Collagen-degrading Matrix Metalloproteinases Research Articles

Compliance of the fish outflow tract is altered by thermal acclimation through connective tissue remodelling.

To protect the gill capillaries from high systolic pulse pressure, the fish heart contains a compliant non-contractile chamber called the bulbus arteriosus which is part of the outflow tract (OFT) which extends from the ventricle to the ventral aorta. Thermal acclimation alters the form and function of the fish atria and ventricle to ensure appropriate cardiac output at different temperatures, but its impact on the OFT is unknown. Here we used ex vivo pressure–volume curves to demonstrate remodelling of passive stiffness in the rainbow trout (Oncorhynchus mykiss) bulbus arteriosus following more than eight weeks of thermal acclimation to 5, 10 and 18°C. We then combined novel, non-biased Fourier transform infrared spectroscopy with classic histological staining to show that changes in compliance were achieved by changes in tissue collagen-to-elastin ratio. In situ gelatin zymography and SDS-PAGE zymography revealed that collagen remodelling was underpinned, at least in part, by changes in activity and abundance of collagen degrading matrix metalloproteinases. Collectively, we provide the first indication of bulbus arteriosus thermal remodelling in a fish and suggest this remodelling ensures optimal blood flow and blood pressure in the OFT during temperature change.

A novel placental tissue biologic, PTP-001, inhibits inflammatory and catabolic responses in vitro and prevents pain and cartilage degeneration in a rat model of osteoarthritis

Characterization of a novel human placental tissue-derived biologic, PTP-001, which is in development as a candidate therapeutic for the treatment of osteoarthritis symptoms and pathophysiology. Human placental tissues from healthy donors were prepared as a particulate formulation, PTP-001. PTP-001 extracts were assayed for the presence of disease-relevant biofactors which could have beneficial effects in treating osteoarthritis. PTP-001 eluates were tested in human chondrocyte cultures to determine effects on the production of a key collagen-degrading matrix metalloproteinase, MMP-13. PTP-001 eluates were also assessed for anti-inflammatory potential in human monocyte/macrophage cultures, as well as for growth-stimulating anabolic effects in human synoviocytes. The in vivo effects of PTP-001 on joint pain and histopathology were evaluated in a rat model of osteoarthritis induced surgically by destabilization of the medial meniscus. PTP-001 was found to contain an array of beneficial growth factors, cytokines and anti-inflammatory molecules. PTP-001 eluates dose-dependently inhibited the production of chondrocyte MMP-13, and the secretion of proinflammatory cytokines from monocyte/macrophage cultures. PTP-001 eluates also promoted proliferation of cultured synovial cells. In a rat osteoarthritis model, PTP-001 significantly reduced pain responses throughout 6 weeks post-dosing. The magnitude and duration of pain reduction following a single intraarticular treatment with PTP-001 was comparable to that observed for animals treated with a corticosteroid (active control). For rats dosed twice with PTP-001, significant reductions in cartilage histopathology scores were observed. PTP-001 represents a promising biologic treatment for osteoarthritis, with a multi-modal mechanism of action that may contribute to symptom management and disease modification.

Effects of ultra-weak fractal electromagnetic signals on the aqueous phase in living systems: a test-case analysis of molecular rejuvenation markers in fibroblasts

ABSTRACT Skin aging is primarily associated with the alterations in dermal extracellular matrix, in particular a decrease in collagen type-1 content. Recent studies have shown that collagen-degrading matrix metalloproteinase (MMP-1) is produced by fibroblasts in response to chronoaging, which in human dermal fibroblasts leads to the release of proinflammatory cytokines. Past studies showed that anti-inflammatory capabilities could be induced via non-chemical means. One of these methods makes use of ultra-weak fractal electromagnetic (uwf-EM) signals. Such ultra-/very-low frequency (U/VLF) signals (few nT in intensity and within 0.5–30 kHz) interact with aqueous solutions in living systems. The fractal nature of such EM-signals relates to the self-similar property by which a “cut-out” and magnified piece of this signal reveals again the original. Thus, the aim of this study is twofold, to i) investigate the extent of this modulating effect using Human Dermal Fibroblasts (HDF)-cells, and ii) analyse molecular rejuvenation markers therein. We could demonstrate that a 10 min uwf-EM exposure (prior to incubation) increases type-1 collagen and modulates elastin in human fibroblasts cultured up to 96 h, while at the same time reduces IL-6, TNF-α and MMP-1 (the later three being statistically significant). Such up- respectively down-regulation of corresponding genes are strong indicators of an EM-induced hormetic effect that influences the epigenomic landscape of HDFs. In the Appendix, we present, in the framework of Quantum Field Theory (QFT), water as a biphasic liquid and how its coherent fraction can be affected by uwf-EM signals while at the same time resolving the “kT paradox”.

Macro- and micromechanical remodelling in the fish atrium is associated with regulation of collagen 1 alpha 3 chain expression

Numerous pathologies lead to remodelling of the mammalian ventricle, often associated with fibrosis. Recent work in fish has shown that fibrotic remodelling of the ventricle is ‘reversible’, changing seasonally as temperature-induced changes in blood viscosity alter haemodynamic load on the heart. The atrial response to varying haemodynamic load is less understood in mammals and completely unexplored in non-mammalian vertebrates. To investigate atrial remodelling, rainbow trout were chronically cooled (from 10 ± 1 to 5 ± 1 °C) and chronically warmed (from 10 ± 1 to 18 ± 1 °C) for a minimum of 8 weeks. We assessed the functional effects on compliance using ex vivo heart preparations and atomic force microscopy nano-indentation and found chronic cold increased passive stiffness of the whole atrium and micromechanical stiffness of tissue sections. We then performed histological, biochemical and molecular assays to probe the mechanisms underlying functional remodelling of the atrial tissue. We found cooling resulted in collagen deposition which was associated with an upregulation of collagen-promoting genes, including the fish-specific collagen I alpha 3 chain, and a reduction in gelatinase activity of collagen-degrading matrix metalloproteinases (MMPs). Finally, we found that cooling reduced mRNA expression of cardiac growth factors and hypertrophic markers. Following long-term warming, there was an opposing response to that seen with cooling; however, these changes were more moderate. Our findings suggest that chronic cooling causes atrial dilation and increased myocardial stiffness in trout atria analogous to pathological states defined by changes in preload or afterload of the mammalian atria. The reversal of this phenotype following chronic warming is particularly interesting as it suggests that typically pathological features of mammalian atrial remodelling may oscillate seasonally in the fish, revealing a more dynamic and plastic atrial remodelling response.

Platelet factor 4 upregulates matrix metalloproteinase-1 production in gingival fibroblasts.

Periodontitis is a highly prevalent chronic inflammatory disease that causes tooth loss, morbidity and confers an increased risk for systemic disease. Tissue destruction during periodontitis is due in large part to collagen-degrading matrix metalloproteinases (MMPs) released by resident cells of the periodontium in response to proinflammatory cytokines. Platelets are immune-competent blood cells with a newly recognized role in chronic inflammation; however, their role in the pathogenesis of periodontitis is undefined. Consequently, the objective of this study was to assess the effect of platelet factor 4 (PF4), a major platelet-derived cytokine, on MMP-1 (collagenase) expression in human gingival fibroblasts (HGFs). HGFs were cultured in the presence or absence of recombinant PF4. Pro-MMP-1 secretion was quantified by enzyme-linked immunosorbent assay analysis of the cell culture supernatants. MMP-1 transcription was quantified by real-time polymerase chain reaction. Regulation of MMP-1 production by the p44/42 MAP kinase (MAPK) signaling pathway was examined in the presence or absence of PF4. Exposure to PF4 caused a ~ 2-3-fold increase in MMP-1 transcription and secretion from cultured HGFs. PF4 treatment also enhanced phosphorylation of p44/42 MAPK, which has been previously shown to induce MMP-1 expression in fibroblasts. Blockade of p44/42 MAPK signaling with the cell-permeant inhibitors PD98059 and PD184352 abrogated PF4-induced pro-MMP-1 transcription upregulation and release from cultured HGFs. We conclude that PF4 upregulates MMP-1 expression in HGFs in a p44/42 MAPK-dependent manner. These findings point to a previously unidentified role for platelets in the pathogenesis of periodontal diseases.

Spent coffee ground extract suppresses ultraviolet B-induced photoaging in hairless mice

The aim of this study is to evaluate the effect of spent coffee ground (SCG) ethanol extract on UVB-induced skin aging in hairless mice. An ethanol extract of SCG (ESCG) was prepared using the residue remaining after extraction of oil from roasted SCG. High performance liquid chromatography (HPLC) analysis showed that the content of caffeine (41.58±0.54μg/mg) was higher than that of chlorogenic acid isomers (~9.17μg/mg) in ESCG. ESCG significantly decreased the UVB-induced intracellular reactive oxygen species in HaCaT cells. UVB-induced wrinkle formation in mice dorsal skin was effectively reduced by ESCG administration; high dose of ESCG (5g/L) caused the reduction of wrinkle area by 30% compared with UVB-treated control (UVBC). This result correlated with the ESCG-mediated decrease in epidermis thickness (25%). In addition, ESCG administration significantly reduced transdermal water loss (20%) and erythema formation (35%) derived from UVB exposure. Collagen type I (COL-1) level in dorsal skin was effectively recovered by ESCG administration. These results were supported by down-regulation of collagen-degrading matrix metalloproteinase 2 (MMP2) and 9 (MMP9) expressions. Our results indicate that ESCG protects mouse skin from UVB-induced photoaging by suppressing the expression of matrix metalloproteinases. Our study suggests that ESCG may be anti-photoaging agent.

Mechanical Stretching of Mouse Calvarial Osteoblasts In Vitro Models Changes in MMP-2 and MMP-9 Expression at the Bone-Implant Interface.

Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress.

The CYP2E1 inhibitor DDC up-regulates MMP-1 expression in hepatic stellate cells via an ERK1/2- and Akt-dependent mechanism

DDC (diethyldithiocarbamate) could block collagen synthesis in HSC (hepatic stellate cells) through the inhibition of ROS (reactive oxygen species) derived from hepatocyte CYP2E1 (cytochrome P450 2E1). However, the effect of DDC on MMP-1 (matrix metalloproteinase-1), which is the main collagen degrading matrix metalloproteinase, has not been reported. In co-culture experiments, we found that DDC significantly enhanced MMP-1 expression in human HSC (LX-2) that were cultured with hepatocyte C3A cells either expressing or not expressing CYP2E1. The levels of both proenzyme and active MMP-1 enzyme were up-regulated in LX-2 cells, accompanied by elevated enzyme activity of MMP-1 and decreased collagen I, in both LX-2 cells and the culture medium. H2O2 treatment abrogated DDC-induced MMP-1 up-regulation and collagen I decrease, while catalase treatment slightly up-regulated MMP-1 expression. These data suggested that the decrease in ROS by DDC was partially responsible for the MMP-1 up-regulation. ERK1/2 (extracellular signal-regulated kinase 1/2), Akt (protein kinase B) and p38 were significantly activated by DDC. The ERK1/2 inhibitor (U0126) and Akt inhibitor (T3830) abrogated the DDC-induced MMP-1 up-regulation. In addition, a p38 inhibitor (SB203580) improved MMP-1 up-regulation through the stimulation of ERK1/2. Our data indicate that DDC significantly up-regulates the expression of MMP-1 in LX-2 cells which results in greater MMP-1 enzyme activity and decreased collagen I. The enhancement of MMP-1 expression by DDC was associated with H2O2 inhibition and coordinated regulation by the ERK1/2 and Akt pathways. These data provide some new insights into treatment strategies for hepatic fibrosis.

Hydrogen sulfide and sodium nitroprusside compete to activate/deactivate MMPs in bone tissue homogenates

BackgroundBone microvascular remodeling is the primary predictor of bone structure and function. Remodeling by its very nature implies synthesis and degradation of the extracellular matrix. Normally, 50% of total protein in the vessel wall is elastin. During remodeling, elastin is degraded by specialized matrix metalloproteinases (MMPs). Because the turnover of elastin is 1000-fold slower than that of collagen, most of the elastin is replaced by stiffer collagen. Stiffer vessels impose pressure on the aortic valve, causing regurgitation and increased pulse pressure. On the other hand, high MMP activity will cause vascular dilatation, leading to aneurysm. Therefore, balanced constitutive remodeling is necessary for adequate bone structure and function. Interestingly, collagen-degrading MMPs are involved in various pathological conditions, including osteoporosis, osteoarthritis, and cardiovascular disease. Sodium nitroprusside is a nitric oxide donor that could potentially alter MMP activity via vasodilation in vivo, but can also produce peroxynitrite, which activates MMPs by combining with superoxide. Moreover, hydrogen sulfide is a known antioxidant as well as a vasodilator, and is also speculated to contribute directly to MMP activity. We hypothesized that hydrogen sulfide reduced activity of MMP in ex vivo bone tissue homogenates and that sodium nitroprusside would increase MMP activity in vitro.MethodsWe surgically removed the tibia and femur from anesthetized mice, and prepared bone tissue homogenates using a mortar and pestle, measured the protein concentration with a spectrophotometer, and detected MMP activity using gelatin gel zymography.ResultsOur data showed increased MMP activity at a sodium nitroprusside concentration of 1 μM, and MMP activity increased exponentially. There was a decrease in MMP activity with increasing hydrogen sulfide, beginning at 16 μM (P < 0.01) and continuing to 40 μM. Moreover, sodium nitroprusside 3 μM was able to overcome the decrease in MMP activity that occurred with hydrogen sulfide 40 μM; this resulted in a more pronounced exponential increase in MMP activity.ConclusionThere are several substances that can potentially be used to decrease MMP activity and to alleviate pathological remodeling by MMPs.

Antiphotoaging effect of chitooligosaccharides on human dermal fibroblasts

In the present study, the effect of 3-5 kDa chitooligosaccharide (COS) on homeostasis between the expression of collagen-degrading matrix metalloproteinases (MMPs) and collagen synthesis was investigated using ultraviolet (UV)-A irradiated dermal fibroblasts. UV protection imparted by 3-5 kDa COS was measured by examining the UV absorption spectrum. Collagenase MMP secretion was examined using an enzyme-linked immunosorbent assay. The levels of collagenases and collagen synthetic markers were determined by employing the reverse transcriptase-polymerase chain reaction and Western blot analysis. The 3-5 kDa COS not only absorbed UV-A and UV-B light but also inhibited collagenase (MMP-1, MMP-8, and MMP-13) and gelatinase (MMP-2 and MMP-9) MMP expression. The suppression of MMP expression was found to be due to an increase in expression of the tissue inhibitors of MMP (TIMP)-1 and TIMP-2. Treatment with 3-5 kDa COS enhanced collagen synthetic markers such as procollagen, type I, III, and IV collagens in UV-A-irradiated dermal fibroblasts. Furthermore, the effects of 3-5 kDa COS on collagen degradation and collagen synthesis in UV-A irradiated dermal fibroblasts were regulated via the inhibition of activating protein-1 (AP-1) signaling. Our results suggest that 3-5 kDa COS can be used to develop as topical applications for antiphotoaging cosmeceuticals as it enhances collagen synthesis.

Matrix metalloproteinase–13 is required for osteocytic perilacunar remodeling and maintains bone fracture resistance

Like bone mass, bone quality is specified in development, actively maintained postnatally, and disrupted by disease. The roles of osteoblasts, osteoclasts, and osteocytes in the regulation of bone mass are increasingly well defined. However, the cellular and molecular mechanisms by which bone quality is regulated remain unclear. Proteins that remodel bone extracellular matrix, such as the collagen-degrading matrix metalloproteinase (MMP)-13, are likely candidates to regulate bone quality. Using MMP-13-deficient mice, we examined the role of MMP-13 in the remodeling and maintenance of bone matrix and subsequent fracture resistance. Throughout the diaphysis of MMP-13-deficient tibiae, we observed elevated nonenzymatic cross-linking and concentric regions of hypermineralization, collagen disorganization, and canalicular malformation. These defects localize to the same mid-cortical bone regions where osteocyte lacunae and canaliculi exhibit MMP-13 and tartrate-resistant acid phosphatase (TRAP) expression, as well as the osteocyte marker sclerostin. Despite otherwise normal measures of osteoclast and osteoblast function, dynamic histomorphometry revealed that remodeling of osteocyte lacunae is impaired in MMP-13(-/-) bone. Analysis of MMP-13(-/-) mice and their wild-type littermates in normal and lactating conditions showed that MMP-13 is not only required for lactation-induced osteocyte perilacunar remodeling, but also for the maintenance of bone quality. The loss of MMP-13, and the resulting defects in perilacunar remodeling and matrix organization, compromise MMP-13(-/-) bone fracture toughness and postyield behavior. Taken together, these findings demonstrate that osteocyte perilacunar remodeling of mid-cortical bone matrix requires MMP-13 and is essential for the maintenance of bone quality.

Abstract 9910: Chronic Inflammatory Responses to Polymer Can Evoke Both Late Restenosis and Very Late Stent Thrombosis After Sirolimus-Eluting Stent Implantation in Human Coronary Arteries

Background: Sirolimus-eluting stents (SES) can reduce the rate of restenosis by marked inhibition of neointimal hyperplasia. However, late adverse events occurring more than 1 year (Y) after SES implantation, in-stent late luminal loss (LLL) and very late stent thrombosis (VLST), have been reported. The detailed mechanisms of these events are poorly understood at present. Methods and Results: Postmortem histopathological investigation of late vascular responses of coronary arteries was performed in 32 consecutive patients (pts) more than 1 Y after SES implantation. Although most simple lesions with a single SES showed gradual endothelial coverage (80 to 90%) after 1 Y, there was considerable inflammatory reaction involving T lymphocytes (T cells), macrophages (MΦ) and multinucleated giant cells adjacent to the struts. Furthermore, 14 cases showed necrotic core formation with numerous circumferential cholesterol clefts and pronounced infiltration by lipid-laden MΦ that were immunoreactive for collagen-degrading matrix metalloproteinases around the struts. In 3 of these arteries, the luminal surface was focally ruptured and massive thrombi composed of platelets and fibrin were observed. Furthermore, in 2 pts who died of VLST more than 2 Y after SES implantation, the arterial wall of SES segments was aneurysmally dilated with an extensive inflammatory infiltrate involving the intima, media and adventitia consisting predominantly of T cells and eosinophils, and occasional giant cells. Furthermore, 2 other pts developing coronary aneurysms in the stented segments demonstrated diffuse restenosis. Granulomatous inflammatory reaction with mild to moderate inflammatory cell infiltration but remarkable smooth muscle cell proliferation was clearly observed in the restenotic regions in these pts. Conclusions: These findings suggest that the polymer of SES can evoke significant chronic destructive and/or proliferative inflammatory responses followed by severe adverse clinical events such as LLL or VLST, depending on the intensity of inflammation. Although relatively milder inflammatory changes induce intimal hyperplasia, vessel wall destruction by severe inflammation can lead to aneurysm formation in extreme cases.

A multi-component herbal preparation (PADMA 28) improves structure/function of corticosteroid-treated skin, leading to improved wound healing of subsequently induced abrasion wounds in rats

PADMA 28 is a multi-component herbal mixture formulated according to an ancient Tibetan recipe. PADMA 28 is known to stimulate collagen production and reduced levels of collagen-degrading matrix metalloproteinases (MMPs). The goal of the present study was to determine whether topical treatment of rat skin with PADMA 28 would improve skin structure/function, and whether subsequently induced abrasion wounds would heal more rapidly in skin that had been pretreated with PADMA 28. Hairless rats were exposed to a potent topical corticosteroid (Temovate) in combination with either DMSO alone or with PADMA 28 given topically. At the end of the treatment period, superficial wounds were created in the skin, and time to wound closure was assessed. Collagen production and matrix-degrading MMPs were assessed. Abrasion wounds in skin that had been pretreated with PADMA 28 healed more rapidly than did wounds in Temovate plus DMSO-treated skin. Under conditions in which improved wound healing was observed, there was an increased collagen production and decreased MMP expression, but no significant epidermal hyperplasia and no evidence of skin irritation. The ability to stimulate collagen production and inhibit collagen-degrading enzymes in skin and facilitate more rapid wound closure without irritation should provide a rationale for development of the herbal preparation as a "skin-repair" agent.

Ultraviolet Irradiation Induces CYR61/CCN1, a Mediator of Collagen Homeostasis, through Activation of Transcription Factor AP-1 in Human Skin Fibroblasts

UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

Cysteine Cathepsins in Human Dentin-Pulp Complex

Introduction Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-β) on cathepsin expression was also analyzed. Methods Human odontoblasts and pulp tissue were cultured with and without TGF-β, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. Results Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-β. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. Conclusions These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging.

Stanniocalcin 2 promotes invasion and is associated with metastatic stages in neuroblastoma

Stanniocalcin 2 (STC2) is a secreted glycoprotein of as yet unknown functions. We investigated STC2 in human neuroblastoma, the most common solid extra-cranial tumor of infancy. In primary tumor samples, we found that expression of STC2 is associated with the metastatic Stages 4 and 4s and MYCN expression. In vitro, however, we demonstrate that cell proliferation is reduced by STC2 due to an increase in the basal apoptosis rate of the transfected cells. On the other hand, in vitro assays showed that STC2-transfected neuroblastoma cells have an increased invasive potential and display higher activity of collagen-degrading matrix metalloproteinase 2 (MMP2). Using experimental tumors on the chick chorioallantoic membrane (CAM), we observed that STC2 expressing cells show signs of emigration from the solid tumor and destroy blood vessels of the CAM, giving rise to massively bleeding tumors. Erosion of blood vessels was also seen when purified STC2 protein was applied on the CAM. Taken together, we demonstrate a dual role for STC2 in neuroblastoma. It reduces proliferation of tumor cells in vitro, but increases the invasive potential and induces bleeding, and thereby may facilitate early metastasis. The potential of STC2 as a surrogate marker for metastatic neuroblastoma calls for further investigation.

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. MT1-MMP was highly expressed at the pannus-cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP-dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP-dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP-dependent invasion may represent a novel therapeutic strategy for RA.

High-Mobility Group Box 1 Protein Activates Hepatic Stellate Cells In Vitro

Objectives Our previous study noticed remarkably elevated titers of anti–high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis. Methods The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure α-smooth muscle actin (α-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs). Results HMGB1 at concentrations >1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. α-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment. Conclusion Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts.

Chemokine-like functions of MIF in atherosclerosis

The cytokine macrophage migration inhibitory factor (MIF) is a unique pro-inflammatory regulator of many acute and chronic inflammatory diseases. In the pathogenesis of atherosclerosis, chronic inflammation of the arterial wall characterized by chemokine-mediated influx of leukocytes plays a central role. The contribution of MIF to atherosclerotic vascular disease has come into focus of many studies in recent years. MIF is highly expressed in macrophages and endothelial cells of different types of atherosclerotic plaques, and functional studies established the contribution of MIF to lesion progression and plaque inflammation. This proatherogenic effect may partly be explained by the finding that MIF regulates inflammatory cell recruitment to lesion areas. Similar to chemokines, MIF induces integrin-dependent arrest and transmigration of monocytes and T cells. These chemokine-like functions are mediated through interaction of MIF with the chemokine receptors CXCR2 and CXCR4 as a non-canonical ligand. In atherogenic monocyte recruitment, MIF-induced monocyte adhesion involves CD74 and CXCR2, which form a signaling receptor complex. In addition to lesion progression, MIF has been implicated in plaque destabilization, since MIF is predominantly expressed in vulnerable plaques and can induce collagen-degrading matrix metalloproteinases. The latter could be a relevant mechanism in atherosclerotic abdominal aneurysm formation, where MIF expression is correlated with aneurysmal expansion. In summary, MIF has been identified as an important regulator of atherosclerotic vascular disease with exceptional chemokine-like functions. Detailed analysis of the interaction of MIF with its receptors could provide valuable information for drug development for the anti-inflammatory treatment of established and unstable atherosclerosis.

Statine und ihr Einfluss auf entzündliche Prozesse in arteriosklerotischen Plaques bei Patienten mit Stenose der Arteria carotis interna

Arterio-arterial emboli originating from a high-grade stenosis of the internal carotid artery are a common cause for cerebral ischemias. Inflammatory processes are not only pivotal in the development of atherosclerotic vessel wall changes, but also for their clinical destabilization. Inflammatory cells, like macrophages, can turn a chronic high-grade carotid stenosis into a high-risk area for the development of arterial thromboses by way of a complex pathogenesis involving the elevation of proinflammatory factors, biosynthesis of collagen-degrading matrix metalloproteinases and expression of prothrombotic tissue factor. This process could affect the occurrence of perioperative complications during carotid endarterectomies. Statins are potent cholesterol-lowering agents. Among other lipid-independent effects, statins appear to play a significant role in preventing cardiovascular events. A number of studies have shown that statins possess plaque-stabilizing effects and that they improve cerebral autoregulation. A growing evidence supports the preoperative administration of statins in patients with high-grade stenoses of the internal carotid artery.