Collagenase expression and activity is modulated by the interaction of collagen types, hypoxia, and nutrition in human lung cells

Hypoxia not only controls organogenesis, embryogenesis, and wound repair, but also triggers tumor progression and metastasis. Matrix metalloproteinases (MMP), especially gelatinases (MMP-2, MMP-9) regulate the composition and stability of the extracellular matrix (ECM), which affects cell proliferation, migration, and differentiation. This study investigated the effect of hypoxia alone and in combination with ECM compounds and nutrition on MMP-2 and MMP-9 expression, activity, and synthesis in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMC). We also determined the expression of the tissue inhibitors of MMP (TIMP-1, -2). Cells were grown on plastic, collagen-I, collagen-IV, or gelatin and in either starving medium (0.1% serum) or growth medium (5% serum), and were subjected to normoxia or hypoxia (1% O(2)). Collagenases expression was determined by zymography. TIMP-1, -2 expression was assessed by Western blotting and RT-PCR. Depending on serum concentration human lung cells expressed pro-MMP-2 on all substrates. Hypoxia increased pro-MMP-2 expression, on collagen type I or type IV further via Erk1/2 and p38 MAP kinase signaling. MMP-9 was only expressed when cells were grown on collagen type IV and increased with serum concentration, and by hypoxia. TIMP-1 expression was only expressed when cells were grown on collagen type I and was significantly increased by hypoxia, while TIMP-2 expression was unchanged. We demonstrated that the hypoxia, ECM composition, and nutrition, rather than one of these conditions alone, modulate the expression and activity of collagenases and their inhibitors in primary human lung fibroblasts.     

Towards alpha- or beta-D-C-glycosyl compounds by tin-catalyzed addition of glycosyl radicals to acrylonitrile and vinylphosphonate,and flexible reduction of tetra-O-acetyl-alpha-D-glucopyranosyl bromide with cyanoborohydride

Photo-induced radical addition of acetylated alpha-D-glucopyranosyl bromide (1). to acrylonitrile or diethyl vinylphosphonate, in the presence of catalytic amounts of tri-n-butyltin chloride and sodium (or tetra-n-butylammonium) cyanoborohydride in excess, allowed efficient preparations of alpha-configurated nonononitrile and 2-(alpha-D-glucopyranosyl)-ethylphosphonate (79, 70% yields, respectively). These conditions led to 2-(alpha-D-manno-, and galactopyranosyl)-ethylphosphonates in 68 and 76% yields. Similarly, radical addition of acetylated 1-bromo-beta-D-glucopyranosyl chloride (2). to acrylonitrile or diethyl vinylphosphonate afforded mainly intermediate chlorides which, upon radical reduction with excess tri-n-butyltin hydride, afforded the corresponding beta anomers (40 and 38%, respectively) by sequential C-C and C-H bond formation. Stereocontrol relies on the alpha-stereoselective quenching of D-glycopyranos-1-yl radicals. We found also that UV light irradiation of 1 with excess NaBH(3)CN in tert-butanol afforded either 1,3,4,6-tetra-O-acetyl-2-deoxy-alpha-D-arabino-hexopyranose (65% after crystallization) or, when 10% mol thiophenol was added, 2,3,4,6-tetra-O-acetyl-1,5-anhydro-D-glucitol (79%). These are simple, tin-free, and easily controlled conditions, which compare well with known preparations of these reduced sugars.     

Contemporary carbon stocks of mineral forest soils in the Swiss Alps

Soil organic carbon (SOC) has been identified as the main globalterrestrial carbon reservoir, but considerable uncertainty remains as toregional SOC variability and the distribution of C between vegetationand soil. We used gridded forest soil data (8–km × 8–km)representative of Swiss forests in terms of climate and forest typedistribution to analyse spatial patterns of mineral SOC stocks alonggradients in the European Alps for the year 1993. At stand level, meanSOC stocks of 98 t C ha^–1 (N = 168,coefficient of variation: 70%) were obtained for the entiremineral soil profile, 76 t C ha^–1 (N =137, CV: 50%) in 0–30 cm topsoil, and 62 t Cha^–1 (N = 156, CV: 46%) in0–20 cm topsoil. Extrapolating to national scale, we calculatedcontemporary SOC stocks of 110 Tg C (entire mineral soil, standarderror: 6 Tg C), 87 Tg C (0–30 cm topsoil, standarderror: 3.5 Tg C) and 70 Tg C (0–20 cm topsoil, standarderror: 2.5 Tg C) for mineral soils of accessible Swiss forests(1.1399 Mha). According to our estimate, the 0–20 cm layers ofmineral forest soils in Switzerland store about half of the Csequestered by forest trees (136 Tg C) and more than five times morethan organic horizons (13.2 Tg C).At stand level, regression analyses on the entire data set yielded nostrong climatic or topographic signature for forest SOC stocks in top(0–20 cm) and entire mineral soils across the Alps, despite thewide range of values of site parameters. Similarly, geostatisticalanalyses revealed no clear spatial trends for SOC in Switzerland at thescale of sampling. Using subsets, biotic, abiotic controls andcategorial variables (forest type, region) explained nearly 60%of the SOC variability in topsoil mineral layers (0–20 cm) forbroadleaf stands (N = 56), but only little of thevariability in needleleaf stands (N = 91,R ² = 0.23 for topsoil layers).Considerable uncertainties remain in assessments of SOC stocks, due tounquantified errors in soil density and rock fraction, lack of data onwithin-site SOC variability and missing or poorly quantifiedenvironmental control parameters. Considering further spatial SOCvariability, replicate pointwise soil sampling at 8–km × 8–kmresolution without organic horizons will thus hardly allow to detectchanges in SOC stocks in strongly heterogeneous mountain landscapes.     

Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling

Thiazolidinediones (TZDs) are insulin-sensitizing agents that also decrease systemic blood pressure, attenuate the formation of atherosclerotic lesions, and block remodeling of injured arterial walls. Recently, TZDs were shown to prevent pulmonary arterial (PA) remodeling in rats treated with monocrotaline. Presently we report studies testing the ability of the TZD rosiglitazone (ROSI) to attenuate pathological arterial remodeling in the lung and prevent the development of pulmonary hypertension (PH) in rats subjected to chronic hypoxia. PA remodeling was reduced in ROSI-treated animals exposed to hypoxia compared with animals exposed to hypoxia alone. ROSI treatment blocked muscularization of distal pulmonary arterioles and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia. Decreased PA remodeling in ROSI-treated animals was associated with decreased smooth muscle cell proliferation, decreased collagen and elastin deposition, and increased matrix metalloproteinase-2 activity in the PA wall. Cells expressing the c-Kit cell surface marker were observed in the PA adventitia of untreated animals exposed to hypoxia but not in ROSI-treated hypoxic rats. Right ventricular hypertrophy and cardiomyocyte hypertrophy were also blunted in ROSI-treated hypoxic animals. Interestingly, mean PA pressures were elevated equally in the untreated and ROSI-treated groups, indicating that ROSI had no effect on the development of PH. However, mean PA pressure was normalized acutely in both groups of hypoxia-exposed animals by Fasudil, an agent that inhibits RhoA/Rho kinase-mediated vasoconstriction. We conclude that ROSI can attenuate and reverse PA remodeling and neomuscularization associated with hypoxic PH. However, this agent fails to block the development of PH, apparently because of its inability to repress sustained Rho kinase-mediated arterial vasoconstriction.     

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.     

Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy

Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.Congenital obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children (1). The most frequently found cause of congenital obstructive nephropathy is ureteropelvic junction (UPJ)¹ obstruction with an estimated incidence of 1 in 1000–1500 births. Milder forms of UPJ obstruction often progress to the spontaneous resolution of the pathology over time. This has led to a watchful waiting approach with surgical intervention only if renal deterioration is detected (2). Although this medical surveillance prevents unnecessary surgery, it mostly relies on invasive follow-up. Consequently with the aim to reduce this invasive follow-up, several groups have initiated research to identify noninvasive urinary biomarkers of UPJ obstruction using both targeted and nontargeted (e.g. proteome analysis based) strategies. Targeted strategies including urinary cytokine expression analyses failed to clearly determine the need for surgery in UPJ obstruction (3, 4). On the other hand, untargeted strategies have been more successful and by using urinary proteomics, biomarkers for renal and non-renal diseases have been identified (5–9). Using urinary peptidome analysis, we identified and validated a urinary peptide panel that predicted the clinical outcome of newborns with UPJ obstruction with 97% accuracy several months in advance (3, 10). An independent small-scale study confirmed the efficiency of this biomarker panel (7). These studies indicate the potential of urinary proteomics to predict the clinical fate of patients with UPJ obstruction. Although these endogenous urinary peptide biomarkers are of great potential clinical value, sequencing of these biomarkers mainly identified collagen fragments that are less informative on the pathophysiology of the disease. In contrast, studies of the high molecular weight urinary proteome (i.e. proteins) might be more informative on the pathophysiology of disease. Different approaches have been used in the past to characterize the urinary proteome, either by 2D-gel electrophoresis coupled to mass spectrometry (11, 12) or reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis (13–16). In-depth proteome analysis using extensive fractionation of the sample and high resolution, fast sequencing mass spectrometers have reported the identification of >2000 proteins in normal human urine (13, 15, 16). Here, we applied quantitative high-resolution label free LC-MS/MS analysis for the identification of urinary proteins associated to UPJ obstruction in newborns. Among a number of proteins uniquely associated with severe UPJ obstruction, we identified Arginase 1, not previously recognized in UPJ obstruction. Using an independent larger cohort, we further verified reduced urinary abundance of Arginase 1 using both Western blot and multiple reaction monitoring (MRM). Using the mouse model of obstructive nephropathy, we observed that the expression of arginases is modulated in situ in obstructed kidneys. Further gene expression analysis of the arginase pathway allowed us to hypothesize for a role of arginases in the development of fibrotic lesions in obstructive nephropathy.     

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.     

Body context and posture affect mental imagery of hands

Different visual stimuli have been shown to recruit different mental imagery strategies. However the role of specific visual stimuli properties related to body context and posture in mental imagery is still under debate. Aiming to dissociate the behavioural correlates of mental processing of visual stimuli characterized by different body context, in the present study we investigated whether the mental rotation of stimuli showing either hands as attached to a body (hands-on-body) or not (hands-only), would be based on different mechanisms. We further examined the effects of postural changes on the mental rotation of both stimuli. Thirty healthy volunteers verbally judged the laterality of rotated hands-only and hands-on-body stimuli presented from the dorsum- or the palm-view, while positioning their hands on their knees (front postural condition) or behind their back (back postural condition). Mental rotation of hands-only, but not of hands-on-body, was modulated by the stimulus view and orientation. Additionally, only the hands-only stimuli were mentally rotated at different speeds according to the postural conditions. This indicates that different stimulus-related mechanisms are recruited in mental rotation by changing the bodily context in which a particular body part is presented. The present data suggest that, with respect to hands-only, mental rotation of hands-on-body is less dependent on biomechanical constraints and proprioceptive input. We interpret our results as evidence for preferential processing of visual- rather than kinesthetic-based mechanisms during mental transformation of hands-on-body and hands-only, respectively.     

White Matter Reorganization and Functional Response after Focal Cerebral Ischemia in the Rat

After stroke, the brain has shown to be able to achieve spontaneous functional recovery despite severe cerebral damage. This phenomenon is poorly understood. To address this issue, focal transient ischemia was induced by 60 min middle cerebral artery occlusion in Wistar rats. The evolution of stroke was followed using two magnetic resonance imaging modalities: diffusion spectrum imaging (acquired before, one and four weeks after stroke) and functional magnetic resonance imaging (acquired before and five weeks after stroke). To confirm the imaging observations, immunohistochemical staining for myelin, astrocytes and macrophages/microglia was added. At four weeks after stroke, a focal alteration of the diffusion anisotropy was observed between the ipsilesional ventricle and the lesion area. Using tractography this perturbation was identified as reorganization of the ipsilesional internal capsule. Functional imaging at five weeks after ischemia demonstrated activation of the primary sensorimotor cortex in both hemispheres in all rats except one animal lacking a functional response in the ipsilesional cortex. Furthermore, fiber tracking showed a transhemispheric fiber connection through the corpus callosum, which-in the rat without functional recovery-was lost. Our study shows the influence of the internal capsule reorganization, combined with inter-hemispheric connections though the corpus callosum, on the functional activation of the brain from stroke. In conclusion, tractography opens a new door to non-invasively investigate the structural correlates of lack of functional recovery after stroke.