RESUMO
While the cardiovascular system is a primary target of organophosphorus flame retardants (OPFRs), particularly aryl-OPFRs, it is still exclusive whether the diisodecyl phenyl phosphate (DIDPP), widely used and broadly present in the environment at high concentrations, elicits atherosclerosis effects. Liver X receptors (LXRs) play a direct role in regulating the formation of atherosclerotic lesions. This study was the first to demonstrate that DIDPP acts as an LXRα ligand and functions as an LXRα antagonist with a half-maximal inhibitory concentration of 16.2 µM. We showed that treatment of an in vitro macrophage model with 1 to 10 µM of DIDPP resulted in the downregulation of direct targets of LXRα, namely ABCA1, ABCG1 and SR-B1, thereby leading to a 7.9-13.2 % reduction in cholesterol efflux. This caused dose-dependent, 24.1-43.1 % increases in the staining intensity of foam cells in the macrophage model. This atherosclerotic effect of DIDPP was proposed to be due to its antagonism of LXRα activity, as DIDPP treatment did not alter cholesterol influx. In conclusion, the findings of this study demonstrate that exposure to DIDPP may be a risk factor for atherosclerosis due to the LXRα-antagonistic activity of DIDPP and its ubiquity in the environment.
Assuntos
Células Espumosas , Receptores X do Fígado , Receptores X do Fígado/metabolismo , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Animais , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Organofosfatos/farmacologia , Organofosfatos/toxicidade , Camundongos , Humanos , Retardadores de Chama/toxicidade , Retardadores de Chama/farmacologia , Células RAW 264.7 , Receptores Depuradores Classe B/metabolismoRESUMO
Immunotoxicity remains a major hindrance to chemotherapy in cancer therapy. Nanocarriers may alleviate the immunotoxicity, but the optimal design remains unclear. Here, we created two variants of maytansine (DM1)-loaded synthetic high-density lipoproteins (D-sHDL) with either physically entrapped (ED-sHDL) or chemically conjugated (CD-sHDL) DM1. We found that CD-sHDL showed less accumulation in the tumor draining lymph nodes (DLNs) and femur, resulting in a lower toxicity against myeloid cells than ED-sHDL via avoiding scavenger receptor class B type 1 (SR-B1)-mediated DM1 transportation into the granulocyte-monocyte progenitors and dendritic cells. Therefore, higher densities of lymphocytes in the tumors, DLNs, and blood were recorded in mice receiving CD-sHDL, leading to a better efficacy and immune memory of CD-sHDL against colon cancer. Furthermore, liposomes with conjugated DM1 (CD-Lipo) showed lower immunotoxicity than those with entrapped drug (ED-Lipo) through the same mechanism after apolipoprotein opsonization. Our findings highlight the critical role of drug loading patterns in dictating the biological fate and activity of nanomedicine.
Assuntos
Nanopartículas , Animais , Nanopartículas/química , Camundongos , Linhagem Celular Tumoral , Humanos , Receptores Depuradores Classe B/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Lipoproteínas HDL/metabolismo , Portadores de Fármacos/química , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Lipossomos/química , Lipídeos/químicaRESUMO
Arterial macrophage cholesterol accumulation and impaired cholesterol efflux lead to foam cell formation and the development of atherosclerosis. Modified lipoproteins interact with toll-like receptors (TLR), causing an increased inflammatory response and altered cholesterol homeostasis. We aimed to determine the effects of TLR antagonists on cholesterol efflux and foam cell formation in human macrophages. Stimulated monocytes were treated with TLR antagonists (MIP2), and the cholesterol efflux transporter expression and foam cell formation were analyzed. The administration of MIP2 attenuated the foam cell formation induced by lipopolysaccharides (LPS) and oxidized low-density lipoproteins (ox-LDL) in stimulated THP-1 cells (p < 0.001). The expression of ATP-binding cassette transporters A (ABCA)-1, ABCG-1, scavenger receptor (SR)-B1, liver X receptor (LXR)-α, and peroxisome proliferator-activated receptor (PPAR)-γ mRNA and proteins were increased (p < 0.001) following MIP2 administration. A concentration-dependent decrease in the phosphorylation of p65, p38, and JNK was also observed following MIP2 administration. Moreover, an inhibition of p65 phosphorylation enhanced the expression of ABCA1, ABCG1, SR-B1, and LXR-α. TLR inhibition promoted the cholesterol efflux pathway by increasing the expression of ABCA-1, ABCG-1, and SR-B1, thereby reducing foam cell formation. Our results suggest a potential role of the p65/NF-kB/LXR-α/ABCA1 axis in TLR-mediated cholesterol homeostasis.
Assuntos
Transportador 1 de Cassete de Ligação de ATP , Colesterol , Células Espumosas , Lipoproteínas LDL , Receptores X do Fígado , Receptores Toll-Like , Humanos , Células Espumosas/metabolismo , Células Espumosas/efeitos dos fármacos , Colesterol/metabolismo , Receptores X do Fígado/metabolismo , Receptores Toll-Like/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacologia , PPAR gama/metabolismo , Células THP-1 , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Lipopolissacarídeos/farmacologia , Receptores Depuradores Classe B/metabolismo , Receptores Depuradores Classe B/genéticaRESUMO
As an escalating public health issue, obesity and overweight conditions are predispositions to various diseases and are exacerbated by concurrent chronic inflammation. Nonetheless, extant antiobesity pharmaceuticals (quercetin, capsaicin, catecholamine, etc.) manifest constrained efficacy alongside systemic toxic effects. Effective therapeutic approaches that selectively target adipose tissue, thereby enhancing local energy expenditure, surmounting the limitations of prevailing antiobesity modalities are highly expected. In this context, we developed a temperature-sensitive hydrogel loaded with recombinant high-density lipoprotein (rHDL) to achieve targeted delivery of resveratrol, an adipose browning activator, to adipose tissue. rHDL exhibits self-regulation on fat cell metabolism and demonstrates natural targeting toward scavenger receptor class B type I (SR-BI), which is highly expressed by fat cells, thereby achieving a synergistic effect for the treatment of obesity. Additionally, the dispersion of rHDL@Res in temperature-sensitive hydrogels, coupled with the regulation of their degradation and drug release rate, facilitated sustainable drug release at local adipose tissues over an extended period. Following 24 days' treatment regimen, obese mice exhibited improved metabolic status, resulting in a reduction of 68.2% of their inguinal white adipose tissue (ingWAT). Specifically, rHDL@Res/gel facilitated the conversion of fatty acids to phospholipids (PA, PC), expediting fat mobilization, mitigating triglyceride accumulation, and therefore facilitating adipose tissue reduction. Furthermore, rHDL@Res/gel demonstrated efficacy in attenuating obesity-induced inflammation and fostering angiogenesis in ingWAT. Collectively, this engineered local fat reduction platform demonstrated heightened effectiveness and safety through simultaneously targeting adipocytes, promoting WAT browning, regulating lipid metabolism, and controlling inflammation, showing promise for adipose-targeted therapy.
Assuntos
Tecido Adiposo , Lipoproteínas HDL , Animais , Camundongos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Tecido Adiposo/metabolismo , Proteínas Recombinantes , Resveratrol/farmacologia , Resveratrol/química , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Hidrogéis/química , Camundongos Endogâmicos C57BL , Humanos , Masculino , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/química , Sistemas de Liberação de Medicamentos , Receptores Depuradores Classe B/metabolismoAssuntos
Doença da Artéria Coronariana , Modelos Animais de Doenças , Camundongos Knockout , Receptores de LDL , Receptores Depuradores Classe B , Animais , Doença da Artéria Coronariana/genética , Receptores de LDL/genética , Receptores de LDL/deficiência , Receptores de LDL/metabolismo , Camundongos , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , HumanosRESUMO
BACKGROUND: Carotenoids are fat-soluble phytochemicals with biological roles, including ultraviolet protective functions in skin. Spectroscopic skin carotenoid measurements can also serve as a noninvasive biomarker for carotenoid consumption. Single-nucleotide polymorphisms (SNPs) in metabolic genes are associated with human plasma carotenoid concentrations; however, their relationships with skin carotenoid concentrations are unknown. OBJECTIVES: The objective of this study was to determine the relationship between 13 candidate SNPs with skin and plasma carotenoid concentrations before and after a carotenoid-rich tomato juice intervention. METHODS: In this randomized, controlled trial, participants (n = 80) were provided with lycopene-rich vegetable juice providing low (13.1 mg), medium (23.9 mg), and high (31.0 mg) daily total carotenoid doses for 8 wk. Plasma carotenoid concentrations were measured by high-pressure liquid chromatography, and skin carotenoid score was assessed by reflection spectroscopy (Veggie Meter) at baseline and the end-of-study time point. Thirteen candidate SNPs in 5 genes (BCO1, CD36, SCARB1, SETD7, and ABCA1) were genotyped from blood using PCR-based assays. Mixed models tested the effects of the intervention, study time point, interaction between intervention and study time point, and SNP genotype on skin and plasma carotenoids throughout the study. Baseline carotenoid intake, body mass index, gender, and age are covariates in all models. RESULTS: The genotype of CD36 rs1527479 (P = 0.0490) was significantly associated with skin carotenoid concentrations when baseline and the final week of the intervention were evaluated. Genotypes for BCO1 rs7500996 (P = 0.0067) and CD36 rs1527479 (P = 0.0018) were significant predictors of skin carotenoid concentrations in a combined SNP model. CONCLUSIONS: These novel associations between SNPs and skin carotenoid concentrations expand on the understanding of how genetic variation affects interindividual variation in skin carotenoid phenotypes in humans. This trial was registered at clinicaltrials.gov as NCT03202043.
Assuntos
Carotenoides , Sucos de Frutas e Vegetais , Genótipo , Licopeno , Polimorfismo de Nucleotídeo Único , Pele , Humanos , Carotenoides/sangue , Carotenoides/metabolismo , Masculino , Feminino , Pele/metabolismo , Pele/química , Adulto , Pessoa de Meia-Idade , Sucos de Frutas e Vegetais/análise , Solanum lycopersicum/genética , Solanum lycopersicum/química , Adulto Jovem , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , beta-Caroteno 15,15'-Mono-OxigenaseRESUMO
The high-affinity IgE receptor FcεRI is the mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the FcεRI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE resulting in degranulation and proinflammatory cytokine production. In comparison to the T- and B-cell receptor complexes, for which several co-receptors orchestrating the initial signaling events have been described, information is scarce about FcεRI-associated proteins. Additionally, it is unclear how FcεRI signaling synergizes with input from other receptors and how regulators affect this synergistic response. We found that the HDL receptor SR-BI (gene name: Scarb1/SCARB1) is expressed in MCs, functionally associates with FcεRI, and regulates the plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains. This impacted the activation of MCs upon co-stimulation of the FcεRI with receptors known to synergize with FcεRI signaling. Amongst them, we investigated the co-activation of the FcεRI with the receptor tyrosine kinase KIT, the IL-33 receptor, and GPCRs activated by adenosine or PGE2. Scarb1-deficient bone marrow-derived MCs showed reduced cytokine secretion upon co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating respective MC activation. Mimicking Scarb1 deficiency by cholesterol depletion employing MßCD, we identified PKB and PLCγ1 as cholesterol-sensitive proteins downstream of FcεRI activation in bone marrow-derived MCs. When MCs were co-stimulated with stem cell factor (SCF) and Ag, PLCγ1 activation was boosted, which could be mitigated by cholesterol depletion and SR-BI inhibition. Similarly, SR-BI inhibition attenuated the synergistic response to PGE2 and anti-IgE in the human ROSAKIT WT MC line, suggesting that SR-BI is a crucial regulator of synergistic MC activation.
Assuntos
Membrana Celular , Colesterol , Mastócitos , Receptores de IgE , Transdução de Sinais , Mastócitos/imunologia , Mastócitos/metabolismo , Colesterol/metabolismo , Animais , Receptores de IgE/metabolismo , Receptores de IgE/imunologia , Camundongos , Transdução de Sinais/imunologia , Membrana Celular/metabolismo , Receptores Depuradores Classe B/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/imunologia , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Degranulação Celular/imunologia , Camundongos Knockout , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Fosfolipase C gama/metabolismoRESUMO
HDLs carry sphingosine-1-phosphate (S1P) and stimulate signaling pathways in different cells including macrophages and endothelial cells, involved in atherosclerotic plaque development. HDL signaling via S1P relies on the HDL receptor scavenger receptor class B, type I (SR-B1) and the sphingosine-1-phosphate receptor 1 (S1PR1), which interact when both are heterologously overexpressed in the HEK293 cell line. In this study, we set out to test if SR-B1 and S1PR1 interacted in primary murine macrophages in culture and atherosclerotic plaques. We used knock-in mice that endogenously expressed S1PR1 tagged with eGFP-(S1pr1eGFP/eGFP mice), combined with proximity ligation analysis to demonstrate that HDL stimulates the physical interaction between SR-B1 and S1PR1 in primary macrophages, that this is dependent on HDL-associated S1P and can be blocked by an inhibitor of SR-B1's lipid transfer activity or an antagonist of S1PR1. We also demonstrate that a synthetic S1PR1-selective agonist, SEW2871, stimulates the interaction between SR-B1 and S1PR1 and that this was also blocked by an inhibitor of SR-B1's lipid transport activity. Furthermore, we detected abundant SR-B1/S1PR1 complexes in atherosclerotic plaques of S1pr1eGFP/eGFP mice that also lacked apolipoprotein E. Treatment of mice with the S1PR1 antagonist, Ex26, for 12 h disrupted the SR-B1-S1PR1 interaction in atherosclerotic plaques. These findings demonstrate that SR-B1 and S1PR1 form ligand-dependent complexes both in cultured primary macrophages and within atherosclerotic plaques in mice and provide mechanistic insight into how SR-B1 and S1PR1 participate in mediating HDL signaling to activate atheroprotective responses in macrophages.
Assuntos
Macrófagos , Placa Aterosclerótica , Receptores Depuradores Classe B , Receptores de Esfingosina-1-Fosfato , Animais , Receptores de Esfingosina-1-Fosfato/metabolismo , Macrófagos/metabolismo , Camundongos , Receptores Depuradores Classe B/metabolismo , Receptores Depuradores Classe B/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Ligantes , Humanos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Lisofosfolipídeos/metabolismo , Lipoproteínas HDL/metabolismo , Camundongos Endogâmicos C57BL , Tiofenos/farmacologia , OxidiazóisRESUMO
Atherosclerosis results from the deposition and oxidation of LDL and immune cell infiltration in the sub-arterial space leading to arterial occlusion. Studies have shown that transcytosis transports circulating LDL across endothelial cells lining blood vessels. LDL transcytosis is initiated by binding to either scavenger receptor B1 (SR-B1) or activin A receptor-like kinase 1 on the apical side of endothelial cells leading to its transit and release on the basolateral side. HDL is thought to partly protect individuals from atherosclerosis due to its ability to remove excess cholesterol and act as an antioxidant. Apolipoprotein A1 (APOA1), an HDL constituent, can bind to SR-B1, raising the possibility that APOA1/HDL can compete with LDL for SR-B1 binding, thereby limiting LDL deposition in the sub-arterial space. To examine this possibility, we used in vitro approaches to quantify the internalization and transcytosis of fluorescent LDL in coronary endothelial cells. Using microscale thermophoresis and affinity capture, we find that SR-B1 and APOA1 interact and that binding is enhanced when using the cardioprotective variant of APOA1 termed Milano (APOA1-Milano). In male mice, transiently increasing the levels of HDL reduced the acute deposition of fluorescently labeled LDL in the atheroprone inner curvature of the aorta. Reduced LDL deposition was also observed when increasing circulating wild-type APOA1 or the APOA1-Milano variant, with a more robust inhibition from the APOA1-Milano. The results suggest that HDL may limit SR-B1-mediated LDL transcytosis and deposition, adding to the mechanisms by which it can act as an atheroprotective particle.
Assuntos
Apolipoproteína A-I , Lipoproteínas HDL , Lipoproteínas LDL , Transcitose , Animais , Humanos , Masculino , Camundongos , Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Ligação Proteica , Receptores Depuradores Classe B/metabolismoRESUMO
Understanding how cells process nanoparticles is crucial to optimize nanomedicine efficacy. However, characterizing cellular pathways is challenging, especially if non-canonical mechanisms are involved. In this Article a genome-wide forward genetic screening based on insertional mutagenesis is applied to discover receptors and proteins involved in the intracellular accumulation (uptake and intracellular processing) of silica nanoparticles. The nanoparticles are covered by a human serum corona known to target the low-density lipoprotein receptor (LDLR). By sorting cells with reduced nanoparticle accumulation and deep sequencing after each sorting, 80 enriched genes are identified. We find that, as well as LDLR, the scavenger receptor SCARB1 also mediates nanoparticle accumulation. Additionally, heparan sulfate acts as a specific nanoparticle receptor, and its role varies depending on cell and nanoparticle type. Furthermore, some of the identified targets affect nanoparticle trafficking to the lysosomes. These results show the potential of genetic screening to characterize nanoparticle pathways. Additionally, they indicate that corona-coated nanoparticles are internalized via multiple receptors.
Assuntos
Nanopartículas , Receptores de LDL , Dióxido de Silício , Humanos , Nanopartículas/química , Receptores de LDL/metabolismo , Receptores de LDL/genética , Dióxido de Silício/química , Dióxido de Silício/metabolismo , Testes Genéticos/métodos , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Heparitina Sulfato/metabolismo , Coroa de Proteína/metabolismo , Coroa de Proteína/química , Lisossomos/metabolismo , Mutagênese InsercionalRESUMO
Circular RNAs (circRNAs) exhibit essential regulation in the malignant development of hepatocellular carcinoma (HCC). This study aims to investigate the physiological mechanisms of circ_0029343 encoded by scavenger receptor class B member 1 (SCARB1) involved in the growth and metastasis of HCC. Differentially expressed mRNAs in HCC were obtained, followed by the prediction of target genes of differentially expressed miRNAs and gene ontology and kyoto encyclopedia of genes and genomes analysis on the differentially expressed mRNAs. Moreover, the regulatory relationship between circRNAs encoded by SCARB1 and differentially expressed miRNAs was predicted. In vitro cell experiments were performed to verify the effects of circ_0029343, miR-486-5p, and SRSF3 on the malignant features of HCC cells using the gain- or loss-of-function experiments. Finally, the effects of circ_0029343 on the growth and metastasis of HCC cells in xenograft mouse models were also explored. It was found that miR-486-5p might interact with seven circRNAs encoded by SCARB1, and its possible downstream target gene was SRSF3. Moreover, SRSF3 was associated with the splicing of various RNA. circ_0029343 could sponge miR-486-5p to up-regulate SRSF3 and activate PDGF-PDGFRB (platelet-derived growth factor and its receptor, receptor beta) signaling pathway by inducing p73 splicing, thus promoting the proliferation, migration, and invasion and inhibiting apoptosis of HCC cells. In vivo, animal experiments further confirmed that overexpression of circ_0029343 could promote the growth and metastasis of HCC cells in nude mice. circ_0029343 encoded by SCARB1 may induce p73 splicing and activate the PDGF-PDGFRB signaling pathway through the miR-486-5p/SRSF3 axis, thus promoting the growth and metastasis of HCC cells.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs , Humanos , Animais , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , RNA Circular/genética , RNA Circular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Nus , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Platinum (Pt)-based chemotherapy is the main treatment for ovarian cancer (OC); however, most patients develop Pt resistance (Pt-R). This work shows that Pt-R OC cells increase intracellular cholesterol through uptake via the HDL receptor, scavenger receptor type B-1 (SR-B1). SR-B1 blockade using synthetic cholesterol-poor HDL-like nanoparticles (HDL NPs) diminished cholesterol uptake leading to cell death and inhibition of tumor growth. Reduced cholesterol accumulation in cancer cells induces lipid oxidative stress through the reduction of glutathione peroxidase 4 (GPx4) leading to ferroptosis. In turn, GPx4 depletion induces decreased cholesterol uptake through SR-B1 and re-sensitizes OC cells to Pt. Mechanistically, GPx4 knockdown causes lower expression of the histone acetyltransferase EP300, leading to reduced deposition of histone H3 lysine 27 acetylation (H3K27Ac) on the sterol regulatory element binding transcription factor 2 (SREBF2) promoter and suppressing expression of this key transcription factor involved in the regulation of cholesterol metabolism. SREBF2 downregulation leads to decreased SR-B1 expression and diminished cholesterol uptake. Thus, chemoresistance and cancer cell survival under high ROS burden obligates high GPx4 and SR-B1 expression through SREBF2. Targeting SR-B1 to modulate cholesterol uptake inhibits this axis and causes ferroptosis in vitro and in vivo in Pt-R OC.
Assuntos
Nanopartículas , Neoplasias Ovarianas , Humanos , Feminino , Receptores Depuradores Classe B/metabolismo , Colesterol/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , OxirreduçãoRESUMO
AIMS: The entry of lipoproteins from blood into the arterial wall is a rate-limiting step in atherosclerosis. It is controversial whether this happens by filtration or regulated transendothelial transport.Because sphingosine-1-phosphate (S1P) preserves the endothelial barrier, we investigated in vivo and in vitro, whether S1P and its cognate S1P-receptor 3 (S1P3) regulate the transendothelial transport of lipoproteins. METHODS AND RESULTS: Compared to apoE-haploinsufficient mice (CTRL), apoE-haploinsufficient mice with additional endothelium-specific knock-in of S1P3 (S1P3-iECKI) showed decreased transport of LDL and Evan's Blue but increased transport of HDL from blood into the peritoneal cave. After 30 weeks of high-fat diet feeding, S1P3-iECKI mice had lower levels of non-HDL-cholesterol and less atherosclerosis than CTRL mice. In vitro stimulation with an S1P3 agonist increased the transport of 125I-HDL but decreased the transport of 125I-LDL through human aortic endothelial cells (HAECs). Conversely, inhibition or knock-down of S1P3 decreased the transport of 125I-HDL but increased the transport of 125I-LDL. Silencing of SCARB1 encoding scavenger receptor B1 (SR-BI) abrogated the stimulation of 125I-HDL transport by the S1P3 agonist. The transendothelial transport of 125I-LDL was decreased by silencing of SCARB1 or ACVLR1 encoding activin-like kinase 1 but not by interference with LDLR. None of the three knock-downs prevented the stimulatory effect of S1P3 inhibition on transendothelial 125I-LDL transport. CONCLUSION: S1P3 regulates the transendothelial transport of HDL and LDL oppositely by SR-BI-dependent and SR-BI-independent mechanisms, respectively. This divergence supports a contention that lipoproteins pass the endothelial barrier by specifically regulated mechanisms rather than passive filtration.
Assuntos
Aterosclerose , Células Endoteliais , Lipoproteínas HDL , Lipoproteínas LDL , Transporte Proteico , Receptores de Esfingosina-1-Fosfato , Animais , Humanos , Camundongos , Aterosclerose/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Transporte Biológico , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Lisofosfolipídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Receptores Depuradores Classe B/metabolismo , Receptores Depuradores Classe B/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Transporte Proteico/genéticaRESUMO
The high-density lipoprotein (HDL)-associated enzyme paraoxonase 1 (PON1) is expressed almost exclusively in the liver and is then transported by HDL to the peripheral tissues. The lipophilic nature of PON1 enables its easy exchange between the lipoprotein and cell membranes in a process that is dependent on the HDL receptor scavenger receptor class B, type 1 (SR-B1). In endothelial cells, PON1 binding to the cell membrane leads to its internalization by endocytosis and subsequent lysosomal degradation. PON1 is a "promiscuous" enzyme with unusually broad substrate specificity in vitro, but its actual function and substrate are still unknown. The enzyme requires a lipid environment and becomes completely inactive upon delipidation. However, when PON1 binds HDL, its active site faces the lipoprotein's core and is inaccessible to external substrates. Hence, the HDL-bound PON1 is inactive toward substrates outside the particle's lipid core and is rapidly degraded and becomes inactive upon internalization. Consequently, the enzyme is only active in the cell membrane during its transit from HDL to the cytoplasm. To assign a function to PON1, we investigated whether it is a palmitoyl-protein thioesterase (PPT) that can hydrolyze the palmitoyl moieties of membrane proteins involved in HDL and cholesterol transport, such as SR-B1, ABCA1, or their neighboring caveola proteins to facilitate the release of HDL or trigger its endocytosis. This study shows that PON1 can hydrolyze palmitoyl-cysteine thioester bonds in vitro, has direct or indirect PPT activity in vivo, and can significantly decrease the presence of SR-B1 in the endothelial membrane.
Assuntos
Arildialquilfosfatase , Membrana Celular , Lipoproteínas HDL , Receptores Depuradores Classe B , Tioléster Hidrolases , Arildialquilfosfatase/metabolismo , Arildialquilfosfatase/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Humanos , Membrana Celular/metabolismo , Receptores Depuradores Classe B/metabolismo , Receptores Depuradores Classe B/genética , Lipoproteínas HDL/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais da Veia Umbilical Humana , Animais , Endocitose/fisiologiaRESUMO
The objective of this study was to observe the infection of human cytomegalovirus (HCMV) to human umbilical vein endothelial cells, and its effect on the expression of single-stranded DNA-binding protein (SSBP1) and on lipid metabolism in endothelial cells. We screened the differential expression of mRNAs after HCMV infection by suppression subtractive hybridization and the expression levels of SSBP1 mRNA and protein after HCMV infection by real-time PCR and western blot. After verification of successful infection by indirect immunofluorescent staining and RT-PCR, we found a differential expression of lipid metabolism-related genes including LDLR, SCARB, CETP, HMGCR, ApoB and LPL induced by HCMV infection. The expression levels of SSBP1 mRNA and protein after HCMV infection were significantly down-regulated. Furthermore, we found that upregulation of SSBP1 inhibited the expression of atherosclerosis-associated LDLR, SCARB, HMGCR, CETP as well as the accumulation of lipids in the cells. The results showed that the inhibition of SSBP1 by HCMV infection promotes lipid accumulation in the cells.
Assuntos
Humanos , Infecções por Citomegalovirus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/virologia , Metabolismo dos Lipídeos/fisiologia , Proteínas Mitocondriais/metabolismo , Aterosclerose/metabolismo , Aterosclerose/virologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Colesterol/análise , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Hidroximetilglutaril-CoA Redutases/metabolismo , Metabolismo dos Lipídeos/genética , Proteínas Mitocondriais/genética , Receptores de LDL/metabolismo , Receptores Depuradores Classe B/metabolismo , Fatores de TempoRESUMO
Background: Plasma high density lipoproteins (HDL) are involved in reverse cholesterol transport mediated by the scavenger receptor class B type I (SR-BI). Nicotinic acid increases HDL cholesterol levels, even though its specific impact on SR-BI dependent-cellular cholesterol transport remains unknown. Aim: To determine the effect of nicotinic acid on HDL particle functionality in cholesterol efflux and uptake mediated by SR-BI in cultured cells in hypoalphalipoproteinemic patients. Material and Methods: In a pilot study, eight patients with low HDL (≤ 40 mg/dL) were treated with extended release nicotinic acid. HDL cholesterol and phospholipid levels, HDL2 and HDL3 fractions and HDL particle sizes were measured at baseline and post-therapy. Before and after nicotinic acid treatment, HDL particles were used for cholesterol transport studies in cells transfected with SR-BI. Results: Nicotinic acid treatment raised total HDL cholesterol and phospholipids, HDL2 levels as well as HDL particle size. Nicotinic acid significantly increased HDL cholesterol efflux and uptake capacity mediated by SR-BI in cultured cells. Conclusions: Nicotinic acid therapy increases SR-BI-dependent HDL cholesterol transport in cultured cells, establishing a new cellular mechanism by which this lipid-lowering drug appears to modulate HDL metabolism in patients with hypoalphalipoproteinemia.