Caveolin-1, a binding protein of CD26, is essential for the anti-inflammatory effects of dipeptidyl peptidase-4 inhibitors on human and mouse macrophages.

We previously reported that inhibition of dipeptidyl peptidase (DPP)-4, the catalytic site of CD26, prevents atherosclerosis in animal models through suppression of inflammation; however, the underlying molecular mechanisms have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae located on the surface of the cellular membrane, has been reported to modulate inflammatory responses by binding to CD26 in T cells. In this study, we investigated the role of Cav-1 in the suppression of inflammation mediated by the DPP-4 inhibitor, teneligliptin, using mouse and human macrophages. Mouse peritoneal macrophages were isolated from Cav-1+/+ and Cav-1-/- mice after stimulation with 3% thioglycolate. Inflammation was induced by the toll-like receptor (TLR)4 agonist, lipopolysaccharide (LPS), isolated from Escherichia coli. The expression of pro-inflammatory cytokines was determined using reverse transcription-polymerase chain reaction. Co-expression of Cav-1 and CD26 was detected using immunohistochemistry in both mouse and human macrophages. Teneligliptin treatment (10 nmol/L) suppressed the LPS-induced expression of interleukin (IL)-6 (70%) and tumor necrosis factor-α (37%) in peritoneal macrophages isolated from Cav-1+/+ mice. However, teneligliptin did not have any effect on the macrophages from Cav-1-/- mice. In human monocyte/macrophage U937 cells, teneligliptin treatment suppressed LPS-induced expression of pro-inflammatory cytokines in a dose-dependent manner (1-10 nmol/L). These anti-inflammatory effects of teneligliptin were mimicked by gene knockdown of Cav-1 or CD26 using small interfering RNA transfection. Furthermore, neutralization of these molecules using an antibody against CD26 or Cav-1 also showed similar suppression. Teneligliptin treatment specifically inhibited TLR4 and TLR5 agonist-mediated inflammatory responses, and suppressed LPS-induced phosphorylation of IL-1 receptor-associated kinase 4, a downstream molecule of TLR4. Next, we determined whether teneligliptin could directly inhibit the physical interaction between Cav-1 and CD26 using the Biacore system. Binding of CD26 to Cav-1 protein was detected. Unexpectedly, teneligliptin also bound to Cav-1, but did not interfere with CD26-Cav-1 binding, suggesting that teneligliptin competes with CD26 for binding to Cav-1. In conclusion, we demonstrated that Cav-1 is a target molecule for DPP-4 inhibitors in the suppression of TLR4-mediated inflammation in mouse and human macrophages.

Vasoprotective effects of urocortin 1 against atherosclerosis in vitro and in vivo.

AIM: Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis. METHODS: We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice. RESULTS: Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe-/- mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions. CONCLUSIONS: This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.

A glucagon-like peptide-1 analog liraglutide suppresses macrophage foam cell formation and atherosclerosis.

Macrophage foam cell formation, characterized by cholesterol ester accumulation catalyzed by acyl-CoA:cholesterol acyltransferase 1 (ACAT1), is the hallmark of early atherogenesis. We previously demonstrated the suppressive effects of incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), on the development of atherosclerotic lesions in apolipoprotein E-deficient (apoE(-/-)) mice. The present study was performed to evaluate the suppressive effects of these incretins and GLP-1 analogs, such as exendin-4 and liraglutide, on human macrophage foam cell formation in vitro and those of liraglutide on atherosclerotic lesion development in apoE(-/-) mice. We investigated the suppressive effects of GLP-1, GIP, exendin-4, and liraglutide against oxidized low-density lipoprotein (oxLDL)-induced foam cell formation in primary cultured human monocyte-derived macrophages. Seventeen-week-old apoE(-/-) mice were administered a long-acting GLP-1 analog liraglutide by osmotic mini-pumps for 4 weeks. Aortic atherosclerosis, oxLDL-induced foam cell formation, and related gene expression in exudate peritoneal macrophages were determined in vivo and ex vivo. Receptors for GLP-1 and GIP were expressed at high levels in human aortic smooth muscle cells and monocytes, but at relatively low levels in human macrophages and foam cells. GLP-1, GIP, exendin-4, and liraglutide significantly suppressed oxLDL-induced foam cell formation mainly associated with ACAT1 down-regulation in human monocyte-derived macrophages. The infusion of liraglutide into apoE(-/-) mice significantly retarded atherosclerotic lesions with monocyte/macrophage infiltration in the aortic wall and suppressed foam cell formation and ACAT1 expression in macrophages. These findings indicate that liraglutide could prevent the development of atherosclerotic lesions by suppressing macrophage foam cell formation mainly associated with ACAT1 down-regulation.

Preventive effect of dipeptidyl peptidase-4 inhibitor on atherosclerosis is mainly attributable to incretin's actions in nondiabetic and diabetic apolipoprotein E-null mice.

AIM: Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe (-/-)) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). METHODS: Nontreated Apoe (-/-) mice, streptozotocin-induced diabetic Apoe (-/-) mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9-39), the GIP receptor blocker, (Pro(3))GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined. RESULTS: Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe (-/-) mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe (-/-) mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9-39) or (Pro(3))GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9-39)+(Pro(3))GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation. CONCLUSIONS: Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.

Glucose-dependent insulinotropic polypeptide prevents the progression of macrophage-driven atherosclerosis in diabetic apolipoprotein E-null mice.

AIM: We recently reported that glucose-dependent insulinotropic polypeptide (GIP) prevents the development of atherosclerosis in apolipoprotein E-null (Apoe(-/-)) mice. GIP receptors (GIPRs) are found to be severely down-regulated in diabetic animals. We examined whether GIP can exert anti-atherogenic effects in diabetes. METHODS: Nondiabetic Apoe(-/-) mice, streptozotocin-induced diabetic Apoe(-/-) mice, and db/db mice were administered GIP (25 nmol/kg/day) or saline (vehicle) through osmotic mini-pumps for 4 weeks. The animals were assessed for aortic atherosclerosis and for oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages. RESULTS: Diabetic Apoe(-/-) mice of 21 weeks of age exhibited more advanced atherosclerosis than nondiabetic Apoe(-/-) mice of the same age. GIP infusion in diabetic Apoe(-/-) mice increased plasma total GIP levels by 4-fold without improving plasma insulin, glucose, or lipid profiles. GIP infusion significantly suppressed macrophage-driven atherosclerotic lesions, but this effect was abolished by co-infusions with [Pro(3)]GIP, a GIPR antagonist. Foam cell formation was stimulated by 3-fold in diabetic Apoe(-/-) mice compared with their nondiabetic counterparts, but this effect was halved by GIP infusion. GIP infusion also attenuated the foam cell formation in db/db mice. In vitro treatment with GIP (1 nM) reduced foam cell formation by 15% in macrophages from diabetic Apoe(-/-) mice, and this attenuating effect was weaker than that attained by the same treatment of macrophages from nondiabetic counterparts (35%). While GIPR expression was reduced by only about a half in macrophages from diabetic mice, it was reduced much more dramatically in pancreatic islets from the same animals. Incubation with high glucose (500 mg/dl) for 9-10 days markedly reduced GIPR expression in pancreatic islet cells, but not in macrophages. CONCLUSIONS: Long-term infusion of GIP conferred significant anti-atherogenic effects in diabetic mice even though the GIPR expression in macrophages was mildly down-regulated in the diabetic state.

Effects of PKF275-055, a dipeptidyl peptidase-4 inhibitor, on the development of atherosclerotic lesions in apolipoprotein E-null mice.

We recently discovered that glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide can both prevent the development of atherosclerosis in apolipoprotein E-null (Apoe(-/-)) mice. In the present study, we attempted to extend these findings to orally administered dipeptidyl peptidase (DPP)-4 inhibitor. Seventeen-week-old Apoe(-/-) mice fed an atherogenic diet were administered a DPP-4 inhibitor, vildagliptin analogue (PKF275-055 [PKF], 100 µm/[kg d]), in drinking water over a period of 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation were determined. Orally administered PKF increased plasma levels of active glucagon-like peptide-1 by 3.5-fold, increased total glucose-dependent insulinotropic polypeptide levels by 2-fold, reduced body weight by 13%, and reduced plasma cholesterol levels by 30%. Compared with drinking water controls, PKF significantly suppressed total aortic atherosclerotic lesions, atheromatous plaque in the aortic root, and macrophage accumulation in the aortic wall by 30% to 40% (P < .001). None of these changes were associated with the PKF-induced reductions in body weight and plasma cholesterol levels. Foam cell formation was suppressed by 40% in the exudate peritoneal macrophages obtained from the PKF-treated mice. The DPP-4 inhibitor prevents the development of atherosclerotic lesions by suppressing macrophage foam cell formation.

Intact glucagon-like peptide-1 levels are not decreased in Japanese patients with type 2 diabetes.

Impaired secretion of glucagon-like peptide 1 (GLP-1) has been suggested to contribute to the deficient incretin effect in patients with type 2 diabetes mellitus (T2DM). Recent studies, however, have not always supported this notion. Since Japanese patients with T2DM usually have severe impairment in the earlyphase of insulin secretion, the measurement of incretin secretions in Japanese T2DM patients would be useful for assessing the association between incretin levels and insulin secretion. We conducted an oral glucose tolerance test (75 g) (OGTT) and meal tolerance test (480 kcal) (MTT) for subjects with normal glucose tolerance (NGT, n=12), subjects with impaired glucose tolerance (IGT, n=7), and T2DM patients (n=21). The tests were carried out over 120-min study periods on separate occasions. Intact GLP-1, GIP, and dipeptidyl peptidase (DPP)-IV were measured by ELISA. T2DM exhibited an impaired early phase of insulin secretion and a reduction in glucagon suppression. There were no significant differences in GLP-1 or GIP levels at each sampling time among NGT, IGT, and T2DM after the ingestions; hence the incremental areas under the curve (IAUC) for the three groups were quite similar. The levels of DPP-IV, a limiting enzyme for the degradation of incretins, were comparable among the three groups. The GLP-1-IAUC was not correlated with IAUCs of insulin, C-peptide, or glucagon determined by the OGTT or the MTT. We concluded that intact GLP-1 levels are comparable between non-diabetics and T2DM, suggesting that impaired insulin secretion in Japanese T2DM is not attributable to defect in GLP-1 secretion.

Synergistic induction of CX3CL1 by TNF alpha and IFN gamma in osteoblasts from rheumatoid arthritis: involvement of NF-kappa B and STAT-1 signaling pathways.

To explore the regulation of CX3CL1 in inflammatory bone diseases, CX3CL1 expression by osteoblasts (OB) was examined. Human OB isolated from rheumatoid arthritis (RA) patients, osteoarthritis patients, and normal individuals were incubated in the presence of cytokines. Soluble CX3CL1 levels were determined with an enzyme-linked immunosorbent assay. Expression of CX3CL1 mRNA was examined using quantitative real-time polymerase chain reaction. Although tumor necrosis factor (TNF)-α or interferon (IFN)-γ alone RA OB induced negligible CX3CL1 secretion, the combination of TNF-α and IFN-γ induced dramatic increases in both soluble CX3CL1 protein and mRNA transcripts. This synergistic effect was more pronounced in OB from RA than in OB from either osteoarthritis or normal individuals. The expression of CX3CL1 was markedly reduced by specific inhibitors of the nuclear factor-κB (NF-κB) or STAT-1 transcription factor. These findings suggest that osteoblasts are an important cellular source of CX3CL1 and may play roles in inflammatory bone/joint diseases.

Role of RIG-I, MDA-5, and PKR on the expression of inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells.

BACKGROUND: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and reported that dsRNA stimulates expression of inflammatory chemokines through a receptor of dsRNA Toll-like receptor (TLR) 3 in airway epithelial cells. In this study, we focused our study on the role of other receptors for dsRNA, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and double-stranded RNA-dependent protein kinase (PKR). METHODS: Airway epithelial cell BEAS-2B was cultured in vitro. Expression of target RNA and protein were analyzed by PCR and ELISA. To analyze the role of receptors for dsRNA, knockdown of theses genes was performed with short interfering RNA (siRNA). RESULTS: We first investigated the effects of chloroquine, an inhibitor of lysosomal acidification, on the expression of chemokines. Preincubation with 100 microM chloroquine significantly inhibited the expression of mRNA for RANTES, IP-10, and IL-8, stimulated by poly I:C, indicating that poly I:C may react with a receptor expressed inside the cells. RIG-I, MDA-5, and PKR are supposed to be expressed inside the airway epithelial cells. However, the expression of chemokines stimulated with poly I:C was not significantly inhibited for these putative receptors in the cells which were transfected with siRNA. CONCLUSIONS: Synthetic dsRNA poly I:C stimulates the expression of inflammatory chemokines in airway epithelial cells, but the putative receptors for dsRNA such as RIG-I, MDA-5, or PKR may not play pivotal roles in this process. TLR3 may play a major role as reported previously.

Differential regulation of chemokine expression by Th1 and Th2 cytokines and mechanisms of eotaxin/CCL-11 expression in human airway smooth muscle cells.

BACKGROUND: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells. METHODS: Human ASM cells were cultured in vitro and treated with IL-4, interferon-gamma (IFNgamma), and tumor necrosis factor-alpha (TNFalpha). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay. RESULTS: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNgamma stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter. CONCLUSIONS: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Inhibiting lentiviral replication by HEXIM1, a cellular negative regulator of the CDK9/cyclin T complex.

OBJECTIVE: Tat-dependent transcriptional elongation is crucial for the replication of HIV-1 and depends on positive transcription elongation factor b complex (P-TEFb), composed of cyclin dependent kinase 9 (CDK9) and cyclin T. Hexamethylene bisacetamide-induced protein 1 (HEXIM1) inhibits P-TEFb in cooperation with 7SK RNA, but direct evidence that this inhibition limits the replication of HIV-1 has been lacking. In the present study we examined whether the expression of FLAG-tagged HEXIM1 (HEXIM1-f) affected lentiviral replication in human T cell lines. METHODS: HEXIM1-f was introduced to five human T cell lines, relevant host for HIV-1, by murine leukemia virus vector and cells expressing HEXIM1-f were collected by fluorescence activated cell sorter. The lentiviral replication kinetics in HEXIM1-f-expressing cells was compared with that in green fluorescent protein (GFP)-expressing cells. RESULTS: HIV-1 and simian immunodeficiency virus replicated less efficiently in HEXIM1-f-expressing cells than in GFP-expressing cells of the five T cell lines tested. The viral revertants were not immediately selected in culture. In contrast, the replication of vaccinia virus, adenovirus, and herpes simplex virus type 1 was not limited. The quantitative PCR analyses revealed that the early phase of viral life cycle was not blocked by HEXIM1. On the other hand, Tat-dependent transcription in HEXIM1-f-expressing cells was substantially repressed as compared with that in GFP-expressing cells. CONCLUSION: These data indicate that HEXIM1 is a host factor that negatively regulates lentiviral replication specifically. Elucidating the regulatory mechanism of HEXIM1 might lead to ways to control lentiviral replication.