Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat1–3 and are retained in the nucleus4, 5. The mutant RNA exerts a toxic gain-of-function6, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target mRNAs7, 8. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are extraordinarily sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUGexp RNA in skeletal muscle, correcting the physiological, histopathologic, and transcriptomic features of the disease. The effect was sustained for up to one year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat-1, a long noncoding RNA (lncRNA) that is retained in the nucleus9. These results provide a general strategy to correct RNA gain-of-function and modulate the expression of expanded repeats, lncRNAs, and other transcripts with prolonged nuclear residence.

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.Sepsis remains the leading cause of mortality of patients in intensive care units, causing at least 210,000 deaths annually in the United States1. Much of the pathology of sepsis has been attributed to a hyper-reaction of the inflammatory system to the invading pathogens, a condition called 'systemic inflammatory response syndrome' 2 . During the early phases of sepsis, systemic concentrations of inflammatory cytokines and chemokines rapidly increase and the endothelium is activated to cause vascular leakage and septic shock. In late-stage sepsis, the clotting cascade is triggered by the damaged endothelium, leading to disseminated intravascular coagulation (DIC) and multiorgan failure 3, 4. The vascular damage is caused by many sepsis-related factors, including bacterial endotoxin, tumor

Abstract-Thrombosis associated with the pathophysiological activation of platelets and vascular cells has brought thrombin and its receptors to the forefront of cardiovascular medicine. Thrombin signaling through the protease-activated receptors (PARs) has been shown to influence a wide range of physiological responses including platelet activation, intimal hyperplasia, inflammation, and maintenance of vascular tone and barrier function. The thrombin receptors PAR1 and PAR4 can be effectively targeted in animals in which acute or prolonged exposure to thrombin leads to thrombosis and/or restenosis. In the present study, we describe the molecular and pharmacological basis of small-molecule inhibitors that target PAR1. In addition, we discuss a new class of cell-penetrating inhibitors, termed pepducins, that provide insight into previously unidentified roles of PAR1 and PAR4 in protease signaling. Key Words: arteries Ⅲ endothelium Ⅲ inhibitors Ⅲ platelets Ⅲ receptors Ⅲ signal transduction Ⅲ thrombosis P rotease-activated receptors (PARs) play critical roles in coagulation, inflammation, and vascular homeostasis. [1][2][3][4][5] Proteases that are produced during vascular injury exert many of their cellular effects by cleaving and activating the PARs. Thrombin-dependent platelet activation and aggregation have been shown to be heightened in the setting of angioplasty and stenting, which may cause clinical complications including acute myocardial infarction and death. 6 -8 The high-affinity thrombin receptor PAR1 has long been recognized as an obvious candidate for therapeutic intervention in patients with acute coronary syndromes. It is not yet known, however, whether targeting only PAR1 will achieve sufficient therapeutic efficacy because of the presence of a more recently identified second thrombin receptor named PAR4. 9 -12 PAR1 and PAR2 (a trypsin but not a thrombin receptor) have also been shown to affect other cardiovascular functions such as vasoreactivity and cardiomyocyte hypertrophy.The purpose of the present review is to help the clinical reader understand why PARs are essential for the maintenance of normal vascular integrity. This review will focus on the potential therapeutic utility of targeting the PARs in thrombosis, atherosclerosis, and restenosis. Historically, the PARs have been recalcitrant to the development of peptidomimetic-based antagonists; however, recent PAR1 drug candidates based on natural products are now entering large-scale clinical trials for treatment of patients with acute coronary syndromes. In an orthogonal approach, PARs have also been blocked on the inside of the cell with the use of cell-penetrating pepducins that prevent signaling to internally located G proteins. [13][14][15][16][17] Proof-of-concept experiments in acute thrombosis models point to novel antiplatelet therapies that could potentially benefit patients at risk for acute thrombosis. The Role of PARs in Normal Platelet FunctionPlatelets are essential for proper blood coagulation. Initiation of a platelet thromb...

Background-Thrombin is the most potent agonist of platelets and plays a critical role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, protease-activated receptor (PAR) 1 and PAR4; however, there are no therapeutic strategies that effectively target both receptors. Methods and Results-Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small-molecule antagonists and pepducins was effective against carotid artery occlusion. Coimmunoprecipitation and fluorescence resonance energy transfer studies revealed that PAR1 and PAR4 associate as a heterodimeric complex in human platelets and fibroblasts. PAR1-PAR4 cofactoring was shown by acceleration of thrombin cleavage and signaling of PAR4 on coexpression with PAR1. Conclusions-We show that PAR1 and PAR4 form a stable heterodimer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 complex may present a novel therapeutic opportunity to prevent arterial thrombosis.

Matrix metalloproteases (MMPs) play many important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis and cancer. Traditionally, MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival, however their mode of action remains unknown. We demonstrated that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1 which promotes aggregation through PAR1. Unexpectedly, we found that MMP-1 cleaved PAR1 at a novel site which strongly activated Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 greatly curtailed thrombogenesis under arterial flow conditions and inhibited thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a new target for the prevention of arterial thrombosis.

We describe a new therapeutic approach for the treatment of lethal sepsis using cell-penetrating lipopeptides-termed pepducins-that target either individual or multiple chemokine receptors. Interleukin-8 (IL-8), a ligand for the CXCR1 and CXCR2 receptors, is the most potent endogenous proinflammatory chemokine in sepsis. IL-8 levels rise in blood and lung fluids to activate neutrophils and other cells, and correlate with shock, lung injury and high mortality. We show that pepducins derived from either the i1 or i3 intracellular loops of CXCR1 and CXCR2 prevent the IL-8 response of both receptors and reverse the lethal sequelae of sepsis, including disseminated intravascular coagulation and multi-organ failure in mice. Conversely, pepducins selective for CXCR4 cause a massive leukocytosis that does not affect survival. CXCR1 and CXCR2 pepducins conferred nearly 100% survival even when treatment was postponed, suggesting that our approach might be beneficial in the setting of advanced disease.

The protease-activated receptors are tethered ligand G proteincoupled receptors that are activated by proteolytic cleavage of the extracellular domain of the receptor. The archetypic protease-activated receptor PAR1 strongly activates G q signaling pathways, but very little is known regarding the mechanism of signal transference between receptor and internally located G protein. The recent x-ray structure of rhodopsin revealed the presence of a highly conserved amphipathic 8th helix that is likely to be physically interposed between receptor and G protein. We found that the analogous 8th helix region of PAR1 was critical for activation of G q -dependent signaling. Engineering an 8th helix ␣-aneurysm with a downwardsdirected alanine residue markedly interfered with signal transference to G q . The 8th helix-anchoring cysteine palmitoylation sites were important for the affinity of ligand-dependent G protein coupling but did not affect the maximal signal. A network of H-bond and ionic interactions was found to connect the N-terminal portion of the 8th helix to the nearby NPXXY motif on transmembrane helix 7 and also to the adjacent intracellular loop-1. Disruption of these pairwise interactions caused additive defects in coupling to G protein, indicating that the transmembrane 7-8th helix-i1 loop may move in a coordinated manner to transfer the signal from PAR1 to G protein. This "7-8-1" interaction network was found to be prevalent in G protein-coupled receptors involved in endothelial signaling and angiogenesis.Proteolytic cleavage of the G protein-coupled protease-activated receptors (PARs) 2 activates an extraordinarily diverse array of physiologic responses. These include platelet aggregation and cell-cell adhesion, proliferation/apoptosis, protease homing, cancer invasion, angiogenesis, and the hemostatic and inflammatory responses to vascular injury (1-10). Four protease-activated receptors have been identified: PAR1, PAR2, PAR3, and PAR4. A distinguishing feature of the PARs is that they all contain a preligand sequence located within their extracellular N-terminal domains (1). PAR1 is cleaved by thrombin and other proteases at the Arg 41 -Ser 42 bond to create a fresh 42 SFLLRN 47 N terminus that acts as an intramolecular ligand (11,12). NMR studies indicate that, following cleavage by thrombin, the PAR1 ligand region undergoes a large conformational change and docks to ligand-binding site(s), one of which is located in the N-terminal extracellular domain (13). Synthetic peptides (e.g. SFLLRN) corresponding to the freshly cleaved N terminus can displace the tethered ligand from the ligand binding site(s) and fully activate PAR1 in an intermolecular mode (13).Following intra-or intermolecular liganding, the transmembrane domains of the PARs undergo a conformational change that is transmitted to the intracellular loops (i1-i3) and C-terminal i4 domain. It is these intracellular loops and domains that communicate with the plasma membrane-associated heterotrimeric G proteins. PAR1 can interact with three of t...

Inflammation is traditionally viewed as a physiological reaction to tissue injury. Leukocytes contribute to the inflammatory response by the secretion of cytotoxic and pro‐inflammatory compounds, by phagocytotic activity and by targeted attack of foreign antigens. Leukocyte accumulation in tissues is important for the initial response to injury. However, the overzealous accumulation of leukocytes in tissues also contributes to a wide variety of diseases, such as atherosclerosis, chronic inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, vasculitis, systemic inflammatory response syndrome, juvenile diabetes and psoriasis. Many therapeutic interventions target immune cells after they have already migrated to the site of inflammation. This review addresses different therapeutic strategies, used to reduce or prevent leukocyte–endothelial cell interactions and communication, in order to limit the progression of inflammatory diseases.