Increased lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA 2 is a causative agent. Here we show that selective inhibition of Lp-PLA 2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA 2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA 2 inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke.Atherosclerosis, the most common cause of myocardial infarction, stroke and cardiovascular death, is an inflammatory-immunomodulatory disease 1,2 . A key early step in its development is the accumulation and subsequent oxidation of low-density lipoproteins COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturemedicine/. Lp-PLA 2 , also known as platelet-activating factor acetylhydrolase or type VIIA PLA 2 , is a calcium-independent phospholipase A 2 . In humans, Lp-PLA 2 is secreted by leukocytes and is associated with circulating LDL and macrophages in atherosclerotic plaques. Although some have hypothesized that Lp-PLA 2 has a protective role in atherosclerotic lesion development 9,10 , the preponderance of recent data suggests that Lp-PLA 2 has an active role in atherosclerotic development and progression [11][12][13] . Elevated circulating Lp-PLA 2 activity predicts increased cardiovascular risk 14 . A proatherogenic role for Lp-PLA 2 has been postulated on the basis of its ability to generate two key proinflammatory mediators, lysophosphatidylcholine (LPC) and oxidized nonesterified fatty acids (oxNEFAs), through the cleavage of oxidized or polar phospholipids generated during LDL oxidation 15,16 . Evidence exists for a regulatory role of these proinflammatory lipids, particularly of LPC 12,13,17 , in promoting atherosclerotic plaque development that can ultimately lead to the formation of a necrotic core. These steps include recruitment and activation of leukocytes 12,18 , induction of apoptosis 12,19 and impaired removal of dead cells 20,21 . The demonstration that Lp-PLA 2 is highly upregulated in macrophages undergoing apoptosis within the necrotic core and fibrous cap of vulnerable and ruptured plaques, ...

A description of cellular factors that govern alternative splicing of pre-mRNA is largely incomplete. In the case of the rat preprotachykinin gene, splicing of the alternative exon E4 occurs by a poorly understood mechanism in which exon selection is under the positive control of U1 snRNP. Because the binding of U1 snRNP to the 5' splice site of E4 is coincident with the selection of the 3' splice site of E4, this mechanism would appear to involve interactions that bridge across the exon. In this work, a UV cross-linking strategy was used to identify possible RNA-protein interactions involved in the proposed exon-bridging model. Of particular interest is a prominent 61-kD protein, p61, that binds to the 3' splice site of E4 in a manner that is clearly facilitated by a downstream 5' splice site and U1 snRNP particles. The identity of p61 is the essential splicing factor U2AF65, on the basis of copurification and selective binding to polypyrimidine tracts. These results indicate a model in which exon selection is positively regulated by the communication of U1 snRNP and U2AF65. That is, a natural deficiency in binding U2AF65 to the 3' splice site that leads to exon skipping might be overcome by a mechanism in which U1 snRNP facilitates the binding of U2AF65 through a network of template-directed and exon-bridging interactions.[Key Words: RNA-protein binding; U1 snRNP; U2AF65; alternative splicing; exon selection] Received May 8, 1992; revised version accepted October 2, 1992.Alternative splicing of pre-mRNA is a widespread process that is largely responsible for the diversity of polypeptides expressed in mammalian cells, yet a molecular understanding of the mechanisms and factors that govern this process is lacking. A central question of interest is how to explain the regulation of splice site selection that leads to the observed patterns of alternative 5', or 3' splice site selection, mutually exclusive exon selection, or controlled exon skipping (Andreadis et al. 1987;Maniatis 1991).Our current understanding, from studies of single iniron-containing pre-mRNA substrates, is that splice site recognition is achieved by the assembly of a spliceosome complex. The spliceosome is known to contain the small nuclear ribonucleoprotein (snRNP) particles U1, U2, U5, and U4 + U6, as well as additional protein factors Steitz et al. 1988;Green 1991;Guthrie 1991). In one of the earliest spliceosome assembly events, the 5' splice site at the upstream boundary of the intron is recognized by direct base-pairing with a 6-to 9-nucleotide stretch at the 5' end of U1 small nuclear ~Affifiated with btCB Graduate Program, Brown University, Providence, Rhode Island 02912 USA. 2Corresponding author.RNA (snRNA) (Zhuang and Weiner 1986;Siliciano and Guthrie 1988;Rosbash and Seraphin 1991). This RNA base-pairing interaction appears to be further stabilized by the involvement of a U1 snRNP-specific protein, U1-C (Heinrichs et al. 1990). Interestingly, engineered nucleotide changes in the 5' end of U1 snRNA have been shown to alter 5' splice s...

The activation of the TRPM8 channel, a member of the large class of TRP ion channels, has been reported to be involved in overactive bladder and painful bladder syndrome, although an endogenous activator has not been identified. In this study, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB) was evaluated as a TRPM8 channel blocker and used as a tool to evaluate the effects of this class of ion channel blocker on volume-induced bladder contraction and nociceptive reflex responses to noxious bladder distension in the rat. AMTB inhibits icilin-induced TRPM8 channel activation as measured in a Ca(2+) influx assay, with a pIC(50) of 6.23. In the anesthetized rat, intravenous administration of AMTB (3 mg/kg) decreased the frequency of volume-induced bladder contractions, without reducing the amplitude of contraction. The nociceptive response was measured by analyzing both visceromotor reflex (VMR) and cardiovascular (pressor) responses to urinary bladder distension (UBD) under 1% isoflurane. AMTB (10 mg/kg) significantly attenuated reflex responses to noxious UBD to 5.42 and 56.51% of the maximal VMR response and pressor response, respectively. The ID50 value on VMR response was 2.42 +/- 0.46 mg/kg. These results demonstrate that TRPM8 channel blocker can act on the bladder afferent pathway to attenuate the bladder micturition reflex and nociceptive reflex responses in the rat. Targeting TRPM8 channel may provide a new therapeutic opportunity for overactive bladder and painful bladder syndrome.

RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B52 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.Multiprotein assemblies drive a variety of highly regulated biological processes. To better understand and control such processes, novel reagents are needed to modulate functions of specific proteins in cells and whole organisms. An ideal reagent would, (i) like an antibody, be made to order specifically for a particular protein, (ii) like a small organic molecule, be able to rapidly target a specific protein domain within cells, and (iii) like a conditional allele, be able to exert its effect in whole organisms, but (iv) also be targetable to specific tissues, cells, or stages of development.Although many strategies exist for inactivating genes or gene products, use of RNA aptamers (1) presents several compelling advantages. Extremely rare RNAs that have high affinity for specific proteins can be selected in vitro (SELEX) (2, 3). Genetically controlled induction of such high affinity RNA aptamers could provide a means of rapidly inactivating a specific domain of a protein and thereby assessing its primary function and mechanism of action in vivo. Alternatively, continuous expression of specific RNA aptamers after stable gene transfer could achieve a long-term alteration in the activity of a target protein. In yeast, the constitutive expression of an aptamer against RNA polymerase II caused cell growth defects under conditions in which the RNA polymerase level was artificially reduced (4). Although this study demonstrated the potential of RNA aptamers as inhibitors of protein function in vivo, several advances are needed to apply this approach to multicellular organisms.Previously, we selected and characterized RNA aptamers to B52, a regulator of RNA splicing in Drosophila melanogaster. B52, also known as SRp55, is a member of the Drosophila SR protein family, a group of nuclear proteins that are essential for pre-mRNA splicing and influence splice site choice (5, 6). B52 contains two RNA recognition motifs in its N-terminal half and a domain rich in serine-arginine dipeptide repeats in its C-terminal half (7). RNA aptamers that bind B52 with high affinity (K d ϭ 20-50 nM) and specificity were selected from a large pool of RNAs (8). The B52 binding sites (BBS) on members of this nonclonally derived family of RNA aptamers not only have a ''conserved'' consensus sequen...

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.Most metazoan genes are transcribed to yield pre-mRNAs containing introns that are excised and exons that are joined to create a functional mRNA (for a review, see reference 39). This pre-mRNA splicing process is catalyzed by the spliceosome, a multicomponent protein-RNA complex consisting of small nuclear ribonuclear protein particles (snRNPs) and nonsnRNP proteins. The accurate selection and pairing of corresponding 5Ј and 3Ј splice sites occur when these factors are assembled onto the pre-mRNA in an orderly manner (for a review, see reference 32). A family of non-snRNP protein factors, termed SR proteins, have been identified as both essential splicing factors and modulators of alternative splicing that function in early spliceosome assembly (for reviews, see references 13 and 30).SR proteins are structurally and functionally related and evolutionarily conserved. This family comprises at least six members, designated SRp20, SRp30a, SRp30b, SRp40, SRp55, and SRp75, which are conserved from Drosophila melanogaster to humans (50). Among them, the human SRp30a (SF2/ASF) (16,17,25,26) and SRp30b (SC35) (14, 15) have been the most intensively studied. In addition, three other human SR proteins, SRp30c (38), p54 (8), and 9G8 (6), have been cloned recently. For Drosophila, only two SR proteins have been cloned. They are homologous to the human SRp55 and SRp20 and are designated B52 (7) and RBP1 (24), respectively. Different SR proteins appear to have overlapping but distinct functions in constitutive and alternative splicing reactions in vitro. Although individual SR proteins are able to complement splicing-deficient cytoplasmic S100 extracts (31, 50), their activities vary when they are presented with different premRNA substrates (12). In alternative splicing, one SR protein may promote preferential usage of one splice site, whe...