Increased Gelatinase Activity Research Articles

Brain matrix metalloproteinase-9 activity is altered in the corticosterone mouse model of depression

Major depressive disorder is a highly prevalent psychiatric condition. Metalloproteinase 9 (MMP-9), a gelatinase involved in synaptic plasticity, learning and memory processes, is elevated in both chronic stress animal models and human peripheral blood samples of depressed patients. In this study we have evaluated the MMP-9 activity and protein expression in brain areas relevant to depression using the chronic corticosterone mouse model of depression. These mice show a depressive- and anxious-like behaviour. The MMP-9 activity and protein levels are significantly elevated in both the hippocampus and the cortex, and nectin-3 levels are lower in these brain areas in this model. In particular, these mice display an increased gelatinase activity in the CA1 and CA3 subfields of the hippocampus and in the internal layer of the prefrontal cortex. Moreover, the immobility time in the tail suspension test presents a positive correlation with the cortical MMP-9 activity, and a negative correlation with nectin-3 levels.In conclusion, the chronic corticosterone model of depression leads to an increase in the protein expression and activity of MMP-9 and a reduction of its substrate nectin-3 in relevant areas implicated in this disease. The MMP-9 activity correlates with behavioural despair in this model of depression. All these findings support the role of MMP-9 in the pathophysiology of depression, and as a putative target to develop novel antidepressant drugs.

Cigarette Smoke Condensate Exposure Induces Receptor for Advanced Glycation End-Products (RAGE)-Dependent Sterile Inflammation in Amniotic Epithelial Cells.

Maternal smoking is a risk factor of preterm prelabor rupture of the fetal membranes (pPROM), which is responsible for 30% of preterm births worldwide. Cigarettes induce oxidative stress and inflammation, mechanisms both implicated in fetal membranes (FM) weakening. We hypothesized that the receptor for advanced glycation end-products (RAGE) and its ligands can result in cigarette-dependent inflammation. FM explants and amniotic epithelial cells (AECs) were treated with cigarette smoke condensate (CSC), combined or not with RAGE antagonist peptide (RAP), an inhibitor of RAGE. Cell suffering was evaluated by measuring lactate dehydrogenase (LDH) medium-release. Extracellular HMGB1 (a RAGE ligand) release by amnion and choriodecidua explants were checked by western blot. NF-κB pathway induction was determined by a luciferase gene reporter assay, and inflammation was evaluated by cytokine RT-qPCR and protein quantification. Gelatinase activity was assessed using a specific assay. CSC induced cell suffering and HMGB1 secretion only in the amnion, which is directly associated with a RAGE-dependent response. CSC also affected AECs by inducing inflammation (cytokine release and NFκB activation) and gelatinase activity through RAGE engagement, which was linked to an increase in extracellular matrix degradation. This RAGE dependent CSC-induced inflammation associated with an increase of gelatinase activity could explain a pathological FM weakening directly linked to pPROM.

Морфометрический анализ тканевых структур слизистой оболочки десен крыс под действием инфракрасного лазерного излучения

The aim of the study — was a comparative study of the adaptive responses of tissue structures and gelatinases of the gingival mucosa (SOD) in healthy rats to the effect of different modes of low-intensity laser radiation (NILI) in the infrared range. Materials and methods of research: the work was performed on 55 laboratory mature female rats using histological, immunohistochemical, zymographic, morphometric and statistical methods. The results of the study: the obtained data confirm the development of biological effects in response to the action of NILI: absorption of photon energy by intracellular components стим stimulation of adaptation reactions: increased activity of enzymes and TC function, activation of microcirculation. Conclusions: 1. After 1 minute and on the 1st day after exposure to NILI with a wavelength of 970 nm and a power of 0.5 W, a more significant increase in the number of TC with VEGFR1 expression, degranulated forms of TC, including with the 1st, 2nd and 3rd degrees of degranulation, the number, diameter of blood vessels and the area occupied by them, and an increase in gelatinase activity was recorded up to 5 days compared to similar indicators at the specified periods after exposure to NILI with a wavelength of 890 nm and a capacity of 25 MW. 2. The presence of a direct strong correlation between the parameters of TC, blood vessels and the activity of gelatinases indicates the development of interrelated physiological processes of adaptation in SOD tissues in response to the action of various parameters of the NILI infrared range. 3. The immunohistochemical method allows for specific detection, precise localization, and objective morphometric analysis of TC with VEGFR1 expression in SOD of control and experimental rats.

The systemic influence of chronic smoking on skin structure and mechanical function.

One of the major functions of human skin is to provide protection from the environment. Although we cannot entirely avoid, for example, sun exposure, it is likely that exposure to other environmental factors could affect cutaneous function. A number of studies have identified smoking as one such factor that leads to both facial wrinkle formation and a decline in skin function. In addition to the direct physical effects of tobacco smoke on skin, its inhalation has additional profound systemic effects for the smoker. The adverse effects on the respiratory and cardiovascular systems from smoking are well known. Central to the pathological changes associated with smoking is the elastic fibre, a key component of the extracellular matrices of lungs. In this study we examined the systemic effect of chronic smoking (>40 cigarettes/day; >5 years) on the histology of the cutaneous elastic fibre system, the nanostructure and mechanics of one of its key components, the fibrillin-rich microfibril, and the micromechanical stiffness of the dermis and epidermis. We show that photoprotected skin of chronic smokers exhibits significant remodelling of the elastic fibre network (both elastin and fibrillin-rich microfibrils) as compared to the skin of age- and sex-matched non-smokers. This remodelling is not associated with increased gelatinase activity (as identified by in situ zymography). Histological remodelling is accompanied by significant ultrastructural changes to extracted fibrillin-rich microfibrils. Finally, using scanning acoustic microscopy, we demonstrated that chronic smoking significantly increases the stiffness of both the dermis and the epidermis. Taken together, these data suggest an unappreciated systemic effect of chronic inhalation of tobacco smoke on the cutaneous elastic fibre network. Such changes may in part underlie the skin wrinkling and loss of skin elasticity associated with smoking. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Insulin promotes invasion and migration of KRASG12D mutant HPNE cells by upregulating MMP-2 gelatinolytic activity via ERK- and PI3K-dependent signalling.

ObjectivesHyperinsulinemia is a risk factor for pancreatic cancer, but the function of insulin in carcinogenesis is unclear, so this study aimed to elucidate the carcinogenic effects of insulin and the synergistic effect with the KRAS mutation in the early stage of pancreatic cancer.Materials and methodsA pair of immortalized human pancreatic duct‐derived cells, hTERT‐HPNE E6/E7/st (HPNE) and its oncogenic KRASG12D variant, hTERT‐HPNE E6/E7/KRASG12D/st (HPNE‐mut‐KRAS), were used to investigate the effect of insulin. Cell proliferation, migration and invasion were assessed using Cell Counting Kit‐8 and transwell assays, respectively. The expression of E‐cadherin, N‐cadherin, vimentin and matrix metalloproteinases (MMP‐2, MMP‐7 and MMP‐9) was evaluated by Western blotting and/or qRT‐PCR. The gelatinase activity of MMP‐2 and MMP‐9 in conditioned media was detected using gelatin zymography. The phosphorylation status of AKT, GSK3β, p38, JNK and ERK1/2 MAPK was determined by Western blotting.ResultsThe migration and invasion ability of HPNE cells was increased after the introduction of the mutated KRAS gene, together with an increased expression of MMP‐2. These effects were further enhanced by the simultaneous administration of insulin. The use of MMP‐2 siRNA confirmed that MMP‐2 was involved in the regulation of cell invasion. Furthermore, there was a concentration‐ and time‐dependent increase in gelatinase activity after insulin treatment, which could be reversed by an insulin receptor tyrosine kinase inhibitor (HNMPA‐(AM)3). In addition, insulin markedly enhanced the phosphorylation of PI3K/AKT, p38, JNK and ERK1/2 MAPK pathways, with wortmannin or LY294002 (a PI3K‐specific inhibitor) and PD98059 (a MEK1‐specific inhibitor) significantly inhibiting the insulin‐induced increase in MMP‐2 gelatinolytic activity.ConclusionsTaken together, these results suggest that insulin induced migration and invasion in HPNE and HPNE‐mut‐KRAS through PI3K/AKT and ERK1/2 activation, with MMP‐2 gelatinolytic activity playing a vital role in this process. These findings may provide a new therapeutic target for preventing carcinogenesis and the evolution of pancreatic cancer with a background of hyperinsulinemia.

Animal Models of Steatosis (NAFLD) and Steatohepatitis (NASH) Exhibit Hepatic Lobe-Specific Gelatinases Activity and Oxidative Stress.

Animal models of obstructive cholestasis and ischemia/reperfusion damage have revealed the functional heterogeneity of liver lobes. This study evaluates this heterogeneity in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) rat models. Twelve-week-old Obese and Lean male Zucker rats were used for NAFLD. Eight-week-old male Wistar rats fed with 8-week methionine-choline-deficient (MCD) diet and relative control diet were used for NASH. Gelatinase (MMP-2; MMP-9) activity and protein levels, tissue inhibitors of metalloproteinase (TIMPs), reactive oxygen species (ROS), and thiobarbituric acid-reactive substances (TBARS) were evaluated in the left (LL), median (ML), and right liver (RL) lobes. Serum hepatic enzymes and TNF-alpha were assessed. An increase in gelatinase activity in the NASH model occurred in RL compared with ML. TIMP-1 and TIMP-2 displayed the same trend in RL as ML and LL. Control diet RL showed higher MMP-9 activity compared with ML and LL. No significant lobar differences in MMP-2 activity were detected in the NAFLD model. MMP-9 activity was not detectable in Zucker rats. TIMP-1 was lower in LL when compared with ML while no lobar differences were detectable for TIMP-2 in either Obese or Lean Zucker rats. Control diet rats exhibited higher ROS formation in LL versus RL. Significant increases in TBARS levels were observed in LL versus ML and RL in control and MCD rats. The same trend for ROS and TBARS was found in Obese and Lean Zucker rats. An increased serum TNF-alpha occurred in MCD rats. A lobar difference was detected for MMPs, TIMPs, ROS, and TBARS in both MCD and Zucker rats. Higher MMP activation in RL and higher oxidative stress in the LL, compared with the other lobes studied, supports growing evidence for functional heterogeneity among the liver lobes occurring certainly in both NAFLD and NASH rats.

Antimetastatic effect of epigenetic drugs, hydralazine and valproic acid, in Ras-transformed NIH 3T3 cells.

IntroductionMetastasis involves the accumulation of genetic and epigenetic alterations leading to activation of prometastatic genes and inactivation of antimetastatic genes. Among epigenetic alterations, DNA hypermethylation and histone hypoacetylation are the focus of intense translational research because their pharmacological inhibition has been shown to produce antineoplastic activity in a variety of experimental models.AimsThis study aimed to evaluate the antimetastatic effect of the DNA-methylation inhibitor, hydralazine, and the histone deacetylase inhibitor, valproic acid.MethodsNIH 3T3-Ras murine cells were treated with hydralazine and valproic acid to evaluate their effects upon cell proliferation, cell motility, chemotaxis, gelatinase activity, and gene expression. Lung metastases were developed by intravenous injection of NIH 3T3-Ras cells in BALB/c nu/nu mice and then treated with the drug combination.ResultsTreatment induced a growth-inhibitory effect on NIH 3T3-Ras cells, showed a trend toward increased gelatinase activity of MMP2 and MMP9, and inhibited chemotaxis and cell motility. The combination led to a strong antimetastatic effect in lungs of nude mice.ConclusionHydralazine and valproic acid, two repositioned drugs as epigenetic agents, exhibit antimetastatic effects in vitro and in vivo and hold potential for cancer treatment.

Morphological Alterations and Increased Gelatinase Activity in the Superficial Digital Flexor Tendon of Chickens During Growth and Maturation.

The superficial digital flexor tendon (SDFT) connects the superficial digital flexor muscle to the digits and its main function is to participate in digit flexion. The SDFT presents different regions along its length, which adapt to different biomechanical forces. During growth and maturation, the tendon may present changes in the regions subjected to compression and tension, with variations in the composition of the extracellular matrix (ECM), in the arrangement of collagen fibers and cellularity. With the purpose of analyzing the morphological and biochemical alterations of ECM of tendons during the growth and maturation, Gallus domesticus were euthanized at 1, 5, and 8 months of age and their SDFT were divided into regions of tension/compression (Sp) and tension (Sd). From 1 month of age, the Sp region already presented fibrocartilage characteristics with cells similar to chondrocytes. At 5 and 8 months, the Sd region displayed formation of a new structure similar to bone matrix, and intense metachromasia. The animals of 5 and 8 months presented an increase in MMP-2 and -9 activities and a lower number of cells when compared with the animals of 1 month, in both regions. In conclusion, structural and biochemical alterations occur during the maturation process of the SDFT, involving a decrease in the number of cells and changes in the degradation and composition of the ECM. Anat Rec, 302:964-972, 2019. © 2018 Wiley Periodicals, Inc.

Radiotherapy Activates and Protease Inhibitors Inactivate Matrix Metalloproteinases in the Dentinoenamel Junction of Permanent Teeth

The objectives of this study were to investigate changes in the activity and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in permanent teeth with or without exposure to radiotherapy, and the role of proteinase inhibitors in their inactivation. In situ zymography and immunofluorescence assays were performed to evaluate the activity and expression of two key gelatinases (MMP-2 and MMP-9) in sections of permanent molars, assigned to irradiated and nonirradiated subgroups. Dental fragments were exposed to radiation at a dose of 2 Gy fractions for 5 consecutive days until a cumulative dose of 60 Gy was reached. To evaluate the effect of protease inhibitors on MMPs, teeth were immersed in 0.5 mL of 0.12% chlorhexidine digluconate (CHX), 0.05% sodium fluoride (NaF), 400 μM polyphenol epigallocatechin-3-gallate (EGCG), or distilled water (control) for 1 h. Fluorescence in the dentinoenamel junction (DEJ) was evaluated in 3 areas of the tooth: cervical, cuspal, and pit. These regions were photographed using a fluorescence microscope at 1.25× and 5× magnifications. Results were analyzed using the D’Agostino-Person normality test, and the Kruskal-Wallis, Dunn, and Wilcoxon tests for intergroup and paired comparisons (α = 0.05). The fluorescence intensity/mm² in the DEJ at the three regions studied was higher in the irradiated teeth (p < 0.05) than in the nonirradiated teeth, revealing regions of expression of MMP-2 and MMP-9 by immunofluorescence. Postradiotherapy treatment with different solutions (CHX, NaF, and EGCG) led to lower fluorescence intensity/mm² in irradiated teeth than in the control group (distilled water; p < 0.05), as a result of MMP inactivation. In conclusion, irradiation increased gelatinase activity in all regions of the DEJ. Treatment with 0.12% CHX, 0.05% NaF, and 400 μM polyphenol EGCG postradiotherapy inactivated enzyme activity.

Transforming growth factor-β1 induces differentiation of rainbow trout (Oncorhynchus mykiss) cardiac fibroblasts into myofibroblasts.

The collagen content of the rainbow trout heart increases in response to cold acclimation and decreases with acclimation to warm temperatures. This ability to remodel the myocardial extracellular matrix (ECM) makes these fish useful models to study the cellular pathways involved in collagen regulation in the vertebrate heart. Remodelling of the ECM in the mammalian heart is regulated, in part, by myofibroblasts which arise from pre-existing fibroblasts in response to transforming growth factor-β1 (TGF-β1). We have previously demonstrated that treatment of cultured rainbow trout cardiac fibroblasts with human TGF-β1 causes an increase in collagen production. Here, we showed that repetitive treatment of rainbow trout cardiac fibroblasts with a physiologically relevant concentration of human recombinant TGF-β1 results in a ∼29-fold increase in phosphorylated small mothers against decapentaplegic 2 (pSmad2); a 2.9-fold increase in vinculin protein, a 1.2-fold increase in cellular size and a 3-fold increase in filamentous actin (F-actin). These are common markers of the transition of fibroblasts to myofibroblasts. Cells treated with TGF-β1 also had highly organized cytoskeletal α-smooth muscle actin, as well as increased transcript abundances of mmp-9, timp-2 and col1a1 Furthermore, using gelatin zymography, we demonstrated that TGF-β1 treatment causes a 5.3-fold increase in gelatinase activity. Together, these results suggest that trout cardiac fibroblasts have the capacity to differentiate into myofibroblasts and that this cell type can increase extracellular collagen turnover via gelatinase activity. Cardiac myofibroblasts are, therefore, likely involved in the remodelling of the cardiac ECM in the trout heart during thermal acclimation.

Inhibition of collagen synthesis by IWR-1 in normal and keloid-derived skin fibroblasts

AimsKeloid is a benign tumor that is characterized by the hyperproliferation of dermal fibroblasts and excessive deposition of extracellular matrix (ECM) especially the collagen. Aberrant activation of Wnt/β-catenin signaling is implicated in the pathogenesis of keloid. In this study, we investigated the effects of IWR-1, a small molecule inhibitor for Wnt/β-catenin signaling via the inhibition of tankyrase, on production of collagen and matrix metalloproteinase (MMP) in dermal fibroblasts. Main methodsWe cultured human normal skin- and keloid-derived fibroblasts, then treated with IWR-1. The effects of IWR-1 on collagen and MMP production were determined by Western blot, ELISA and zymography. Key findingsIWR-1 significantly suppressed the proliferation and migration of both the normal and keloid fibroblasts. IWR-1 also inhibited the production and secretion of type I collagen from the fibroblasts. In addition, IWR-1 significantly increased the expression of MMPs, such as MMP-1, MMP-3 and MMP-13, along with the increase of gelatinase activity. These results suggest that inhibitory effect of IWR-1 on collagen production may be related with the increased MMP activity. SignificanceThis study provides the possible action mechanism of IWR-1 on regulation of collagen expression, on which to base further investigation for preventing skin fibrotic diseases such as keloid.

Abstract 654: Loss of SPRR3 In ApoE -/- Mice Leads to Increased Atheroma Vulnerability and Evidence of Plaque Rupture and Cardiac Infarcts

Atheroma rupture is the leading cause of myocardial infarction. While studies have examined inflammatory cell-mediated effects on plaque vulnerability, less is known about the role of vascular smooth muscle cells (VSMCs) or specific molecular players in the maintenance of atheroma stability. We reported that loss of small proline-rich repeat protein 3 (SPRR3), enriched in atheroma VSMCs, leads to increased VSMC death and significantly accelerates atherosclerosis progression in ApoE -/- mice. Here, we show that loss of SPRR3 promotes features in plaques of brachiocephalic arteries common to unstable lesions, such as increased necrotic core size, reduced cap collagen content, and reduced VSMC content. Moreover, ApoE -/- mice lacking SPRR3 develop coronary artery lesions with advanced features, including intraplaque hemorrhage. In addition, Sprr3 -/- ApoE -/- mice fed a high-fat diet for 6 months develop spontaneous myocardial infarction. In vitro , SPRR3 deficient VSMCs show reduced expression of procollagen type I, an event associated with Akt activation. SPRR3-deficient VSMCs also show increased expression of MMP2 transcripts, and aortic root lesions of Sprr3 -/- ApoE -/- mice have increased gelatinase activity consistent with MMP2 activation. Our data demonstrate that SPRR3 loss in ApoE -/- mice decreases VSMC survival and collagen I synthesis while increasing MMP2 synthesis and activity, resulting in atheroma instability with evidence of downstream myocardial infarction. Taken together the results present the Sprr3 -/- ApoE -/- mouse as an experimental model of plaque rupture. This model will be used for additional experimental studies including in vivo genetic modulation of the Akt pathway as well as in vitro studies to determine phenotypic outcome, i.e. coronary arterial lesions, myocardial infarction, VSMC survival and collagen synthesis. We hope to establish a mechanistic link between altered Akt signaling and matrix integrity in the context of atheroma rupture, as well as potentially use SPRR3 as a molecular marker which could lead to detection of plaque instability as well as therapeutic intervention methodologies.

Aryl Hydrocarbon Receptor Activation by TCDD Modulates Expression of Extracellular Matrix Remodeling Genes during Experimental Liver Fibrosis.

The aryl hydrocarbon receptor (AhR) is a soluble, ligand-activated transcription factor that mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence implicates the AhR in regulating extracellular matrix (ECM) homeostasis. We recently reported that TCDD increased necroinflammation and myofibroblast activation during liver injury elicited by carbon tetrachloride (CCl4). However, TCDD did not increase collagen deposition or exacerbate fibrosis in CCl4-treated mice, which raises the possibility that TCDD may enhance ECM turnover. The goal of this study was to determine how TCDD impacts ECM remodeling gene expression in the liver. Male C57BL/6 mice were treated for 8 weeks with 0.5 mL/kg CCl4, and TCDD (20 μg/kg) was administered during the last two weeks. Results indicate that TCDD increased mRNA levels of procollagen types I, III, IV, and VI and the collagen processing molecules HSP47 and lysyl oxidase. TCDD also increased gelatinase activity and mRNA levels of matrix metalloproteinase- (MMP-) 3, MMP-8, MMP-9, and MMP-13. Furthermore, TCDD modulated expression of genes in the plasminogen activator/plasmin system, which regulates MMP activation, and it also increased TIMP1 gene expression. These findings support the notion that AhR activation by TCDD dysregulates ECM remodeling gene expression and may facilitate ECM metabolism despite increased liver injury.

Improving the stability of chitosan–gelatin-based hydrogels for cell delivery using transglutaminase and controlled release of doxycycline

Although local cell delivery is an option to repair tissues, particularly using chitosan-based hydrogels, significant attrition of injected cells prior to engraftment has been a problem. To address this problem, we explored the possibility of stabilizing the chitosan-gelatin (CG) injectable hydrogels using (1) controlled release of doxycycline (DOX) to prevent premature degradation due to increased gelatinase activity (MMP-2 and MMP-9), and (2) transglutaminase (TG) to in situ cross-link gelatin to improve the mechanical stability. We prepared DOX-loaded PLGA nanoparticles, loaded into the CG hydrogels, measured DOX release for 5 days, and modeled using a single-compartmental assumption. Next, we assessed the influence of TG and DOX on hydrogel compression properties by incubating hydrogels for 7 days in PBS. We evaluated the effect of these changes on retention of fibroblasts and alterations in MMP-2/MMP-9 activity by seeding 500,000 fibroblasts for 5 days. These results showed that 90 % of DOX released from cross-linked CG hydrogels after 4 days, unlike CG hydrogels where 90 % of DOX was released within the first day. Addition of TG enhanced the CG hydrogel stability significantly. More than 60 % of seeded fibroblasts were recovered from the CG-TG hydrogels at day 5, unlike 40 % recovered from CG-hydrogels. Inhibition of MMP-2/MMP-9 were observed. In summary, controlled release of DOX from CG hydrogels cross-linked with TG shows a significant potential as a carrier for cell delivery.

Abstract 4154: Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells

Abstract We have recently established a zoledronic acid-resistant prostate cancer cell line, DU145R80,which exhibits higher invasive capability compared to parental DU145 cells, and epithelial-to-mesenchymal transition (EMT)[Milone MR, Cell Death Dis. 2013]. To investigate the mechanism by which the cells become invasive, we compared DU145R80 and DU145 cells by a proteomic approach, unveiling a signaling network that links the interior of the nucleus to changes in cytoskeleton dynamics and to the extracellular matrix, dictating prostate cancer aggressiveness [Milone MR Oncotarget 2014].Cytoskeletal modifications allow cells to gain a more migratory/invasive behavior and to interact with the surrounding microenvironment triggering different kind of biological phenomena, including the formation of large oncosomes (LO), a bioactive class of extracellular vesicles (EVs) [Di Vizio D, Cancer Res. 2009]. In this study, we found a higher amount of spontaneously shed LO along with an increased gelatinase activity from DU145R80 cells compared to the parental counterpart. By applying low speed and discontinuous gradient ultracentrifugation to both DU145 and DU145R80 cell media, we obtained a pure preparation of LO, floating at a 1.15 g/mL density fraction and positive for LO markers such as as Cav-1, Ck18, GAPDH. Moreover, we treated DU145 parental cells with pure preparations of LO from either DU145 or DU145R80 cells and performed an invasion assay, showing that LO from the resistant, aggressive DU145R80 cell line increase the invading ability of DU145. Treatment of DU145 cells with LO originating from the parental cell line itself did not result in increased invasinevess, suggesting a specific role for EVs from aggressive cells. In addition, in order to investigate LO content, we focused our attention on a cell surface glycoprotein, CUB domain-containing protein 1 (CDCP-1), putatively linked to the network of proteins identified in DU145R80 cells and whose role in cancer is still under debate. Our results demonstrate that CDCP-1 expression is significantly reduced in DU145R80 compared to DU145 cells, suggesting a tumor-protective role for the protein in prostate cancer. We also observed, for the first time, that the CDCP-1 expression pattern displayed by both cell lines was reflected in their derived LO. These data intriguingly suggest that CDCP-1 may change its expression pattern upon modifications in tumor cells properties, playing a different role in different stages of cancer development. Overall, these findings highlight LO as a new biological component in the development of aggressive features by prostate cancer cells, connecting molecular alterations to changes in the ability to interact with the surrounding environment, and suggested new prognostic markers and/or therapeutical targets. Citation Format: Chiara Ciardiello, Valentina R. Minciacchi, Mariana Reis-Sobreiro, Maria R. Milone, Biagio Pucci, Rita Lombardi, Francesca Bruzzese, Dolores Di Vizio, Alfredo Budillon. Large oncosomes derived from the aggressive prostate cancer sub-line, DU145R80, can modify the biological behavior of the parental DU145 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2015-4154

Matrix metalloproteinase-9 expression is enhanced in renal parietal epithelial cells of zucker diabetic Fatty rats and is induced by albumin in in vitro primary parietal cell culture.

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether and how MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. In situ zymography, immunostaining and Western blot for renal MMP gelatinolytic activity and MMP-9 protein expression were performed in Zucker lean and Zucker diabetic rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of diabetic rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells (PECs) expressing claudin-1 in the diabetic kidneys. Interestingly, increased parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the sclerotic area of affected glomeruli in diabetic rats. Additionally, urinary excretion of podocyte marker proteins was significantly increased in association with the levels of MMP-9 and albumin in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-9, primary cultured rat glomerular PECs were incubated with rat serum albumin (0.25 - 1 mg/ml) for 24 - 48 hrs. MMP-9 mRNA levels were significantly increased following albumin treatment. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein and activity in culture supernatants of PECs. Moreover, albumin activated p44/42 mitogen-activated protein kinase (MAPK) in PECs. Inhibition of p44/42 MAPK suppressed albumin-induced MMP-9 secretion from glomerular PECs. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a loss of adjacent podocytes in progressive diabetic nephropathy. Albumin overload may induce MMP-9 expression and secretion by PECs via the activation of p44/42 MAPK pathway.

Abstract 549: An Up-regulation Of Matrix Metalloproteinase-9 In Parietal Epithelial Cells Is Associated With A Progressive Podocyte Loss In Diabetic Glomerulopathy

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in the remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. Immunostaining, in situ zymography and Western blot for renal protein expression and MMP activity were evaluated in Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of Fa/Fa rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells expressing claudin-1 in the diabetic kidneys. Interestingly, an increase in parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the adjacent area. Additionally, urinary excretion of nephrin and podocalyxin was progressively increased in association with the levels of MMP-9 and total protein in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-2 and MMP-9, primary cultured rat glomerular parietal epithelial cells were incubated with exogenous albumin. MMP-9 mRNA levels were significantly increased by 306%, 565%, and 829% (n=4-6), respectively, when the cells were exposed to rat serum albumin (0.25, 0.5 and 1 mg/ml) for 24 hour. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein (7.4-, 14.1-, and 21.1-fold, respectively) and activity (3.2-, 4.5-, and 6.7-fold, respectively) in the culture supernatants of parietal epithelial cells. In contrast, albumin treatment did not significantly alter the expression level and enzymatic activity of MMP-2 in cultured parietal epithelial cells. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a progressive loss of adjacent podocytes during the development of diabetic glomerulopathy. Albumin overload also induces MMP-9 production in primary cultured glomerular epithelial cells.

Curcumin increases gelatinase activity in human neutrophils by a p38 mitogen-activated protein kinase (MAPK)-independent mechanism

Curcumin has been found to possess anti-inflammatory activities and neutrophils, key players in inflammation, were previously found to be important targets to curcumin in a few studies. For example, curcumin was found to induce apoptosis in neutrophils by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. However, the role of curcumin on the biology of neutrophils is still poorly defined. To study the role of curcumin on neutrophil degranulation and to determine the role of p38 MAPK, human neutrophils were freshly isolated from healthy individuals and incubated in vitro with curcumin. Degranulation was studied at three levels: surface expression of granule markers by flow cytometry; release of matrix metallopeptidase-9 (MMP-9 or gelatinase B) enzyme into supernatants by Western blot; and gelatinase B activity by zymography. Activation of p38 MAPK was studied by monitoring its tyrosine phosphorylation levels by western blot and its role by the utilization of a pharmacological inhibitor. The results indicate that curcumin increased the cell surface expression of CD35 (secretory vesicle), CD63 (azurophilic granules), and CD66b (gelatinase granules) in neutrophils. Also, curcumin increased the release and enzymatic activity of gelatinase B in the extracellular milieu and activated p38 MAP kinase in these cells. However, in contrast to fMLP, curcumin-induced enzymatic activity and secretion of gelatinase B were not reversed by use of a p38 inhibitor. Finally, it was found that curcumin was able to enhance phagocytosis. Taken together, the results here demonstrate that curcumin induced degranulation in human neutrophils and that the increased gelatinase activity is not dependent on p38 MAPK activation. Therefore, degranulation is another human neutrophil function that could be modulated by curcumin, as well as phagocytosis.

Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury

Remote ischemic preconditioning (RIPC) is a phenomenon, whereby short episodes of non-lethal ischemia to an organ or tissue exert protection against ischemia/reperfusion injury in a distant organ. However, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms within the target organ and the released factors. Here we established a human cell culture model to investigate cellular and molecular effects of RIPC and to identify factors responsible for RIPC-mediated intestinal protection.Human umbilical vein cells (HUVEC) were exposed to repeated episodes of hypoxia (3×15min) and conditioned culture media (CM) were collected after 24h. Human intestinal cells (CaCo-2) were cultured with or without CM and subjected to 90min of hypoxia/reoxygenation injury. Reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, hydrogen peroxide measurements and lactate dehydrogenase (LDH) assays were performed.In HUVEC cultures hypoxic conditioning did not influence the profile of secreted proteins but led to an increased gelatinase activity (P<0.05) in CM. In CaCo-2 cultures 90min of hypoxia/reoxygenation resulted in morphological signs of cell damage, increased LDH levels (P<0.001) and elevated levels of hydrogen peroxide (P<0.01). Incubation of CaCo-2 cells with CM reduced the hypoxia-induced signs of cell damage and LDH release (P<0.01) and abrogated the hypoxia-induced increase of hydrogen peroxide. These events were associated with an enhanced phosphorylation status of the prosurvival kinase Erk1/2 (P<0.05) but not Akt and STAT-5.Taken together, CM of hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury. The established culture model may help to unravel RIPC-mediated cellular events and to identify molecules released by RIPC.

Protective effect of melatonin against transient global cerebral ischemia-induced neuronal cell damage via inhibition of matrix metalloproteinase-9

AimsMelatonin possesses various pharmacological effects including neuroprotective effects against brain ischemia. Post-ischemic increases in matrix metalloproteinase-9 (MMP-9) expression and activity mainly contribute to neuronal damage by degradation of the extracellular matrix. This study was designed to examine whether melatonin has a neuroprotective effect and an influence on MMP-9 in transient global brain ischemia. Main methodsMice were subjected to 20min of global brain ischemia and sacrificed 72h later. Melatonin (30mg/kg) was administered 30min before and 2h after ischemia as well as once daily until sacrifice. Key findingsHippocampal pyramidal cell damage after ischemia was significantly decreased by melatonin. As observed by zymography, melatonin inhibited the increase of MMP-9 activity after ischemia. In the brain sections, the increased gelatinase activity was mainly observed in the hippocampus after ischemia and melatonin also reduced gelatinase activity. The laminin and NeuN expression levels were reduced in the hippocampal CA1 and CA2 regions after ischemia, and melatonin reduced laminin degradation and neuronal loss. A TUNEL assay demonstrated that there were TUNEL-positive cells in the hippocampus and the number of TUNEL-positive cells was significantly decreased by melatonin. There was no difference in the ischemia-induced hippocampal neuronal damage between the vehicle- and melatonin-treated groups of MMP-9 knock-out mice. SignificanceThese data demonstrate that melatonin suppressed the occurrence of neuronal injury, which might be partly due to its inhibitory effects on MMP-9 in addition to its anti-oxidative effects. MMP-9 may be an important key target of melatonin in neuroprotection against global ischemia.