Prognostic value of matrix metalloproteinase-2 protein and matrix metalloproteinase-9 protein in colorectal cancer: a meta-analysis.

INTRODUCTION: Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are critical components of the extracellular matrix (ECM) in colorectal cancer (CRC). We aimed to evaluate the prognostic value of MMP-2 and MMP-9 in patients with CRC. METHODS: We performed a meta-analysis of cohort studies with available data on the effect of MMP-2 and MMP-9 expression on both disease-free survival (DFS) and overall survival (OS) by the risk ratios (RRs) with their 95% confidence intervals (CIs). Studies were subgrouped based on the different tissue types, including cancer tissue and normal tissue, and the subgroup effect of MMP expression in different tissues was analyzed through meta-regression. To ensure the quality and reduce the risk of bias, the Newcastle‒Ottawa Scale (NOS) was used to assess the included studies. A sensitivity analysis was randomly performed to assess the potential impact of each study on our results. RESULTS: Eighteen trials were selected (Table 1) and included a total of 3944 patients. According to our primary meta-analysis, the expression of MMP-2 was significantly associated with a decrease in OS (RR = 1.75, 95% CI = 1.34 to 2.29, P < 0.001) and DFS (RR = 2.62, 95% CI = 1.25 to 5.49, P < 0.001), and the expression of MMP-9 was not significantly associated with a decrease in OS (RR = 1.48, 95% CI = 0.97 to 2.24, P = 0.069) or DFS (RR = 1.60, 95% CI = 0.87 to 2.94, P = 0.133). According to the subgroup analysis of MMPs in different tissues, high MMP-2 expression in cancer tissue (RR = 1.90, 95% CI = 1.29 to 2.79) and normal tissue (RR = 1.59, 95% CI = 1.17 to 2.17) were significant indicators of poor OS. High MMP-2 expression in cancer tissue was significant indicator of poor DFS (RR = 2.12, 95% CI = 1.09 to 4.11). MMP-9 expression was also associated with poor OS (RR = 1.40, 95% CI = 0.85 to 2.29), but the difference in OS between the high and low expression groups was not statistically significant. CONCLUSIONS: High MMP-2 expression, especially in cancer tissue, is significantly associated with both poor DFS and poor OS in patients with CRC. High MMP-9 expression tended to indicate a poor prognosis of CRC but the correlation was not significant.

LIGHT protein: A novel gingival crevicular fluid biomarker associated with increased probing depth after periodontal surgery.

AIM: To evaluate the protein profiles in gingival crevicular fluid (GCF) in relation to clinical outcomes after periodontal surgery and examine if any selected proteins affect the mRNA expression of pro-inflammatory cytokines in human gingival fibroblasts. MATERIALS AND METHODS: This exploratory study included 21 consecutive patients with periodontitis. GCF was collected, and the protein pattern (n = 92) and clinical parameters were evaluated prior to surgery and 3, 6 and 12 months after surgery. Fibroblastic gene expression was analysed by real-time quantitative polymerase chain reaction. RESULTS: Surgical treatment reduced periodontal pocket depth (PPD) and changed the GCF protein pattern. Twelve months after surgery, 17% of the pockets showed an increase in PPD. Levels of a number of proteins in the GCF decreased after surgical treatment but increased with early signs of tissue destruction, with LIGHT being one of the proteins that showed the strongest association. Furthermore, LIGHT up-regulated the mRNA expression of pro-inflammatory cytokines interleukin (IL)-6, IL-8 and MMP9 in human gingival fibroblasts. CONCLUSIONS: LIGHT can potentially detect subjects at high risk of periodontitis recurrence after surgical treatment. Moreover, LIGHT induces the expression of inflammatory cytokines and tissue-degrading enzymes in gingival fibroblasts.

Vaginal matrix metalloproteinase-9 (MMP-9) as a potential early predictor of preterm birth.

OBJECTIVES: To evaluate the differences in vaginal matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMPs) in pregnant patients with a history of prior preterm birth compared with controls. METHODS: A prospective cohort pilot study recruited patients during prenatal care with history of prior spontaneous preterm birth (high-risk group) or no history of preterm birth (low-risk/controls). Inclusion criteria were singleton gestation at 11-16 weeks and between 18 and 55 years of age. Exclusion criteria were diabetes mellitus, hypertension, diseases affecting the immune response or acute vaginitis. A vaginal wash was performed at time of enrollment, and patients were followed through delivery. Samples were analyzed using semi-quantitative analysis of MMPS and TIMPS. The study was approved by the IRB and a p-value <0.05 was considered significant. RESULTS: A total of 48 pregnant patients were recruited: 16 with a history of preterm birth (high-risk group) and 32 with no history of preterm birth (low-risk group/controls). Groups were similar in age, race, BMI, and delivery mode. The high-risk group had more multiparous women (100 vs. 68.8 %; p=0.02), a greater preterm birth rate (31.2 vs. 6.3 %; p=0.02), and a lower birth weight (2,885 ± 898 g vs. 3,480 ± 473 g; p=0.02). Levels of vaginal MMP-9 were greater in high-risk patients than low-risk patients (74.9 % ± 27.0 vs. 49.4 % ± 31.1; p=0.01). When dividing the cohort into patients that had a spontaneous preterm birth (7/48, 14.6 %) vs. those with a term delivery (41/48, 85.4 %), the vaginal MMP-9 remained elevated in the cohort that experienced a preterm birth (85.46 %+19.79 vs. 53.20 %+31.47; p=0.01). There were no differences in the other MMPS and in TIMPs between high and low-risk groups. CONCLUSIONS: There was an increase in vaginal MMP-9 during early pregnancy in those at high risk for preterm birth and in those who delivered preterm, regardless of prior pregnancy outcome. Vaginal MMP-9 may have potential as a marker of increased risk of preterm birth.

Correlation of amniotic fluid inflammatory markers with preterm birth: a meta-analysis.

BACKGROUND: The relationship between amniotic fluid inflammatory biomarkers and preterm birth in second- or third-trimester pregnancy has been a focus, and understanding the correlation between these markers and preterm birth is important for early identification and intervention in preterm birth. The aim of this study was to explore potential inflammatory biomarkers in second- or third-trimester pregnancy amniotic fluid associated with preterm birth. METHODS: On November 30, 2023, we searched literature involved the influence of second- or third-trimester pregnancy amniotic fluid inflammatory biomarkers on preterm birth through PubMed, Web of Science, Embase, Scope, CNKI, WanFang, VIP and China Biomedical Databases. The search languages were Chinese and English. Included outcomes indexes were combined utility analysis via R software. RESULTS: A total of 11 articles were included in the combined utility analysis. This combined analysis revealed significant differences in several inflammatory biomarkers in amniotic fluid between the two groups (MD = 6.87, 95%CI: 0.26 - 13.47, P < 0.01); the difference in amniotic fluid IL-6 between the two groups (MD = 5.73, 95%CI: 3.13-8.32, P < 0.01); the difference in amniotic fluid IL-10 between the two groups (MD = 0.11, 95%CI: -3.26-3.48, P < 0.01); the difference in amniotic fluid CRP between the two groups (MD = 21.34, 95%CI: 11.69-30.89, P < 0.01); the difference in amniotic fluid MCP-1 between the two groups (MD = 312.14, 95%CI: 211.34-412.97, P < 0.01); the difference in the amniotic fluid MMP-9 between the two groups (MD = 0.86, 95%CI: -0.10-1.82, P < 0.01); and the difference in TNF-α in amniotic fluid between the two groups (MD = 22.78, 95%CI: -5.05-50.61, P < 0.01). CONCLUSIONS: The inflammatory biomarkers IL-1ß, IL-6, IL-10, CRP, TNFα, MCP-1 and MMP-9 in the amniotic fluid of patients in the second- or third-trimester pregnancy were all correlated with preterm birth.

The premature foetus has many serious complications in the near and long term because of the immature organs, which is related to the long-term incidence of cerebral palsy, developmental delay and retinopathy of prematurity, which is the main cause of perinatal foetal death. Preterm birth cases are accompanied by infection of pathogenic microorganisms in amniotic cavity, which then leads to inflammatory reaction in amniotic cavity. However, research on the correlation between inflammatory markers and preterm birth has shown certain complexity and differences. The results of this meta-analysis show that the inflammatory biomarkers interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and interleukin-10 (IL-10), C-reactive protein (CRP), tumour necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) in amniotic fluid of patients in the second- or third-trimester pregnancy are significant between the preterm birth group and the control group, and the expression level of inflammatory factors in amniotic fluid of patients in the preterm birth group is elevated, thus suggesting that these inflammatory factors may be able to predict preterm birth.

[A preliminary study on the tear matrix metalloproteinase 9 point-of-care assay using a domestic kit].

Objective: To compare the point-of-care assays for tear matrix metalloproteinase 9 (MMP-9) using domestic and InflammaDry kits, and to evaluate the feasibility of diagnosing dry eye with the domestic kit. Methods: It was a cross-sectional study. Thirty dry eye patients and 30 age-and sex-matched normal volunteers were continuously enrolled in this cross-sectional study from June 2022 to July 2022. Both domestic and InflammaDry kits were used to detect the tear MMP-9 levels. The positive rates were recorded for qualitative analysis, and the gray ratios of bands (the gray value of detection bands to that of control bands) were collected for quantitative analysis. The correlations of MMP-9 levels with age, ocular surface disease index, fluorescence tear break-up time, tear meniscus height, Schirmer's Ⅰ test score, corneal fluorescein staining score, and meibomian gland dropout were analyzed. The Mann-Whitney U test, paired Chi-square test, Kappa test, and Spearman's correlation coefficient were used for statistical analysis. Results: There were 14 males and 16 females (30 eyes) in the control group, and their age was (39.37±19.55) years. In the dry eye group, 11 males and 19 females (30 eyes), aged (46.87±17.85) years, had moderate to severe dry eye. The positive rates of MMP-9 in tear fluid were significantly different between dry eye patients (InflammaDry: 86.67%; domestic kit: 70.00%) and controls (InflammaDry: 16.67%, P<0.001; domestic kit: 6.67%, P<0.001). Although the sensitivity of the domestic kit was lower than that of the InflammaDry kit (70.0% vs. 86.7%, P=0.001), the specificity was higher (93.3% vs. 83.3%, P=0.001). In dry eye patients, the positive coincidence rate was 80.7% (21/26), the negative coincidence rate was 100% (4/4), and the total coincidence rate was 83.3% (25/30), with no significant difference between the two kits (McNemar test: χ2=3.20, P>0.05), and the results of both kits were consistent (Kappa=0.53, P=0.001). The Spearman's correlation coefficient showed the gray ratios using both kits were positively correlated with the corneal fluorescein staining score (InflammaDry: ρ=0.48, P<0.05; domestic kit: ρ=0.52, P=0.003). Conclusion: The performances of the domestic and InflammaDry kits are consistent in the point-of-care assay for tear MMP-9, and the domestic kit has lower sensitivity but higher specificity.

Discovery, validation, and diagnostic ability of multiple protein-based biomarkers in saliva and gingival crevicular fluid to distinguish between health and periodontal diseases.

AIM: To discover and validate differential protein biomarker expression in saliva and gingival crevicular fluid (GCF) to discriminate objectively between periodontal health and plaque-induced periodontal disease states. MATERIALS AND METHODS: One-hundred and ninety participants were recruited from two centres (Birmingham and Newcastle upon Tyne, UK) comprising healthy, gingivitis, periodontitis, and edentulous donors. Samples from the Birmingham cohort were analysed by quantitative mass spectrometry proteomics for biomarker discovery. Shortlisted candidate proteins were then verified by enzyme-linked immunosorbent assay in both cohorts. Leave-one-out cross validation logistic regression analysis was used to identify the best performing biomarker panels. RESULTS: Ninety-five proteins were identified in both GCF and saliva samples, and 15 candidate proteins were selected based upon differences discovered between the donor groups. The best performing panels to distinguish between: health or gingivitis and periodontitis contained matrix metalloproteinase-9 (MMP9), S100A8, alpha-1-acid glycoprotein (A1AGP), pyruvate kinase, and age (area under the curve [AUC] 0.970); health and gingivitis contained MMP9, S100A8, A1AGP, and pyruvate kinase, but not age (AUC 0.768); and mild to moderate and advanced periodontitis contained MMP9, S100A8, A1AGP, pyruvate kinase, and age (AUC 0.789). CONCLUSIONS: Biomarker panels containing four proteins with and without age as a further parameter can distinguish between periodontal health and disease states.

Molecular Mechanisms of RSV and Air Pollution Interaction: A Scoping Review.

RSV is one of the major infectious agents in paediatrics, and its relationship with air pollution is frequently observed. However, the molecular basis of this interaction is sparsely reported. We sought to systematically review the existing body of literature and identify the knowledge gaps to answer the question: which molecular mechanisms are implied in the air pollutants-RSV interaction? Online databases were searched for original studies published before August 2022 focusing on molecular mechanisms of the interaction. The studies were charted and a narrative synthesis was based upon three expected directions of influence: a facilitated viral entry, an altered viral replication, and an inappropriate host reaction. We identified 25 studies published between 1993 and 2020 (without a noticeable increase in the number of studies) that were performed in human (n = 12), animal (n = 10) or mixed (n = 3) models, and analysed mainly cigarette smoke (n = 11), particulate matter (n = 4), nanoparticles (n = 3), and carbon black (n = 2). The data on a damage to the epithelial barrier supports the hypothesis of facilitated viral entry; one study also reported accelerated viral entry upon an RSV conjugation to particulate matter. Air pollution may result in the predominance of necrosis over apoptosis, and, as an effect, an increased viral load was reported. Similarly, air pollution mitigates epithelium function with decreased IFN-γ and Clara cell secretory protein levels and decreased immune response. Immune response might also be diminished due to a decreased viral uptake by alveolar macrophages and a suppressed function of dendritic cells. On the other hand, an exuberant inflammatory response might be triggered by air pollution and provoke airway hyperresponsiveness (AHR), prolonged lung infiltration, and tissue remodeling, including a formation of emphysema. AHR is mediated mostly by increased IFN-γ and RANTES concentrations, while the risk of emphysema was related to the activation of the IL-17 → MCP-1 → MMP-9 → MMP-12 axis. There is a significant lack of evidence on the molecular basics of the RSV-air pollution interaction, which may present a serious problem with regards to future actions against air pollution effects. The major knowledge gaps concern air pollutants (mostly the influence of cigarette smoke was investigated), the mechanisms facilitating an acute infection or a worse disease course (since it might help plan short-term, especially non-pharmacological, interventions), and the mechanisms of an inadequate response to the infection (which may lead to a prolonged course of an acute infection and long-term sequelae). Thus far, the evidence is insufficient regarding the broadness and complexity of the interaction, and future studies should focus on common mechanisms stimulated by various air pollutants and a comparison of influence of the different contaminants at various concentrations.

Features of the content of matrix metalloproteinases ММР-9 and ММР-2 in mixed saliva of young individuals with dental caries against the background of different level of 25(OH) vitamin D in the body.

The content ofММР-9 and ММР-2 in oral fluid of 105 individuals between the ages of 19 and 23 has been researched.Of these, 42 people are individuals with dental caries and normal level of the active form of vitamin Din serum (25(OH)D >30ng/mL) and 42 people - with 25(OH)D <30 ng/mL level.The control group was composed of 21 individuals with low DMFt index (1,5) and a normal level of 25(OH)D in blood. It has been established that the level of ММР-9 in mixed salivaincreases against the background of dental caries,while the content of ММР-9 and ММР-2 increasessignificantlyamidthe lack and deficiency of25(OH)Din the body. Inverse correlations between the 25(OH)D level in serum and the value ofmatrix metalloproteinasesin saliva have been revealed: noticeable - with the amount of MMP-9 and moderate- with the concentration of MMP-2.

The Elk1/MMP-9 axis regulates E-cadherin and occludin in ventilator-induced lung injury.

BACKGROUND: Ventilator-induced lung injury (VILI) is a common complication in the treatment of respiratory diseases with high morbidity and mortality. ETS-domain containing protein (Elk1) and Matrix metalloproteinase (MMP) 9 are involved in VILI, but the roles have not been fully elucidated. This study examined the mechanisms of the activation of MMP-9 and Elk1 regulating barrier function in VILI in vitro and in vivo. METHODS: For the in vitro study, Mouse lung epithelial cells (MLE-12) were pre-treated with Elk1 siRNA or MMP-9 siRNA for 48 h prior to cyclic stretch at 20% for 4 h. For the in vivo study, C57BL/6 mice were pre-treated with Elk1 siRNA or MMP-9 siRNA for 72 h prior to 4 h of mechanical ventilation. The expressions of Elk1, MMP-9, Tissue inhibitor of metalloproteinase 1 (TIMP-1), E-cadherin, and occludin were measured by Western blotting. The intracellular distribution of E-cadherin and occludin was shown by immunofluorescence. The degree of pulmonary edema and lung injury were evaluated by Hematoxylin-eosin (HE) staining, lung injury scores, Wet/Dry (W/D) weight ratio, total cell counts, and Evans blue dye. RESULTS: 20% cyclic stretch and high tidal volume increases the expressions of Elk1, MMP-9, and TIMP-1, increases the ratio of MMP-9/TIMP-1, decreases the E-cadherin and occludin level. Elk1 siRNA or MMP-9 siRNA reverses the degradations of E-cadherin, occludin, and the ratio of MMP-9/TIMP-1 caused by cyclic stretch. Elk1 siRNA decreases the MMP-9 level with or not 20% cyclic stretch and high tidal volume. CONCLUSIONS: The results demonstrate mechanical stretch damages the tight junctions and aggravates the permeability in VILI, Elk1 plays an important role in affecting the tight junctions and permeability by regulating the balance of MMP-9 and TIMP-1, thus indicating the therapeutic potential of Elk1 to treat VILI.

Sphingosine-1-phosphate Receptor-1 Promotes Vascular Invasion and EMT in Hepatocellular Carcinoma.

BACKGROUND: It remains unknown whether epithelial-mesenchymal transition (EMT)-mediated vascular invasion and cancer stemness are associated with sphingosine-1-phosphate receptor-1 (S1PR1) expression in human hepatocellular carcinoma (HCC). The aim of this study was to investigate the correlation between S1PR1 expression and prognosis of patients with primary HCC and to define the potential of S1PR as a therapeutic target. MATERIALS AND METHODS: We investigated 108 patients who underwent primary surgical resection for HCC treatment. Expression of S1PR1 and EMT markers was analyzed to predict prognosis of patients with HCC. Furthermore, three-dimensional organotypic culture, anoikis assay, and cell invasion were performed to validate the association of S1PR1 with EMT and cancer stemness. RESULTS: Among patients with HCC, the high S1PR1 expression group had significantly shorter overall survival than the low expression group. Moreover, high S1PR1 expression was significantly associated with shorter recurrence-free survival, increased risk of portal and hepatic vein invasion, and intrahepatic metastasis. Multivariate analyses revealed that S1PR1 overexpression was an independent prognostic factor in patients with HCC. S1PR1 overexpression positively correlated with vimentin and MMP-9 expression and negatively correlated with E-cadherin. In addition, S1PR1 overexpression induced EMT and enhanced tumor invasion and cancer stemness. CONCLUSIONS: S1PR1 overexpression, via EMT-induced vascular invasion and increased cancer stem cell properties, establishes a metastatic niche, enhances the capacity of hematogenous metastasis, and associates with poor outcomes in patients with HCC. Hence, S1PR1 may serve as a therapeutic target for patients with HCC with vascular invasion.

Complex challenges of estimating the age and vitality of muscle wounds: a study with matrix metalloproteinases and their inhibitors on animal and human tissue samples.

The estimation of wound age and wound vitality is a recurring task in forensic routine work and has been subject of forensic research for a long time. By now, an unrestrictedly reliable marker or set of markers has not been found. In a study on myocardial infarctions, matrix metalloproteinases (MMP) 2 and 9 as well as tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) were detected immunohistochemically in mechanically wounded myocardium (ECG electrodes, vessel ligations). Against this background, the potency of MMP-9, MMP-2, and TIMP-1 as markers for the estimation of wound age and wound vitality was tested in a broad approach with human tissue samples drawn during autopsies and with an animal model, the isolated perfused Langendorff heart. The study comprised samples of injured human skeletal muscle, injured human myocardium, rats' hearts with vital wounds, and rats' hearts with postmortem-inflicted wounds that were all stained immunohistochemically. The results showed great scattering, leading to the conclusion that MMP-2, MMP-9, and TIMP-1 are not suitable for wound age estimation. Merely the results for TIMP-1 suggested that this marker might be able to differentiate between vital and postmortem-inflicted wounds. With a view to the promising results of the preceding study, the results underline the necessity to test possible markers of wound age/wound vitality on a large and diverse sample set.

Mild hyperhomocysteinemia induces blood-brain barrier dysfunction but not neuroinflammation in the cerebral cortex and hippocampus of wild-type mice.

This study explored the potential effects of mild hyperhomocysteinemia (HHcy) on the blood-brain barrier (BBB) and neuroinflammation. Seven-week-old male wild-type C57BL/6 mice were fed normal mouse chow (the control group) or a methionine-enriched diet (the HHcy group) for 14 weeks. Mice in the HHcy group exhibited a slight increase in serum Hcy levels (13.56 ± 0.61 µmol/L). Activation of the ERK signaling pathway, up-regulation of matrix metalloproteinase-9 (MMP-9), and degradation of tight junction proteins (occludin and claudin-5) were observed in both the cerebral cortex and hippocampus of mice with mild HHcy. However, microglia were not activated and the levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were not changed in either the cerebral cortex or hippocampus of mice with mild HHcy. Moreover, the signaling activity of STAT3 also did not differ significantly between the two groups. These findings demonstrate that the BBB is highly vulnerable to homocysteine insult. Even a slight increase in serum homocysteine levels up-regulates MMP-9 expression and disrupts the BBB integrity. Meanwhile, microglia activation or the STAT3 pathway might not contribute to the effects of mild HHcy on the brain.

MMP-9-mediated regulation of hypoxia-reperfusion injury-related neutrophil inflammation in an in vitro proximal tubular cell model.

BACKGROUND: Hypoxia-reperfusion (HR) and inflammation are causes of renal allograft injury. Pathological evidence has indicated that ischemia followed by reperfusion leads to the proteolysis and destruction of the extracellular matrix (ECM) in renal tubular epithelial cells. Matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, play roles in cleaving and reshaping the ECM. Acute accumulation of MMP-9 secreted from neutrophils promotes the incidence of inflammation and exacerbates graft trauma. Our goal was to investigate the activities of MMP-9/MMP-2 and their correlation with HR injury and neutrophil-related inflammation in renal proximal tubular cells. METHODS: This model was established by placing HK-2 cells under hypoxic conditions (5% CO2, 1% O2) for 6 h and then exposing them to reperfusion (5% CO2, 21% O2) for 12 h in a tri-gas incubator. The cell culture medium was collected for culturing polymorphonuclear leukocytes (PMNs). BB-94 (MMP-9 inhibitor) was added to the culture medium in the inhibitor group. RESULTS: Flow cytometry showed a significant increase in reactive oxygen species (ROS) levels in HK-2 cells from the HR injury group. MMP-9 expression was significantly increased and MMP-2 expression was significantly decreased in HK-2 cells from the HR group. MMP-9 and MPO expression were significantly increased in the HR group, while MPO expression was significantly decreased in the PMN inhibitor group. CONCLUSIONS: The outcomes indicated that MMP-9 and MMP-2 are important components of an underlying pathophysiological mechanism of injury following HR. MMP-9 inhibition may be a potential approach to mitigateHR injury.

Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

Deciphering the Functional Role of RIPK4 in Melanoma.

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1ß level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4high) or independent (RIPK4low) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

Simultaneous Sensing of Multiple Cancer Biomarkers by a Single DNA Nanoprobe in a Nanopore.

Both vascular endothelial growth factor (VEGF) and matrix metallopeptidase-9 (MMP-9) are key biomarkers in tumor angiogenesis. Determination of the overexpression of the two biomarkers would provide valuable information on the progression of tumor growth and metastasis, but their simultaneous quantification by a single probe is unprecedented. Here, we develop a triplex DNA-based nanoprobe for simultaneously quantifying VEGF and MMP-9 using an α-hemolysin nanopore. A DNA aptamer is used as the triplex molecular beacon (tMB) loop to bind VEGF, and a stem-forming oligonucleotide modified with a short peptide is used to recognize MMP-9. The sequential presence of VEGF and MMP-9 could also be identified by different patterns of current events. Besides, the characteristic current events generated by the DNA probe possess pH-dependent patterns that can be used to reflect the environmental pH. Success in the construction of such DNA nanoprobes will greatly facilitate the investigation of the mechanisms of different tumor angiogenesis processes and provide a useful approach for cancer diagnosis.

Elevated MMP9 expression in breast cancer is a predictor of shorter patient survival.

PURPOSE: MMP9 is a matricellular protein associated with extracellular matrix (ECM) remodelling, that promotes tumour progression, and modulates the activity of cell adhesion molecules and cytokines. This study aims to assess the prognostic value of MMP9 and its association with cytoskeletal modulators in early-stage invasive breast cancer (BC). METHODS: MMP9 expression was evaluated by immunohistochemistry using a well-characterised series of primary BC patients with long-term clinical follow-up. Association with clinicopathological factors, patient outcome and ECM remodelling BC-biomarkers were investigated. METABRIC dataset, BC-GenExMiner v4.0 and TCGA were used for the external validation of MMP9 expression. GSEA gene enrichment analyses were used to evaluate MMP9 associated pathways. RESULTS: MMP9 immunopositivity was observed in the stroma and cytoplasm of BC cells. Elevated MMP9 protein levels were associated with high tumour grade, high Nottingham Prognostic Index, and hormonal receptor negativity. Elevated MMP9 protein expression correlated significantly with cytokeratin 17 (Ck17), Epidermal Growth Factor Receptor (EGFR), proliferation (Ki67) biomarkers, cell surface adhesion receptor (CD44) and cell division control protein 42 (CDC42). Cytoplasmic MMP9 expression was an independent prognostic factor associated with shorter BC-specific survival. In the external validation cohorts, MMP9 expression was also associated with poor patients' outcome. Transcriptomic analysis confirmed a positive association between MMP9 and ECM remodelling biomarkers. GSEA analysis supports MMP9 association with ECM and cytoskeletal pathways. CONCLUSION: This study provides evidence for the prognostic value of MMP9 in BC. Further functional studies to decipher the role of MMP9 and its association with cytoskeletal modulators in BC progression are warranted.

Magnetic resonance imaging assessment of proteolytic enzyme concentrations and biologic properties of intraluminal thrombus in abdominal aortic aneurysms.

OBJECTIVE: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.

LPS-induced inflammation disorders bone modeling and remodeling by inhibiting angiogenesis and disordering osteogenesis in chickens.

Inflammation plays a negative role in the growth and development of bone. However, the underlining mechanisms of inflammation caused abnormal bone development and even bone disease are still poorly understood, especially in chickens. In this study, we explored the influence of inflammation on bone formation in broilers for the first time by using lipopolysaccharide (LPS) to establish systemic inflammatory models in chickens with tibia as the research object. The measurements of production and tibial parameters showed an inefficient production performance and lower growth rate in LPS group. We also found a large amount of platelets, inflammatory cells in chickens' blood and higher levels of inflammatory factors in serum after LPS injection, meanwhile, increase in thrombus, chondrocyte nucleolysis, and osteoclasts and a reduction in blood vessels were observed in growth plate through histological observation. The qPCR analysis showed that the mRNA expression levels of NF-κB, TLR4, TF, TPO, and its receptor C-MPL enhanced, while VEGFA was inhibited in LPS group. In addition, in OPG/RANKL system, OPG was decreased while RANKL enhanced. It was also observed that the mRNA levels of MMP-9 and its inducing factor CD147 enhanced in LPS group. The western blot results were basically in consistent with mRNA test. Thus, we infer that inflammation can inhibit bone modeling and remodeling by affecting angiogenesis and osteogenesis, and result in negative effect on bone formation furtherly.

Inhibition of VEGF (Vascular Endothelial Growth Factor)-A or its Receptor Activity Suppresses Experimental Aneurysm Progression in the Aortic Elastase Infusion Model.

OBJECTIVE: We examined the pathogenic significance of VEGF (vascular endothelial growth factor)-A in experimental abdominal aortic aneurysms (AAAs) and the translational value of pharmacological VEGF-A or its receptor inhibition in aneurysm suppression. Approaches and Results: AAAs were created in male C57BL/6J mice via intra-aortic elastase infusion. Soluble VEGFR (VEGF receptor)-2 extracellular ligand-binding domain (delivered in Ad [adenovirus]-VEGFR-2), anti-VEGF-A mAb (monoclonal antibody), and sunitinib were used to sequester VEGF-A, neutralize VEGF-A, and inhibit receptor tyrosine kinase activity, respectively. Influences on AAAs were assessed using ultrasonography and histopathology. In vitro transwell migration and quantitative reverse transcription polymerase chain reaction assays were used to assess myeloid cell chemotaxis and mRNA expression, respectively. Abundant VEGF-A mRNA and VEGF-A-positive cells were present in aneurysmal aortae. Sequestration of VEGF-A by Ad-VEGFR-2 prevented AAA formation, with attenuation of medial elastolysis and smooth muscle depletion, mural angiogenesis and monocyte/macrophage infiltration. Treatment with anti-VEGF-A mAb prevented AAA formation without affecting further progression of established AAAs. Sunitinib therapy substantially mitigated both AAA formation and further progression of established AAAs, attenuated aneurysmal aortic MMP2 (matrix metalloproteinase) and MMP9 protein expression, inhibited inflammatory monocyte and neutrophil chemotaxis to VEGF-A, and reduced MMP2, MMP9, and VEGF-A mRNA expression in macrophages and smooth muscle cells in vitro. Additionally, sunitinib treatment reduced circulating monocytes in aneurysmal mice. CONCLUSIONS: VEGF-A and its receptors contribute to experimental AAA formation by suppressing mural angiogenesis, MMP and VEGF-A production, myeloid cell chemotaxis, and circulating monocytes. Pharmacological inhibition of receptor tyrosine kinases by sunitinib or related compounds may provide novel opportunities for clinical aneurysm suppression.