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1.
Cardiovasc Diabetol ; 21(1): 154, 2022 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-35962339

RESUMEN

BACKGROUND: Reduced cholesterol efflux capacity (CEC) of HDLs is likely to increase cardiovascular risk in type 1 diabetes (T1D). We aimed to assess whether improvement of glycemic control in T1D patients is associated with changes in CEC in relation with changes in carbamylation of HDLs. METHODS: In this open-label trial, 27 uncontrolled T1D patients were given a three-month standard medical intervention to improve glycemic control. HDL fraction was isolated from plasma, and CEC was measured on THP-1 macrophages. Carbamylation of HDLs was evaluated by an immunoassay. Control HDLs from healthy subjects were carbamylated in vitro with potassium cyanate. RESULTS: HbA1c decreased from 11.4% [10.2-12.9] (median [1st-3rd quartiles]) at baseline to 8.1% [6.6-9.0] after the three-month intervention (P < 0.00001). The CEC of HDLs increased after intervention in 19 (70%) patients (P = 0.038). At the same time, the carbamylation of HDLs decreased in 22 (82%) patients after intervention (P = 0.014). The increase in CEC significantly correlated with the decrease in carbamylated HDLs (r = -0.411, P = 0.034), even after adjustment for the change in HbA1c (ß = -0.527, P = 0.003). In vitro carbamylation of control HDLs decreased CEC by 13% (P = 0.041) and 23% (P = 0.021) using 1 and 10 mmol/L of potassium cyanate, respectively. CONCLUSIONS: The improvement of CEC in relation to a decrease in the carbamylation of HDLs may likely contribute to the beneficial cardiovascular effect of glycemic control in T1D patients. TRIAL REGISTRATION: NCT02816099 ClinicalTrials.gov.


Asunto(s)
Diabetes Mellitus Tipo 1 , HDL-Colesterol , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Control Glucémico , Humanos , Lipoproteínas HDL , Carbamilación de Proteína
2.
J Transl Med ; 18(1): 457, 2020 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-33272291

RESUMEN

BACKGROUND: Although immune modulation is a promising therapeutic avenue in coronavirus disease 2019 (COVID-19), the most relevant targets remain to be found. COVID-19 has peculiar characteristics and outcomes, suggesting a unique immunopathogenesis. METHODS: Thirty-six immunocompetent non-COVID-19 and 27 COVID-19 patients with severe pneumonia were prospectively enrolled in a single center, most requiring intensive care. Clinical and biological characteristics (including T cell phenotype and function and plasma concentrations of 30 cytokines) and outcomes were compared. RESULTS: At similar baseline respiratory severity, COVID-19 patients required mechanical ventilation for significantly longer than non-COVID-19 patients (15 [7-22] vs. 4 (0-15) days; p = 0.0049). COVID-19 patients had lower levels of most classical inflammatory cytokines (G-CSF, CCL20, IL-1ß, IL-2, IL-6, IL-8, IL-15, TNF-α, TGF-ß), but higher plasma concentrations of CXCL10, GM-CSF and CCL5, compared to non-COVID-19 patients. COVID-19 patients displayed similar T-cell exhaustion to non-COVID-19 patients, but with a more unbalanced inflammatory/anti-inflammatory cytokine response (IL-6/IL-10 and TNF-α/IL-10 ratios). Principal component analysis identified two main patterns, with a clear distinction between non-COVID-19 and COVID-19 patients. Multivariate regression analysis confirmed that GM-CSF, CXCL10 and IL-10 levels were independently associated with the duration of mechanical ventilation. CONCLUSION: We identified a unique cytokine response, with higher plasma GM-CSF and CXCL10 in COVID-19 patients that were independently associated with the longer duration of mechanical ventilation. These cytokines could represent the dysregulated immune response in severe COVID-19, as well as promising therapeutic targets. ClinicalTrials.gov: NCT03505281.


Asunto(s)
COVID-19/diagnóstico , COVID-19/inmunología , Inmunidad Innata/fisiología , Neumonía Viral/diagnóstico , Neumonía Viral/inmunología , Anciano , Anciano de 80 o más Años , COVID-19/mortalidad , COVID-19/terapia , Cuidados Críticos , Femenino , Francia/epidemiología , Humanos , Inmunofenotipificación , Activación de Linfocitos/fisiología , Masculino , Persona de Mediana Edad , Neumonía Viral/mortalidad , Neumonía Viral/terapia , Pronóstico , Respiración Artificial , SARS-CoV-2/fisiología , Índice de Severidad de la Enfermedad
3.
J Lipid Res ; 59(1): 25-34, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29150495

RESUMEN

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.


Asunto(s)
Lipoproteínas HDL/química , Lipoproteínas HDL/farmacología , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Vasodilatación/efectos de los fármacos , Voluntarios Sanos , Humanos , Lipoproteínas HDL/sangre , Lisofosfolípidos/sangre , Esfingosina/sangre , Esfingosina/metabolismo
4.
Arterioscler Thromb Vasc Biol ; 37(5): 804-811, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28360087

RESUMEN

OBJECTIVE: High-density lipoprotein (HDL) from nondiabetic patients with metabolic syndrome (MetS) displays abnormalities in their lipidome, such as triglyceride enrichment and sphingosine-1-phosphate depletion. We hypothesized that these abnormalities could impair the ability of HDL to stimulate endothelial nitric oxide synthase (eNOS). APPROACH AND RESULTS: Compared with HDL from control subjects, HDL from normoglycemic patients with MetS was 39% richer in triglycerides (P<0.01) and 15% poorer in sphingosine-1-phosphate (P<0.05; n=23 in each group). eNOS activity, assessed by the conversion of L-[3H]arginine to L-[3H]citrulline, was 69% lower in human umbilical vein endothelial cells incubated with HDL from MetS patients than in cells incubated with HDL from controls (P<0.0001). In addition, the activating phosphorylation of eNOS at serine (Ser) 1177 and of Akt (protein kinase B) at Ser473 was 37% (P<0.001) and 39% (P<0.05) lower, respectively, with HDL from MetS patients. Sphingosine-1-phosphate enrichment of HDL from MetS patients restored their ability to stimulate eNOS activity (P<0.05), in relation with a significant increase in eNOS phosphorylation at Ser1177 (P<0.05) and in Akt phosphorylation at Ser473 (P=0.05). By contrast, triglyceride enrichment of HDL from control subjects did not modify eNOS activity (P=0.90) and phosphorylation at Ser1177 (P=0.87). CONCLUSIONS: We provide evidence that the activation of eNOS by HDL is decreased in MetS patients before the appearance of diabetes mellitus and that sphingosine-1-phosphate depletion of HDL is the main factor responsible for this defect. This has important consequences on the impairment of HDL functionality and antiatherogenic properties in these patients.


Asunto(s)
Diabetes Mellitus/enzimología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Lipoproteínas HDL/sangre , Lisofosfolípidos/sangre , Síndrome Metabólico/enzimología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Esfingosina/análogos & derivados , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Diabetes Mellitus/sangre , Diabetes Mellitus/diagnóstico , Progresión de la Enfermedad , Activación Enzimática , Femenino , Humanos , Masculino , Síndrome Metabólico/sangre , Síndrome Metabólico/diagnóstico , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingosina/sangre
6.
Heart Vessels ; 30(6): 845-9, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25031153

RESUMEN

Several current diseases are associated with an increase in the oxidation of HDL, which is likely to impair their functionality. Our aim was to identify whether oxidation could change the protective effect of HDL against the deleterious effect on vasoreactivity induced by oxidative stress. HDL from healthy subjects were oxidized in vitro by Cu(2+), and the ability of oxidized HDL to counteract the inhibitory effect of oxidized LDL on acetylcholine-induced vasodilation was tested on isolated rabbit aorta rings. Oxidation of HDL was evidenced by the increase in the 7-oxysterols/cholesterol ratio (3.20 ± 1.12 vs 0.02 ± 0.01 % in native HDL, p < 0.05). Oxidized LDL inhibited endothelium-dependent vasodilation (E max = 50.2 ± 5.0 vs 92.5 ± 1.7 % for incubation in Kreb's buffer, p < 0.05) and native HDL counteracted this inhibition (E max = 72.4 ± 4.8 vs 50.2 ± 5.0 % p < 0.05). At the opposite, oxidized HDL had no effect on oxidized LDL-induced inhibition on endothelium-dependent vasorelaxation (E max = 53.7 ± 4.8 vs 50.2 ± 5.0 %, NS). HDL oxidation is associated with a decreased ability of HDL to remove 7-oxysterols from oxidized LDL. In conclusion, these results show that oxidation of HDL induces the loss of their protective effect against endothelial dysfunction, which could promote atherosclerosis in diseases associated with increased oxidative stress.


Asunto(s)
Acetilcolina/uso terapéutico , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Estrés Oxidativo/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Animales , Aorta/efectos de los fármacos , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Conejos
7.
Metabolites ; 13(2)2023 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-36837872

RESUMEN

Alterations affecting high-density lipoproteins (HDLs) are one of the various abnormalities observed in dyslipidemia in type 2 diabetes mellitus (T2DM) and obesity. Kinetic studies have demonstrated that the catabolism of HDL particles is accelerated. Both the size and the lipidome and proteome of HDL particles are significantly modified, which likely contributes to some of the functional defects of HDLs. Studies on cholesterol efflux capacity have yielded heterogeneous results, ranging from a defect to an improvement. Several studies indicate that HDLs are less able to inhibit the nuclear factor kappa-B (NF-κB) proinflammatory pathway, and subsequently, the adhesion of monocytes on endothelium and their recruitment into the subendothelial space. In addition, the antioxidative function of HDL particles is diminished, thus facilitating the deleterious effects of oxidized low-density lipoproteins on vasculature. Lastly, the HDL-induced activation of endothelial nitric oxide synthase is less effective in T2DM and metabolic syndrome, contributing to several HDL functional defects, such as an impaired capacity to promote vasodilatation and endothelium repair, and difficulty counteracting the production of reactive oxygen species and inflammation.

8.
J Clin Endocrinol Metab ; 107(9): e3816-e3823, 2022 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-35647758

RESUMEN

OBJECTIVE: To assess whether, in type 2 diabetes (T2D) patients, lipidomic abnormalities in high-density lipoprotein (HDL) are associated with impaired cholesterol efflux capacity and anti-inflammatory effect, 2 pro-atherogenic abnormalities. DESIGN AND METHODS: This is a secondary analysis of the Lira-NAFLD study, including 20 T2D patients at T0 and 25 control subjects. Using liquid chromatography/tandem mass spectrometry, we quantified 110 species of the main HDL phospholipids and sphingolipids. Cholesterol efflux capacity was measured on THP-1 macrophages. The anti-inflammatory effect of HDL was measured as their ability to inhibit the tumor necrosis factor α (TNFα)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) on human vascular endothelial cells (HUVECs). RESULTS: The cholesterol-to-triglyceride ratio was decreased in HDL from T2D patients compared with controls (-46%, P = 0.00008). As expressed relative to apolipoprotein AI, the amounts of phosphatidylcholines, sphingomyelins, and sphingosine-1-phosphate were similar in HDL from T2D patients and controls. Phosphatidylethanolamine-based plasmalogens and ceramides (Cer) were, respectively, 27% (P = 0.038) and 24% (P = 0.053) lower in HDL from T2D patients than in HDL from controls, whereas phosphatidylethanolamines were 41% higher (P = 0.026). Cholesterol efflux capacity of apoB-depleted plasma was similar in T2D patients and controls (36.2 ±â€…4.3 vs 35.5 ±â€…2.8%, P = 0.59). The ability of HDL to inhibit the TNFα-induced expression of both VCAM-1 and ICAM-1 at the surface of HUVECs was similar in T2D patients and controls (-70.6 ±â€…16.5 vs -63.5 ±â€…18.7%, P = 0.14; and -62.1 ±â€…13.2 vs -54.7 ±â€…17.7%, P = 0.16, respectively). CONCLUSION: Despite lipidomic abnormalities, the cholesterol efflux and anti-inflammatory capacities of HDL are preserved in T2D patients.


Asunto(s)
Diabetes Mellitus Tipo 2 , Antiinflamatorios/metabolismo , HDL-Colesterol/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Lipidómica , Lipoproteínas HDL/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo
9.
FASEB J ; 24(9): 3544-54, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20418497

RESUMEN

Hexaacyl lipopolysaccharide (LPS) aggregates in aqueous media, but its partially deacylated lipid A moiety forms monomers with weaker toxicity. Because plasma phospholipid transfer protein (PLTP) transfers hexaacyl LPS, its impact on metabolism and biological activity of triacyl lipid A in mice was addressed. Triacyl lipid A bound readily to plasma high-density lipoproteins (HDLs) when active PLTP was expressed [HDL-associated lipid A after 4.5 h: 59.1+/-16.0% of total in wild-type (WT) vs. 32.5+/-10.3% in PLTP-deficient mice, P<0.05]. In the opposite to hexaacyl LPS, plasma residence time of lipid A was extended by PLTP, and proinflammatory cytokines were produced in higher amounts in WT than PLTP(-/-) mice (remaining lipid A after 8 h: 53+/-12 vs. 35+/-7%, and IL6 concentration after 4.5 h: 45.5+/-5.9 vs. 14.6+/-7.8 ng/ml, respectively; P<0.05 in all cases). After 1 wk, onset of B16-induced melanoma was observed in only 30% of lipid A-treated WT mice, whereas >80% of the untreated WT, untreated PLTP-deficient, or lipid A-treated PLTP-deficient animals bore tumors (P<0.05 in all cases). It is concluded that PLTP is essential in mediating the association of triacyl lipid A with lipoproteins, leading to extension of its residence time and to magnification of its proinflammatory and anticancer properties.


Asunto(s)
Regulación de la Expresión Génica , Inmunidad Innata/fisiología , Lípido A/inmunología , Lípido A/farmacología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Quimiocina CCL2/sangre , Citocinas/sangre , Citometría de Flujo , Inmunidad Innata/genética , Interferón gamma/sangre , Interleucina-10/sangre , Interleucina-6/sangre , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Ratones Mutantes , Proteínas de Transferencia de Fosfolípidos/genética , Factor de Necrosis Tumoral alfa/sangre
10.
Arterioscler Thromb Vasc Biol ; 30(12): 2452-7, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20864671

RESUMEN

OBJECTIVE: Earlier in vitro studies suggested a putative role for the plasma phospholipid transfer protein (PLTP) in the modulation of blood coagulation. The effect of PLTP expression on blood coagulation under both basal and oxidative stress conditions was compared here in wild-type and PLTP-deficient (PLTP-/-) mice. METHODS AND RESULTS: Under basal conditions, PLTP deficiency was associated with an extended tail bleeding time despite a significant depletion of vascular α-tocopherol content and an impairment of endothelial function. When acute oxidative stress was generated in vivo in the brain vasculature, the steady state levels of oxidized lipid derivatives, the extent of blood vessel occlusion, and the volume of ischemic lesions were more severe in wild-type than in PLTP-/- mice. CONCLUSIONS: In addition to its recognized hyperlipidemic, proinflammatory, and proatherogenic properties, PLTP increases blood coagulation and worsens the extent of ischemic lesions in response to acute oxidative stress. Thus, PLTP arises here as a cardiovascular risk factor for the late thrombotic events occurring in the acute phase of atherosclerosis.


Asunto(s)
Coagulación Sanguínea , Infarto Cerebral/prevención & control , Endotelio Vascular/metabolismo , Trombosis Intracraneal/prevención & control , Estrés Oxidativo , Proteínas de Transferencia de Fosfolípidos/deficiencia , Animales , Tiempo de Sangría , Infarto Cerebral/sangre , Infarto Cerebral/genética , Infarto Cerebral/patología , Infarto Cerebral/fisiopatología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Trombosis Intracraneal/sangre , Trombosis Intracraneal/genética , Trombosis Intracraneal/patología , Trombosis Intracraneal/fisiopatología , Ácidos Linoleicos/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Proteínas de Transferencia de Fosfolípidos/genética , Vasodilatadores/farmacología , alfa-Tocoferol/sangre
11.
Cell Biol Toxicol ; 25(2): 127-39, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18317936

RESUMEN

Oxysterols found in oxidized low-density lipoproteins are probably involved in the appearance of atheroma; some are cytotoxic and some able to induce cytokine secretion. An oxysterol-induced interleukin-8 (IL-8) secretion in human monocytes/macrophages has been previously noticed, but the mechanisms remained unclear. In this paper, we investigated the signaling pathways leading to the induction of IL-8 secretion in monocytic THP-1 cells treated with 7beta-hydroxycholesterol, a cytototoxic oxysterol, or with 25-hydroxycholesterol, an oxysterol non-cytotoxic toward this cell line. The oxysterol-induced IL-8 secretion appears to be a calcium-dependent phenomenon as shown by the use of calcium channel blockers, which strongly decreased IL-8 secretion and IL-8 messenger RNA (mRNA) levels. Fluo-3 staining used in flow cytometry and video microscopy revealed an oxysterol-induced Ca(2+) influx, varying according to the oxysterol studied, leading to the activation of the MEK/ERK1/2 pathway as demonstrated by Western blot analysis. ERK activation led to an increase of c-fos mRNA and/or an activation of c-fos. Luciferase reporter gene assay using constructs of the human IL-8 gene promoter and Transam assay revealed the involvement of the AP-1 transcription factor in oxysterol-dependent IL-8 secretion. These results demonstrate that oxysterol-induced IL-8 secretion is a calcium-dependent phenomenon involving the MEK/ERK1/2 pathway leading to the activation of IL-8 gene via AP-1 (c-fos).


Asunto(s)
Calcio/metabolismo , Hidroxicolesteroles/farmacología , Interleucina-8/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Monocitos/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Humanos , Hidroxicolesteroles/metabolismo , Interleucina-8/genética , Lipoproteínas LDL/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Nifedipino/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción AP-1/metabolismo , Verapamilo/farmacología
12.
FEBS J ; 272(12): 3093-104, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15955068

RESUMEN

Oxysterols, and particularly 7-ketocholesterol, appear to be strongly involved in the physiopathology of atherosclerosis. These molecules are suspected to be cytotoxic to the cells of the vascular wall and monocytes/macrophages, particularly by inducing apoptosis. Previous studies have demonstrated that 7-ketocholesterol-induced apoptosis is triggered by a sustained increase of cytosolic-free Ca2+, which elicits the mitochondrial pathway of apoptosis by activation of the calcium-dependent phosphatase calcineurin, leading to dephosphorylation of the 'BH3 only' protein BAD. However, thorough study of the results suggests that other pathways are implicated in 7-ketocholesterol-induced cytotoxicity. In this study, we demonstrate the involvement of two other calcium-dependent pathways during 7-ketocholesterol-induced apoptosis. The activation of the MEK-->ERK pathway by the calcium-dependent tyrosine kinase PYK 2, a survival pathway which delays apoptosis as shown by the use of the MEK inhibitor U0126, and a pathway involving another pro-apoptotic BH3 only protein, Bim. Indeed, 7-ketocholesterol treatment of human monocytic THP-1 cells induces the release of Bim-LC8 from the microtubule-associated dynein motor complex, and its association with Bcl-2. Therefore, it appears that 7-ketocholesterol-induced apoptosis is a complex phenomenon resulting from calcium-dependent activation of several pro-apoptotic pathways and also one survival pathway.


Asunto(s)
Apoptosis/fisiología , Calcio/metabolismo , Cetocolesteroles/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Proteína 11 Similar a Bcl2 , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Células Cultivadas , Receptor con Dominio Discoidina 1 , Dineínas/efectos de los fármacos , Dineínas/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasa 2 de Adhesión Focal , Humanos , Cetocolesteroles/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/metabolismo , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteína Letal Asociada a bcl
13.
Diabetes Care ; 26(5): 1540-4, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12716819

RESUMEN

OBJECTIVE: In type 2 diabetic patients with poor metabolic control, kinetic studies have demonstrated that LDL fractional catabolic rate (FCR) is slowed down, whereas it is normalized on insulin therapy. This study was designed to analyze whether variations in the expression of LDL receptors at the cell surface could explain the results observed in kinetic studies. RESEARCH DESIGN AND METHODS: LDL receptors were quantified at the surface of mononuclear cells in fresh fasting blood samples by a flow cytometry method in 21 control subjects and 21 type 2 diabetic patients before and 3 months after the introduction of insulin therapy and concomitant removal of oral antidiabetic drugs. RESULTS: Before insulin treatment, monocyte LDL receptor expression was reduced by 41% (6,439 +/- 2,310 vs. 10,846 +/- 2,764 receptors per monocyte, P < 0.001) in type 2 diabetic patients compared with control subjects. It increased by 57% after 3 months of insulin therapy (10,096 +/- 5,657 vs. 6,439 +/- 2,310, P < 0.01) and was similar to that observed in control subjects. CONCLUSIONS: Our results suggest that insulin plays an important role in the in vivo expression of LDL receptors. Moreover, modulations in the expression of LDL receptors in type 2 diabetic patients either with poor metabolic control or on insulin therapy are likely to contribute to the variations of LDL FCR demonstrated by kinetic studies under those circumstances.


Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Insulina/uso terapéutico , Receptores de LDL/sangre , Administración Oral , Anciano , Glucemia/metabolismo , Membrana Celular/metabolismo , Colesterol/sangre , Femenino , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/uso terapéutico , Insulina/sangre , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Valores de Referencia , Insuficiencia del Tratamiento , Triglicéridos/sangre
14.
Br J Pharmacol ; 137(5): 655-62, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12381679

RESUMEN

Among components of oxidized low density lipoproteins, cholesterol derivatives oxidized in position 7 inhibit endothelium-dependent arterial relaxation by decreasing the release of the main endothelium-derived relaxing factor, nitric oxide (NO). The aim of the present study was to bring new insights into the molecular mechanism by which 7-ketocholesterol can block the endothelium-dependent arterial relaxation. Superoxide dismutase did not prevent the inhibitory effect of 7-ketocholesterol on endothelium-dependent relaxation, and consistent observations were made whether superoxide dismutase was conjugated or not to polyethylene glycol. In addition, neither glutathione supplementation, nor oxypurinol, i.e. a xanthine oxidase inhibitor could reverse the effect of 7-ketocholesterol, indicating that NO was not inactivated by superoxide anion. A direct alteration of the activity of the calcium-dependent NO synthase could also be ruled out, since identical relaxing effects of the calcium ionophore A23187 were observed whether arterial rings were treated or not with 7-ketocholesterol. 4 Whereas the above observations come in support of an early, inhibitory action of 7-ketocholesterol, the specific blockade of one given subtype of membrane receptors could be discarded, and similar inhibitions were observed when either muscarinic or purinergic receptors were stimulated. Finally, the blockade of protein kinase C activity by chelerythrine arose as the sole relevant tool in preventing the effect of 7-ketocholesterol on the endothelium-dependent relaxation of rabbit aortic rings. In addition, complementary studies on cultured bovine aortic endothelial cells came in direct support of the ability of 7-ketocholesterol to activate PKC. In conclusion, 7-ketocholesterol that is present in human hypercholesterolaemic plasma, in atherosclerotic arteries, and in many processed foods can block the release of NO by vascular endothelial cells through its ability to activate PKC.


Asunto(s)
Aorta Abdominal/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Cetocolesteroles/farmacología , Proteína Quinasa C/metabolismo , Vasodilatación/efectos de los fármacos , Animales , Aorta Abdominal/enzimología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/enzimología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Técnicas In Vitro , Proteína Quinasa C/antagonistas & inhibidores , Conejos , Vasodilatación/fisiología
15.
Biochem Pharmacol ; 64(3): 527-41, 2002 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-12147305

RESUMEN

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol) 7beta-hydroxycholesterol and 7-ketocholesterol are potent inducers of cell death and probably play central roles in atherosclerosis. As suggested by our previous investigations, 7-ketocholesterol might be a causative agent of vascular damage by inducing apoptosis and enhancing superoxide anion (O2*-) production. To determine the precise relationships between cytotoxicity and oxidative stress, the ability of oxysterols oxidized at C7 to induce apoptosis, to stimulate O2*- production and to promote lipid peroxidation was compared with different pro-apoptotic chemicals: antitumoral drugs (VB, Ara-C, CHX, and VP-16) and STS. All compounds, except 7alpha-hydroxycholesterol, induced apoptosis characterized by the occurrence of cells with fragmented and/or condensed nuclei, loss of mitochondrial potential, caspase-3 activation, PARP degradation, and internucleosomal DNA fragmentation. The highest proportion of apoptotic cells was found with antitumoral drugs and STS, whereas the highest overproduction of O2*- detected before and after the loss of mitochondrial potential was obtained with 7beta-hydroxycholesterol and 7-ketocholesterol. Overproduction of O2*- was always correlated with enhanced lipid peroxidation. Vit E was only capable to significantly counteract apoptosis and oxidative stress induced by 7beta-hydroxycholesterol, 7-ketocholesterol, VB and STS. By electron and fluorescence microscopy, myelin figures evocating autophagic vacuoles were barely observed under treatment with 7beta-hydroxycholesterol and 7-ketocholesterol, and their formation occurring before the loss of mitochondrial potential was reduced by Vit E. In the presence of 7alpha-hydroxycholesterol, no enhancement of O2*- production, no lipid peroxidation, and no formation of myelin figures were observed. Collectively, our data demonstrate, that there can be a more or less important stimulation of oxidative stress during apoptosis. They also suggest that enhancement of O2*- production associated with lipid peroxidation during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis could contribute to in vivo vascular injury, and that myelin figures could constitute suitable markers of oxysterol-induced cell death.


Asunto(s)
Hidroxicolesteroles/farmacología , Cetocolesteroles/farmacología , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Análisis de Varianza , Apoptosis , Caspasa 3 , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , ADN/efectos de los fármacos , ADN/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Mitocondrias/fisiología , Mitocondrias/ultraestructura , Vaina de Mielina/química , Estrés Oxidativo/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células U937
16.
J Clin Endocrinol Metab ; 99(10): E2015-9, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25137425

RESUMEN

CONTEXT: High-density lipoproteins (HDLs) from type 2 diabetic patients are unable to counteract the inhibitory effect of oxidized low-density lipoproteins (ox-LDLs) on vasorelaxation. We hypothesized that glitazones, which improve glycemic control and dyslipidemia, could correct this abnormality. OBJECTIVES AND DESIGN: We compared the ability of HDL from controls (n = 12) and from type 2 diabetic patients before and after 6 months of treatment with either rosiglitazone (n = 11) or pioglitazone (n = 8) to counteract the inhibitory effect of ox-LDL on vasodilatation of rabbit aorta rings. RESULTS: Rosiglitazone induced a decrease in hemoglobin A1c (7.7% ± 1.1% vs 9.8% ± 1.0%, P = .003) and an increase in HDL cholesterol (1.14 ± 0.32 vs 0.98 ± 0.24 mmol/L, P = .033). Pioglitazone induced a decrease in hemoglobin A1c (8.3% ± 2.5% vs 9.5% ± 3.2%, P = .068) and serum triglycerides (1.58 ± 0.89 vs 2.03 ± 0.70 mmol/L, P = .069) and an increase in HDL cholesterol (1.39 ± 0.22 vs 1.14 ± 0.22 mmol/L, P = .018). The triglyceride content of HDL was unchanged by rosiglitazone and was decreased by 25% (P = .068) by pioglitazone. HDL from controls counteracted the inhibitory effect of ox-LDL on vasodilatation (maximal relaxation [Emax] = 74.4% ± 3.5% vs 51.9% ± 3.3%, P = .0029), whereas HDL from type 2 diabetic patients did not (Emax = 51.7% ± 5.8% vs 52.3% ± 4.6% [P = .66] and 52.7% ± 5.5% vs 51.9% ± 4.5% [P = .78] for the rosiglitazone and pioglitazone group, respectively). Rosiglitazone or pioglitazone did not improve Emax (58.6% ± 5.9% vs 52.3% ± 4.6% [P = .15] and 49.3% ± 6.5% vs 51.9% ± 4.5% [P = .48], respectively). CONCLUSION: Glitazones increased the concentration of HDL cholesterol without restoring the ability of HDL particles to protect the endothelium from oxidative stress-induced dysfunction, meaning that HDL remained dysfunctional with impaired antiatherogenic properties.


Asunto(s)
HDL-Colesterol/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Lipoproteínas HDL/sangre , Tiazolidinedionas/uso terapéutico , Vasodilatación/fisiología , Anciano , Animales , Aorta/efectos de los fármacos , Aorta/fisiología , Dislipidemias/tratamiento farmacológico , Dislipidemias/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Femenino , Humanos , Hipoglucemiantes/uso terapéutico , Lipoproteínas LDL/sangre , Masculino , Persona de Mediana Edad , Pioglitazona , Conejos , Rosiglitazona , Vasodilatación/efectos de los fármacos
17.
J Biol Chem ; 284(23): 15826-34, 2009 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-19351882

RESUMEN

Cholesterol oxides, in particular 7-ketocholesterol, are proatherogenic compounds that induce cell death in the vascular wall when localized in lipid raft domains of the cell membrane. Deleterious effects of 7-ketocholesterol can be prevented by vitamin E, but the molecular mechanism involved is unclear. In this study, unlike gamma-tocopherol, the alpha-tocopherol vitamin E form was found to prevent 7-ketocholesterol-mediated apoptosis of A7R5 smooth muscle cells. To be operative, alpha-tocopherol needed to be added to the cells before 7-ketocholesterol, and its anti-apoptotic effect was reduced and even suppressed when added together or after 7-ketocholesterol, respectively. Both pre- and co-treatment of the cells with alpha-tocopherol resulted in the redistribution of 7-ketocholesterol out of the sphingolipid/cholesterol-enriched (lipid raft) domains. In turn, fewer amounts of alpha-tocopherol associated with lipid rafts on 7-ketocholesterol-pretreated cells compared with untreated cells, with no prevention of cell death in this case. In further support of the implication of lipid raft domains, the dephosphorylation/inactivation of Akt-PKB was involved in the 7-ketocholesterol-induced apoptosis. Akt-PKB dephosphorylation was prevented by alpha-tocopherol, but not gamma-tocopherol pretreatment.


Asunto(s)
Muerte Celular/fisiología , Colesterol/metabolismo , Cetocolesteroles/metabolismo , Microdominios de Membrana/metabolismo , Músculo Liso Vascular/fisiología , Esfingolípidos/metabolismo , Vitamina E/farmacología , alfa-Tocoferol/metabolismo , Aorta , Línea Celular , Permeabilidad de la Membrana Celular , Humanos , Peróxido de Hidrógeno/metabolismo , Potenciales de la Membrana , Membranas Mitocondriales/fisiología , Músculo Liso Vascular/citología , Oxidación-Reducción , Tocoferoles/metabolismo
18.
J Biol Chem ; 283(27): 18702-10, 2008 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-18458077

RESUMEN

Lipopolysaccharides (LPS) are components of Gram-negative bacteria. The cellular response from the host to LPS is mediated through stepwise interactions involving the lipopolysaccharide-binding protein (LBP), CD14, and MD-2, which produces the rearrangement of TLR4. In addition to LBP, the lipid transfer/lipopolysaccharide-binding protein gene family includes the phospholipid transfer protein (PLTP). Here we show that the intravascular redistribution of LPS from the plasma lipoprotein-free fraction toward circulating lipoproteins is delayed in PLTP-deficient mice. In agreement with earlier in vitro studies, which predicted the neutralization of the endotoxic properties of LPS when associated with lipoproteins, significant increases in the plasma concentration of proinflammatory cytokines were found in PLTP-deficient as compared with wild type mice. Similar inflammatory damage occurred in tissues from wild type and PLTP-deficient mice 24 h after one single intraperitoneal injection of LPS but with a more severe accumulation of red blood cells in glomeruli of LPS-injected PLTP-deficient mice. Complementary ex vivo experiments on isolated splenocytes from wild type and PLTP-deficient mice further supported the ability of cell-derived PLTP to prevent LPS-mediated inflammation and cytotoxicity when combined with lipoprotein acceptors. Finally, PLTP deficiency in mice led to a significant increase in LPS-induced mortality. It is concluded that increasing circulating levels of PLTP may constitute a new and promising strategy in preventing endotoxic shock.


Asunto(s)
Citocinas/sangre , Endotoxemia/sangre , Mediadores de Inflamación/sangre , Lipopolisacáridos/toxicidad , Proteínas de Transferencia de Fosfolípidos/sangre , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Fase Aguda/genética , Animales , Proteínas Portadoras/sangre , Proteínas Portadoras/genética , Citocinas/genética , Endotoxemia/inducido químicamente , Endotoxemia/genética , Endotoxemia/patología , Inflamación/sangre , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Receptores de Lipopolisacáridos/sangre , Receptores de Lipopolisacáridos/genética , Antígeno 96 de los Linfocitos/sangre , Antígeno 96 de los Linfocitos/genética , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Bazo/metabolismo , Bazo/patología , Factores de Tiempo
19.
Am J Physiol Endocrinol Metab ; 293(1): E416-20, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17473053

RESUMEN

The LDL receptor (LDL-R) has been proposed as the viral receptor for Hepatitis C virus (HCV). This hypothesis has been based exclusively on in vitro studies. In human mononuclear cells, LDL-R gene expression has been demonstrated to be parallel and be coordinately regulated to gene expression in the human liver. The purpose of the current study was to determine the mononuclear cell surface expression of the LDL receptor in patients with HCV chronic infection according to viral load. Sixty-eight consecutive untreated chronic hepatitis C patients were studied to determine the mononuclear cell surface expression of the LDL-R. LDL-Rs were quantified at the surface of mononuclear cells in fresh blood samples taken after fasting using flow cytometry. LDL-R expression was significantly associated with LDL-cholesterol (r = -0.25; P = 0.03) and HCV-viral load (r = 0.37, P = 0.002). In multivariate analysis, the LDL-R expression was significantly associated with HCV viral load, whereas genotype, age, body mass index, and fibrosis were not. In conclusion, our data provided by a human study, suggest that the LDL-R may be one of the receptors implicated in HCV replication.


Asunto(s)
Hepacivirus , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/virología , Receptores de LDL/metabolismo , Carga Viral , Adulto , Anciano , Antígenos de Superficie/metabolismo , Femenino , Hepacivirus/fisiología , Hepatitis C Crónica/sangre , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Replicación Viral
20.
Cytometry A ; 52(2): 57-69, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12655649

RESUMEN

BACKGROUND: Time-lapse video microscopy was used to determine whether mitochondrial and nuclear changes (decrease in mitochondrial transmembrane potential, condensation, and/or fragmentation of the nuclei, morphologic features typical of apoptosis) occurring during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells took place before or after the loss of cell adhesion. In addition, changes in actin organization were followed by conventional fluorescence microscopy. METHODS: Morphologic, functional, and spatial changes at the mitochondrial level were investigated with 3,3'-dihexyloxacarbocyanine iodide and/or MitoTracker Red, and nuclear morphology was characterized by staining with Hoechst 33342. Actin fibers, which are major components of the filament network of the cytoskeleton, were visualized with phalloidin linked to fluorescein. The numbers of adherent and nonadherent cells were determined by cell counting. RESULTS: 7-Ketocholesterol-induced cell death was associated with a rapid alteration of actin fibers, a loss of intercellular junctions, and cell shape modifications. Analysis of mitochondrial transmembrane potential showed successively a hyperpolarization and a more or less pronounced progressive decrease followed by a dramatic drop associated with an increase in Hoechst 33342 staining, reflecting chromatin condensation and morphologic changes in the nuclei. CONCLUSIONS: During cell death induced by 7-ketocholesterol in A7R5 rat smooth muscle cells, the different methods of microscopy allowed us to establish that alterations of actin fibers and mitochondrial dysfunctions occurred before condensation and/or fragmentation of the nuclei, which preceded the loss of cell adhesion.


Asunto(s)
Inhibidores Enzimáticos/toxicidad , Citometría de Imagen/métodos , Cetocolesteroles/toxicidad , Músculo Liso Vascular/efectos de los fármacos , Actinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Recuento de Células , Muerte Celular/efectos de los fármacos , Fragmentación del ADN , Potenciales de la Membrana/efectos de los fármacos , Microscopía Fluorescente , Microscopía por Video , Mitocondrias/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ratas , Ratas Endogámicas , Factores de Tiempo
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