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1.
Oncotarget ; 9(40): 25764-25780, 2018 May 25.
Article in English | MEDLINE | ID: mdl-29899820

ABSTRACT

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

2.
J Pharm Sci ; 106(10): 2946-2954, 2017 10.
Article in English | MEDLINE | ID: mdl-28576695

ABSTRACT

An increasing number of therapeutic proteins are being developed for delivery through the subcutaneous (SC) route of administration. Relative to intravenous (IV) administration, the SC route offers more convenience to patients, flexibility in dosing, and potential to reduce health care costs. There is a perception that SC administration can pose a higher immunogenicity risk than IV administration for a given protein. To evaluate whether there is a difference in therapeutic protein immunogenicity associated with administration routes, a more detailed understanding of the interactions with the immune system by each route is needed. Few approved therapeutic proteins have available clinical immunogenicity data sets in the public domain that represent both IV and SC administration routes. This has prevented a direct comparison of the 2 routes of administration across a large sample size. Of the 6 marketed products where SC and IV route-related incidences of anti-drug antibody (ADA) were available, 4 were associated with higher immunogenicity incidence with SC. In other cases, there was no apparent difference between the SC and IV routes. Overall, the ADA incidence was low (<15%) with no impact on safety or efficacy. The challenges associated with identifying specific risk factors unique to SC administration are discussed.


Subject(s)
Antibodies/immunology , Antibody Formation/immunology , Proteins/immunology , Administration, Intravenous/methods , Animals , Humans , Injections, Intravenous/methods , Injections, Subcutaneous/methods , Risk Factors
3.
J Immunol ; 188(12): 6287-99, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22566565

ABSTRACT

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin-dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.


Subject(s)
Endothelial Cells/metabolism , Inflammation/metabolism , Interleukin-17/metabolism , Neutrophil Infiltration/immunology , Tumor Necrosis Factor-alpha/metabolism , Animals , Chemokines/biosynthesis , Endothelial Cells/immunology , Flow Cytometry , Inflammation/immunology , Interleukin-17/immunology , Leukocyte Rolling/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/immunology
4.
J Immunol ; 188(10): 4876-84, 2012 May 15.
Article in English | MEDLINE | ID: mdl-22491251

ABSTRACT

PD-1, a member of the CD28 family of immune regulatory molecules, is expressed on activated T cells, interacts with its ligands, PD-L1/B7-H1 and PD-L2/B7-DC, on other cells, and delivers inhibitory signals to the T cell. We studied the role of this pathway in modulating autoreactive T cell responses in two models of myocarditis. In a CD8(+) T cell-mediated adoptive transfer model, we found that compared with Pd1(+/+) CD8(+) T cells, Pd1(-/-) CD8(+) T cells cause enhanced disease, with increased inflammatory infiltrate, particularly rich in neutrophils. Additionally, we show enhanced proliferation in vivo and enhanced cytotoxic activity of PD-1-deficient T lymphocytes against myocardial endothelial cells in vitro. In experimental autoimmune myocarditis, a disease model dependent on CD4(+) T cells, we show that mice lacking PD-1 develop enhanced disease compared with wild-type mice. PD-1-deficient mice displayed increased inflammation, enhanced serum markers of myocardial damage, and an increased infiltration of inflammatory cells, including CD8(+) T cells. Together, these studies show that PD-1 plays an important role in limiting T cell responses in the heart.


Subject(s)
Muscle Cells/immunology , Muscle Cells/pathology , Myocarditis/immunology , Myocarditis/pathology , Programmed Cell Death 1 Receptor/physiology , T-Lymphocyte Subsets/immunology , Amino Acid Sequence , Animals , Cell Movement/genetics , Cell Movement/immunology , Cells, Cultured , Coculture Techniques , Disease Models, Animal , Female , Inflammation/genetics , Inflammation/pathology , Inflammation/prevention & control , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Myocarditis/genetics , Programmed Cell Death 1 Receptor/deficiency , Programmed Cell Death 1 Receptor/genetics , T-Lymphocyte Subsets/pathology
5.
Atherosclerosis ; 222(1): 59-66, 2012 May.
Article in English | MEDLINE | ID: mdl-22410122

ABSTRACT

OBJECTIVE: Secondhand smoke (SS) induces chronic infection of endothelial cells by Chlamydia pneumoniae (Cpn) in vitro. We investigated the in vivo effect on atherosclerosis following exposure to SS and infection with Cpn both independently and in combination in ApoE-/- mice. METHODS AND RESULTS: Plaques were largest in the combined SS+Cpn-exposed mice with 12-57% greater cross-sectional area compared with all other groups (P<0.03). Quantitative RT-PCR (qRT-PCR) from aortic roots revealed a synergistic upregulation of both OX40L (CD134L) and MyD88 in SS+Cpn mice (P<0.05). This upregulation occurred despite decreased numbers of macrophage, dendritic cell, CD4 T cell and smooth-muscle-cell infiltrates as determined by quantitative IHC and qRT-PCR. To elucidate whether enhanced apoptosis correlated with reduced plaque cellularity, area of Tdt-mediated dUTP nick labeling positive (TUNEL+) cells and expression of key bridging molecules necessary for efferocytosis (Mertk, Tgm2, FasL and C1qa) were examined. In SS+Cpn mice, there was an increase of the area of TUNEL+ cells in plaque cores (P<0.001) and a downregulation of efferocytosis gene expression (P<0.05). Systemic expression of cytokines in sera (Luminex) showed no differences between groups, suggesting that focal disease mechanisms within the plaque predominated. CONCLUSIONS: The combination of SS exposure and Cpn infection enhanced atherosclerosis more than either variable did independently by activating inflammatory cells and by promoting growth and maturation of lesions via defective phagocytic clearance and accumulation of apoptotic cells.


Subject(s)
Atherosclerosis/immunology , Chlamydophila Infections/immunology , Tobacco Smoke Pollution , Animals , Apolipoproteins E/deficiency , Chlamydophila pneumoniae/immunology , Female , In Situ Nick-End Labeling , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/metabolism , OX40 Ligand , Phagocytosis/immunology , Tumor Necrosis Factors/metabolism
6.
J Immunol ; 188(3): 1421-30, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22219321

ABSTRACT

T cell subset-specific migration to inflammatory sites is tightly regulated and involves interaction of the T cells with the endothelium. Th17 cells often appear at different inflammatory sites than Th1 cells, or both subsets appear at the same sites but at different times. Differences in T cell subset adhesion to endothelium may contribute to subset-specific migratory behavior, but this possibility has not been well studied. We examined the adhesion of mouse Th17 cells to endothelial adhesion molecules and endothelium under flow in vitro and to microvessels in vivo and we characterized their migratory phenotype by flow cytometry and quantitative RT-PCR. More Th17 than Th1 cells interacted with E-selectin. Fewer Th17 than Th1 cells bound to TNF-α-activated E-selectin-deficient endothelial cells, and intravital microscopy studies demonstrated that Th17 cells engage in more rolling interactions with TNF-α-treated microvessels than Th1 cells in wild-type mice but not in E-selectin-deficient mice. Th17 adhesion to ICAM-1 was dependent on integrin activation by CCL20, the ligand for CCR6, which is highly expressed by Th17 cells. In an air pouch model of inflammation, CCL20 triggered recruitment of Th17 but not Th1 cells. These data provide evidence that E-selectin- and ICAM-1-dependent adhesion of Th17 and Th1 cells with endothelium are quantitatively different.


Subject(s)
Cell Adhesion/immunology , Endothelium, Vascular/immunology , Th1 Cells/physiology , Animals , Chemokine CCL20/metabolism , E-Selectin/immunology , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiology , Intercellular Adhesion Molecule-1/immunology , Mice , Th17 Cells
7.
J Immunol ; 187(7): 3521-9, 2011 Oct 01.
Article in English | MEDLINE | ID: mdl-21873519

ABSTRACT

The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg, as well as their direct influence at the site of inflammation, remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3(+) adaptive/inducible Treg (iTreg), but not naturally occurring Treg, efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial Ag presentation or immobilized anti-CD3ε, Foxp3(+) iTreg suppressed TNF-α- and IL-1ß-mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGF-ß-induced activin receptor-like kinase 5 signaling in endothelial cells. In addition, Foxp3(+) iTreg adhered to inflamed endothelium in vivo, and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3(+) iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.


Subject(s)
Chemotaxis, Leukocyte/immunology , Endothelium, Vascular/immunology , Inflammation/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Adhesion/immunology , Cell Separation , Endothelium, Vascular/metabolism , Flow Cytometry , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Gene Knock-In Techniques , Mice , Mice, Inbred C57BL , T-Lymphocytes, Regulatory/metabolism
8.
Circulation ; 124(2): 185-95, 2011 Jul 12.
Article in English | MEDLINE | ID: mdl-21690490

ABSTRACT

BACKGROUND: Regulatory T cells (Treg) are present in atherosclerotic lesions and can modulate disease. In this study we characterized changes in Treg responses associated with prolonged hypercholesterolemia and lesion progression. METHODS AND RESULTS: Low-density lipoprotein receptor null mice in which Treg express green fluorescent protein were fed a control or cholesterol-rich diet, and green fluorescent protein-positive cells were enumerated in lymphoid tissues and in aorta. Splenic Treg numbers increased after 4, 8, and 20 weeks in cholesterol-diet-fed mice. However, the number of circulating and lesional Treg peaked at 4 weeks and decreased significantly at 8 and 20 weeks, concomitant with increased numbers of CD4(+) effector T cells and increased lesion size over this period. Treg expression of selectin ligands and their ability to bind to aortic endothelium decreased after prolonged hypercholesterolemia, and apoptosis of lesional Treg increased. After 4 weeks of cholesterol-rich diet, a switch to a control diet for 4 weeks reduced serum cholesterol and stopped lesion growth, and the high aortic Treg content was maintained, compared with mice fed a cholesterol diet for 8 weeks. After the diet reversal, the splenic Treg retained the phenotype of Treg after 4 weeks of cholesterol diet. CONCLUSIONS: Prolonged hypercholesterolemia impairs Treg but not effector T cell accumulation in lesions, but reversal of hypercholesterolemia can prevent loss of lesional Treg. Therefore, cholesterol-lowering therapies may induce dynamic and beneficial changes in Treg:effector T cell ratios in atherosclerotic lesions.


Subject(s)
Cholesterol, Dietary/blood , Cholesterol/blood , Diet, Atherogenic , Endothelium, Vascular/metabolism , Hypercholesterolemia/blood , T-Lymphocytes, Regulatory/metabolism , Animals , Atherosclerosis/blood , Atherosclerosis/immunology , Atherosclerosis/therapy , CD4 Lymphocyte Count , Cholesterol/adverse effects , Cholesterol/pharmacology , Cholesterol, Dietary/adverse effects , Cholesterol, Dietary/pharmacology , Hypercholesterolemia/genetics , Hypercholesterolemia/immunology , Hypercholesterolemia/therapy , Mice , Mice, Knockout , Receptors, LDL/genetics , Receptors, LDL/metabolism , T-Lymphocytes, Regulatory/immunology
9.
Arterioscler Thromb Vasc Biol ; 31(5): 1100-7, 2011 May.
Article in English | MEDLINE | ID: mdl-21393583

ABSTRACT

OBJECTIVE: Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals on interaction with its 2 ligands, PD-L1 and PD-L2. We studied the contribution of the PD-1 pathway to regulation of T cells that promote atherosclerotic lesion formation and inflammation. METHODS AND RESULTS: We show that compared with Ldlr-/- control mice, Pd1-/-Ldlr-/- mice developed larger lesions with more abundant CD4+ and CD8+ T cells and macrophages, accompanied by higher levels of serum tumor necrosis factor-α. Iliac lymph node T cells from Pd1-/-Ldlr-/- mice proliferated more to αCD3 or oxidized low-density lipoprotein stimulation compared with controls. CD8+ T cells from Pd1-/-Ldlr-/- mice displayed more cytotoxic activity compared with controls in vivo and in vitro. Administration of a blocking anti-PD-1 antibody increased lesional inflammation in hypercholesterolemic Ldlr-/- mice with more lesional T cells and more activated T cells in paraaortic lymph nodes. The changes in lesional T-cell content when PD-1 was absent or blocked were also observed in bone marrow chimeric Ldlr-/- mice lacking PD-L1 and PD-L2 on hematopoietic cells. CONCLUSIONS: PD-1 has an important role in downregulating proatherogenic T-cell responses, and blockade of this molecule for treatment of viral infections or cancer may increase risk of cardiovascular complications.


Subject(s)
Antigens, Surface/metabolism , Apoptosis Regulatory Proteins/metabolism , Atherosclerosis/metabolism , Inflammation/metabolism , Lymphocyte Activation , T-Lymphocyte Subsets/metabolism , Animals , Antibodies, Blocking/pharmacology , Antigens, Surface/genetics , Apoptosis , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Atherosclerosis/genetics , Atherosclerosis/immunology , Atherosclerosis/pathology , B7-1 Antigen/genetics , B7-1 Antigen/metabolism , B7-H1 Antigen , Bone Marrow Transplantation , CD3 Complex/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Genes, T-Cell Receptor , Genotype , Hypercholesterolemia/genetics , Hypercholesterolemia/immunology , Hypercholesterolemia/metabolism , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Lipoproteins, LDL/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphocyte Activation/drug effects , Macrophages/immunology , Macrophages/metabolism , Male , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptides/deficiency , Peptides/genetics , Phenotype , Programmed Cell Death 1 Ligand 2 Protein , Programmed Cell Death 1 Receptor , Receptors, LDL/deficiency , Receptors, LDL/genetics , Signal Transduction
10.
Curr Opin Lipidol ; 22(3): 165-70, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21412153

ABSTRACT

PURPOSE OF REVIEW: Statins have diverse effects on the cellular mediators of inflammation and immunity that may be partially responsible for their efficacy in preventing cardiovascular disease, and which have encouraged their use in treating immune/inflammatory diseases. We discuss a selection of recently published studies that provide new insights into the mechanisms by which statins exert anti-inflammatory effects. RECENT FINDINGS: Statins have a variety of direct effects on the gene expression and function of cells of both the innate and adaptive immune systems, including endothelial cells, macrophages, dendritic cells and T cells. Many of these effects are related to statin blockade of GTPase isoprenylation, as has been shown in older literature, although newly identified cell type-specific downstream pathways of GTPase have been described. Recently published analyses of data from clinical trials have also provided further evidence that statin therapy has anti-inflammatory effects and benefits independent of lowering cholesterol. SUMMARY: Ongoing research continues to strengthen the case that statins can modulate immune responses by several mechanisms, independent of lowering blood cholesterol. A major challenge for investigators will be to determine how to take advantage of these new mechanistic insights to improve treatment of cardiovascular disease and primary immune/inflammatory disorders.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Adaptive Immunity/drug effects , Animals , Clinical Trials as Topic , Humans , Immunity, Innate/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
12.
Arterioscler Thromb Vasc Biol ; 30(12): 2604-10, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20864668

ABSTRACT

OBJECTIVE: To gain insights into mechanisms by which intimal hyperplasia interferes with the repair process by investigating expression and function of the catalytic telomerase reverse transcriptase (TERT) subunit after vascular injury. METHODS AND RESULTS: Functional telomerase is essential to the replicative longevity of vascular cells. We found that TERT was de novo activated in the intima of injured arteries, involving activation of the nuclear factor κB pathway. Stimulation of the isolated intimal smooth muscle cell (SMC) by basic fibroblast growth factor or tumor necrosis factor α resulted in increased TERT activity. This depends on the activation of c-Myc signaling because mutation of the E-box in the promoter or overexpression of mitotic arrest deficient 1 (MAD1), a c-Myc competitor, abrogated the transcriptional activity. Inhibition of nuclear factor κB in both intimal SMCs and the injured artery attenuated TERT transcriptional activity through reduction of c-Myc expression. Pharmacological blockade of TERT led to SMC senescence. Finally, depletion of telomerase function in mice resulted in severe intimal SMC senescence after vascular injury. CONCLUSIONS: These results support a model in which vascular injury induces de novo expression of TERT in intimal SMCs via activation of nuclear factor κB and upregulation of c-Myc. The resumed TERT activity is critical for intimal hyperplasia.


Subject(s)
Carotid Artery Injuries/enzymology , Cell Proliferation , Cellular Senescence , Myocytes, Smooth Muscle/enzymology , NF-kappa B/metabolism , Telomerase/metabolism , Transcriptional Activation , Tunica Intima/enzymology , Aminobenzoates/pharmacology , Animals , Binding Sites , Carotid Artery Injuries/genetics , Carotid Artery Injuries/pathology , Cell Proliferation/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Fibroblast Growth Factor 2/metabolism , Gene Expression Regulation, Enzymologic , Hyperplasia , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Male , Mice , Mice, Knockout , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Naphthalenes/pharmacology , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/metabolism , RNA/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Telomerase/antagonists & inhibitors , Telomerase/deficiency , Telomerase/genetics , Transduction, Genetic , Tumor Necrosis Factor-alpha/metabolism , Tunica Intima/drug effects , Tunica Intima/pathology
13.
J Clin Invest ; 120(6): 1961-70, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20440076

ABSTRACT

The transcription factor Krüppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodulatory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/prenylation-dependent pathway. Statins also diminished T cell proliferation and IFN-gamma expression. shRNA blockade of KLF2 expression in human T cells increased IFN-gamma expression and prevented statin-induced IFN-gamma reduction. In a mouse model of myocarditis induced by heart antigen-specific CD8+ T cells, both statin treatment of the T cells and retrovirally mediated overexpression of KLF2 in the T cells had similar ameliorating effects on disease induction. We conclude that statins reduce inflammatory functions and pathogenic activity of T cells through KLF2-dependent mechanisms, and this pathway may be a potential therapeutic target for cardiovascular diseases.


Subject(s)
Gene Expression/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Kruppel-Like Transcription Factors/metabolism , T-Lymphocytes/metabolism , Transcription Factors/metabolism , Acyl Coenzyme A , Animals , Endothelial Cells/drug effects , Endothelial Cells/immunology , Endothelial Cells/metabolism , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/immunology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/immunology , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Prenylation , T-Lymphocytes/immunology , Transcription Factors/genetics , Transcription Factors/immunology
14.
Circ Res ; 106(6): 1040-51, 2010 Apr 02.
Article in English | MEDLINE | ID: mdl-20133903

ABSTRACT

RATIONALE: The multiligand RAGE (receptor for advanced glycation end products) contributes to atherosclerosis in apolipoprotein (Apo)E-null mice. OBJECTIVE: To delineate the specific mechanisms by which RAGE accelerated atherosclerosis, we performed Affymetrix gene expression arrays on aortas of nondiabetic and diabetic ApoE-null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but that we would be able to identify the genes likely involved in diabetes- and RAGE-dependent atherogenesis. METHODS AND RESULTS: We report that there is very little overlap of the genes that are differentially expressed both in the onset of diabetes in ApoE-null mice, and in the effect of RAGE deletion in diabetic ApoE-null mice. Pathway-Express analysis revealed that the transforming growth factor-beta pathway and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE-null mice, and the mechanism by which deletion of RAGE ameliorates this effect. Quantitative polymerase chain reaction studies, Western blotting, and confocal microscopy in aortic tissue and in primary cultures of murine aortic smooth muscle cells supported these findings. CONCLUSIONS: Taken together, our work suggests that RAGE-dependent acceleration of atherosclerosis in ApoE-null mice is dependent, at least in part, on the action of the ROCK1 (rho-associated protein kinase 1) branch of the transforming growth factor-beta pathway.


Subject(s)
Aortic Diseases/etiology , Apolipoproteins E/deficiency , Atherosclerosis/etiology , Diabetes Mellitus, Experimental/complications , Diabetic Angiopathies/etiology , Receptors, Immunologic/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolism , rho-Associated Kinases/metabolism , Animals , Aorta/enzymology , Aorta/pathology , Aortic Diseases/enzymology , Aortic Diseases/genetics , Aortic Diseases/pathology , Apolipoproteins E/genetics , Atherosclerosis/enzymology , Atherosclerosis/genetics , Atherosclerosis/pathology , Blotting, Western , Cell Movement , Cell Proliferation , Cells, Cultured , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , Diabetic Angiopathies/enzymology , Diabetic Angiopathies/genetics , Diabetic Angiopathies/pathology , Disease Progression , Enzyme Activation , Focal Adhesions/enzymology , Gene Expression Profiling/methods , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/pathology , Oligonucleotide Array Sequence Analysis , Receptor for Advanced Glycation End Products , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Thrombospondin 1/metabolism , Time Factors , Transforming Growth Factor beta/genetics , rho-Associated Kinases/genetics
15.
J Clin Invest ; 118(1): 183-94, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18079965

ABSTRACT

Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall.


Subject(s)
Apolipoproteins E , Atherosclerosis/metabolism , Endothelium, Vascular/metabolism , Glycation End Products, Advanced/metabolism , Receptors, Immunologic/immunology , Animals , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/pathology , Disease Models, Animal , Endothelium, Vascular/injuries , Endothelium, Vascular/pathology , Epitopes/genetics , Epitopes/metabolism , Glycation End Products, Advanced/genetics , HMGB1 Protein/genetics , HMGB1 Protein/metabolism , Humans , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Leukocyte L1 Antigen Complex/genetics , Leukocyte L1 Antigen Complex/metabolism , Ligands , Mice , Mice, Knockout , Receptor for Advanced Glycation End Products , Receptors, Immunologic/genetics
16.
Am J Pathol ; 169(6): 2245-53, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17148685

ABSTRACT

Neutrophil gelatinase-associated lipocalin (NGAL) has recently emerged as an important modulator of cell homeostasis. Elevated plasma NGAL levels, possibly because of activation of blood leukocytes, are associated with atherosclerosis. However, little is known about induction of NGAL expression in blood vessels. Using a rat carotid artery injury model, we found that NGAL was highly induced in the intima after angioplasty but was attenuated by adenovirus-mediated expression of a dominant-negative mutant of inhibitor of nuclear factor (NF)-kappaB kinase beta (dnIKKbeta). Expression of NGAL mRNA and protein was also up-regulated in an NF-kappaB-dependent manner in rat and human vascular smooth muscle cells (SMCs) in response to interleukin-1beta stimulation. Rat SMC-produced NGAL was present as mono- and homomeric forms in the cytosol and in a complex containing matrix metalloproteinase-9 (MMP-9) after secretion. In agreement with levels of NGAL, proteolytic activity of MMP-9 was markedly high in the intima of injured vessels and in the culture supernatant of activated intimal SMCs but was reduced in the vessels transduced with dnIKKbeta. The present study reveals a previously unrecognized vascular response to an-gioplastic injury, characterized by NF-kappaB-dependent expression of NGAL in vascular SMCs. Further-more, SMC-produced NGAL interacts with MMP-9, a mechanism by which NGAL may modulate MMP-9 proteolytic activity in the vascular repair process.


Subject(s)
Acute-Phase Proteins/metabolism , Carotid Artery Injuries/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cells, Cultured , Humans , I-kappa B Kinase/physiology , Interleukin-1beta/pharmacology , Lipocalin-2 , Lipocalins , Male , Matrix Metalloproteinase 9/metabolism , Myocytes, Smooth Muscle/metabolism , Rats , Rats, Sprague-Dawley
17.
Proc Natl Acad Sci U S A ; 102(48): 17501-6, 2005 Nov 29.
Article in English | MEDLINE | ID: mdl-16293697

ABSTRACT

Leukotriene B(4) (LTB(4)), a potent leukocyte chemoattractant derived from the 5-lipoxygenase metabolism of arachidonic acid, exerts its action by means of specific cell surface receptors, denoted BLT(1) and BLT(2). In this study, BLT(1) receptor proteins were detected in human carotid artery atherosclerotic plaques, colocalizing with markers for macrophages, endothelial cells, and vascular smooth muscle cells (SMC). Challenge of human coronary artery SMC with either LTB(4) or U75302, a partial agonist that is selective for the BLT(1) receptor, induced an approximately 4-fold increase of whole-cell currents by using the patch-clamp technique, indicating that these cells express functional BLT(1) receptors. LTB(4) induced migration and proliferation of SMC in vitro, and treatment with the BLT receptor antagonist BIIL 284 (10 mg/kg, once daily) for 14 days after carotid artery balloon injury in vivo inhibited intimal hyperplasia in rats. In the latter model, SMC derived from the intima exhibited increased levels of BLT(1) receptor mRNA compared with medial SMC. BLT receptor up-regulation in the intima in vivo, as well as that induced by IL-1beta in vitro, were prevented by transfection with a dominant-negative form of Ikappa kinase beta carried by adenovirus, indicating that BLT(1) receptor expression depends on NF-kappaBeta. These results show that LTB(4) activates functional BLT(1) receptors on vascular SMC, inducing chemotaxis and proliferation, and that BLT(1) receptors were up-regulated through an Ikappa kinase beta/NF-kappaB-dependent pathway. Inhibition of LTB(4)/BLT(1) signaling during the response to vascular injury reduced intimal hyperplasia, suggesting this pathway as a possible target for therapy.


Subject(s)
Atherosclerosis/metabolism , Carotid Artery Injuries/metabolism , Leukotriene B4/pharmacology , Muscle, Smooth, Vascular/metabolism , Receptors, Leukotriene B4/metabolism , Receptors, Purinergic P2/metabolism , Signal Transduction/physiology , Tunica Intima/metabolism , Up-Regulation/drug effects , Amidines/pharmacology , Analysis of Variance , Animals , Blotting, Western , Carbamates/pharmacology , Cell Movement/drug effects , Electrophysiology , Fatty Alcohols/pharmacology , Glycols/pharmacology , Humans , Hyperplasia/metabolism , Male , Muscle, Smooth, Vascular/cytology , NF-kappa B/metabolism , Patch-Clamp Techniques , Polymerase Chain Reaction , Purinergic P2 Receptor Agonists , Purinergic P2 Receptor Antagonists , Rats , Rats, Sprague-Dawley , Receptors, Leukotriene B4/agonists , Receptors, Leukotriene B4/antagonists & inhibitors , Signal Transduction/drug effects
18.
FASEB J ; 19(10): 1293-5, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15939736

ABSTRACT

Nuclear factor-kappaB (NF-kappaB)-mediated vascular inflammation is a prominent characteristic of atherogenesis and restenosis. We noted that angioplastic injury to carotid artery elicited two phases of NF-kappaB activation characterized by an early activation in the arterial media and a late activation coupled with high levels of inhibitor of IkappaB kinase (IKK) activity in intima. These findings prompted us to elucidate the role for the different phases of NF-kappaB activation and IKK in the progress of vascular repair. Our results show that blockade of the early NF-kappaB activation by perivascular administration of pyrrolidine dithiocarbamate transiently attenuates the expression of proinflammatory genes in the injured vessels but does not affect intimal formation. Interruption of IKKbeta by overexpressing a dominant-negative IKKbeta in the injured artery effectively inhibited the late phase of NF-kappaB activation, resulting in down-regulation of inducible nitric oxide synthase, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 expression in conjunction with a 36% reduction in intima size, albeit with a lack of inhibitory effect on the early NF-kappaB activation. Collectively, these findings show that the IKKbeta-mediated late-phase NF-kappaB activation contributes to intimal hyperplasia and the accompanied vascular inflammatory responses.


Subject(s)
I-kappa B Kinase/physiology , NF-kappa B/physiology , Signal Transduction/physiology , Tunica Intima/pathology , Vasculitis/etiology , Angioplasty/adverse effects , Animals , Apoptosis , Carotid Arteries/pathology , Cell Proliferation , Hyperplasia , Male , Muscle, Smooth, Vascular/pathology , Rats , Rats, Sprague-Dawley
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