TIMP-1 Protein Research Articles

Effects of Metallic and Carbon-Based Nanomaterials on Human Pancreatic Cancer Cell Lines AsPC-1 and BxPC-3.

Pancreatic cancer, due to its asymptomatic development and drug-resistance, is difficult to cure. As many metallic and carbon-based nanomaterials have shown anticancer properties, we decided to investigate their potential use as anticancer agents against human pancreatic adenocarcinoma. The objective of the study was to evaluate the toxic properties of the following nanomaterials: silver (Ag), gold (Au), platinum (Pt), graphene oxide (GO), diamond (ND), and fullerenol (C60(OH)40) against the cell lines BxPC-3, AsPC-1, HFFF-2, and HS-5. The potential cytotoxic properties were evaluated by the assessment of the cell morphology, cell viability, and cell membrane damage. The cancer cell responses to GO and ND were analysed by determination of changes in the levels of 40 different pro-inflammatory proteins. Our studies revealed that the highest cytotoxicity was obtained after the ND treatment. Moreover, BxPC-3 cells were more sensitive to ND than AsPC-1 cells due to the ND-induced ROS production. Furthermore, in both of the cancer cell lines, ND caused an increased level of IL-8 and a decreased level of TIMP-2, whereas GO caused only decreased levels of TIMP-2 and ICAM-1 proteins. This work provides important data on the toxicity of various nanoparticles against pancreatic adenocarcinoma cell lines.

Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall

Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

TIMP1 and TIMP2 Downregulate TGFβ Induced Decidual-like Phenotype in Natural Killer Cells.

Simple SummaryCancer patients are characterized by NK cells with altered surface markers, such as CD56 brightness, CD9, CD49a (pro-angiogenic) and PD-1, and TIM-3 (exhaustion), that favor immune escape. Transforming growth factor-beta (TGFβ) is a major tumor-derived cytokine that favors cancer growth and supports pro-angiogenic activities in NK cells by inducing pro-angiogenic molecules. TIMP-1 and TIMP-2 play a crucial role in extracellular matrix (ECM) regulation, wound healing, pregnancy and cancer, and there is increasing evidence that they are immune-modulatory. We found that recombinant TIMP-1 and -2 can partially contrast the induction of pro-tumor/pro-angiogenic decidual-like polarization of NK cells by TGFβ.Natural Killer (NK) cells have been found to be anergic, exhausted and pro-angiogenic in cancers. NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like-phenotype, together with decreased levels of NKG2D activation marker, increased levels of TIM-3 exhaustion marker, similar to cancer-associated NK cells. Tissue inhibitors of metalloproteases (TIMPs) exert dual roles in cancer. The role of TIMPs in modulating immune cells is a very novel concept, and the present is the first report studying their ability to contrast TGFβ action on NK cells. Here, we investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFβ. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. We found that TIMP1 and TIMP2 were effective in interfering with TGFβ induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFβ in increasing the percentage of CD56bright, CD16−, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFβ and decreased the TGFβ upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFβ and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Treatment with recombinant TIMP-1 or TIMP-2 showed a trend, although not statistically significant, to decrease CD49a+ and TIM-3+ expression and increase NKG2D in peripheral blood NK cells exposed to conditioned media from colon cancer cell lines. Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization. Given the heterogeneity of released factors within the TME, it is clear that TGFβ stimulation represents a model to prove TIMP’s new properties, but it cannot be envisaged as a soloist NK cell polarizing agent. Therefore, further studies from the scientific community will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic–therapeutic roles.

Advanced image analysis-based evaluation of protein antibody microarray chemiluminescence signal improves glioma type identification by blood serum proteins concentrations

Background and ObjectiveGliomas are the most common brain tumors usually classified as benign low-grade or aggressive high-grade glioma. One of the promising possibilities of glioma diagnostics and tumor type identification could be based on concentration measurements of glioma secreted proteins in blood. However, several published approaches of quantitative proteomic analysis emphasize limits of one single protein to be used as biomarker of these types of tumors. Simultaneous multi-protein concentrations analysis giving antibody array–based methods suffer from poor measurement accuracy due to technical limitations of imaging systems. MethodsWe applied Principal Component Analysis (PCA) for series of repeated antibody array chemiluminescence images to extract the component representing relative values of protein concentrations, free from zero-mean noise and uneven background illumination – main factors corrupting evaluation result. ResultsThe proposed method increased accuracy of protein concentration estimates at least 2-fold. Decision tree classifier applied to the relative concentration values of three proteins TIMP-1, PAI-1 and NCAM-1 estimated by proposed image analysis method effectively distinguished between low-grade glioma, high-grade glioma and healthy control subjects showing validation accuracy of 74.9% with the highest positive predictive value of 81.2% for high grade glioma and 57.1% for low grade glioma cases. ConclusionsPCA-based image processing could be applied in protein antibody microarray and other multitarget detection/evaluation investigations to increase estimation accuracy.

THE RELATIONSHIP BETWEEN THE EXPRESSION LEVELS OF TISSUE INHIBITOR OF METALLOPROTEINASES-3 (TIMP3) AND SEVERITY OF ATHEROSCLEROSIS

Objective: TIMP3 is one of tissue inhibitors of metalloproteinases (TIMPs), which binds to the components of the extracellular matrix, and has crucial roles in atherosclerogenesis and adipose tissue differentiation. In this study, it was aimed to determine the effects of TIMP3 gene expression levels on severity of atherosclerosis in different tissues. Material and Methods: The first group of the study (evaluated for coronary artery disease) were cases classified as high and low plaque scores according to the degree and location of atherosclerotic lesions. In the second group (post-mortem cases) were male cadavers who died due to coronary heart disease (CHD, n=26) and non-cardiac trauma (T-nonP, n=4). The TIMP3 expression levels were examined in leukocyte and peri-coronary epicardial adipose tissues (EAT) samples (n=69 and n=34, respectively) of the first group and EAT and coronary artery samples (n=12 and n=30, respectively) of the second group using quantitative RT-PCR. In addition, the protein expressions of TIMP3 were analysed on artery sections by immunofluorestaining. Results: In the post-mortem study group, the TIMP3 expression levels were found to increase in no plaque segments of arteries of cases with CHD (CHD-nonP) compared to advanced atherosclerotic arteries (CHD-P) and normal arteries of T-nonP cases (p=0.01 and p=0.05, respectively). The TIMP3 expressions in EATs and leukocytes were not statistically significant between the study groups. In addition, TIMP3 protein was detected in normal arteries, peri-coronary EATs and mostly in macrophages-rich areas in advanced atherosclerotic arteries. Conclusion: TIMP3 expression levels increase in normal coronary arterial segments of cases with CHD, which depict a protective role of TIMP3 in development of atherosclerosis.

The expression of matrix metalloproteinases and their tissue inhibitors in the vein wall following superficial venous thrombosis.

Superficial venous thrombosis (SVT) is the complications of varicose great saphenous veins (VGSVs), but its pathogenesis remains unclear. This study was designed to measure the changes in expression of matrix metalloproteinases (MMPs) and the tissue inhibitor of metalloproteinases (TIMPs) from SVT, VGSVs, and great saphenous veins (GSVs). In the venous walls of the three groups, the expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 proteins, protein-positive expression ratios, mRNA expression, and protein expression were determined by immunohistochemistry, polymerase chain reaction, and western blot. The MMP-2, MMP-9, TIMP-1, and TIMP-2 protein-positive expression ratios, mRNA and protein expression in the SVT group were significantly higher than those in the VGSV and the GSV groups. The corresponding expression in the VGSV group were significantly higher than those in the GSV group. Disequilibrium of MMPs and TIMPs in SVT wall occurs due to underlying high hydrostatic pressure and inflammation. These results suggested that MMPs and TIMPs participate in the process of venous wall remodeling.

Effects of Dapagliflozin on Renal Interstitial Fibrosis in Diabetic Rats through Smad3, TIMP1 and MMP24 Pathway

Objective: This research was designed to probe into the effects of Dapagliflozin on renal interstitial fibrosis in diabetic rats through Smad3, TIMP1 and MMP24 pathway. Methods: Rats were bought to establish models, and then intervened by Dapagliflozin. Human mesangial cell lines (HMCs) stimulated by high glucose were purchased, and the Smad3, TIMP1 and MMP24 levels in rats after modeling and Dapagliflozin intervention were detected. The Smad3, TIMP1 and MMP24 protein expression in kidney tissue was examined after the rats were killed, and the expression in an intervention group (IG) and a blank group (BG) were analyzed. The cells were divided into three groups: Dapagliflozin intervention (Group 1), TGF-P1/Smad3 pathway inhibitor SIS3 intervention (Group 2) and no intervention (Group 3). The TIMP1 and MMP24 levels were assessed. Results: The Smad3 and MMP24 levels in group A were higher than those in other two groups (p &lt; 0.05), while those of TIMP1 were lower (p &lt; 0.05). Compared with pre-intervention, the Smad3 and MMP24 levels in groups A and B decreased (p &lt; 0.05), while those of TIMP1 increased (p &lt; 0.05). The Smad3 and MMP24 protein levels in groups A and B were higher than those in other two groups (p &lt; 0.05), while those of TIMP1 was lower (p &lt; 0.05). Compared with the BG, the Smad3 and MMP24 expression in the IG was lower (p &lt; 0.05) and that of TIMP1 was higher (p &lt; 0.05). The TIMP1 expression in Group 3 was lower (p &lt; 0.05) and that of MMP24 was higher than those in Groups 1 and 2 (p &lt; 0.05). Conclusion: Dapagliflozin can treat diabetic renal interstitial fibrosis by inhibiting TGF-P1/Smad3 signaling pathway, decreasing MMP24 and increasing TIMP1.

In vitro antimetastatic activity of Momordica balsamina crude acetone extract in HT-29 human colon cancer cells.

Plant-derived compounds and/or extracts have proven to be beneficial for the treatment of a broad spectrum of cancers with minimal side effects. In this study, we investigated whether a crude acetone extract of Momordica balsamina (MBE) can interfere with the metastatic ability of HT-29 colorectal cancer (CRC) cells. The phytochemical composition of MBE was determined by ultra-performance liquid chromatography and cytotoxic effects by the MTT and acridine orange/ethidium bromide staining assays. The effect of MBE on the formation of reactive oxygen species was assessed using the DCFH2 -DA assay. Wound healing assay, transwell cell invasion assay, cell adhesion assay, and the extracellular matrix-cell adhesion array were used to assess the antimetastatic effects of MBE. The effect of MBE on the expression of TNF-α, NF-κB, TIMP-3, MMP-2, and MMP-9 was assessed by western blot analysis. Our results showed that MBE consists of a mixture of compounds without a known anticancer activity in CRC and exhibits cytotoxicity against HT-29 cells. MBE also suppressed reactive oxygen species formation, cell invasion, cell migration, and cell adhesion. The reduction of cell invasion was associated with the downregulation of TNF-α, NF-κB, MMP2, and MMP9 and upregulation of TIMP-3 proteins. We concluded that MBE inhibits the metastatic ability of HT-29 CRC cells in vitro.

Abstract 3159: Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients

Abstract Tissue inhibitors of metalloproteases (TIMPs) have been reported to exert dual roles in cancer, based on the different cancer type and interactions with the tumor microenvironments. As inhibitors of Matrix metalloproteases (MMP) they downregulate invasion, metastasis and angiogenesis. However, TIMP1 levels have been found increased in patients with different types of cancer. Natural Killer cells have been found to be phenotypically and functionally compromised in several solid and hematological cancers. We were the first in characterizing NK pro-angiogenic phenotype in the peripheral blood and tumor tissues of patients with Non-small Cell Lung cancer (NSCLC), colorectal cancers (CRC) and in pleural effusion (PE) of patients with metastatic cancers. Also, we found that pro-angiogenic NK cells in the peripheral blood of colorectal cancer patients release of TIMP-1 and TIMP-2, in a STAT3/STAT5-independent manner. Here, we investigated the effects of TIMP-1 and TIMP-2 recombinant proteins, using a TGFβ-mediated NK cell polarization model, in vitro. The effects of TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptides, was determined by multicolor flow cytometry for surface antigens and cytokine productions, on cytolytic NK cells isolated from healthy subjects. Commercially available antibody membrane arrays was used to determine the modulation of the secretome, on FACS-sorted NK cells, from healthy subjects, following 72 hours of exposure to TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptide. We found that NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like phenotype, together with decreased levels of the activation marker NKG2D. Administration of TIMP1 or TIMP2 peptides was effective in interfering with TGFβ-induced NK cell polarization towards the CD56brightCD9+CD49a+ decidual-like phenotype and in restoration of the levels of the NKG2D activation marker. We also observed that TGFβ-polarized NK cell, when co-treated with TIMP1 or TIMP2, exhibit a trend toward decreased production of VEGF and increased production of the anti-tumor cytokines IFNγ and TNFα Our results suggest a potential role of TIMPs in contrasting the angiogenic switch in NK cells and suggest a possible use of TIMPs peptides in the re-education of anergic/pro-angiogenic NK cells in cancer patients. Citation Format: Matteo Gallazzi, Antonino Bruno, Douglas M. Noonan, William G. Stetler-Stevenson, Adriana Albini. Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3159.

Serum protein triplet TGF-\u03b21, TIMP-1, and YKL-40 serve as diagnostic and prognostic profile for astrocytoma

Astrocytoma is the most common glial tumour of the CNS. The most malignant form is grade IV Astrocytoma, also called Glioblastoma. Due to its heterogeneity, aggressiveness and lethal nature scientists are trying to find less invasive methods for early prediction of tumour onset, recurrence, response to therapy and patients’ survival. Here, applying decision tree classification algorithm we performed astrocytoma specific protein profile analysis on serum proteins TIMP-1, active and latent form of TGF-β1, IP-10, ANGPT-1, OPN, and YKL-40 using enzyme-linked immunosorbent detection assay (ELISA). Results have demonstrated that astrocytoma specific profile consisted of three proteins—active form of TGF-β1, TIMP-1 and YKL-40 and was able to correctly classify 78.0% (103/132) of sample and 83.3% (60/72) of astrocytoma sample. Calculating decision tree algorithm associated with astrocytoma patient survival, prediction model reached an accuracy of 83.3% (60/72). All together these results indicate that glioma detection and prediction from patient serum using glioma associated proteins and applying mathematical classification tools could be achieved, and applying more comprehensive research further could be implemented in clinic.

Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action.

Xanthohumol (XN), the main prenylated flavonoid from hop cones, has been recently reported to exert significant proapoptotic, anti-proliferative, and growth inhibitory effects against lung cancer in both in vitro and in vivo studies. However, its anti-metastatic potential towards this malignancy is still unrevealed. Previously, we indicated that the human lung adenocarcinoma A549 cell line was sensitive to XN treatment. Therefore, using the same tumour cell model, we have studied the influence of XN on the phorbol-12-myristate-13-acetate (PMA)-induced cell migration and invasion. The effects of XN on the expression/activity of pro-invasive MMP-9 and MMP-2 and the expression of MMP inhibitors, i.e., TIMP-1 and TIMP-2 (anti-angiogenic factors), were evaluated. Additionally, the influence of XN on the production of the key pro-angiogenic cytokine, i.e., VEGF, and the release of TGF-β, which is both a pro-angiogenic cytokine and an epithelial-mesenchymal transition (EMT) stimulator, was studied. Furthermore, the influence of XN on the expression of EMT-associated proteins such as E-cadherin and α-E-catenin (epithelial markers), vimentin and N-cadherin (mesenchymal markers), and Snail-1 (transcriptional repressor of E-cadherin) was studied. To elucidate the molecular mechanism underpinning the XN-mediated inhibition of metastatic progression in PMA-activated cells, the phosphorylation levels of AKT, FAK, and ERK1/2 kinases, which are signalling molecules involved in EMT program activation, were assayed. The results showed that XN in non-cytotoxic concentrations impaired the PMA-driven migratory and invasive capacity of A549 cells by decreasing the level of expression of MMP-9 and concomitantly increasing the expression of the TIMP-1 protein, i.e., a specific blocker of pro-MMP-9 activation. Moreover, XN decreased the PMA-induced production of VEGF and TGF-β. Furthermore, the XN-treatment counteracted the PMA-induced EMT of the A549 cells by the upregulation of E-cadherin and α-E-catenin and the downregulation of N-cadherin, vimentin, and Snail-1 expression. The proposed mechanism underlying the anti-invasive XN activity involved the inhibition of the ERK/MAPK pathway and suppression of FAK and PI3/AKT signalling. Our results suggesting migrastatic properties of XN against lung cancer cells require further verification in in vivo assays.

Expression of Matrix Metalloproteinases and Tissue Inhibitor of Matrix Metalloproteinases during Apical Periodontitis Development

IntroductionMatrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) are considered important mediators of the periapical immune response to infection. This study aimed to clarify the putative relationship between MMPs and TIMPs by elucidating the activity of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 in the temporal development of apical periodontitis (AP) in mice. MethodsAP was induced in the lower first molars of 30 male Kunming mice. The animals were randomly killed at 0, 7, 14, 28, 60, and 90 days after pulp exposure. The jaws were removed and subjected to quantitative real-time reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. ResultsThe MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 messenger RNA and protein expression levels increased with periapical inflammation progression (P < .05). The MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 messenger RNA and protein expression levels increased during the acute and chronic stages of periapical lesions, with less MMP-2 and MMP-9 expression levels at the chronic stage (P < .05). The MMP-8 expression increased at the chronic stage of inflammation (P < .05) but not at the acute stage. Immunostained MMP-2 and TIMP-1 were observed in all experimental periods. ConclusionsMMP-1, MMP-2, MMP-8, MMP-9, TIMP-1, and TIMP-2 were expressed in all periapical samples with varying levels between them. MMP expression could be related to TIMP expression in the temporal development of AP.

QUINAPRIL AMELIORATES CARDIAC REMODELING IN DIABETIC RATS VIA SUPPRESSION OF EGR1 AND STAT3 ACTIVATION

Objective: RAAS over activation in diabetes may induce myocardial hypertrophy and extracellular matrix accumulation even without hypertension, and ACE-inhibitors are widely used to manage blood pressure. STAT3 activation is known to promote experimental kidney remodeling, but its role in diabetic myocardium has not been elucidated. Experimental evidence shows that EGR1 contributes to myocardial remodeling, yet the effect of ACE-inhibition on myocardial EGR1 in diabetes is still unknown. Thus, we investigated whether the ACE-inhibitor quinapril could influence myocardial EGR1 and STAT3 activation in diabetic rats. Design and method: Male Sprague-Dawley rats (250–300 g, n=12) were ip. injected with 60 mg/kg streptozotocin to induce type-1 diabetes (DM). Half of DM rats was treated with 50 mg/kg/day quinapril in drinking water (Q) for 8 weeks. Non-diabetic littermates served as negative controls (CTL). Experimental groups were as follows (n = 6/group): 1) CTL; 2) DM; 3) DM+Q. At harvest, plasma renin activity (PRA) was assessed and myocardium was processed for histology, immunohistochemistry, mRNA and protein expression analyses. Data are presented as mean ± SD, analyzed using Kruskal-Wallis test with Dunn's post-hoc test. Results: PRA was elevated in diabetic groups regardless of treatment (CTL:1.3 ± 0.2, DM:2.2 ± 1.0, DM+Q:1.8 ± 1.1 ng/ml/h, p &lt; 0.05), accompanied by myocardial (pro)renin receptor (PRR) mRNA overexpression (fold expression, CTL:1.0 ± 0,2, DM:1.8 ± 0.9, DM+Q:1.7 ± 0.5, p &lt; 0.05). However, quinapril reduced collagen-1 mRNA expression (CTL:1.0 ± 0.3, DM:1.3 ± 0.4, DM+Q:0.7 ± 0.3, p &lt; 0.01) and fibronectin immunoreactivity, accompanied by reduced matrix remodeling shown by the expression of both tissue inhibitor of metalloproteases TIMP1 and TIMP2 (TIMP1, CTL:0.9 ± 0.1, DM:1.6 ± 0.7, DM+Q:1.0 ± 0.5, p &lt; 0.05; TIMP2, CTL:1.0 ± 0.1, DM:1.8 ± 0.5, DM+Q:1.1 ± 0.4, p &lt; 0.05). Immunoblots showed that quinapril also attenuated diabetes induced EGR1 protein expression (CTL:1.0 ± 0.4, DM:1.8 ± 0.5, DM+Q:0.5 ± 0.2, p &lt; 0.01) and STAT3 phosphorylation (CTL:1.0 ± 0.1, DM:1.4 ± 0.2, DM+Q:0.6 ± 0.1, p &lt; 0.01), accompanied by reduced TIMP1 protein expression (CTL:0.9 ± 0.1, DM:1.5 ± 0.3, DM+Q:0.9 ± 0.3, p &lt; 0.05) that correlated with STAT3 activity (R-square: 0.4, p = 0.03). Conclusions: In our rat model of type-1 diabetes, chronic quinapril treatment significantly attenuated cardiac remodeling via its direct inhibitory effect on the profibrotic myocardial EGR1 and STAT3. These results underscore the tissue-specific, blood pressure-independent beneficial effects of ACE inhibition in diabetes.

Ginsenoside Rg1 ameliorates blood–brain barrier disruption and traumatic brain injury via attenuating macrophages derived exosomes miR-21 release

During the traumatic brain injury (TBI), improved expression of circulatory miR-21 serves as a diagnostic feature. Low levels of exosome-miR-21 in the brain can effectively improve neuroinflammation and blood–brain barrier (BBB) permeability, reduce nerve apoptosis, restore neural function and ameliorate TBI. We evaluated the role of macrophage derived exosomes-miR-21 (M-Exos-miR-21) in disrupting BBB, deteriorating TBI, and Rg1 interventions. IL-1β-induced macrophages (IIM)-Exos-miR-21 can activate NF-κB signaling pathway and induce the expressions of MMP-1, -3 and -9 and downregulate the levels of tight junction proteins (TJPs) deteriorating the BBB. Rg1 reduced miR-21-5p content in IIM-Exos (RIIM-Exos). The interaction of NMIIA–HSP90 controlled the release of Exos-miR-21, this interaction was restricted by Rg1. Rg1 could inhibit the Exos-miR-21 release in peripheral blood flow to brain, enhancing TIMP3 protein expression, MMPs proteolysis, and restricting TJPs degradation thus protected the BBB integrity. Conclusively, Rg1 can improve the cerebrovascular endothelial injury and hold the therapeutic potential against TBI disease.

Identification and development of a novel invasion-related gene signature for prognosis prediction in colon adenocarcinoma

The overall survival of metastatic colon adenocarcinoma (COAD) remains poor, so it is important to explore the mechanisms of metastasis and invasion. This study aimed to identify invasion-related genetic markers for prognosis prediction in patients with COAD. Three molecular subtypes (C1, C2, and C3) were obtained based on 97 metastasis-related genes in 365 COAD samples from The Cancer Genome Atlas (TCGA). A total of 983 differentially expressed genes (DEGs) were identified among the different subtypes by using the limma package. A 6-gene signature (ITLN1, HOXD9, TSPAN11, GPRC5B, TIMP1, and CXCL13) was constructed via Lasso-Cox analysis. The signature showed strong robustness and could be used in the training, testing, and external validation (GSE17537) cohorts with stable predictive efficiency. Compared with other published signatures, our model showed better performance in predicting outcomes. Pan-cancer expression analysis results showed that ITLN1, TSPAN11, CXCL13, and GPRC5B were downregulated and TIMP1 was upregulated in most tumor samples, including COAD, which was consistent with the results of the TCGA and GEO cohorts. Western blot analysis and immunohistochemistry were performed to validate protein expression. Tumor immune infiltration analysis results showed that TSPAN11, GPRC5B, TIMP1, and CXCL13 protein levels were significantly positively correlated with CD4+ T cells, macrophages, neutrophils, and dendritic cells. Further, the TIMP1 and CXCL13 proteins were significantly related to the tumor immune infiltration of CD8+ T cells. We recommend using our signature as a molecular prognostic classifier to assess the prognostic risk of patients with COAD.

RETRACTED ARTICLE: Effects of miR-672 on the angiogenesis of adipose-derived mesenchymal stem cells during bone regeneration

BackgroundSufficient vascular network plays an important role in the repair of bone defects. Bone morphogenetic protein 2 (BMP2) being a key regulator of angiogenesis has attracted the attention of researchers. In addition, evidence has suggested that BMP2 coordinates with microRNAs (miRNAs) to form intracellular networks regulating mesenchymal stem cells (MSCs) angiogenesis. Elucidating the underlying mechanisms that are regulating adipose-derived mesenchymal stem cells (ADSCs) angiogenesis might provide more effective method to enhance bone regeneration.MethodsWe identified the specific miRNA in rat ADSCs during BMP2-induced angiogenesis and chose the most significant differentially expressed miRNA, miR-672. Three lentiviral system named Lenti-miR-672, Lenti-as-miR-672, and Lenti-miR-NC were transduced into the ADSCs individually. Then, the quantitative real-time polymerase chain reaction (qPCR), western blotting, and blood vessel formation analysis were performed to investigate the effects of miR-672 on ADSCs angiogenesis. Bioinformation platforms were used to screen the potential target of miR-672. Small interfering RNA (siRNA) against TIMP2 (si-TIMP2) mRNA were obtained from GenePharma, and then si-TIMP2 miRNA and miR-672 were co-transfected into ADSCs to detect the effects of TIMP2 on angiogenesis. Calcium phosphate cement (CPC) scaffolds that seeded the lentiviral-modified ADSCs were constructed to test the vascularized bone regeneration in vivo.ResultsOur data showed that after the angiogenesis of ADSCs induced by BMP2, miR-672 was the most significantly upregulated miRNA. Overexpression of miR-672 promoted the angiogenesis of ADSCs, while knockdown of miR-672 repressed the angiogenesis of ADSCs. The bioinformation prediction showed that TIMP2 might be the one of miR-672′ potential targets. TIMP2 protein expression was gradually decreased in ADSCs with overexpressed miR-672. And the angiogenic factors were upregulated in the ADSCs which were transduced with si-TIMP2. Then, the CPC scaffolds coupled the miR-672-modified ADSCs and showed the good potential in vascularized bone regeneration. The overexpressed miR-672 could greatly enhance the blood vessel volume and Microfil-labeled blood vessel numbers in newly formed bone.ConclusionBMP2 could promote the angiogenesis of ADSCs through stimulating the expression of miR-672 in ADSCs. miR-672 acted as a positive regulator on the angiogenesis of ADSCs, and incorporating the miR-672-modified ADSCs in the CPC could significantly promote the vascularization and the bone regeneration.

Response to trastuzumab and investigation of expression profiles of matrix metalloproteinase-related proteins in primary breast cancer stem cells.

Breast cancer (BC) is the leading cause of cancer deaths in women. One of the reasons for the failure of BC treatment is reportedly the ineffectiveness of chemotherapeutic drugs against breast cancer stem-like cells (BCSCs). HER2 receptors have an important role in the self-renewal of BCSCs. Matrix metalloproteinase (MMP) and cytokine levels were found to be higher in BCSCs, which demonstrates their potential metastatic capacity. Therefore, the aim of this study was to evaluate the response of BCSCs to trastuzumab and to investigate the MMP levels in primary breast cancer cells and HER2+ BCSCs. Tumour tissue samples were obtained during surgical intervention from ten breast cancer patients, and primary culture cells were established from these tissues. Four major molecular subgroups were sorted from the primary culture: HER2+ BCSCs (CD44+CD24-HER2+), HER2- BCSCs (CD44+CD24-HER2-), HER2- primary culture cells (CD44+CD24+HER2-) and triple positive primary culture cells (CD44+CD24+HER2+). These cells were cultured and treated with trastuzumab, paclitaxel, carboplatin, and the combination of those three drugs for 96h. Cellular responses to these drugs were determined by XTT cytotoxicity test. MMPs and cytokine array analysis showed that MMPs and TIMP-1, TIMP-2 proteins were expressed more in HER2+ BCSCs than in primary culture. HER2- BCSCs were more resistant to drugs than HER2+ BCSCs. Our findings suggest that the presence of HER2- BCSCs may be responsible for primary trastuzumab resistance in HER2+ BC cell population. Further studies investigating the function of MMPs are needed for drug targeting of BCSCs.

Long-term exposure to butyric acid induces excessive production of matrix metalloproteases in human gingival fibroblasts

ObjectiveThe purpose of this study was to clarify the relationship between bacteria-induced butyric acid and periodontal disease progression. DesignNormal human gingival fibroblasts were exposed to butyric acid (0, 1, 5, 10, and 15 mM) adjusted to a pH of 7.2–7.4 using sodium hydroxide for 0–96 h and cell viability was evaluated. In addition, the effects of butyric acid on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in gingival fibroblasts were analyzed by real-time RT-PCR, ELISA, western blotting, and stromelysin zymography. ResultsButyric acid reduced the viability of gingival fibroblasts in a concentration- and time-dependent manner. Furthermore, butyric acid promoted production of MMP-1, MMP-3, and MMP-10 in gingival fibroblasts and suppressed TIMP-2 protein production. ConclusionsButyric acid promoted overproduction of MMPs, resulting in a disruption of the balance between MMPs and TIMPs expression in gingival fibroblasts. Our study suggests that the butyric acid produced by causative bacteria stimulates excessive MMP expression in periodontal tissue, leading to destruction of the tissue.

Modified Simiaowan prevents articular cartilage injury in experimental gouty arthritis by negative regulation of STAT3 pathway

Ethnopharmacological relevanceModified Simiaowan (MSW) is a traditional Chinese medicine formula that is composed of six herbs. It has been widely used in the treatment of gouty arthritis. Aim of the studyThis study was designed to investigate the effect of MSW on gouty arthritis and explore the possible mechanisms. Material and methodsThe rat gouty arthritis model was established by intra-articular injection of Monosodium Urate (MSU) crystal, and then treated with MSW for 5 days. The perimeter of the knee joints was measured in a time-dependent manner and serum samples were collected for the detection of TNF-α, IL-1β, and IL-6 protein levels by ELISA. The protein expressions of MMP-3, TIMP-3, STAT3, and p-STAT3 in cartilage tissues and C28/I2 cells were detected by Western blot, and the levels of proteoglycan in primary chondrocytes and cartilage tissues were determined by toluidine blue staining. In addition, AG490 and IL-6 were used in vitro to explore the function of IL-6/STAT3 pathway in the protective effect of MSU. ResultsMSW reduced the joint swelling rate in gouty arthritis model and inhibited MSU induced up-regulation of IL-1β, TNF-α, and IL-6 protein levels in serum and synovial fluid. IL-1β induced an increase in p-STAT3 and MMP-3 protein expression in C28/I2 cells, as well as a decrease in TIMP-3. MSW serum inhibited the protein expression changes induced by IL-1β in vitro. Furthermore, inhibition of STAT3 signaling negated the effect of MSW serum on p-STAT3, MMP-3, and TIMP-3 protein levels in C28/I2 cells. MSW also increased the content of proteoglycan significantly both in vivo and in vitro. ConclusionOur data indicated that MSW protected rats from MSU-induced experimental gouty arthritis and IL-1β/IL-6/STAT3 pathway played an essential role in the protective effect of MSU against GA.

IOX1 protects from TGF-β induced fibrosis in LX-2 cells via the regulation of extracellular matrix protein expression.

The aim of the present study was to investigate the effect of the histone H3K9 demethylase inhibitor, IOX1, on the mechanism of hepatic fibrosis in TGF-β-induced human hepatic stellate LX-2 cells. Cellular proliferation, apoptosis, histone H3K9 dimethylation (H3K9me2), protein expression of extracellular matrix (ECM)-related proteins α-smooth muscle actin (SMA), type I collagen (Col I), MMP-1 and TIMP-1 were measured. H3K9me2 levels in the promoter region of ECM-related genes were detected by real-time cell analysis (RTCA), flow cytometry, western blotting and chromatin immunoprecipitation (ChIP) in LX-2 cells. IOX1 significantly inhibited cell proliferation and the IC50 of IOX1 was 100 µM in cells treated with IOX1 for 48 h. IOX1 significantly induced apoptosis in LX-2 cells in a concentration-dependent manner. In addition, different concentration of IOX1 increased the level of H3K9me2 and downregulated the expression of α-SMA, Col I, MMP-1 and TIMP-1 in TGF-β-induced LX-2 cells. ChIP measurements indicated that H3K9me2 levels in the promotor region of the corresponding genes were increased in TGF-β-induced LX-2 cells. IOX1 may elevate H3K9me2 in the promotor region of Col I, MMP-1, and TIMP-1 genes to regulate α-SMA, Col I, MMP-1 and TIMP-1 protein expression to induce cell apoptosis, inhibit LX-2 cell proliferation and oppose hepatic fibrotic activity.