Extracellular Matrix-degrading Enzymes Research Articles

Potential prevention effects of Rubus occidentalis seed on UVB-induced MMP-1 production and procollagen degradation in CCD-986sk cells

UV exposure induces matrix metalloproteinases (MMPs) and extracellular matrix-degrading enzymes expression. We studied the protective effect of Rubus occidentalis seed against UVB-generated skin photoaging using human fibroblast cells (CCD-986sk). We used an ELISA kit to measure the supernatents of ...

Coupled Hybrid Continuum-Discrete Model of Tumor Angiogenesis and Growth.

The processes governing tumor growth and angiogenesis are codependent. To study the relationship between them, we proposed a coupled hybrid continuum-discrete model. In this model, tumor cells, their microenvironment (extracellular matrixes, matrix-degrading enzymes, and tumor angiogenic factors), and their network of blood vessels, described by a series of discrete points, were considered. The results of numerical simulation reveal the process of tumor growth and the change in microenvironment from avascular to vascular stage, indicating that the network of blood vessels develops gradually as the tumor grows. Our findings also reveal that a tumor is divided into three regions: necrotic, semi-necrotic, and well-vascularized. The results agree well with the previous relevant studies and physiological facts, and this model represents a platform for further investigations of tumor therapy.

Enhancement of Epidermal Basement Membrane Formation by Synthetic Inhibitors of Extracellular Matrix-degrading Enzymes

Enhancement of Epidermal Basement Membrane Formation by Synthetic Inhibitors of Extracellular Matrix-degrading Enzymes

Residual biomasses and protein hydrolysates of three green microalgae species exhibit antioxidant and anti-aging activity

It is widely recognized that microalgae represent a promising strategy in the development of next-generation biofuels; however, their economic viability remains questionable. Several studies have shown microalgal biomass is a relatively unexplored source of natural components with nutraceutical activities; indeed, some authors state that the remaining residue after oil extraction from the microalgae biomass, known as microalgae residual biomass (MRB), could have outstanding biological activities. Therefore, in the present study, we analyzed the antioxidant capacity and anti-aging potential of MRB and protein hydrolysates of three green microalgae species: Dunaliella tertiolecta, Tetraselmis suecica, and Nannochloropsis sp. Antioxidant capacity, determined through oxygen radical absorbance capacity (ORAC), radical cation activity (ABTS), and radical scavenging activity (DPPH), of the three MRBs was good overall, with D. tertiolecta and Nannochloropsis having the highest antioxidant capacity. Interestingly, their protein hydrolysates showed markedly higher antioxidant capacities than their MRBs at the concentrations tested. Anti-aging potential, determined by the inhibition of the activity of two extracellular matrix-degrading enzymes, elastase and hyaluronidase, was found to be higher in D. tertiolecta and Nannochloropsis biomasses for anti-elastase activity; however, no significant differences between microalgae species were found in anti-hyaluronidase activity. For the protein hydrolysates, Nannochloropsis had the highest anti-elastase and anti-hyaluronidase activity. In conclusion, selected MRBs and their protein hydrolysates exhibited a good antioxidant capacity and/or anti-aging potential overall, indicating the economical importance of MRBs in the nutraceutical and nutricosmetic industries. Overall, this study contributes to convert microalgae biofuel production into a more sustainable and economically feasible process, through the comprehensive utilization of residual biomass.

Recent advances in renal cell carcinoma from a pathological point of view.

The purpose of this article is to review the recent advances in renal cell carcinoma (RCC) from a pathological point of view. Because the genetic features and morphological characteristics have become major criteria for the classification of RCC, special techniques, such as immunohistochemistry, are essential to the differential diagnosis of renal tumors. Metastasis is frequently observed among the RCC patients with curative nephrectomy, and extracellular matrix-degrading enzymes, such as matrix metalloproteinases (MMP) and heparanase, play a key role in invasion and metastasis of RCC. Snail and Slug, transcription factors of epithelial-mesenchymal transition (EMT), accelerate cancer cell invasion through downregulation of E-cadherin and up-regulation of MMP. Therapies targeted at the vascular endothelial growth factor pathway have become the standard treatment of metastatic RCC. Although they lead to tumor shrinkage mainly by inhibiting angiogenesis, they have typically been associated with drug resistance. The mechanism of the resistance remains largely unknown, but complex events including re-activation of angiogenesis, EMT and cancer stem cells, and immune escape are implicated in the refractory response to the therapy. Recent advances of the research on RCC have caused the changes of classification and therapy, and pathologists should take overall view of these as integrated pathology.

Matrix Metalloproteinase Expression in the Rat Myometrium During Pregnancy, Term Labor, and Postpartum.

Pregnancy, spontaneous term labor (TL), and postpartum (PP) involution are associated with changes in the cellular and extracellular matrix composition of the uterus. Both the uterine smooth muscle (myometrium) and the infiltrating peripheral blood leukocytes involved in the activation of labor secrete extracellular matrix-degrading enzymes (matrix metalloproteinases, MMPs) that can modulate cellular behavior and barrier function. MMP expression is induced by mechanical stretch in several tissues. We hypothesized that the expression and activity of myometrial MMPs and their tissue inhibitors (TIMPs) are modulated in preparation for TL and PP involution and are regulated by mechanical stretch of uterine walls imposed by the growing fetus. Myometrial tissues were collected from bilaterally and unilaterally pregnant rats across gestation, TL, and PP. Total RNA and proteins were subjected to real-time PCR and immunoblotting, respectively, and tissue localization and activity was examined by immunohistochemistry and in situ zymography. We found that Mmp7, Mmp11, and Mmp12 mRNA levels were upregulated during TL and PP, while Mmp2, Mmp3, Mmp8, Mmp9, Mmp10, and Mmp13 mRNAs were only upregulated during PP. Timp1–Timp4 were stably expressed throughout gestation with some fluctuations PP. Active MMP2 was induced in the empty uterine horn during gestation and in the gravid PP uterus, suggesting negative regulation by biological mechanical stretch. We conclude that specific subsets of uterine MMPs are differentially regulated in the rat myometrium in preparation for two major events: TL and PP uterine involution.

Mathematical Modelling of a Brain Tumour Initiation and Early Development: A Coupled Model of Glioblastoma Growth, Pre-Existing Vessel Co-Option, Angiogenesis and Blood Perfusion.

We propose a coupled mathematical modelling system to investigate glioblastoma growth in response to dynamic changes in chemical and haemodynamic microenvironments caused by pre-existing vessel co-option, remodelling, collapse and angiogenesis. A typical tree-like architecture network with different orders for vessel diameter is designed to model pre-existing vasculature in host tissue. The chemical substances including oxygen, vascular endothelial growth factor, extra-cellular matrix and matrix degradation enzymes are calculated based on the haemodynamic environment which is obtained by coupled modelling of intravascular blood flow with interstitial fluid flow. The haemodynamic changes, including vessel diameter and permeability, are introduced to reflect a series of pathological characteristics of abnormal tumour vessels including vessel dilation, leakage, angiogenesis, regression and collapse. Migrating cells are included as a new phenotype to describe the migration behaviour of malignant tumour cells. The simulation focuses on the avascular phase of tumour development and stops at an early phase of angiogenesis. The model is able to demonstrate the main features of glioblastoma growth in this phase such as the formation of pseudopalisades, cell migration along the host vessels, the pre-existing vasculature co-option, angiogenesis and remodelling. The model also enables us to examine the influence of initial conditions and local environment on the early phase of glioblastoma growth.

Hedgehog signalling does not stimulate cartilage catabolism and is inhibited by Interleukin-1β.

BackgroundIn osteoarthritis, chondrocytes adopt an abnormal hypertrophic morphology and upregulate the expression of the extracellular matrix-degrading enzymes, MMP-13 and ADAMTS-5. The activation of the hedgehog signalling pathway has been established in osteoarthritis and is thought to influence both of these processes. However, the role of this pathway in the initiation and progression of osteoarthritis is unclear as previous studies have been unable to isolate the effects of hedgehog pathway activation from other pathological processes. In this study we test the hypothesis that hedgehog pathway activation causes cartilage degradation in healthy cartilage and in an in vitro model of inflammatory arthritis.MethodsIsolated articular chondrocytes from the bovine metacarpal-phalangeal joint were stimulated for up to 24 hours with the agonist, recombinant Indian hedgehog (r-Ihh). ADAMTS-5 and MMP-13 gene expression was quantified by real-time PCR. In addition, healthy bovine cartilage explants were treated with r-Ihh or the hedgehog antagonist, cyclopamine, and sGAG release into the media was measured over 72 hours. Studies were repeated using chondrocytes and cartilage explants from human knee joint. Finally, studies were conducted to determine the effect of hedgehog pathway activation on matrix catabolism in the presence of the pro-inflammatory cytokine, IL-1β.ResultsAddition of r-Ihh activated hedgehog signalling, confirmed by upregulation of Gli1 and Ptch1 expression, but did not increase ADAMTS-5 or MMP-13 expression in bovine or human chondrocytes. Furthermore, r-Ihh did not induce sGAG release in healthy bovine or human cartilage explants. IL-1β treatment induced sGAG release, but this response was not altered by the stimulation or inhibition of hedgehog signalling. Hedgehog pathway activation was downregulated by IL-1β. Conversely, r-Ihh weakly suppressed IL-1β-induced ADAMTS-5 expression.ConclusionOur results show for the first time that Indian hedgehog does not cause extracellular matrix degradation in healthy ex vivo cartilage or in the presence of IL-1β and that IL-1β downregulates Indian hedgehog induced signalling. Thus, we suggest reported hedgehog induced matrix catabolism in osteoarthritis must be due to its interaction with pathological factors other than IL-1β. Hence, hedgehog signalling and its downstream effects are highly context-dependent.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0891-z) contains supplementary material, which is available to authorized users.

Abstract 16994: RNA Editing Controls Gene Expression in Atherosclerotic Heart Disease by Enabling HuR-mediated Posttranscriptional Regulation

Background: Adenosine to inosine (A-to-I) RNA editing is catalysed by ADARs (adenosine deaminases acting on RNA) and is an important posttranscriptional regulator of RNA metabolism. Although RNA editing is essential for life, its role in cardiovascular disease is unknown. Methods and Results: Next generation RNA-sequencing (RNA-seq) of human endothelial cells revealed that ADAR1 edits almost 25% of endothelial transcriptome, regulates gene expression (135 transcripts were at least 2-fold regulated after ADAR1 knockdown) and controls endothelial cell function. Among the highest edited transcripts is cathepsin S (CTSS), an extracellular matrix degradation enzyme with an established role in cardiovascular and inflammatory diseases. The number of edited nucleotides in the 3’UTR of CTSS mRNA was increased under hypoxic conditions and was strongly associated with increased CTSS expression (r=0.971, P=0.029). Most importantly, absence of RNA editing resulted in a 3-fold decrease of CTSS mRNA expression under hypoxic conditions. RNA-seq analysis of 8 patients with coronary heart disease (CHD) and 4 healthy subjects revealed that A-to-I RNA editing of CTSS 3’UTR is significantly increased in patients with CHD compared to control healthy subjects (P<0.001). Most importantly, a strong association was observed between RNA editing in the CTSS 3’UTR and mRNA expression in our cohort (r>0.7, P<0.001). Mechanistically, RNA-immunoprecipitation, mRNA stability, luciferase reporter assays, mutagenesis experiments and NMR spectroscopy revealed that silencing of ADAR1 completely inhibited RNA editing of the 3’UTR of CTSS mRNA, which consequently affected CTSS RNA secondary structure, HuR binding to the CTSS 3’UTR and finally CTSS mRNA stability and protein expression (P<0.001 for all). Taken into consideration that 82% of edited genes in 3’UTR are enriched for HuR binding sites, RNA editing may critically regulate HuR-mediated posttranscriptional regulation and thus gene expression. Conclusion: This study suggests a novel primate-specific mechanism that may account for the increased cathepsin S expression in ischemic/inflammatory diseases and it may serve as a prototypical example for the evaluation of RNA-based mechanisms in health and disease.

Oxidative stress inhibits the proliferation, induces premature senescence and promotes a catabolic phenotype in human nucleus pulposus intervertebral disc cells.

Aged and degenerated intervertebral discs are characterised by a significant increase in the number of senescent cells, which may be associated with the deterioration of this tissue due to their catabolic phenotype. On the other hand, carboxymethyl-lysine has been found to be accumulated with ageing in the proteins of the disc, evidencing the existence of oxidative stress in this tissue. Accordingly, here we investigated the effect of oxidative stress on the physiology of human nucleus pulposus cells. Hydrogen peroxide (H2O2) at subcytotoxic concentrations transiently increased the intracellular levels of reactive oxygen species, activated the p38 MAPK, ERKs, JNKs and Akt signalling pathways and induced the nuclear translocation of NF-κΒ and Nrf2. It also provoked DNA damage and triggered a DNA repair response by activating the ATM-Chk2-p53-p21(WAF1)-pRb pathway, ultimately resulting in a G1 cell cycle delay and the decrease of cells' proliferation. Prolonged exposure to H2O2 led to premature cellular senescence, as characterised by the inhibition of proliferation, the enhanced senescence-associated β galactosidase staining and the over-expression of known molecular markers, without though a significant decrease in the chromosome telomere length. H2O2-senescent cells were found to possess a catabolic phenotype, mainly characterised by the up-regulation of extracellular matrix-degrading enzymes (MMP-1, -2, -9 and ADAMTS-5) and the down-regulation of their inhibitors (TIMPs), as well as of several proteoglycans, including aggrecan, the major component of the nucleus pulposus. The senescent phenotype could be reversed by N-acetyl-L-cysteine, supporting the use of antioxidants for the improvement of disc physiology and the deceleration of disc degeneration.

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.

Phosphocitrate reduced cartilage degeneration in non-calcification induced osteoarthritis

Purpose: It was believed that phosphocitrate (PC) exerted its disease modifying effect on calcification-induced osteoarthritis (OA) by inhibiting the formation of calcium crystals within the joints. However, recent studies suggest that PC exerts its disease modifying effect on OA, at least in part, through a crystal-independent mechanism. This study sought to investigate the disease-modifying effect of PC on partial-meniscectomy induced OA or non-calcification induced OA and test the hypothesis that PC is not only potentially a disease modifying drug for calcification-induced OA therapy but also potentially a disease-modifying drug for non-calcification-inducted OA therapy. Methods: Male Hartley guinea pigs of 4 weeks old were subjected to intraperitoneal injections of PC and physiological saline respectively. Two months later, partial medial meniscectomy was performed on the right knee of all guinea pigs to remove the anterior horn (calcification site) of the medical meniscus. After the surgery, injections of PC and saline were resumed. Five months later, these guinea pigs were euthanized and hind limbs were collected. Meniscal calcification and cartilage degeneration was examined with digital x-ray, Indian ink and safranin O staining. Matrix metaloproteinse-13 (MMP-13), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5), chemokine (C-C motif) ligand 5 (CCL5), cyclooxygenase 2 (Cox-2) and type X collagen were examined with immunostaining. Results were are expressed as the mean ± standard deviation. The differences between PC-treated and untreated groups were analyzed using Student’s t test or Wilcoxon rank sum test. Results: Meniscal calcification in the medial meniscus was absent in the partial-meniscectomy performed right knees. PC treatment significantly reduced the severity of cartilage degeneration and cartilage thinning in the partial-meniscectomy induced OA or non-calcification-induced OA. The reductions in cartilage degeneration and cartilage thinning were accompanied with significantly decreased protein levels of MMP-13, ADAMTS5 and CCL5. Conclusions: PC is not only potentially a disease modifying drug for calcification-induced OA therapy but also potentially a disease-modifying drug for non-calcification-inducted OA therapy. PC exerts its disease modifying activity on non-calcification-induced OA mainly by targeting the production of extracellular matrix-degrading enzymes through a crystal-independent mechanism. These findings provide further support for the development of PC or its analogues as disease-modifying drugs for human OA therapy.Figure 2. Mean histological scores of the tibia plateaus. Left bar group: Mean histological scores of the medial tibia plateaus of the rights knees in the untreated and PC-treated guinea pigs. Right bar group: Mean histological scores of the lateral tibia plateaus of the right knees in the untreated and PC-treated guinea pigs. * = P < 0.05 PC-treated versus the untreated guinea pigs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3. Cartilage thickness. Left: thickness of the medial tibia cartilage in the untreated guinea pigs. Right: thickness of the medial tibia cartilage in the PC-treated guinea pigs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4. Immunostaining scores. Left bar group: MMP-15 immunostaining. Middle bar group: ADAMTS5 immunostaining. Right bar group: CCL-5 immunostaining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Highlights of This Issue

Inhibitors of the enzyme CYP17A1 such as abiraterone are initially effective in men with castration-resistant prostate cancer because they markedly decrease androgen synthesis, but upstream substrates, including progesterone, are increased. Chen and colleagues sequenced the androgen receptor (AR) in a series of tumor biopsies from men who developed resistance to CYP17A1 inhibitors and identified the T878A mutation, which confers responsiveness to alternative ligands, including progesterone, in 4 of 19 cases. These findings indicate that at least a subset of abiraterone-resistant tumors remain AR driven and identifies mutations that can exploit alternative ligands as a mechanism of resistance to abiraterone.Chemotherapy-induced cognitive impairment (CICI) has been recognized as a clinically significant problem in patients undergoing chemotherapy; however, little is known about the mechanisms and diagnostic measures of CICI. The phosphorylated form of the high-molecular-weight neurofilament heavy subunit NF-H (pNF-H), one of the major structural proteins in axon, has potential as an effective biomarker of neuronal damage. In this study, Natori and colleagues assessed serum pNF-H level in breast cancer patients treated with chemotherapy and found that it was increased in a cumulative dose-dependent manner, suggesting the potential application of pNF-H as a biomarker of neural damage after chemotherapy.Oncolytic viruses infect tumors cells while sparing normal tissues; however, efficacy of virotherapy in the clinic has been hindered by poor tumor targeting and intratumoral dissemination of oncolytic viruses. To tackle simultaneously these drawbacks, the authors present an oncolytic adenovirus, VCN-01, which combines a retargeting modification of the capsid with expression of the extracellular matrix-degrading enzyme hyaluronidase. The pRB-based replication selectivity of VCN-01 allows for a wide applicability in different tumor types. The reported favorable efficacy-toxicity preclinical profile supported two ongoing clinical trials of VCN-01, by intratumoral or intravenous administration, for the treatment of pancreatic cancer and other cancer types.K-Ras is the mostly widely dysregulated oncogene in non–small cell lung cancers, somatically point-mutated in ∼30% of adenocarcinomas. To assess the effect of Ras mutation status on prognostic markers for lung cancer, Starmans and colleagues designed targeted and permutation studies and compared biomarker accuracy in RAS-wildtype and RAS-mutant tumours. They find that RAS-mutant tumors are generally more difficult to prognose. Rather than creating a single prognostic biomarker, it is more effective to stratify patients by Ras-status and subject each group to an independent biomarker. These studies provide a template for integrated DNA and RNA biomarkers, as well as providing a new strategy for more accurately personalizing therapy in lung cancer.

Potential roles of cytokines and chemokines in human intervertebral disc degeneration: interleukin-1 is a master regulator of catabolic processes

These studies investigated cytokine and chemokine receptor profiles in nucleus pulposus (NP) cells, and the effects of receptor stimulation on mRNA levels of extracellular matrix (ECM) components, degrading enzymes and cytokine and chemokine expression. Immunohistochemistry (IHC) was performed to localise expression of CD4, CCR1, CXCR1 and CXCR2 in human NP tissue samples. Effects of cytokine and chemokine stimulation was performed to investigate effects related to ECM remodelling and modulation of cytokine and chemokine mRNA expression. IHC identified CD4, CCR1, CXCR1 and CXCR2 expression by NP cells. Differential expression profiles were observed for CD4 and CXCR2 in tissue samples from degenerate and infiltrated IVDs. Invitro stimulations of primary human NP cultures with IL-16, CCL2, CCL3, CCL7 or CXCL8 did not identify any modulatory effects on parameters associated with ECM remodelling or expression of other cytokines and chemokines. Conversely, IL-1 was seen to modulate ECM remodelling and expression of all other cytokines and chemokines investigated. This study demonstrates for the first time that NP cells express a number of cytokine and chemokine receptors and thus could respond in an autocrine or paracrine manner to cytokines and chemokines produced by NP cells, particularly during tissue degeneration. However, this study failed to demonstrate regulatory effects on ECM genes and degradative enzymes or other cytokines and chemokines for any target investigated, with the exception of IL-1. This suggests that IL-1 is a master regulator within the IVD and may exert regulatory potential over a plethora of other cytokines and chemokines.

Matrix Metalloproteinase-9 Is Essential for Physiological Beta Cell Function and Islet Vascularization in Adult Mice

The availability of paracrine factors in the islets of Langerhans, and the constitution of the beta cell basement membrane can both be affected by proteolytic enzymes. This study aimed to investigate the effects of the extracellular matrix-degrading enzyme gelatinase B/matrix metalloproteinase-9 (Mmp-9) on islet function in mice. Islet function of Mmp9-deficient (Mmp9(-/-)) mice and their wild-type littermates was evaluated both in vivo and in vitro. The pancreata of Mmp9(-/-) mice did not differ from wild type in islet mass or distribution. However, Mmp9(-/-) mice had an impaired response to a glucose load in vivo, with lower serum insulin levels. The glucose-stimulated insulin secretion was reduced also in vitro in isolated Mmp9(-/-) islets. The vascular density of Mmp9(-/-) islets was lower, and the capillaries had fewer fenestrations, whereas the islet blood flow was threefold higher. These alterations could partly be explained by compensatory changes in the expression of matrix-related proteins. This in-depth investigation of the effects of the loss of MMP-9 function on pancreatic islets uncovers a deteriorated beta cell function that is primarily due to a shift in the beta cell phenotype, but also due to islet vascular aberrations. This likely reflects the importance of a normal islet matrix turnover exerted by MMP-9, and concomitant release of paracrine factors sequestered on the matrix.

Influence of gut microbiota-derived ellagitannins' metabolites urolithins on pro-inflammatory activities of human neutrophils.

Ellagitannin-rich products exhibit beneficial influence in the case of inflammation-associated diseases. Urolithins, metabolites of ellagitannins produced by gut microbiota, in contrary to high molecular weight hydrophilic parental polyphenols, possess well established bioavailability. Because of the important role of neutrophils in progression of inflammation, the influence of urolithins on their pro-inflammatory functions was tested. Urolithin B at a concentration of 20 µM showed significant inhibition of interleukin 8 and extracellular matrix-degrading enzyme MMP-9 production. It was also significantly active in prevention of cytochalasin A/formyl-met-leu-phenylalanine-triggered selectin CD62L shedding. Urolithin C was the only active compound towards inhibition of elastase release from cytochalasin A/formyl-met-leu-phenylalanine-stimulated neutrophils with 39.0 ± 15.9% inhibition at a concentration of 5 µM. Myeloperoxidase release was inhibited by urolithins A and C (at 20 µM by 46.7 ± 16.1 and 63.8 ± 8.6%, respectively). Urolithin A was the most potent reactive oxygen species release inhibitor both in formyl-met-leu-phenylalanine and 4β-phorbol-12β-myristate-R13-acetate-stimulated neutrophils. At the concentration of 1 µM, it caused reactive oxygen species level decrease by 42.6 ± 26.6 and 53.7 ± 16.0%, respectively. Urolithins can specifically modulate inflammatory functions of neutrophils, and thus could contribute to the beneficial health effects of ellagitannin-rich medicinal plant materials and food products.

Caspase 3 Silencing Inhibits Biomechanical Overload–Induced Intervertebral Disk Degeneration

Intervertebral disk (IVD) degeneration causes debilitating low back pain in much of the worldwide population. No efficient treatment exists because of an unclear pathogenesis. One characteristic event early in such degeneration is the apoptosis of nucleus pulposus (NP) cells embedded in IVDs. Excessive biomechanical loading may also be a major etiology of IVD degeneration. The present study used in vitro and in vivo models of compressive loading to elucidate the underlying mechanism of IVD degeneration. In addition, we investigated whether the inhibition of apoptosis is a potential clinical therapeutic strategy for the treatment of IVD degeneration induced by biomechanical stress. A TUNEL assay showed that NP cell-agarose three-dimensional composite cultures subjected to uniaxial, unconfined, static, compressive loading exhibited a time-dependent increase in apoptosis. Western blot analysis revealed the up-regulation of several extracellular matrix-degrading enzymes and down-regulation of tissue inhibitor of metalloproteinase 1. These responses to compressive loading were all significantly inhibited by caspase 3 siRNA. In the in vivo model of compressive loading-induced IVD degeneration, a single local injection of caspase 3 siRNA significantly inhibited IVD degeneration by magnetic resonance imaging, histological findings, IHC, and TUNEL assay. The present study suggests that caspase 3 siRNA attenuates overload-induced IVD degeneration by inhibiting NP cell apoptosis and the expression of matrix-degrading enzymes.

CXCL12-CXCR4 Promotes Proliferation and Invasion of Pancreatic Cancer Cells

CXCL12 exerts a wide variety of chemotactic effects on cells. Evidence indicates that CXCL12, in conjunction with its receptor, CXCR4, promotes invasion and metastasis of tumor cells. Our objective was to explore whether the CXCL12-CXCR4 biological axis might influence biological behavior of pancreatic cancer cells. Miapaca-2 human pancreatic cancer cells were cultured under three different conditions: normal medium (control), medium + recombinant CXCL12 (CXCL12 group), or medium + CXCR4-inhibitor AMD3100 (AMD3100 group). RT-PCR was applied to detect mRNA expression levels of CXCL12, CXCR4, matrix metalloproteinase 2 (MMP-2), MMP-9, and human urokinase plasminogen activator (uPA). Additionally, cell proliferation and invasion were performed using CCK-8 colorimetry and transwell invasion assays, respectively. CXCL12 was not expressed in Miapaca-2 cells, but CXCR4 was detected, indicating that these cells are capable of receiving signals from CXCL12. Expression of extracellular matrix-degrading enzymes MMP-2, MMP- 9, and uPA was upregulated in cells exposed to exogenous CXCL12 (P<0.05). Additionally, both proliferation and invasion of pancreatic cancer cells were enhanced in the presence of exogenous CXCL12, but AMD3100 intervention effectively inhibited these processes (P<0.05). The CXCL12-CXCR4 biological axis plays an important role in promoting proliferation and invasion of pancreatic cancer cells.

Viper venom-induced oxidative stress and activation of inflammatory cytokines: a therapeutic approach for overlooked issues of snakebite management

The snakebite mortality rate has been significantly reduced due to effective anti-venin therapy. The intravenously infused anti-venom will neutralize free and target-bound toxins but fails to neutralize venom-induced inflammation and oxidative stress, as the antigen-antibody complex itself is pro-inflammatory. Therefore, an auxiliary therapy is necessary to treat secondary/overlooked envenomation complications. Blood samples from healthy donors were treated with viper venom (100 μg/ml) for 2 h. The venom-induced inflammation, oxidative damage and effect of crocin pre-treatment were determined by assessing the serum levels of cytoplasmic, lysosomal and oxidative stress markers along with pro-inflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and cyclo-oxygenase (COX)-2. Significantly increased stress markers, cytoplasmic, lysosomal and extracellular matrix-degrading enzymes as well as the pro-inflammatory mediators TNF-α, IL-1β, IL-6 and COX-2 indicated increased cellular damage but significantly reduced oxidative damage and inflammation in crocin pre-treated groups. The data clearly suggest that venom-induced oxidative stress and inflammation is also responsible for oxidative burst and cell death in the circulation, which may worsen even after anti-venin therapy. Hence, the current study demands a supportive therapy in addition to anti-venin therapy to neutralize the overlooked issues of snakebite.

Anti-invasion Effect of Crebanine and &lt;i&gt;O&lt;/i&gt;-Methylbulbocapnine from &lt;i&gt;Stephania venosa&lt;/i&gt; &lt;i&gt;via&lt;/i&gt; Down-Regulated Matrix Metalloproteinases and Urokinase Plasminogen Activator

The alkaloids isolated from Stephania venosa (S. venosa) have been shown to inhibit the proliferation and to induce the apoptosis of cancer cells. However, the anti-metastatic effect of the alkaloids on cancer cell invasion is unknown. In this study, we investigated the anti-invasive properties of four alkaloids from S. venosa, crebanine (CN), O-methylbulbocapnine (OMBC), tetrahydropalmatine (THP), and N-methyltetrahydropalmatine (NMTHP), in HT1080 human fibrosacroma cells. Treatment of the cells with 15 µg/mL of CN and OMBC reduced the chemo-invasion of HT1080 cells to 45 and 50%, respectively, whereas THP and NMTHP had a negative effect. On the other hand, CN and OMBC had no effect on cell migration. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) are the extracellular matrix (ECM) degradation enzymes that play an important role in cancer cell metastasis. Results from zymography and western blot analysis showed that CN and OMBC comparatively reduced MMP-2, MMP-9, MT1-MMP and uPA expression in a dose-dependent manner. However, CN and OMBC had no effect on the activity of collagenase, MMP-2 and MMP-9. We also found that CN and OMBC reduced the nuclear translocation and DNA binding activity of nuclear factor kappa B (NF-κB), which is the expressed mediator of ECM degradation enzymes. These findings demonstrated that CN and OMBC mediated HT1080 cell invasion by the reduction of MMP-2, MMP-9, uPA and MT1-MMP expression, possibly by targeting of NF-κB signaling pathway in the HT1080 cells.