Antoni Paul

C-Reactive Protein Accelerates the Progression of Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Plasma C-reactive protein (CRP) concentration is a strong predictor of atherosclerosis. However, to date, there is no in vivo evidence that CRP is proatherogenic. Methods and Results—We studied the effect of human CRP transgene (tg) expression, under basal and turpentine-stimulated conditions, on atherosclerosis in apolipoprotein (apo) E−/− mice. Aortic atherosclerotic lesions in 29-week-old male mice were 48% larger (P <0.02) in turpentine-treated mice and 34% larger (P <0.05) in untreated CRPtg+/0/apoE−/− mice. Turpentine treatment per se did not affect the extent of atherosclerosis in CRP transgenic or nontransgenic apoE−/− mice. Transgenic mice exhibited lower plasma complement C3 but increased deposition of CRP and C3 in the lesions, which suggests that CRP stimulated activation of complement within the lesion. There was more intense and widespread vascular cell adhesion molecule-1 and collagen staining in the lesions of CRPtg+/0/apoE−/− mice than in CRPtg0/0/apoE−/− littermates. Lesions of CRPtg+/0/apoE−/− mice contained increased angiotensin type 1 receptor (AT1-R) transcripts and displayed increased AT1-R immunostaining compared with those of CRPtg0/0/apoE−/− mice. There was no difference in blood pressure in the 2 types of mice, which indicates that the proatherogenic effect of CRP-associated AT1-R overexpression is local and not mediated by its hypertensive properties. Conclusions—Human CRP transgene expression causes accelerated aortic atherosclerosis in apoE−/− mice. CRP was detected in the lesion, which was associated with increased C3 deposition and increased AT1-R, vascular cell adhesion molecule-1, and collagen expression. These data document a proatherogenic role for CRP in vivo.

Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.

ABSTRACT Adipose differentiation-related protein (ADFP; also known as ADRP or adipophilin), is a lipid droplet (LD) protein found in most cells and tissues. ADFP expression is strongly induced in cells with increased lipid load. We have inactivated the Adfp gene in mice to better understand its role in lipid accumulation. The Adfp-deficient mice have unaltered adipose differentiation or lipolysis in vitro or in vivo. Importantly, they display a 60% reduction in hepatic triglyceride (TG) and are resistant to diet-induced fatty liver. To determine the mechanism for the reduced hepatic TG content, we measured hepatic lipogenesis, very-low-density lipoprotein (VLDL) secretion, and lipid uptake and utilization, all of which parameters were shown to be similar between mutant and wild-type mice. The finding of similar VLDL output in the presence of a reduction in total TG in the Adfp-deficient liver is explained by the retention of TG in the microsomes where VLDL is assembled. Given that lipid droplets are thought to form from the outer leaflet of the microsomal membrane, the reduction of TG in the cytosol with concomitant accumulation of TG in the microsome of Adfp−/− cells suggests that ADFP may facilitate the formation of new LDs. In the absence of ADFP, impairment of LD formation is associated with the accumulation of microsomal TG but a reduction in TG in other subcellular compartments.

Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.

The transcription factor Neurogenin3 (Ngn3) is required for islet-cell type specification. Here, we show that hepatic gene transfer of Ngn3 transiently induces insulin in terminally differentiated hepatocytes but fails to transdifferentiate them, i.e., switch their lineage into islet cells. However, Ngn3 leads to long-term diabetes reversal in mice due to the emergence of periportal islet-like cell clusters. These neo-islets display glycemia-regulated insulin, beta-cell-specific transcripts, and an islet-specific transcription cascade, and they produce all four major islet hormones. They appear to arise from hepatic progenitor cells, most likely endoderm-derived oval cells. Thus, transfer of a single lineage-defining transcription factor, Ngn3, is sufficient to induce cell-lineage switching from a hepatic to an islet lineage in these progenitor cells, a process consistent with transdetermination, i.e, lineage switching in lineage-determined, but not terminally differentiated, cells. This paradigm of induced transdetermination of receptive progenitor cells in vivo may be generally applicable to therapeutic organogenesis for multiple diseases, including diabetes.

Functional role of CD11c+ monocytes in atherogenesis associated with hypercholesterolemia

Background— Monocyte activation and migration into the arterial wall are key events in atherogenesis associated with hypercholesterolemia. CD11c/CD18, a &bgr;2 integrin expressed on human monocytes and a subset of mouse monocytes, has been shown to play a distinct role in human monocyte adhesion on endothelial cells, but the regulation of CD11c in hypercholesterolemia and its role in atherogenesis are unknown. Methods and Results— Mice genetically deficient in CD11c were generated and crossbred with apolipoprotein E (apoE)−/− mice to generate CD11c−/−/apoE−/− mice. Using flow cytometry, we examined CD11c on blood leukocytes in apoE−/− hypercholesterolemic mice and found that compared with wild-type and apoE−/− mice on a normal diet, apoE−/− mice on a Western high-fat diet had increased CD11c+ monocytes. Circulating CD11c+ monocytes from apoE−/− mice fed a high-fat diet exhibited cytoplasmic lipid vacuoles and expressed higher levels of CD11b and CD29. Deficiency of CD11c decreased firm arrest of mouse monocytes on vascular cell adhesion molecule-1 and E-selectin in a shear flow assay, reduced monocyte/macrophage accumulation in atherosclerotic lesions, and decreased atherosclerosis development in apoE−/− mice on a high-fat diet. Conclusions— CD11c, which increases on blood monocytes during hypercholesterolemia, plays an important role in monocyte recruitment and atherosclerosis development in an apoE−/− mouse model of hypercholesterolemia.

ArticleNeurogenin3 Is Sufficient for Transdetermination of Hepatic Progenitor Cells into Neo-Islets In Vivo but Not Transdifferentiation of Hepatocytes

The transcription factor Neurogenin3 (Ngn3) is required for islet-cell type specification. Here, we show that hepatic gene transfer of Ngn3 transiently induces insulin in terminally differentiated hepatocytes but fails to transdifferentiate them, i.e., switch their lineage into islet cells. However, Ngn3 leads to long-term diabetes reversal in mice due to the emergence of periportal islet-like cell clusters. These neo-islets display glycemia-regulated insulin, beta-cell-specific transcripts, and an islet-specific transcription cascade, and they produce all four major islet hormones. They appear to arise from hepatic progenitor cells, most likely endoderm-derived oval cells. Thus, transfer of a single lineage-defining transcription factor, Ngn3, is sufficient to induce cell-lineage switching from a hepatic to an islet lineage in these progenitor cells, a process consistent with transdetermination, i.e, lineage switching in lineage-determined, but not terminally differentiated, cells. This paradigm of induced transdetermination of receptive progenitor cells in vivo may be generally applicable to therapeutic organogenesis for multiple diseases, including diabetes.

Deficiency of Adipose Differentiation-Related Protein Impairs Foam Cell Formation and Protects Against Atherosclerosis

Foam cells are a hallmark of atherosclerosis. However, it is unclear whether foam cell formation per se protects against atherosclerosis or fuels it. In this study, we investigated the role of adipose differentiation-related protein (ADFP), a major lipid droplet protein (LDP), in the regulation of foam cell formation and atherosclerosis. We show that ADFP expression facilitates foam cell formation induced by modified lipoproteins in mouse macrophages in vitro. We show further that Adfp gene inactivation in apolipoprotein E–deficient (ApoE−/−) mice reduces the number of lipid droplets in foam cells in atherosclerotic lesions and protects the mice against atherosclerosis. Moreover, transplantation of ADFP-null bone marrow-derived cells effectively attenuated atherosclerosis in ApoE−/− mice. Deficiency of ADFP did not cause a detectable compensatory increase in the other PAT domain proteins in macrophages in vitro or in vivo. Mechanistically, ADFP enables the macrophage to maintain its lipid content by hindering lipid efflux. We detected no significant difference in lesion composition or in multiple parameters of inflammation in macrophages or in their phagocytic activity between mice with and without ADFP. In conclusion, Adfp inactivation in ApoE−/− background protects against atherosclerosis and appears to be a relatively pure model of impaired foam cell formation.

Low-Cholesterol and High-Fat Diets Reduce Atherosclerotic Lesion Development in ApoE-Knockout Mice

We have investigated the effect of most common oils used in human nutrition on the development of atherosclerosis in apoE-knockout mice. Seven groups of animals, separated according to sex, were fed for 10 weeks either chow diet or the chow diet 10% (wt/wt) enriched with different oils (palm, coconut, 2 types of olive oil, and 2 types of sunflower oil) without addition of cholesterol. At the end of this period, plasma lipid parameters were measured and vascular lesions scored. None of the diets induced changes in plasma cholesterol concentrations, whereas plasma triglycerides were uniformly reduced in all diet groups. Some diets caused significant reductions in the size of atherosclerotic lesions in males and others in females; males responded most to sunflower oils and females to palm oil and one olive oil (II). The lesion reduction in males consuming sunflower oils was associated with the decrease of triglycerides in triglyceride-rich lipoproteins, whereas the decrease in females consuming olive oil II or palm oil was accompanied by an increase in plasma apoA-I. The increase in plasma apoA-I in the latter condition, is mainly due to overexpression of hepatic message elicited by a mechanism independent of apoE ligand. The data suggest that the different diets modulate lesion development in a gender specific manner and by different mechanisms and that the development of atherosclerosis, due to genetic deficiencies, may be modulated by nutritional maneuvers that may be implemented in human nutrition.

The continuous administration of aspirin attenuates atherosclerosis in apolipoprotein E-deficient mice

Aspirin reduces the incidence of thrombotic occlusive events. Classically this has been thought to be due to the platelet inhibitory action of aspirin but it has recently been shown that inflammation plays a predominant role in the initiation and progression of lesions in atherosclerosis. In humans, treatment with aspirin reduces cardiovascular risk and slows carotid plaque growth in a dose-dependent fashion. We have explored this issue in Apo E-deficient mice on a high-fat, high cholesterol diet which provided these animals with a continuous administration of 500 microg/day of acetylsalicylic acid in the drinking water. After 10 weeks of treatment, the size of the atherosclerotic lesion at the aortic sinus had reduced by 35%. At the end of the trial there were no significant changes in either plasma lipids or in the quantitative distribution among lipoproteins. Likewise, the total antioxidant status and the resistance of plasma to oxidation in vitro was similar and there was no change in the distribution of iron deposits and in the relative composition of plasma pro-oxidants and antioxidants, or in the concentration of plasma in ferritin. Therefore, it is our hypothesis that the antiinflammatory effect is responsible for the reduction in lesion size. We propose that antiinflammatory molecules which do not cause gastrointestinal complications should be tested in humans to determine long-term efficacy in the attenuation of atherosclerosis.

Paraoxonase Gln-Arg(192) and Leu-Met(55) gene polymorphisms and enzyme activity in a population with a low rate of coronary heart disease

OBJECTIVES To assess whether paraoxonase (PON1) polymorphisms at positions 55 and 192 and/or their phenotypic expressions influence the risk of myocardial infarction (MI) in Spanish population. DESIGN AND METHODS Two hundred and fifteen male survivors of a MI and their age-matched controls were included in the study. Lipids, apolipoproteins (apo) A-I and B, PON1 activity on paraoxon and phenylacetate and PON1 polymorphisms were determined. RESULTS Genotype distribution was similar in patients and controls. Enzyme activities were lower in patients, but multiple logistic regression analysis did not show any independent association with a higher risk of MI. CONCLUSION None of the PON1 polymorphisms or their corresponding measured activities are independent risk factors for MI in our population.

Gene Therapy with Neurogenin 3 and Betacellulin Reverses Major Metabolic Problems in Insulin-Deficient Diabetic Mice

Insulin deficiency in type 1 diabetes leads to disruptions in glucose, lipid, and ketone metabolism with resultant hyperglycemia, hyperlipidemia, and ketonemia. Exogenous insulin and hepatic insulin gene therapy cannot mimic the robust glucose-stimulated insulin secretion (GSIS) from native pancreatic islets. Gene therapy of streptozotocin-diabetic mice with neurogenin 3 (Ngn3) and betacellulin (Btc) leads to the induction of periportal oval cell-derived neo-islets that exhibit GSIS. We hence hypothesized that this gene therapy regimen may lead to a complete correction of the glucose and lipid metabolic abnormalities associated with insulin deficiency; we further hypothesized that the neo-islets formed in response to Ngn3-Btc gene delivery may display an ultrastructure and transcription profile similar to that of pancreatic islets. We injected streptozotocin-diabetic mice with helper-dependent adenoviral vectors carrying Ngn3 and Btc, which restored GSIS and reversed hyperglycemia in these animals. The treatment also normalized hepatic glucose secretion and reversed ketonemia. Furthermore, it restored hepatic glycogen content and reinstated hepatic lipogenesis-related gene transcripts back to nondiabetic levels. By transmission electron microscopy, the neo-islets displayed electron-dense granules that were similar in appearance to those in pancreatic islets. Finally, using RNA obtained by laser capture microdissection of the periportal neo-islets and normal pancreatic islets, we found that the neo-islets and pancreatic islets exhibited a very similar transcription profile on microarray-based transcriptome analysis. Taken together, this indicates that Ngn3-Btc gene therapy corrects the underlying dysregulated glucose and lipid metabolism in insulin-deficient diabetic mice by inducing neo-islets in the liver that are similar to pancreatic islets in structure and gene expression profile.