RESUMO
The liver is the most common site of metastatic disease1. Although this metastatic tropism may reflect the mechanical trapping of circulating tumour cells, liver metastasis is also dependent, at least in part, on the formation of a 'pro-metastatic' niche that supports the spread of tumour cells to the liver2,3. The mechanisms that direct the formation of this niche are poorly understood. Here we show that hepatocytes coordinate myeloid cell accumulation and fibrosis within the liver and, in doing so, increase the susceptibility of the liver to metastatic seeding and outgrowth. During early pancreatic tumorigenesis in mice, hepatocytes show activation of signal transducer and activator of transcription 3 (STAT3) signalling and increased production of serum amyloid A1 and A2 (referred to collectively as SAA). Overexpression of SAA by hepatocytes also occurs in patients with pancreatic and colorectal cancers that have metastasized to the liver, and many patients with locally advanced and metastatic disease show increases in circulating SAA. Activation of STAT3 in hepatocytes and the subsequent production of SAA depend on the release of interleukin 6 (IL-6) into the circulation by non-malignant cells. Genetic ablation or blockade of components of IL-6-STAT3-SAA signalling prevents the establishment of a pro-metastatic niche and inhibits liver metastasis. Our data identify an intercellular network underpinned by hepatocytes that forms the basis of a pro-metastatic niche in the liver, and identify new therapeutic targets.
Assuntos
Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Fígado/patologia , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Animais , Carcinoma Ductal Pancreático/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/secundário , Feminino , Interleucina-6/metabolismo , Masculino , Camundongos , Fator de Transcrição STAT3/metabolismo , Proteína Amiloide A Sérica/metabolismoRESUMO
Serum amyloid A (SAA) is predictive of CVD in humans and causes atherosclerosis in mice. SAA has many proatherogenic effects in vitro. However, HDL, the major carrier of SAA in the circulation, masks these effects. The remodeling of HDL by cholesteryl ester transfer protein (CETP) liberates SAA restoring its proinflammatory activity. Here, we investigated whether deficiency of SAA suppresses the previously described proatherogenic effect of CETP. ApoE-/- mice and apoE-/- mice deficient in the three acute-phase isoforms of SAA (SAA1.1, SAA2.1, and SAA3; "apoE-/- SAA-TKO") with and without adeno-associated virus-mediated expression of CETP were studied. There was no effect of CETP expression or SAA genotype on plasma lipids or inflammatory markers. Atherosclerotic lesion area in the aortic arch of apoE-/- mice was 5.9 ± 1.2%; CETP expression significantly increased atherosclerosis in apoE-/- mice (13.1 ± 2.2%). However, atherosclerotic lesion area in the aortic arch of apoE-/- SAA-TKO mice (5.1 ± 1.1%) was not significantly increased by CETP expression (6.2 ± 0.9%). The increased atherosclerosis in apoE-/- mice expressing CETP was associated with markedly increased SAA immunostaining in aortic root sections. Thus, SAA augments the atherogenic effects of CETP, which suggests that inhibiting CETP may be of particular benefit in patients with high SAA.
Assuntos
Aterosclerose , Proteínas de Transferência de Ésteres de Colesterol , Humanos , Camundongos , Animais , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteína Amiloide A Sérica/metabolismo , Aterosclerose/metabolismo , Apolipoproteínas E/metabolismo , Aorta/metabolismoRESUMO
BACKGROUND: Obesity increases the risk for human abdominal aortic aneurysms (AAAs) and enhances Ang II (angiotensin II)-induced AAA formation in C57BL/6J mice. Obesity is also associated with increases in perivascular fat that expresses proinflammatory markers including SAA (serum amyloid A). We previously reported that deficiency of SAA significantly reduces Ang II-induced inflammation and AAA in hyperlipidemic apoE-deficient mice. In this study. we investigated whether adipose tissue-derived SAA plays a role in Ang II-induced AAA in obese C57BL/6J mice. METHODS: The development of AAA was compared between male C57BL/6J mice (wild type), C57BL/6J mice lacking SAA1.1, SAA2.1, and SAA3 (TKO); and TKO mice harboring a doxycycline-inducible, adipocyte-specific SAA1.1 transgene (TKO-Tgfat; SAA expressed only in fat). All mice were fed an obesogenic diet and doxycycline to induce SAA transgene expression and infused with Ang II to induce AAA. RESULTS: In response to Ang II infusion, SAA expression was significantly increased in perivascular fat of obese C57BL/6J mice. Maximal luminal diameters of the abdominal aorta were determined by ultrasound before and after Ang II infusion, which indicated a significant increase in aortic luminal diameters in wild type and TKO-TGfat mice but not in TKO mice. Adipocyte-specific SAA expression was associated with MMP (matrix metalloproteinase) activity and macrophage infiltration in abdominal aortas of Ang II-infused obese mice. CONCLUSIONS: We demonstrate for the first time that SAA deficiency protects obese C57BL/6J mice from Ang II-induced AAA. SAA expression only in adipocytes is sufficient to cause AAA in obese mice infused with Ang II.
Assuntos
Angiotensina II , Aneurisma da Aorta Abdominal , Adipócitos/metabolismo , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Apolipoproteínas E/genética , Modelos Animais de Doenças , Doxiciclina/efeitos adversos , Masculino , Metaloproteinases da Matriz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/complicações , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismoRESUMO
Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.
Assuntos
Lesão Pulmonar , Sepse , Animais , Camundongos , Lesão Pulmonar/patologia , Proteína Amiloide A Sérica/genética , Sepse/patologia , Pulmão/patologia , Quimiocinas , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
OBJECTIVE: Abdominal aortic aneurysm (AAA) is a common progressive disease and a significant cause of morbidity and mortality. Prior investigations have shown that diabetes mellitus (DM) may be relatively protective of AAA incidence and growth. The Non-invasive Treatment of Aortic Aneurysm Clinical Trial (N-TA3CT) is a contemporary study of small AAA growth that provides a unique opportunity to validate and explore the effect of DM on AAA. Confirming the effect of DM on AAA growth in this study may present opportunities to explore for clues to potential biologic mechanisms as well as inform current patient management. METHODS: This is a secondary analysis examining the association of diabetes and aneurysm growth within N-TA3CT: a placebo-controlled multicenter trial of doxycycline in 261 patients with AAA maximum transverse diameters (MTDs) between 3.5 and 5 cm. The primary outcome is the change in the MTD from baseline as determined by computed tomography (CT) scans obtained during the trial. Secondary outcome is the growth pattern of the AAA. Baseline characteristics and growth patterns were assessed with t tests (continuous) or χ2 tests (categorical). Unadjusted and adjusted longitudinal analyses were performed with a repeated measures linear mixed model to compare AAA growth rates between patients with and without diabetes. RESULTS: Of 261 patients, 250 subjects had sufficient imaging and were included in this study. There were 56 patients (22.4%) with diabetes and 194 (77.6%) without. Diabetes was associated with higher body mass index and increased rates of hypercholesterolemia and coronary artery disease (P < .05). Diabetes was also associated with increased frequency of treatment for atherosclerosis and hypertension including treatment with statin, angiotensin-converting enzyme inhibitor, angiotensin II receptor blocker, anti-platelet, and diuretic therapy (P < .05). Baseline MTD was not significantly different between those with (4.32 cm) and without DM (4.30 cm). Median growth rate for patients with diabetes was 0.12 cm/y (interquartile range, 0.07-0.22 cm/y) and 0.19 cm/y (interquartile range, 0.12-0.27 cm/y) in patients without DM, which was significantly different on unadjusted analysis (P < .0001). Diabetes remained significantly associated with AAA growth after adjustment for other relevant clinical factors (coef, -0.057; P < .0001). CONCLUSIONS: Patients with diabetes have more than a 35% reduction in the median growth rates of AAA despite more severe concomitant vascular comorbidities and similar initial sizes of aneurysms. This effect persists and remains robust after adjusted analysis; and slower growth rates may delay the time to reach repair threshold. Rapid growth (>0.5 cm/y) is infrequent in patients with DM.
Assuntos
Aneurisma da Aorta Abdominal , Diabetes Mellitus , Hipertensão , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/epidemiologia , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/epidemiologia , Humanos , Fatores de Risco , Tomografia Computadorizada por Raios XRESUMO
PURPOSE OF REVIEW: Serum amyloid A (SAA) is a highly sensitive acute phase reactant that has been linked to a number of chronic inflammatory diseases. During a systemic inflammatory response, liver-derived SAA is primarily found on high-density lipoprotein (HDL). The purpose of this review is to discuss recent literature addressing the pathophysiological functions of SAA and the significance of its association with HDL. RECENT FINDINGS: Studies in gene-targeted mice establish that SAA contributes to atherosclerosis and some metastatic cancers. Accumulating evidence indicates that the lipidation state of SAA profoundly affects its bioactivities, with lipid-poor, but not HDL-associated, SAA capable of inducing inflammatory responses in vitro and in vivo. Factors that modulate the equilibrium between lipid-free and HDL-associated SAA have been identified. HDL may serve to limit SAA's bioactivities in vivo. Understanding the factors leading to the release of systemic SAA from HDL may provide insights into chronic disease mechanisms.
Assuntos
Aterosclerose , Lipoproteínas HDL , Animais , Aterosclerose/genética , Humanos , Fígado , Camundongos , Proteína Amiloide A SéricaRESUMO
Aortic structure and function are controlled by the coordinated actions of different aortic cells and the extracellular matrix. Several pathways have been identified that control the aortic wall in a cell-type-specific manner and play diverse roles in various phases of aortic injury, repair, and remodeling. This complexity of signaling in the aortic wall poses challenges to the development of therapeutic strategies for treating aortic aneurysms and dissections. Here, in part II of this Recent Highlights series on aortic aneurysms and dissections, we will summarize recent studies published in Arteriosclerosis, Thrombosis, and Vascular Biology that have contributed to our knowledge of the signaling pathway-related mechanisms of aortic aneurysms and dissections.
Assuntos
Aneurisma Aórtico/metabolismo , Dissecção Aórtica/metabolismo , Matriz Extracelular/metabolismo , Transdução de Sinais , Dissecção Aórtica/tratamento farmacológico , Aneurisma Aórtico/tratamento farmacológico , Humanos , Mutação , Receptores de Angiotensina/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
The aortic wall is composed of highly dynamic cell populations and extracellular matrix. In response to changes in the biomechanical environment, aortic cells and extracellular matrix modulate their structure and functions to increase aortic wall strength and meet the hemodynamic demand. Compromise in the structural and functional integrity of aortic components leads to aortic degeneration, biomechanical failure, and the development of aortic aneurysms and dissections (AAD). A better understanding of the molecular pathogenesis of AAD will facilitate the development of effective medications to treat these conditions. Here, we summarize recent findings on AAD published in ATVB. In this issue, we focus on the dynamics of aortic cells and extracellular matrix in AAD; in the next issue, we will focus on the role of signaling pathways in AAD.
Assuntos
Aorta/patologia , Aneurisma Aórtico/patologia , Dissecção Aórtica/patologia , Matriz Extracelular/patologia , Dissecção Aórtica/metabolismo , Dissecção Aórtica/fisiopatologia , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/fisiopatologia , Dilatação Patológica , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Hemodinâmica , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Remodelação VascularRESUMO
Liver-derived serum amyloid A (SAA) is present in plasma where it is mainly associated with HDL and from which it is cleared more rapidly than are the other major HDL-associated apolipoproteins. Although evidence suggests that lipid-free and HDL-associated forms of SAA have different activities, the pathways by which SAA associates and disassociates with HDL are poorly understood. In this study, we investigated SAA lipidation by hepatocytes and how this lipidation relates to the formation of nascent HDL particles. We also examined hepatocyte-mediated clearance of lipid-free and HDL-associated SAA. We prepared hepatocytes from mice injected with lipopolysaccharide or an SAA-expressing adenoviral vector. Alternatively, we incubated primary hepatocytes from SAA-deficient mice with purified SAA. We analyzed conditioned media to determine the lipidation status of endogenously produced and exogenously added SAA. Examining the migration of lipidated species, we found that SAA is lipidated and forms nascent particles that are distinct from apoA-I-containing particles and that apoA-I lipidation is unaltered when SAA is overexpressed or added to the cells, indicating that SAA is not incorporated into apoA-I-containing HDL during HDL biogenesis. Like apoA-I formation, generation of SAA-containing particles was dependent on ABCA1, but not on scavenger receptor class B type I. Hepatocytes degraded significantly more SAA than apoA-I. Taken together, our results indicate that SAA's lipidation and metabolism by the liver is independent of apoA-I and that SAA is not incorporated into HDL during HDL biogenesis.
Assuntos
Lipoproteínas HDL/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Apolipoproteína A-I/deficiência , Apolipoproteína A-I/metabolismo , Hepatócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Amiloide A Sérica/deficiência , Proteína Amiloide A Sérica/genéticaRESUMO
Serum amyloid A (SAA) is a high-density apolipoprotein whose plasma levels can increase more than 1000-fold during a severe acute-phase inflammatory response and are more modestly elevated in chronic inflammation. SAA is thought to play important roles in innate immunity, but its biological activities have not been completely delineated. We previously reported that SAA deficiency protects mice from developing abdominal aortic aneurysms (AAAs) induced by chronic angiotensin II (AngII) infusion. Here, we report that SAA is required for AngII-induced increases in interleukin-1ß (IL-1ß), a potent proinflammatory cytokine that is tightly controlled by the Nod-like receptor protein 3 (NLRP3) inflammasome and caspase-1 and has been implicated in both human and mouse AAAs. We determined that purified SAA stimulates IL-1ß secretion in murine J774 and bone marrow-derived macrophages through a mechanism that depends on NLRP3 expression and caspase-1 activity, but is independent of P2X7 nucleotide receptor (P2X7R) activation. Inhibiting reactive oxygen species (ROS) by N-acetyl-l-cysteine or mito-TEMPO and inhibiting activation of cathepsin B by CA-074 blocked SAA-mediated inflammasome activation and IL-1ß secretion. Moreover, inhibiting cellular potassium efflux with glyburide or increasing extracellular potassium also significantly reduced SAA-mediated IL-1ß secretion. Of note, incorporating SAA into high-density lipoprotein (HDL) prior to its use in cell treatments completely abolished its ability to stimulate ROS generation and inflammasome activation. These results provide detailed insights into SAA-mediated IL-1ß production and highlight HDL's role in regulating SAA's proinflammatory effects.
Assuntos
Inflamassomos/metabolismo , Inflamação/imunologia , Lipoproteínas HDL/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Amiloide A Sérica/metabolismo , Proteína Amiloide A Sérica/fisiologia , Animais , Caspase 1/metabolismo , Catepsina B/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína Amiloide A Sérica/genética , Transdução de SinaisRESUMO
Objective- SAA (serum amyloid A) is a family of acute-phase reactants that have proinflammatory and proatherogenic activities. SAA is more lipophilic than apoA-I (apolipoprotein A-I), and during an acute-phase response, <10% of plasma SAA is found lipid-free. In most reports, SAA is found exclusively associated with high-density lipoprotein; however, we and others have reported SAA on apoB (apolipoprotein B)-containing lipoproteins in both mice and humans. The goal of this study was to determine whether SAA is an exchangeable apolipoprotein. Approach and Results- Delipidated human SAA was incubated with SAA-free human lipoproteins; then, samples were reisolated by fast protein liquid chromatography, and SAA analyzed by ELISA and immunoblot. Both in vitro and in vivo, we show that SAA associates with any lipoprotein and does not remain in a lipid-free form. Although SAA is preferentially found on high-density lipoprotein, it can exchange between lipoproteins. In the presence of CETP (cholesterol ester transfer protein), there is greater exchange of SAA between lipoproteins. Subjects with diabetes mellitus, but not those with metabolic syndrome, showed altered SAA lipoprotein distribution postprandially. Proteoglycan-mediated lipoprotein retention is thought to be an underlying mechanism for atherosclerosis development. SAA has a proteoglycan-binding domain. Lipoproteins containing SAA had increased proteoglycan binding compared with SAA-free lipoproteins. Conclusions- Thus, SAA is an exchangeable apolipoprotein and increases apoB-containing lipoproteins' proteoglycan binding. We and others have previously reported the presence of SAA on low-density lipoprotein in individuals with obesity, diabetes mellitus, and metabolic syndrome. We propose that the presence of SAA on apoB-containing lipoproteins may contribute to cardiovascular disease development in these populations.
Assuntos
Apolipoproteínas/metabolismo , Proteína Amiloide A Sérica/metabolismo , Idoso , Animais , Apolipoproteína B-100/metabolismo , Apolipoproteínas/sangue , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Diabetes Mellitus/sangue , Feminino , Humanos , Lipoproteínas HDL/metabolismo , Masculino , Síndrome Metabólica/sangue , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Obesidade/sangue , Período Pós-Prandial , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoglicanas/metabolismo , Proteína Amiloide A Sérica/deficiência , Proteína Amiloide A Sérica/genéticaRESUMO
BACKGROUND: Surgical resection is the only curative treatment option for gastric cancer. Despite widespread adoption of multimodality perioperative treatment strategies, 5-year overall survival rates remain low. In patients with advanced gastric or gastroesophageal junction adenocarcinoma, pembrolizumab has demonstrated promising efficacy and manageable safety as monotherapy in previously treated patients and as first-line therapy in combination with cisplatin and 5-fluorouracil. Combining chemotherapy with pembrolizumab in the neoadjuvant/adjuvant setting may benefit patients with locally advanced, resectable disease. AIM: To describe the design and rationale for the global, multicenter, randomized, double-blind, Phase III KEYNOTE-585 study to evaluate the efficacy and safety of pembrolizumab plus chemotherapy compared with placebo plus chemotherapy as neoadjuvant/adjuvant treatment for localized gastric or gastroesophageal junction adenocarcinoma. ClinicalTrials.gov : NCT03221426.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ensaios Clínicos Fase III como Assunto , Neoplasias Gástricas/terapia , Adulto , Quimioterapia Adjuvante/métodos , Intervalo Livre de Doença , Método Duplo-Cego , Feminino , Gastrectomia , Humanos , Masculino , Estudos Multicêntricos como Assunto , Terapia Neoadjuvante/métodos , Assistência Perioperatória/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Análise de Sobrevida , Adulto JovemRESUMO
Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (â¼2,500-fold vs. â¼6,000-fold, respectively) and fat (â¼400-fold vs. â¼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were â¼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas â¼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that â¼100% of plasma SAA1.1 was recovered in HDL compared with only â¼50% of SAA2.1 and â¼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation.
Assuntos
Reação de Fase Aguda/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Células Cultivadas , Lipopolissacarídeos/farmacologia , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Amiloide A Sérica/deficiência , Proteína Amiloide A Sérica/genéticaRESUMO
The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism.
Assuntos
HDL-Colesterol/metabolismo , Lipoproteínas HDL/sangue , Isoformas de Proteínas/genética , Proteína Amiloide A Sérica/genética , Reação de Fase Aguda/metabolismo , Animais , Apolipoproteína A-I/sangue , Apolipoproteína A-I/química , Apolipoproteína A-II/sangue , Apolipoproteína A-II/química , Apolipoproteína A-II/metabolismo , Humanos , Lipoproteínas HDL/química , Camundongos , Isoformas de Proteínas/química , Receptores Depuradores Classe B/sangue , Receptores Depuradores Classe B/química , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismoRESUMO
Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA2 is produced as a pro-enzyme (pro-GX sPLA2) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA2 activation have not been identified. We previously reported that GX sPLA2 negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA2 expression construct (FLAG-pro-GX sPLA2), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA2 processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA2 processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA2 processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA2 to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA2 processing and sPLA2 activity in Y1 cells, and it significantly attenuated GX sPLA2-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA2 is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.
Assuntos
Glândulas Suprarrenais/metabolismo , Furina/metabolismo , Fosfolipases A2 Secretórias/metabolismo , Pró-Proteína Convertases/metabolismo , Esteroides/biossíntese , Glândulas Suprarrenais/citologia , Hormônio Adrenocorticotrópico/farmacologia , Animais , Sequência de Bases , Primers do DNA , Ativação Enzimática , Camundongos , Reação em Cadeia da Polimerase , ProteóliseRESUMO
OBJECTIVE: Rupture of abdominal aortic aneurysm (AAA), a major cause of death in the aged population, is characterized by vascular inflammation and matrix degradation. Serum amyloid A (SAA), an acute-phase reactant linked to inflammation and matrix metalloproteinase induction, correlates with aortic dimensions before aneurysm formation in humans. We investigated whether SAA deficiency in mice affects AAA formation during angiotensin II (Ang II) infusion. APPROACH AND RESULTS: Plasma SAA increased ≈60-fold in apoE(-/-) mice 24 hours after intraperitoneal Ang II injection (100 µg/kg; n=4) and ≈15-fold after chronic 28-day Ang II infusion (1000 ng/kg per minute; n=9). AAA incidence and severity after 28-day Ang II infusion was significantly reduced in apoE(-/-) mice lacking both acute-phase SAA isoforms (SAAKO; n=20) compared with apoE(-/-) mice (SAAWT; n=20) as assessed by in vivo ultrasound and ex vivo morphometric analyses, despite a significant increase in systolic blood pressure in SAAKO mice compared with SAAWT mice after Ang II infusion. Atherosclerotic lesion area of the aortic arch was similar in SAAKO and SAAWT mice after 28-day Ang II infusion. Immunostaining detected SAA in AAA tissues of Ang II-infused SAAWT mice that colocalized with macrophages, elastin breaks, and enhanced matrix metalloproteinase activity. Matrix metalloproteinase-2 activity was significantly lower in aortas of SAAKO mice compared with SAAWT mice after 10-day Ang II infusion. CONCLUSIONS: Lack of endogenous acute-phase SAA protects against experimental AAA through a mechanism that may involve reduced matrix metalloproteinase-2 activity.
Assuntos
Angiotensina II/farmacologia , Aneurisma da Aorta Abdominal/prevenção & controle , Apolipoproteínas E/deficiência , Metaloproteinase 2 da Matriz/metabolismo , Proteína Amiloide A Sérica/deficiência , Animais , Aneurisma da Aorta Abdominal/patologia , Biomarcadores/sangue , Modelos Animais de Doenças , Elastina/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Sensibilidade e Especificidade , Proteína Amiloide A Sérica/metabolismoAssuntos
American Heart Association/organização & administração , Doenças da Aorta , Pesquisa Biomédica/organização & administração , Doença Arterial Periférica , Pesquisadores/organização & administração , Comitês Consultivos/organização & administração , American Heart Association/economia , Animais , Doenças da Aorta/diagnóstico , Doenças da Aorta/mortalidade , Doenças da Aorta/fisiopatologia , Doenças da Aorta/terapia , Pesquisa Biomédica/economia , Humanos , Doença Arterial Periférica/diagnóstico , Doença Arterial Periférica/mortalidade , Doença Arterial Periférica/fisiopatologia , Doença Arterial Periférica/terapia , Pesquisadores/economia , Apoio à Pesquisa como Assunto/organização & administração , Estados UnidosRESUMO
Serum amyloid A (SAA) has a number of proatherogenic effects including induction of vascular proteoglycans. Chronically elevated SAA was recently shown to increase atherosclerosis in mice. The purpose of this study was to determine whether a brief increase in SAA similarly increased atherosclerosis in a murine model. The recombination activating gene 1-deficient (rag1(-/-)) × apolipoprotein E-deficient (apoe(-/-)) and apoe(-/-) male mice were injected, multiple times or just once respectively, with an adenoviral vector encoding human SAA1 (ad-SAA); the injected mice and controls were maintained on chow for 12-16 weeks. Mice receiving multiple injections of ad-SAA, in which SAA elevation was sustained, had increased atherosclerosis compared with controls. Strikingly, mice receiving only a single injection of ad-SAA, in which SAA was only briefly elevated, also had increased atherosclerosis compared with controls. Using in vitro studies, we demonstrate that SAA treatment leads to increased LDL retention, and that prevention of transforming growth factor beta (TGF-ß) signaling prevents SAA-induced increases in LDL retention and SAA-induced increases in vascular biglycan content. We propose that SAA increases atherosclerosis development via induction of TGF-ß, increased vascular biglycan content, and increased LDL retention. These data suggest that even short-term inflammation with concomitant increase in SAA may increase the risk of developing CVD.
Assuntos
Aterosclerose/sangue , Proteína Amiloide A Sérica/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteoglicanas/metabolismo , Proteína Amiloide A Sérica/genética , Fator de Crescimento Transformador betaRESUMO
Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA2 is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA2-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA2 overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA2 (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA2 on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA2 plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.