Degradation Of Extracellular Matrix Proteins Research Articles

Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE(2) synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.

Matrix metalloproteinase levels in peritoneal effluents were increased in a patient with appendicitis undergoing continuous ambulatory peritoneal dialysis

A differential diagnosis of common bacterial peritonitis and appendicitis is difficult in continuous ambulatory peritoneal dialysis (CAPD) patients, and thus the definite diagnosis of appendicitis is often delayed. In this case, a 60-year-old man undergoing CAPD was at first diagnosed with bacterial peritonitis but not appendicitis, and antibiotics were administered. The number of leukocytes in the peritoneal effluent decreased mildly, but the level of C-reactive protein continued to be high and the pain aggravated. When the catheter was removed, suppurative appendicitis was confirmed for the first time. Levels of matrix metalloproteinase (MMP)-2 and -9 in peritoneal effluents were markedly high. Appendicitis should be diagnosed as early as possible because MMPs directly injure the peritoneum via degradation of extracellular matrix proteins. Future studies in a greater numbers of cases of appendicitis are required.

MMP-9 mediates colitis associated cancer in mice through Notch-1 via p53 activation

While individuals with Inflammatory Bowel Disease (IBD) have long been appreciated to be at increased risk of developing colon cancer, the mechanisms underlying colitis-associated cancer (CAC) are poorly understood. Matrix metalloproteinases (MMPs) are zinc-dependent neutral endopeptidases that participate in degradation of extra-cellular matrix proteins that occurs in both normal tissue remodeling and a variety of pathological processes including inflammation and cancer. MMP-9 is the predominant MMP that mediates inflammation. In accordance, MMP-9 protein expression and activity is undetectable in most healthy adult tissues including the intestine but is highly expressed in a variety of inflammatory states. We have previously demonstrated that epithelial-derived MMP-9 is upregulated during IBD, mediates tissue damage during colitis and regulates goblet cell differentiation through proteolytic cleavage of Notch-1 in colon. We have also demonstrated that despite being a mediator of pro-inflammatory response, MMP-9 plays a protective role and acts as a tumor suppressor in CAC. The aim of the present study is to understand how MMP-9 protects from CAC. C57/B6 wild type (WT) mice (n=18/group) were administered with a single dose of azoxymethane (AOM, 7.6mg/kg, i.p.). One week later they were exposed to dextran sulfate sodium (DSS, 3% in drinking water). Mice were given 2 cycles DSS of one week each and with two weeks apart. In the AOM/DSS model, WT animals were given vehicle or γ-secretase inhibitor- DAPT (Difluorophenacetyl-L-alanyl-S-phenylglycine t-butyl ester, 10 mol/kg i.p.) at every day of each DSS treatment (during acute phase of colitis for 5 days). Mice were sacrificed on day 56. We analyzed embryonic fibroblasts isolated from wild-type and MMP9-/- mice and HCT116 cells that were stably transfected with MMP9. Mice induced with CAC were more sensitive to colon cancer upon inhibition of Notch-1 as evidenced by increased polyp number (WT-vehicle3±0.82, WT-DAPT=6.4±1.39). In addition, increased erosion of mucosal layer, infiltration of neutrophils and dysplasia were seen in the polyps of DAPT treated mice compared to vehicle both induced with CAC. Western blot analysis indicated significantly decreased protein expression (in percent) of NICD (89.0±20.0), p53 (35.0±20.0), p21WAF1/Cip1 (57.0±9.0), Bax-1 (65.0±7.0) and caspase-3 (90.0±10.0) with Notch-1 inhibition compared to placebo group mice both induced with CAC. TUNEL staining showed decreased apoptosis in the colons of mice treated with DAPT (0.61±0.26) compared to vehicle group (14.36±1.12) mice both induced with CAC. We analyzed embryonic fibroblasts isolated from wild-type and MMP9-/- mice and HCT116 cells that were stably transfected with MMP9. MMP-9 mediated activation of Notch-1 signaling effects p53 expression and modulates apoptosis and cell cycle arrest, and thereby acts as a tumor suppressor in CAC. This study elucidates the key role for MMP-9 in malignant transformation of the colonic epithelium in the context of inflammation which may provide new diagnostic and therapeutic approaches.

PAI‐1 in tissue fibrosis

Fibrosis is defined as a fibroproliferative or abnormal fibroblast activation-related disease. Deregulation of wound healing leads to hyperactivation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, the pathological manifestation of fibrosis. The accumulation of excessive levels of collagen in the ECM depends on two factors: an increased rate of collagen synthesis and or decreased rate of collagen degradation by cellular proteolytic activities. The urokinase/tissue type plasminogen activator (uPA/tPA) and plasmin play significant roles in the cellular proteolytic degradation of ECM proteins and the maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs rely mostly on the activity of a potent inhibitor of uPA/tPA, plasminogen activator inhibitor-1 (PAI-1). Under normal physiologic conditions, PAI-1 controls the activities of uPA/tPA/plasmin/MMP proteolytic activities and thus maintains the tissue homeostasis. During wound healing, elevated levels of PAI-1 inhibit uPA/tPA/plasmin and plasmin-dependent MMP activities, and, thus, help expedite wound healing. In contrast to this scenario, under pathologic conditions, excessive PAI-1 contributes to excessive accumulation of collagen and other ECM protein in the wound area, and thus preserves scarring. While the level of PAI-1 is significantly elevated in fibrotic tissues, lack of PAI-1 protects different organs from fibrosis in response to injury-related profibrotic signals. Thus, PAI-1 is implicated in the pathology of fibrosis in different organs including the heart, lung, kidney, liver, and skin. Paradoxically, PAI-1 deficiency promotes spontaneous cardiac-selective fibrosis. In this review, we discuss the significance of PAI-1 in the pathogenesis of fibrosis in multiple organs.

Vascular remodelling in human skeletal muscle

Exercise-induced angiogenesis in skeletal muscle involves both non-sprouting and sprouting angiogenesis and results from the integrated responses of multiple systems and stimuli. VEGF-A (vascular endothelial growth factor A) levels are increased in exercised muscle and have been demonstrated to be critical for exercise-induced capillary growth. Only limited information is available regarding the role of other angiogenic and angiostatic factors in exercise, but changes in the angiopoietin family following repetitive bouts of exercise occur in a pattern that is favourable for angiogenesis. Results from other angiogenic model systems, indicate that miRNAs (microRNAs) are important factors in the regulation of angiogenesis and thus to explore their role as regulators of exercise induced angiogenesis will be an important avenue of study in the future. ECM (extracellular matrix) remodelling and activation of MMPs (matrix metalloproteinases) are, to some extent, overlooked players in skeletal muscle adaptation. Degradation of ECM proteins liberates angiogenic factors from immobilized matrix stores and make cell migration possible. In fact, it is known that MMPs become activated by a single bout of exercise in humans, rapid interstitial changes occur long before any changes in gene transcription could result in protein synthesis and inhibition of MMP activity completely abolishes sprouting angiogenesis. A growing body of evidence suggests that circulating and resident progenitor cells, in addition to other cell types located in skeletal muscle tissue, participate in skeletal muscle angiogenesis by various mechanisms. However, more studies are needed before these can be confirmed as mechanisms of exercise-induced capillary growth.

Urokinase-Type Plasminogen Activator Receptor (uPAR, CD87) Regulates Integrin Redistribution in Endothelial Cells Via Interaction with Low Density Lipoprotein Receptor- (LDLR-) Like Proteins

Abstract 5315angiogenesis by degradation of extracellular matrix proteins as well as induction of intracellular signal transduction. We recently could demonstrate that in VEGF-stimulated endothelial cells pro-uPA becomes activated, which leads to uPAR-complex formation, it's internalization and redistribution of uPAR to newly formed focal adhesions ad the leading edge of migrating endothelial cells. Thereby, uPAR surface expression is tightly transcriptional regulated via the Density Enhanced Phosphatase-1 (DEP-1), but also via the LDLR-family members, which regulate subcellular uPAR distribution.Here, we describe a mechanisms by which uPAR-internalization regulates integrin redistribution. We have characterized a novel binding motif on uPAR domain 3 for LDLR-protein interaction by using affinity chromatography as well as co-immunoprecipitation experiments. To proof a functional relevance of a direct uPAR/LDLR protein interaction, we reconstituted either uPAR mutants (mutL3/uPAR), lacking the binding site for LDLR-proteins, or wild type uPAR into endothelial cells derived from uPAR−/− mice. Reconstitution of mutL3/uPAR was incapable to redistribute uPAR as well as integrins during VEGF-induced endothelial cell migration when compared to wild type uPAR reconstitutes. The functional importance of uPAR / LDLR interaction was further reflected by the use of an inhibitory peptide (P1) interfering with uPAR/LDLR-protein interaction, which functionally reverted full length uPAR reconstitution, or the chaperon Receptor Associated Protein (RAP), a high affinity ligand for LDLR-proteins, which prevents uPAR/LDLR interactions. Thus, interfering with uPAR/LDLR-protein interaction at different levels led to an impaired endothelial cell spreading behavior on integrin-adhesive matrix proteins as well as a reduced pY576 FAK phosphorylation upon endothelial cell adhesion, leading to an reduced migratory response towards VEGF.These data suggest a central role of uPAR/LDLR-protein interaction in VEGF-induced endothelial cell migration via induction of integrin redistribution. Thus, uPAR/LDLR interaction might represent a novel therapeutic target in angiogenesis-related diseases. Disclosures:No relevant conflicts of interest to declare.

Contribution of Cystatin C Gene Polymorphisms to Cerebral White Matter Lesions

Background: Vascular remodeling plays an important role in the development of arteriosclerosis and any of the resulting white matter lesions in the brain. An imbalance between cysteine proteases and the cysteine protease inhibitor cystatin C (CST3) may exacerbate vascular remodeling through degradation of extracellular matrix proteins. Therefore, we evaluated the association between functional polymorphisms in the CST3 gene and the development of cerebral white matter lesions. Methods: In a total of 2,676 participants, 3 CST3 genepolymorphisms were genotyped in 92 cases with severe deep white matter hyperintensity (DWMH), and 184 subjects were randomly selected age- and sex-matched controls without any signs of DWMH. The genetic effects of these polymorphisms on DWMH and plasma CST3 levels were examined. CST3 expression vectors were transfected into an astrocytoma cell line and the expression level of CST3 mRNA was analyzed by quantitative RT-PCR. Intracellular and secreted levels of CST3 in the cell culture were quantified by Western blot and ELISA, respectively. Results: A significant association was found between one CST3 gene haplotype and DWMH (p = 0.002). This haplotype was also associated with lower plasma CST3 levels (p = 0.01). An in vitro transfection study revealed that the +148A allele, which is included in the risk haplotype, significantly reduced the secretion and increased the intracellular accumulation of CST3; however, it had no effect on the mRNA expression. Conclusions: Our study shows that polymorphisms in the CST3 gene are significantly associated with the likelihood of DWMH. Substitution of A for G at +148 of the CST3 gene decreased the extracellular availability of CST3 in vitro, which might result in the activation of protease activity.

Elevated MMP‐7 levels in patients with systemic sclerosis: correlation with pulmonary involvement

Fibrosis is characterized by an excessive accumulation of connective tissue because of an imbalance between synthesis and degradation of extracellular matrix proteins. Systemic sclerosis (SSc) is a prototypic chronic inflammatory disease leading to a severe fibrosis of the skin and many internal organs. QUESTIONS ADDRESSED: We investigated whether serum MMP-7 levels reflect the activity of the fibrotic reaction in systemic sclerosis. Serum samples were obtained from 123 patients with systemic sclerosis. MMP-serum levels of all patients with SSc were compared with age-matched healthy controls. Significantly increased median serum MMP-7 levels were found in patients with SSc when compared with controls. The median MMP-7 serum level of patients with lung fibrosis (LF) was significantly higher compared with those without LF. Accordingly, patients with dyspnea and DLCO (diffusion capacity of the lung for carbon monoxide) levels below 60% showed significantly higher median MMP-7 levels. Elevated MMP-7 levels are associated with an advanced stage of SSc and LF. These data suggest that in SSc MMP-7 is involved in the process of fibrotic tissue remodelling.

Cysteine Cathepsins and the Skeleton

Cysteine cathepsins are lysosomal proteases with housekeeping as well as tissue-specific functions. One of their specialized functions includes the degradation of extracellular matrix proteins and the regulation of the immune response. In recent years, this protease family received increasing attention as drug targets for bone and cartilage-related diseases. Cathepsin K is identified as the major osteoclast protease with a potent and unique collagenase activity. The protease is responsible for the bulk of collagen degradation in bone remodeling and plays a significant role in the regulation of osteoclast activity. Major efforts in the pharmaceutical industry led to the development of highly potent and specific cathepsin K inhibitors for clinical trials in osteoporosis. Odanacatib, a nitrile-based non-lysosomotropic inhibitor is presently in phase III trials. The compound is well tolerated and showed efficacy regarding the increase of bone mineral density, and reduction of serum and urinary markers of bone resorption. Moreover, cathepsin inhibitors do less or not interfere with the bone formation process. Cathepsin K is also a drug target in arthritic diseases where it is likely involved in type II collagen and glycoprotein degradation. Cathepsin S, which lacks the collagenase activity, is involved in MHC class II antigen presentation in inflammatory and auto-immune related joint diseases and its inhibitors have the potential to represent a new class of anti-inflammatory drugs.

Morphea-like skin reactions in patients treated with the cathepsin K inhibitor balicatib

In a multicenter clinical trial in North America and Europe that tested the cathepsin K (catK) inhibitor balicatib for the treatment of osteoporosis, several patients developed hardening of the skin. We sought to characterize these observed adverse events. Patients with skin hardening were examined by a local dermatologist. All of those patients except one had at least one biopsy specimen taken from affected skin, which was read by local and two central dermatopathologists. Workup was directed for consideration of systemic scleroderma. Nine patients of 709 treated with balicatib developed skin hardening and were given a diagnosis of morphea-like skin changes. No such events were observed in patients taking placebo or the lowest balicatib dose. After discontinuation of balicatib, skin changes resolved completely in 8 and partially in one patient. Each patient was seen by a different dermatologist in 6 different countries. These observations are likely dose-related adverse effects of balicatib. Although catK was originally thought to be expressed only in osteoclasts, it has more recently also been found in lung and dermal fibroblasts and been implicated in the degradation of the extracellular matrix in the lung and the skin. It is therefore plausible that the observed dermal fibrosis in balicatib-treated patients is a result of impaired degradation of extracellular matrix proteins and may represent a class effect of catK inhibitors. We recommend that further exploration of catK inhibition for the treatment of osteoporosis or cancer should include monitoring for similar adverse effects.

Abstract 3808: The functional role of extracellular keratin complexes in cancer

Abstract Research in our group is focused on identifying improved predictive molecular markers for cancer, specifically, lung and prostate cancers. Unlike other cancers, there are no molecular markers for either the diagnosis or prognosis of lung cancer; biopsy and imaging modalities are the only available options. For prostate cancer, serum PSA, Gleason score, stage, and surgical margins are used for diagnosis and prognosis; however, these criteria are less than precise in predicting disease outcome, with only 10% specificity at the 90% sensitivity level. We are investigating the appearance of a particular antibody present in the sera of lung cancer and prostate cancer patients through an ELISA-based test. This antibody binds to an epitope of the C-terminus of keratin 8 (K8). In ongoing clinical trials at M. D. Anderson Cancer Center Orlando, we are able to identify serum samples from patients diagnosed with cancer when compared to control samples. These observations have led us to investigate the mechanism by which this K8 antibody appears in cancer patients. We hypothesize that the antibody is produced in response to K8 that aberrantly appears on the surface of epithelial cancer cells. Keratins are intermediate filament proteins that are contained within normal epithelia, including prostate and lung. Published reports, however, have placed epitopes of K8 on the surfaces of a variety of epithelial cancer cell lines. Our results from flow cytometry and immunofluorescence experiments demonstrate the extracellular expression of K8 (eK8) on some, but not all, cancer cell lines. This differential expression of eK8 may be an important marker of cancer prognosis. We have also found that K8 expression in primary human prostate tumors and lung tumors differs significantly from normal tissue. These observations led us to investigate the functional role of K8 in cancer. Due to a terminal lysine, eK8 is a potential binding site for plasminogen, the activation of which is implicated in tumor progression and metastasis. We found that plasminogen exhibited a specific binding interaction with the C-terminus of K8. We also confirmed that K8 interacts with urokinase plasminogen activator (uPA), which suggests that K8 may serve as a docking site for plasminogen activation system (PAS) components, facilitating the activation of plasmin. We found that the presence of K8 accelerated the activation rate of plasminogen by urokinase plasminogen activator (uPA). Furthermore, the presence of K8 accelerated activated plasmin-mediated degradation of extracellular matrix proteins. We hypothesize that eK8 present on epithelial prostate and lung cancer cells works in conjunction with components of the plasminogen activation system, thereby playing an integral role in tumor migration and in vivo dissemination. The results of this work may help distinguish lethal from indolent cancer, as well as enabling effective monitoring of treatment response, thus improving patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3808. doi:10.1158/1538-7445.AM2011-3808

Extracellular matrix remodeling of the umbilical cord in pre-eclampsia as a risk factor for fetal hypertension.

The human umbilical cord forms a connection between the placenta and the foetus. It is composed of two arteries and one vein surrounded by Wharton's jelly. Pre-eclampsia is accompanied by extensive remodeling of extracellular matrix of umbilical cord. Matrix metalloproteinases (MMPs) are engaged in degradation of extracellular matrix proteins and activation/inactivation of certain cytokines and enzymes. These enzymes will probably play a central role in the release of matrix-embedded cytokines and growth factors. MMP-2 (gelatinase A) is the main collagenolytic enzyme of both umbilical artery and vein. Other metalloproteinases are present in several times lower amounts. Reduced activity of collagen-degrading enzymes may be a factor, which enhances the accumulation of collagen and some other proteins in the pre-eclamptic umbilical cord tissues. It seems to be possible that similar alterations occur in other fetal blood vessels. It may result in an increase in peripheral resistance as well as an increase in the blood pressure in the fetal vascular system. Some observations suggest that the raised pressure may persist after birth. Pre-eclampsia may be a factor that evokes an initiation of hypertension in utero and its amplification through childhood and adulthood.

Cystatin C Deficiency Promotes Epidermal Dysplasia in K14-HPV16 Transgenic Mice

BackgroundCysteine protease cathepsins are important in extracellular matrix protein degradation, cell apoptosis, and angiogenesis. Mice lacking cathepsins are protected from tumor progression in several animal models, suggesting that the regulation of cathepsin activities controls the growth of various malignant tumors.Methods and ResultsWe tested the role of cathepsins using a mouse model of multistage epithelial carcinogenesis, in which the human keratin-14 promoter/enhancer drove the expression of human papillomavirus type 16 (HPV16) early region E6/E7 transgenes. During the progression of premalignant dysplasia, we observed increased expression of cysteine protease cathepsin S, but concomitantly reduced expression of cathepsin endogenous inhibitor cystatin C in the skin tissue extract. Absence of cystatin C in these transgenic mice resulted in more progression of dysplasia to carcinoma in situ on the face, ear, chest, and tail. Chest and ear skin extract real time PCR and immunoblot analysis, mouse serum sample ELISA, tissue immunohistological analysis, and tissue extract-mediated in vitro elastinolysis and collagenolysis assays demonstrated that cystatin C deficiency significantly increased cathepsin expression and activity. In skin from both the chest and ear, we found that the absence of cystatin C reduced epithelial cell apoptosis but increased proliferation. From the same tissue preparations, we detected significantly higher levels of pro-angiogenic laminin 5-derived γ2 peptides and concurrently increased neovascularization in cystatin C-deficient mice, compared to those from wild-type control mice.ConclusionEnhanced cathepsin expression and activity in cystatin C-deficient mice contributed to the progression of dysplasia by altering premalignant tissue epithelial proliferation, apoptosis, and neovascularization.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

BackgroundEnvenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption.Methodology/Principal FindingsIn this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues.Conclusions/SignificanceThese results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

AP-1 (Fra-1/c-Jun)-mediated Induction of Expression of Matrix Metalloproteinase-2 Is Required for 15(S)-Hydroxyeicosatetraenoic Acid-induced Angiogenesis

To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.

Urinary hyaluronidase activity in rats infected with Blastocystis hominis—evidence for invasion?

The fact whether Blastocystis hominis can invade has always been in question. Apart from a few sporadic studies such as that done on gnotobiotic guinea pigs which showed surface invasion and mucosal inflammation of the host's intestine caused by B. hominis infection, no real documentation of invasion has been proven. Studies have shown that hyaluronidase is secreted during the penetration into the host's skin and gut by nematode parasites. Hyaluronidase activity in protozoa namely Entamoeba histolytica has also been described previously. This study attempts to determine hyaluronidase in urine samples of B. hominis-infected rats. The presence of hyaluronidase in urine provides an indirect evidence of invasion by B. hominis into colonic epithelium causing the degradation of extracellular matrix proteins namely hyaluronic acid (HA). HA is depolymerized by hyaluronidase which may be used by organisms to invade one another. In this study, the levels of urinary hyaluronidase of Sprague-Dawley rats infected with B. hominis were monitored for 30 days. Hyaluronidase levels in the infected rats were significantly higher on days 28 and 30 compared to the day before inoculation (P < 0.01 and P < 0.05, respectively). During this stage, parasitic burden in infected stools was also at a high level. Proinflammatory cytokines, interleukin-6 and interleukin-8, were also significantly higher (P < 0.05) in the serum of infected rats. The study demonstrates that since no other pathogen was present and that amoeboid forms of the parasites have been shown to exist previously, the elevated levels of hyaluronidase in this preliminary finding suggests that the organism is capable of having invasion or penetration activity in the hosts' intestine.

PTU-077 TIMP-1 inhibits pancreatic stellate cell apoptosis: implications for pancreatic fibrosis: Abstract PTU-077

<h3>Introduction</h3> Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix proteins. Pancreatic stellate cells (PSC) have the capacity to synthesise both matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinase (TIMPs). These may play a key role in pancreatic fibrogenesis by protecting extracellular matrix from degradation and supporting the survival of fibrogenic PSC. Therefore it was hypothesised that TIMP-1 has proproliferative and anti-apoptotic effects on culture activated PSC. <h3>Methods</h3> Culture activated rat PSC were used between passages 2 and 4 and serum deprived prior to all experiments. Apoptosis was induced with cycloheximide (50 mm/l) in the presence or absence of recombinant rat TIMP-1 (1–200 ng/ml). Apoptosis was quantified using acridine orange and counting of apoptotic bodies. PSC proliferation was determined by [³H]-thymidine incorporation. Culture-activated rat PSC were transfected with TIMP-1 and a negative control siRNA by electroporation. Cytotoxicity of siRNA was assessed by lactate dehydrogenase activity assay. Culture supernatants were collected to quantitate TIMP-1 protein knockdown following electroporation. <h3>Results</h3> TIMP-1 showed a dose dependent inhibition of cycloheximide induced PSC apoptosis. TIMP-1 at 100 ng/ml and 200 ng/ml significantly reduced apoptosis by up to 61% at 6 h, and up to 74% at 24 h (see table). Although there was a trend towards TIMP-1 stimulation of increased proliferation, this did not reach statistical significance. TIMP-1 electroporation was an effective means of silencing PSC TIMP-1 expression. Peak knockdown of target TIMP-1 protein was 77% at 24 h for TIMP-1 compared to negative control siRNA (p&lt;0.05). There was no increase in cell toxicity in TIMP-1 siRNA treated cells compared with electroporation alone. <h3>Conclusion</h3> These data suggest that TIMP-1 reduces PSC apoptosis in the presence of cycloheximide, with no significant effect on proliferation. These data also suggest that siRNA are an effective means of silencing TIMP-1 in PSC. This may represent an important therapeutic target in the treatment of pancreatic fibrosis.

MMP-2 genetic variant and plaque features of instability

More than 80 different proteins, glycoproteins, collagens, proteoglycans and glycosaminoglycans, constitute the extra cellular matrix (ECM), the complex structure surrounding cells in solid tissues and organs. Tissue remodeling, i.e. the changes in amount, type and architecture of these proteins is critical in important physiological events like growth, development and reproduction as in disease. ECM protein degradation is largely (but not exclusively) dependent on a class of neutral endoproteases called MMPs.

Anticytokine Therapy for Osteoarthritis

Several recent in vitro investigations and experimental studies performed in animal models of osteoarthritis (OA) sustained the previously held view that interleukin (IL)-1 or tumour necrosis factor-alpha (TNFalpha) disrupt the metabolism of synovial joint tissues. The evidence to date indicates that, in addition to IL-1 and TNFalpha, other pro-inflammatory cytokines, including IL-6, members of the IL-6 protein superfamily, IL-7, IL-17 and IL-18, can also promote articular cartilage extracellular matrix protein degradation or synergize with other cytokines to amplify and accelerate cartilage destruction. Most importantly, many of these cytokines have been implicated in causing synovial tissue activation and damage to subchondral bone as well as altering cartilage homeostasis in spontaneously occurring or surgically induced animal models of OA and in transgenic mice genetically primed to develop OA. In this regard, these pro-inflammatory cytokines may also play a significant role in the pathogenesis of human OA. However, attempts to modify the progression of human OA in well designed, controlled clinical trials with an IL-1 receptor antagonist protein (IRAP) have not been successful. Several anabolic cytokines (also termed growth factors), including transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF), have also been proposed as regulators of skeletal long bone growth and development as well as cartilage and bone homeostasis. TGF-beta, IGF-1 and FGF-2, in particular, have been characterized as potential chondroprotective agents. Thus, enzymatic disruption and removal of these growth factors from cartilage extracellular matrix proteins, as in the case of TGF-beta and FGF-2, or disruption of their function, as in the case of the enhanced binding of free IGF-1 with IGF binding proteins in OA joint synovial fluid, may compromise and ultimately be responsible for the inadequate repair of articular cartilage in OA. An improved understanding of the cellular and molecular mechanisms by which pro-inflammatory and/or anabolic cytokines alter both the structure and function of synovial joints may eventually result in the commercial development of disease-modifying OA drugs (DMOADs). Since the prevalence of OA is high in the elderly population, future development of DMOADs must also take into account potential differences in the way DMOADs would be metabolized in the older individual compared with younger people.