Simvastatin reduces neointimal thickening in low-density lipoprotein receptor-deficient mice after experimental angioplasty without changing plasma lipids.

BACKGROUND: Statins exert antiinflammatory and antiproliferative actions independent of cholesterol lowering. To determine whether these actions might affect neointimal formation, we investigated the effect of simvastatin on the response to experimental angioplasty in LDL receptor-deficient (LDLR-/-) mice, a model of hypercholesterolemia in which changes in plasma lipids are not observed in response to simvastatin. METHODS AND RESULTS: Carotid artery dilation (2.5 atm) and complete endothelial denudation were performed in male C57BL/6J LDLR-/- mice treated with low-dose (2 mg/kg) or high-dose (20 mg/kg) simvastatin or vehicle subcutaneously 72 hours before and then daily after injury. After 7 and 28 days, intimal and medial sizes were measured and the intima to media area ratio (I:M) was calculated. Total plasma cholesterol and triglyceride levels were similar in simvastatin- and vehicle-treated mice. Intimal thickening and I:M were reduced significantly by low- and high-dose simvastatin compared with vehicle alone. Simvastatin treatment was associated with reduced cellular proliferation (BrdU), leukocyte accumulation (CD45), and platelet-derived growth factor-induced phosphorylation of the survival factor Akt and increased apoptosis after injury. CONCLUSIONS: Simvastatin modulates vascular repair after injury in the absence of lipid-lowering effects. Although the mechanisms are not yet established, additional research may lead to new understanding of the actions of statins and novel therapeutic interventions for preventing restenosis.

Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38.

Early events in the response of cells to lipopolysaccharide (LPS) include activation of NF-kappaB and stress-activated MAP kinase p38. Recent studies have shown that the human Toll-like receptor 2 (TLR2) mediates activation of NF-kappaB in response to commercial preparations of LPS (comLPS), membrane lipoproteins, and Gram-positive bacterial products. Here, we show that expression of TLR2 in human embryonic kidney 293 cells enabled p38 phosphorylation in response to comLPS, a synthetic bacterial lipoprotein, and B. subtilis. Activation of p38 was confirmed by an in vitro kinase assay using ATF2 as substrate and by an assay measuring activation of the downstream effector of p38, MAP kinase-activated protein kinase in cells. Thus, TLR2 initiated the signaling pathway for p38 in response to bacterial products.

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.

TLR2 recognizes a bacterial lipopeptide through direct binding.

The TLRs play an important role in the initiation of cellular innate immune responses to a wide range of bacterial products, including LPS and lipoproteins. Although rapid progress has been made on signaling functions of activated TLRs, the molecular mechanisms that lead to TLR activation are still poorly understood. We report in this study that the extracellular domain of TLR2 interacts directly with synthetic bacterial lipopeptide (sBLP), a potent analog of bacterial lipoproteins. Using fluorescently labeled sBLP complexed to soluble recombinant CD14 (rsCD14), we observed specific binding of sBLP to the surface of cells expressing TLR2 transgenes and to a recombinant soluble form of the TLR2 ectodomain. TLR2-mediated binding of sBLP at the cell surface did not require prior induction of intracellular signals. In addition, using a chimeric TLR2/TLR4 construct, we showed that the leucine-rich region of TLR2 carries the specificity for binding of the agonist and for initiating signaling. Specific binding of fluorescent sBLP to purified sTLR2 required sCD14. However, sCD14 was not part of the complex formed by soluble TLR2 and sBLP. Together, these data provide evidence that TLR2 recognizes sBLP through direct binding.

Small molecule antagonists of complement receptor type 3 block adhesion and adhesion-dependent oxidative burst in human polymorphonuclear leukocytes.

The leukocyte integrin complement receptor type 3 (CR3, Mac-1, CD11b/CD18) is the predominant beta(2) integrin receptor of polymorphonuclear leukocytes (PMNs). This cell surface receptor plays a central role in innate immunity against pathogens as well as being a major cellular effector of inflammation and tissue injury. Two small molecules, compounds 1 and 2, have been identified, that interact with CR3 and prevent CR3 from binding to its natural ligand, C3bi. Compounds 1 and 2 have IC(50) values of 0.14 and 0.33 microM, respectively, for the inhibition of binding of monomeric C3bi-alkaline phosphatase to immobilized CR3. Both compounds also inhibit binding of CR3 to biotinylated sheep red blood cells opsonized with C3bi, with IC(50) values in the micromolar range. Inhibition of ligand binding by the compounds is not easily reversed and requires light, suggesting the formation of a covalent adduct through photoactivation. Compounds 1 and 2 also inhibit adhesion of human PMNs to fibrinogen in response to tumor necrosis factor (TNF) or PMA, with IC(50) values of 2.5 to >10 microM. They block the adhesion-dependent production of H(2)O(2) stimulated by TNF or phorbol 12-myristate 13-acetate (PMA) with IC(50) values of 0.2 to 0.8 microM and 1 to 3 microM, respectively. Limited structure-activity relationship studies based on compound 2 indicate the importance of the two benzothiazole rings, an ethyl side chain, and the length of the carbon chain linking the rings. Further modification of these groups may help in making compounds appropriate for in vivo studies.

Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis.

Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.

Deficiency in sPLA(2) does not affect HDL levels or atherosclerosis in mice.

Secretory non-pancreatic phospholipase A(2) (sPLA(2)) has been implicated in inflammation and has been found in human atherosclerotic lesions. To test the effect of sPLA(2) deficiency on atherosclerosis, C57BL/Ks mice (apoE(+/+) and PLA(2)(++) were bred with C57BL/6 apoE knockout mice which are sPLA(2)(--) due to a spontaneous mutation. Sibling pairs of mice (apoE(--)/sPLA(2)(++) and apoE(--)/sPLA(2)(--)) on high fat Western diets were dissected at 22 weeks. In vitro enzyme assays confirmed higher serum sPLA(2) activity in the sPLA(2)(++) compared to sPLA(2)(--) for both sexes, while sPLA(2)(--) males had slightly higher serum cholesterol and phospholipids. Analysis of lipoprotein profiles by FPLC showed no effect of sPLA(2) genotype on any measured parameters. Atherosclerosis was quantitated by assaying cholesterol in aortic extracts. Male sPLA(2) trended slightly higher than sPLA(2)(++) with no statistical significance. Female sPLA(2)(++) and sPLA(2)(--) mice showed no significant differences in any of the measured parameters. These results suggest that the endogenous mouse sPLA(2) gene does not significantly affect HDL or atherosclerosis in mice.

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow.

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to alpha(4)beta(1) or alpha(4)beta(7) on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human alpha(4)beta(7)-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC(50) values of 0.1 and 1 microM, respectively. Intravital microscopy was used to quantitate alpha(4)-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn(2+) to activate alpha(4)beta(7) binding to ligand, anti-alpha(4) monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-beta(1) mAb did not block adhesion, demonstrating that this interaction was dependent on alpha(4)beta(7). TR14035 blocked adhesion to HEVs [ED(50) of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, alpha(4)beta(7)/alpha(4)beta(1) antagonists blocked alpha(4)beta(7)-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.

A small molecule alpha4beta1/alpha4beta7 antagonist differentiates between the low-affinity states of alpha4beta1 and alpha4beta7: characterization of divalent cation dependence.

An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated alpha4beta7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily alpha4beta1 with a KD of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four alpha4beta1/alpha4beta7 antagonists to block binding of activated alpha4beta1 or alpha4beta7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to alpha4beta1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha4beta1 and alpha4beta7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.