Anastasia Z. Kalea

Abstract: OBJECTIVE: To quantify food consumption (based on food group classification) during several time periods in a sample of adolescents and to identify potential differences in food patterns between normal-weight and overweight participants. DESIGN: Cross-sectional study. Participants were classified as normal weight and overweight/obese. Dietary intake was assessed by 3-day food records and analyzed at the food group level for 7 different time periods. To exploit the time dimension of the data and to further control for the influence of other covariates, the authors estimated a random effects regression model for panel data. PARTICIPANTS: One hundred twenty students. SETTING: Two high schools. RESULTS: In multivariate analysis, no statistically significant differences were found between normal-weight and overweight adolescents with regard to their consumption patterns throughout the day. However, compared to females, male participants had higher intake of all food groups with the exception of fruit, and adolescents engaging in vigorous exercise tended to consume more servings from all food groups during any given time period. CONCLUSIONS AND IMPLICATIONS: No preferential use of some food or food groups in specific day periods was found among overweight compared to normal-weight high school students.

Abstract: Apelin is a peptide that has been identified as the endogenous ligand for the receptor APJ. The apelin/APJ system may be an important factor in the regulation of vascular tone and cardiovascular function. Studies on cultured cells and small animal models have revealed that apelin and APJ are localized in cardiomyocytes and vascular cells. The infusion of apelin affects vascular tone and blood pressure, with both central and peripheral actions. In clinical conditions such as heart failure and atherosclerosis, the gene expression of APJ and apelin, as well as the levels of circulating apelin, may be altered. The only known active homolog of ACE, ACE2, hydrolyzes apelin with similar potency to angiotensin II and, therefore, is responsible for the degradation of both peptides. Emerging data on a potential interaction between the two pathways suggest that the function of apelin/APJ in the vasculature may be relevant to cardiovascular disease, and identifying how this system is regulated could be useful clinically.

Abstract: Aging is inevitably accompanied by gradual and irreversible innate endothelial dysfunction. In this study, we tested the hypothesis that accentuation of glucose metabolism via the aldose reductase (AR) pathway contributes to age-related vascular dysfunction. AR protein and activity levels were significantly increased in aged vs. young aortic homogenates from Fischer 344 rats. Immunostaining revealed that the principal site of increased AR protein was the aortic endothelium as well as smooth muscle cells. Studies revealed that endothelial-dependent relaxation (EDR) in response to acetylcholine was impaired in aged rats compared to young rats and that treatment with the AR inhibitor (ARI) zopolrestat significantly improved EDR in aged rats. Methylglyoxal (MG), a key precursor of advanced glycation endproducts (AGEs), was significantly increased in the aortas of aged rats vs. young rats. Consistent with central roles for AR in generation of MG in aging, ARI treatment significantly reduced MG levels in aged rat aorta to those in young rats. Treatment of aged rats with soluble(s) RAGE, a soluble form of the chief signal transduction receptor for AGEs, RAGE, significantly improved EDR in aged rats, thus establishing the contribution of age-related increases in AGEs to endothelial dysfunction. These findings reveal that significant increases in AR expression and activity in aged rat vasculature linked to endothelial dysfunction may be mitigated, at least in part, via ARI and that aging-linked increased flux via AR generates AGEs; species which transduce endothelial injury consequent to their interaction with RAGE. These data demonstrate for the first time that AR mediates aging-related vascular dysfunction, at least in part, via RAGE.

Abstract: BACKGROUND AND AIMS: We have previously reported that wild blueberry (Vaccinium angustifolium)-enriched diets (WB) attenuate aortic adrenergic response through endothelial-mediated pathways. The duration of dietary intervention necessary to induce the positive changes on vasomotor tone has not been studied to date. Thus, our objective was to investigate the temporal effect of WB consumption on vascular function and reactivity in Sprague-Dawley (SD) rat aorta after 4 and 7 weeks of dietary treatment. METHODS AND RESULTS: Forty male SD rats were randomly assigned to a control (AIN-93) (C) or a WB diet for 4 or 7 weeks. Vascular ring studies were conducted in 3-mm isolated rat aortic rings to investigate vasoconstriction induced by six doses of the alpha(1)-adrenergic agonist, l-phenylephrine (Phe, 10(-8)-3x10(-6) M) alone or in the presence of the NOS inhibitor, l-N(G)-monomethyl-arginine (l-NMMA, 10(-4)M). The maximum force of contraction (F(max)) and vessel sensitivity (pD(2)) were determined. Analysis of variance revealed no significant differences on F(max) after 4 weeks of the WB diet but only a significant increase in pD(2) in the absence of l-NMMA. Seven week WB consumption significantly attenuated contraction in response to l-Phe and resulted in lower pD(2). Inhibition of NOS induced a significant increase in the constrictor response in both diet groups at both time periods, with the WB group fed for 7 weeks having the greater response. CONCLUSION: Thus wild blueberries incorporated into the diet at 8% w/w positively affect vascular smooth muscle contractility and sensitivity but these effects are evident only after 7 weeks of WB consumption.

Abstract: The effect of a wild blueberry-enriched diet on vasoconstriction and vasorelaxation was examined in the adult, 20-week-old spontaneously hypertensive rat (SHR) after 8 weeks of a control (C) or an 8% wild blueberry (WB) diet. Nitric oxide (NO)- and cyclooxygenase (COX)-mediated aortic responses were examined ex vivo with the agonists L-phenylephrine (Phe) and acetylcholine (Ach), in the absence or presence of the NO synthase (NOS) inhibitor L-NG-monomethyl arginine (L-NMMA) or the COX inhibitor mefenamic acid (MFA). The vasoconstriction elicited by Phe was reduced in the WB group, attributed to the NO pathway, favoring a lower vascular tone under basal conditions. Acetylcholine-induced vasorelaxation in the WB group was possibly mediated through the COX, but not the NO pathway. These findings document the potential of wild blueberries to modify major pathways of vasomotor control and improve the vascular tone in the adult SHR with endothelial dysfunction.

Abstract: We have previously reported on the positive effects of wild blueberries on arterial contractile response to alpha(1) adrenergic stimuli and on endothelium-mediated vasorelaxation. Our present study was designed to evaluate the effects of the dietary enrichment with wild blueberries on aortic function and reactivity in the developmental phase of essential hypertension in spontaneously hypertensive rats (SHR). We investigated the possible influence blueberries may have on the acetylcholine (Ach)-induced endothelium-dependent vasorelaxation and phenylephrine-induced vasoconstriction in young SHRs, as well as the contribution of the nitric oxide (NO) synthase and cyclooxygenase (COX) pathways in each of the above responses in an animal model with dysfunctional endothelium. Vascular ring studies were conducted in 3-mm isolated rat aortic ring preparations to investigate vasoconstriction induced by l-Phenylephrine (Phe, 10(-8) to 3x10(-6)M) and vasorelaxation induced by acetylcholine (Ach, 10(-9) to 3x10(-6)M). The major findings of our study were that in Phe-induced vasoconstriction, SHR-BB aortas relaxed to a greater degree in comparison to controls when mefenamic acid (MFA) was present and that the incubation with this COX inhibitor failed to restore - and in fact decreased - the maximum vasodilator response to Ach, in comparison to controls. Our vessel reactivity index (pD(2)) observations indicate that blueberries appear to modulate cell membrane-agonist (Ach) interactions primarily in response to Ach in the young SHR model, but not to the alpha(1) adrenoreceptor agonist. Incorporating wild blueberries in the diet seems to affect the endothelium-dependent vasorelaxation by modulating alternative metabolic pathway(s) (such as affecting the production/activity of COX-derived products) in the young SHR aorta.

Abstract: Receptor for advanced glycation end products (RAGE) and its ligands are overexpressed in multiple cancers. RAGE has been implicated in tumorigenesis and metastasis, but little is known of the mechanisms involved. In this study, we define a specific functional role for an alternate splice variant termed RAGE splice variant 1 (RAGEv1), which encodes a soluble endogenous form of the receptor that inhibits tumorigenesis. RAGEv1 was downregulated in lung, prostate, and brain tumors relative to control matched tissues. Overexpressing RAGEv1 in tumor cells altered RAGE ligand stimulation of several novel classes of genes that are critical in tumorigenesis and metastasis. Additionally, RAGEv1 inhibited tumor formation, cell invasion, and angiogenesis induced by RAGE ligand signaling. Analysis of signal transduction pathways underlying these effects revealed marked suppression of c-jun-NH(2)-kinase (JNK) pathway signaling, and JNK inhibition suppressed signaling through the RAGE pathway. Tumors expressing RAGEv1 were significantly smaller than wild-type tumors and displayed prominently reduced activation of JNK. Our results identify RAGEv1 as a novel suppressor, the study of which may offer new cancer therapeutic directions.

Abstract: RAGE [receptor for AGEs (advanced glycation end-products)] plays an important role in the development and progression of vascular disease. Studies in cultured cells and small animal models of disease have clearly demonstrated that RAGE is central to the pathogenesis of vascular disease of the macro- and micro-vessels in both the diabetic and non-diabetic state. Emerging results from human clinical studies have revealed that levels of circulating soluble RAGE in the plasma may reflect the presence and/or extent of vascular disease state. Additionally, genetic variants of the RAGE gene (AGER in HUGO nomenclature) have been associated with vascular disease risk. Combining RAGE circulating protein levels and the presence of particular RAGE polymorphisms may be a useful clinical tool for the prediction of individuals at risk for vascular disease. Therapeutic intervention targeted at the RAGE gene may therefore be a useful means of treating pathologies of the vasculature.

Abstract: The alternative splicing of pre-mRNAs is a critical mechanism in genomic complexity, disease, and development. Studies of the receptor for advanced glycation end-products (RAGE) indicate that this gene undergoes a variety of splice events in humans. However, no studies have extensively analyzed the tissue distribution in other species or compared evolutionary differences of RAGE isoforms. Because the majority of studies probing RAGE function have been performed in murine models, we therefore performed studies to identify and characterize the splice variants of the murine RAGE gene, and we compared these to human isoforms. Here, using mouse tissues, we identified numerous splice variants including changes in the extracellular domain or the removal of the transmembrane and cytoplasmic domains, which produce soluble splice isoforms. Comparison of splice variants between humans and mice revealed homologous regions in the RAGE gene that undergo splicing as well as key species-specific mechanisms of splicing. Further analysis of tissue splice variant distribution in mice revealed major differences between lung, kidney, heart, and brain. To probe the potential impact of disease-like pathological states, we studied diabetic mice and report that RAGE splice variation changed dramatically, resulting in an increase in production of soluble RAGE (sRAGE) splice variants, which were not associated with detectable levels of sRAGE in murine plasma. In conclusion, we have determined that the murine RAGE gene undergoes extensive splicing with distinct splice isoforms being uniquely distributed in different tissues. These differences in RAGE splicing in both physiological and pathogenic states further expand our understanding of the biological repertoire of this receptor in health and disease.

Abstract: OBJECTIVE: The progression of diabetes is associated with profound endothelial dysfunction. We tested the hypothesis that cellular stress would be detectable in ECs retrieved from arterial and venous vessels of diabetic mice. METHOD: We describe a method for direct isolation of well-characterised aortic and venous ECs from mice in which cells are not subjected to propagation in culture. RESULTS: Gene expression profiling, confirmed by real-time PCR, revealed a progressive increase in markers of injury within two main gene families, EC activation and EC apoptosis, in aortic and venous ECs recovered from diabetic versus non-diabetic mice. In short-term diabetes, Il1b mRNA transcripts were higher in aortic and venous ECs of diabetic mice versus controls. In long-term diabetes, casp-1 mRNA transcripts were higher in aortic and venous ECs of diabetic mice versus controls. CONCLUSION: These data suggest that diabetes imparts diffuse endothelial perturbation in the arterial and venous endothelium.

Abstract: We have previously reported that consumption of blueberry-enriched (BB) diets attenuates the arterial contractile response to alpha(1)-adrenergic stimuli and affects vasomotor tone via endothelium-related pathways. The present study was designed to evaluate vascular function and responsiveness in aortas of weanling male Sprague-Dawley rats fed a control (C) or a BB diet for 7 weeks. Vascular ring studies were conducted in 3-mm isolated rat aortic ring preparations to investigate vasoconstriction induced by L-phenylephrine (Phe) (10(-8)-3 x 10(-6) M) and vasorelaxation induced by acetylcholine (ACh) (10(-8)-3 x 10(-6) M). Agonists were used alone and in the presence of nitric oxide (NO) synthase and cyclooxygenase (COX) inhibitors. We observed a significantly diminished vasoconstrictor response to Phe in aortic rings from rats fed the BB diet. Inhibition of NO synthase but not COX caused a significant increase in the constrictor response in both dietary groups, with the BB group having the greater response. Similarly, the participation of the NO pathway in endothelium-dependent vasorelaxation induced by ACh was greater in the rats fed a BB diet, while COX inhibition showed no effect on maximum ACh-induced vasorelaxation in any diet group. The vessel sensitivity of BB aortic rings to the vasoconstrictor and vasodilator was significantly reduced when compared to controls. We have concluded that diets enriched with blueberries, fed for 7 weeks in Sprague-Dawley rats, seem to affect NO metabolic pathways in the aorta at basal and stimulated levels.

Abstract: Endothelial dysfunction is a key triggering event in atherosclerosis. Following the entry of lipoproteins into the vessel wall, their rapid modification results in the generation of advanced glycation endproduct epitopes and subsequent infiltration of inflammatory cells. These inflammatory cells release receptor for advanced glycation endproduct (RAGE) ligands, specifically S100/calgranulins and high-mobility group box 1, which sustain vascular injury. Here, we demonstrate critical roles for RAGE and its ligands in vascular inflammation, endothelial dysfunction, and atherosclerotic plaque development in a mouse model of atherosclerosis, apoE-/- mice. Experiments in primary aortic endothelial cells isolated from mice and in cultured human aortic endothelial cells revealed the central role of JNK signaling in transducing the impact of RAGE ligands on inflammation. These data highlight unifying mechanisms whereby endothelial RAGE and its ligands mediate vascular and inflammatory stresses that culminate in atherosclerosis in the vulnerable vessel wall.

Abstract: Cellular migration is a fundamental process linked to diverse pathological states such as diabetes and its complications, atherosclerosis, inflammation, and cancer. The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule which binds distinct ligands that accumulate in these settings. RAGE-ligand interaction evokes central changes in key biological properties of cells, including proliferation, generation of inflammatory mediators, and migration. Although RAGE-dependent signal transduction is critically dependent on its short cytoplasmic domain, to date the proximate mechanism by which this RAGE domain engages and stimulates cytoplasmic signaling pathways has yet to be identified. Here we show that the RAGE cytoplasmic domain interacts with Diaphanous-1 (Dia-1) both in vitro and in vivo. We employed the human RAGE cytoplasmic domain as "bait" in the yeast two-hybrid assay and identified the formin homology (FH1) domain of Dia-1 as a potential binding partner of this RAGE domain. Immunoprecipitation studies revealed that the RAGE cytoplasmic domain interacts with the FH1 domain of Dia-1. Down-regulation of Dia-1 expression by RNA interference blocks RAGE-mediated activation of Rac-1 and Cdc42 and, in parallel, RAGE ligand-stimulated cellular migration. Taken together, these findings indicate that the interaction of the RAGE cytoplasmic domain with Dia-1 is required to transduce extracellular environmental cues evoked by binding of RAGE ligands to their cell surface receptor, a chief consequence of which is Rac-1 and Cdc42 activation and cellular migration. Because RAGE and Dia-1 are implicated in the regulation of inflammatory, vascular, and transformed cell migration, these findings highlight this interaction as a novel target for therapeutic intervention in inflammation, atherosclerosis, diabetes, and cancer.

Abstract: The receptor for advanced glycation end-products (RAGE) is a single-transmembrane, multiligand receptor of the immunoglobulin superfamily. RAGE up-regulation is implicated in numerous pathological states including vascular disease, diabetes, cancer, and neurodegeneration. The understanding of the regulation of RAGE is important in both disease pathogenesis and normal homeostasis. Here, we demonstrate the characterization and identification of human RAGE splice variants by analysis of RAGE cDNA from tissue and cells. We identified a vast range of splice forms that lead to changes in the protein coding region of RAGE, which we have classified according to the Human Gene Nomenclature Committee (HGNC). These resulted in protein changes in the ligand-binding domain of RAGE or the removal of the transmembrane domain and cytosolic tail. Analysis of splice variants for premature termination codons reveals approximately 50% of identified variants are targeted to the nonsense-mediated mRNA decay pathway. Expression analysis revealed the RAGE_v1 variant to be the primary secreted soluble isoform of RAGE. Taken together, identification of functional splice variants of RAGE underscores the biological diversity of the RAGE gene and will aid in the understanding of the gene in the normal and pathological state.

Abstract: We examined the effect of dietary Mn on the composition and structure of heparan sulfate (HS) glycosaminoglycans (GAGs) of rat aorta. Animals were randomly assigned to either a Mn deficient (MnD), adequate (MnA) or supplemented (MnS) diet (Mn<1, 10-15 and 45-50 ppm, respectively). After 15 weeks, aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition of the HS fractions was determined by HPCE following treatment with heparin lyases I, II and III. In MnS aortas we observed increased concentration of total GalAGs and decreased concentration of HS and HA, when compared to MnA aortas. Aortas from MnD and MnA rats appeared to have similar distribution of individual GAGs. Heparan sulfate chains of MnS aortas contained higher (41%) concentration of non-sulfated units compared to MnA ones. Variable amounts of trisulfated and disulfated units were found only in MnD and MnA groups but not in MnS. Our results demonstrate that HS biosynthesis in the rat aorta undergoes marked structural modifications that depend upon dietary Mn intake. The reduced expression and undersulfation of HSPGs with Mn supplementation might indicate a reduced ability of vascular cells to interact with biologically active molecules such as growth factors. Alterations in cell-membrane binding ability to a variety of extracellular ligands might affect signal-transduction pathways and arterial functional properties.

Abstract: Previously we showed that manganese (Mn) deficiency enhances the arterial contractile response to alpha(1) adrenergic stimuli and affects vasomotor tone. The aim of this study was to test the hypothesis that dietary Mn deficiency inhibits the vasodilation pathways of rat aorta. Vascular ring studies were conducted in aortic rings from weanling male Sprague-Dawley rats that were fed either a Mn deficient (MnD) or a Mn adequate/control diet (MnA) (<1 and 12 mg/kg Mn, respectively) for a 14-wk period. We investigated endothelium-dependent vasodilation induced by acetylcholine (Ach; 10(-8) to 3 x 10(-6) mol/L) in isolated 3-mm aortic rings precontracted with l-phenylephrine (l-Phe; 10(-6) mol/L). Seven concentrations of Ach were used in the presence or absence of inhibitors of nitric oxide synthase and cyclo-oxygenase. After a second precontraction, 8 doses of sodium nitroprusside (SNP; 10(-8) to 10(-5) mol/L) were added to assess endothelium-independent vasodilation. We observed a decrease in Ach-induced and SNP-induced vasodilation in MnD rat aortas when compared with MnA rat aortas (P </= 0.05). Vessel sensitivity of MnD and MnA aortas to Ach was similar. The addition of l-arginine had no effect on nitric oxide-mediated vasodilation in either group. Nitric oxide synthase-inhibition blunted endothelium-dependent vasodilation to the same degree for both diet groups. Cyclo-oxygenase inhibition enhanced both Ach-induced and SNP-induced vasodilation of MnD rings compared with MnA aortic rings (P </= 0.05). Manganese inhibits the synthesis or activity of a prostanoid-derived vasoconstrictor, which seems to be present at basal and at stimulated levels. This effect is independent of membrane-related events. Our results provide further information on the critical role of Mn on vasomotor tone.

Abstract: It has been documented that increased intake of polyphenols may provide protection against coronary heart disease and stroke. Blueberries (Vaccinium angustifolium) are one of the richest sources of antioxidants among fruits and vegetables. Phenolic compounds from berry extracts inhibit human low density lipoprotein and liposome oxidation. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are structural components of aortas with great structural diversity. Their interaction with compounds such as enzymes, cytokines, growth factors, proteins and lipoproteins and their subsequent role in degenerative diseases has been documented. We investigated the effects of a diet rich in blueberries on the content and structure of GAGs. Sprague-Dawley rats were fed either a control (C) or a blueberry (B) diet for 13 weeks. Aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition was determined by high-performance capillary electrophoresis following enzymatic degradation. A 13% higher amount of total GAGs in aortas of B-fed rats was attributed to a higher content of GalAGs (67%). Determination of the sulfated disaccharides showed an overall lower concentration of oversulfated disaccharides in both HS and GalAG populations in the aortas of the B group. Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.

Abstract: We examined the effect of dietary manganese (Mn) on the vascular contractile machinery in rat thoracic aortas. Weanling male Sprague-Dawley rats were fed either an Mn-deficient (MnD), Mn-adequate (MnA) or Mn-supplemented (MnS) diet (<1, 10-15 and 45-50 ppm Mn, respectively). After 15 weeks on the diets the rats were sacrificed and 3-mm aortic rings were contracted in six cumulative doses of the alpha(1) adrenergic receptor agonist L-phenylephrine (l-Phe, 10(-8) to 3 x 10(-6) M) under 1.5-g preload and relaxed with one dose of acetylcholine (3 x 10(-6) M) to assess intact endothelium. The maximal force (F(max)) of contraction and relaxation, as well as the vessel sensitivity (pD(2)) were determined. Manganese deficiency, assessed by hepatic Mn content, significantly lowered the rate of animal growth. A two-way analysis of variance revealed that MnS animals developed lower F(max) when contracted with L-Phe compared with the MnD and MnA animals (P</=.001). Thus, dietary Mn at levels of 45-50 ppm affects the contractile machinery by reducing maximal vessel contraction to an alpha(1) adrenergic agonist. The observed pD(2) was significantly greater in the MnD group compared with the MnA and MnS animals (P</=.001). Thus, restriction of dietary Mn affects vascular sensitivity to the alpha(1) adrenergic receptor. Our results demonstrate for the first time that dietary Mn influences the receptor signaling pathways and contractile machinery of vascular smooth muscle cells in response to an alpha(1) adrenergic receptor.

Abstract: A334 xD;