RESUMO
Patients with systemic autoimmune diseases show increased incidence of atherosclerosis. However, the contribution of proatherogenic factors to autoimmunity remains unclear. We found that atherogenic mice (herein referred to as LDb mice) exhibited increased serum interleukin-17, which was associated with increased numbers of T helper 17 (Th17) cells in secondary lymphoid organs. The environment within LDb mice was substantially favorable for Th17 cell polarization of autoreactive T cells during homeostatic proliferation, which was considerably inhibited by antibodies directed against oxidized low-density lipoprotein (oxLDL). Moreover, the uptake of oxLDL induced dendritic-cell-mediated Th17 cell polarization by triggering IL-6 production in a process dependent on TLR4, CD36, and MyD88. Furthermore, self-reactive CD4(+) T cells that expanded in the presence of oxLDL induced more profound experimental autoimmune encephalomyelitis. These findings demonstrate that proatherogenic factors promote the polarization and inflammatory function of autoimmune Th17 cells, which could be critical for the pathogenesis of atherosclerosis and other related autoimmune diseases.
Assuntos
Aterosclerose/imunologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Interleucina-17/metabolismo , Células Th17/imunologia , Transferência Adotiva , Animais , Anticorpos Bloqueadores/metabolismo , Aterosclerose/genética , Autoimunidade , Antígenos CD36/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Interleucina-6/metabolismo , Lipoproteínas LDL/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: The cAMP second messenger system, a major stress-response pathway, plays essential roles in normal cardiovascular functions and in pathogenesis of heart diseases. Here, we test the hypothesis that the Epac1 (exchange protein directly activated by cAMP 1) acts as a major downstream effector of cAMP signaling to promote atherogenesis and represents a novel therapeutic target. Approach and Results: To ascertain Epac1's function in atherosclerosis development, a triple knockout mouse model (LTe) was generated by crossing Epac1-/- mice with atherosclerosis-prone LDb mice lacking both Ldlr and Apobec1. Deletion of Epac1 led to a significant reduction of atherosclerotic lesion formation as measured by postmortem staining, accompanied by attenuated macrophage/foam cell infiltrations within atherosclerotic plaques as determined by immunofluorescence staining in LTe animals compared with LDb littermates. Primary bone marrow-derived macrophages were isolated from Epac1-null and wild-type mice to investigate the role of Epac1 in lipid uptake and foam cell formation. ox-LDLs (oxidized low-density lipoproteins) stimulation of bone marrow-derived macrophages led to elevated intracellular cAMP and Epac1 levels, whereas an Epac-specific agonist, increased lipid accumulation in wild-type, but not Epac1-null, bone marrow-derived macrophages. Mechanistically, Epac1 acts through PKC (protein kinase C) to upregulate LOX-1 (ox-LDL receptor 1), a major scavenger receptor for ox-LDL uptake, exerting a feedforward mechanism with ox-LDL to increase lipid uptake and propel foam cell formation and atherogenesis. CONCLUSIONS: Our study demonstrates a fundamental role of cAMP/Epac1 signaling in vascular remodeling by promoting ox-LDL uptake and foam cell formation during atherosclerosis lesion development. Therefore, Epac1 represents a promising, unexplored therapeutic target for atherosclerosis.
Assuntos
Aorta Torácica/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Células Espumosas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Placa Aterosclerótica , Receptores Depuradores Classe E/metabolismo , Desaminase APOBEC-1/deficiência , Desaminase APOBEC-1/genética , Animais , Aorta Torácica/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/patologia , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Células Espumosas/patologia , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/deficiência , Receptores de LDL/genética , Sistemas do Segundo Mensageiro , Células THP-1 , Remodelação VascularRESUMO
OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.
Assuntos
Colesterol/sangue , Hepatócitos/metabolismo , Fígado/metabolismo , Lisossomos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Receptores de LDL/metabolismo , Animais , Dieta Hiperlipídica , Regulação para Baixo , Feminino , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatases de Fosfoinositídeos/genética , Transporte Proteico , Proteólise , Receptores de LDL/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) interacts directly with cytoplasmic apoB and prevents its degradation via the autophagosome/lysosome pathway. This process affects VLDL and LDL production and influences atherogenesis. Here, we investigated the molecular machinery by which PCSK9 modulates autophagy and affects atherogenesis. We backcrossed Pcsk9-/- mice with atherosclerosis-prone Ldlr-/-Apobec1-/- (LDb) mice to generate Ldlr-/-Apobec1-/-Pcsk9-/- (LTp) mice. Deletion of PCSK9 resulted in decreased hepatic apoB secretion, increased autophagic flux, and decreased plasma levels of IDL and LDL particles. The LDLs from LTp mice (LTp-LDLs) were less atherogenic and contained less cholesteryl ester and phospholipids than LDb-LDLs. Moreover LTp-LDLs induced lower endothelial expression of the genes encoding TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1ß, Beclin-1, p62, and TRAF6 Collectively, these effects were associated with substantially less atherosclerosis development (>4-fold) in LTp mice. The absence of PCSK9 in LDb mice results in decreased lipid and apoB levels, fewer atherogenic LDLs, and marked reduction of atherosclerosis. The effect on atherogenesis may be mediated in part by the effects of modified LDLs on endothelial cell receptors and proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDL receptor activity.
Assuntos
Apolipoproteínas B/metabolismo , Aterosclerose/metabolismo , Endotélio Vascular/metabolismo , Pró-Proteína Convertase 9/metabolismo , Animais , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pró-Proteína Convertase 9/deficiênciaRESUMO
PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases plasma cholesterol levels by promoting LDL receptor degradation. Current antibody inhibitors block the interaction between PCSK9 and LDL receptors, significantly decrease plasma cholesterol levels, and provide beneficial clinical outcomes. To reduce the action of PCSK9 in plasma, a novel strategy that will produce a panel of non-native, conformationally-altered isomers of PCSK9 (X-PCSK9) to develop active immunotherapy targeting of native PCSK9 and inhibiting/blocking the interaction of PCSK9 with LDL receptor, thus decreasing plasma cholesterol levels is proposed. The authors used the scrambled disulfide bond technique to generate conformationally-altered isomers of the catalytic domain of mouse PCSK9. The focus was on the immune response of four X-isomers and their effects on plasma cholesterol and triglyceride levels in both C57BL/6J and Apoe-/- mice. The authors showed that the four immunogens produced significant immunogenicity against native PCSK9 to day 120 after immunization of C57BL/6J and Apoe-/- mice. This resulted in significantly decreased plasma cholesterol levels in C57BL/6J mice, and to a lesser degree in Apoe-/- mice. The X-PCSK9-B1 treated mice had increased LDL receptor mRNA and protein levels at day 120 after treatment. Thus, this study provides a new, potentially promising approach that uses long-term immunotherapy for a treatment of hypercholesterolemia.
Assuntos
Domínio Catalítico , Metabolismo dos Lipídeos , Pró-Proteína Convertase 9/química , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/genética , Sequência de Aminoácidos , Animais , Anticorpos , Cromatografia Líquida , Dissulfetos/química , Expressão Gênica , Imunidade , Imunização , Isomerismo , Lipídeos/sangue , Camundongos , Camundongos Knockout , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/isolamento & purificação , Dobramento de Proteína , RNA Mensageiro , Receptores de LDL/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization. AB-FUBINACA is an example of one of the new indazole-carboxamide synthetic cannabinoids introduced in the past year. Even though AB-FUBINACA has become increasingly prominent in forensic drug and toxicology specimens analyses, little is known about the pharmacology of this substance. To study its metabolic fate, we utilized Wistar rats to study the oxidative products of AB-FUBINACA in urine and its effect on gene expressions in liver and heart. Rats were injected with 5 mg/kg of AB-FUBINACA each day for 5 days. Urine samples were collected every day at the same time. On day 5 after treatment, we collected the organs such as liver and heart. The urine samples were analyzed by mass spectrometry, which revealed several putative metabolites and positioning of the hydroxyl addition on the molecule. We used quantitative PCR gene expression array to analyze the hepatotoxicity and cardiotoxicity on these rats and confirmed by real-time quantitative RT-PCR. We identified three genes significantly associated with dysfunction of oxidation and inflammation. Our study reports in vivo metabolites of AB-FUBINACA in urine and its effect on the gene expressions in liver and heart.
Assuntos
Canabinoides/urina , Drogas Desenhadas/farmacocinética , Indazóis/urina , Fígado/efeitos dos fármacos , Metaboloma , Animais , Biotransformação , Canabinoides/farmacocinética , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Indazóis/farmacocinética , Fígado/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVE: To examine infiltration of blood foamy monocytes, containing intracellular lipid droplets, into early atherosclerotic lesions and its contribution to development of nascent atherosclerosis. APPROACH AND RESULTS: In apoE(-/-) mice fed Western high-fat diet (WD), >10% of circulating monocytes became foamy monocytes at 3 days on WD and >20% of monocytes at 1 week. Foamy monocytes also formed early in blood of Ldlr(-/-)Apobec1(-/-) (LDb) mice on WD. Based on CD11c and CD36, mouse monocytes were categorized as CD11c(-)CD36(-), CD11c(-)CD36(+), and CD11c(+)CD36(+). The majority of foamy monocytes were CD11c(+)CD36(+), whereas most nonfoamy monocytes were CD11c(-)CD36(-) or CD11c(-)CD36(+) in apoE(-/-) mice on WD. In wild-type mice, CD11c(+)CD36(+) and CD11c(-)CD36(+), but few CD11c(-)CD36(-), monocytes took up cholesteryl ester-rich very low-density lipoproteins (CE-VLDLs) isolated from apoE(-/-) mice on WD, and CE-VLDL uptake accelerated CD11c(-)CD36(+) to CD11c(+)CD36(+) monocyte differentiation. Ablation of CD36 decreased monocyte uptake of CE-VLDLs. Intravenous injection of DiI-CE-VLDLs in apoE(-/-) mice on WD specifically labeled CD11c(+)CD36(+) foamy monocytes, which infiltrated into nascent atherosclerotic lesions and became CD11c(+) cells that were selectively localized in atherosclerotic lesions. CD11c deficiency reduced foamy monocyte infiltration into atherosclerotic lesions. Specific and consistent depletion of foamy monocytes (for 3 weeks) by daily intravenous injections of low-dose clodrosome reduced development of nascent atherosclerosis. CONCLUSIONS: Foamy monocytes, which form early in blood of mice with hypercholesterolemia, infiltrate into early atherosclerotic lesions in a CD11c-dependent manner and play crucial roles in nascent atherosclerosis development.
Assuntos
Aorta/patologia , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , VLDL-Colesterol/metabolismo , Hipercolesterolemia/metabolismo , Monócitos/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/metabolismo , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Antígeno CD11c/genética , Antígenos CD36/deficiência , Antígenos CD36/genética , Linhagem Celular , Colesterol na Dieta , Dieta Hiperlipídica , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/genética , Hipercolesterolemia/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
Atherosclerosis, a deadly disease insufficiently addressed by cholesterol-lowering drugs, needs new therapeutic strategies. Fortilin, a 172-amino acid multifunctional polypeptide, binds p53 and blocks its transcriptional activation of Bax, thereby exerting potent antiapoptotic activity. Although fortilin-overexpressing mice reportedly exhibit hypertension and accelerated atherosclerosis, it remains unknown if fortilin, not hypertension, facilitates atherosclerosis. Our objective was to test the hypothesis that fortilin in and of itself facilitates atherosclerosis by protecting macrophages against apoptosis. We generated fortilin-deficient (fortilin(+/-)) mice and wild-type counterparts (fortilin(+/+)) on a LDL receptor (Ldlr)(-/-) apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (Apobec1)(-/-) hypercholesterolemic genetic background, incubated them for 10 mo on a normal chow diet, and assessed the degree and extent of atherosclerosis. Despite similar blood pressure and lipid profiles, fortilin(+/-) mice exhibited significantly less atherosclerosis in their aortae than their fortilin(+/+) littermate controls. Quantitative immunostaining and flow cytometry analyses showed that the atherosclerotic lesions of fortilin(+/-) mice contained fewer macrophages than those of fortilin(+/+) mice. In addition, there were more apoptotic cells in the intima of fortilin(+/-) mice than in the intima of fortilin(+/+) mice. Furthermore, peritoneal macrophages from fortilin(+/-) mice expressed more Bax and underwent increased apoptosis, both at the baseline level and in response to oxidized LDL. Finally, hypercholesterolemic sera from Ldlr(-/-)Apobec1(-/-) mice induced fortilin in peritoneal macrophages more robustly than sera from control mice. In conclusion, fortilin, induced in the proatherosclerotic microenvironment in macrophages, protects macrophages against Bax-induced apoptosis, allows them to propagate, and accelerates atherosclerosis. Anti-fortilin therapy thus may represent a promising next generation antiatherosclerotic therapeutic strategy.
Assuntos
Doenças da Aorta/metabolismo , Apoptose , Aterosclerose/metabolismo , Biomarcadores Tumorais/metabolismo , Macrófagos Peritoneais/metabolismo , Desaminase APOBEC-1 , Animais , Doenças da Aorta/etiologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/fisiopatologia , Aterosclerose/etiologia , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Biomarcadores Tumorais/deficiência , Biomarcadores Tumorais/genética , Pressão Sanguínea , Colesterol/sangue , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Modelos Animais de Doenças , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Placa Aterosclerótica , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína Tumoral 1 Controlada por Tradução , Proteína X Associada a bcl-2/metabolismoRESUMO
OBJECTIVE: proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified. METHODS AND RESULTS: We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (Ldlr(-/-) and Ldlr(-/-)Apobec1(-/-)). Irrespective of LDLR expression, mice transduced with the PCSK9 gene invariably exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB. Consistent with these findings, the levels of very-low-density lipoprotein and LDL were also increased whereas high-density lipoprotein levels were unchanged. Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in hepatocytes. The PCSK9/apoB interaction resulted in increased production of apoB, possibly through the inhibition of intracellular apoB degradation via the autophagosome/lysosome pathway. CONCLUSIONS: We propose a new role for PCSK9 that involves shuttling between apoB and LDLR. The present study thus provides new insights into the action of PCSK9 in regulating apoB metabolism. Furthermore, our results indicate that targeting PCSK9 expression represents a new paradigm in therapeutic intervention against hyperlipidemia.
Assuntos
Apolipoproteínas B/fisiologia , Pró-Proteína Convertases/fisiologia , Receptores de LDL/fisiologia , Serina Endopeptidases/fisiologia , Animais , Apolipoproteínas B/sangue , Autofagia , Colesterol/sangue , Células Hep G2 , Humanos , Lipoproteínas VLDL/sangue , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Triglicerídeos/sangueRESUMO
While macrophages take up modified LDL to form foam cells and multiply to develop fatty streaks, vascular smooth muscle cells (VSMC) migrate from the media to intima, secrete extracellular matrix, and increase the volume of atherosclerotic lesions. A medicinal plant Garcinia dulcis has been used in traditional Thai medicine for centuries to treat various chronic human diseases. Morelloflavone, a biflavonoid and an active ingredient of the plant, has been shown to inhibit VSMC migration through its inhibition of multiple migration-related kinases such as focal adhesion kinase, c-Src, ERK, and RhoA. However, the exact role of morelloflavone in atherosclerogenesis was unknown. We fed Ldlr(-/-)Apobec1(-/-) mice with either normal chow or chow containing 0.003% morelloflavone for 8 mo and assessed the extent of atherosclerosis by the en face and cross-sectional analyses. A cell composition analysis of atherosclerotic tissue was carried out using immunohistochemical staining. Oral morelloflavone therapy significantly reduced the atherosclerotic areas of the mouse aortas (a 26% reduction), without changing plasma lipid profiles or weights. Immunohistochemical analyses showed that morelloflavone reduced the number of VSMC in the atherosclerotic lesion while it did not change the density of macrophages in the lesion or the percentages of proliferating and apoptotic cells. Oral, low-dose, morelloflavone therapy retards atherosclerogenesis by limiting the migration of VSMC into the intima in the mouse model of human atherosclerosis. Upon further investigation, morelloflavone may be found to be a novel oral antiatherosclerotic agent and a viable addition to the conventional therapies such as statins in humans.
Assuntos
Aorta/efeitos dos fármacos , Aterosclerose/tratamento farmacológico , Biflavonoides/uso terapêutico , Desaminase APOBEC-1 , Animais , Aorta/patologia , Aterosclerose/patologia , Biflavonoides/farmacologia , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Lipídeos/sangue , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fosfolipases A/antagonistas & inibidores , Receptores de LDL/genética , Receptores de LDL/metabolismo , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologiaRESUMO
We previously demonstrated that atherogenic Ldlr -/- Apobec1 -/- (LDb) double knockout mice lacking both low-density lipoprotein receptor (LDLR) and apolipoprotein B mRNA-editing catalytic polypeptide-1 (Apobec1) had increased serum IL-17 levels, with T cell programming shifted towards Th17 cells. In this study, we assessed the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in T cell programming and atherogenesis. We deleted the Pcsk9 gene from LDb mice to generate Ldlr -/- Apobec1 -/- Pcsk9 -/- (LTp) triple knockout mice. Atherosclerosis in the aortic sinus and aorta were quantitated. Lymphoid cells were analyzed by flow cytometry, ELISA and real-time PCR. Despite of dyslipidemia, LTp mice developed barely detectable atherosclerotic lesions. The IL-17, was very low in plasma and barely detectable in the aortic sinus in the LTp mice. In the spleen, the number of CD4+CD8- cells and splenocytes were much lower in the LDb mice than LTp mice, whereas, the IL-17-producing cells of γδTCR+ T cells and effector memory CD4+ T cells (CD44hiCD4+) in the spleen were significantly higher in the LDb mice than in the LTp mice. The Rorc mRNA expression levels were elevated in LDb mice compared to LTp mice. When re-stimulated with an anti-CD3 Ab, CD44hiCD4+ T cells from LDb mice secreted more IL-17 than those from LTp mice. T cells from LDb mice (with PCSK9) produce more IL-17 at basal and stimulated conditions when compared with LTp mice (without PCSK9). Despite the dyslipidemic profile and the lack of LDLR, atherogenesis is markedly reduced in LTp mice. These results suggest that PCSK9 is associated with changes in T cell programming that contributes to the development of atherosclerosis.
RESUMO
Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases. However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage. In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo. In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages. Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation. Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominant-negative mutant of GEFT inhibits this process. Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes. In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.
Assuntos
Adipogenia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Regeneração , Células 3T3-L1 , Adipogenia/genética , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Citoplasma/química , Ativação Enzimática , Técnicas de Transferência de Genes , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Camundongos , Desenvolvimento Muscular/genética , Proteína MyoD/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Regeneração/genética , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Transcrição Gênica , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Cardiovascular diseases are often accompanied by elevated LDL particles and endothelial dysfunction. We have examined the possibility of concurrently reducing LDL levels and modulating endothelial function using a single helper-dependent adenovirus vector system to simultaneously express the apolipoprotein B mRNA editing enzyme (Apobec1) and the scavenger receptor, class B, type I (SR-BI) genes under the control of separate promoters (designated HD-C2). Apobec1 edits apoB mRNA at nucleotide C-6666 to produce truncated apoB48 and is normally expressed in small intestine only. SR-BI is a receptor for multiple ligands with distinct tissue-specific functions. Expression of Apobec1 in HepG2 cells resulted in apoB mRNA editing, leading to decreased apoB100 abundance (to 6% of control) and the appearance of apoB48. Editing of apoB mRNA in HepG2 cells resulted in decline in apoB mRNA levels of 50%. This was probably the result of nonsense-mediated decay of edited message, since over-expression of Apobec1 increased neither Apobec1 complementary factor (ACF) mRNA nor protein abundance. Over-expression of SR-BI in human endothelial cells activated endothelial nitric oxide synthase (eNOS) activity by phosphorylation of eNOS at residue Ser-1177 in the presence of HDL, leading to increased production of the anti-atherogenic molecule nitric oxide (NO). Taken together, this study demonstrates that using one vector delivery system to express two genes in two different cell types results in the cell-specific beneficial effects of decreasing apoB100 production and increasing eNOS activities. This combined gene expression approach may provide an improved therapeutic strategy by targeting multiple sites in the mechanism of cardiovascular injury.
Assuntos
Apolipoproteínas B/genética , Aterosclerose/metabolismo , Citidina Desaminase/genética , Expressão Gênica , Terapia Genética , RNA Mensageiro/genética , Receptores Depuradores Classe B/genética , Desaminase APOBEC-1 , Adenoviridae/genética , Animais , Aterosclerose/genética , Aterosclerose/terapia , Western Blotting , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Citometria de Fluxo , Vetores Genéticos , Humanos , Técnicas In Vitro , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
BACKGROUND: The proliferative response of vascular smooth muscle cells (VSMCs) to various growth stimuli is critical for atherosclerosis and postangioplasty restenosis. Although tumor suppressor protein p53 plays a critical role in the elimination of cancerous cells, recent genetic studies have indicated that it also protects against atherosclerosis and restenosis. METHODS AND RESULTS: We examined the levels of p53 protein in normal VSMCs before and after serum stimulation. The p53 protein levels increased robustly on stimulation. Upregulated p53 protein was capable of binding to the p53 consensus sequence, as shown by electrophoretic mobility shift assay. In addition, p53 upregulation was associated with increases in the transcript and protein levels of p21WAF1/CIP1 and Bax, as shown by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Furthermore, the upregulation of p21WAF1/CIP1 and Bax was followed by cell-cycle arrest and apoptosis induction, as shown by 5-bromo-2'-dUTP incorporation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, respectively. Finally, double-staining analyses showed that the majority of p53-expressing cells also expressed p21WAF1/CIP1 and Bax proteins. CONCLUSIONS: p53 protein expression in quiescent VSMCs is paradoxically increased by application of a growth stimulus. Through the mediation of p21WAF1/CIP1 and Bax, the induced p53 protein negatively regulates the growth of dividing VSMCs, thereby minimizing the inappropriate accumulation of VSMCs. Therefore, p53 may be a negative regulator of VSMC growth.
Assuntos
Proteínas Sanguíneas/farmacologia , Retroalimentação Fisiológica/fisiologia , Músculo Liso Vascular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/fisiologia , Apoptose/efeitos dos fármacos , Bromodesoxiuridina , Causalidade , Contagem de Células , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Sequência Consenso/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fase S/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2RESUMO
Atherosclerosis is a complex disease that gene and environment interaction influences the progression of atherosclerotic lesion development. Our laboratory used mice lacking both the low density lipoprotein (LDL) receptor and Apobec1 genes (LDLR-/-Apobec1-/-, designated LDb) to investigate gene-gene interaction and the influence of an environmental factor (high-fat diet) on gene networks. LDb mice (males and females) at 5 months of age were fed a chow or high fat diet for 3-month. The mice on a chow diet had elevated plasma cholesterol and triglyceride levels and developed atherosclerosis. Feeding a high-fat diet accelerated the development of lesions >1.5-fold. We performed microarray analysis of the expression of 12442 murine genes in the livers of these animals, which identified 54 genes in males and 77 genes in females were significantly perturbed by the high-fat diet. Moreover, most of these genes (>70%) were upregulated. The results suggested that glycolysis, fat transport, and steroid hormone biosynthesis pathways were upregulated, probably to compensate for the high fat intake. Furthermore, a batch of stress-responsive genes was upregulated. The study also shows a dynamic cellular communication network including T cells, neutrophils, and monocytes/macrophages, which related to inflammatory and immune/complement responses. Importantly, this study discovered that many genes involved in calcium signaling and bone formation were up regulated. Alizarin Red S staining was used to detect calcium deposits in the region of atherosclerotic lesions. Real-time quantitative RT-PCR and Western blot analyses provided verification of the gene expression levels. In conclusion, this study demonstrated the global differential gene expression profiles, which are influenced by feeding a high fat diet to LDb mice. The results of the study provide new insights into the significance of calcification in atherogenesis.
Assuntos
Apolipoproteínas B/genética , Arteriosclerose/genética , Sinalização do Cálcio/genética , Citidina Desaminase/genética , Expressão Gênica , Fígado/metabolismo , Processamento Pós-Transcricional do RNA/genética , Receptores de LDL/genética , Desaminase APOBEC-1 , Animais , Aorta/metabolismo , Aorta/patologia , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Cálcio/metabolismo , Dieta Aterogênica , Gorduras na Dieta/administração & dosagem , Feminino , Perfilação da Expressão Gênica , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Regulação para CimaRESUMO
BACKGROUND: In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. METHODS: We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. RESULTS: The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant. CONCLUSION: This study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.
RESUMO
Target substrate-specific hammerhead ribozyme cleaves the specific mRNA efficiently and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. The goal of this study is to demonstrate that long-term reduction of apoB gene expression using hammerhead ribozyme would result in inhibition of atherosclerosis development. We designed two hammerhead ribozymes targeted at the nucleotides of apoB mRNA GUC(2326) (designated RB1) and GUA(6679) (designated RB15), and we used self-complementary adeno-associated virus 8.2 (scAAV8.2) vector to deliver these active ribozymes of RB1, RB15, combination of RB1/RB15, and an inactive hammerhead ribozyme RB15 mutant to atherosclerosis-prone LDb mice (Ldlr(-/-)Apobec1(-/-)). LDb mice lack both low density lipoproteins (LDL) receptor (Ldlr(-/-)) and apoB mRNA editing enzyme (Apobec1(-/-)) genes and develop atherosclerosis spontaneously. After the RB1, RB15, or combination of RB1/RB15 ribozymes treatment, the LDb mice had significantly decreased plasma triglyceride and apoB levels, resulting in markedly decreased of atherosclerotic lesions, Furthermore, the active ribozymes treatment decreased the levels of diacylglycerol acyltransferase 1 (Dgat1) mRNA and the levels of multiple diacylglycerol (DAG) molecular species. These results provide the first evidence that decreased apoB levels results to reduction of Dgat1 expression and triglyceride levels (TAG), which had a significant impact on the development of atherosclerosis.Molecular Therapy-Nucleic Acids (2013) 2, e125; doi:10.1038/mtna.2013.53; published online 1 October 2013.
RESUMO
OBJECTIVE: Atherosclerosis is influenced by the interaction of environmental and genetic susceptibility risk factors. We used global microarray expression profiling to investigate differentially regulated genes in aorta during development of atherosclerosis in a susceptible genetically modified mouse model in response to the interaction between risk factors including hyperlipidemic genotype, shear stress, diet, and age. METHODS AND RESULTS: In this study we investigated transcriptional changes in lesion-prone and lesion-resistant regions of aortas in genetically modified mice lacking both genes of the LDL receptor and the apolipoprotein B mRNA editing enzyme (LDb; Ldlr(-/-)Apobec1(-/-)). Risk factors including hyperlipidemic genotype (LDb vs. C57BL/6 wildtype), shear stress (lesion-prone vs. lesion resistant aortic regions), diet (chow vs. Western high-fat), and age (2- vs. 8-months) were studied. We hybridized aortic RNA samples with microarray chips containing probes for 45,000 mouse genes and expressed sequence tags (ESTs). Overall, the differentially expressed genes were components of 20 metabolic and physiological pathways. Notably, calcium signaling is the major pathway identified with differential regulation of 30 genes within this pathway. We also found differential expression of calcium-signaling genes in cultured primary endothelial cells from lesion-prone and lesion-resistant arterial regions (LDb mice vs. C57BL/6 controls), providing further support for involvement of calcium signaling in the pathogenesis of atherosclerosis. Moreover, we demonstrated protein expression of genes in the calcium-signaling pathway using Western blot analysis and immunofluorescence. CONCLUSIONS: Our results suggest that calcium signaling may play an important role in regulation of genes expressed in aorta during development of atherosclerosis. Calcium signaling may act via mechanistic responses to genetic, mechanical, and environmental insults that trigger an imbalance of intracellular calcium homeostasis, resulting in altered biological processes leading to lesion development.
Assuntos
Aterosclerose/metabolismo , Cálcio/metabolismo , Regulação da Expressão Gênica , Ração Animal , Animais , Aorta/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Genótipo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Risco , Transdução de SinaisRESUMO
Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-gamma secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-gamma mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7- and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-gamma synthesis during infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Células Epiteliais/metabolismo , Intestino Delgado/citologia , Leucossialina/metabolismo , Infecções por Reoviridae/metabolismo , Regulação para Cima/genética , Animais , Anticorpos Monoclonais , Antígeno B7-1/metabolismo , Proliferação de Células , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Citometria de Fluxo , Ligante Coestimulador de Linfócitos T Induzíveis , Proteína Coestimuladora de Linfócitos T Induzíveis , Interferon gama/biossíntese , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Animal models provide vital tools to explicate the pathogenesis of atherosclerosis. Accordingly, we established two atherosclerosis-prone mice models: (i) mice lacking the LDL (low-density lipoprotein) receptor (LDLR) and the ability to edit apo (apolipoprotein) B mRNA (Apobec1; designated LDb : LDLR-/- Apobec1-/-), and (ii) mice with the LDb background, who also overexpressed human apoB100 (designated LTp : LDLR-/- Apobec1-/- ERhB+/+). Both LDb and LTp mice had markedly elevated levels of LDL and increased levels of NEFAs (non-esterified fatty acids) compared with C57BL/6 wild-type mice. However, fasting glucose and insulin levels in both animals were not different than those in C57BL/6 wild-type mice. It has been suggested that PAF-AH (platelet-activating factor acetylhydrolase) increases susceptibility to vascular disease. Both LDb and LTp mice had significantly higher PAF-AH mRNA levels compared with C57BL/6 wild-type mice. PAF-AH gene expression was also significantly influenced by age and sex. Interestingly, PAF-AH mRNA levels were significantly higher in both LTp male and female mice than in the LDb mice. This increased PAF-AH gene expression was associated with elevated plasma PAF-AH enzyme activities ( LTp > LDb > C57BL/6 ). Moreover, a greater proportion of PAF-AH activity was associated with the apoB-containing lipoproteins: 29% in LTp and 13% in LDb mice compared with C57BL/6 wild-type animals (6.7%). This may explain why LTp mice developed more atherosclerotic lesions than LDb mice by 8 months of age. In summary, increased plasma NEFAs, PAF-AH mRNA and enzyme activities are associated with accelerated atherogenesis in these animal models.