MMP-9 mRNA Research Articles

Physalis angulata reduces the progression of chronic experimental periodontitis by immunomodulatory mechanisms

Ethnopharmacological relevancePhysalis angulata is an herb found in tropical and subtropical regions of the world; it is widely applied in popular medicine due to the therapeutic properties of the whole plant and its parts. Extracts and infusions of this plant have been extensively applied in folk medicine worldwide to treat inflammatory and immune-mediated diseases, including oral inflammatory conditions such as sore throat and gingivitis. Aim of the studyThe present study was designed to investigate the protective effects of the ethanolic extract of P. angulata (EEPA) in a murine model of chronic periodontitis, aiming to corroborate its traditional use as an anti-inflammatory and immunomodulatory agent, and to point out possible mechanisms involved in these effects. Materials and methodsEEPA was obtained from the stems of P. angulata collected in Belém (PA, Brazil). Chronic periodontitis was induced in male C57BL/6 mice by 12 administrations of lipopolysaccharide (LPS; 20 μg/1μL) into the gingival papilla in the course of 28 days. Starting from the 15th day after the first LPS injection, mice were daily treated with EEPA (50 or 100 mg/kg), nimesulide (25 mg/kg, reference drug), or vehicle by oral route for 14 days. At the end of the experimental period, alveolar bone loss was evaluated along with the gingival expression of biomarkers of periodontitis and cytokines by RT-q-PCR and ELISA. Hematological and biochemical parameters suggestive of systemic toxicity were also evaluated. The transcriptional activity of NF-κB was investigated using the luciferase assay in macrophages. ResultsMice with chronic experimental periodontitis suffered alveolar bone loss that was prevented by the treatment with EEPA (50 or 100 mg/kg) or nimesulide (25 mg/kg). EEPA (50 and 100 mg/kg) and nimesulide (25 mg/kg) reduced mRNA levels of MMP-9 mRNA, but not of TIMP-1 in gingival tissue of periodontitis-induced mice. Both treatments also reduced the production of the pro-inflammatory cytokines IL-1β and IL-6. The treatment with EEPA (100 mg/kg) increased the production of the anti-inflammatory cytokine TGF-β. No hematological or biochemical alterations were caused by the daily treatment with EEPA. In vitro luciferase assay suggested that a putative mechanism of EEPA is reducing the transcriptional activity of NF-κB. ConclusionsEEPA exhibited a disease-modifying effect in the chronic experimental periodontitis, along with unidentifiable systemic toxicity. This work corroborates the traditional use of P. angulata in oral inflammatory conditions and provides mechanistic hypotheses to explain its therapeutic effects.

Post-transcriptional regulation of MMP2 mRNA by its interaction with miR-20a and Nucleolin in breast cancer cell lines.

Matrix-metalloproteinase-2 (MMP2) is a foremost MMP, governing invasion of breast cancer cells during metastasis. miR-20a was reported to induce mesenchymal to epithelial transition in MDA-MB-231 cells and its endogenous expression varies directly with invasiveness of breast cancer cells. The inverse and direct correlation of invasiveness with miR-20a and Nucleolin respectively led us to study the post-transcriptional regulation of MMP2 by miR-20a and mRNA stabilizing protein, Nucleolin. Thus, understanding the mechanism of its regulation will enable modification of the invasion potential. MMP2 was found to be higher in MDA-MB-231 than MCF-7 cells both at RNA and protein levels. RNA-protein co-immunoprecipitation assay with Argonaute 2 revealed that MMP2 undergoes miRNA-mediated post-transcriptional regulation. miR-20a decreased MMP2 expression as well as its enzymatic activity as found by zymogram assay. Reporter assay showed that miR-20a directly binds to its putative binding site in MMP2 3'-UTR as per in silico prediction. miR-20a additionally impeded MMP2 mRNA stability, and binding of stabilizing trans-factor Nucleolin to its 3'-UTR was confirmed by RNA-protein co-immunoprecipitation assay. Partial down-regulation of Nucleolin by Si-RNA resulted in the downregulation of MMP2and Nucleolin over-expression rescued the inhibitory effect of miR-20a on MMP2 expression. Delineating the mechanism of post-transcriptional regulation of MMP2, two of its potent regulators, miR-20a and Nucleolin were identified. It was established for the first time that MMP2 is a direct target of miR-20a. The results also elucidated that Nucleolin binds to MMP2 3' UTR and its abundance affects MMP2 expression.

English

The aims of this work were to study growth, isothiocyanate (ITC), bioactive content, antioxidant activity and anticancer activity of mustard green (MG) microgreens grown from seeds treated with cold plasma at 21 and 23 kV for 5 min. Microgreens from plasma-treated seeds at 23 kV showed almost 2-fold increased ITC content (1.57 ± 0.05 mmol/100 g DW) compared to MG from seeds without plasma (control), showed the highest total phenolic content (TPC) (6.76 ± 0.14 mg GAE/g DW) and total flavonoid content (TFC) (0.16±0.01 mg RE/g DW). However, MG plasma-treated seeds at 21 kV showed the highest antioxidant activity from 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay (3.51 ± 0.38 mg TE/g DW). Allyl isothiocyanate and 3-butenyl isothiocyanate were the dominant ITCs in MG. The highest cytotoxicities using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against MCF-7 (IC50 of 32.44 ± 1.64 μg/mL) and HepG2 (IC50 of 28.58 ± 1.04 μg/mL) after 72 h exposure were found in MG from plasma-treated seeds at 23 kV and MG from control seeds, respectively. However, MG from plasma-treated seeds at 21 kV exhibited the highest antiproliferative effect against MCF-7 (IC50 of 23.23 ± 0.23 μg/mL) and HepG2 (IC50 of 20.44 ± 0.56 μg/mL) for 14 days and also the most potent antimigratory effect. MG from cold plasma inhibited MMP-9 protein expression in both cancers indicating antimigratory property. MG from cold plasma also significantly reduced MMP-9 mRNA expression in both cancers when compared to the control and untreated cells. In conclusion, cold plasma treatment on seeds seemed to be an innovative tool to enhance ITC, TPC, TFC and anticancer properties of MG microgreens for better health implications. © 2021 Friends Science Publishers

Stepwise candidate drug screening for myopia control by using zebrafish, mouse, and Golden Syrian Hamster myopia models.

BackgroundWe developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia.MethodsA morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-β2 (TGF-β2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice.FindingsIn the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes.InterpretationStepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials.FundingThis research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare.

Evaluation of Virola oleifera activity in musculoskeletal pathologies: Inhibition of human multiple myeloma cells proliferation and combination therapy with dexamethasone or bortezomib

Ethnopharmacological relevanceVirola oleifera (Schott) A.C. Smith, Myristicaceae, has been widely used in traditional medicine in Brazil to treat rheumatic pain, joint tumours, skin diseases, halitosis, bronchial asthma, haemorrhoids, and intestinal worms. Recently, research data showed the antioxidant properties in several oxidative stress-related models. However, there is no experimental evidence supporting its potential use in managing rheumatic diseases and bone malignancies. Aims of the studyTo evaluate the therapeutic potential of the resin from Virola oleifera in joint and bone diseases, namely arthritis, osteosarcoma, chondrosarcoma, and multiple myeloma. Materials and methodsTo determine Virola oleifera resin (VO) effects on arthritis-associated inflammation and cartilage degradation, the LPS-induced NO production, and mRNA and protein expression of ADAMTS5, MMP13, COL2, and ACAN, were evaluated in chondrocytes (ATDC5 and TC28 cell lines). The cytotoxic effects of VO (0.05–50 μg/ml) on multiple myeloma (ARH-77), osteosarcoma (SAOS-2), and chondrosarcoma (SW-1353) cell lines were analysed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The VO effects, combined with dexamethasone or bortezomib, were evaluated in a multiple myeloma cell line. The mechanisms of VO, alone or in combination with bortezomib, were determined by cell cycle analysis through flow cytometry, while expression levels of p-Akt/Akt, p-ERK/ERK, p-p38/p38 MAPK, Bax, Bcl-2, and cleaved-caspase-3/caspase-3 proteins by Western blot. ResultsVO had no significant effect on LPS-induced NO production in chondrocytes at non-cytotoxic concentrations. VO treatment diminished the mRNA levels of metalloproteinases and ECM components; however, any significant effect was observed on the protein expression levels. The cell viability of a multiple myeloma cell line was strongly reduced by VO treatment in a dose- and time-dependent manner, while osteosarcoma and chondrosarcoma cell lines viability was significantly affected only by the highest dose assessed. In multiple myeloma cells, VO leads to G2/M cell cycle arrest. Furthermore, it synergizes with dexamethasone by increasing cell toxicity. Finally, VO reverts bortezomib activity by counteracting ERK1/2, Bax, and caspase-3 activation. ConclusionsThe current work supports the ethnopharmacological use of Virola oleifera (Schott) A.C. Smith in bone and joint diseases, but there is no evidence for the amelioration of arthritis-associated inflammatory or catabolic processes. Our data also supports the potential use of Virola oleifera as adjuvant therapy to optimize the pharmacologic effects of current chemotherapeutic drugs. However, possible herb-drug interactions should be considered before clinical application.

Citrulluside H and citrulluside T from young watermelon fruit attenuate ultraviolet B radiation-induced matrix metalloproteinase expression through the scavenging of generated reactive oxygen species in human dermal fibroblasts.

Prolonged exposure to UV radiation disrupts the oxidative stress defense systems; in the absence of adequate reactive oxygen species (ROS)-scavenging mechanisms, the ROS produced cause oxidative damage to DNA, proteins, carbohydrates, and lipids. Thus, ROS elimination can protect against photoaging and possibly skin cancer. We recently isolated two novel phenolic glycosides (citrulluside H and citrulluside T) with antioxidative activity from young watermelon (Citrullus lanatus) fruits. In the present study, we evaluated the protective effect of these compounds against UV-B-induced photodamage in normal human dermal fibroblasts (NHDFs). NHDFs were exposed to UV-B irradiation at 25mJ/cm2 . Matrix metalloproteinase (MMP)-1 and MMP-3 mRNA levels were measured by quantitative real-time PCR. MMP protein expression, as well as MAPK and NF-κB signaling, was investigated by Western blot analysis. Activation protein-1 (AP-1) transcriptional activity was determined using a luciferase reporter assay. Intracellular ROS levels were measured using the CellROX™ Deep Red Reagent. Citrulluside H and citrulluside T significantly downregulated the protein and mRNA expression of MMP-1 and MMP-3, which are associated with the onset of skin wrinkle formation. Importantly, inhibition of UV-B-induced MMP expression in NHDFs by citrulluside H and citrulluside T was primarily due to the attenuation of MAPK/AP-1 and NF-κB signaling via scavenging of the generated ROS. Our findings delineated both citrulluside H and citrulluside T as potential agents for alleviating UV-induced skin photoaging.

Abstract PD15-02: Cx3cl1-cx3cr1 axis in liver metastasis of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a breast cancer subtype with increased risk of distant metastasis, especially to visceral organs. However, the detailed molecular mechanism underlying the visceral metastasis of TNBC is unknown. We used three patient-derived xenograft models derived from TNBC tumors with different metastatic capacities. PDX1 and PDX2 model developed distant metastasis in only lung and liver, respectively. In contrast, PDX3 tumor metastasized to both lung and liver. RNA sequencing of the primary tumor and distant metastatic sites of these three PDX models was performed. The gene expression profiles of the primary tumors of the three models showed minimally overlapping genes. However, the microenvironment gene expression profiles, measured by using the sequencing reads aligned to mouse genome, showed distinct difference between the models. Among the differentially expressed genes, we identified Cx3cr1 as a significantly upregulated gene in the liver microenvironment of the PDX models that metastasized to liver. Next, we used 4T1 syngeneic mouse mammary carcinoma model to validate the Cx3cr1 in the mouse liver tissues. Interestingly, the Cx3cr1 up-regulation occurred during the pre-metastatic period, and Cx3cl1, the ligand of the Cx3cr1, was also up-regulated in the liver tissue prior to the development of metastasis suggesting the Cx3cr1 regulates the formation of pre-metastatic niche. Cx3cl1, the ligand of the Cx3cr1, was also up-regulated in the liver tissue prior to the development of metastasis. These data suggest that Cx3cl1 and Cx3cr1-mediated tumor-microenvironment interaction is critical in developing liver metastasis in TNBC. Cx3cl1 increased the invasion and migration of Raw264.7 monocyte/macrophage cells and this effect was abrogated by the Cx3cr1 silencing in Raw264.7 cells or treating Cx3cl1 neutralizing antibody. Pathway analysis of the RNA sequencing data showed that genes involved in extracellular matrix remodeling was significantly dysregulated in the liver tissues including Lysyl Oxidase (Lox). Cx3cl1 treatment in Raw264.7 cells resulted in increased expression of Lox and MMP9 mRNA in three-dimensional culture system. The Raw264.7 cells caused increased invasion of 4T1 cells in vitro. In conclusion, our data suggest that Cx3cl1-Cx3cr1 axis plays critical role during the liver metastasis of TNBC. Citation Format: Woohang Heo, Hye-Youn Son, Ju hee Kim, Kyu Jin Lee, Myeonghee Jeon, Songbin Li, Jeehee Jang, Dong-Young Noh, Wonsik Han, Hyeong-Gon Moon. Cx3cl1-cx3cr1 axis in liver metastasis of triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-02.

SIRT6 enhances telomerase activity to protect against DNA damage and senescence in hypertrophic ligamentum flavum cells from lumbar spinal stenosis patients.

Lumbar spinal stenosis (LSS) is a condition wherein patients exhibit age-related fibrosis, elastin-to-collagen ratio reductions, and ligamentum flavum hypertrophy. This study was designed to assess the relationship between SIRT6 and telomerase activity in hypertrophic ligamentum flavum (LFH) cells from LSS patients. We observed significant reductions in SIRT6, TPP1, and POT1 protein levels as well as increases in telomerase reverse transcriptase (TERT) levels and telomerase activity in LFH tissues relative to non- hypertrophic ligamentum flavum (LFN) tissues. When SIRT6 was overexpressed in these LFH cells, this was associated with significant increases in telomerase activity and a significant reduction in fibrosis-related protein expression. These effects were reversed, however, when telomerase activity was inactivated by hTERT knockdown in these same cells. SIRT6 overexpression was further found to reduce the frequency of senescence-associated β-galactosidase (SA-β-Gal)-positive LFH cells and to decrease p16, MMP3, and L1 mRNA levels and telomere dysfunction-induced foci (TIFs) in LFH cells. In contrast, hTERT knockdown-induced telomerase inactivation eliminated these SIRT6-dependent effects. Overall, our results indicate that SIRT6 functions as a key protective factor that prevents cellular senescence and telomere dysfunction in ligamentum flavum cells, with this effect being at least partially attributable to SIRT6-dependent telomerase activation.

Blackcurrant (Ribes nigrum L.) Extract Exerts Potential Vasculoprotective Effects in Ovariectomized Rats, Including Prevention of Elastin Degradation and Pathological Vascular Remodeling.

Estrogen exerts cardioprotective effects in menopausal women. Phytoestrogens are plant-derived substances exhibiting estrogenic activity that could beneficially affect vascular health. We previously demonstrated that blackcurrant (Ribes nigrum L.) extract (BCE) treatment exerted beneficial effects on vascular health via phytoestrogenic activity in ovariectomized (OVX) rats, which are widely used as menopausal animal models. Here, we examined whether BCE treatment reduced elastin degradation and prevented pathological vascular remodeling in OVX rats fed a regular diet (OVX Control) or a 3% BCE-supplemented diet (OVX BCE), compared with sham surgery rats fed a regular diet (Sham) for 3 months. The results indicated a lower staining intensity of elastic fibers, greater elastin fragmentation, and higher α-smooth muscle actin protein expression in OVX Control rats than in OVX BCE and Sham rats. Pathological vascular remodeling was only observed in OVX Control rats. Additionally, we investigated matrix metalloproteinase (MMP)-12 mRNA expression levels to elucidate the mechanism underlying elastin degradation, revealing significantly upregulated MMP-12 mRNA expression in OVX Control rats compared with that in Sham and OVX BCE rats. Together, we identify BCE as exerting a vascular protective effect through reduced MMP-12 expression and vascular smooth muscle cell proliferation. To our knowledge, this is the first report indicating that BCE might protect against elastin degradation and pathological vascular remodeling during menopause.

Expression of matrix metalloproteinases (MMPs) and their inhibitor (TIMP) genes on mRNA and protein levels in oral squamous cell carcinoma

Introduction. To investigate the mRNA and protein expression of MMP-2, MMP-9, MMP-7 and their tissue inhibitor TIMP-2 in tissue specimens with oral squamous cell cancer (OSCC) and in healthy tissues. Material and methods. The expression genes of MMP-2, MMP-9, MMP-7 and TIMP-2 on mRNA levels were detected by the RT-PCR method in 31 samples with oral squamous cell carcinoma and in 31 healthy, control tissues. Secondly, the concentration of the analysed metalloproteinases and their inhibitor was assessed in tumor and non-tumor tissues using the enzyme-linked immunosorbent assay (ELISA) method. Results. The mean values of gene expression of MMP-2, MMP-7, MMP-9 and TIMP in tissues with oral squamous cell cancer were significantly higher in comparison to normal ones (p < 0.0001). Similar observations were found for concentration levels of analysed MMPs and TIMP in tissues with and without oral cancer (p < 0.0001). Conclusions. The present study demonstrated that MMP-2, MMP-7, MMP-9 and TIMP-2 gene expression on protein and mRNA levels is higher in oral squamous cell carcinoma tissues than in healthy control tissues. This may suggest that MMPs and TIMP play an important role in tumorogenesis. We did not observe any correlation between the clinicopathological characteristics of patients with OSCC and expression levels of MMPs and TIMP.

A Human Cellular Model for Colorectal Anastomotic Repair: The Effect of Localization and Transforming Growth Factor-β1 Treatment on Collagen Deposition and Biomarkers.

Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-β1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90+), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-β1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-β1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-β1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-β1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.

Synergistic effects of magnesium ions and simvastatin on attenuation of high-fat diet-induced bone loss

IntroductionMagnesium (Mg) has a prophylactic potential against the onset of hyperlipidemia. Similar to statin, Mg is recommended as lipid-lowering medication for hypercholesterolemia and concomitantly exhibits an association with increased bone mass. The combination of statin with Mg ions (Mg2+) may be able to alleviate the high-fat diet (HFD)-induced bone loss and reduce the side-effects of statin. This study aimed to explore the feasibility of combined Mg2+ with simvastatin (SIM) for treating HFD-induced bone loss in mice and the involving mechanisms. Materials and methodsC57BL/6 male mice were fed with a HFD or a normal-fat diet (NFD). Mice were intraperitoneally injected SIM and/or orally received water with additional Mg2+ until sacrificed. Enzyme-linked immunosorbent assay was performed to measure cytokines and cholesterol in serum and liver lysates. Bone mineral density (BMD) and microarchitecture were assessed by micro-computed tomography (μCT) in different groups. The adipogenesis in palmitate pre-treated HepG2 cells was performed under various treatments. ResultsμCT analysis showed that the trabecular bone mass was significantly lower in the HFD-fed group than that in NFD-fed group since week 8. The cortical thickness in HFD-fed group had a significant decrease at week 24, as compared with NFD-fed group. The combination of Mg2+ and SIM significantly attenuated the trabecular bone loss in HFD-fed mice via arresting the osteoclast formation and bone resorption. Besides, such combination also reduced the hepatocytic synthesis of cholesterol and inhibited matrix metallopeptidase 13 (Mmp13) mRNA expression in pre-osteoclasts. ConclusionsThe combination of Mg2+ and SIM shows a synergistic effect on attenuating the HFD-induced bone loss. Our current formulation may be a cost-effective alternative treatment to be indicated for obesity-related bone loss.

Resveratrol inhibits matrix metalloproteinase-1 and -3 expression by suppressing of p300/NFκB acetylation in TNF-α-treated human dermal fibroblasts

The aim of this study was to explore the signaling pathways associated with the effects of tumor necrosis factor alpha (TNF-α) on matrix metalloproteinase-1 (MMP-1) and MMP-3 expression in the human dermal fibroblast cell line CCD-966SK. TNF-α upregulated MMP-1 and MMP-3 mRNA and protein expression, and NFκB/p65 activation was found to be involved in TNF-α-induced MMP-1 and MMP-3 upregulation. TNF-α induced p65 phosphorylation at Ser536 and acetylation at Lys310. p300 knockdown suppressed TNF-α-induced p65 acetylation and reduced MMP-1 and MMP-3 expression in TNF-α-treated cells, but did not greatly restore MMP-1 and MMP-3 expression when p65 phosphorylation was inhibited by Bay11-7082 (IκBα inhibitor). NF-κB/luciferase reporter assay revealed that p300-mediated p65 acetylation was crucial for TNF-α-induced nuclear factor-kappa B (NF-κB) transcriptional activity. The chromatin immunoprecipitation (ChIP) assay indicated that TNF-α increased p300 recruitment to the MMP-1 and MMP-3 promoter regions surrounding the NFκB-binding site. Resveratrol notably inhibited TNF-α-induced MMP-1 and MMP-3 upregulation and abrogated TNF-α-induced p65 acetylation, leading to the downregulation of MMP-1 and MMP-3 expression in TNF-α-treated cells. Our data indicate that TNF-α-induced p300-mediated p65 acetylation leads to the upregulation of MMP-1 and MMP-3 expression in dermal fibroblasts, whereas resveratrol reduces this TNF-α-induced upregulation by downregulating p300 expression.

TREM-1 orchestrates angiotensin II-induced monocyte trafficking and promotes experimental abdominal aortic aneurysm.

The triggering receptor expressed on myeloid cells 1 (TREM-1) drives inflammatory responses in several cardiovascular diseases but its role in abdominal aortic aneurysm (AAA) remains unknown. Our objective was to explore the role of TREM-1 in a mouse model of angiotensin II-induced (AngII-induced) AAA. TREM-1 expression was detected in mouse aortic aneurysm and colocalized with macrophages. Trem1 gene deletion (Apoe-/-Trem1-/-), as well as TREM-1 pharmacological blockade with LR-12 peptide, limited both AAA development and severity. Trem1 gene deletion attenuated the inflammatory response in the aorta, with a reduction of Il1b, Tnfa, Mmp2, and Mmp9 mRNA expression, and led to a decreased macrophage content due to a reduction of Ly6Chi classical monocyte trafficking. Conversely, antibody-mediated TREM-1 stimulation exacerbated Ly6Chi monocyte aorta infiltration after AngII infusion through CD62L upregulation and promoted proinflammatory signature in the aorta, resulting in worsening AAA severity. AngII infusion stimulated TREM-1 expression and activation on Ly6Chi monocytes through AngII receptor type I (AT1R). In human AAA, TREM-1 was detected and TREM1 mRNA expression correlated with SELL mRNA expression. Finally, circulating levels of sTREM-1 were increased in patients with AAA when compared with patients without AAA. In conclusion, TREM-1 is involved in AAA pathophysiology and may represent a promising therapeutic target in humans.

RBM3 Increases Cell Survival but Disrupts Tight Junction of Microvascular Endothelial Cells in Acute Lung Injury

BackgroundRNA-binding motif protein 3 (RBM3) is an important cold shock protein, which also responds to hypothermia or hypoxia. RBM3 is involved into multiple physiologic processes, such as promoting cell survival. However, its expression and function in acute lung injury (ALI) have not been reported. MethodsA mouse ALI model was established by lipopolysaccharides (LPS) treatment. The RBM3 and cold inducible RNA-binding protein mRNA levels were examined by RT-qPCR, and MMP9 mRNA stability was determined by actinomycin D assay. RBM3 and MMP9 mRNA was tested by RNA immunoprecipitation (RIP assay). RBM3 overexpression or silent stable cell lines were established using recombinant lentivirus and subsequently used for cell survival and tight junction measurements. ResultsIn this study, we found that RBM3, rather than cold inducible RNA-binding protein, was upregulated in lung tissue of ALI mice. RBM3 was increased in human pulmonary microvascular endothelial cells (HPMVECs) in response to LPS treatment, which is modulated by the NF-κB signaling pathway. Furthermore, RBM3 could reduce cell apoptosis induced by LPS, probably through suppressing p53 expression. Because increased permeability of HPMVECs leads to pulmonary edema in ALI, we subsequently examined the effect of RBM3 on cell tight junctions. Unexpectedly, RBM3 decreased the expression of tight junction protein zonula occludens-1 and increased cell permeability, and RBM3 overexpression increased MMP9 mRNA stability. Furthermore, RIP assay confirmed the interaction between RBM3 and MMP9 mRNA, possibly explaining the contribution of RBM3 to increase cell permeability. ConclusionsRBM3 seems to act as a "double-edged sword" in ALI, that RBM3 alleviates cell apoptosis but increases HPMVEC permeability in ALI.

Potential role of M2 TAMs around lymphatic vessels during lymphatic invasion in papillary thyroid carcinoma

The aim of this study was to examine whether lymphatic invasion in papillary thyroid carcinoma (PTC) occurs when tumour-associated macrophages (TAMs) injure lymphatic vessels together with cancer cells. While there was no difference in the lymphatic vessel density in PTC and follicular thyroid carcinoma (FTC), the number of TAMs around the lymphatic vessels was increased in PTC compared to that in FTC. In particular, TAMs were observed together with cancer cells in lymphatic invasive lesions, and the number of M2 cells inside and outside the lymphatic vessels showed a significant correlation. MMP-2 mRNA was expressed in nonneoplastic stromal cells as well as cancer cells, and double immunofluorescence staining confirmed M2 positivity. Consequently, this study reveals that M2 TAMs around lymphatic vessels within the tumour border of PTC may be associated with the lymphatic invasion of cancer cells. This study represents a step forward in elucidating the mechanism of lymphatic invasion.

TIMELESS inhibits breast cancer cell invasion and metastasis by down-regulating the expression of MMP9

Breast cancer is the first killer leading to female death, and tumor metastasis is one of the important factors leading to the death of patients, but the specific mechanism of breast cancer metastasis is not very clear at present. Our study showed that overexpression of TIMELESS could significantly inhibit the invasion and metastasis of breast cancer cells ZR-75-30 and the assembly of F-actin protein. On the contrary, knockdown of TIMELESS promoted the invasion and metastasis of breast cancer cells. Further study revealed that TIMELESS overexpression decreased the mRNA and protein levels of MMP9. Furthermore, TIMELESS could interact with p65, leading to repress the association of p65 and its acetyltransferase CBP and down-regulating the acetylation level of p65, which inhibited the activation of NF-κB signal pathway. In conclusion, our research showed that TIMELESS may repress the invasion and metastasis of breast cancer cells via inhibiting the acetylation of p65, inhibiting the activation of NF-κB, thus down-regulating the expression of MMP9, and then inhibiting the invasion and metastasis of breast cancer cells.

The pathomechanism of human myxomatous valvular degeneration at the mechanical and cellular level.

The purpose of this study was to explore the pathomechanism of human myxomatous valve degeneration by investigating changes in the phenotype of valvular cells, the metabolism of the extracellular matrix and their mechanical properties. Mitral valve specimens were harvested from patients who had undergone valve replacement, and divided into two groups: patients with a myxomatous mitral valve and a control group. Histological investigation showed that the morphology of the extracellular matrix was looser and less coordinated in myxomatous valves than in controls. α-SMA (α-smooth muscle actin) and Vimentin were positive and DNA (deoxyribonucleic acid) assay of leaflets and expression of SMemb (embryonic smooth muscle myosin heavy chain), MMP-13 (matrix Metalloproteinases-13), MMP-1 mRNA (messenger Ribonucleic Acid) of the myxomatous valves were increased while the hydroxyproline content, expression of TIMP-1 (tissue inhibitor of metalloproteinase-1) mRNA and mechanical properties were decreased compared with controls. Compared to the quiescent interstitial cells in non-myxomatous valves, interstitial cells in myxomatous valves exhibit myofibroblast activation and express excessive levels of matrix metalloproteinases. The balance between MMP/TIMP was disrupted. We conclude that overactivation of VICs (Valvular interstitial cells) and the imbalance of MMP/TIMP could be important features of the pathomechanism of myxomatous mitral valve degeneration.

Circular RNA circDLC1 inhibits MMP1-mediated liver cancer progression via interaction with HuR.

Rationale: circular RNAs (circRNAs) have been demonstrated to play a crucial role in cancer progression. KIAA1429, a key component of the m6A methyltransferase complex, has recently been reported to promote hepatocellular carcinoma (HCC) progression by regulating the m6A methylation. The aim of present study is to investigate the role of circular RNAs in KIAA1429-mediated HCC progression.Methods: RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (m6A-seq) were utilized to identify KIAA1429-regulated circRNAs. The effects of circDLC1 on proliferation and metastasis of hepatoma cells were examined in vitro and in vivo. RT-qPCR was used to measure the expression of circDLC1 in HCC tissues and hepatoma cells. RNA FISH, RIP assays and biotin-labeled RNA pull-down were used to investigate the downstream effector of circDLC1. The downstream targets of circDLC1 were identified using RNA-seq.Results: Our data demonstrated that circDLC1 was downregulated in HCC tissues and closely relevant to favorable prognosis. Overexpression of circDLC1 inhibited the proliferation and motility of hepatoma cells in vitro and in vivo, while silencing of circDLC1 played the opposite role. Mechanistic investigations revealed that circDLC1 could bind to RNA-binding protein HuR, which subsequently reduced the interaction between HuR and MMP1 mRNAs, and thus inhibited the expression of MMP1, ultimately contributing to inhibition of HCC progression.Conclusion: Our work suggests that circDLC1, a downstream target of KIAA1429, is a promising prognostic marker for HCC patients, and the circDLC1-HuR-MMP1 axis may serve as a potential therapeutic target for HCC treatment.

SUMOylation of IGF2BP2 promotes vasculogenic mimicry of glioma via regulating OIP5-AS1/miR-495-3p axis.

Rationale: Glioma is the most common primary malignant tumor of human central nervous system, and its rich vascular characteristics make anti-angiogenic therapy become a therapeutic hotspot. However, the existence of glioma VM makes the anti-angiogenic therapy ineffective. SUMOylation is a post-translational modification that affects cell tumorigenicity by regulating the expression and activity of substrate proteins.Methods: The binding and modification of IGF2BP2 and SUMO1 were identified using Ni2+-NTA agarose bead pull-down assays, CO-IP and western blot; and in vitro SUMOylation assays combined with immunoprecipitation and immunofluorescence staining were performed to explore the detail affects and regulations of the SUMOylation on IGF2BP2. RT-PCR and western blot were used to detect the expression levels of IGF2BP2, OIP5-AS1, and miR-495-3p in glioma tissues and cell lines. CCK-8 assays, cell transwell assays, and three-dimensional cell culture methods were used for evaluating the function of IGF2BP2, OIP5-AS1, miR-495-3p, HIF1A and MMP14 in biological behaviors of glioma cells. Meantime, RIP and luciferase reporter assays were used for inquiring into the interactions among IGF2BP2, OIP5-AS1, miR-495-3p, HIF1A and MMP14. Eventually, the tumor xenografts in nude mice further as certained the effects of IGF2BP2 SUMOylation on glioma cells.Results: This study proved that IGF2BP2 mainly binds to SUMO1 and was SUMOylated at the lysine residues K497, K505 and K509 sites, which can be reduced by SENP1. SUMOylation increased IGF2BP2 protein expression and blocked its degradation through ubiquitin-proteasome pathway, thereby increasing its stability. The expressions of IGF2BP2 and OIP5-AS1 were up-regulated and the expression of miR-495-3p was down-regulated in both glioma tissues and cells. IGF2BP2 enhances the stability of OIP5-AS1, thereby increasing the binding of OIP5-AS1 to miR-495-3p, weakening the binding of miR-495-3p to the 3'UTR of HIF1A and MMP14 mRNA, and ultimately promoting the formation of VM in glioma.Conclusions: This study first revealed that SUMOylation of IGF2BP2 regulated OIP5-AS1/miR-495-3p axis to promote VM formation in glioma cells and xenografts growth in nude mice, providing a new idea for molecular targeted therapy of glioma.