Daping Fan

MicroRNA-155 Deficiency Results in Decreased Macrophage Inflammation and Attenuated Atherogenesis in Apolipoprotein E–Deficient Mice

Objective—microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. Approach and Results—Quantitative real-time polymerase chain reaction showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was correlated positively with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to lipopolysaccharide through targeting suppressor of cytokine signaling-1 and impaired cholesterol efflux from acetylated low-density lipoprotein–loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses and enhanced cholesterol efflux possibly via enhancing lipid loading–induced macrophage autophagy. We next examined the atherogenesis in apolipoprotein E–deficient (apoE−/−) and miR155−/−/apoE−/− (double knockout) mice fed a Western diet. Compared with apoE−/− mice, the double knockout mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells and reduced numbers of Th17 cells and CD11b+/Ly6Chigh cells in the spleen of double knockout mice. Peritoneal macrophages from the double knockout mice had significantly reduced proinflammatory cytokine expression and secretion both in the absence and presence of lipopolysaccharide stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed a bone marrow transplantation study. Deficiency of miR155 in bone marrow–derived cells suppressed atherogenesis in apoE−/− mice, demonstrating that hematopoietic cell–derived miR155 plays a critical role. Conclusions—miR155 deficiency attenuates atherogenesis in apoE−/− mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an antiatherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis.

Serum Proprotein Convertase Subtilisin/Kexin Type 9 and Cell Surface Low-Density Lipoprotein Receptor Evidence for a Reciprocal Regulation

Background— Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation. Methods and Results— To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B–containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR−/− mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR+/− mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR−/− recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression. Conclusions— Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.

Self-Association of Human PCSK9 Correlates with Its LDLR-Degrading Activity

Genetic studies have demonstrated an important role for proprotein convertase subtilisin/kexin type 9 (PCSK9) as a determinant of plasma cholesterol levels. However, the underlying molecular mechanism is not completely understood. To this end, we have generated a mammalian cell expression system for human PCSK9 and its mutants and produced transgenic mice expressing human PCSK9. HEK293T cells transfected with the human PCSK9 DNA construct expressed and secreted PCSK9 and displayed decreased LDLR levels; functional PCSK9 protein was purified from the conditioned medium. In vitro studies showed that PCSK9 self-associated in a concentration-, temperature-, and pH-dependent manner. A mixture of PCSK9 monomers, dimers, and trimers displayed an enhanced LDLR degrading activity compared to monomeric PCSK9. A gain-of-function mutant, D374Y, displayed greatly increased self-association compared to wild-type PCSK9. Moreover, we demonstrated that the catalytic domain of PCSK9 is responsible for the self-association. Self-association of PCSK9 was enhanced by incubation with mouse apoE-/- VLDL and inhibited by incubation with both human and mouse HDL. When PCSK9 protein was incubated with total serum, it partially associated with LDL and HDL but not with VLDL. In transgenic mice, PCSK9 also associated with LDL and HDL but not with VLDL. We conclude that self-association is an intrinsic property of PCSK9, correlated to its LDLR-degrading activity and affected by plasma lipoproteins. These results provide a basis for developing strategies to manipulate PCSK9 activity in the circulation for the treatment of hypercholesterolemia.

Characterization of Sparstolonin B, a Chinese Herb-derived Compound, as a Selective Toll-like Receptor Antagonist with Potent Anti-inflammatory Properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.

Local effects of human PCSK9 on the atherosclerotic lesion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low‐density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE−/− and LDLR−/− mice, which were then placed on a high‐fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow‐derived PCSK9 progressively accumulated in lesions of apoE−/− recipient mice, while increasing the infiltration of Ly6Chi inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b‐ and Ly6Chi‐positive cell numbers in spleens of apoE−/− mice. In vitro, expression of hPCSK9 in LPS‐stimulated macrophages increased mRNA levels of the pro‐inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti‐inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR‐dependent, as PCSK9 protein was not detected in lesions of LDLR−/− recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR‐dependent but cholesterol‐independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction. Copyright

MicroRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis

MicroRNAs (miRNAs) play important roles in the regulation of immune responses. There is evidence that miRNAs also participate in the pathogenesis of multiple sclerosis (MS), but how the miRNAs regulate the pathogenesis of MS is still under investigation. The identification of new members of the miRNA family associated with the pathogenesis of MS could facilitate early diagnosis and treatment. Here, we show that the level of miRNA let‐7e is significantly upregulated in EAE, an animal model of MS using miRNA array and quantitative real‐time PCR. The expression of let‐7e was mainly in CD4+ T cells and infiltrated mononuclear cells of CNS, and highly correlated with the development of EAE. We found that let‐7e silencing in vivo inhibited encephalitogenic Th1 and Th17 cells and attenuated EAE, with reciprocal increase of Th2 cells; overexpression of let‐7e enhanced Th1 and Th17 cells and aggravated EAE. We also identified IL‐10 as one of the functional targets of let‐7e. Together, we propose that let‐7e is a new miRNA involved in the regulation of encephalitogenic T‐cell differentiation and the pathogenesis of EAE.

Human apolipoprotein E peptides inhibit hepatitis C virus entry by blocking virus binding

Hepatitis C virus (HCV) entry is a multiple‐step process involving a number of host factors and hence represents a promising target for new antiviral drug development. In search of novel inhibitors of HCV infection, we found that a human apolipoprotein E (apoE) peptide, hEP, containing both a receptor binding fragment and a lipid binding fragment of apoE specifically blocked the entry of cell culture grown HCV (HCVcc) at submicromolar concentrations. hEP caused little cytotoxicity in vitro and remained active even if left 24 hours in cell culture. Interestingly, hEP inhibited neither human immunodeficiency virus (HIV)‐HCV pseudotypes (HCVpp) nor HIV and Dengue virus (DENV) infection. Further characterization mapped the anti‐HCV activity to a 32‐residue region that harbors the receptor binding domain of apoE, but this fragment must contain a cysteine residue at the N‐terminus to mediate dimer formation. The anti‐HCV activity of the peptide appears to be dependent on both its length and sequence and correlates with its ability to bind lipids. Finally, we demonstrated that the apoE‐derived peptides directly blocked the binding of both HCVcc and patient serum‐derived virus to hepatoma cells as well as primary human hepatocytes. Conclusion: apoE peptides potently inhibit HCV infection and suggest a direct role of apoE in mediating HCV entry. Our findings also highlight the potential of developing apoE mimetic peptides as novel HCV entry inhibitors by targeting HCV‐host interactions. (HEPATOLOGY 2012)

Novel Domain Interaction Regulates Secretion of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Protein

Background: The lack of understanding of the structure-function relation of PCSK9 hinders efforts to develop small molecule inhibitors. Results: The prodomains of C-terminal domain deletion PCSK9 mutants enable secretion of prodomain deletion mutants. Conclusion: An interaction between the prodomain and C-terminal domain regulates the secretion of PCSK9. Significance: PCSK9 may be inhibited by disrupting the interaction between the prodomain and C-terminal domain. PCSK9 (proprotein convertase subtilisin/kexin type 9) has emerged as a novel therapeutic target for hypercholesterolemia due to its LDL receptor (LDLR)-reducing activity. Although its structure has been solved, the lack of a detailed understanding of the structure-function relation hinders efforts to develop small molecule inhibitors. In this study, we used mutagenesis and transfection approaches to investigate the roles of the prodomain (PD) and the C-terminal domain (CD) and its modules (CM1–3) in the secretion and function of PCSK9. Deletion of PD residues 31–40, 41–50, or 51–60 did not affect the self-cleavage, secretion, or LDLR-degrading activity of PCSK9, whereas deletion of residues 61–70 abolished all of these functions. Deletion of the entire CD protein did not impair PCSK9 self-cleavage or secretion but completely abolished LDLR-degrading activity. Deletion of any one or two of the CD modules did not affect self-cleavage but influenced secretion and LDLR-reducing activity. Furthermore, in cotransfection experiments, a secretion-defective PD deletion mutant (ΔPD) was efficiently secreted in the presence of CD deletion mutants. This was due to the transfer of PD from the cotransfected CD mutants to the ΔPD mutant. Finally, we found that a discrete CD protein fragment competed with full-length PCSK9 for binding to LDLR in vitro and attenuated PCSK9-mediated hypercholesterolemia in mice. These results show a previously unrecognized domain interaction as a critical determinant in PCSK9 secretion and function. This knowledge should fuel efforts to develop novel approaches to PCSK9 inhibition.

miR-155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health‐care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR‐155, a microRNA, is up‐regulated in various inflammatory disease states, including IBD, and is a positive regulator of T‐cell responses. To date, no reports have defined a function for miR‐155 with regard to cellular responses in IBD. Using an acute experimental colitis model, we found that miR‐155−/− mice, as compared to wild‐type control mice, have decreased clinical scores, a reversal of colitis‐associated pathogenesis, and reduced systemic and mucosal inflammatory cytokines. The increased frequency of CD4+ lymphocytes in the spleen and lamina propria with dextran sodium sulphate induction was decreased in miR‐155−/− mice. Similarly, miR‐155 deficiency abrogated the increased numbers of interferon‐γ expressing CD4+ T cells typically observed in wild‐type mice in this model. The frequency of systemic and mucosal T helper type 17‐, CCR9‐expressing CD4+ T cells was also reduced in miR‐155−/− mice compared with control mice. These findings strongly support a role for miR‐155 in facilitating pro‐inflammatory cellular responses in this model of IBD. Loss of miR‐155 also results in decreases in T helper type 1/type 17, CD11b+, and CD11c+ cells, which correlated with reduced clinical scores and severity of disease. miR‐155 may serve as a potential therapeutic target for the treatment of IBD.

Ursolic acid promotes cancer cell death by inducing Atg5-dependent autophagy

Ursolic acid (UA) has been reported to possess anticancer activities. Although some of the anticancer activities of UA have been explained by its apoptosis‐inducing properties, the mechanisms underlying its anticancer actions are largely unknown. We have found that UA‐activated autophagy induced cytotoxicity and reduced tumor growth of cervical cancer cells TC‐1 in a concentration‐dependent manner. UA did not induce apoptosis of TC‐1 cells in vitro as determined by annexin V/propidium iodide staining, DNA fragmentation, and Western blot analysis of the apoptosis‐related proteins. We found that UA increased punctate staining of light chain 3 (LC3), which is an autophagy marker. LC3II, the processed form of LC3I which is formed during the formation of double membranes, was induced by UA treatment. These results were further confirmed by transmission electron microscopy. Wortmannin, an inhibitor of autophagy, and a small interfering RNA (siRNA) for autophagy‐related genes (Atg5) reduced LC3II and simultaneously increased the survival of TC‐1 cells treated with UA. We also found that LC3II was significantly reduced and that survival was increased in Atg5−/− mouse embryonic fibroblast (MEF) cells compared to Atg5+/+ MEF cells under UA treatment. However, silencing BECN1 by siRNA affected neither the expression of LC3II nor the survival of TC‐1 cells under UA treatment. These results suggest that autophagy is a major mechanism by which UA kills TC‐1 cells. It is Atg5 rather than BECN1 that plays a crucial role in UA‐induced autophagic cell death in TC‐1 cells. The activation of autophagy by UA may become a potential cancer therapeutic strategy complementing the apoptosis‐based therapies. Furthermore, regulation of Atg5 may improve the efficacy of UA in cancer treatment.