TIMP-1 Protein Research Articles

Comparison of cotton swab to Whatman cellulose paper as media for examination of biomarkers from diabetic ulcers

IntroductionChronic wounds, including diabetic foot ulcers, require proper diagnostics. Proper wound care must be supported by proper diagnostic procedures, one of which is the diagnosis of biomarkers that accelerate and inhibit wound healing, consistent for wound analysis. Many researchers have stated the importance of gelatinase, collagenase and matrix metalloprotease (MMP) levels and their role in wound healing. A balance between vascular endothelial growth factor (VEGF), inflammatory cytokines, MMPs and tissue matrix metalloprotease inhibitors (TIMPs) is indispensable in the wound healing process. Previous test results showed Whatmann had the ability to carry out cytokines and proteases in diabetic foot ulcer (DFU). The purpose of this study was to compare the sampling technique with the cotton swab technique and use Whatman cellulose paper as a preliminary study. The biomarkers taken were VEGF, interleukin 6 (IL-6), MMP-9 and TIMP-1 as representatives of growth factors, markers of inflammation and protease enzymes. The aim of the study was to determine the standard for taking commonly used media practicality for the extraction of growth factor (GF) and protein from the wound surface.Material and methodsSix DFU subjects were included in this study, and divided into two groups based on the method of topical extraction: the cotton group and the Whatman paper group. The swab method is to use a cotton swab on the center of the wound and rotate it about 3 times until the cotton swab is wet. The Whatman method involves sticking Whatman paper to the wound surface, applying light pressure and leaving it for 15 min. Cotton swabs and Whatman paper were placed in Eppendorf tubes with 1.5 cm3 of NaCl and processed by making lysates in an integrated laboratory. Furthermore, cotton swabs and Whatman paper containing wound fluid in the Eppendorf tube were centrifuged immediately at 10,000 rpm for 3 min at room temperature. The resulting cell-free supernatant was subjected to analysis by the ELISA method. VEGF, interleukin-6 and MMP-9 and TIMP-1 proteins were calculated by statistic analysis. Descriptive statistics were used to present the variable location and type of injury. Quantitative variables are summarized as mean with standard deviation (SD). The relationship between levels of biomarkers obtained from concurrently collected wound fluids (cotton swab and Whatman) was assessed using an independent t test analysis using SPSS 2015 software, version 20.0ResultsThere was a significant difference in material collection from cotton swabs compared to the material collection method with Whatman paper for MMP-9 protein (p = 0.028) and TIMP-1 (p = 0.030). Meanwhile, for VEGF and IL-6, there were no insignificant differences between the two methods of taking the material on the diabetic foot ulcers surface.ConclusionsDiagnostic wound fluid in diabetic foot ulcers using a non-pressure wound swab (Levine technique) can be considered for the collection of wound fluid from diabetic foot ulcers for ELISA examination as a guideline for further treatment.

Assessment of the correlation between oxidative stress and expression of MMP-2, TIMP-1 and COX-2 in human aortic smooth muscle cells.

IntroductionSmooth muscle cells (SMCs) are considered to be the main producer of matrix metalloproteinase-2 (MMP-2) participating primarily in extracellular matrix (ECM) remodeling. Any disturbances in ECM structure may underlie the pathogenesis of many cardiovascular diseases and contribute to angiogenesis, cancer development, invasion or metastasis. The purpose of the study was to examine the effect of oxidative stress on the expression of MMP-2, its tissue inhibitor type 1 (TIMP-1) and cyclooxygenase-2 (COX-2) in human aortic smooth muscle cells (HASMCs).Material and methodsHASMCs were treated with exogenously applied H2O2 or TNF-α. N-acetylcysteine (NAC) was used as an antioxidant. Gene expression levels were measured by real-time PCR and the protein levels were determined using ELISA assay.ResultsThe studies revealed no association between oxidative stress and either mRNA quantity or protein secretion of MMP-2 and TIMP-1. However, we found markedly reduced (p < 0.001) MMP-2 secretion in cells incubated with NAC. HASMCs stimulated with TNF-α demonstrated a significantly increased COX-2 mRNA level as well as enzyme activity. H2O2-induced cells showed lowered COX-2 activity in comparison to untreated cells. MMP-2 and TIMP-1 expression did not change after COX-2 inhibition with DuP-697.ConclusionsWe did not find any effect of oxidative stress on expression of MMP-2 and TIMP-1 in HASMCs. However, COX-2 mRNA and protein level were elevated in these conditions. There was no correlation between COX-2 activity and MMP-2 and TIMP-1 mRNA expression or protein secretion.

Effect of baicalin on expression of fibrogenic factors TGF-β1, mmp2 and timp2 in tissue of mice with pulmonary fibrosis

To investigate the effect of baicalin extracted from Qinbai Qingfei Concentrated Pills on the expressions of TGF-β1, mmp2 and timp2 in mice with pulmonary fibrosis induced by bleomycin. The Biacore technique was used to detect the specific binding between Qinbai Qingfei Concentrated Pills and TGF-β1, and the affinity components were enriched, regenerated and recovered by Biacore fishing. Then ultra-performance liquid chromatography and quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS) were used to determine whether the monomer was baicalin. Biacore was used to verify the affinity kinetics of baicalin, which was validated by pharmacodynamics in vivo. Totally 30 BALB/C mice were randomly divided into three groups: baicalin group, blank group and model group. The blank group was given sodium chloride injection(0.08 mL·kg~(-1)), while the model group and the baicalin group were injected with 4 mg·kg~(-1) bleomycin. The localization of TGF-β1, mmp2 and timp2 protein in the cells and the mRNA expressions of TGF-β1, mmp2 and timp2 were detected by RT-PCR 14 days later. The results of Biacore affinity analysis showed that the peak of binding response between Qinbai Qingfei Concentrated Pills and TGF-β1 protein reached 1 524.0 RU, with specific binding. The affinity constant K_D of baicalin and TGF-β1 was 1.620 06 μmol·L~(-1), which was determined by SPR kinetic analysis, suggesting a stable binding between baicalin and TGF-β1, which verified the results of angulation. The results of immunohistochemistry showed that the deposition of cellulose in baicalin group was significantly less than that in model group, the mRNA expressions of TGF-β1, mmp2 and timp2 were decreased in baicalin solution compared with the model group. Baicalin combined with TGF-β1 could inhibit the expressions of mmp2 and timp2 and delay the progress of pulmonary fibrosis.

Tissue Inhibitor of Matrix Metalloproteinase 2-Loaded Polylactic Acid-Polyethylene Glycol Nanoparticles Directly Regulates Renal Tubular Epithelial Cell Proliferation via Protein Tyrosine Kinase KIT.

TIMP2 has been previously reported to be associated with acute kidney injury (AKI); however, the underlying mechanism remains unclear. Therefore, the present study investigated the regulation of TIMP2 in human tubular epithelial cells HK-2 cells. Proliferation of HK-2 cells treated by TIMP2 at normal expression level was detected. GST pulldown and mass spectrometry were performed to investigate the interacting protein of TIMP2 and immunofluorescence test was used to determine the location of the protein in HK-2 cells. Cell viability as well as the expression level of CCND1, C-FOS, MAPK1 and P-MAPK1 were detected in the samples treated by overexpressed TIMP2 and the inhibitor of the interacting protein KIT. TIMP2 significantly inhibited cell proliferation compared with the control and BB-94-treated groups (P < 0.05). KIT was identified as the interacting protein of TIMP2, and was located in both the cytoplasm and membrane of HK-2 cells. Inhibited KIT and the overexpressed of TIMP2 both significantly suppressed cell proliferation and decreased the expression levels of CCND1, MAPK1, and P-MAPK1 compared with the control (P < 0.05). No significant difference was observed in cell proliferation and the expression level of aforementioned proteins between overexpressed TIMP2 and KIT-inhibited group. The results revealed that TIMP2 regulates cell proliferation by reducing the expression levels of CCND1, MAPK1, and P-MAPK1 via KIT, indicating that TIMP2 directly regulates cell proliferation without inhibiting matrix metalloproteinases in AKI. Furthermore, PLA-PEG nanoparticles successfully transported TIMP2 to the target with no significant effect on cell proliferation.

Effect of eCG treatment on gene expression of selected matrix metalloproteinases (MMP-2, MMP-7, MMP-9, MMP-10, and MMP-13) and the tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3) in the chicken ovary

Several metalloproteinases (MMPs) are present and functional in the chicken ovary and regulate the extracellular matrix (ECM) during follicle development, ovulation, atresia, and regression. The regulation of the abundance of MMPs in avian ovarian follicles, however, is largely unknown. The aim of the present study was to examine effects of equine chorionic gonadotropin (eCG) on abundance of selected MMPs and relevant tissue inhibitors of MMPs (TIMPs) in the hen ovary. The MMP-2 and MMP-9 activity was also determined. Results indicated there were effects of eCG on abundances of MMP-2, MMP-7, MMP-9, MMP-10, MMP-13, TIMP-2, and TIMP-3 mRNA transcript and/or protein relative abundances in white, yellowish, small yellow, and the largest yellow preovulatory (F3-F1) ovarian follicles. The response to eCG depended on the stage of follicle development, layer of follicular wall, and the type of MMPs or TIMPs affected by eCG. Furthermore, there was a pause in egg laying when eCG was administered and there were morphological changes in the ovary following eCG treatment that were associated with alterations in MMP-2 and MMP-9 activity. In general, the results indicate that eCG, which has primarily follicle stimulating hormone (FSH)-like bioactivities, is a negative regulator of MMP abundance and activity in the largest yellow preovulatory follicles. Results from the present study indicate the gonadotropins, especially FSH, by the regulation of transcription, translation, and/or activity of proteins of the MMP system have effects on the mechanisms that underlie ECM remodeling and cell function throughout ovarian follicle development in the chicken ovary.

Turnera diffusa extract attenuates profibrotic, extracellular matrix and mitochondrial markers in activated human hepatic stellate cells (HSC)

Introduction and objectivesHepatic fibrosis is characterized by the accumulation of extracellular matrix which includes the accumulation of α-smooth muscle actin (α-SMA), collagen type I (COL1α1), as well as remodeling induced by metalloproteinases and tissue inhibitor of metalloproteinase (TIMPs), where hepatic stellate cells (HSCs) play a central role. In addition, the transcription factor SNAI1 (which participates in epithelial-mesenchymal transition, EMT) and mitofusin 2 (MFN2, a mitochondrial marker) plays an important role in chronic liver disease. Turnera diffusa (TD), a Mexican endemic plant, has been shown to possess antioxidant and hepatoprotective activity in vitro. We treated human HSC (LX2 cells) with a methanolic extract of Turnera diffusa (METD) to evaluate the mechanism involved in its hepatoprotective effect measured as fibrosis modulation, EMT, and mitochondrial markers. Materials and methodsHSC LX-2 cells were treated with METD (100 and 200ng/mL) alone or combined with TGF-β (10ng/mL) at different time points (24, 48, and 72h). α-SMA, COL1α1, MMP2, TIMP1, SNAI1, and MFN2 mRNAs and protein levels were determined by real-time quantitative PCR and Western Blot analysis. ResultsWe found that METD decreases COL1α1-mRNA, α-SMA, and TIMP1 protein expression in LX2 cells treated with and TGF-β. This treatment also decreases MFN2 and TIMP1 protein expression and induces overexpression of MMP2-mRNA. ConclusionsOur results suggest that a methanolic extract of Turnera diffusa is associated with an antifibrotic effect by decreasing profibrotic and mitochondrial markers together with the possible induction of apoptosis through SNAI1 expression in activated HSC cells.

Bicuspid aortopathy – molecular involvement of microRNAs and MMP-TIMP

Objectives We aimed to elucidate the correlation between expression patterns of aortic tissue microRNAs and the aortopathy formation in bicuspid aortic valve (BAV) disease. Methods All 65 patients who underwent elective aortic valve repair/replacement +/− proximal aortic replacement due to BAV disease with or without concomitant aortic aneurysm were identified from our BAV registry. Aortic tissue was collected intraoperatively from the ascending aorta at the greater and lesser curvature. Aortic tissue microRNAs analysis included 11 microRNAs (miR-1, miR-17, miR-18a, miR-19a, miR-20a, miR-21, miR-29b, miR-106a, miR-133a, miR-143 and miR-145). Furthermore, analysis of MMP2, TIMP1/2 mRNA and the protein expression was subsequently performed. The primary study endpoint was the correlation between microRNAs and MMP2, TIMP1/2 mRNA/protein expression. Results We found a significant association between miR-133a and TIMP1 mRNA (r = 0.870, p < 0.001), an inverse correlation between miR-143a and MMP2 protein expression (r= −0.614, p = 0.044) and a positive correlation between miR-133a and TIMP-2 protein expression (r = 0.583, p = 0.036) at the greater curvature. Conclusion Our findings indicate that aortic tissue microRNAs may reflect remodelling processes of the proximal aorta in BAV aortopathy. Specific aortic tissue microRNAs may exert their regulatory effects on the aortopathy through their impact on MMPs/TIMPs homeostasis at the level of the greater curvature.

Atrial matrix remodeling in atrial fibrillation patients with aortic stenosis

BackgroundThis study aimed to evaluate atrium extracellular matrix remodeling in atrial fibrillation (AF) patients with severe aortic stenosis, through histological fibrosis quantification and extracellular matrix gene expression analysis, as well as serum quantification of selected protein targets.MethodsA posthoc analysis of a prospective study was performed in a cohort of aortic stenosis patients. Between 2014 and 2019, 56 patients with severe aortic stenosis submitted to aortic valve replacement surgery in a tertiary hospital were selected.ResultsFibrosis was significantly increased in the AF group when compared to sinus rhythm (SR) patients (p = 0.024). Moreover, cardiomyocyte area was significantly higher in AF patients versus SR patients (p = 0.008). Conversely, collagen III gene expression was increased in AF patients (p = 0.038). TIMP1 was less expressed in the atria of AF patients. MMP16/TIMP4 ratio was significantly decreased in AF patients (p = 0.006). TIMP1 (p = 0.004) and TIMP2 (p = 0.012) were significantly increased in the serum of AF patients. Aortic valve maximum (p = 0.0159) and mean (p = 0.031) gradients demonstrated a negative association with serum TIMP1.ConclusionsAtrial fibrillation patients with severe aortic stenosis present increased atrial fibrosis and collagen type III synthesis, with extracellular matrix remodelling demonstrated by a decrease in the MMP16/TIMP4 ratio, along with an increased serum TIMP1 and TIMP2 proteins.

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Extracellular Matrix Remodeling during Left Ventricular Diastolic Dysfunction and Heart Failure with Preserved Ejection Fraction: A Systematic Review and Meta-Analysis.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are pivotal regulators of extracellular matrix (ECM) composition and could, due to their dynamic activity, function as prognostic tools for fibrosis and cardiac function in left ventricular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction (HFpEF). We conducted a systematic review on experimental animal models of LVDD and HFpEF published in MEDLINE or Embase. Twenty-three studies were included with a total of 36 comparisons that reported established LVDD, quantification of cardiac fibrosis and cardiac MMP or TIMP expression or activity. LVDD/HFpEF models were divided based on underlying pathology: hemodynamic overload (17 comparisons), metabolic alteration (16 comparisons) or ageing (3 comparisons). Meta-analysis showed that echocardiographic parameters were not consistently altered in LVDD/HFpEF with invasive hemodynamic measurements better representing LVDD. Increased myocardial fibrotic area indicated comparable characteristics between hemodynamic and metabolic models. Regarding MMPs and TIMPs; MMP2 and MMP9 activity and protein and TIMP1 protein levels were mainly enhanced in hemodynamic models. In most cases only mRNA was assessed and there were no correlations between cardiac tissue and plasma levels. Female gender, a known risk factor for LVDD and HFpEF, was underrepresented. Novel studies should detail relevant model characteristics and focus on MMP and TIMP protein expression and activity to identify predictive circulating markers in cardiac ECM remodeling.

Identification, Validation, and Functional Annotations of Genome-Wide Profile Variation between Melanocytic Nevus and Malignant Melanoma

Cutaneous melanoma (CM) is known as an aggressive malignant cancer; some of which are directly derived from melanocytic nevi, which have been attracting growing attention from the last decades. This study focused on comprehensive identification, validation, and functional annotations of prognostic differentially expressed genes (DEGs) between melanocytic nevus and malignant melanoma in genome-wide profiles. DEGs were obtained using three chip datasets from GEO database to identify after standardization annotation. A total of 73 DEGs were identified as possible candidate prognostic biomarkers between melanocytic nevus and malignant melanoma. In addition, survival curves indicated that six hub genes, including FABP5, IVL, KRT6A, KRT15, KRT16, and TIMP2, were significant prognostic signatures for CM and of significant value to predict transformation from nevi to melanoma. Furthermore, immunohistochemistry staining was performed to validate differential expression levels and prognostic implications of six hub genes between CM tissue and nevus tissues from the First Affiliated Hospital of Soochow University cohort. It suggested that significantly elevated FABP5, IVL, KRT6A, KRT15, KRT16, and TIMP2 proteins expressed in the CM than in the nevus tissues. Functional enrichment and significant pathways of the six significant hub genes indicated that the mostly involved hallmarks include the P53 pathway, K-ras signaling, estrogen response late, and estrogen response early. In summary, this study identified significant DEGs participating in the process of malignant transformation from nevus to melanoma tissues based on comprehensive genomic profiles. Transcription profiles of FABP5, IVL, KRT6A, KRT15, KRT16, and TIMP2 provided clues of prognostic implications, which might help us evaluate malignant potential of nevus and underlying carcinogenesis progress from melanocytic nevus to melanoma.

MMP13 and TIMP1 are functional markers for two different potential modes of action by mesenchymal stem/stromal cells when treating osteoarthritis

Mesenchymal stem cells (MSCs) have been investigated as a potential injectable therapy for the treatment of knee osteoarthritis, with some evidence of success in preliminary human trials. However, optimization and scale-up of this therapeutic approach depends on the identification of functional markers that are linked to their mechanism of action. One possible mechanism is through their chondrogenic differentiation and direct role in neo-cartilage synthesis. Alternatively, they could remain undifferentiated and act through the release of trophic factors that stimulate endogenous repair processes within the joint. Here, we show that extensive in vitro aging of bone marrow-derived human MSCs leads to loss of chondrogenesis but no reduction in trophic repair, thereby separating out the two modes of action. By integrating transcriptomic and proteomic data using Ingenuity Pathway Analysis, we found that reduced chondrogenesis with passage is linked to downregulation of the FOXM1 signaling pathway while maintenance of trophic repair is linked to CXCL12. In an attempt at developing functional markers of MSC potency, we identified loss of mRNA expression for MMP13 as correlating with loss of chondrogenic potential of MSCs and continued secretion of high levels of TIMP1 protein as correlating with the maintenance of trophic repair capacity. Since an allogeneic injectable osteoar therapy would require extensive cell expansion in vitro, we conclude that early passage MMP13+ , TIMP1-secretinghigh MSCs should be used for autologous OA therapies designed to act through engraftment and chondrogenesis, while later passage MMP13- , TIMP1-secretinghigh MSCs could be exploited for allogeneic OA therapies designed to act through trophic repair.

Role of MANF, TIMP-2, and Irisin Proteins in Aging

The issues of increasing life span and significant improvement in its quality are relevant for modern gerontology. The review discusses one of the proposed methods to slow the aging of an organism via the transfusion of blood or plasma from young people to senile ones. Information on the structure, properties, and functions of TIMP-2, MANF, and irisin protein growth factors, which are contained in large amounts in the blood of young organisms, is presented. Experiments on animals showed an increase in their life span and improvement of their cognitive abilities under the effect of these factors. They are of great importance for the enhancement of neurogenesis in the brain, improvement of the cardiovascular and immune system functions, and cancer prevention. The relationship between the high content of irisin in the blood of young organisms and telomere lengthening, its role in eliminating signs of Alzheimer’s disease, mouse modeling, and the elimination of cerebrovascular ischemia were revealed. The administration of umbilical cord blood with a high TIMP-2 content to old mice improves synaptic plasticity and cognitive functions. Further research on the geroprotection effects of blood transfusions from young organisms to aging ones shows promise for the creation of drugs based on these proteins for the treatment of neurodegenerative diseases and cerebrovascular pathology.

Human mesenchymal stem cells respond differentially to platelet preparations and synthesize hyaluronic acid in nucleus pulposus extracellular matrix

BACKGROUND CONTEXTIn recent years, autologous platelet-rich plasma (PRP) and bone marrow aspirate concentrate (BMAC) have been used as treatments for disc-related pain. A better understanding of the effects of leukocyte-rich (LR) versus leukocyte poor (LP-) PRP on bone marrow derived human mesenchymal stem/progenitor cells (hMSCs) is likely to improve future research studies, clinical practice and care for patients with chronic discogenic back pain. PURPOSEThe primary aim of this study is to determine the effects of LR-PRP and LP-PRP on the proliferation and migration of hMSCs in pig nucleus pulposus (NP) extracellular matrix (ECM). The secondary aim is to characterize hMSC-dependent expression of the matrix remodeling enzymes metalloproteinases MMP-2, MMP-3, MMP-9 and tissue inhibitor of metalloproteinases TIMP-2, and to determine whether transplanted hMSCs can synthesize hyaluronic acid (HA). STUDY DESIGNControlled laboratory study. METHODSBone marrow-derived culture expanded hMSCs were seeded onto pig NP and cultured with LR-PRP, LP-PRP or serum/platelet releasate (PR). The same conditions without hMSCs were used as controls. hMSC proliferation, migration and dispersion was assessed via fluorescent microscopy, while HA synthesis, MMP-2, MMP-3, MMP-9, and TIMP-2 protein levels were assessed via enzyme linked immunosorbent assay. All funding was provided by a 501c(3) research foundation and does not have any commercial or sponsorship interests. RESULTSLP-PRP and PR cultures resulted in higher hMSC proliferation, migration, dispersion, and MMP-2 expression. LP-PRP cultures resulted in the highest HA production. LR-PRP cultures resulted in lower hMSC proliferation, negligible migration and dispersion, increased MMP-9 expression and lower HA production. CONCLUSIONSHuman bone marrow-derived hMSCs seeded onto pig NP ECM are capable of synthesizing HA, indicating a transition towards a NP cell phenotype. This process was most enhanced by LP-PRP and marked by increased hMSC proliferation, MMP-2 production, HA synthesis and reduced MMP-9 levels. CLINICAL SIGNIFICANCELP-PRP and PR, with or without hMSCs, may provide better outcomes than LR-PRP in lab investigations and clinical trials for discogenic pain. Bone marrow-derived hMSCs may hold promise as a treatment for disc degeneration.

Icariside II attenuates cerebral ischemia/reperfusion-induced blood-brain barrier dysfunction in rats via regulating the balance of MMP9/TIMP1.

Cerebral ischemia/reperfusion (I/R) results in harmful consequences during ischemic stroke, especially the disruption of the blood-brain barrier (BBB), which leads to severe hemorrhagic transformation through aggravation of edema and brain hemorrhage. Our previous study demonstrated that icariside II (ICS II), which is derived from Herba Epimedii, attenuates cerebral I/R injury by inhibiting the GSK-3β-mediated activation of autophagy both in vitro and in vivo. However, the effect of ICS II on the BBB remains unclear. Thus, in this study, we investigated the regulation of BBB integrity by ICS II after cerebral I/R injury and further explored the underlying mechanism in rats. Cerebral I/R injury was induced by middle cerebral artery occlusion (MCAO), and the treatment groups were administered ICS II at a dose of 16 mg/kg by gavage twice a day for 3 days. The results showed that ICS II effectively prevented BBB disruption, as evidenced by Evans Blue staining. Moreover, ICS II not only significantly reduced the expression of MMP2/9 but also increased TIMP1 and tight junction protein (occludin, claudin 5, and ZO 1) expression. Intriguingly, ICS II may directly bind to both MMP2 and MMP9, as evidenced by molecular docking. In addition, ICS II also inhibited cerebral I/R-induced apoptosis and ameliorated the Bax/Bcl-2 ratio and cleaved-caspase 3 level. Collectively, our findings reveal that ICS II significantly ameliorates I/R-induced BBB disruption and neuronal apoptosis in MCAO rats by regulating the MMP9/TIMP1 balance and inhibiting the caspase 3-dependent apoptosis pathway.

217-LB: A Novel GPR119 Agonist, DA-1241, Improves Hepatic Inflammation and Fibrosis in ob-NASH Mice

GPR119 activation is known to inhibit de novo lipogenesis in hepatocytes, but it still remains elusive if and how GPR119 agonists can affect the pathogenesis of hepatic inflammation and fibrosis in NASH. Herein, we report the therapeutic potential of DA-1241 for NASH, especially for hepatic inflammation and fibrosis. DA-1241 was given to ob/ob mice with western diet for 10 weeks. Plasma and liver tissue samples were tested for biochemical and histological analyses. Human monocyte cells and primary hepatic stellate cells were used for in vitro proof-of-concept. At the end of experiment, ob/ob NASH mice distinctly exhibited increased plasma total cholesterol and hepatic lipid accumulation, but plasma glucose and triglyceride values were within normal range. After 10-weeks of treatment, DA-1241 significantly blocked the progression of hepatic inflammation and fibrosis compared to the NASH control (-39% and -64% for NAS and fibrosis score; p&amp;lt;0.05), while MBX-2982, a GPR119 agonist under clinical development, failed to reach statistical significance at the same dose (-19.4% and -29%; p&amp;gt;0.05). Moreover, plasma total glucagon-like peptide-1 levels were increased by DA-1241 treatment, but not by MBX-2982. Plasma ALT and AST levels were significantly lower in DA-1241-treated mice, which were much lower than those of MBX-2982-treated mice. Of note, DA-1241 were more efficacious than MBX-2982 in reducing the hepatic expression of fibrotic proteins such as type 1 collagen, α-SMA, and TIMP1, and pro-inflammatory proteins including CCL2, TNFα. To explore its underlying modes of action in vitro, THP-1 macrophages or primary hepatic stellate cells were activated by LPS or TGFβ, respectively. DA-1241 inhibited the differentiation and activation of macrophages and blocked stellate cells activation. Our findings suggest that DA-1241 has a therapeutic potential for NASH, which may be partly attributed to blocking macrophage and stellate cell activation in addition to the inhibition of lipogenesis. Disclosure H. Park: Employee; Self; Dong-A ST Co., Ltd. B. Lee: Employee; Self; DONG-A ST Co,Ltd. Y. Chae: Employee; Self; Dong-A ST Co., LTD. M. Kim: Employee; Self; Dong-A ST Co., Ltd.

Fructose Metabolism and Regulation of Extracellular Matrix Protein Gene Expression in Activated Macrophages

Abstract Objectives Recently fructose has been linked to the development of Nonalcoholic Fatty Liver Disease (NAFLD), but the mechanisms behind the progression remain to be elucidated. Kupffer cells have been identified as regulators of hepatic inflammation and extracellular matrix (ECM) proteins. Activated macrophages are known to express tissue inhibitor of metalloproteinase (TIMP1), which inhibits matrix metalloproteinase 9 (MMP9) activity of reconstructing ECM which leads to fibrosis. In humans and mouse models, TIMP1 expression is positively correlated with the progression of NAFLD. Based on these findings, we hypothesize that fructose regulates TIMP1 gene expression in activated proinflammatory macrophages. Methods Using an in vitro model of J774 macrophages and primary Kupffer cells, cells were treated to induce an M1 phenotype in the presence of glucose or fructose. Cells were harvested for RNA and RT-PCR was conducted to measure ECM gene expression. Isolated Kupffer cells were collected from C57BL/6J mice fed a high fat diet (HFD) supplemented with 30% fructose or 30% glucose and analyzed for ECM expression. To examine fructose uptake and intra- and extracellular metabolites, mass spectrophotometry and nuclear magnetic resonance was conducted. Results Timp1 gene expression was significantly increased in J77.4 cells treated with fructose compared to glucose. Surprisingly, fructose treatment decreased Mmp9 gene expression. Likewise, fructose treatment increased TIMP1 protein expression in isolated Kupffer cells. In vivo, isolated hepatic macrophages from mice fed HF and high fructose diet had elevated Timp1 gene expression compared to mice fed high glucose diet. Extracellular levels of lactate decreased by 1.5-fold in fructose treated J77.4 cells compared to glucose. Metabolites involved in the TCA cycle and mitochondrial function were decreased when treated with fructose compared to glucose in non-activated macrophages. However, fructose treatment increased intracellular methanol and acetate levels in M1 macrophages compared to glucose. Conclusions Our data suggest that fructose upregulates Timp1 expression and possibly decreases mitochondrial function while increasing acetate and methanol production, identifying new mechanisms by which fructose drives the progression of NAFLD. Funding Sources Kenan Institute North Carolina University.

Chondrogenic potency is lost but trophic repair is maintained by highly passaged mesenchymal stem/stromal cells

Purpose: Bone marrow-derived mesenchymal stromal cells (MSCs) are defined as stem cells primarily because of their capacity for multipotent differentiation, however more recently they have been shown also to have trophic properties that may be important for promoting tissue repair through the release of regulatory molecules that can promote regeneration through the stimulation of endogenous cells, whilst also inhibiting immune/inflammatory processes. Each of these modes of action may be important when developing therapeutic strategies for the treatment of osteoarthritic joints using injectable MSCs. Since a viable MSC therapy is likely to require the use of highly expanded populations of allogenic cells, we have undertaken a detailed analysis of how the differentiation and trophic properties of MSCs change with in vitro ageing of the cells. Methods: MSCs were cultured from passage until senescence. Cells from multiple passages were tested for their potency when used either for chondrogenesis (cartilage tissue engineering assay) or trophic repair. Two methods were used as determinants of trophic repair: (i) a meniscal cartilage integration assay in which undifferentiated human MSCs on a collagen scaffold were used to induce integration of two pieces of sheep meniscal cartilage and (ii) an immunoregulation assay in which undifferentiated MSCs were used to suppress the proliferation of stimulated T-cells. Genomic and proteomic screening was used to identify potential markers of MSCS with capacity for differentiation and trophic repair. Results: MSCs from 4 different donors were found to proliferate for a minimum of 16 passages before growth arrest with cells from one donor continuing to divide even at passage 30 (Figure 1). The chondrogenic potency of these MSCs fell significantly for all four donors, when measured as either the dry weight of cartilage tissue engineering or the Glycosaminoglycans content of the engineered cartilage (Figure 2). However, there was no reduction in trophic repair whether measured as integration of meniscal cartilage or suppression of T-cell proliferation (Figure 3). Combined genomic and proteomic analysis demonstrated that earlier passage cells with both chondrogenic and trophic potency are positive for both the MMP13 gene and TIMP-1 protein, whereas later passage cells with poor chondrogenic potency but strong trophic potency are negative for the MMP13 gene but positive for the TIMP-1 protein (Figure 4). Conclusions: We have demonstrated clear differences in the regulation of chondrogenic potency and trophic properties of MSCs. The measurement of MMP13 gene and TIMP-1 protein markers will facilitate the design of future therapeutic strategies that exploit either of one these modes of action.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Upregulation of matrix metalloproteinase 9 (MMP9)/tissue inhibitor of metalloproteinase 1 (TIMP1) and MMP2/TIMP2 ratios may be involved in lipopolysaccharide-induced acute lung injury.

ObjectiveThis study aimed to examine the changes and significance of matrix metalloproteinase 9 (MMP9), MMP2, tissue inhibitor of metalloproteinase 1 (TIMP1), and TIMP2 in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI).MethodsWistar rats were randomly divided into a control group (injected with saline) and an ALI group (injected with LPS), then subdivided into four time points (2, 6, 12, and 24 hours). Serum tumor necrosis factor alpha and interleukin-6 levels were detected by ELISA to investigate the inflammatory reaction after LPS injection. The degree of ALI was determined by hematoxylin–eosin staining of lung tissue, the lung wet/dry weight ratio, and pulmonary permeability index. Changes in lung MMP and TIMP protein and mRNA levels were detected by western blotting and quantitative real-time polymerase chain reaction.ResultsChanges in the ratios of MMP9/TIMP1 and MMP2/TIMP2 were consistent with and strongly positively associated with the lung wet/dry weight ratio, the pulmonary permeability index, and serum tumor necrosis factor alpha and interleukin-6 levels in the ALI group.ConclusionALI induced by LPS may be related to upregulation of MMP9/TIMP1 and MMP2/TIMP2 ratios.

Effect of bFGF on the growth and matrix turnover of stem cells from human apical papilla: Role of MEK/ERK signaling

Basic fibroblast growth factor (bFGF) exhibits multiple biological functions in various tissues. Stem cells from apical papilla (SCAP) can be isolated from human apical papilla tissues in developmental teeth of children. The purposes of this study were to investigate the expression of FGF receptors (FGFRs) and the effects of bFGF on SCAP and related MEK/ERK signaling. SCAP cells were treated under different concentrations of bFGF with or without U0126 (an inhibitor of MEK/ERK). Expression of FGFR1 and FGFR2 in SCAP was analyzed by RT-PCR. Cell proliferation was measured by MTT assay. The expressions of type I collagen, cdc 2, cyclin B1, TIMP-1 and p-ERK proteins were examined by Western blot. SCAP cells expressed FGFR1 and FGFR2. Exposure of SCAP to bFGF enhanced cell proliferation, and the expression cyclinB1, cdc 2, and TIMP-1, but not type I collagen. U0126 pretreatment and co-incubation attenuated the bFGF-induced proliferation, cdc2, cyclin B1 and TIMP-1 proteins' expression, but not type I collagen in SCAP. SCAP cells express FGFRs. bFGF may stimulate proliferation and affect the matrix turnover of SCAP cells, possibly via stimulation of FGFRs and MEK/ERK signaling pathway. These results are useful for clinical therapies for apexogenesis and regeneration of pulpo-dentin complex.

N6-methyladenosine ALKBH5 promotes non-small cell lung cancer progress by regulating TIMP3 stability

Mounting evidence demonstrates that N6-methyladenosine (m6A) play critical roles of m6A in the epigenetic regulation, especially for human cancer. The m6A modification is installed by methyltransferase and erased demethylases, leading to the significant modification for gene expression and cell fate. Here, we investigated the biological roles and mechanism of demethylase alkylation repair homolog protein 5 (ALKBH5) in the non-small cell lung cancer (NSCLC). Results revealed that ALKBH5 was ectopically up-regulated in the NSCLC tissue and cells, and closely correlated with the poor prognosis. Functionally, ALKBH5 promoted the proliferation and reduced apoptosis of NSCLC cells in vitro, and knockdown of ALKBH5 repressed the tumor growth in vivo. Mechanistically, RNA immunoprecipitation sequencing (RIP-Seq) revealed that ALKBH5 targeted the TIMP3. Moreover, ALKBH5 repressed TIMP3 mRNA stability and protein production. In conclusion, the present research confirmed the ALKBH5/TIMP3 pathway in the NSCLC oncogenesis progress, providing a novel insight for the epitranscriptome and potential therapeutic target for NSCLC.