highlight the crucial role of MMP2 and TIMP2 in the modulation of oxidative stress-induced cellular responses in LECs. Collectively, TIMP2 alleviates H2O2-induced lens epithelial cell viability damage, apoptosis, DNA damage and cell senescence in LECs by inhibiting MMP2.

Thrombopoietin mimetic reduces mouse lung inflammation and fibrosis after radiation by attenuating activated endothelial phenotypes.

Radiation-induced lung injury (RILI) initiates radiation pneumonitis and progresses to fibrosis as the main side effect experienced by patients with lung cancer treated with radiotherapy. There is no effective drug for RILI. Sustained vascular activation is a major contributor to the establishment of chronic disease. Here, using a whole thoracic irradiation (WTI) mouse model, we investigated the mechanisms and effectiveness of thrombopoietin mimetic (TPOm) for preventing RILI. We demonstrated that administering TPOm 24 hours before irradiation decreased histologic lung injury score, apoptosis, vascular permeability, expression of proinflammatory cytokines, and neutrophil infiltration in the lungs of mice 2 weeks after WTI. We described the expression of c-MPL, a TPO receptor, in mouse primary pulmonary microvascular endothelial cells, showing that TPOm reduced endothelial cell-neutrophil adhesion by inhibiting ICAM-1 expression. Seven months after WTI, TPOm-treated lung exhibited less collagen deposition and expression of MMP-9, TIMP-1, IL-6, TGF-β, and p21. Moreover, TPOm improved lung vascular structure, lung density, and respiration rate, leading to a prolonged survival time after WTI. Single-cell RNA sequencing analysis of lungs 2 weeks after WTI revealed that TPOm shifted populations of capillary endothelial cells toward a less activated and more homeostatic phenotype. Taken together, TPOm is protective for RILI by inhibiting endothelial cell activation.

Quercetin Alleviates Scleral Remodeling Through Inhibiting the PERK-EIF2α Axis in Experiment Myopia.

This study aims to investigate the effect of quercetin (QUE) on scleral remodeling by inhibiting the PERK-EIF2α signaling pathway and to evaluate its potential role in slowing myopia. Lens-induced myopia (LIM) guinea pigs were obtained and treated with QUE. After 4 and 6 weeks of treatments, ocular biological measurements were conducted. Hematoxylin and eosin (H&E) staining was used to observe the changes in scleral morphology and thickness, and Masson staining was used to examine scleral collagen fiber arrangement. Quantitative PCR (qPCR) and Western bolt were utilized to detect the mRNA and protein expression of PERK, EIF2α, MMP-2, TIMP-2, and collagen I in the scleral tissues. Calcium ion flow in each group was measured using noninvasive micro-test technology, and reactive oxygen species levels were detected by flow cytometry. Compared with the LIM group, the ocular measurements showed that the refractive errors and axial length of the eyes were significantly reduced in the LIM+QUE group (P < 0.01). H&E and Masson staining showed that sclera in the LIM+QUE group was thickened, collagen was dense, and the fiber gap was reduced. In the LIM+QUE group, the expression levels of PERK, EIF2α, and MMP-2 were decreased, whereas the expression levels of TIMP-2 and collagen I were increased. Calcium release and reactive oxygen species (ROS) in the LIM+QUE group were decreased. Quercetin ameliorates scleral remodeling in myopic guinea pigs by inhibiting the PERK-EIF2α signaling pathway, thereby alleviating the progression of myopia. These findings provide new experimental evidence for the potential application of quercetin in myopia prevention and treatment.

Evidence of Browning and Inflammation Features in Visceral Adipose Tissue of Women with Endometriosis

Patients with endometriosis tend to have a low body mass index, suggesting an inverse relationship between body fat and risk of disease. This is supported by evidence that miRNAs differentially expressed in endometriosis induce browning of pre-adipocytes in vitro. Thus, we hypothesize that endometriosis may underlie adipose tissue (AT) dysfunction and browning. Identify inflammation and browning processes in AT collected from endometriosis patients. Visceral and subcutaneous AT samples were obtained during endometriosis (n = 32) or uterine myoma (n = 14; controls) surgery. Blood catecholamines were determined by high-performance liquid chromatography while IL-6 and TGF-β levels were quantified by ELISA. Adipocyte cross-sectional areas were analyzed in H&E-stained sections by computer-assisted morphometry. Macrophages (F4/80; Galectin-3) and browning activation (UCP-1; PGC-1α) in tissues were identified by dual label immunofluorescence. Expression of inflammatory (IL-6; MCP-1; Galectin-3; CD206; TIMP1; TGF-β) and browning-related (UCP-1; PGC-1α; DIO2; CITED1; CIDEA; TMEM26; TBX1; PRDM16; PPAR-γ) molecules in AT were assessed by RT-PCR and Western blotting. Compared to controls, patients presented smaller adipocytes, especially in VAT, and lower norepinephrine levels. Serum IL-6, but not TGF-β, was increased in patients. UCP-1, PGC-1α, IL-6, and MCP-1 were upregulated in VAT from endometriosis women, which also evidenced a reduction of CD206, relative to controls. However, no differences were found in mRNA expression of IL-6, TIMP1, and TGF-β nor Galectin-3 protein levels. In SAT, protein expression remained unchanged between patients and controls. Our findings support an endometriosis' role as a pro-catabolic state along with local signals of VAT browning and inflammation.

Enhancing human capillary tube network assembly and maturation through upregulated expression of pericyte-derived TIMP-3.

In this study, we identify and characterize new molecular determinants that optimize human capillary tube network assembly. Our lab has previously reported a novel, serum free-defined 3D co-culture model using human endothelial cells (ECs) and human pericytes whereby EC-lined tubes form and co-assemble with pericytes, but when these cultures are maintained at or beyond 5 days, tubes become progressively wider and unstable. To address this issue, we generated novel human pericytes that carry a tissue inhibitor of metalloproteinase (TIMP)-3 transgene which can be upregulated following doxycycline addition. EC-pericyte co-cultures established in the presence of doxycycline demonstrated marked enhancement of capillary network assembly including dramatic narrowing of capillary tube widths to an average of 8µm (physiologic capillary tube width), increased tube lengths, increased tube branching, and robust stimulation of basement membrane matrix assembly, particularly with collagen type IV and fibronectin deposition compared to controls. These substantial changes depend not only on induction of pericyte TIMP-3, but also on recruitment of pericytes to EC tubes. Blockade of pericyte recruitment prevents these dramatic capillary network alterations suggesting that EC-pericyte interactions and induction of pericyte TIMP-3 are necessary together to coordinate and facilitate capillary assembly and maturation. Overall, this work is critical for our basic understanding of capillary formation, but also for the ability to reproducibly generate stabilized networks of capillary tubes.

MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, MUC1 and CD29 Expression in Endometrial Polyps at Implantation Window Compared to Neighbouring Normal Endometrium

MMP-1, MMP-2, MMP-9, TIMP-1, TIMP-2, MUC1 and CD29 Expression in Endometrial Polyps at Implantation Window Compared to Neighbouring Normal Endometrium

Jun modulates endoplasmic reticulum stress-associated ferroptosis in dorsal root ganglia neurons during neuropathic pain by regulating Timp1

Neuropathic pain (NP) is a complex disorder caused by lesions or diseases affecting the somatosensory nervous system, severely impacting patients' quality of life. Recent studies suggest ferroptosis may be involved in NP induction, but its precise mechanisms remain unclear. We used GO and KEGG pathway enrichment analyses to functionally annotate ferroptosis-related differentially expressed genes (FRDs). Through STRING and the maximum cluster centrality (MCC) algorithm, we identified five hub FRDs (Jun, Timp1, Egfr, Cdkn1a, Cdkn2a). Single-cell analysis revealed significant expression of Jun and Timp1 in neurons. Our study confirmed the association between ferroptosis and endoplasmic reticulum stress (ERS) in NP and validated changes in hub FRD expression across various NP animal models. In vitro experiments demonstrated that Jun regulates neuronal ferroptosis and ERS, particularly by modulating Timp1 expression. Transcription factor prediction and JASPAR binding site analysis elucidated the regulatory network involving Jun. ROC curve analysis of external datasets highlighted the diagnostic potential of hub FRDs and ERS-related differentially expressed genes (ERSRDs) in NP. Using the Comparative Toxicogenomics Database (CTD), we identified estradiol (E2) as a potential therapeutic drug targeting hub FRDs and ERSRDs. Molecular docking predicted its binding sites with Jun and Timp1, and in vivo experiments confirmed that E2 alleviated NP and reversed the expression of Jun and Timp1. This study underscores the crucial role of Jun and Timp1 in the interplay between ferroptosis and ERS, offering new insights and promising avenues for NP treatment.

Matrix metalloproteinases mediate influenza A-associated shedding of the alveolar epithelial glycocalyx.

The alveolar epithelium is protected by a heparan sulfate-rich, glycosaminoglycan layer called the epithelial glycocalyx. It is cleaved in patients with acute respiratory distress syndrome (ARDS) and in murine models of influenza A (IAV) infection, shedding fragments into the airspace from the cell surface. Glycocalyx shedding results in increased permeability of the alveolar-capillary barrier, amplifying acute lung injury. The mechanisms underlying alveolar epithelial glycocalyx shedding in IAV infection are unknown. We hypothesized that induction of host sheddases such as matrix metalloproteinases (MMPs) during IAV infection results in glycocalyx shedding and increased lung injury. We measured glycocalyx shedding and lung injury during IAV infection with and without treatment with the pan-MMP inhibitor Ilomastat (ILO) and in an MMP-7 knock out (MMP-7KO) mouse. C57BL/6 or MMP-7KO male and female mice were given IAV A/PR/8/34 (H1N1) at 30,000 PFU/mouse or PBS intratracheally. For some experiments, C56BL/6 mice were infected in the presence of ILO (100mg/kg) or vehicle given daily by IP injection. Bronchoalveolar lavage (BAL) and lung tissue were collected on day 1, 3, and 7 for analysis of glycocalyx shedding (BAL Syndecan-1) and lung injury (histology, BAL protein, BAL cytokines, BAL immune cell infiltrates, BAL RAGE). Expression and localization of the sheddase MMP-7 and its inhibitor TIMP-1 was examined by RNAScope. For in vitro experiments, MLE-12 mouse lung epithelial cells were cultured and treated with active or heat-inactivated heparinase (2.5 U/mL) prior to infection with IAV (MOI 1) and viral load and MMP-7 and TIMP-1 expression analyzed. IAV infection caused shedding of the epithelial glycocalyx into the BAL. Inhibition of MMPs with ILO reduced glycocalyx shedding by 36% (p = 0.0051) and reduced lung epithelial injury by 40% (p = 0.0404). ILO also reduced viral load by 68% (p = 0.027), despite having no significant effect on lung cytokine production. Both MMP-7 and its inhibitor TIMP-1 were upregulated in IAV infected mice: MMP-7 colocalized with IAV, while TIMP-1 was limited to cells adjacent to infection. However, MMP-7KO mice had similar glycocalyx shedding, epithelial injury, and viral load compared to WT littermates, suggesting redundancy in MMP sheddase function in the lung. In vitro, heparinase treatment before infection led to a 52% increase in viral load (p = 0.0038) without altering MMP-7 or TIMP-1 protein levels. Glycocalyx shedding and MMPs play key roles in IAV-induced epithelial injury, with significant impact on IAV viral load. Further studies are needed to understand which specific MMPs regulate lung epithelial glycocalyx shedding.

Human hypofunctional NCF1 variants promote pulmonary fibrosis in the bleomycin-induced mouse model and patients with systemic sclerosis via expansion of SPP1+ monocytes-derived macrophages

ObjectiveWe assessed the role of a systemic lupus erythematosus causal hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in systemic sclerosis (SSc).MethodsAssociation of NCF1-H90 with SSc was performed in...

Greater TIMP-1 protein levels and neointimal formation represent sex-dependent cellular events limiting aortic vessel expansion in female rats.

Fragmentation/loss of the structural protein elastin represents the precipitating event translating to aortic expansion and subsequent aneurysm formation. The present study tested the hypothesis that greater protein expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and neointimal growth secondary to a reduction of medial elastin content represent sex-dependent events limiting aortic vessel expansion in females. TIMP-1 protein levels were higher in the ascending aorta of female versus male patients diagnosed with a bicuspid aortic valve (BAV). The latter paradigm was recapitulated in the aorta of adult male and female rats complemented by greater TIMP-2 expression in females. CaCl2 (0.5 M) treatment of the infrarenal aorta of adult male and female rats increased the in situ vessel diameter and expansion was significantly smaller in females despite a comparable reduction of medial elastin content. The preferential appearance of a neointimal region of the CaCl2-treated infrarenal aorta of female rats may explain in part the smaller in situ expansion and neointimal growth correlated positively with the % change of the in situ diameter. Neointimal formation was secondary to a significant increase in the density of medial/neointimal vascular smooth muscle cells (VSMCs) that re-entered the G2-M phase whereas VSMC cell cycle re-entry was attenuated in the CaCl2-treated infrarenal aorta of male rats. Thus, greater TIMP-1 expression in the aorta of female BAV patients may prevent excessive elastin fragmentation and preferential neointimal growth following CaCl2-treatment of the infrarenal aorta of female rats represents a sex-dependent biological event limiting vessel expansion secondary to a significant loss of the structural protein.

SIRT1 mediates the inflammatory response of macrophages and regulates the TIMP3/ADAM17 pathway in atherosclerosis

ObjectiveMacrophage polarization and the resulting phenotype have versatile roles in atherosclerosis. The study aims to decipher the role of SIRT1 in regulating macrophage phenotypes and atherosclerosis development. MethodsTwo mouse lines of SIRT1△Mac/ApoE−/− and SIRT1fl/fl/ApoE−/− were fed with high-fat diet to generate atherosclerotic lesion. Mouse peritoneal macrophages were isolated and transfected with SIRT1-overexpressing vector or vector-null. ResultsThe SIRT1△Mac/ApoE−/− mice exhibited greater atherosclerotic lesions, stronger immunofluorescence staining for M1-like macrophage marker, iNOS, and weaker immunofluorescence staining for M2-like macrophage marker, Arginase-1, than the SIRT1fl/fl/ApoE−/− littermates. The gene expressions of M1 markers (IL-1β, IL-6, and iNOS) were increased and those of M2 markers (IL-10 and Arg-1) decreased in both aortic roots and peritoneal macrophages from SIRT1△Mac/ApoE−/− mice, whereas SIRT1 overexpression rectified the changes in M1/M2 expression. A declined expression of TIMP3 with an increased expression of ADAM17 was noted in SIRT1△Mac/ApoE−/− macrophages, whereas SIRT1 overexpression rescued TIMP3 expression and inhibited ADAM17 expression. ConclusionOur data suggest that SIRT1 deficiency may promote macrophage M1 polarization and regulate the TIMP3/ADAM17 pathway thus favoring atherosclerosis development, indicating an anti-atherosclerotic role of macrophage SIRT1.

Single-Cell RNA Sequencing Reveals Transcriptional Signatures and Cell-Cell Communication in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a major cause of vision loss in workingage individuals worldwide. Cell-to-cell communication between retinal cells and retinal pigment epithelial cells (RPEs) in DR is still unclear, so this study aimed to generate a single-cell atlas and identify receptor‒ligand communication between retinal cells and RPEs. A mouse single-cell RNA sequencing (scRNA-seq) dataset was retrieved from the GEO database (GSE178121) and was further analyzed with the R package Seurat. Cell cluster annotation was performed to further analyze cell‒cell communication. The differentially expressed genes (DEGs) in RPEs were explored through pathway enrichment analysis and the protein‒ protein interaction (PPI) network. Core genes in the PPI were verified by quantitative PCR in ARPE-19 cells. We observed an increased proportion of RPEs in STZ mice. Although some overall intercellular communication pathways did not differ significantly in the STZ and control groups, RPEs relayed significantly more signals in the STZ group. In addition, THBS1, ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 were found to be the core DEGs of the PPI network in RPEs. qPCR results showed that the expression of ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 was higher and consistent with scRNA-seq results in ARPE-19 cells under hyperglycemic conditions. Our study, for the first time, investigated how signals that RPEs relay to and from other cells underly the progression of DR based on scRNA-seq. These signaling pathways and hub genes may provide new insights into DR mechanisms and therapeutic targets.

Estrogen-dependent gene regulation: Molecular basis ofTIMP-1 as a sex-specific biomarker for acute lung injury.

Increased circulating tissue inhibitor of metalloproteinases-1 (TIMP-1) levels have been observed in patients with acute lung injury (ALI). However, the sex-specific regulation of TIMP-1 and the underlying molecular mechanisms have not been well elucidated. In this study, we found that plasma TIMP-1 levels were significantly higher in COVID-19 and H1N1 patients compared with those in healthy subjects (n = 25). TIMP-1 concentrations were significantly different between males and females in each disease group. Among female but not male patients, TIMP-1 levels significantly correlated with the PaO2/FiO2 ratio and hospital length of stay. Using the mouse model of ALI induced by the H1N1 virus, we found that TIMP-1 is strikingly induced in PDGFRα-positive cells in the murine lungs. Moreover, female mice showed a higher Timp-1 expression in the lungs on day 3 postinfection. Mechanistically, we observed that estrogen can upregulate TIMP-1 expression in lung fibroblasts, not epithelial cells. In addition, overexpression of estrogen receptor α (ERα) increased the TIMP-1 promoter activity. In summary, TIMP-1 is an estrogen-responsive gene, and its promoter activity is regulated by ERα. Circulating TIMP-1 may serve as a sex-specific marker, reflecting the severity and worst outcomes in female patients with SARS-CoV2- and IAV-related ALI.

A narrative review of genes associated with liver fibrosis in biliary atresia.

Biliary atresia (BA) is characterized by biliary inflammation and obstruction. In the later phase, liver fibrosis occurs. Although the etiology of BA is believed to be multi-factorial, genetic predisposition has been proposed to play a critical role in the pathogenesis. This review aimed to provide an updated summary of the genes that have been reported to be involved in BA-associated liver fibrosis. The review was conducted via evaluation of MalaCards (BA disease: MalaCards-research articles, drugs, genes, clinical trials) which is a universally applied website including various human disease database. The database of genes that are involved in liver fibrosis were studied. Thirty-one genes that are associated with BA according to the disease relevance score were reviewed after further evaluations. Eleven genes (GPT, NR1H4, TGF-B1, MMP7, CCN2, TIMP1, SPP1, ADD3, KRT7, ADD3-AS1, SOX9) that are specific and with a potential association with liver fibrosis were selected for detailed description. Increased expression of GPT, TGF-B1, MMP7, CCN2, TIMP1, SPP1, ADD3, KRT7 and ADD3-AS1 maybe associated with the development of liver fibrosis in BA patients, while the expression of NR1H4 and SOX9 are more likely to suppress liver fibrosis. Current scientific evidence using gene database has revealed a close association between genetic anomalies and the pathogenesis of liver fibrosis in BA. With a better understanding of these anomalies, therapy targeting these related genes may be a new therapeutic approach to alleviate liver fibrosis in BA.

Expression of matrix metalloproteinases and their inhibitors in corneal stromal fibroblasts and keratocytes from healthy and keratoconus corneas.

To examine the in-vitro expression of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in corneal stromal cells by distinguishing between fibroblasts and keratocytes of healthy and keratoconus (KC) corneas. Stromal cells were isolated from healthy and KC corneas (n = 8). A normal-glucose, serum-containing cell culture medium (NGSC-medium) was used for cultivation of healthy human corneal fibroblasts (HCFs) and KC human corneal fibroblasts (KC-HCFs). In order to obtain a keratocyte phenotype, the initial cultivation with NGSC-medium was changed to a low-glucose, serum-free cell culture medium for healthy (Keratocytes) and KC cells (KC-Keratocytes). Gene and protein expression of MMP-1, -2, -3, -7, -9 and TIMP-1, -2, -3 were measured by quantitative PCR and Enzyme-Linked Immunosorbent Assay (ELISA) from the cell culture supernatant. KC-HCFs demonstrated a lower mRNA gene expression for MMP-2 compared to HCFs. In contrast to their respective fibroblast groups (either HCFs or KC-HCFs), Keratocytes showed a higher mRNA gene expression of TIMP-3, whereas TIMP-1 mRNA gene expression was lower in Keratocytes and KC-Keratocytes. Protein analysis of the cell culture supernatant revealed lower concentrations of MMP-1 in KC-HCFs compared to HCFs. Compared to Keratocytes, TIMP-1 concentrations was lower in the cell culture supernatant of KC-Keratocytes. In HCFs and KC-HCFs, protein levels of MMP-1 and TIMP-1 were higher and MMP-2 was lower compared to Keratocytes and KC-Keratocytes, respectively. This study indicates an imbalance in MMP and TIMP expression between healthy and diseased cells. Furthermore, differences in the expression of MMPs and TIMPs exist between corneal fibroblasts and keratocytes, which could influence the specific proteolytic metabolism in-vivo and contribute to the progression of KC.

Exploration of N6-methyladenosine modification in ascorbic acid 2-glucoside constructed stem cell sheets.

The aim of this study was to explore the mechanism of bone marrow stem cells (BMSCs) sheets constructed with different doses of Ascorbic acid 2-glucoside (AA-2G) in conjunction with N6-methyladenosine (m6A)-associated epigenetic genes analysing transcriptome sequencing data. Experimental groups of BMSCs induced by different AA-2G concentrations were set up, and the tissue structures were observed by histological staining of cell slices and scanning electron microscopy. Expression patterns of DEGs were analysed using short-time sequence expression mining software, and DEGs associated with m6A were selected for gene ontology analysis and pathway analysis. The protein-protein interaction (PPI) network of DEGs was analysed and gene functions were predicted using the search tool of the Retrieve Interacting Genes database. There were 464 up-regulated DEGs and 303 down-regulated DEGs between the control and high-dose AA-2G treatment groups, and 175 up-regulated DEGs and 37 down-regulated DEGs between the low and high-dose AA-2G treatment groups. The profile 7 exhibited a gradual increase in gene expression levels over AA-2G concentration. In contrast, profile 0 exhibited a gradual decrease in gene expression levels over AA-2G concentration. In the PPI network of m6A-related DEGs in profile 7, the cluster of metallopeptidase inhibitor 1 (Timp1), intercellular adhesion molecule 1(Icam1), insulin-like growth factor 1 (Igf1), matrix metallopeptidase 2(Mmp2), serpin family E member 1 (Serpine1), C-X-C motif chemokine ligand 2 (Cxcl2), galectin 3(Lgals3) and angiopoietin-1(Angpt1) was the top hub gene cluster. The expression of all hub genes was significantly increased after AA-2G intervention (P < 0.05), and the expression of Igf1 and Timp1 increased with increasing intervention concentration. The m6A epigenetic modifications were involved in the AA-2G-induced formation of BMSCs. Igf1, Serpine1 and Cxcl2 in DEGs were enriched for tissue repair, promotion of endothelial and epithelial proliferation and regulation of apoptosis.

The association between exosomal proteins and the efficacy of thermoradiochemotherapy in overweight/obese rectal cancer patients: a pilot prospective cohort study

Background: Overweight and especially obesity are associated with the risk of the development and progression of colorectal cancer. It can be assumed that there are multifaceted interactions between the tumor and adipose tissue during anti-tumor treatment. Cancer cells secrete exosomes, extracellular vesicles affecting the microenvironment of the tumor and promoting its progression or regression. The presence of transcription/translation/folding factors (heat shock proteins (HSPs), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in exosomes secreted by irradiated cells and cells exposed to hyperthermia, indicates the cell adaptation to the thermal and radiation stress. Aim: To analyze the MMPs, TIMP1, and HSPs on CD9-positive (CD9+) exosomes, as well as on exosomes of adipocytic origin (FABP4+) in rectal cancer patients with overweight/obesity under thermoradiochemotherapy (TRCT) and their association with the immediate treatment efficacy. Methods: Since 2021, 20 patients (of those 8 men; median age 59.0 [52.0; 63.0] years, median body mass index 29.6 [28.5; 33.1] kg/m2) with morphologically verified rectal cancer (T3-4N0M0 and T3-4N1M0, differentiation grade G1–G3) have been participating in the study. They were treated with TRCT: external gamma therapy (2 Gy, 1 fraction/day, 5 days/week, total focal dose 54 Gy), chemotherapy with capecitabine (825 mg/m2 twice daily) combined with local hyperthermia (42–44 °C, 60 min, 3 times/week, 10 sessions). The TRCT efficacy was assessed by RECIST 1.1 and ESGAR criteria. Blood samples for exosomes were taken from the patients at baseline (point 1), in the middle of the treatment course (point 2), at 6 to 10 weeks after the end of TRCT (point 3), and at 6 months after point 1 (point 4). Small extracellular vesicles were isolated from plasma by ultrafiltration with double ultracentrifugation. The isolated exosomes were characterized by transmission electronic microscopy, flow cytometry and nanoparticle trajectory analysis (NTA). Results: TRCT resulted in complete tumor regression in 13/20 of the rectal cancer patients and partial regression or stabilization in 7/20. Four subpopulations of CD9+ and FABP4+ exosomes associated with the TRCT efficacy were identified (CD9+MMP2+, СD9+MMP2+9+TIMP1+, СD9+MMP2+9+TIMP1-, and FABP4+MMP2+9-TIMP1+). Compared to the CD9+ exosomes, the adipocytic vesicles had higher MMP2 expression (p = 0.026); however, the adipocyte vesicles subpopulation were virtually free of vesicles with combined MMP2 and MMP9 gelatinase expression. The HSPs expression by circulating exosomes at various TRCT steps was associated neither with direct treatment efficacy nor with the vesicle type. Conclusion: The expression of MMPs and TIMP1 on CD9+ and FABP4+ exosomes is associated with TRCT efficacy. In the future, vesicular markers could be used to build prognostic models, to identify patient groups with an unfavorable prognosis, and to personalize treatment and follow-up.

Shatavarin-IV, a steroidal saponin from Asparagus racemosus, inhibits cell cycle progression and epithelial-to-mesenchymal transition in AGS cells under hyperglycemic conditions

Gastric cancer (GC)-diabetes co-morbidity is nowadays growing into a rising concern. However, no separate treatment procedures have been outlined for such patients. Phytochemicals and their derivatives can therefore be used as therapeutics as they have greater effectiveness, reduced toxicity, and a reduced likelihood of developing multi-drug resistance in cancer treatments. The present study intended to assess the therapeutic efficacy of Shatavarin-IV – a major steroidal saponin from the roots of Asparagus racemosus, in human gastric adenocarcinoma cell line under hyperglycemic conditions and explore its mechanism of action in controlling GC progression. For the present study, AGS cells were incubated in high glucose-containing media and the effects of Shatavarin-IV therein have been evaluated. Cell proliferation, confocal microscopic imaging, flow-cytometric analysis for cell cycle and apoptosis, immunoblotting, zymography, reverse zymography, wound-healing, colony formation, and invasion assays were performed. Shatavarin-IV has a prominent effect on AGS cell proliferation; with IC50 of 2.463 µ M under hyperglycemic conditions. Shatavarin-IV induces cell cycle arrest at the G0/G1 phase, thereby preventing hyperglycemia-induced excessive cell proliferation that later on leads to apoptotic cell death at 36 h of incubation. Shatavarin-IV further inhibits the migratory and invasive potential of AGS cells by altering the expression patterns of different EMT markers. It also inhibits MMP-9 while promoting TIMP-1 activity and expression; thereby regulating ECM turnover. This is the first report demonstrating the therapeutic efficacy of Shatavarin-IV against AGS cells grown in hyperglycemic conditions, implicating new insights into the treatment paradigm of patients with GC-diabetes co-morbidity.

The Foxo1-YAP-Notch1 axis reprograms STING-mediated innate immunity in NASH progression

Innate immune activation is critical for initiating hepatic inflammation during nonalcoholic steatohepatitis (NASH) progression. However, the mechanisms by which immunoregulatory molecules recognize lipogenic, fibrotic, and inflammatory signals remain unclear. Here, we show that high-fat diet (HFD)-induced oxidative stress activates Foxo1, YAP, and Notch1 signaling in hepatic macrophages. Macrophage Foxo1 deficiency (Foxo1M-KO) ameliorated hepatic inflammation, steatosis, and fibrosis, with reduced STING, TBK1, and NF-κB activation in HFD-challenged livers. However, Foxo1 and YAP double knockout (Foxo1/YAPM-DKO) or Foxo1 and Notch1 double knockout (Foxo1/Notch1M-DKO) promoted STING function and exacerbated HFD-induced liver injury. Interestingly, Foxo1M-KO strongly reduced TGF-β1 release from palmitic acid (PA)- and oleic acid (OA)-stimulated Kupffer cells and decreased Col1α1, CCL2, and Timp1 expression but increased MMP1 expression in primary hepatic stellate cells (HSCs) after coculture with Kupffer cells. Notably, PA and OA challenge in Kupffer cells augmented LIMD1 and LATS1 colocalization and interaction, which induced YAP nuclear translocation. Foxo1M-KO activated PGC-1α and increased nuclear YAP activity, modulating mitochondrial biogenesis. Using chromatin immunoprecipitation (ChIP) coupled with massively parallel sequencing (ChIP-Seq) and in situ RNA hybridization, we found that NICD colocalizes with YAP and targets Mb21d1 (cGAS), while YAP functions as a novel coactivator of the NICD, which is crucial for reprogramming STING function in NASH progression. These findings highlight the importance of the macrophage Foxo1–YAP–Notch1 axis as a key molecular regulator that controls lipid metabolism, inflammation, and innate immunity in NASH.

Protease expression in the human and rat cumulus-oocyte complex during the periovulatory period: a role in cumulus-oocyte complex migration†.

The migratory and matrix-invading capacities of the cumulus-oocyte complex have been shown to be important for the ovulatory process. In metastatic cancers, these capacities are due to increased expression of proteases, however, there is limited information on protease expression in the cumulus-oocyte complexes. The present study examined cumulus-oocyte complex expression of plasmins, matrix metalloproteases, and A Disintegrin and Metalloproteinase with Thrombospondin Motifs family members in the rat and human. In the rat, human chorionic gonadotropin (hCG) administration increased cumulus-oocyte complex expression of Mmp2, Mmp9, Mmp13, Mmp14, Mmp16, Adamts1, and the protease inhibitors Timp1, Timp3, and Serpine1 by 8-12h. This ovulatory induction of proteases in vivo could be mimicked by forskolin and ampiregulin treatment of cultured rat cumulus-oocyte complexes with increases observed in Mmp2, Mmp13, Mmp14, Mmp16, Mmp19, Plat, and the protease inhibitors Timp1, Timp3, and Serpine1. Comparison of expression between rat cumulus-oocyte complexes and granulosa cells at the time of ovulation showed decreased Mmp9 and increased Mmp13, Mmp14, Mmp16, Adamts1, Timp1, and Timp3 expression in the cumulus-oocyte complexes. In human, comparison of expression between cumulus and granulosa cells at the time of in vitro fertilization retrieval showed decreased MMP1, MMP2, MMP9, and ADAMTS1, while expression of MMP16, TIMP1, and TIMP3 were increased. Treatment of expanding rat cumulus-oocyte complexes with a broad spectrum matrix metalloproteases inhibitor, GM6001, significantly reduced the migration of cumulus cells in vitro. These data provide evidence that multiple proteases and their inhibitors are expressed in the cumulus-oocyte complex and play an important role in imparting the migratory phenotype of the cumulus-oocyte complex at the time of ovulation. Summary Sentence Multiple proteases and their inhibitors are induced in the cumulus-oocyte complex (COC) during the periovulatory period and potentially play an important role in imparting the migratory phenotype of the COC at the time of ovulation.