Felicia Antohe

Increased uptake of folate conjugates by activated macrophages in experimental hyperlipemia

In the pathogenesis of atherosclerosis, macrophages become activated and play a crucial role in plaque formation. Activated synovial macrophages have recently been shown to express receptors for folic acid. We have determined whether activated macrophages also over-express folate receptor (FR) in atherosclerosis. Most normal cells express little or no FR, and, if FR is present on activated macrophages, folate-linked compounds and drugs could be selectively targeted to those cells that do express FR. To evaluate the FR on macrophages of atherosclerotic animals, golden Syrian hamsters were maintained on a hyperlipidemic diet until extensive vascular lesions had developed. Uptake of folic acid conjugated to fluorescent tags was then examined in tissue fragments from lesion-prone areas, and peritoneal activated macrophages were harvested from the same animals. Spectrofluorimetric and fluorescence microscopic analyses showed a significantly greater uptake of folate-conjugates by peritoneal macrophages of hyperlipidemic hamsters compared with those of hamsters fed a normal or folate-deficient diet. Systemically administered folate-fluorescent conjugates were found to accumulate as bright spots in protrusions of atherosclerotic plaques populated by macrophages, whereas a low level of fluorescence was detected uniformly dispersed across the lesion. The uptake of the folate conjugate by U937 macrophage cells grown in a high-lipid culture medium was significantly higher than in controls. Our data thus indicate that hyperlipidemic conditions induce an increased uptake of folate attributable to the over-expression of FRs on activated macrophages. This increase in FR expression can be exploited to deliver folate-linked compounds selectively to atherosclerotic lesions.

Hyperlipidemia stimulates the extracellular release of the nuclear high mobility group box 1 protein

Our aim was to evaluate the effect of hyperlipidemia on the activation of endogenous alarmin, the high mobility group box 1 (HMGB1) protein, related to systemic inflammation associated with the progression of experimental atherosclerosis and to establish whether statin treatment regulates the HMGB1 signaling pathway. Hyperlipidemia was induced in vivo in golden Syrian hamsters and in monocyte cell culture (U937) by feeding the animals with a high-fat Western diet and by exposing the cells to hyperlipidemic serum. Blood samples, heart, lung and cells were harvested for biochemical, morphological, Western blot, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses. The data revealed that, in the atherosclerotic animal model, the protein HMGB1 and its gene expression were increased and that fluvastatin treatment significantly reduced the release of HMGB1 into the extracellular space. The cell culture experiments demonstrated the relocation of HMGB1 protein from the nucleus to cytoplasm under hyperlipidemic stress. The high level of detected HMGB1 correlated positively with the up-regulation of the advanced glycation end product receptors (RAGE) in the lung tissue from hyperlipidemic animals. During hyperlipidemic stress, the AKT signaling pathway could be activated by HMGB1-RAGE interaction. These results support the existence of a direct correlation between experimentally induced hyperlipidemia and the extracellular release of HMGB1 protein; this might be controlled by statin treatment. Moreover, the data suggest new potentials for statin therapy, with improved effects on patients with systemic inflammation induced by hyperlipidemia.

Heart microvessels and aortic endothelial cells express the 15 kDa heart-type fatty acid-binding proteins.

Due to their hydrophobic nature, free fatty acids require carriers for transport across and within the cells. The endothelial layer is the first barrier to be traversed by the fatty acids, from the plasma to the underlying cells and tissues. We tried to find out whether cytosolic fatty acid-binding proteins (FABPs) are present in the endothelium of large vessels (aortic endothelial cells) and small vessels (myocardial capillaries) using the following experimental approaches: (i) loading the delipidated aortic endothelial cell (EC) homogenate and the heart cytosolic proteins and membrane proteins with [14C]palmitate or [14C]oleate, respectively, followed by autoradiographic detection of electrophoretically separated bands; (ii) detection by immunoprecipitation of heart-type FABP (H-FABP) using an affinity-purified antibody raised against bovine H-FABP (anti-H-FABP), and (iii) localization of FABP by indirect immunofluorescence and gold-immunocytochemistry applied to cultured EC and to thick and thin frozen sections of mouse heart. The results showed that: (i) within the EC homogenate proteins that express affinity for [14C]palmitate have an apparent Mr of 15000, and 40000-45000, that correspond as molecular mass to cytosolic and membrane FABPs, respectively. Similar affinity was found by incubation with [14C]oleate, that binds to a protein of Mr 15000 in the heart cytosol, and to a 40-45 kDa protein in the membrane fraction; (ii) anti-H-FABP immunoprecipitated specifically a cytosolic 15 kDa peptide (H-FABP); (iii) by indirect immunofluorescence, cytosolic H-FABP was localized on heart microvessels and myocytes and also in cultured aortic EC where intense spotted fluorescence characteristic for cytosolic antigens was present; (iv) by immunocytochemistry, H-FABP was detected in the EC cytoplasm, and in close proximity to the cytoplasmic aspect of plasmalemma and vesicle membranes. Together the data attest the presence of the 15 kDa, heart-type FABP in the endothelium of aorta and heart microvessels.

Upregulation of caveolin-1 expression is associated with structural modifications of endothelial cells in diabetic lung.

Diabetes and the associated hyperglycemia affect pulmonary physiology and biochemistry inducing endothelial impairment, as the first step in lung vascular dysfunction. Caveolin-1, a characteristic protein of endothelial caveolae, acts as a scaffolding protein involved in signal transduction, cholesterol homeostasis, and vesicular trafficking. To document the effect of hyperglycemia on lung endothelial cells, we designed experiments on streptozotocin-induced diabetes and on double transgenic diabetic mice and investigated (1) the early morphological changes occurring in endothelial cells, (2) the ACE activity and cholesterol content of caveolae-rich membrane microdomains, and (3) the protein and gene expression of caveolin-1. We provide evidence that in diabetic lung, the endothelial cell displays an increased number of caveolae and enlarged surface area and a well-developed synthetic machinery, changes that correlate with an overall augmented ACE activity and cholesterol content and overexpression (gene and protein) of caveolin-1. Targeting the endothelial cell surface molecules modulated by hyperglycemia, such as caveolin-1 and ACE could be an additional therapeutic strategy in diabetes.

High-fat diet alters protein composition of detergent-resistant membrane microdomains

A high-lipid diet is one of the main risk factors in atherosclerosis and can induce changes in the composition of plasma membrane microdomains. In response, important functions such as vesicle trafficking, protein docking, signaling and receptor recognition are significantly altered. In particular, interactions of heat-shock proteins (Hsps), acting as danger signals, with components of the membrane microdomains can influence signaling pathways and the inflammatory response of cells. Our study focuses on the composition of detergent-resistant membrane (DRM) isolated from ApoE−/− mice fed a standard or high-fat diet with and without fluvastatin treatment versus appropriate controls. Biochemical studies, immunoblotting and liquid chromatography mass spectrometric analysis were performed to investigate whether the structural components (such as caveolin and cavin) of the detergent-resistant microdomains were correlated with the expression and secretion of stress-inducible Hsps (Hsp70 and Hsp90) and AKT phosphorylation in experimental atherosclerosis. ApoE-/− mice challenged with a high-fat diet developed extensive atherosclerotic plaques in lesion-prone areas. DRM harvested from hyperlipidemic animals showed a modified biochemical composition with cholesterol, glycerolipids, caveolin-1 and phospho-AKT being up-regulated, whereas cavin-1 and dynamin were down-regulated. The data also demonstrated the co-fractionation of Hsps with caveolin-1 in isolated DRM, expression being positively correlated with their secretion into blood serum. Statin therapy significantly attenuated the processes induced by the development of atherosclerosis in ApoE−/− mice under a high-fat diet. Thus, high-lipid stress induces profound changes in DRM biochemistry and modifies the cellular response, supporting the systemic inflammatory onset of atherosclerosis.

Hyperlipidemia induces endothelial-derived foam cells in culture.

Endothelial cells (ECs) play a major role in the pathophysiology of various diseases, conditions in which stress proteins are most probably involved. Both in humans and in experimental models, hyperlipidemia induces early alterations of plasma components that in turn have a profound effect on EC. Activated ECs change their basal characteristics becoming more permeable to lipoproteins, increasing the synthesis of their basal lamina, and express new adhesion molecules; the cells are “activated”. In lesion-prone areas, the ECs are the first cells to experience the impact of hyperlipidemia. In this study, human ECs were activated by exposure to serum from hyperlipidemic human subjects. In this condition, the EC gradually become loaded with lipid droplets and turn into endothelial-derived foam cells. The EC-derived foam cells express adhesion molecules (VCAM-1, VLA-4), show enhanced intracellular Ca2+ release, and demonstrate high level of heat shock proteins (Hsp27, Hsp70, and Hsp90). In this study, we bring evidence that the EC-derived foam cells in culture proved to be an useful model to identify the multiple changes induced in activated ECs under hyperlipidemic stress. On the basis of these considerations, future studies using this model system will help to elucidate the molecular basis of the modulator role of molecular chaperones (Hsp) in atherosclerosis under various environmental conditions.

Transcytosis of Albumin in Endothelial Cells is Brefeldin A—Independent

To determine whether in endothelial cells (EC) the pathways of endocytosis and transcytosis of macromolecules interconnect, the effect of Brefeldin A (BFA) on these processes was tested. To this purpose EC were grown to confluence on plastic culture dishes or on cell culture chamber inserts placed into corresponding wells, so as to obtain a dual chamber system. The cells maintained the typical characteristics of EC and had an electrical resistance in the range of 30-60 Ohm.cm2. Transendothelial transport of albumin conjugated to the fluorochrom Texas Red (Alb-TR) and of horseradish peroxidase (HRP) added to the upper compartment, in the absence or presence of BFA (0-25 micrograms/ml), was evaluated in aliquots collected from the lower compartment. At different time intervals, quantitative data were obtained by fluorimetry and spectrophotometry. In other experiments transcytosis of Alb-TR was examined in the presence of 100 microM forskolin (an inhibitor of BFA effect). The endocytosis of Alb-TR and HRP was evaluated by incubating EC with the probes, and the internalized tracers determined in the cell lysate using the methods described above. The results showed that BFA has no significant effect on transcytosis of albumin and HRP. In contradistinction, BFA (5 micrograms/ml) reduced markedly endocytosis of HRP (by 47%). Forskolin has no effect on transcytosis. The data indicate that the BFA-induced perturbance in the endocytic route does not affect the transcytotic pathway of albumin, and suggest that in EC, transcytosis of macromolecules may represent a shortcut for rapid and direct transport of some plasma molecules across the cell.

Evidence for thyroxine transport by the lung and heart capillary endothelium

The uptake and transport of carrier-bound thyroxine by the endothelium were investigated by perfusing through the heart and lung of young rats radiolabeled thyroxine bound to prealbumin ([125I]T4Pa) or serum ([125I]T4S). In addition these complexes were tagged to 5-nm gold particles to obtain quantitative (radioassay) and qualitative (autoradiography) data from the same experiment. The complexes (prewarmed at 37 degrees) were perfused in situ at various concentrations (1 to 50 muCi/ml) for time intervals ranging from 5 to 30 min. After thorough washing of the unbound probe, tissue fragments were either measured for total uptake in a gamma counter or processed for electron microscopy autoradiography. The results showed that both the lung and heart take up [125I]T4 complexes by a process that is saturable at low hormone concentration; uptake is completed by free T4 and Pa. In specimens perfused with double-labeled complexes (iodinated and tagged to gold) autoradiography revealed that silver grains and gold particles colocalize predominantly on endothelial plasmalemmal vesicles. The probe appeared first in vesicles open to the capillary lumen (5 min) and further on in vesicles apparently free within the cytoplasm or open to the abluminal front. At 30 min, only silver grains seem to be present in the pericapillary space, on the alveolar epithelial cells, as well as on the nucleus and mitochondria of heart myocytes. The findings suggest that (1) T4Pa uptake by the endothelial cell (EC) is a specific process (possibly via specific binding sites); (2) T4Pa is taken up and transported across capillary EC by plasmalemmal vesicles; (3) in the pericapillary space T4 seems to dissociate from its carrier.

Fluvastatin reduces the high mobility group box 1 protein expression in hyperlipidemia.

Hyperlipidemia characterized by high levels of cholesterol and triglyceride is considered to be a risk factor for atherosclerosis. The inflammatory process accompanies the development of the atherosclerotic disease and represents a response of the arterial wall to hyperlipidemic insult. The inflammatory process is presently monitored by measuring well-known blood markers. Among them, the high mobility box 1 (HMGB1) protein has been recently documented to be an inflammatory molecule that mediates the response to infection, injury and inflammation [1–5]. One of the most frequently prescribed treatments for cardiovascular diseases is statin therapy which, beyond lowering LDL cholesterol has also anti-inflammatory effects. Therefore, in order to evaluate the HMGB1 protein expression during the development of atherosclerotic plaques under hyperlipidemic diet and to establish whether the statin treatment regulates the HMGB1 signalling pathway, 18 healthy male golden Syrian hamsters were randomly divided into three experimental groups: 1) control (C) fed with standard diet, 2) hyperlipidemic (H) fed with standard diet supplemented with 3% cholesterol and 15% butter for six months and 3) treated hyperlipidemic (Ht) hamsters fed for threemonths like the H group; after that, they received standard diet and fluvastatin sodium (0.075 mg/g bw/day) administered by gavage for another three months. To validate the experimental atherosclerosis model, 5 μm cryosections from each animal were performed from the aortic root area at

Alarmins in chronic noncommunicable diseases: Atherosclerosis, diabetes and cancer

There is a wide range of pathological conditions proved to be associated with inflammation. The inflammatory process offers protection against harmful stimuli such as induced cell injury and tissues damage by means of specialized mediators and cells. Alarmins, also known as endogenous danger signals or damage-associated molecular patterns (DAMPs) molecules, are critical players of immune response to tissue suffering. In many inflammatory and autoimmune conditions, alarmins are released into the extracellular milieu and bind to specific receptors to stimulate and promote activation of innate immune cells, cell differentiation, cell death or secretion of inflammatory mediators. This paper, based on biochemical and mass spectrometry proteomic data, highlights the role of heat shock proteins (HSPs), high-mobility group box 1 (HMGB1) protein and S100 proteins as main alarmins involved in the maintaining and amplifying inflammation in atherosclerosis, diabetes and cancer. BIOLOGICAL SIGNIFICANCE This paper, based on biochemical and mass spectrometry proteomic data, highlights the role of the heat shock proteins (HSPs), high-mobility group box 1 (HMGB1) protein and S100 proteins as main alarmins involved in maintaining and amplifying atherosclerosis, diabetes and cancer inflammation.