Tissue Inhibitor Of Metalloprotease Research Articles

Curcumin relieves the arecoline-induced fibrosis of oral mucosal fibroblasts via inhibiting HIF-1α/TGF-β/CTGF signaling pathway: an in vitro study.

Oral submacosal fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis. The current treatments for OSF have failed to achieve the desired curative effect. Here, we propose that curcumin has excellent therapeutic effect on OSF and explore its specific mechanism. Transwell assay was performed to detected cell migration. Flow cytometry was used to measured apoptosis. And MTT assay was performed to test cell viability. Gene and protein levels were detected by quantitative real-time polymerase chain reaction (qPCR) and western blotting. Our results displayed that curcumin treatment reduced fibrosis-related molecules (collagen type I alpha 1, collagen type III alpha 1, tissue inhibitor of metalloprotease 2) in arecoline-treated oral mucosal fibroblasts and elevated matrix metalloproteinase 2 expression. Additionally, curcumin could suppress cell proliferation and migration, and enhance the apoptosis of arecoline-treated normal oral mucosal fibroblasts. Most importantly, the hypoxia-inducible factor-1α (HIF-1α), transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) expressions in arecoline-treated normal oral mucosal fibroblasts were reduced after exposure to curcumin, whereas the activation of HIF-1α/TGF-β/CTGF axis reversed curcumin's effect on improving fibrosis of arecoline-treated normal oral mucosal fibroblasts. Therefore, curcumin alleviated oral submucosal fibrosis via inhibiting HIF-1α/TGF-β/CTGF axis. In summary, curcumin effectively inhibited the migration and proliferation and promoted apoptosis in arecoline-induced normal oral mucosal fibroblasts by inactivating HIF-1α/TGF-β/CTGF pathway. And curcumin might be a potential therapeutic drug for OSF treatment.

Relationship between Metalloprotease-7 and -14 and Tissue Inhibitor of Metalloprotease 1 Expression by Mucosal Stromal Cells and Colorectal Cancer Development in Inflammatory Bowel Disease.

Colorectal carcinoma (CRC) associated with inflammatory bowel disease (IBD) is an example of an inflammation-related cancer. Matrix metalloproteases (MMP) are known to be associated with both processes. The aim of the study was to compare the expression of MMP-7, MMP-14 and tissue inhibitor of metalloproteases-1 (TIMP-1) in sporadic CRC- and IBD-associated CRC, and to compare the expression in inflamed and non-inflamed colonic tissue samples from IBD patients without or with associated CRC. An immunohistochemical study of MMP-7, -14 and TIMP-1 was performed on sporadic CRC (n = 86), IBD-associated CRC (n = 23) and colorectal mucosa of non-tumor samples from IBD patients without (n = 47) and with (n = 23) associated CRC. These factors were more frequently expressed by cancer-associated fibroblasts (CAF) from IBD-associated CRC than by CAF from CRC not associated with IBD. Regarding the inflamed tissue of IBD patients, Crohn’s disease (CD) patients with CRC development showed a higher expression of MMP-14 by fibroblasts and by mononuclear inflammatory cells (MICs) than CD patients without CRC development. In non-inflamed tissue samples, MMP-7 associated with fibroblasts and MICs, and TIMP-1 associated with MICs, were more frequently expressed in CD patients with CRC development than in CD patients without CRC development. Our data suggest that these factor expressions by stromal cells may be biological markers of CRC development risk in IBD patients.

Chondroprotective effect of Alpinia oxyphylla extract in experimentally induced cartilage degradation in rabbit articular cartilage explants.

Alpinia oxyphylla is a widely used medicinal herb for diarrhea, gastralgia, tumors, hypertention, and cerebrovascular disorders. Here, we evaluated the chondroprotective effect of A.oxyphylla dried fruit ethanol extract (AOE) against cartilage degradation in rabbit articular cartilage explants. Treatment of interleukin-1α (IL-1α) and plasminogen increased degraded collagen release in culture supernatants, but pretreatment of AOE (50, 100, 200µg/ml) inhibited the collagen release in dose-dependent manner. To examine the mechanism of action of AOE on chondroprotection, the level of matrix metalloproteinases-3 (MMP-3), matrix metalloproteinases-13 (MMP-13), tissue inhibitor of metalloprotease-1 (TIMP-1), and inflammatory mediators like prostaglandin E2 (PGE2 ) and nitric oxide (NO) was evaluated. AOE inhibited upregulation of MMP-3 and MMP-13 and downregulation of TIMP-1 and also reduced increase of PGE2 and NO level induced by exposure of IL-1α and plasminogen. These results indicate that AOE show chondroprotective effect through inhibiting collagen degradation via regulating MMPs, TIMP-1, and inflammatory mediators. PRACTICAL APPLICATIONS: Osteoarthritis (OA) is a one of the most common chronic disorders in elderly persons. Because the regenerative power of joint articular cartilage is very low, treatment of OA is difficult to expect complete recovery. Therefore, there is a need to develop a therapeutic agent that can safely and effectively inhibit the cartilage destruction. For the first time, we exhibited the inhibitory effect of AOE on collagen degradation through regulating MMPs and TIMP-1 in articular cartilage explants. These findings support AOE could be used as herbal therapeutic application for protecting articular cartilage to prevent OA.

Sphingosine kinases negatively regulate the expression of matrix metalloproteases (MMP1 and MMP3) and their inhibitor TIMP3 genes via sphingosine 1-phosphate in extravillous trophoblasts.

PurposeExtracellular matrix remodeling is essential for extravillous trophoblast (EVT) cell migration and invasion during placental development and regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs). Sphingosine kinases (SPHK1 and SPHK2) synthesize sphingosine‐1‐phosphate (S1P), which works either intracellularly or extracellularly via its receptors S1PR1‐5 in an autocrine or paracrine manner. The role of SPHKs/S1P in regulating the expression of MMPs and TIMPs in EVT is mostly unknown and forms the primary objective of the study.MethodsHTR‐8/SVneo cells were used as a model of EVT. To inhibit the expression of SPHKs, cells were treated with specific inhibitors, SK1‐I and SKI‐II, or gene‐specific siRNAs. The expressions of MMPs and TIMPs were estimated by qPCR.ResultsWe demonstrated that SPHK1, MMP1‐3, and TIMP1‐3 were highly expressed in HTR‐8/SVneo cells. We found that treatment of cells with SK1‐I, SKI‐II, and knockdown of SPHK1 or SPHK2 increased the expression of MMP1, MMP3, and TIMP3. The addition of extracellular S1P inhibits the upregulation of MMPs and TIMPs in treated cells.ConclusionsSPHKs negatively regulate the expression of MMP1, MMP3, and TIMP3. The level of intracellular S1P acts as a negative feedback switch for MMP1, MMP3, and TIMP3 expression in EVT cells.

Associations between TGF-β1 Levels and Markers of Hemolysis, Inflammation, and Tissue Remodeling in Pediatric Sickle Cell Patients.

Transforming growth factor beta (TGF-β) is a cytokine with important involvement in biological processes related to the pathogenesis of sickle cell disease (SCD), including endothelial and vascular dysfunction, inflammation, and hematopoietic homeostasis. This study is aimed at investigating associations between levels of TGF-β1 and classical laboratory biomarkers and inflammatory mediators, as well as the tissue inhibitor of metalloproteases-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9), in pediatric patients (n = 123) with SCD in steady state: 84 with sickle cell anemia (HbSS) and 39 with hemoglobin SC disease (HbSC). A healthy control (HC) group of 59 individuals was also included. Hematological and biochemical analyses were carried out using electronic methods. TGF-β1, TIMP-1, and MMP-9 plasma quantifications were performed by ELISA. TGF-β1 plasma levels were higher in HbSS individuals than in HbSC and HC. In individuals with HbSS, TGF-β1 levels were positively correlated with red blood cells, hemoglobin, hematocrit, platelets, and TIMP-1. In addition, HbSS individuals with TGF-β1 levels above the median (≥72.29 ng/mL) also presented increased monocyte counts and decreased albumin levels. In patients with HbSC, TGF-β1 levels were positively correlated with leukocytes, eosinophils, lymphocytes, monocytes, and platelets, as well as levels of TIMP-1, VLDL-C, triglycerides, heme, and AST. Additionally, HbSC individuals with TGF-β1 levels above the median (≥47.80 ng/mL) presented increased leukocyte and platelet counts, as well as increased levels of triglycerides, VLDL-C, MMP-9, and TIMP-1, and decreased HDL-C. Our findings suggest that TGF-β1 may play important roles in vascular remodeling, vasculopathy, angiogenesis, and inflammation in pediatric patients with SCD.

Quantitative Proteomics Reveals Changes Induced by TIMP-3 on Cell Membrane Composition and Novel Metalloprotease Substrates.

Ectodomain shedding is a key mechanism of several biological processes, including cell-communication. Disintegrin and metalloproteinases (ADAMs), together with the membrane-type matrix metalloproteinases, play a pivotal role in shedding transmembrane proteins. Aberrant shedding is associated to several pathological conditions, including arthritis. Tissue inhibitor of metalloproteases 3 (TIMP-3), an endogenous inhibitor of ADAMs and matrix metalloproteases (MMPs), has been proven to be beneficial in such diseases. Thus, strategies to increase TIMP-3 bioavailability in the tissue have been sought for development of therapeutics. Nevertheless, high levels of TIMP-3 may lead to mechanism-based side-effects, as its overall effects on cell behavior are still unknown. In this study, we used a high-resolution mass-spectrometry-based workflow to analyze alterations induced by sustained expression of TIMP-3 in the cell surfaceome. In agreement with its multifunctional properties, TIMP-3 induced changes on the protein composition of the cell surface. We found that TIMP-3 had differential effects on metalloproteinase substrates, with several that accumulated in TIMP-3-overexpressing cells. In addition, our study identified potentially novel ADAM substrates, including ADAM15, whose levels at the cell surface are regulated by the inhibitor. In conclusion, our study reveals that high levels of TIMP-3 induce modifications in the cell surfaceome and identifies molecular pathways that can be deregulated via TIMP-3-based therapies.

Dermal fibroblasts cultured from donors with type 2 diabetes mellitus retain an epigenetic memory associated with poor wound healing responses

The prevalence of Type 2 diabetes mellitus (T2DM) is escalating globally. Patients suffer from multiple complications including the development of chronic wounds that can lead to amputation. These wounds are characterised by an inflammatory environment including elevated tumour necrosis factor alpha (TNF-α). Dermal fibroblasts (DF) are critical for effective wound healing, so we sought to establish whether there were any differences in DF cultured from T2DM donors or those without diabetes (ND-DF). ND- and T2DM-DF when cultured similarly in vitro secreted comparable concentrations of TNF-α. Functionally, pre-treatment with TNF-α reduced the proliferation of ND-DF and transiently altered ND-DF morphology; however, T2DM-DF were resistant to these TNF-α induced changes. In contrast, TNF-α inhibited ND- and T2DM-DF migration and matrix metalloprotease expression to the same degree, although T2DM-DF expressed significantly higher levels of tissue inhibitor of metalloproteases (TIMP)-2. Finally, TNF-α significantly increased the secretion of pro-inflammatory cytokines (including CCL2, CXCL1 and SERPINE1) in ND-DF, whilst this effect in T2DM-DF was blunted, presumably due to the tendency to higher baseline pro-inflammatory cytokine expression observed in this cell type. Collectively, these data demonstrate that T2DM-DF exhibit a selective loss of responsiveness to TNF-α, particularly regarding proliferative and secretory functions. This highlights important phenotypic changes in T2DM-DF that may explain the susceptibility to chronic wounds in these patients.

The predictive value of TIMP-2 and IGFBP7 for kidney failure and 30-day mortality after elective cardiac surgery

Acute kidney injury (AKI) is an important risk factor for chronic kidney disease, renal replacement therapy (RRT), and mortality. However, predicting AKI with currently available markers remains problematic. We assessed the predictive value of urinary tissue inhibitor of metalloprotease-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) regarding the need for RRT, and 30-day mortality, in elective cardiac surgery patients. In 344 elective cardiac surgery patients, we measured urinary TIMP-2 and IGFBP7 and serum creatinine at baseline and directly after surgery. Discrimination of both urinary biomarkers was assessed by the C-statistic. Model improvement for each biomarker when added to a basic model containing serum creatinine and duration of surgery was tested by the net-reclassification index (cf-NRI) and integrated discrimination index (IDI). At baseline, mean age was 66 years and 67% were men. Of all patients, 22 required RRT following surgery. IGFBP7 pre- and post-surgery and change in TIMP-2 during surgery predicted RRT with a C-statistic of about 0.80. However, a simple model including baseline serum creatinine and duration of surgery had a C-statistic of 0.92, which was improved to 0.93 upon addition of post-surgery TIMP-2 or IGFBP7, with statistically significant cf-NRIs but non-significant IDIs. Post-surgery TIMP-2 and IGFBP predicted 30-day mortality, with C-statistics of 0.74 and 0.80. In conclusion, in elective cardiac surgery patients, pre- and peri-operative clinical variables were highly discriminating about which patients required RRT after surgery. Nonetheless, in elective cardiac surgery patients, urinary TIMP-2 and IGFBP7 improved prediction of RRT and 30-day mortality post-surgery.

Tissue Inhibitor of Metalloprotease-1 (TIMP-1) Regulates Adipogenesis of Adipose-derived Stem Cells (ASCs) via the Wnt Signaling Pathway in an MMP-independent Manner.

Tissue inhibitor of metalloprotease-1 (TIMP-1) is a tissue inhibitor of matrix metalloproteinases (MMPs). It however exerts multiple effects on biological processes, such as cell growth, proliferation, differentiation and apoptosis, in an MMP-independent manner. This study aimed to examine the role of TIMP-1 in adipogenesis of adipose-derived stem cells (ASCs) and the underlying mechanism. We knocked down the TIMP-1 gene in ASCs through lentiviral vectors encoding TIMP-1 small interfering RNA (siRNA), and then found that the knockdown of TIMP-1 in ASCs promoted the adipogenic differentiation of stem cells and inhibited the Wnt/β-catenin signaling pathway in ASCs. We also noted that mutant TIMP-1 without the inhibitory activity on MMPs promoted the activation of Wnt/β-catenin pathway as well as the recombinant wild type TIMP-1 did, which indicated that the effect of TIMP-1 on Wnt/β-catenin pathway was MMP-independent. Our study suggested that TIMP-1 negatively regulated the adipogenesis of ASCs via the Wnt/β-catenin signaling pathway in an MMP-independent manner.

Establishment and Evaluation of an In Vitro System for Biophysical Stimulation of Human Osteoblasts.

While several studies investigated the effects of mechanical or electrical stimulation on osseointegration and bone fracture healing, little is known about the molecular and cellular impact of combined biophysical stimulation on peri-implant osseointegration. Therefore, we established an in vitro system, capable of applying shear stress and electric fields simultaneously. Capacitively coupled electric fields were used for electrical stimulation, while roughened Ti6Al4V bodies conducted harmonically oscillating micromotions on collagen scaffolds seeded with human osteoblasts. Different variations of single and combined stimulation were applied for three days, while samples loaded with Ti6Al4V bodies and untreated samples served as control. Metabolic activity, expression of osteogenic markers and bone remodeling markers were investigated. While combined stimulation showed no substantial benefit compared to sole mechanical stimulation, we observed that 25 µm micromotions applied by roughened Ti6Al4V bodies led to a significant increase in gene expression of osteocalcin and tissue inhibitor of metalloprotease 1. Additionally, we found an increase in metabolic activity and expression of bone remodeling markers with reduced procollagen type 1 synthesis after 100 mVRMS electrical stimulation. We were able to trigger specific cellular behaviors using different biophysical stimuli. In future studies, different variations of electrical stimulation will be combined with interfacial micromotions.

Direct Delivery of Apatite Nanoparticle-Encapsulated siRNA Targeting TIMP-1 for Intractable Abnormal Scars

Hypertrophic scars (HSs) and keloids are histologically characterized by excessive extracellular matrix (ECM) deposition. ECM deposition depends on the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteases (TIMPs). TIMP-1 has been linked to ECM degradation and is therefore a promising therapeutic strategy. In this study, we generated super carbonate apatite (sCA) nanoparticle-encapsulated TIMP-1 small interfering RNA (siRNA) (siTIMP1) preparations and examined the effect of local injections on mouse HSs and on ex vivo-cultured keloids. The sCA-siTIMP1 injections significantly reduced scar formation, scar cross-sectional areas, collagen densities, and collagen types I and III levels in the lesions. None of the mice died or exhibited abnormal endpoints. Apatite accumulation was not detected in the other organs. In an ex vivo keloid tissue culture system, sCA-siTIMP1 injections reduced the thickness and complexity of collagen bundles. Our results showed that topical sCA-siTIMP1 injections during mechanical stress-induced HS development reduced scar size. When keloids were injected three times with sCA-siTIMP1 during 6 days, keloidal collagen levels decreased substantially. Accordingly, sCA-siRNA delivery may be an effective approach for keloid treatment, and further investigations are needed to enable its practical use.

Diagnostic Role of Saliva in Oral Cancers

Saliva is a noninvasive diagnostic fluid alternative containing almost all the compounds found in blood, oral and tumoral cells. The aim of this study is to analyze the correlations occurred between different key biomarkers involved in different stages of oral cancer, such as inflammation markers - interleukin 6 (IL-6), extracellular collagen degradation markers - matrix metalloprotease 9 (MMP-9) and tissue inhibitors of metalloprotease 2 (TIMP-2).This study included a lot of 30 patients diagnosed with oral cancer at the Department of Oro Maxillo-Facial Surgery, University Hospital of Dental Medicine Prof. Dr. Dan Teodorescu. The patients were included in this study after informed consent was obtained. Salivary biomarker analysis was made using ELISA methods, biochemical or colorimetric assays. Uric acid (UA) levels were positively correlated with total antioxidant capacity (TAC) levels (r = 0.78). TAC levels were negatively correlated with IL-6 levels (r = -0.66). MMP-9 levels were positively correlated with the Ki-67protein (also known as MKI67) levels (r = 0.65) and negatively correlated with TIMP-2 levels (r = -0.71). We can conclude upon the results that salivary research may lead to the usage of saliva as a quick and reliable screening and diagnostic tool for oral cancer.

High-Affinity Binding of LDL Receptor-Related Protein 1 to Matrix Metalloprotease 1 Requires Protease:Inhibitor Complex Formation.

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

Tumoral and stromal expression of MMP-2, MMP-9, MMP-14, TIMP-1, TIMP-2, and VEGF-A in cervical cancer patient survival: a competing risk analysis

BackgroundExpression of matrix metalloproteases 2, 9 and 14 (MMP-2, MMP-9, MMP-14), tissue inhibitors of metalloprotease 1 and 2 (TIMP-1, TIMP-2) and vascular endothelial growth factor A (VEGF-A) is involved in tumor invasion and metastasis via extracellular matrix degradation and angiogenesis. This study aimed to assess whether the expression of MMP-2, MMP-9, MMP-14, TIMP-1, and TIMP-2 in tumors and in the adjacent stroma is associated with cervical cancer prognosis.MethodsThis study analyzed a retrospective cohort of 64 patients. Protein expression was previously obtained by immunohistochemistry from biopsies containing both tumor and stroma. The expression and percentage of stained cells were categorized as high or low according to the cutoff points by using ROC curves. The follow-up data was collected from diagnosis to the last clinical visit. Clinical status categorized as alive without disease, alive with disease, death due to other causes, and death from the disease. The relative risk of death from the disease was evaluated according to the proteins expression using a cause-specific Cox regression model with a 95% confidence interval (95%CI). For the significant associations (p < 0.05), survival curves of patients with low and high expression were plotted for the competing risk survival curve analyses.ResultsHigh expression levels of stromal MMP-2 (RR; 95%CI: 3.91; 1.17–13.02) and stromal TIMP-2 (RR, 95%CI: 8.67; 1.15–65.27) were associated with a greater relative risk of death from the disease and with lower survival (p = 0.03; p = 0.04) than lower expression levels. Low expression levels of stromal MMP-9 (RR, 95%CI: 0.19; 0.05–0.65) and tumoral MMP-9 (HR, 95%CI: 0.19; 0.04–0.90) were protective factors against death from the disease and were associated with poorer survival.ConclusionsHigh expression levels of MMP-2 and TIMP-2 in the stroma were significantly associated with poor survival in cervical cancer patients. High expression of MMP-9 was associated with a favorable cervical cancer prognosis.

Urinary cell cycle arrest proteins urinary tissue inhibitor of metalloprotease 2 and insulin-like growth factor binding protein 7 predict acute kidney injury after severe trauma: A prospective observational study.

Recognition and clinical diagnosis of acute kidney injury (AKI) after trauma is difficult. The majority of trauma patients do not have a known true baseline creatinine, which makes application of the guidelines set forth by the international guidelines difficult to apply. Use of alternative biomarkers of renal dysfunction in trauma patients may be beneficial. We hypothesized that urinary tissue inhibitor of metalloprotease 2 (TIMP-2) × insulin-like growth factor binding protein 7 (IGFBP-7) would accurately predict AKI development in severely injured trauma patients. A prospective observational study of adult (≥16 years old) trauma intensive care unit (ICU) patients was performed between September 2018 to March 2019. Urine was collected on ICU admission and was measured for TIMP-2 × IGFBP-7. Univariate, multivariable, and receiver operating characteristic curve analyses were performed using the optimal threshold generated by a Youden index. Of 88 included patients, 75% were male, with a median injury severity score was 27 (interquartile range [IQR], 17-34), and age of 40 years (IQR, 28-54 years). Early AKI developed in 39 patients (44%), and of those, 7 (8%) required dialysis within 48 hours. Patients without early AKI had a TIMP-2 × IGFBP-7 of 0.17 U (IQR, 0.1-0.3 U), while patients with early AKI had a TIMP-2 × IGFBP-7 of 0.46 U (IQR, 0.17-1.29 U; p < 0.001). On multivariable analyses, TIMP-2 × IGFBP-7 was associated with AKI development (p = 0.02) and need for dialysis (p = 0.03). Using the optimal threshold 0.33 U to predict AKI, the area under the receiver operating characteristic curve was 0.731, with an accuracy of 0.75, sensitivity of 0.72, and specificity of 0.78. Urinary TIMP-2 × IGFBP-7 measured on ICU admission accurately predicted 48-hour AKI and was independently associated with AKI and dialysis requirement after trauma and is a promising screening tool for treatment. Prognostic, prospective, observational study, level III.

Serum biomarkers that predict lean mass loss over bed rest in older adults: An exploratory study

BackgroundLean mass (LM) loss during extended bed rest contributes to long term functional decline in older adults. Identifying blood biomarkers that predict a hospitalized individual’s risk of losing LM could allow for timely intervention. MethodsLM from 19 healthy subjects (age 60–76 y, 4 males, 15 females), who were confined to 10 days of complete bed rest, was measured pre- and post-bed rest. One hundred eighty-seven biomarkers from pre-bed rest fasted serum samples were obtained from all evaluable subjects (n = 18), analyzed using multiplexed immunoassay array and pooled. Decision tree analysis was used to identify pre-bed rest markers that predict LM loss over bed rest. ResultsSixty-three markers were excluded due to being below assay detection limits. One pair of markers, Tissue inhibitor of metalloprotease-1 (TIMP1) and tenascin C (TNC), were found to correlate with percent change in total LM over bed rest: [R2 = 0.71, all subjects; R2 = 0.76, females]. Subjects with pre-bed rest TIMP1 ≥ 141 ng/ml had the highest loss of total LM over bed rest, whereas subjects with pre-bed rest TIMP1 < 141 and TNC ≥ 461 ng/ml maintained total LM over bed rest. An additional marker set was found to correlate with percent change in leg LM loss over bed rest: matrix metalloprotease-3 (MMP3) and apolipoprotein A2 (APOA2) [R2 = 0.59, females]. Females with pre-bed rest MMP3 < 6.93 ng/ml had the highest loss of leg LM over bed rest. Whereas females with pre-bed rest MMP3 ≥ 6.93 and ApoA2 < 276 ng/ml, maintained leg lean mass at the end of bed rest. ConclusionsPanels of blood biomarkers associated with the muscle extracellular matrix may predict the likelihood for LM loss over extended bed rest.

P0681TIMP-1 DEFICIENCY DAMPENS RENAL GALECTIN-3 RESPONSE IN DIABETES

Abstract Background and Aims Overexpression of tissue inhibitors of metalloproteases (TIMPs) are a hallmark of renal fibrosis, and elevated TIMP-1 has been reported in experimental and human diabetes. Also, renal galectin-3 (Lgals3) overproduction might link macrophages to fibrosis progression. However, the possible association of renal Lgals3 with TIMP-1 in diabetes is still unclear. Thus, we investigated nephropathy and renal Lgals3 in type-1 diabetic wild type and TIMP-1 knockout (KO) mice. Method Type-1 diabetes was induced in 6 week-old male TIMP-1 KO (DM KO, n=7) and wild type (DM, n=6) mice with daily intraperitoneal streptozotocin (50 mg/kg/day) injections for 5 consecutive days. Non-diabetic controls (CTL, n=5) were injected with vehicle. Fasting blood glucose was monitored, and after 8 weeks kidneys were analyzed for histology and mRNA expression. Results Both diabetic groups developed similar hyperglycemia (CTL: 6±2, DM: 29±5, DM KO: 33±7 mmol/l, p&amp;lt;0.01). However, serum creatinine was elevated only in wild type diabetic mice (CTL: 9±2, DM: 47±28, DM KO: 11±2 ug/dl, p&amp;lt;0.001). Histology revealed significant tubular damage in DM mice (score: CTL: 0.4±0.1, DM: 2.3±0.2, DM KO: 1.6±0.2, p&amp;lt;0.05), accompanied by 10-fold lipocalin-2 (CTL: 1.0±0.3, DM: 10.9±6.2, DM KO: 3.3±1.7, p&amp;lt;0.05) and 2-fold collagen-1 overexpression (CTL: 1.0±0.2, DM: 1.9±0.7, DM KO: 1.2±0.4, p&amp;lt;0.05), practically absent in diabetic KO kidneys. Similarly, TIMP-1 deficiency was associated with twofold decrease in renal Lgal3 (CTL: 1.0±0.6, DM: 2.0±1.1, DM KO: 0.8±0.3, p&amp;lt;0.05) and tenfold decrease in CCL2 expression levels (CTL: 1.0±0.3, DM: 20.2±4.2, DM KO: 2.3±1.5, p&amp;lt;0.01) as compared to DM. Conclusion In our model of type-1 diabetes, TIMP-1 deficiency attenuated the development of renal fibrotic and inflammatory response by reducing galectin-3 and CCL2 (MCP-1), preserved tubular integrity and renal function. This might implicate TIMP-1 to be a possible therapeutic target in the future.

ERBB2 gene expression silencing involved in ovarian cancer cell migration and invasion through mediating MAPK1/MAPK3 signaling pathway.

To explore possible mechanism of ERBB2 gene expression silencing mediating mitogen-activated protein kinase 1/mitogen-activated protein kinase 3 (MAPK1/MAPK3) signaling pathway on proliferation, migration, and invasion of ovarian cancer cells. A total of 240 cancer specimens were collected in patients with epithelial ovarian cancer intraoperatively in our hospital from January 2015 to January 2018. Expressions of ERBB2, MAPK1, and MAPK3 in tissues were detected by immunohistochemistry. Following the culture of ovarian cancer cell lines, target cell line with high expression of ERBB2 was screened by qRT-PCR. Cell grouping was performed with four groups after transfection, including Blank group, negative control (NC) group, ERBB2 shRNA group, and ERBB2 overexpression group (shorted as ERBB2 group). The expression levels of ERBB2, MAPK1, MAPK3, vascular endothelial growth factor (VEGF), metalloproteases-2 (MMP-2), and tissue inhibitor of metalloproteases-2 (TIMP-2) were detected by qRT-PCR in different transfection groups, followed by the detection of protein expressions with Western blot. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to test the proliferation activity of each group after transfection, while transwell assay and scratch test explored cell invasion and migration in each group, respectively. Immunohistochemistry showed that the positive rates of ERBB2, MAPK1, and MAPK3 in ovarian cancer tissues were significantly increased than those in adjacent normal epithelial tissues. In the cell experiment, ERBB2 gene was highly expressed in SKOV3 ovarian cancer cell line. There was no significant difference in each index between Blank group and NC group (p > 0.05). Compared with Blank group and NC group, the expression levels of ERBB2, MAPK1, MAPK3, VEGF, and MMP-2 in ERBB2 shRNA group decreased significantly, TIMP-2 increased markedly, and proliferation, invasion, and migration abilities of cells decreased markedly after transfection, showing statistically significant differences (All p < 0.05). By contrast, the expression levels of ERBB2, MAPK1, MAPK3, VEGF, and MMP-2 increased remarkably in ERBB2 group, while TIMP-2 decreased significantly, and cell proliferation, invasion, and migration ability increased evidently after transfection, with statistically significant differences (All p < 0.05). Silencing ERBB2 gene expression may inhibit the activation of MAPK1/MAPK3 signaling pathway and thus suppress the proliferation, invasion, and migration of ovarian cancer cells. Overexpression of ERBB2 gene can reverse those trends, which in turn support the role of ERBB2 gene expression silencing in molecular targeted therapy of ovarian cancer.

Comparative analysis of the expression of metalloproteases (MMP-2, MMP-9, MMP-11 and MMP-13) and the tissue inhibitor of metalloprotease 3 (TIMP-3) between previous negative biopsies and radical prostatectomies

Metalloproteases (MMPs) and tissue inhibitor of metalloprotease-3 (TIMP-3) have been associated to the risk of having cancer and tumor aggressiveness. When facing the difficulties of prostate cancer diagnosis, the expression of MMPs and TIMP-3 in negative biopsies could be helpful to evaluate a diagnostic suspicion. Our objective is to carry out a comparative study of the expression of MMPs and TIMP-3 in previous negative biopsies and radical prostatectomies (RP). Material and methodsRetrospective analysis of a hospital-based cohort including 21 patients with suspicion of prostate carcinoma, whose expressions of MMP-2, 9, 11 and 13 and TIMP-3 were evaluated by immunohistochemistry in the tumor area from previous negative biopsies and RP. ResultsImmunohistochemical staining values (Score) for MMPs (-11 and -13) and TIMP-3 showed no significant differences when comparing the areas of negative biopsies where tumors subsequently developed with those of the RP. However, we did observe a significant difference in the increased expression of MMP-2 (P=0.002) and MMP-9 (P=0.001) in the tumor area of the RP with respect to the corresponding area of the previous negative biopsy. ConclusionsOur data indicate a higher overall expression of MMP-2 and MMP-9 in the tumor area of the RP compared to the corresponding areas of the negative previous biopsy, which seems to be associated to the process of malignant transformation.

Análisis comparativo de la expresión de metaloproteasas (MMP-2, MMP-9, MMP-11 y MMP-13) y del inhibidor tisular de la metaloproteasa 3 (TIMP-3) entre las biopsias previas negativas y las prostatectomías radicales

Metalloproteases (MMPs) and tissue inhibitor of metalloprotease-3 (TIMP-3) have been associated to the risk of having cancer and tumor aggressiveness. When facing the difficulties of prostate cancer diagnosis, the expression of MMPs and TIMP-3 in negative biopsies could be helpful to evaluate a diagnostic suspicion. Our objective is to carry out a comparative study of the expression of MMPs and TIMP-3 in previous negative biopsies and radical prostatectomies (RP). Material and methodsRetrospective analysis of a hospital-based cohort including 21 patients with suspicion of prostate carcinoma, whose expressions of MMP-2, 9, 11 and 13 and TIMP-3 were evaluated by immunohistochemistry in the tumor area from previous negative biopsies and RP. ResultsImmunohistochemical staining values (Score) for MMPs (-11 and -13) and TIMP-3 showed no significant differences when comparing the areas of negative biopsies where tumors subsequently developed with those of the RP. However, we did observe a significant difference in the increased expression of MMP-2 (P=.002) and MMP-9 (P=.001) in the tumor area of the RP with respect to the corresponding area of the previous negative biopsy. ConclusionsOur data indicate a higher overall expression of MMP-2 and MMP-9 in the tumor area of the RP compared to the corresponding areas of the negative previous biopsy, which seems to be associated to the process of malignant transformation.