Design, synthesis and biological evaluation of novel cyclic malonamide derivatives as selective RIPK1 inhibitors.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) plays a key role in cell death and inflammation. RIPK1 is a well-established therapeutic target, due to the presence of a unique kinase-regulating allosteric pocket, which enables selective inhibition. Herein we used GSK2982772 as our starting point in our discovery campaign. Applying isosteric replacement, we successfully identified the malonamide scaffold, instead of the well-established serine template. Further structural optimization led to the design and synthesis of a series of analog inhibitors. The enantiomers of the most promising compound were tested on 97 different kinases. The active enantiomer proved to be kinase selective.

6-Aryl-quinazolines as novel GABAB receptor positive allosteric modulators.

The systemic use of GABAB orthosteric agonist baclofen might be limited due to its detrimental properties: sedation and motor impairment. In contrast, GABAB positive allosteric modulators produce less adverse effects. Using BHF-177 as a starting point, we found a new active scaffold: the 6-aryl-quinazoline scaffold. Further elaborating the scaffold, we identified several in vitro and in vivo active compounds.

Palmitoylation of the ciliary GTPase ARL13b is necessary for its stability and its role in cilia formation.

Primary cilia are hairlike extensions of the plasma membrane of most mammalian cells that serve specialized signaling functions. To traffic properly to cilia, multiple cilia proteins rely on palmitoylation, the post-translational attachment of a saturated 16-carbon lipid. However, details regarding the mechanism of how palmitoylation affects cilia protein localization and function are unknown. Herein, we investigated the protein ADP-ribosylation factor-like GTPase 13b (ARL13b) as a model palmitoylated ciliary protein. Using biochemical, cellular, and in vivo studies, we found that ARL13b palmitoylation occurs in vivo in mouse kidneys and that it is required for trafficking to and function within cilia. Myristoylation, a 14-carbon lipid, is shown to largely substitute for palmitoylation with regard to cilia localization of ARL13b, but not with regard to its function within cilia. The functional importance of palmitoylation results in part from a dramatic increase in ARL13b stability, which is not observed with myristoylation. Additional results show that blockade of depalmitoylation slows the degradation of ARL13b that occurs during cilia resorption, raising the possibility that the sensitivity of ARL13b stability to palmitoylation may be exploited by the cell to accelerate degradation of ARL13b by depalmitoylating it. Together, the results show that palmitoylation plays a unique and critical role in controlling the localization, stability, abundance, and thus function of ARL13b. Pharmacological manipulation of protein palmitoylation may be a strategy to alter cilia function.

Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis.

Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of ß1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of ß1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo.

The Protein Acyl Transferase ZDHHC21 Modulates α1 Adrenergic Receptor Function and Regulates Hemodynamics.

OBJECTIVE: Palmitoylation, the reversible addition of the lipid palmitate to a cysteine, can alter protein localization, stability, and function. The ZDHHC family of protein acyl transferases catalyzes palmitoylation of numerous proteins. The role of ZDHHC enzymes in intact tissue and in vivo is largely unknown. Herein, we characterize vascular functions in a mouse that expresses a nonfunctional ZDHHC21 (F233Δ). APPROACH AND RESULTS: Physiological studies of isolated aortae and mesenteric arteries from F233Δ mice revealed an unexpected defect in responsiveness to phenylephrine, an α1 adrenergic receptor agonist. In vivo, F233Δ mice displayed a blunted response to infusion of phenylephrine, and they were found to have elevated catecholamine levels and elevated vascular α1 adrenergic receptor gene expression. Telemetry studies showed that the F233Δ mice were tachycardic and hypotensive at baseline, consistent with diminished vascular tone. In biochemical studies, ZDHHC21 was shown to palmitoylate the α1D adrenoceptor and to interact with it in a molecular complex, thus suggesting a possible molecular mechanism by which the receptor can be regulated by ZDHHC21. CONCLUSIONS: Together, the data support a model in which ZDHHC21 F233Δ diminishes the function of vascular α1 adrenergic receptors, leading to reduced vascular tone, which manifests in vivo as hypotension and tachycardia. This is to our knowledge the first demonstration of a ZDHHC isoform affecting vascular function in vivo and identifies a novel molecular mode of regulation of vascular tone and blood pressure.

Synthesis and pKa determination of new enantiopure dimethyl-substituted acridino-crown ethers containing a carboxyl group: Useful candidates for enantiomeric recognition studies.

New enantiopure dimethyl-substituted acridino-18-crown-6 and acridino-21-crown-7 ethers containing a carboxyl group at position 9 of the acridine ring [(S,S)-8, (S,S)-9, (R,R)-10] were synthesized. The pKa values of the new crown ethers [(S,S)-8, (S,S)-9, (R,R)-10] and of an earlier reported macrocycle [(R,R)-2] were determined by UV-pH titrations. Crown ether (S,S)-8 was attached to silica gel by covalent bonds and the enantiomeric separation ability of the newly prepared chiral stationary phase [(S,S)-CSP-12] was studied by high-performance liquid chromatography (HPLC). Homochiral preference was observed and the best separation was achieved for the enantiomers of 1-NEA. Ligands (S,S)-9 and (R,R)-10 are precursors of enantioselective sensor and selector molecules for the enantiomers of protonated primary amines, amino acids, and their derivatives.

Reticulon 4 is necessary for endoplasmic reticulum tubulation, STIM1-Orai1 coupling, and store-operated calcium entry.

Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca(2+) levels via SOCE and therefor are less susceptible to Ca(2+) overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca(2+) homeostasis.

Saponin monomer 13 of dwarf lilyturf tuber (DT-13) protects serum withdrawal-induced apoptosis through PI3K/Akt in HUVEC.

Dwarf lilyturf tuber is widely used in clinics to prevent cardiovascular diseases. DT-13, the saponin monomer 13 of dwarf lilyturf tuber, shows protective activities in anti-thrombosis, anti-inflammation, and cardioprotective. However, little is known about the cellular function of DT-13 in cardiovascular system. Vascular endothelial cells (EC) are important to maintain the integrity of the vasculature throughout entire body. Dysregulation of EC may lead to pathophysiological processes of numerous cardiovascular diseases. We thus tested the function of DT-13 in EC. In the present study, we are the first to report that DT-13 has anti-apoptosis activity on human umbilical vein endothelial cells (HUVEC), potentially through down regulation of cleaved caspase-3 and cleaved PARP expression. DT-13 also increased mitochondrial membrane potential. To explore the potential mechanism, we investigated the effect of DT-13 on Akt and MAPK pathways and found that DT-13 was involved in Akt signaling confirmed by using PI3K/Akt inhibitor LY294002. Thus, DT-13 could improve survival of EC and therefore be a potential clinical use in the treatment of cardiovascular diseases.

Reticulon 4B (Nogo-B) is a novel regulator of hepatic fibrosis.

UNLABELLED: Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. Its function in the liver is not understood. The aim of this study was to characterize Nogo-B in liver fibrosis and cirrhosis. Nogo-B distribution was assessed in normal and cirrhotic human liver sections. We also determined the levels of liver fibrosis in wild-type (WT) and Nogo-A/B knockout (NGB KO) mice after sham operation or bile duct ligation (BDL). To investigate the mechanisms of Nogo-B's involvement in fibrosis, hepatic stellate cells were isolated from WT and NGB KO mice and transformed into myofibroblasts. Portal pressure was measured to test whether Nogo-B gene deletion could ameliorate portal hypertension. In normal livers, Nogo-B expression was found in nonparenchymal cells, whereas its expression in hepatocytes was minimal. Nogo-B staining was significantly elevated in cirrhotic livers. Fibrosis was significantly increased in WT mice 4 weeks after BDL compared with NGB KO mice. The absence of Nogo-B significantly reduced phosphorylation of Smad2 levels upon transforming growth factor ß (TGF-ß) stimulation. Reconstitution of the Nogo-B gene into NGB KO fibroblasts restored Smad2 phosphorylation. Four weeks after BDL, portal pressure was significantly increased in WT mice by 47%, compared with sham-operated controls (P = 0.03), whereas such an increase in portal pressure was not observed in NGB KO mice (P = NS). CONCLUSION: Nogo-B regulates liver fibrosis, at least in part, by facilitating the TGFß/Smad2 signaling pathway in myofibroblasts. Because absence of Nogo-B ameliorates liver fibrosis and portal hypertension, Nogo-B blockade may be a potential therapeutic target in fibrosis/cirrhosis.

Bacterial DNA activates endothelial cells and promotes neutrophil adherence through TLR9 signaling.

TLR9 detects bacterial DNA (CpG DNA) and elicits both innate and adoptive immunity. Recent evidence indicates that TLR9 is expressed in more diverse cell types than initially thought. In this study, we report that HUVECs constitutively express TLR9 and selectively recognize unmethylated CpG motifs in bacterial DNA and synthetic immune stimulatory CpG oligodeoxynucleotides. HUVECs respond to CpG DNA with rapid phosphorylation of IkappaB-alpha and NF-kappaB-mediated gene transcription and surface expression of the adhesion molecules ICAM-1 and E-selectin independent of MAPK signaling. The telomere-derived TLR9 inhibitory oligonucleotide 5'-TTT AGG GTT AGG GTT AGG G-3', agents that block endosomal acidification such as chloroquine and bafilomycin A, and NF-kappaB inhibitors abrogated CpG DNA-induced signaling. HUVEC activation by CpG DNA led to markedly enhanced neutrophil adhesion under nonstatic conditions that was further enhanced when neutrophils were stimulated with CpG DNA. The adhesive interactions were blocked by Abs against CD18 and, to a lesser degree, by anti-E-selectin and anti-L-selectin Abs. Our findings demonstrate that bacterial DNA promotes beta(2) integrin and E-selectin-mediated HUVEC-neutrophil adherence, and indicate the ability of CpG DNA to initiate and/or maintain the inflammatory response.

Myeloperoxidase delays neutrophil apoptosis through CD11b/CD18 integrins and prolongs inflammation.

Polymorphonuclear neutrophil granulocytes have a central role in innate immunity and their programmed cell death and removal are critical for efficient resolution of acute inflammation. Myeloperoxidase (MPO), a heme protein abundantly expressed in neutrophils, is generally associated with killing of bacteria and oxidative tissue injury. Because MPO also binds to neutrophils, we investigated whether MPO could affect the lifespan of neutrophils. Here, we report that MPO independent of its catalytic activity through signaling via the adhesion molecule CD11b/CD18 rescued human neutrophils from constitutive apoptosis and prolonged their life span. MPO evoked a transient concurrent activation of extracellular signal-regulated kinase and Akt, leading to phosphorylation of Bad at both Ser112 and Ser136, prevention of mitochondrial dysfunction, and subsequent activation of caspase-3. Consistently, pharmacological inhibition of extracellular signal-regulated kinase, Akt, or caspase-3 reversed the antiapoptosis action of MPO. Acute increases in plasma MPO delayed murine neutrophil apoptosis assayed ex vivo. In a mouse model of self-resolving inflammation, MPO also prolonged the duration of carrageenan-induced acute lung injury, as evidenced by enhanced alveolar permeability and accumulation of neutrophils parallel with suppression of neutrophil apoptosis. Our results indicate that MPO functions as a survival signal for neutrophils and thereby contribute to prolongation of inflammation.

Absence of Akt1 reduces vascular smooth muscle cell migration and survival and induces features of plaque vulnerability and cardiac dysfunction during atherosclerosis.

OBJECTIVE: Deletion of Akt1 leads to severe atherosclerosis and occlusive coronary artery disease. Vascular smooth muscle cells (VSMCs) are an important component of atherosclerotic plaques, responsible for promoting plaque stability in advanced lesions. Fibrous caps of unstable plaques contain less collagen and ECM components and fewer VSMCs than caps from stable lesions. Here, we investigated the role of Akt1 in VSMC proliferation, migration, and oxidative stress-induced apoptosis. In addition, we also characterized the atherosclerotic plaque morphology and cardiac function in an atherosclerosis-prone mouse model deficient in Akt1. METHODS AND RESULTS: Absence of Akt1 reduces VSMC proliferation and migration. Mechanistically, the proliferation and migratory phenotype found in Akt1-null VSMCs was linked to reduced Rac-1 activity and MMP-2 secretion. Serum starvation and stress-induced apoptosis was enhanced in Akt1 null VSMCs as determined by flow cytometry using Annexin V/PI staining. Immunohistochemical analysis of atherosclerotic plaques from Akt1(-/-ApoE-/-) mice showed a dramatic increase in plaque vulnerability characteristics such as enlarged necrotic core and reduced fibrous cap and collagen content. Finally, we show evidence of myocardial infarcts and cardiac dysfunction in Akt1(-/-ApoE-/-) mice analyzed by immunohistochemistry and echocardiography, respectively. CONCLUSIONS: Akt1 is essential for VSMC proliferation, migration, and protection against oxidative stress-induced apoptosis. Absence of Akt1 induces features of plaque vulnerability and cardiac dysfunction in a mouse model of atherosclerosis.

15-epi-lipoxin A4 inhibits myeloperoxidase signaling and enhances resolution of acute lung injury.

RATIONALE: Apoptosis is essential for removal of neutrophils from inflamed tissues and efficient resolution of inflammation. Myeloperoxidase (MPO), abundantly expressed in neutrophils, not only generates cytotoxic oxidants but also signals through the beta(2) integrin Mac-1 to rescue neutrophils from constitutive apoptosis, thereby prolonging inflammation. OBJECTIVES: Because aspirin-triggered 15-epi-lipoxin A(4) (15-epi-LXA(4)) modulates Mac-1 expression, we investigated the impact of 15-epi-LXA(4) on MPO suppression of neutrophil apoptosis and MPO-mediated neutrophil-dependent acute lung injury. METHODS: Human neutrophils were cultured with MPO with or without 15-epi-LXA(4) to investigate development of apoptosis. Acute lung injury was produced by intratracheal injection of carrageenan plus MPO or intraperitoneal injection of live Escherichia coli in mice, and the animals were treated with 15-epi-LXA(4) at the peak of inflammation. MEASUREMENTS AND MAIN RESULTS: 15-Epi-LXA(4) through down-regulation of Mac-1 expression promoted apoptosis of human neutrophils by attenuating MPO-induced activation of extracellular signal-regulated kinase and Akt-mediated phosphorylation of Bad and by reducing expression of the antiapoptotic protein Mcl-1, thereby aggravating mitochondrial dysfunction. The proapoptotic effect of 15-epi-LXA(4) was dominant over MPO-mediated effects even when it was added at 4 hours post MPO. In mice, treatment with 15-epi-LXA(4) accelerated the resolution of established carrageenan plus MPO-evoked as well as E. coli-induced neutrophil-dependent pulmonary inflammation through redirecting neutrophils to caspase-mediated cell death and facilitating their removal by macrophages. CONCLUSIONS: These results demonstrate that aspirin-triggered 15-epi-LXA(4) enhances resolution of inflammation by overriding the powerful antiapoptosis signal from MPO, thereby demonstrating a hitherto unrecognized mechanism by which aspirin promotes resolution of inflammation.

Opposing regulation of neutrophil apoptosis through the formyl peptide receptor-like 1/lipoxin A4 receptor: implications for resolution of inflammation.

Neutrophils have a central role in innate immunity, and their programmed cell death and removal are critical to the optimal expression as well as to efficient resolution of inflammation. Human neutrophils express the pleiotropic receptor formyl peptide receptor-like 1/lipoxin A4 (LXA(4)) receptor that binds a variety of ligands, including the acute-phase reactant serum amyloid A (SAA), the anti-inflammatory lipids LXA(4) and aspirin-triggered 15-epi-LXA(4) (ATL), and the glucocorticoid-inducible protein annexin 1. In addition to regulation of neutrophil activation and recruitment, these ligands have a profound influence on neutrophil survival and apoptosis with contrasting actions, mediating aggravation or resolution of the inflammatory response. Thus, annexin 1 accelerates, whereas SAA rescues human neutrophils from constitutive apoptosis by preventing mitochondrial dysfunction and subsequent activation of caspase-3. Furthermore, ATL overcomes the antiapoptosis signal from SAA and redirects neutrophils to caspase-mediated cell death. We review recent developments about the molecular basis of these actions and suggest a novel mechanism by which aspirin promotes resolution of acute inflammation and tissue injury.

Neutrophil recognition of bacterial DNA and Toll-like receptor 9-dependent and -independent regulation of neutrophil function.

Neutrophils are essential for host defense and detect the presence of invading microorganisms through recognition of pathogen-associated molecular patterns. Among these receptors are Toll-like receptors (TLRs). Neutrophils express all known TLRs except for TLR3. TLR9, localized intracellularly, is to date the best characterized sensor for bacterial DNA, containing short sequences of unmethylated CpG motifs, though TLR9-independent intracellular DNA recognition mechanism(s) may also exist. Bacterial DNA has profound impact on neutrophil functions; it promotes neutrophil trafficking in vivo, induces chemokine expression, regulates expression of adhesion molecules, enhances phagocyte activity, and rescues neutrophils from constitutive apoptosis. TLR9 stimulation results in alterations in cellular redox balance, peroxynitrite formation, activation of the mitogen-activated protein kinase, PI3-kinase, and Jun N-terminal kinase pathways and/or nuclear factor kappaB and AP-1. These features identify an important role for bacterial DNA and TLR9 signaling in the regulation of neutrophil functions that are critical for optimal expression as well as for resolution of the inflammatory response.

Loss of pentameric symmetry in C-reactive protein induces interleukin-8 secretion through peroxynitrite signaling in human neutrophils.

Plasma levels of C-reactive protein (CRP), nitrotyrosine, and interleukin-8 (IL-8) are known predictors of acute cardiovascular events. Peroxynitrite (ONOO-) may function as an intracellular signal for the production of IL-8; however, it is not known whether CRP regulates these events. Emerging evidence suggests that some bioactivities of CRP are expressed only when the pentameric structure of CRP is lost, resulting in formation of monomeric or modified CRP (mCRP). We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on ONOO- formation and ONOO--mediated IL-8 gene expression in human leukocytes. Incubation of human whole blood or isolated neutrophils with mCRP (0.1 to 100 microg/mL) for 4 hours increased IL-8 gene expression and secretion that was blocked approximately 70% by the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). In neutrophils, mCRP simultaneously increased superoxide production and endothelial nitric oxide synthase-mediated NO formation, leading to enhanced ONOO- formation, and consequently activation of nuclear factor-kappaB and activator protein-1. Native CRP had no detectable effect at 4 hours, whereas it enhanced IL-8 release after a 24-hour incubation that was blocked by L-NAME. An anti-CD16 antibody, but not an anti-CD32 antibody, produced 60% to 70% reductions in mCRP-stimulated NO formation and IL-8 release (both P<0.05). These results suggest that loss of the pentameric symmetry in CRP, resulting in formation of mCRP, leads to IL-8 release from human neutrophils via peroxynitrite-mediated activation of nuclear factor-kappaB and activator protein-1.

Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils.

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K(+) efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K(+) electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24-48 h of culture. High extracellular K(+) (10-100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K(+) efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (DeltaPsi(m)) that preceded development of apoptotic morphology. Changes in DeltaPsi(m) resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K(+) efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K(+) released locally from damaged cells may function as a survival signal for neutrophils.

Opposing effects of C-reactive protein isoforms on shear-induced neutrophil-platelet adhesion and neutrophil aggregation in whole blood.

BACKGROUND: Plasma C-reactive protein (CRP) level is a powerful predictor of cardiovascular events. However, it is not known whether CRP could affect neutrophil-platelet adhesion and neutrophil aggregation, key events in acute coronary syndromes. Emerging in vitro evidence suggests that some bioactivities of CRP are expressed on loss of the pentameric symmetry, resulting in formation of modified or monomeric CRP (mCRP). METHODS AND RESULTS: We studied the impact of human native CRP and bioengineered mCRP that cannot rearrange into the pentameric structure on the kinetics of neutrophil-platelet adhesion and neutrophil aggregation in whole blood subjected to shear (approximately 100 s(-1)) using real-time flow cytometry. Shear resulted in upregulation of platelet P-selectin expression, leading to platelet capture of neutrophils and subsequent neutrophil aggregation, which was dependent on P-selectin, L-selectin, and CD18. Native CRP at clinically relevant concentrations markedly attenuated these changes. The residual amount of neutrophil adhesion was blocked with anti-CD18 or anti-CD11b antibody. By contrast, mCRP concentration-dependently enhanced shear-induced platelet P-selectin expression and increased the rate and extent of formation of both neutrophil-platelet and neutrophil-neutrophil aggregates. Complete abrogation of platelet-neutrophil adhesion and neutrophil aggregation required both anti-P-selectin and anti-CD18 antibodies but not anti-L-selectin antibody. The CRP action was markedly inhibited by an anti-CD32 antibody, whereas the mCRP effects were significantly attenuated by an anti-CD16 antibody. CONCLUSIONS: These results indicate that native CRP inhibits platelet activation and prevents platelet capture of neutrophils, whereas mCRP displays potent prothrombotic activities under low levels of shear. Thus, mCRP rather than native CRP may precipitate acute coronary syndromes.

Conformational rearrangement in C-reactive protein is required for proinflammatory actions on human endothelial cells.

BACKGROUND: C-reactive protein (CRP) has been suggested to actively amplify the inflammatory response underlying coronary heart diseases by directly activating endothelial cells. In this study, we investigated whether loss of the cyclic pentameric structure of CRP, resulting in formation of modified or monomeric CRP (mCRP), is a prerequisite for endothelial cell activation. METHODS AND RESULTS: We examined the impact of native CRP and mCRP on the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), key regulators of leukocyte recruitment, and on the expression of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular adhesion molecule-1 (VCAM-1) in human cultured coronary artery endothelial cells (HCAECs). Incubation with mCRP for 4 hours increased MCP-1 and IL-8 secretion and mRNA levels and expression of ICAM-1, E-selectin, and VCAM-1 protein and mRNA. Significant induction occurred at 1 to 5 microg/mL, reached a maximum at 30 microg/mL, and did not require the presence of serum. Native CRP was without detectable effects at 4 hours, whereas it enhanced cytokine release after a 24-hour incubation. An anti-FcgammaRIII (CD16) but not an anti-FcgammaRII (CD32) antibody produced a 14% to 32% reduction of the mCRP effects (P<0.05). mCRP but not CRP evoked phosphorylation of p38 mitogen-activated protein kinase, and inhibition of this kinase with SB 203580 reversed the effects of mCRP. Furthermore, culture of HCAECs in the presence of SB203580 markedly decreased mCRP-stimulated E-selectin and ICAM-1-dependent adhesion of neutrophils to HCAECs (P<0.001). CONCLUSIONS: Loss of pentameric symmetry in CRP, resulting in formation of mCRP, promotes a proinflammatory HCAEC phenotype through a p38 MAPK-dependent mechanism.

CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytes.

Human neutrophil granulocytes die rapidly, and their survival is contingent upon rescue from programmed cell death by signals from the environment. We now show that a novel signal for delaying neutrophil apoptosis is unmethylated CpG motifs prevalent in bacterial DNA (CpG- DNA). Human neutrophils express toll-like receptor 9 that recognizes these motifs. CpG-DNA, but not mammalian DNA or methylated bacterial DNA, markedly enhanced neutrophil viability by delaying spontaneous apoptosis. Endosomal maturation of CpG-DNA is prerequisite for these actions and was coupled to concurrent activation of the extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase/Akt signaling pathways, leading to phosphorylation of BAD at Ser112 and Ser136, respectively, and to prevention of decreases in mitochondrial transmembrane potential, cytochrome c release and caspase-3 activation. Consistently, pharmacological inhibition of either ERK or phosphatidylinositol 3-kinase partially reversed these actions of CpG-DNA; however, they did not produce additive inhibition. Furthermore, intravenous injection of CpG-DNA (200 microg/kg) into rats evoked slight decreases in blood pressure and induced a modest leukocytosis, whereas it effectively suppressed neutrophil apoptosis as assayed ex vivo. Our results indicate that unmethylated CpG motifs in bacterial DNA promote neutrophil survival by suppressing the apoptotic machinery and may therefore contribute to prolongation and amplification of inflammation.