RESUMEN
The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering an artificial tissue of 11 cm² in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode array measurements. A dense network of CD31âº/eNOS⺠cells was detected as permeating the whole construct. Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC, resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM represent a promising step toward engineering a functional perfusion system, enabling the engineering of vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.
Asunto(s)
Órganos Bioartificiales , Geles/farmacología , Corazón/efectos de los fármacos , Mucosa Intestinal/trasplante , Intestino Delgado/trasplante , Andamios del Tejido/química , Animales , Línea Celular , Movimiento Celular/efectos de los fármacos , Forma de la Célula , Fenómenos Electrofisiológicos/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Mucosa Intestinal/irrigación sanguínea , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/irrigación sanguínea , Intestino Delgado/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/metabolismo , Sus scrofaRESUMEN
AIMS: A problem in generating artificial tissues is supplying nutrients to cells within 3D constructs. The use of a decellularized biological vascularized matrix with preserved pedicles (BioVaM), as a scaffold, appears to aid the generation of perfusable tissue constructs in vitro. To prevent vessel occlusion upon implantation, a functional endothelium must line the graft vessel bed. Here we tested whether the pro-angiogenic factor CCN1 could improve the re-endothelialization of BioVaM in vitro. METHODS AND RESULTS: BioVaM vessel beds were incubated with 100 ng/mL recombinant human CCN1. Human cord blood endothelial cells (hCBEC) were analysed with respect to adhesion behaviour upon CCN1 exposure and seeded onto vessel structures of CCN1 exposed BioVaM (cBioVaM). BioVaMs were fixed in a bioreactor and perfusion cultured for 4 and 14 days (d). BioVaM without CCN1 treatment served as controls. Initial seeding success and endothelialization progression were monitored by fluorescence-labelled hCBEC. During construct cultivation, pH and lactate production were measured. Degree of endothelialization and characterization of seeded cells, with respect to endothelial markers, were investigated histologically. BioVaM vessel structures showed a 78 +/- 17% increase of attached cells when pre-treated with CCN1. Evaluation of re-endothelialization (arbitrary units) was 4.0 +/- 0.8 and 2.6 +/- 0.8 after 4 d, and 5.0 +/- 0.0 and 3.0 +/- 0.5 after 14 d in cBioVaM vs. BioVaM, respectively. On day 14, lactate concentration, an indicator of metabolic activity, was increased 12-fold in cBioVaM relative to BioVaM. A preserved endothelial phenotype of seeded cells was verified in all cultures by acetylated low density lipoprotein uptake and positive immunohistochemistry against von Willebrand factor, endothelial nitric oxide synthase, and CD31. CONCLUSION: Coating of decellularized vessel structures with CCN1 supports adhesion of hCBEC and enhances re-endothelialization of BioVaM. Perfusable, endothelialized constructs may aid in solving the problem of nourishing cells inside 3D tissue-engineered constructs.
Asunto(s)
Proteína 61 Rica en Cisteína/farmacología , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Ingeniería de Tejidos/métodos , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Sangre Fetal/citología , HumanosRESUMEN
Dietary trans-fatty acids are associated with increased risk of cardiovascular disease and have been implicated in the incidence of obesity and type 2 diabetes mellitus (T2DM). It is established that high-fat saturated diets, relative to low-fat diets, induce adiposity and whole-body insulin resistance. Here, we test the hypothesis that markers of an obese, prediabetic state (fatty liver, visceral fat accumulation, insulin resistance) are also worsened with provision of a low-fat diet containing elaidic acid (18:1t), the predominant trans-fatty acid isomer found in the human food supply. Male 8-week-old Sprague-Dawley rats were fed a 10% trans-fatty acid enriched (LF-trans) diet for 8 weeks. At baseline, 3 and 6 weeks, in vivo magnetic resonance spectroscopy (1H-MR) assessed intramyocellular lipid (IMCL) and intrahepatic lipid (IHL) content. Euglycemic-hyperinsulinemic clamps (week 8) determined whole-body and tissue-specific insulin sensitivity followed by high-resolution ex vivo 1H-NMR to assess tissue biochemistry. Rats fed the LF-trans diet were in positive energy balance, largely explained by increased energy intake, and showed significantly increased visceral fat and liver lipid accumulation relative to the low-fat control diet. Net glycogen synthesis was also increased in the LF-trans group. A reduction in glucose disposal, independent of IMCL accumulation was observed in rats fed the LF-trans diet, whereas in rats fed a 45% saturated fat (HF-sat) diet, impaired glucose disposal corresponded to increased IMCLTA. Neither diet induced an increase in IMCLsoleus. These findings imply that trans-fatty acids may alter nutrient handling in liver, adipose tissue, and skeletal muscle and that the mechanism by which trans-fatty acids induce insulin resistance differs from diets enriched with saturated fats.
Asunto(s)
Adiposidad , Dieta con Restricción de Grasas , Resistencia a la Insulina , Síndrome Metabólico/etiología , Obesidad/etiología , Ácido Oléico/metabolismo , Estado Prediabético/etiología , Ácidos Grasos trans/metabolismo , Animales , Glucemia/metabolismo , Ingestión de Energía , Metabolismo Energético , Técnica de Clampeo de la Glucosa , Glucógeno/metabolismo , Hiperfagia/etiología , Hiperfagia/metabolismo , Hiperfagia/fisiopatología , Insulina/sangre , Grasa Intraabdominal/metabolismo , Hígado/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Síndrome Metabólico/metabolismo , Síndrome Metabólico/fisiopatología , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Obesidad/fisiopatología , Ácido Oléico/administración & dosificación , Ácido Oléico/efectos adversos , Ácidos Oléicos , Estado Prediabético/metabolismo , Estado Prediabético/fisiopatología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Ácidos Grasos trans/administración & dosificación , Ácidos Grasos trans/efectos adversosRESUMEN
The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Insulina/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Alimentación Animal , Animales , Proteínas de Unión al ADN/genética , Metabolismo Energético/efectos de los fármacos , Grasas/farmacología , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Glucógeno/biosíntesis , Ligandos , Hígado/metabolismo , Receptores X del Hígado , Masculino , Ratones , Oligopéptidos , Receptores Nucleares Huérfanos , Oxígeno/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Aumento de Peso/efectos de los fármacosRESUMEN
Dietary fatty acids modulate plasma and intracellular cholesterol concentrations. Circulating non-high-density lipoprotein cholesterol (nHDL-C) concentration is determined by rates of hepatic very low-density lipoprotein assembly and secretion, and clearance of subsequent metabolic products. The effect of dietary fat (butter, traditional margarine, soybean oil, and canola oil) was assessed with respect to plasma lipids, hepatic lipid composition, and messenger RNA (mRNA) abundance of low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, sterol regulatory element-binding protein (SREBP) 2, and microsomal triglyceride transfer protein (MTP) in the Golden-Syrian hamster (Charles River Laboratories, Wilmington, MA). Hamsters were fed with a nonpurified diet (6.25 fat g/100 g) with 0.1 g cholesterol/100 g (control diet) or control diet with an additional 10 g experimental fat/100 g for 12 weeks. Hamsters fed with the control diet, unsaturated fats (canola and soybean oils), and margarine, relative to butter, had significantly lower total cholesterol and nHDL-C and triglyceride concentrations. Additional dietary fat, regardless of fatty acid profile, resulted in higher hepatic cholesterol concentrations. In contrast, relative to the control diet-, butter-, or margarine-fed hamsters, these changes were associated with a 4- and 8-fold higher LDL receptor and 5- and 9-fold higher SREBP mRNA abundance, in hamsters fed with canola and soybean oils, respectively. MTP mRNA, a marker of very low-density lipoprotein particle formation, was higher in canola- and soybean oil-fed hamsters relative to the control diet-fed hamsters, although differences were modest. These results suggest that the substitution of canola and soybean oils for butter results in lower nHDL-C concentrations that may be related to increased mRNA abundance of the LDL receptor, SREBP-2, and MTP genes.
Asunto(s)
Proteínas Portadoras/biosíntesis , Grasas de la Dieta/farmacología , Hidroximetilglutaril-CoA Reductasas/biosíntesis , ARN Mensajero/metabolismo , Receptores de LDL/biosíntesis , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Animales , Aorta Abdominal/metabolismo , Apolipoproteína B-100 , Apolipoproteínas B/sangre , Proteínas Portadoras/genética , Colesterol/sangre , Cricetinae , Grasas de la Dieta/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Hígado/metabolismo , Masculino , Mesocricetus , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Distribución Aleatoria , Receptores de LDL/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Triglicéridos/sangreRESUMEN
Dietary fatty acids alter HDL cholesterol concentrations, presumably through mechanisms related to reverse cholesterol transport. The effect of dietary fats (coconut oil, butter, traditional stick margarine, soybean oil, canola oil) differing in fatty acid profile on this antiatherogenic process was assessed with respect to plasma lipids; exogenous and endogenous lecithin-cholesterol acyltransferase (LCAT), cholesterol ester transfer protein (CETP), phospholipid transfer protein (PLTP) activities; and LCAT, apolipoprotein (apo) A-I and scavenger receptor B class-1 (SR-B1) mRNA abundance. Golden-Syrian hamsters were fed a nonpurified (6.25 g/100 g fat) diet containing an additional 10 g/100 g experimental fat and 0.1 g/100 g cholesterol for 6 wk. Canola and soybean oils significantly lowered serum HDL cholesterol concentrations relative to butter. Canola oil, relative to butter, resulted in higher exogenous LCAT activity, and both soybean and canola oils significantly increased hepatic apo A-I and SR-B1 mRNA abundance. Butter, relative to margarine, coconut and soybean oils, significantly increased serum non-HDL cholesterol concentrations. Endogenous and exogenous LCAT, CETP, and PLTP activities did not differ in hamsters fed margarine or saturated fat diets, despite lower hepatic LCAT, apo A-I, and SR-B1 mRNA abundance, suggesting that changes in available substrate and/or modification to the LCAT protein may have been involved in lipoprotein changes. These results suggest that lower HDL cholesterol concentrations, as a result of canola and soybean oil feeding, may not be detrimental due to increases in components involved in the reverse cholesterol transport process in these hamsters and may retard the progression of atherosclerosis.
Asunto(s)
Colesterol en la Dieta/farmacología , HDL-Colesterol/sangre , Grasas de la Dieta/farmacología , Animales , Apolipoproteína A-I/sangre , Mantequilla , HDL-Colesterol/efectos de los fármacos , Cricetinae , Lipoproteínas/sangre , Masculino , Mesocricetus , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Reacción en Cadena de la Polimerasa , Triglicéridos/sangreRESUMEN
Oxidative stress is an important trigger in the complex chain of events leading to neurodegenerative diseases. On the other hand, dietary fatty acids play an essential role in brain function. The objectives of this study were to assess the effect of dietary fat type on vitamin C and vitamin E (alpha-and gamma-tocopherol) concentrations in peripheral and brain tissues and its effect on 8-epiPGF(2)alpha (F(2)-isoprostanes). Male Golden Syrian hamsters (n = 120, 8 wk old) were fed diets enriched in butter, hydrogenated fat (margarine), and canola and soybean oils. After 12 wk, hamsters were deprived of food, anesthetized with isoflurane, and killed via terminal exsanguination. Analyses of vitamins C, E, and 8-epiPGF(2)alpha were performed in peripheral tissues and brain. Hamsters consuming the margarine-enriched diet had lower (P < 0.05) vitamin C and alpha-tocopherol concentrations in liver, plasma, and brain, and higher (P < 0.02) plasma 8-epiPGF(2)alpha than groups fed the butter, and the canola and soybean oil diets. Liver and plasma gamma-tocopherol concentration was higher (P < 0.001) among the groups fed the soybean- and margarine-enriched diets compared with the other groups. alpha-Tocopherol was higher (P < 0.05) and 8-epiPGF(2)alpha lower (P < 0.01) among the groups fed the canola and soybean oil diets compared with the other groups. Across the groups, an inverse correlation between plasma levels of vitamin C and 8-epiPGF(2)alpha (r = -0.37, P = 0.03) and a positive correlation between plasma levels of vitamin C and alpha-tocopherol were observed (r = 0.341, P = 0.003). Hamsters fed the butter-enriched diet had a higher (P < 0.03) plasma uric acid concentration than the other groups. The results of this study provide new evidence concerning the effect of dietary fat on antioxidant status, which is important for the maintenance of good health.
Asunto(s)
Ácido Ascórbico/metabolismo , Encéfalo/fisiología , Grasas de la Dieta/farmacología , Vitamina E/metabolismo , Animales , Ácido Ascórbico/sangre , Biomarcadores/análisis , Encéfalo/efectos de los fármacos , Mantequilla , Cricetinae , Ácidos Grasos Monoinsaturados , Masculino , Margarina , Mesocricetus , Oxidación-Reducción , Aceite de Brassica napus , Vitamina E/sangreRESUMEN
The objective of this study was to characterize two strains of Golden-Syrian hamsters for use in the study of diet-induced changes in lipoprotein metabolism. In Experiment 1, the time course and response to dietary saturated fat was investigated for serum lipoprotein profiles and aortic lesion formation in Golden-Syrian hamsters from Charles River Laboratories, Wilmington, MA (CR) and Bio Breeders, Watertown, MA (F(1)B). Hamsters were fed a nonpurified diet containing 10 g/100 g saturated fat and 0.1 g/100 g dietary cholesterol. After 12 wk, CR hamsters had significantly lower serum total and non-HDL cholesterol (TC and nHDL-C) levels, but higher aortic cholesteryl ester (CE) than the F(1)B hamsters (P < 0.05). In Experiment 2, CR hamsters were fed a nonpurified diet containing 10 g/100 g saturated fat and 0.1, 0.5 or 1 g/100 g dietary cholesterol. After 10 wk of dietary intervention, TC and nHDL-C levels were significantly higher in the 0.5 and 1.0 g/100 g cholesterol groups than in the 0.1 g/100 g cholesterol group. These levels declined after 20 wk of dietary intervention in all groups, potentially reflecting the toxic effect of high cholesterol intakes. CR hamsters fed a 10 g/100 g saturated fat containing 0.1 g/100 g dietary cholesterol for 10 wk appear to be a good model for investigating diet-induced change in plasma lipids.