Low Molecular Weight Form Research Articles

Abstract 634: Apelin as a Novel Therapeutic Target in Preeclampsia

Preeclampsia is a major cause for perinatal fetal and maternal morbidities and mortalities. The endogenous apelin system is an emerging target for the regulation of cardiovascular homeostasis; however, its role in pregnancy is not well understood. Apelin and its receptor are expressed in placental trophoblasts and fetal endothelial cells. We have reported that total immunoreactive apelin was lower in the placenta of preeclamptic women (PRE) compared to normotensive women (NT). The processing of the apelin precursor is complex and may yield a number of bioactive forms of the peptide. Presently, it is not known whether differences exist in the expression of the various molecular forms of apelin in the human placenta and whether the distribution pattern of apelin is altered in PRE. To address this issue, placental samples were pooled from NT (n=4) or PRE (n=4) at 37-38 weeks of gestation and the apelin forms resolved by HPLC coupled to an apelin radioimmunoassay (RIA). Total apelin content was lower in the placenta of PRE compared to NT (4.2 vs. 6.1 pg/mg wet weight). The HPLC analysis revealed negligible levels of apelin-36 from the placentas of NT (0.09 pg/mg wet weight) and PRE (0.11 pg/mg wet weight). A peak of apelin-17 was detected in both NT and PRE samples, but the shorter forms of apelin were more abundant. Therefore, separation conditions were tailored to achieve greater resolution of the lower molecular weight forms of apelin. This analysis revealed that the predominant apelin in both PRE and NT placentas was pyroglutamylated apelin-13 (Pyr-apelin-13; NT: 7.1 and PRE: 6.6 pg/mg wet weight); however, apelin-12 and apelin-13 were lower in PRE vs. NT placentas (1.8 and 22 fold, respectively). Lower expression of apelin-13 may reflect an inability to effectively influence blood pressure in PRE. Indeed, we further show that systemic infusion of apelin-13 (2 mg/kg/day) from day 13 to 20 of gestation reduced mean blood pressure (144±1 vs. 163±5 mmHg, n=4-6, p<0.01) in preeclamptic transgenic rats. In conclusion, we demonstrate that Pyr-apelin-13 is the predominant endogenous isoform of apelin in both NT and PRE placentas, while apelin-13 and apelin-12 are downregulated in PRE placentas. As a novel therapeutic agent, apelin may prevent the development of preeclampsia.

Evodiamine activates AMPK and promotes adiponectin multimerization in 3T3-L1 adipocytes

Adiponectin, an adipokine with insulin-sensitizing effect, is secreted from adipocytes into circulation as high, medium, and low molecular weight (HMW, MMW, and LMW) forms. The HMW adiponectin is more metabolically active and the ratio of HMW adiponectin to total adiponectin directly correlates with insulin sensitivity. Evodiamine is an indole alkaloid found in the traditional Chinese medicinal plant Evodia rutaecarpa. In this study, evodiamine was found to activate AMP-activated protein kinase (AMPK) in both 3T3-L1 adipocytes and 293T cells. Activation of AMPK by evodiamine promoted the assembly of HMW adiponectin and increased the HMW/total ratio of adiponectin in 3T3-L1 adipocytes. The Ca2+-dependent PI3K/Akt/CaMKII-signaling pathway was demonstrated to be involved in evodiamine-induced AMPK activation. This study revealed a novel role of this Ca2+-mediated signaling pathway in promoting the multimerization of adiponectin.

Elucidation of differences in N-glycosylation between different molecular weight forms of recombinant CLEC-2 by LC MALDI tandem MS

C-type lectin-like receptor 2 (CLEC-2) is a newly identified receptor expressed on the platelet surface. It has been reported that CLEC-2 exists as a higher molecular weight (HMW) and a lower molecular weight (LMW) form, which share the same protein core but differ in glycans. The two forms appear to have different ligand-binding abilities, indicating that the differential glycosylation of CLEC-2 possibly produces functionally distinct glycoforms. This study aimed to explore an easy method to directly elucidate the N-glycosylation difference by employing a glycoproteomics approach. The off-line coupling of nano-LC with a MALDI-QIT-TOF mass spectrometer was demonstrated to be capable of sensitive and direct elucidation of the glycosylation difference between HMW and LMW CLEC-2, simultaneously providing information about their oligosaccharide structures and the glycosylation sites. The results reveal that a specific glycosylation site, Asn 134, is differently glycosylated in the two forms, with complex types of bi-antennary, tri-antennary and tetra-antennary, N-linked, fucosylated glycans identified at this site in the HMW form but not in the LMW form. The observed difference in glycosylation might provide new insights into the underlying mechanisms of biological functions of CLEC-2. Because of its simplicity and sensitivity, the method explored in this work suggests that it holds promise as a method of elucidating differences in direct N-glycosylation of target glycoprotein, even in small amount of samples.

Synovial Fluid Lubricant Properties Are Transiently Deficient After Arthroscopic Articular Cartilage Defect Repair With Platelet-Enriched Fibrin Alone and With Mesenchymal Stem Cells

Background:Following various types of naturally occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan, and proteoglycan-4 (PRG4). However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown.Hypothesis:Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin + mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and PRG4 composition.Study Design:Controlled laboratory study.Methods:Equine synovial fluid was aspirated from normal joints at a preinjury state (0 days) and at 10 days and 3 months following fibrin or fibrin + mesenchymal stem cell repair of full-thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and PRG4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high–molecular weight hyaluronan to restore lubrication function.Results:Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to preinjury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months, while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (−64%) and shifted toward lower molecular weight forms, while PRG4 concentration was increased (+210%) in 10-day samples relative to preinjury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high–molecular weight hyaluronan. Addition of high–molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0 mg/mL restored kinetic friction to preinjury levels.Conclusion:Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high–molecular weight hyaluronan.Clinical Relevance:Synovial fluid lubrication is deficient shortly after arthroscopic cartilage repair surgery, and supplementation with high–molecular weight hyaluronan may be beneficial.

Modulation of mAb quality attributes using microliter scale fed-batch cultures.

A high-throughput DoE approach performed in a 96-deepwell plate system was used to explore the impact of media and feed components on main quality attributes of a monoclonal antibody. Six CHO-S derived clonal cell lines expressing the same monoclonal antibody were tested in two different cell culture media with six components added at three different levels. The resulting 384 culture conditions including controls were simultaneously tested in fed-batch conditions, and process performance such as viable cell density, viability, and product titer were monitored. At the end of the culture, supernatants from each condition were purified and the product was analyzed for N-glycan profiles, charge variant distribution, aggregates, and low molecular weight forms. The screening described here provided highly valuable insights into the factors and combination of factors that can be used to modulate the quality attributes of a molecule. The approach also revealed specific intrinsic differences of the selected clonal cell lines - some cell lines were very responsive in terms of changes in performance or quality attributes, whereas others were less affected by the factors tested in this study. Moreover, it indicated to what extent the attributes can be impacted within the selected experimental design space. The outcome correlated well with confirmations performed in larger cell culture volumes such as small-scale bioreactors. Being fast and resource effective, this integrated high-throughput approach can provide information which is particularly useful during early stage cell culture development.

The Role of IKKβ in Venezuelan Equine Encephalitis Virus Infection

Venezuelan equine encephalitis virus (VEEV) belongs to the genus Alphavirus, family Togaviridae. VEEV infection is characterized by extensive inflammation and studies from other laboratories implicated an involvement of the NF-κB cascade in the in vivo pathology. Initial studies indicated that at early time points of VEEV infection, the NF-κB complex was activated in cells infected with the TC-83 strain of VEEV. One upstream kinase that contributes to the phosphorylation of p65 is the IKKβ component of the IKK complex. Our previous studies with Rift valley fever virus, which exhibited early activation of the NF-κB cascade in infected cells, had indicated that the IKKβ component underwent macromolecular reorganization to form a novel low molecular weight form unique to infected cells. This prompted us to investigate if the IKK complex undergoes a comparable macromolecular reorganization in VEEV infection. Size-fractionated VEEV infected cell extracts indicated a macromolecular reorganization of IKKβ in VEEV infected cells that resulted in formation of lower molecular weight complexes. Well-documented inhibitors of IKKβ function, BAY-11-7082, BAY-11-7085 and IKK2 compound IV, were employed to determine whether IKKβ function was required for the production of infectious progeny virus. A decrease in infectious viral particles and viral RNA copies was observed with inhibitor treatment in the attenuated and virulent strains of VEEV infection. In order to further validate the requirement of IKKβ for VEEV replication, we over-expressed IKKβ in cells and observed an increase in viral titers. In contrast, studies carried out using IKKβ−/− cells demonstrated a decrease in VEEV replication. In vivo studies demonstrated that inhibitor treatment of TC-83 infected mice increased their survival. Finally, proteomics studies have revealed that IKKβ may interact with the viral protein nsP3. In conclusion, our studies have revealed that the host IKKβ protein may be critically involved in VEEV replication.

The High Molecular Weight Adiponectin Level is Associated with the Atherogenic Lipoprotein Profiles in Healthy Japanese Males

Circulating adiponectin comprises high, medium and low molecular weight (HMW, MMW, and LMW) forms. Decreased adiponectin levels have been demonstrated to correlate with the atherogenic lipoprotein profile in patients with metabolic syndrome(MS). However, the associations of these isoforms with the atherogenic lipoprotein profiles in the healthy population remain unclear. Apparently healthy male subjects were divided into non-MS(n=132) and MS(n=63) groups. We measured the total, HMW, MMW and LMW adiponectin levels by ELISA, and determined the detailed lipoprotein profiles by high-performance liquid chromatography. The total and HMW adiponectin levels in the MS group were significantly lower than those in the non-MS group(3.8±1.9 vs. 4.9±2.4μg/mL, p＜0.001 and 1.6±1.2 vs. 2.5±1.9μg/mL, p＜0.001, respectively), whereas the MMW and LMW levels did not differ significantly between the groups. The total and HMW adiponectin levels correlated with the atherogenic lipoprotein profiles, including the following: 1) increased cholesterol levels in the small LDL subclasses; 2) decreased cholesterol levels in the larger HDL subclasses; 3) increased triglycerides(TGs) in almost all VLDL and LDL subclasses and 4) decreased TGs in the large HDL and increased TGs in the small HDL subclasses. In addition, a multivariate analysis demonstrated that the HMW adiponectin level was an independent predictor of the small LDL and HDL levels(p=0.02 for both). The HMW, but not MMW or LMW, adiponectin levels are associated with the typical atherogenic lipoprotein profiles, independent of MS components. Measurement of the HMW adiponectin levels could be useful for identifying the atherogenicity in apparently healthy males.

Abstract P6-12-09: Targeting deregulated cyclin E in inflammatory breast cancer via sequential combination therapy

Abstract Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer, accounting for 2-5% of all breast cancers in the US and 10% of breast cancer deaths. Unfortunately, until recently this disease has not been studied extensively at the molecular/cellular level despite the compelling unmet need to develop more effective therapies. In spite of receiving aggressive multidisciplinary therapy (anthracycline-taxane-based neoadjuvant chemotherapy, modified-radical-mastectomy and adjuvant radiation therapy), only about 40% of IBC patients survive 5 years. The goal of this project is to identify new targets that can be exploited pharmacologically either in combination with current therapies or with other novel agents. Previous work has identified tumor-specific low molecular weight forms of cyclin E, termed LMW-E, that are preferentially found in triple negative breast cancers (TNBC) and serve as a poor prognostic biomarker. As a result of cleavage, LMW-E preferentially accumulates in the cytoplasm where it exerts its pleiotropic oncogenic functions including deregulating the cell cycle. Furthermore, we have shown that TNBC cells harboring LMW-E are sensitive to CDK inhibitors. To establish the clinical relevance of targeting the cyclin E/CDK2 axis in IBC, a pilot study was performed to determine the incidence of LMW-E. We performed IHC on 11 post-chemotherapy surgical specimens from MDACC. All tumors expressed cyclin E, 8 of which had predominantly cytoplasmic staining, indicative of LMW-E expression. These results are now being validated in a larger cohort of samples. These promising preliminary results led us to perform in vitro experiments to determine sensitivity to CDK2 inhibitors and characterize cell cycle profiles. Similar to non-IBC breast cancers, IBC tumors can be ER-positive, TNBC or HER2-overexpressing; however, there are only TN-IBC or HER2+ cell lines. We utilized 2 cell lines for these studies, one of which is TN-IBC, the other of which is ER-negative and HER2-overexpressing. These cell lines were treated with a panel of CDK inhibitors to evaluate their sensitivity in both short-term and long-term assays. The long-term assay we developed called the high-throughput survival assay was used to examine combinations of agents for potential synergies using isobologram analysis. We have demonstrated chemotherapy-specific synergistic cell death when cells are first treated with CDK inhibitor in IBC and non-IBC cell lines. Using flow cytometry, we have demonstrated that CDK inhibitors induce a G2/M arrest, which is further increased by sequential treatment with chemotherapy. Furthermore, combination treatment leads to cell death, as measured by sub-G1 DNA content. Taken together, these studies provide solid evidence that targeting CDK2 is a promising strategy in treating IBC tumors that express cyclin E. Beyond these early pre-clinical advances, however, we demonstrated clear sequence-specificity for certain pairs of drugs, implying that future clinical studies should be designed taking into account the mechanism of action of cell cycle targeting drugs. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-09.

T1 TNFα mediates ectodomain shedding of BMPR-II: a mechanism for inflammation as a trigger for pulmonary arterial hypertension

BackgroundMutations in BMP type II receptor (BMPR-II) account for 70% of heritable pulmonary arterial hypertension (PAH) cases, but low penetrance (~20%) in mutation carriers implies a ‘second hit’ is required...

Targeted therapy by gene transfer of a monovalent antibody fragment against the Met oncogenic receptor

Due to the key role played in critical sub-populations, Met is considered a relevant therapeutic target for glioblastoma multiforme and lung cancers. The anti-Met DN30 antibody, engineered to a monovalent Fab (Mv-DN30), proved to be a potent antagonist, inducing physical removal of Met receptor from the cell surface. In this study, we designed a gene therapy approach, challenging Mv-DN30 in preclinical models of Met-driven human glioblastoma and lung carcinoma. Mv-DN30 was delivered by a Tet-inducible-bidirectional lentiviral vector. Gene therapy solved the limitations dictated by the short half-life of the low molecular weight form of the antibody. In vitro, upon doxycycline induction, the transgene: (1) drove synthesis and secretion of the correctly assembled Mv-DN30; (2) triggered the displacement of Met receptor from the surface of target cancer cells; (3) suppressed the Met-mediated invasive growth phenotype. Induction of transgene expression in cancer cells-transplanted either subcutaneously or orthotopically in nude mice-resulted in inhibition of tumor growth. Direct Mv-DN30 gene transfer in nude mice, intra-tumor or systemic, was followed by a therapeutic response. These results provide proof of concept for a gene transfer immunotherapy strategy by a Fab fragment and encourage clinical studies targeting Met-driven cancers with Mv-DN30. Gene transfer allows the continuous in vivo production of therapeutic Fab fragments. Mv-DN30 is an excellent tool for the treatment of Met-driven cancers. Mv-DN30 gene therapy represents an innovative route for Met targeting.

The Succinoglycan Endoglycanase Encoded by exoK Is Required for Efficient Symbiosis of Sinorhizobium meliloti 1021 with the Host Plants Medicago truncatula and Medicago sativa (Alfalfa)

The acidic polysaccharide succinoglycan produced by the nitrogen-fixing rhizobial symbiont Sinorhizobium meliloti 1021 is required for this bacterium to invade the host plant Medicago truncatula and to efficiently invade the host plant M. sativa (alfalfa). The β-glucanase enzyme encoded by exoK has previously been demonstrated to cleave succinoglycan and participate in producing the low molecular weight form of this polysaccharide. Here, we show that exoK is required for efficient S. meliloti invasion of both M. truncatula and alfalfa. Deletion mutants of exoK have a substantial reduction in symbiotic productivity on both of these plant hosts. Insertion mutants of exoK have an even less productive symbiosis than the deletion mutants with the host M. truncatula that is caused by a secondary effect of the insertion itself, and may be due to a polar effect on the expression of the downstream exoLAMON genes.

Distinct reactivity of the commercially available monoclonal antibodies of D-dimer and plasma FDP testing to the molecular variants of fibrin degradation products

Fibrin degradation products (FDP) are an important marker of coagulopathy. We assessed the reactivity of the monoclonal antibodies used in clinical laboratory testing (6 D-dimer reagents, D-dimer-1-6; 4 plasma FDP reagents, plasma FDP-1-4) to quantify FDP using in vitro-generated FDP as well as FDP in clinical samples. The monoclonal antibodies used in D-dimer-1, -2, -5, and -6 reacted poorly to the low molecular weight forms of in vitro-generated FDP. The monoclonal antibodies used in D-dimer-3 and -4 had better reactivity to the low molecular weight forms of in vitro-generated FDP. The monoclonal antibodies used in plasma FDP-2, -3, and -4 reacted well to the high and low molecular weight FDP forms, while the monoclonal antibody in plasma FDP-1 reacted poorly to the low molecular weight FDP forms. Analysis of clinical samples revealed deviations in FDP molecular weight forms in DIC samples. The reactivity of the monoclonal antibodies of laboratory FDP testing to FDP variants in clinical samples was similar to that of in vitro-generated FDP. In conclusion, the monoclonal antibodies used in clinical laboratories to detect FDP have distinct reactivity to the molecular variants of FDP generated in vitro as well as those present in clinical samples. Our findings support the consensus for the standardization of D-dimer and plasma FDP testing.

OC-092 Alteration in Mucosal Keratin 8 Level, Phosphorylation and Relative Ratio to Vimentin Associate with development of Colitis-Associated Cancer

Introduction Intermediate filament(IF) proteins keratin(K) and vimentin(VIM) are cellular cytoskeleton components. Mutations of K8 gene associated with inefficient IF assembly is seen in subset of IBD patients. VIM a marker of epithelial-mesenchymal transformation is associated with aggressive colorectal cancer(CRC). We have previously shown alterations in mucosal IF proteins on mass spectrometry(MS) in UC patients at differing risk of CRC. We aimed to validate changes noted on MS and additionally investigate post-translational modifications in K8. Methods Rectal biopsies were studied from the following groups of patients with UC: quiescent long standing pancolitis(LSPC-20–40years); inactive recent onset UC(ROUC Results Multiple K8 forms were noted; relative to CON, low K8 levels & molecular weight(MW) forms (37kDa) was seen in ROUC and DT (Fig A). Similar to MS results, in ACT there was reduction in K8, K18, K19 & VIM levels with low MW K8 forms (FigB). Vimentin was increased in LSPC, PSC, DR & DT compared to CON (Fig A); progressive increase in VIM:K8 ratio was noted (DT > DR > PSC > LSPC > ROUC- Fig C). K8 phosphorylation was reduced or absent in ACT and ROUC (Fig A&B), with diminished levels noted in PSC, DR and DT compared to LSPC. K8pS23 levels relative to total K8, was comparable in CON and LSPC but showed a reduced ratio in ROUC & dysplasia(Fig D). Conclusion K8 levels and phosphorylation are reduced in acute inflammation and restore/overcompensate in longstanding quiescent disease. This restoration is not apparent in recently inflamed mucosa (ROUC) despite endoscopic and histological remission; with similar changes seen in more aggressive disease. Recurrent bouts of acute inflammation in the mucosa with associated failure of restoration of K8 integrity may be associated with increased colon cancer risk. An altered VIM:K8 ratio may in addition be a surrogate mucosal marker of disease progression. Disclosure of Interest None Declared

PWE-167 Identification of Altered Keratin Level in Cancerized Colonic Fields using Isobaric Tags for Relative and Absolute Quantification (ITRAQ) for Protein Profiling

IntroductionA cancerized field is an area of abnormal tissue in the vicinity of a cancer that macroscopically appears normal, and which may be responsible for neoplastic recurrence. Evidence from keratin-8-deficient...

Hyaluronic acid/CD44 interactions control CD8 T cell memory development to influenza virus infection. (P1450)

Abstract CD44 is upregulated upon T cell activation during an immune response and remains highly expressed on memory T cells, which contribute to protection against challenge infection. However, its role in CD8 T cell response during infection is not well understood. Its main ligand is hyaluronic acid (HA), a glycosaminoglycan component of the extracellular matrix. In the steady state, HA exists at high molecular weight. Upon local inflammation, it is transiently broken down to low molecular weight forms. During influenza infection, we found that CD44-/- CD8 T cells accumulate normally during the expansion phase, but have an intrinsic defect in becoming memory cells. Furthermore, upon reinfection, the few CD44-/- memory CD8 T cells expand less than WT cells. CD44/HA binding is transiently induced in activated T cells, and we show that this interaction is crucial for effector CD8 T cell accumulation during infection. In addition, treatment of infected mice with IRAWB14, an agonist antibody promoting HA/CD44 binding, increased the accumulation of effector CD8 T cells. Correlating with an important role for low molecular weight HA, we found that IRAWB14 treatment is beneficial only if administered during the first phase of the infection, when low molecular weight HA is produced. Our data reveal that HA/CD44 interaction is an early essential and major regulator of the CD8 T cell immune response that controls the development of memory to influenza infection.

OLIGOMERIC ADIPONECTIN FORMS AND THEIR COMPLEXES IN THE BLOOD OF HEALTHY DONORS AND PATIENTS WITH TYPE 2 DIABETES MELLITUS

Adiponectin (Adn) is a protein that circulates in the blood in several oligomeric forms, namely low-, medium-, and high-molecular-weight forms. Adn may serve as a risk factor for type 2 diabetes mellitus (T2DM). The aims of this work were (1) to produce monoclonal antibodies (MAbs) specific to different Adn oligomeric forms, (2) to design immunoassays suitable for measuring the Adn forms present in human blood, and (3) to investigate the changes in Adn forms that occur in patients with T2DM. Gel filtration, fluoroimmunoassays, and Western blotting were utilized as major techniques in this study. MAbs recognizing various oligomeric forms of Adn were obtained. Complexes between Adn and complement component C1q and between the low molecular weight form of Adn and albumin were described in human blood. A decrease in the total Adn and Adn-albumin complex levels in the blood of patients with T2DM and no difference in the levels of the Adn-C1q complex in comparison with healthy volunteers were demonstrated. An Adn94-Adn63 fluoroimmunoassay was selected as the technique that most accurately measured the mass ratio of Adn oligomers in blood samples, and an Adn214-Adn27 assay that measured the low-molecular-weight form of Adn only.

Bioavailability and size-fraction of dissolved organic carbon, nitrogen, and sulfur at the Arbutus Lake watershed, Adirondack Mountains, NY

The heterogeneity of DOM is closely linked with the various sources and the diversity of biogeochemical processes. We studied the spatial and temporal patterns of the quantity (bioavailable [B-] and refractory concentrations by laboratory incubations) and quality (δ13C, aromaticity, and size-fraction by ultrafiltration [low molecular weight (LMW) 1 kDa]) of dissolved organic C (DOC), N (DON), and S (DOS) for surface waters (two upland streams, two wetland-affected streams, and lake outlet) over a 14-month period within the Arbutus Lake watershed in the Adirondack Mountains of New York State, USA. The % BDOC and % BDON of this watershed averages ranged from 6 to 18 % and from 12 to 43 %, respectively. The DOC and DON concentrations increased as water was transported through wetland areas of the Arbutus Lake watershed. DOC and DON constituents in the surface waters passing through a wetland were composed mostly of refractory HMW components (% HMWDOC: 55 %, % HMWDON: 60 %) with a higher level of aromaticity compared to upland streams (% HMWDOC: approximately 35 %, % HMWDON: approximately 30 %). DOS was dominated by the refractory (% BDOS range 6–13 %) and LMW (% LMWDOS range 62–96 %) form and we suggest that bacterial dissimilatory sulfate reduction might play an important role in generating this distinct DOS biogeochemistry. The aromaticity was positively related to total DOC concentration, but negatively to % BDOC. Arbutus Lake DOM was dominated by bioavailable and LMW characteristics, compared to wetland-affected streams where refractory and HMW DOM fractions were more prevalent. Our study suggests the different variability of DOM characteristics among elements (C, N, S) using a “bioavailability-molecular size model” showing a diagenetic perspective due to the relative refractoriness of the LMW DOS. This study also highlights the importance of multiple approaches for understanding DOM biogeochemistry with respect to molecular size, bioavailability, aromaticity, stoichiometry, isotopic values, and elemental concentrations.

Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation

BackgroundThe balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling.MethodsUsing human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1β and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 μg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated.ResultsInflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P< 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 μM, suggesting a class effect.ConclusionThese findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.

Hyaluronan and Layilin Mediate Loss of Airway Epithelial Barrier Function Induced by Cigarette Smoke by Decreasing E-cadherin

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.

Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFβ1 in diabetic eyes undergoing vitrectomy

Angiogenesis in diabetic retinopathy (DR) is a multifactorial process regulated by hypoxia-induced growth factors and inflammatory cytokines. In addition to the angiogenic switch, the proteolytic processing and altered synthesis of the extracellular matrix are critical steps in this disease. This study was performed to evaluate the levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9), angiopoietin-1 and angiopoietin-2 (Ang-1 and Ang-2), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and transforming growth factor-β1 (totalTGFβ1) in the vitreous of diabetic eyes undergoing vitrectomy compared with control eyes operated because of macular hole or pucker. Prospective consecutive controlled observational study performed in the unit of vitreoretinal surgery in Finland during the years 2006-2008. Vitreous samples were collected before the start of the conventional 3-ppp vitrectomy. Vitreous MMP-2 and MMP-9, Ang-1 and Ang-2, VEGF, EPO and TGFβ1 concentrations were measured from 69 patients with Type 1 or 2 diabetes and 40 controls. Comparison of eyes with DR with controls revealed that the mean vitreous concentrations of proMMP-2 (p = 0.0015), totalMMP-2 (p = 0.0011), proMMP-9 (p = 0.00001), totalMMP-9 (p < 0.00001), Ang-2 (p < 0.00001), VEGF (p < 0.00001), EPO (p < 0.00001) and totalTGFβ1 (p = 0.000026) were significantly higher in the former group. A multivariate logistic regression analysis suggested intravitreal Ang-2 concentration being the key marker of PDR (p = 0.00025) (OR = 1507.9). The main new finding is that the intravitreal concentrations of Ang-2 correlated significantly with MMP-9, VEGF, EPO and TGFβ1 levels in diabetic eyes undergoing vitrectomy. Thus, these factors could promote retinal angiogenesis synergistically.