Human Mast Cell Proteome Reveals Unique Lineage, Putative Functions, and Structural Basis for Cell Ablation.

Mast cells are rare tissue-resident cells of importance to human allergies. To understand the structural basis of principle mast cell functions, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast-cell-specific proteome signature, indicative of a unique lineage, only distantly related to other immune cell types, including innate immune cells. Proteome comparison between human and mouse suggested evolutionary conservation of core mast cell functions. In addition to specific proteases and proteins associated with degranulation and proteoglycan biosynthesis, mast cells expressed proteins potentially involved in interactions with neurons and neurotransmitter metabolism, including cell adhesion molecules, ion channels, and G protein coupled receptors. Toward targeted cell ablation in severe allergic diseases, we used MRGPRX2 for mast cell depletion in human skin biopsies. These proteome analyses suggest a unique role of mast cells in the immune system, probably intertwined with the nervous system.

Rare and low-frequency coding variants alter human adult height.

Height is a highly heritable, classic polygenic trait with approximately 700 common associated variants identified through genome-wide association studies so far. Here, we report 83 height-associated coding variants with lower minor-allele frequencies (in the range of 0.1-4.8%) and effects of up to 2 centimetres per allele (such as those in IHH, STC2, AR and CRISPLD2), greater than ten times the average effect of common variants. In functional follow-up studies, rare height-increasing alleles of STC2 (giving an increase of 1-2 centimetres per allele) compromised proteolytic inhibition of PAPP-A and increased cleavage of IGFBP-4 in vitro, resulting in higher bioavailability of insulin-like growth factors. These 83 height-associated variants overlap genes that are mutated in monogenic growth disorders and highlight new biological candidates (such as ADAMTS3, IL11RA and NOX4) and pathways (such as proteoglycan and glycosaminoglycan synthesis) involved in growth. Our results demonstrate that sufficiently large sample sizes can uncover rare and low-frequency variants of moderate-to-large effect associated with polygenic human phenotypes, and that these variants implicate relevant genes and pathways.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation.

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1ß), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1ß. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1ß. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

Endocan, a novel inflammatory marker, is upregulated in human chondrocytes stimulated with IL-1 beta.

Endocan is a circulating proteoglycan, involved in immunity, inflammation, and endothelial function. It has been recently suggested as a biomarker of inflammation, increased angiogenesis, and cancer. In vitro studies have shown that endocan expression could be upregulated by inflammatory cytokines and proangiogenic molecules. High endocan levels were also shown in arthritic joint tissues and particularly in sites characterized by severe inflammation. This study was performed to evaluate endocan expression in chondrocytes stimulated with IL-ß. mRNA and related protein production were measured for endocan, TNF-α, and IL-6. NF-kB activity was also evaluated. IL-1ß treatment induced a significant upregulation of both endocan and the inflammatory parameters as well as NF-kB activity. The treatment of chondrocytes with the specific NF-kB inhibitor before IL-1ß stimulation was able to reduce endocan and the inflammatory markers over-expression. The results of our study indicated that endocan is also expressed in human chondrocytes; furthermore, consistent with previous results in other cell types and tissues, IL-1ß-induced inflammatory response involves the expression of endocan through NF-kB activation. In this context, endocan seems to be an important inflammatory marker associated with the activation of NF-kB pathway.

Exploration of human xylosyltransferase for chemoenzymatic synthesis of proteoglycan linkage region.

Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature, PGs are highly challenging synthetic targets due to the difficulties in integrating the sulfated glycans with the peptide backbone. To expedite the synthesis, herein, the utility of human xylosyltransferase I (XT-I), the enzyme responsible for initiating PG synthesis, has been explored. XT-I was found to be capable of efficiently installing the xylose unit onto a variety of peptide structures on mg scales. Furthermore, an unnatural sugar, i.e., 6-azidoglucose can be transferred by XT-I introducing a reactive handle onto the glycopeptide for selective functionalization. XT-I can be coupled with ß-4-galactosyl transferase-7 for one pot synthesis of glycopeptides bearing galactose-xylose disaccharide, paving the way toward efficient chemoenzymatic synthesis of PG glycopeptides and glycoproteins.

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial-mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

The effect of hydrostatic pressure on proteoglycan production in articular cartilage in vitro: a meta-analysis.

OBJECTIVE: In previous research the use of hydrostatic pressure (HP) has been applied to enhance the formation of engineered cartilage, through the up-regulation of proteoglycan synthesis by mechanotransduction. However, the HP stimulation approach has been shown to vary between studies with a wide disparity in results, including anabolic, catabolic and non-responsive outcomes. To this end, a meta-analysis of HP publications using 3D cultured chondrocytes was performed to elucidate the key experiment factors involved in achieving a mechanotransducive response. DESIGN: The effects of different HP regimes on proteoglycan production were investigated based on the following factors: static vs dynamic application, pressure magnitude, and experiment duration. Meta-analysis was performed on raw data taken from 11 publications which employed either aggrecan gene expression analysis or dimethyl methylene blue colorimetric assay. The measure of effect was calculated based on mean difference using a random effects model. RESULTS: Analysis revealed that a significant anabolic response was most likely achieved when the following factors were employed; a static HP application, a magnitude within the mid-high physiological range of cartilage (≤5-10 MPa) and a study duration of ≥2 weeks. CONCLUSIONS: Thus, we propose that the selection of HP experiment factors can have a significant influence on engineered cartilage development, and that the results of this meta-analysis can be used as a basis for the planning of future HP experiments.

Intravitreal Pharmacokinetic Study of the Antiangiogenic Glycoprotein Opticin.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.

Deficiency of Natriuretic Peptide Receptor 2 Promotes Bicuspid Aortic Valves, Aortic Valve Disease, Left Ventricular Dysfunction, and Ascending Aortic Dilatations in Mice.

RATIONALE: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo. OBJECTIVE: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease. METHODS AND RESULTS: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein-coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2+/- and Npr2+/-;Ldlr-/- mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2+/- fibroblasts was insensitive to CNP treatment, whereas aged Npr2+/- and Npr2+/-;Ldlr-/- mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2+/- and Npr2+/-;Ldlr-/- mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2+/- with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2+/- valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2+/-;Ldlr-/- mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2+/- mice with bicuspid aortic valves than those with tricuspid valves. CONCLUSIONS: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

HMGA2 functions as a competing endogenous RNA to promote lung cancer progression.

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-ß co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-ß signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.

Production, partial purification and characterization of a proteoglycan bioemulsifier from an oleaginous yeast.

In this study, Meyerozyma caribbica, an indigenously isolated oleaginous yeast, produced in media containing glucose a bioemulsifier that was partially characterized as a proteoglycan based on preliminary analysis. Optimization of carbon:nitrogen (C:N) ratio revealed 30:1 as the suitable ratio for enhanced production. Apart from higher emulsification activity (E24: 70-80%), this molecule showed strong emulsion stability over a wide range of pH (2.0-9.0), salinity (0.05%-10%, w/v) and temperature (- 80 °C to + 50 °C). The current study emphasizes on the determination of critical media parameters for improved and stable bioemulsifier production coupled with partial characterization and identification of the molecule. Thus, a proteoglycan-based bioemulsifier with such a stable emulsifying property can serve as a versatile and potential component in food, cosmetics and pharmaceutical formulations.

Selective Elimination of NG2-Expressing Hair Follicle Stem Cells Exacerbates the Sensitization Phase of Contact Dermatitis in a Transgenic Rat Model.

The hair cycle consists of three different phases: anagen (growth), catagen (regression), and telogen (resting). During the anagen phase, hair follicle stem cells (HFSCs) in the bulge and the secondary hair germ proliferate and generate the outer and inner root sheath cells and the hair shafts. We previously identified NG2-immunoreactive (NG2+) cells as HFSCs in both regions of the hair follicles. Recently, the interaction between the hair cycle and the cutaneous immune system has been re-examined under physiological and pathological conditions. However, the roles of NG2+ HFSCs in the skin's immune system remain completely elucidated. In the present study, we investigated whether the elimination of NG2+ HFSCs affects the induction of allergic contact dermatitis, using a herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene system. When the GCV solution was applied to the skin of NG2-HSVtk transgenic (Tg) rats during the depilation-induced anagen phase, NG2+ HFSCs in the Tg rat skin induced apoptotic cell death. Under exposure of a hapten, the selective ablation of NG2+ HFSCs during the anagen phase aggravated the sensitization phase of allergic contact dermatitis. These findings suggest that NG2+ HFSCs and their progeny have immunosuppressive abilities during the anagen phase.

Early embryonic NG2 glia are exclusively gliogenic and do not generate neurons in the brain.

In the central nervous system, the type I transmembrane glycoprotein NG2 (nerve-glia antigen 2) is only expressed by pericytes and oligodendrocyte precursor cells (OPCs). Therefore, OPCs are also termed NG2 glia. Their fate during development has been investigated systematically in several genetically modified mouse models. Consensus exists that postnatal NG2 glia are restricted to the oligodendrocyte (OL) lineage, while, at least in the forebrain, embryonic NG2 glia could also generate astrocytes. In addition, experimental evidence for a neurogenic potential of NG2 glia in the early embryonic brain (before E16.5) has been provided. However, this observation is still controversial. Here, we took advantage of reliable transgene expression in NG2-EYFP and NG2-CreERT2 knock-in mice to study the fate of early embryonic NG2 glia. While pericytes were the main cells with robust NG2 gene activity at E12.5, only a few OPCs expressed NG2 at this early stage of embryogenesis. Subsequently, this proportion of OPCs increased from 3% (E12.5) to 11% and 25% at E14.5 and E17.5, respectively. When Cre DNA recombinase activity was induced at E12.5 and E14.5 and pups were analyzed at postnatal day 0 (P0) and P10, the vast majority of recombined cells, besides pericytes, belonged to the OL lineage cells, with few astrocytes in the ventral forebrain. In other brain regions such as brain stem, cerebellum, and olfactory bulb only OL lineage cells were detected. Therefore, we conclude that NG2 glia from early embryonic brain are restricted to a gliogenic fate and do not differentiate into neurons after birth.

Evaluating the Effects of Injected Calcium Hydroxylapatite on Changes in Human Skin Elastin and Proteoglycan Formation.

BACKGROUND: Previous studies have shown that calcium hydroxylapatite (CaHa) injected intradermally resulted in new collagen production at 6 months after injection, and a possible increase in elastin formation. In addition, a recent study showed the formation of new collagen, elastin, and angiogenesis after injection at 4 and 9 months. OBJECTIVE: To evaluate any changes in the presence of elastic fibers, proteoglycans, and elastin in photodamaged skin after injections with CaHa. METHODS AND MATERIALS: Fifteen subjects underwent a punch biopsy of the right infra-auricular areas before and after injection of CaHa on day 180. Specimens were stained for elastic fibers, elastin, and proteoglycan presence. RESULTS: Quantitative analysis demonstrated a percent change in elastic fibers varying between 29% and 179% at 6 months in comparison with baseline. Subjects showed an increase in elastin between 12% and 66%. Subjects had positive mean percentage change in proteoglycans of 76.27% (t-test of 0.198). CONCLUSION: This is the first study to show that CaHa can increase proteoglycans and echoed previous studies showing it can also have an effect on elastin, which indicates it can induce remodeling of all aspects of the extracellular matrix. Much larger and longer studies are required to confirm its unique impact on collagen, elastin, and proteoglycans.

Conditional Deletion of the L-Type Calcium Channel Cav1.2 in NG2-Positive Cells Impairs Remyelination in Mice.

Exploring the molecular mechanisms that drive the maturation of oligodendrocyte progenitor cells (OPCs) during the remyelination process is essential to developing new therapeutic tools to intervene in demyelinating diseases such as multiple sclerosis. To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for OPC development during remyelination, we generated an inducible conditional knock-out mouse in which the L-VGCC isoform Cav1.2 was deleted in NG2-positive OPCs (Cav1.2KO). Using the cuprizone (CPZ) model of demyelination and mice of either sex, we establish that Cav1.2 deletion in OPCs leads to less efficient remyelination of the adult brain. Specifically, Cav1.2KO OPCs mature slower and produce less myelin than control oligodendrocytes during the recovery period after CPZ intoxication. This reduced remyelination was accompanied by an important decline in the number of myelinating oligodendrocytes and in the rate of OPC proliferation. Furthermore, during the remyelination phase of the CPZ model, the corpus callosum of Cav1.2KO animals presented a significant decrease in the percentage of myelinated axons and a substantial increase in the mean g-ratio of myelinated axons compared with controls. In addition, in a mouse line in which the Cav1.2KO OPCs were identified by a Cre reporter, we establish that Cav1.2KO OPCs display a reduced maturational rate through the entire remyelination process. These results suggest that Ca2+ influx mediated by L-VGCCs in oligodendroglial cells is necessary for normal remyelination and is an essential Ca2+ channel for OPC maturation during the remyelination of the adult brain.SIGNIFICANCE STATEMENT Ion channels implicated in oligodendrocyte differentiation and maturation may induce positive signals for myelin recovery. Voltage-gated Ca2+ channels (VGCCs) are important for normal myelination by acting at several critical steps during oligodendrocyte progenitor cell (OPC) development. To determine whether voltage Ca2+ entry is involved in oligodendrocyte differentiation and remyelination, we used a conditional knockout mouse for VGCCs in OPCs. Our results indicate that VGCCs can modulate oligodendrocyte maturation in the demyelinated brain and suggest that voltage-gated Ca2+ influx in OPCs is critical for remyelination. These findings could lead to novel approaches for obtaining a better understanding of the factors that control OPC maturation in order to stimulate this pool of progenitors to replace myelin in demyelinating diseases.

Glucocorticoids influence versican and chondroitin sulphate proteoglycan levels in the fetal sheep lung.

BACKGROUND: Prenatal glucocorticoid treatment decreases alveolar tissue volumes and facilitates fetal lung maturation, however the mechanisms responsible are largely unknown. This study examines whether changes in versican levels or sulphation patterns of chondroitin sulphate (CS) side chains, are associated with glucocorticoid-induced reductions in peri-alveolar tissue volumes. METHODS: Lung tissue was collected from 1) fetal sheep at 131 ± 0.1 days gestational age (GA) infused with cortisol (122-131d GA) to prematurely induce a pre-parturient-like rise in circulating cortisol, 2) fetal sheep at 143d GA bilaterally adrenalectomised (ADX) at 112d GA to remove endogenous cortisol and 3) fetal sheep at 124d GA in which bolus doses (2 × 11.4 mg) of betamethasone were administered to the pregnant ewe. The level and distribution of versican and CS glycosaminoglycans (GAG) were determined using immunohistochemistry (IHC). Fluorophore assisted carbohydrate electrophoresis (FACE) was used to determine changes in CS sulphation patterns. RESULTS: Cortisol infusion significantly decreased chondrotin-6-sulphate levels (C-6-S) to 16.4 ± 0.7 AU, compared with saline-infused fetuses (18.9 ± 0.7 AU: p = 0.04) but did not significantly alter the level of versican or chondroitin-4-sulphate (C-4-S). ADX significantly increased the level of C-4-S (28.2 ± 2.2 AU), compared with sham-operated fetuses (17.8 ± 2.0 AU; p = 0.006) without altering versican or C-6-S levels. Betamethasone significantly decreased versican, C-4-S and C-6-S in the fetal sheep lung (19.2 ± 0.9 AU, 24.9 ± 1.4 AU and 23.2 ± 1.0 AU, respectively), compared with saline-exposed fetuses (24.3 ± 0.4 AU, p = 0.0004; 33.3±0.6 AU, p = 0.0003; 29.8±1.3 AU, 0.03, respectively). CONCLUSIONS: These results indicate that glucocorticoids alter versican levels and CS side chain microstructure in alveolar lung tissue. Betamethasone appears to have a greater impact on versican and CS side chains than cortisol.

Endocan, sepsis, pneumonia, and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) and hospital-acquired pneumonia (HAP) are major problems of public health in intensive care units (ICUs), occurring in 15% of critically ill patients. Among the factors explaining ARDS development, sepsis is known as a frequent cause. Sepsis, ARDS, and HAP increase morbidity, mortality, length of stay in the ICU, and the overall costs of healthcare. The major challenge remains to identify accurately among critically ill patients those at risk of poor outcomes who could benefit from novel therapies. Endocan is released by the pulmonary endothelium in response to local or systemic injury. It inhibits mainly leukocyte diapedesis rather than leukocyte rolling or adhesion to the endothelial cells both in vitro and in vivo. Endocan was evaluated in 25 clinical reports, including 2454 critically ill patients and 452 healthy controls. The diagnostic value of endocan for sepsis or sepsis severity was equal to procalcitonin but its prognostic value was better. A predictive value for postoperative pneumonia was evidenced in two studies, and a predictive value for ARDS in four studies from three independent centers. This review presents an overview of the structure, expression, and functions of endocan. We also hereby summarize the potential applications of endocan in the prediction and prognosis of ARDS and HAP, as well as in the prognosis of sepsis.

The Role of MicroRNA-181a in Myocardial Fibrosis Following Myocardial Infarction in a Rat Model.

BACKGROUND The role of miR-181a in the development of cardiac disease and in particular, myocardial fibrosis following myocardial infarction (MI) remains unknown. The aim of this study was to explore the role of miR-181a in myocardial fibrosis in a rat model of MI and the expression of TGF-ß receptor III (TßRIII). MATERIAL AND METHODS Forty adult male Wistar rats were randomly divided into an MI model group (n=30) and a control group with (n=10). The rat MI model involved ligating the left anterior descending (LAD) coronary artery in the model group; the control group was treated with a sham operation. Cardiac function was assessed using cardiac ultrasound. Myocardial fibroblasts were extracted from the rat hearts and transfected with a miR-mimic or miR-inhibitor, and cell growth was measured using an MTT assay. The level of miR-181a expression was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blots. RESULTS miR-181a expression was significantly increased during the progression of MI (P<0.05). Over-expression of miR-181a was associated with increased deposition of extracellular matrix (ECM) components, collagen I and fibronectin. This effect was reversed with the use of a miR-181a inhibitor (P<0.05). Upregulation of miR-181a suppressed the expression of TGF-ß receptor III (TßRIII) by binding with 3'-UTR. CONCLUSIONS In this rat model of MI, the findings were that miR-181a had a role in the progression of myocardial fibrosis. The findings require further studies to determine whether miR-181a might provide a novel therapeutic target to limit myocardial fibrosis following MI.

The JCR:LA-cp rat: a novel rodent model of cystic medial necrosis.

Although there are multiple rodent models of the metabolic syndrome, very few develop vascular complications. In contrast, the JCR:LA-cp rat develops both metabolic syndrome and early atherosclerosis in predisposed areas. However, the pathology of the normal vessel wall has not been described. We examined JCR:LA control (+/+) or cp/cp rats fed normal chow diet for 6 or 18 mo. JCR:LA-cp rats developed multiple features of advanced cystic medial necrosis including "cysts," increased collagen formation and proteoglycan deposition around cysts, apoptosis of vascular smooth muscle cells, and spotty medial calcification. These appearances began within 6 mo and were extensive by 18 mo. JCR:LA-cp rats had reduced medial cellularity, increased medial thickness, and vessel hypoxia that was most marked in the adventitia. In conclusion, the normal chow-fed JCR:LA-cp rat represents a novel rodent model of cystic medial necrosis, associated with multiple metabolic abnormalities, vascular smooth muscle cell apoptosis, and vessel hypoxia.NEW & NOTEWORTHY Triggers for cystic medial necrosis (CMN) have been difficult to study due to lack of animal models to recapitulate the pathologies seen in humans. Our study is the first description of CMN in the rat. Thus the JCR:LA-cp rat represents a useful model to investigate the underlying molecular changes leading to the development of CMN.

WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

OBJECTIVE: Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. METHODS: Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. RESULTS: WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. CONCLUSIONS: In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells.