Surface Stoichiometry and Depth Profile of Tix-CuyNz Thin Films Deposited by Magnetron Sputtering.

We report the surface stoichiometry of Tix-CuyNz thin film as a function of film depth. Films are deposited by high power impulse (HiPIMS) and DC magnetron sputtering (DCMS). The composition of Ti, Cu, and N in the deposited film is investigated by X-ray photoelectron spectroscopy (XPS). At a larger depth, the relative composition of Cu and Ti in the film is increased compared to the surface. The amount of adventitious carbon which is present on the film surface strongly decreases with film depth. Deposited films also contain a significant amount of oxygen whose origin is not fully clear. Grazing incidence X-ray diffraction (GIXD) shows a Cu3N phase on the surface, while transmission electron microscopy (TEM) indicates a polycrystalline structure and the presence of a Ti3CuN phase.

Bimetallic gold-silver nanoparticles mediate bacterial killing by disrupting the actin cytoskeleton MreB.

The actin cytoskeleton is required for the maintenance of the cell shape and viability of bacteria. It remains unknown to which extent nanoparticles (NPs) can orchestrate the mechanical instability by disrupting the cytoskeletal network in bacterial cells. Our work demonstrates that Au-Ag NPs disrupt the bacterial actin cytoskeleton specifically, fluidize the inner membrane and lead to killing of bacterial cells. In this study, we have tried to emphasize on the key parameters important for NP-cell interactions and found that the shape, specific elemental surface localization and enhanced electrostatic interaction developed due to the acquired partial positive charge by silver atoms in the aggregated NPs are some of the major factors contributing towards better NP interactions and subsequent cell death. In vivo studies in bacterial cells showed that the NPs exerted a mild perturbation of the membrane potential. However, its most striking effect was on the actin cytoskeleton MreB resulting in morphological changes in the bacterial cell shape from rods to predominantly spheres. Exposure to NPs resulted in the delocalization of MreB patches from the membrane but not the tubulin homologue FtsZ. Concomitant with the redistribution of MreB localization, a dramatic increase of membrane fluid regions was observed. Our studies reveal for the first time that Au-Ag NPs can mediate bacterial killing and disrupt the actin cytoskeletal functions in bacteria.

IONIS-PKKRx a Novel Antisense Inhibitor of Prekallikrein and Bradykinin Production.

Kallikrein is the key contact system mediator responsible for the conversion of high-molecular-weight kininogen into the inflammatory vasodilator peptide bradykinin, a process regulated by C1-esterase inhibitor (C1-INH). In hereditary angioedema (HAE), genetic mutations result in deficient or dysfunctional C1-INH and dysregulation of the contact system leading to recurrent, sometimes fatal, angioedema attacks. IONIS-PKKRx is a second-generation 2'-O-(2-methoxyethyl)-modified chimeric antisense oligonucleotide, designed to bind and selectively reduce prekallikrein (PKK) mRNA in the liver. IONIS-PKKRx demonstrated dose-dependent reduction of human prekallikrein hepatic mRNA and plasma protein in transgenic mice and dose- and time-dependent reductions of plasma PKK in Cynomolgus monkeys. Similar dose-dependent reductions of plasma PKK levels were observed in healthy human volunteers accompanied by decreases in bradykinin generation capacity with an acceptable safety and tolerability profile. These results highlight a novel and specific approach to target PKK for the treatment of HAE and other diseases involving contact system activation and overproduction of bradykinin.

Supramolecular Hydrogel from an Oxidized Byproduct of Tyrosine.

Herein we report for the first time the supramolecular hydrogelation of a derivative of 3-nitrotyrosine (3-NT), which is an oxidized byproduct of tyrosine, produced in the presence of reactive nitrogen species in the cell. The 9-fluorenylmethyloxycarbonyl (Fmoc) derivative of 3-NT, FNT, can form self-supported hydrogels at a wide range of pH values (4.5-8.5) in 50 mM phosphate buffer solutions. Hydrogels prepared at pH 7.0 are yellow, transparent, and thixotropic in nature. A yellow hydrogel was obtained by changing the pH from 4.5 to 8.5. Moreover, the gelation efficiency of the FNT gelator was enhanced by lowering the pH of the buffer solution. pH-Dependent self-assembly properties of the gelator were studied by using UV-vis, fluorescence, and circular dichroism spectroscopy and wide-angle X-ray diffraction techniques. Field-emission-scanning electron microscopy and transmission electron microscopy studies of the self-assembled FNT hydrogel showed a nanofibrillar network structure. Interestingly, the hydrogel showed injectable behavior at physiological pH. The low cytotoxicity value and high antimicrobial properties of the hydrogel indicated that it is a potential material for biomedical applications.

Plasmin Prevents Dystrophic Calcification After Muscle Injury.

Extensive or persistent calcium phosphate deposition within soft tissues after severe traumatic injury or major orthopedic surgery can result in pain and loss of joint function. The pathophysiology of soft tissue calcification, including dystrophic calcification and heterotopic ossification (HO), is poorly understood; consequently, current treatments are suboptimal. Here, we show that plasmin protease activity prevents dystrophic calcification within injured skeletal muscle independent of its canonical fibrinolytic function. After muscle injury, dystrophic calcifications either can be resorbed during the process of tissue healing, persist, or become organized into mature bone (HO). Without sufficient plasmin activity, dystrophic calcifications persist after muscle injury and are sufficient to induce HO. Downregulating the primary inhibitor of plasmin (α2-antiplasmin) or treating with pyrophosphate analogues prevents dystrophic calcification and subsequent HO in vivo. Because plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent soft tissue calcification without adversely affecting systemic bone physiology or concurrent muscle and bone regeneration. © 2016 American Society for Bone and Mineral Research.

Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification.

Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals.

Advances in the medicinal chemistry of antisense oligonucleotide drugs have been instrumental in achieving and optimizing antisense activity in cell types other than hepatocytes, the cell type that is most sensitive to antisense effects following systemic treatment. To broadly characterize the effects of antisense drugs on target messenger RNA (mRNA) levels in different organs and cell types in animals, we have developed a sensitive RNA in situ hybridization technique using the noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a surrogate target. We have used this technique to evaluate the effects of 2'-O-methoxy ethyl (MOE) and constrained ethyl bicyclic nucleic acid (cEt) gapmer antisense oligonucleotides (ASOs). ASO tissue distribution was also characterized using immunohistochemical techniques, and MALAT1 mRNA reductions were confirmed by quantitative real time-polymerase chain reaction. Our findings demonstrate that systemic antisense drug administration in both mice and non-human primates resulted in marked reductions in MALAT1 RNA in many tissues and cell types other than liver including kidney, muscle, lung, adipose, adrenal gland, and peripheral nerve tissue. As expected, ASOs with cEt chemistry were more efficacious than MOE ASO in all tissues examined.

Inhibition of vascular permeability by antisense-mediated inhibition of plasma kallikrein and coagulation factor 12.

Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulation factor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulation factors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

The lncRNA Malat1 is dispensable for mouse development but its transcription plays a cis-regulatory role in the adult.

Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3'-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.

Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding.

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.

Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation.

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Factor Xa binding to annexin 2 mediates signal transduction via protease-activated receptor 1.

The serine protease zymogen factor X is converted to its catalytically active form factor Xa by the binary complex of factor VIIa bound to its cell surface receptor tissue factor (TF) or by the intrinsic Xase complex, which consists of active factors VIII (VIIIa), IX (IXa), factor X, and Ca2+. Factor Xa has procoagulant activity by conversion of prothrombin to thrombin and also induces signal transduction, either alone or in the ternary TF:VIIa:factor Xa coagulation initiation complex. Factor Xa cleaves and activates protease activated receptor (PAR)1 or -2, but factor Xa signaling efficiency varies among cell types. We show here that annexin 2 acts as a receptor for factor Xa on the surface of human umbilical vein endothelial cells and that annexin 2 facilitates factor Xa activation of PAR-1 but does not enhance coagulant function of factor Xa. Overexpression of TF abolishes annexin 2 dependence on factor Xa signaling and diminishes binding to cell surface annexin 2, whereas selectively abolishing TF promotes the annexin 2/factor Xa interaction. We propose that annexin 2 serves to regulate factor Xa signaling specifically in the absence of cell surface TF and may thus play physiological or pathological roles when factor Xa is generated in a TF-depleted environment.

Regulation of macrophage procoagulant responses by the tissue factor cytoplasmic domain in endotoxemia.

Tissue factor (TF) is the primary initiator of coagulation, and the TF pathway mediates signaling through protease-activated receptors (PARs). In sepsis, TF is up-regulated as part of the proinflammatory response in lipopolysaccharide (LPS)-stimulated monocytes leading to systemic coagulation activation. Here we demonstrate that TF cytoplasmic domain-deleted (TF(Delta CT)) mice show enhanced and prolonged systemic coagulation activation relative to wild-type upon LPS challenge. However, TF(Delta CT) mice resolve inflammation earlier and are protected from lethality independent of changes in coagulation. Macrophages from LPS-challenged TF(Delta CT) mice or LPS-stimulated, in vitro-differentiated bone marrow-derived macrophages show increased TF mRNA and functional activity relative to wild-type, identifying up-regulation of macrophage TF expression as a possible cause for the increase in coagulation of TF(Delta CT) mice. Increased TF expression of TF(Delta CT) macrophages does not require PAR2 and is specific for toll-like receptor, but not interferon gamma receptor, signaling. The presence of the TF cytoplasmic domain suppresses ERK1/2 phosphorylation that is reversed by p38 inhibition leading to enhanced TF expression specifically in wild-type but not TF(Delta CT) mice. The present study demonstrates a new role of the TF cytoplasmic domain in an autoregulatory pathway that controls LPS-induced TF expression in macrophages and procoagulant responses in endotoxemia.

A selective tumor microvasculature thrombogen that targets a novel receptor complex in the tumor angiogenic microenvironment.

We have previously shown that part of the heparin-binding domain of the vascular endothelial growth factor (VEGF), designated HBDt, localizes very selectively to surfaces of the endothelial cells of i.t blood vessels. Here, we have coupled the HBDt to the extracellular domain of tissue factor (TFt), to locally initiate the thrombogenic cascade. In tumor-bearing mice, infusion of this HBDt.TFt results in rapid occlusive thrombosis selective only for tumor microvasculature with resultant infarctive destruction of tumors. We now show that infusion of an optimal combination of this HBDt.TFt and its requisite cofactor (factor VIIa) in tumor models results in significant tumor eradication. Binding studies and confocal microscopy indicate that the target for the HBDt.TFt seems to be a trimolecular complex of chondroitin C sulfate proteoglycan, neuropilin-1, and VEGF receptor-2, overexpressed together only in highly angiogenic sites of the tumor microenvironment. The HBDt.TFt was also colocalized with the trimolecular receptor complex in endothelial sprouts from tumor tissues, and its binding inhibited the growth of such sprouts. In vitro, we show that the HBDt structure has its highest affinity for chondroitin 6 sulfate. We show the potential of this HBDt.TFt as a candidate therapeutic and elucidate its target in vivo.

Regulation of tissue factor--mediated initiation of the coagulation cascade by cell surface grp78.

OBJECTIVE: To test the hypothesis that Grp78 negatively regulates cell surface tissue factor (TF) procoagulant activity and whether this is mediated by physical interaction. METHODS AND RESULTS: Biopanning with phage-displayed peptidyl libraries has identified peptide probes that bind selectively in vivo to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds 78-kDa glucose regulated protein (Grp78). Grp78 participates in numerous pathological processes, including the regulation of the coagulation cascade, but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function, we analyzed the effect of Grp78 on TF-mediated procoagulant activity on murine brain endothelial cells (bEND.3) and macrophage-like (RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the surface of endothelium and monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We demonstrate that Grp78 negatively regulates procoagulant activity by interacting physically with the TF extracellular domain on the cell surface. CONCLUSIONS: The evidence indicates that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.

Dexamethasone enhances LPS induction of tissue factor expression in human monocytic cells by increasing tissue factor mRNA stability.

Glucocorticoids, such as dexamethasone (Dex), are used clinically in the treatment of various inflammatory diseases. Dex acts by inhibiting the expression of inflammatory mediators, such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1). It is surprising that Dex enhances bacterial lipopolysaccharide (LPS) induction of tissue factor (TF) expression in human monocytic cells. TF is a transmembrane glycoprotein that activates the coagulation protease cascade. In this study, we analyze the mechanism by which Dex enhances LPS-induced TF expression in human monocytic cells. We found that Dex reduced LPS-induced TF gene transcription but increased the stability of TF mRNA. Dex decreased the stability of MCP-1 mRNA and did not affect TNF-alpha mRNA stability. Finally, we showed that Dex increased the stability of a transcript consisting of the final 297 nucleotides of the TF mRNA in in vitro decay assays. This region contains AU-rich elements that regulate mRNA stability and may mediate the Dex response. Therefore, despite an inhibition of TF gene transcription, Dex enhances TF expression in human monocytic cells by increasing the stability of TF mRNA.

In vivo interrogation of the molecular display of atherosclerotic lesion surfaces.

The endothelial surface of atherosclerotic lesions of ApoE knockout mice was interrogated by in vivo biopanning with a phage-displayed constrained peptidyl library. Through repeated biopanning, 103 peptidyl sequences were identified, many are homologous to known proteins. The sequence CAPGPSKSC contains motifs that are shared by 9.7% of selected peptides. On phage or as a synthetic peptide, this constrained peptide selectively bound to atherosclerotic lesion surfaces of ApoE knockout mice in vivo and of human atherosclerotic lesions ex vivo. A cell-surface protein of approximately 82 kd recognized by this peptide was affinity-purified and determined by mass spectrometry analysis as glucose-regulated protein 78 (Grp78), indicating the surprising presence of this endoplasmic reticulum chaperone on the endothelial cell surface of atherosclerotic lesions. Peptides that mimicked binding functions of their homologues were demonstrated with three peptides homologous to tissue inhibitor of metalloproteinase-2 (TIMP-2), ie, CNHRYMQMC, CNQRHQMSC, and CNNRSDGMC. Phage carrying CNHRYMQMC bound to atherosclerotic lesion endothelium of ApoE knockout mice in vivo. The three peptides bound to endothelial cells in a dose-dependent manner and were inhibited by TIMP-2 protein. These peptides provide a set of probes to interrogate the cell surface repertoire associated with atherogenesis and thrombotic complications.