Matrix Metalloproteinase Gene Expression Research Articles

Hydroxylated polychlorinated biphenyls may affect the thyroid hormone-induced brain development during metamorphosis of Xenopus laevis by disturbing the expression of matrix metalloproteinases.

Thyroid hormones are primarily responsible for the brain development in perinatal mammals. However, this process can be inhibited by external factors such as environmental chemicals. Perinatal mammals are viviparous, which makes direct fetal examination difficult. We used metamorphic amphibians, which exhibit many similarities to perinatal mammals, as an experimental system. Therefore, using metamorphic amphibians, we characterized the gene expression of matrix metalloproteinases, which play an important role in brain development. The expression of many matrix metalloproteinases (mmps) was characteristically induced during metamorphosis. We also found that the expression of many mmps was induced by T3 and markedly inhibited by hydroxylated polychlorinated biphenyls (PCBs). Overall, our findings suggest that hydroxylated PCBs disrupt normal brain development by disturbingthe gene expression of mmps.

Acceleration of Wound Healing in Diabetic Rats through a Novel 3D Organo-Hydrogel Nanocomposite of Polydopamine/TiO2 Nanoparticles and Cu (PDA-TiO2@Cu).

Diabetic wound represents a serious issue with a substantial impact and an exceptionally complex pathology affecting patients' mental health and quality of life. So, we have developed a novel 3D organo-hydrogel nanocomposite of polydopamine/TiO2 nanoparticles and cu (PDA-TiO2@Cu) and examined its efficacy in diabetic wound healing. Forty-five adult male albino rats were divided into normal control rats (non-diabetic rats with non-treated skin wounds), diabetic control rats (diabetic rats with non-treated skin wounds), and organo-hydrogel-treated rats (diabetic wounds treated with topically applied organo- hydrogel once daily). Macroscopic changes of the wound were observed on days 0, 3, 5, 7, and 10 to measure wound diameters. Skin specimens from the wound tissue were taken on days 3, 7, and 10, respectively, and examined histologically and immunohistochemically. Also, the gene expressions of collagen I, Matrix Metalloproteinase-9 (MMP-9), and Epidermal Growth Factor (EGF), and levels of Interleukin 6 (IL-6) and Superoxide Dismutase (SOD) were assessed. Our observed results indicated that the developed patch significantly accelerated the healing time compared to the normal control and diabetic control groups. Moreover, the patchloaded group revealed complete re-epithelization and a highly significant increase in the mean area % of CD31 immunostaining on day 7. The organo-hydrogel-loaded group displayed a significant decrease in gene expression of MMP-9 and a significant increase in gene expression of EGF and collagen I. Additionally, the organo-hydrogel-loaded group exhibited a significant decrease in levels of IL-6 and a significant increase in levels of SOD, compared to the normal diabetic control groups. The organo-hydrogel can be used for treating and decreasing the healing period of diabetic wounds.

Effects of SGLT2-inhibitor on The Expression of MicroRNA-21, Transforming Growth Factor-β1, and Matrix Metalloproteinase-2 in The Process of Cardiac Fibrosis in Hyperglycemic Model Rats

BACKGROUND: Sodium glucose co-transporter-2 inhibitor (SGLT2-i), a new oral antidiabetic drug, has been recommended for its morbidity and mortality benefits in patients with heart failure. This study aimed to determine the effect of acute SGLT2-i therapy on the relative ratio expression of microRNA-21 (miR-21), transforming growth factor-β1 (TGF-β1), and matrix metalloproteinase-2 (MMP-2) in the process of cardiac fibrosis in the hyperglycemia Wistar rat models compared to biguanide (metformin) therapy.METHODS: We used Streptozotocin (STZ) to induce hyperglycemia in Wistar rats (n=31), randomly divided into four groups: negative control (NC, n=4), positive control (PC, n=10), hyperglycemia plus metformin (M, n=8), and hyperglycemia plus empagliflozin (E, n=9). After seven weeks, the rats were sacrificed and the heart tissue was taken for microRNA and messenger RNA (mRNA) extraction, followed by reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) examination. The data was analyzed using One-way ANOVA.RESULTS: Results showed a decreasing trend in the gene expression relative ratio of miR-21 (1.0 vs. 1.9; p=0.079) and TGF-β1 (0.9 vs. 3.2; p=0.145), but a significant increase in MMP-2 gene expression (1.3 vs. 0.7; p=0.002) in the SGLT2-i (empagliflozin) vs. biguanide (metformin) groups.CONCLUSION: Empagliflozin administration may play a significant role in preventing the occurrence of cardiac fibrosis in hyperglycemia.KEYWORDS: sodium glucose co-transporter-2 inhibitor, microRNA-21, transforming growth factor-β1, matrix metalloproteinase-2, cardiac fibrosis

Ranibizumab Modifies the Expression of Metalloproteinases and Their Tissue Inhibitors in Peripheral Blood Mononuclear Cells in Patients with Exudative Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of vision loss in people over 60 years of age. Despite research, the causes of AMD remain unclear. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in AMD development, and anti-vascular endothelial growth factor therapy has revolutionized its treatment. This study aims to analyze the changes in gene expression in MMPs and TIMPS in patients with neovascular AMD before and after three doses of ranibizumab. The study involved 29 patients with neovascular AMD treated with ranibizumab. Peripheral blood mononuclear cells were collected before treatment and 24 h after the third dose of ranibizumab. We assessed MMP and TIMP gene expression profiles through oligonucleotide microarrays and validated selected differential genes using RT-qPCR. A statistically significant change in the expression of six MMP- and TIMP-related genes was observed using oligonucleotide microarray. The mRNA levels of the two genes with the most significant fold changes, MMP15 and TIMP2, were then quantified using RT-qPCR. The results confirmed a statistically significant increase in MMP15 expression and a decrease in TIMP2 levels, although this change was not statistically significant in the group before and after the third dose of ranibizumab. Ranibizumab affects the systemic expression of MMP and TIMP-related genes in patients with neovascular AMD. Results from our exploratory study suggest that MMP15, in particular, may play a role in the treatment response, but further research is necessary.

Evaluation of MMP-9, IL-6, TNF-α levels and peripheral blood mononuclear cells genes expression of MMP-9 and TIMP-1 in Iranian patients with coronary artery disease.

Coronary artery disease (CAD) is the main cause of death and is characterized by atherosclerosis in coronary arteries. Inflammation plays a crucial role in the progression and development of atherosclerosis. The present study consisted of 132 Iranian individuals who underwent coronary angiography, 65 patients with CAD, and 67 controls. The matrix metalloproteinase-9 (MMP-9), TNF-α, IL-6, and vitamin D serum levels were measured by the ELISA technique. The gene expression of MMP-9 and tissue inhibitors of metalloproteinase (TIMP-1) was estimated by real-time PCR assay. A considerable increase in levels and PBMC gene expression of MMP-9 and serum levels of IL-6 and TNF-α were found in CAD patients compared with controls. A significant decrease was detected in vitamin D levels of CAD patients in comparison with controls. A considerable direct correlation was found between MMP-9 levels and MMP-9 and TIMP1 gene expression in CAD patients. MMP-9 levels positively correlated with LDL-C in CAD patients. The correlation between TIMP1 gene expression and IL-6 levels was also negatively significant. There were positive correlations between MMP-9 levels with IL-6 and TNF-α serum levels in CAD patients. This study showed that the interaction between MMPs, TIMP1, and cytokines could play a role in the pathogenesis of atherosclerosis. The present study suggested that high levels of TNF-α and IL-6 and vitamin D deficiency in our studied patients could disturb the MMP-9/TIMP-1 balance and lipid metabolism, leading to plaque formation/ rupture in predisposed CAD patients.

The Protective Role of L-carnitine on Psychosocial Stress-induced Changes in Gene Expression and Protein Levels of Matrix Metalloproteinases, Serum Corticosterone in a Rat Model.

Psychosocial stress (STS) is a common stress in modern societies. Chronic STS is associated with the impairment of a broad range of cognitive functions in humans and with prolonged abnormalities in memory. Matrix metalloproteinases (MMPs), a protein family of zinccontaining endopeptidases, are essential in neuro-inflammation and involved in neurodegenerative diseases. L-Car possessed neuroprotective, antioxidant, and anti-inflammatory properties and was shown to modulate MMPs. The current study aimed to examine the protective effect of L-Carnitine (L-CAR) on STSinduced changes in serum corticosterone levels, MMP-2, -9, and -12 protein and mRNA expression in the hippocampus as a possible mechanism for L-CAR protective effect on STS-induced memory impairment. The chronic STS and L-CAR (300 mg/kg/day, i.p) were simultaneously administered for 6 weeks to adult male Wistar rats. Serum corticosterone and protein levels of MMP-2, -9 and -12 were evaluated using ELISA. Real-Time PCR techniques were used to determine the mRNA levels of MMP-2, -9 and -12 in the hippocampus. The findings showed that serum corticosterone levels and MMP-2 and -9 protein levels were significantly increased (p<0.05) in the STS group compared to the control. Similarly, RT-PCR findings showed that the mRNA of those proteinases significantly increased (p<0.05) following the intruder method. On the other hand, the administration of L-CAR restored the alterations in corticosterone levels and MMPs gene and protein expression induced by chronic STS. STS induced elevations in corticosterone and MMP-2 and -9 levels in the hippocampus. L-CAR, on the other hand, exhibited protective effects against the STS-induced changes in MMP-2 and -9.

Weizmannia coagulans Extracellular Proteins Reduce Skin Acne by Inhibiting Pathogenic Bacteria and Regulating TLR2/TRAF6-Mediated NF-κB and MAPKs Signaling Pathways.

As a probiotic, Weizmannia coagulans (W. coagulans) is often used in food and medicine to regulate intestinal flora and exert anti-inflammatory effects. In this study, the anti-acne efficacy and mechanism of extracellular proteins (YTCY-EPs) from W. coagulans YTCY strain are analyzed. The main components of YTCY-EPs, extracted and separated from the fermentation broth, are peptides ranging from 1.51 to 11.44kDa, accounting for about 80%. Among the peptides identified by LC/MS-MS, YTCY_A-F possess the properties of antimicrobial peptides, while YTCY_1-4 possess antioxidative properties. These peptides have a strong effect on Cutibacterium acnes (C. acnes) and significantly inhibit Staphylococcus aureus. The inhibition rate of biofilm adhesion of YT-EPs to C. acnes reached 50% under the MIC. It was found that YTCY-EPs possess strong antioxidant and anti-inflammatory properties. It can effectively reduce active oxygen nearly 3 times and can reduce the downstream TLR2/NF-κB and MAPKs/AP-1 pathways by regulating the nuclear translocation of NF-κB and AP-1 in vitro. The transcriptional expression of inflammatory cytokines, inflammatory chemokines, and matrix metalloproteinase genes is also regulated, thereby slowing the recruitment of inflammatory cells and the development of inflammation, and increasing keratinocyte mobility. In addition, the expression levels of inflammatory factors and matrix metalloproteinases in the rabbit ears with acne problems that were tested with YTCY-EPs were significantly reduced, and it was obviously observed that the rabbit ear inflammation, acne, and keratinization problems were repaired. The results of this study prove that YTCY-EPs can be used as a potential anti-acne raw material in cosmetics.

Protective Effect of Crocin on Cadmium Induced-Overexpression of Matrix Metalloproteinases (MMPs) Gene Family in Rat Liver

Background: Metal pollutants such as Cadmium are known as carcinogenic agents and correlated with metastatic potential and tumor aggressiveness. Matrix metalloproteinases (MMPs) are taken into account as the focal points of metastasis. Crocin, a carotenoid chemical compound in Saffron, has been proved to have anti-cancerous properties. However, there is no reliable evidence yet to prove its anti-metastatic activity. Objective: The evaluation of the protective effect of Crocin against Cadmium through monitoring the expression of MMP genes. Materials and Methods: The in-silico analysis based on the GEO database was done for purposefully choosing MMPs. At the wet lab step, after 8 weeks of treatment of the animal (40 female Wistar rats divided into four groups of 10) with Cadmium, Crocin, complex (Cadmium+Crocin) and placebo (control), RNA extraction followed by cDNA synthesizing was done from rats’ liver tissues. Using primers for MMP genes (MMP-1, 3, 7, 12 and 13), the relative fold change of gene expression was studied. Ultimately, the statistical analysis determined the difference in gene expression between groups. Results: Cadmium significantly upregulated the selected MMP genes in comparison with controls. In contrast, Crocin significantly downregulated the expression of all studied MMP genes. Complex administration of Cadmium and Crocin displayed a controlled increase of expression in all MMPs except MMP-1, which was lower than the Cadmium group. Conclusion: These results indicated that the elevation of MMPs expression level by Cadmium was significantly suppressed by Crocin. This could promisingly demonstrate that Crocin possesses a modulating role in inhibiting overexpression of MMPs that might be considered as the anti-metastatic agent.

Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture. Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions. Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA. Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged. Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

Human umbilical cord mesenchymal stem cells-derived exosomes exert anti-inflammatory effects on osteoarthritis chondrocytes.

Inflammation of chondrocytes plays a critical role in the occurrence and development of osteoarthritis (OA). Recent evidence indicated exosomes derived from mesenchymal stem cells (MSCs-Exos) exhibit excellent anti-inflammatory ability in many troublesome inflammatory diseases including OA. In the present study, we aimed to explore the role of human umbilical cord-derived MSCs-Exos (hUC-MSCs-Exos) in treating the inflammation of chondrocytes and its related mechanisms. Ultracentrifugation was applied to isolate hUC-MSCs-Exos from the culture supernatant of hUC-MSCs. Two OA-like in vitro inflammation models of human articular chondrocytes induced with interleukin 1β (IL-1β) and co-incubation with macrophage utilizing transwell cell culture inserts were both used to evaluate the anti-inflammatory effects of hUC-MSCs-Exos. The mRNA sequencing of chondrocytes after treatment and microRNA (miRNA) sequencing of hUC-MSCs-Exos were detected and analyzed for possible mechanism analysis. The results of the study confirmed that hUC-MSCs-Exos had a reversed effect of IL-1β on chondrocytes in the expression of collagen type II alpha 1 (COL2A1) and matrix metalloproteinase 13 (MMP13). The addition of hUC-MSCs-Exos to M1 macrophages in the upper chamber showed down-regulation of IL-1β and tumor necrosis factor α (TNF-α), up-regulation of IL-10 and arginase1 (ARG1), and reversed the gene and protein expression of COL2A1 and MMP13 of the chondrocytes seeded in the lower chamber. The results of this study confirmed the anti-inflammatory effects of hUC-MSCs-Exos in the human articular chondrocytes inflammation model. hUC-MSCs-Exos may be used as a potential cell-free treatment strategy for chondrocyte inflammation in OA.

EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells.

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed thatEGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft(CDX)/patient-derived xenograft(PDX) model system is warranted to validate the therapeutic utility of EGCG.

Breathing on chip: Dynamic flow and stretch accelerate mucociliary maturation of airway epithelium in vitro.

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying the effects of dynamic medium and airflow as well as stretch on human primary airway epithelial cell differentiation and maturation, including mucociliary clearance, using an organ-on-chip device. Perfused epithelial cell cultures displayed accelerated maturation and polarization of mucociliary clearance, and changes in specific cell-types when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in an increase in polarization of mucociliary clearance towards the applied flow, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and maturation and that their fine-tuned application could generate models of specific epithelial pathologies, including mucociliary (dys)function.

Assessing effects of mechanical stimulation of fluid shear stress on inducing matrix Metalloproteinase-9 in cultured corneal epithelial cells

Blinking is regarded as mechanical stimulation of fluid shear stress on the corneal epithelial cells. Therefore, we evaluated whether fluid shear stress affects matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human corneal epithelial cells (HCECs). No other study has shown the influence of fluid shear stress on HCECs regarding mRNA expression and the protein levels of MMPs. Cultured HCECs were exposed to shear stress (0, 1.2, 12 dyne/cm2) for 12 and 24 h with the parallel-plate type of flow chamber. Gene expression of MMPs and TIMPs was measured by real-time polymerase reaction. Concentrations of MMP-1 and MMP-9 in cell lysates were determined using bead-based amplified luminescent proximity homogenous assay-linked immunosorbent assay. The expression of MMP-9 and MMP-1 in HCECs exposed to low and high flow for 12 and 24 h, respectively, increased significantly compared with those under static conditions. The expression of MMP-9 in the cells exposed to high flow for 24 h increased significantly compared with those under static and low flow conditions. Levels of MMP-9 in cell lysates exposed to fluid flow for 24 h were elevated significantly with increasing shear stress. Fluid shear stress exerted on HCECs affected MMPs, which was associated with inflammation and pathogenesis. Mechanical stress induced by blinking might influence expression of MMPs on the ocular surface. Further studies are warranted to establish the molecular mechanism of shear stress-induced alternations of MMPs.

Anti-Inflammatory Effects of Dietary Polyphenols through Inhibitory Activity against Metalloproteinases.

Matrix metalloproteinases (MMPs) are zinc-dependent metalloproteinases that play important roles in a variety of diseases, including cancer, cardiovascular disease, diabetes, obesity, and brain diseases. Dietary polyphenols are thought to have a variety of beneficial effects on these diseases characterized by inflammation. Clinical studies have demonstrated that MMPs are in most cases upregulated in various inflammatory diseases, including osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, and Alzheimer's disease. Studies using patient-derived human samples, animal studies, and cellular experiments have suggested that polyphenols may be beneficial against inflammatory diseases by suppressing MMP gene expression and enzyme activity. One important mechanism by which polyphenols exert their activity is the downregulation of reactive oxygen species that promote MMP expression. Another important mechanism is the direct binding of polyphenols to MMPs and their inhibition of enzyme activity. Molecular docking analyses have provided a structural basis for the interaction between polyphenols and MMPs and will help to explore new polyphenol-based drugs with anti-inflammatory properties.

NETosis promotes chronic inflammation and fibrosis in systemic lupus erythematosus and COVID-19

Pulmonary fibrosis, a serious complication of systemic lupus erythematosus (SLE) and coronavirus disease 2019 (COVID-19), leads to irreversible lung damage. However, the underlying mechanism of this condition remains unclear. In this study, we revealed the landscape of transcriptional changes in lung biopsies from individuals with SLE, COVID-19-induced pulmonary fibrosis, and idiopathic pulmonary fibrosis (IPF) using histopathology and RNA sequencing, respectively. Despite the diverse etiologies of these diseases, lung expression of matrix metalloproteinase genes in these diseases showed similar patterns. Particularly, the differentially expressed genes were significantly enriched in the pathway of neutrophil extracellular trap formation, showing similar enrichment signature between SLE and COVID-19. The abundance of Neutrophil extracellular traps (NETs) was much higher in the lungs of individuals with SLE and COVID-19 compared to those with IPF. In-depth transcriptome analyses revealed that NETs formation pathway promotes epithelial-mesenchymal transition (EMT). Furthermore, stimulation with NETs significantly up-regulated α-SMA, Twist, Snail protein expression, while decreasing the expression of E-cadherin protein in vitro. This indicates that NETosis promotes EMT in lung epithelial cells. Given drugs that are efficacious in degrading damaged NETs or inhibiting NETs production, we identified a few drug targets that were aberrantly expressed in both SLE and COVID-19. Among these targets, the JAK2 inhibitor Tofacitinib could effectively disrupted the process of NETs and reversed NET-induced EMT in lung epithelial cells. These findings support that the NETs/EMT axis, activated by SLE and COVID-19, contributes to the progression of pulmonary fibrosis. Our study also highlights that JAK2 as a potential target for the treatment of fibrosis in these diseases.

Effect of miR-4270 Suppression on Migration in Hepatocellular Carcinoma Cell Line (HepG2)

Liver transplantation and surgical resection are two major strategies for treatment of hepatocellular carcinoma (HCC) patients. One approach to treating HCC is the suppression of metastasis to other tissues. Herein, we aimed to study the effect of miR-4270 inhibitor on migration of HepG2 cells as well as activity of matrix metalloproteinase (MMP) these cells in order to find a strategy to suppress metastasis in future. HepG2 cells were treated with 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 nM of miR-4270 inhibitor, and then the cell viability was measured by trypan blue staining. Afterwards, cell migration and MMP activity of HepG2 cells were assessed by wound healing assay and zymography, respectively. The MMP gene expression was determined by real-time reverse transcription polymerase chain reaction. Results showed that miR-4270 inhibitor decreased the cell viability of HepG2 cells in a concentration-dependent manner. Also, inhibition of the miR-4270 reduced invasion, MMP activity, and expression of MMP genes in HepG2 cells, respectively. Our findings suggest that miR-4270 inhibitor decreases in vitro migration, which could help find a new approach for HCC therapy patients.

The application of mechanical load onto mouse tendons by magnetic restraining represses Mmp-3 expression

ObjectivesMechanical loading is crucial for tendon matrix homeostasis. Under-stimulation of tendon tissue promotes matrix degradation and ultimately tendon failure. In this study, we examined the expression of tendon matrix molecules and matrix-degrading enzymes (matrix metalloproteinases) in stress-deprived tail tendons and compared to tendons that were mechanically loaded by a simple restraining method.Data descriptionIsolated mouse tail fascicles were either floated or restrained by magnets in cell culture media for 24 h. The gene expression of tendon matrix molecules and matrix metalloproteinases in the tendon fascicles of mouse tails were examined by real-time RT-PCR. Stress deprivation of tail tendons increase Mmp3 mRNA levels. Restraining tendons represses these increases in Mmp3. The gene expression response to restraining was specific to Mmp3 at 24 h as we did not observe mRNA level changes in other matrix related genes that we examined (Col1, Col3, Tnc, Acan, and Mmp13). To elucidate, the mechanisms that may regulate load transmission in tendon tissue, we examined filamentous (F-)actin staining and nuclear morphology. As compared to stress deprived tendons, restrained tendons had greater staining for F-actin. The nuclei of restrained tendons are smaller and more elongated. These results indicate that mechanical loading regulates specific gene expression potentially through F-actin regulation of nuclear morphology. A further understanding on the mechanisms involved in regulating Mmp3 gene expression may lead to new strategies to prevent tendon degeneration.

1602-P: Galectin-3 Inhibition Protects ApoE Knockout Mice against Western Diet–Induced Nonalcoholic Steatohepatitis and Glucose Intolerance

Nonalcoholic Fatty Liver Disease represents a broad spectrum of histological abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma. NASH is characterized by steatosis, hepatic inflammation, hepatocellular ballooning, and fibrosis. Therefore, the aim of this work was to evaluate the effect of modified citrus pectin (MCP), inhibitor of galectin-3, in ApoE KO mice fed a western diet (WD). ApoE KO mice were divided into 3 groups: standard diet-fed (SD) group, WD-fed group, and WD-fed + MCP-treated group (WD+MCP). WD-fed mice displayed increased hepatic triacylglycerides (TAG-290%), and plasma ALT (115%) and AST (130%) compared with SD group. MCP treatment decreased hepatic TAG (30%) in WD-fed mice, plasma ALT and AST were reverted. WD-fed mice also displayed increased hepatic collagen deposition (18%), and MCP treatment inhibited this increase. These results were similar with hydroxyproline concentration in liver. WD-fed mice showed an impaired glucose tolerance compared with SD-fed mice, while MCP treatment improved glucose tolerance in WD-fed mice. Also, WD-fed mice presented increased fasting insulin (140%) compared with SD-fed mice, and MCP treatment reverted this increase by 100%. Gene expression of F4/80 was increased by 76% in the WD group compared with SD group, however had not significant difference between WD+MCP and SD group. Gene expression of matrix metalloproteinase 2 was increased by 5-fold in WD group compared with SD group, while this increase was inhibited with MCP treatment. Also, it was observed an increase in JNK phosphorylation by 3-fold in WD group, and MCP treatment inhibited this increase. Taken together, we confirm WD-fed ApoE KO mice as a suitable experimental model of NASH, and the inhibition of galectin-3 by the treatment with MCP protects ApoE KO mice against the development of NASH and glucose intolerance. Disclosure L. Araujo: None. F. Sucupira: None. C.C.B. Dias: None. A. Amaral: None. J. Camporez: None. Funding São Paulo Research Foundation (FAPESP)

1485-P: The Involvement of ANGPTL4 in Periodontitis and the Influence of High Glucose

Periodontitis is a chronic inflammatory disease in which periodontal tissue is destroyed by bacterial infection. Periodontal disease is considered a comorbidity of diabetes due to its high prevalence and severity. However, the molecular mechanisms how diabetes aggravates periodontitis are still unclear. In this study, we investigated the involvement of Angiopoietin-like proteins (ANGPTLs) in periodontitis and the impacts of high glucose. Experimental periodontitis was induced in 6-week-old male Sprague-Dawley rats by placing a nylon suture around the neck of the maxillary second molar. Induction of periodontitis significantly increased the expression of ANGPTL4 in the gingiva. To clarify the involvement of ANGPTL4 in periodontitis, the human gingival fibroblasts (hGFs) were stimulated with LPS of Porphyromonas gingivalis (Pg-LPS), the bacterium most frequently isolated from inflamed sites of adult periodontitis. Pg-LPS significantly increased the gene and protein expression of ANGPTL4 in hGFs but not the gene expression of other ANGPTLs or ANGPTL receptors. Recombinant human ANGPTL4 significantly increased matrix metalloproteinase 13 (MMP13) gene expression in hGFs. We also confirmed that MMP13 expression was increased in the gingiva during experimental periodontitis. Pg-LPS induced MMP13 gene expression in hGFs. Next, we investigated the impacts of high glucose in Pg-LPS-stimulated hGFs. The expression of ANGPTL4 by Pg-LPS was significantly increased in high glucose condition compared with that in normal glucose condition. These results suggest the pivotal role of ANGPTL4 in periodontitis, especially under the high glucose condition. Disclosure S.Kondo: None. M.Ito: None. S.Sasajima: None. T.Matsubara: None. A.Mitani: None. K.Naruse: None. K.Kojima: None. N.Nakamura: None. M.Miyabe: None. T.Kikuchi: None. T.Ohno: None. N.Sawada: None. T.Minato: None. T.Saiki: None.

Inhibition of tumor-specific angiogenesis by AS1411 aptamer functionalized Withaferin A loaded PEGylated nanoliposomes by targeting nucleolin

Angiogenesis is a vital process for tumor growth and metastasis. Inhibition of angiogenesis is a promising strategy in cancer treatment. In this study, we analyzed the anti-angiogenic activity of AS1411 functionalized Withaferin A encapsulated PEGylated nanoliposomes (ALW) using both in vitro and in vivo models. AS1411 aptamer functionalized nanoliposomes are an efficient drug delivery system for carrying chemotherapeutic agents to target cancer cells, and Withaferin A (WA) is a steroidal lactone known for potent anti-angiogenic activity. ALW showed significant inhibition in the migration and tube formation of endothelial cells, which are critical events in angiogenesis. In vivo angiogenesis study using ALW showed remarkable inhibition of tumor-directed capillary formation by altered serum cytokines, VEGF, GM-CSF, and NO levels. ALW treatment downregulated the gene expression of Matrix metalloproteinase (MMP)-2, MMP-9, VEGF, NF-kB and upregulated the expression of tissue inhibitor of metalloproteinase (TIMP)-1. Our results demonstrate that ALW inhibits tumor-specific angiogenesis by gene expression of NF-κB, VEGF, MMP-2, and MMP-9. The present study shows that using ALW can offer an attractive strategy for inhibiting tumor angiogenesis.