Arterial wall thickness and blood pressure in children who were born small for gestational age: correlation with umbilical cord high-sensitivity C-reactive protein.

BACKGROUND: Small for gestational age (SGA) infants have an increased risk of later cardiovascular disease. At birth, high sensitivity-C reactive protein (hs-CRP), a prognostic marker of cardiovascular disease, is significantly higher in SGA than in appropriate for gestational age (AGA) infants. AIM: To measure aortic and carotid intima-media thickness (aIMT, cIMT) and blood pressure (BP) in children (aged 3-5 years) who were born SGA and AGA, and to assess the correlation between hs-CRP concentrations obtained at birth and these haemodynamic variables. METHODS: Umbilical cord hs-CRP concentrations were obtained in 38 neonates. In the same subjects aged 3-5 years, aIMT and cIMT were measured by high-resolution ultrasound scan, in the dorsal arterial wall. Anthropometric variables and BPs were obtained for each child. RESULTS: Maximum (median 0.700 mm, range 0.500-1.080 vs 0.650 mm, 0.400-0.860; p = 0.32) aIMTs were similar between children who were born SGA (n = 17) and AGA (n = 21), respectively. Concentrations of hs-CRP were not correlated with IMTs. In children who were born SGA, systolic BP was significantly correlated with umbilical cord hs-CRP concentrations (r = 0.60; p = 0.009). CONCLUSIONS: Children who were born SGA have a higher, although not significant, aortic thickening than those who were born AGA. Umbilical cord hs-CRP concentrations do not seem to be involved in this process. Instead, hs-CRP concentrations were significantly related to systolic BP values in children who were born SGA, suggesting that hs-CRP at birth could be associated with sympathetic system hyperactivity and with the stress response during childhood.

Occult atherosclerosis and physical vascular examination: a simple strategy to avoid inadequate cardiovascular prevention and under-use of diagnostic vascular guidelines in outpatients. A multicenter study by Angiology Care Units in North-Eastern Italy.

AIM: Recent studies show a high prevalence of inadequate secondary prevention in a subset of the US population at highest risk for stroke and acute myocardial infarction. METHODS: The present investigation evaluated subjects older than 50 years of age attending four Angiology Care Units in Northern Italy. The adequacy of risk factor (hypertension, body weight, cigarette smoking and hypercholesterolemia) control was in particular analyzed, and a search was made for occult atherosclerotic lesions during a thorough physical examination. Finally, adherence to diagnostic vascular guidelines was also evaluated. RESULTS: Twenty-two percent out of 483 patients enrolled in this study were found to have unexpected atherosclerotic lesions, 61.9% of the patients with a history of hypertension, 10.6% showed an inadequate control of blood pressure levels, 55% presented poor lipid control, 16.6% had not stopped smoking and 45.7% were overweight. The physical examination revealed that 13.8% of the patients had cervical bruits, 6.3% had aortic hyperpulsatility and 8.5% were lacking lower limb pulses, not previously diagnosed. It was found that in almost half of the participating patients diagnostic vascular guidelines were not being followed. CONCLUSION: This study shows a high prevalence of inadequate primary and secondary prevention and under-use of diagnostic vascular guidelines in the care of high-risk patients (older than 50 years with diabetes, smokers, etc.). Considerable efforts are required to effectively implement risk factor modification strategies and, with regard to Angiology Care Units, to correctly search for occult atherosclerotic lesions in high-risk patients.

Changes in FAT/CD36, UCP2, UCP3 and GLUT4 gene expression during lipid infusion in rat skeletal and heart muscle.

OBJECTIVE: It has been reported that an increased availability of free fatty acids (NEFA) not only interferes with glucose utilization in insulin-dependent tissues, but may also result in an uncoupling effect of heart metabolism. We aimed therefore to investigate the effect of an increased availability of NEFA on gene expression of proteins involved in transmembrane fatty acid (FAT/CD36) and glucose (GLUT4) transport and of the uncoupling proteins UCP2 and 3 at the heart and skeletal muscle level. STUDY DESIGN: Euglycemic hyperinsulinemic clamp was performed after 24 h Intralipid(R) plus heparin or saline infusion in lean Zucker rats. Skeletal and heart muscle glucose utilization was calculated by 2-deoxy-[1-(3)H]-D-glucose technique. Quantification of FAT/CD36, GLUT4, UCP2 and UCP3 mRNAs was obtained by Northern blot analysis or RT-PCR. RESULTS: In Intralipid(R) plus heparin infused animals a significant decrease in insulin-mediated glucose uptake was observed both in the heart (22.62+/-2.04 vs 10.37+/-2.33 ng/mg/min; P<0.01) and in soleus muscle (13.46+/-1.53 vs 6.84+/-2.58 ng/mg/min; P<0.05). FAT/CD36 mRNA was significantly increased in skeletal muscle tissue (+117.4+/-16.3%, P<0.05), while no differences were found at the heart level in respect to saline infused rats. A clear decrease of GLUT4 mRNA was observed in both tissues. The 24 h infusion of fat emulsion resulted in a clear enhancement of UCP2 and UCP3 mRNA levels in the heart (99.5+/-15.3 and 80+/-4%) and in the skeletal muscle (291.5+/-24.7 and 146.9+/-12.7%). CONCLUSIONS: As a result of the increased availability of NEFA, FAT/CD36 gene expression increases in skeletal muscle, but not at the heart level. The augmented lipid fuel supply is responsible for the depression of insulin-mediated glucose transport and for the increase of UCP2 and 3 gene expression in both skeletal and heart muscle.

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs.

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.

Differential functions of members of the low density lipoprotein receptor family suggested by their distinct endocytosis rates.

The low density lipoprotein receptor (LDLR) family is composed of a class of cell surface endocytic receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2), and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its chimeric receptor constructs, and full-length VLDLR and apoER2 stably expressed in LRP-null Chinese hamster ovary cells. We find that the initial endocytosis rates mediated by different cytoplasmic tails are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR and apoER2 exhibit least endocytosis function. Compared with the tail of LRP, the tails of the LDLR and megalin display significantly lower levels of endocytosis rates. Ligand degradation analyses strongly support differential endocytosis rates initiated by these receptors. Interestingly apoER2, which has recently been shown to mediate intracellular signal transduction, exhibited the lowest level of ligand degradation efficiency. These results thus suggest that the endocytic functions of members of the LDLR family are distinct and that certain receptors in this family may play their main roles in areas other than receptor-mediated endocytosis.

Identification of a major cyclic AMP-dependent protein kinase A phosphorylation site within the cytoplasmic tail of the low-density lipoprotein receptor-related protein: implication for receptor-mediated endocytosis.

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor that belongs to the LDL receptor family. Recently, studies have revealed new roles of LDL receptor family members as transducers of extracellular signals. Our previous studies have demonstrated LRP phosphorylation within its cytoplasmic tail, but the nature of LRP phosphorylation and its potential function was unknown. In the present study using both in vivo and in vitro analysis, we found that LRP phosphorylation is mediated by the cAMP-dependent protein kinase A (PKA). Using site-directed mutagenesis and LRP minireceptor constructs, we further identified the predominant LRP phosphorylation site at serine 76 of its cytoplasmic tail. Finally, we demonstrated that mutations of serine 76, which abolish LRP phosphorylation by PKA, result in a decrease in the initial endocytosis rate of LRP and a lower efficiency in delivery of ligand for degradation. Thus, the role of PKA phosphorylation of LRP in receptor-mediated endocytosis may provide a mechanism by which the endocytic function of LRP can be regulated by external signals.

Expression of alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) in rat microglial cells.

Low density lipoprotein receptor-related protein (LRP) participates in the uptake and degradation of several ligands implicated in neuronal pathophysiology including apolipoprotein E (apoE), activated alpha(2) -macroglobulin (alpha(2)M*) and beta-amyloid precursor protein (APP). The receptor is expressed in a variety of tissues. In the brain LRP is present in pyramidal-type neurons in cortical and hippocampal regions and in astrocytes that are activated as a result of injury or neoplasmic transformation. As LRP is expressed in the monocyte/macrophage cell system, we were interested in examining whether LRP is expressed in microglia. We isolated glial cells from the brain of neonatal rats and LRP was immunodetected both in microglial cells and in astrocytes expressing glial fibrillar acidic protein (GFAP). Microglial cells were able to bind and internalize LRP-specific ligand, alpha(2)M*. The internalization was inhibitable by RAP, with a Kd of 1.7 nM. The expression of LRP was up-regulated by dexamethasone, and down-regulated by lipopolysaccharide (LPS), gamma interferon (IFN-gamma) or a combination of both. LRP was less sensitive to dexamethasone in activated astrocytes than in microglia. We provided the first analysis of LRP expression and regulation in microglia. Our results open the possibility that microglial cells could be related to the participation of LRP and its ligands in different pathophysiological states in brain.

The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein.

All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.

Role of rap in the biogenesis of lipoprotein receptors.

The LDL receptor gene family is composed of several endocytic receptors that share structural homology and function in cellular uptake of various ligands including lipoprotein particles. The complex structure of these lipoprotein receptors is highlighted by the presence of clusters of cysteine-rich ligand-binding repeats. An important feature that is shared by all these receptors is the inhibition of ligand interaction by a 39-kDa receptor-associated protein (RAP). Recent studies have shown that under physiological conditions RAP serves as a molecular chaperone to assist the folding of lipoprotein receptors and their safe passage through the secretory pathway. Several non-exclusive models have been proposed regarding the molecular mechanisms of RAP function as an antagonist for ligand interaction with the receptors and as a molecular chaperone within the early secretory pathway. Elucidation of these mechanisms may provide insights into how biogenesis of lipoprotein receptors can be regulated via the expression of RAP under physiological and pathological conditions.

Mannose receptor is present in a functional state in rat microglial cells.

We studied the expression of the mannose receptor (ManR) in rat microglial cells. Microglial cells are the central nervous system resident macrophages, key participants of the innate immune response. ManR is a differentiation marker and a relevant glycoprotein for the phagocytic and endocytic function of macrophages. Because there is evidence suggesting that ManR could mediate some of the nonenzymatic effects of acetilcholinesterase (AchE) and the enzyme seems to be involved in Alzheimer's disease (AD), we looked for ManR in microglia, evaluating the functionality of the receptor. We isolated microglial cells from the brain of 2-day-old neonatal rats. Microglial cells, identified by their specific staining with the lectin Griffonia simplicifolia, expressed ManR, being detected by immunocytochemistry, Western blot, and immunoprecipitation. Microglial ManR was downregulated by lipopolysaccharide (LPS) and upregulated by dexamethasone, as described for peripheral macrophages. Microglial ManR was functional and able to internalize horseradish peroxidase (HRP), a known ManR ligand, in a mannan-inhibitable manner. The presence of a functional ManR in microglia opens the possibility that ManR could participate in multiple physiologic and pathologic conditions in the central nervous system (CNS), including inflammation, ischaemia, and neurodegenerative diseases such as AD.

Acute effects of exercise on circulating leptin in lean and genetically obese fa/fa rats.

Mechanisms of regulation of plasma leptin in lean and genetically obese animals are not completely understood. In particular a relation has been proposed between energy metabolism and leptin. However, it is not clear how energy expenditure and leptin are related under exercise in lean and obese animals. To clarify these aspects we investigated lean and genetically obese (fa/fa) Zucker rats undergoing a single bout (30 min) of swimming and measured several biochemical and hormonal parameters of energy metabolism and leptin changes throughout the study. Moreover ob-gene expression in adipose tissue was also measured. Our results showed that plasma leptin is decreased by 30% at the end of exercise in lean animals while resulting unaffected in obese animals. Leptin changes in lean rats are concomitant with the peak of NEFA and glycerol release from adipose tissue rather than with the reduction of plasma insulin. Ob-gene expression in adipose tissue was markedly increased in fa/fa compared to lean rats, but was not modified by exercise both in lean and obese animals. In conclusion our data show that leptin changes during exercise are related to lipolytic events in adipose tissue and support a link between leptin and energy expenditure.

Cellular and molecular basis of estrogen's neuroprotection. Potential relevance for Alzheimer's disease.

Alzheimer's disease (AD) is one of the most common types of dementia among the aged population, with a higher prevalence in women. The reason for this latter observation remained unsolved for years, but recent studies have provided evidence that a lack of circulating estrogen in postmenopausal women could be a relevant factor. Moreover, follow-up studies among postmenopausal women who had received estrogen-replacement therapy (ERT), suggested that they had a markedly reduced risk of developing AD. In addition, studies among older women who already had AD indeed confirmed that a decrease in estrogen levels was likely to be an important factor in triggering the pathogenesis of the disease. In this review article, we will discuss the evidence suggesting that estrogen may have a protective role against AD, mainly through its action as: a trophic factor for cholinergic neurons, a modulator for the expression of apolipoprotein E (ApoE) in the brain, an antioxidant compound decreasing the neuronal damage caused by oxidative stress, and a promoter of the physiological nonamyloidogenic processing of the amyloid precursor protein (APP), decreasing the production of the amyloid-beta-peptide (A beta), a key factor in the pathogenesis of AD.

Apical sorting of hepatitis B surface antigen (HBsAg) is independent of N-glycosylation and glycosylphosphatidylinositol-anchored protein segregation.

We have used the hepatitis B surface antigen (HBsAg) as a tool to explore mechanisms by which polarized epithelial cells address specific proteins to their apical domain. It recently has been proposed that N-glycans can serve as apical signals recognized by lectin-like sorting receptors in the trans-Golgi network. We found, however, conclusive evidence that the HBsAg follows an apical pathway not mediated by N-glycan signaling. Neither tunicamycin treatment nor replacement of its single glycosylated residue, Asn-146, altered its predominant (>85%) apical secretion from transfected Madin-Darby canine kidney cells (MDCK). Although HBsAg is known to be secreted as a lipoprotein particle, our results suggest that the exocytic machinery involved in its N-glycan-independent pathway overlaps, at least partially, with that of other apically targeted proteins, including the endogenous gp80, as judged by the effects of brefeldin A. We also tested whether its sorting behavior could be ascribed to association with glycosylphosphatidylinositol (GPI)-anchored proteins, which, together with glycosphingolipids, primarily are targeted to the apical domain of MDCK cells. HBsAg was preferentially secreted from the apices of transfected Fisher rat thyroid cells, which, in contrast to MDCK cells, address GPI-proteins and glycosphingolipids to their basal domain. Moreover, complete inhibition of GPI biogenesis by mannosamine treatment did not impair the HBsAg apical secretion, discarding the possibility that HBsAg could be "hitchhiking" with a newly synthesized GPI-protein. Thus, the HBsAg provides a unique model system to search for yet-unknown apical sorting mechanisms that could depend on proteinaceous targeting signals interacting with cognate trans-Golgi network receptors that are at present unidentified.

Hepatic production of very low density lipoprotein, catabolism of low density lipoprotein, biliary lipid secretion, and bile salt synthesis in rats fed a bean (Phaseolus vulgaris) diet.

Rats fed a bean diet develop a significant hypocholesterolemia. The catabolism of low density lipoprotein (LDL; d 1.019-1.063 g/ml) was studied in vivo and in vitro in the isolated perfused liver of rats fed either a casein or a bean diet. The clearance of LDL was significantly increased by 100% from 0.38 +/- 0.04 to 0.63 +/- 0.04 ml/h x 100 g body wt in vivo in the bean-fed rat. Similarly, the clearance of homologous and heterologous (human) LDL was also increased by 100% in the isolated perfused liver of bean-fed animals. Spleen, kidney, and hepatic cholesterogenesis was increased by 150% in these animals. Bile salt synthesis was increased from 1.54 +/- 0.02 to 2.84 +/- 0.09 nmol/min x g liver wt (P < 0.02) and biliary cholesterol output by 200% from 0.81 +/- 0.03 to 2.18 +/- 0.04 nmol/min x g (P < 0.02) in the isolated perfused liver of rats fed a bean diet. These results explained the depletion of hepatic cholesterol and were consistent with the LDL turnover studies, suggesting that apoB/E receptor activity was increased in these animals. ApoB and triglyceride secretion in the d < 1.060 g/ml lipoprotein fraction of liver perfusates remained normal in the bean-fed rats. In contrast, total sinusoidal cholesterol output isolated in the d < 1.060 g/ml fraction significantly decreased by 100% after 90 min of perfusion. Cholesterol output in the d > 1.060 g/ml lipoprotein fraction was unmodified by the bean diet. These data demonstrate that key metabolic pathways of hepatic cholesterol are modified in the bean-fed rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of biliary lipids from the hepatocyte.

A significant amount of biliary cholesterol is carried in unilamellar-phospholipid (lecithin) vesicles, in both supersaturated human hepatic bile and unsaturated rat bile. This fact supports the concept that biliary cholesterol is normally secreted in phospholipid vesicles from the hepatocyte into the canaliculus. The fundamental aspects of biliary lipid secretion relate first to the quantitative determinants of hepatocytic cholesterol secretion into the bile and, second, to the cell biology of this process. There is a tight curvilinear coupling between the rates of bile acids and biliary lipid secretion in all animal species. The hydrophobicity of the bile acid pool may modify this cosecretory mechanism in that more hydrophobic bile acids recruit more phospholipid and cholesterol per mole of bile acid secreted into the bile. The quantitative significance of this effect, however, is relatively minor. In contrast, intrahepatic determinants, such as the rates of hepatic cholesterol esterification and very low density lipoprotein production modulated by dietary factors, may markedly change the amount of cholesterol carried in vesicles into the bile. Recent studies provide strong evidence to support the concept that biliary cholesterol output is also modulated by the amount of free cholesterol available in specific regions of the endoplasmic reticulum for recruitment by the bile acid cosecretory mechanism. The origin of biliary lipids is in the smooth endoplasmic reticulum membranes. The intracellular transport and the canalicular secretory mechanism of the precursor of biliary lipid vesicles is mostly unknown. Two theories related to the cell biology of biliary lipid secretion are discussed in this article, the fusion-budding model and the exocytotic model.

[Characterization of lipoprotein catabolism in biliary cholesterol hypersecretion conditions in rats]. / Caracterización del catabolismo lipoproteico en condiciones de hipersecreción biliar de colesterol en la rata.

The liver represents the principal pathway for sterol excretion from the organism. In addition, the major proportion of serum lipoproteins is catabolized in the liver. It is known that diosgenin and bean diet markedly induce biliary cholesterol output. In this series of studies we characterized the catabolism of several lipoprotein particles in animals fed diosgenin or a bean-rich diet (biliary cholesterol output is increased greater than 300% in these animals). Human low density lipoprotein (hLDL) and rat high density lipoprotein apo-E free (rHDL) were labeled with 125I. Rat chylomicrons were labeled with cholesterol-(3H)-oleate. hLDL clearance increased from 381 +/- 39 to 628 +/- 44 (microliters/h x 100 g body wt) (p less than 0.005) in bean-fed rats. The half life (t1/2) decreased from 12.4 +/- 1 to 9.8 +/- 0.7 h (p less than 0.005) in these rats. The clearance of rHDL apo-E free increased from 579 +/- 8 to 680 +/- 36 (microliters/h x 100 g body wt) (p less than 0.05) in diosgenin-fed animals. The t1/2 significantly decreased from 8.2 +/- 0.7 h to 7.0 +/- 0.3 h (p less than 0.05) in these rats. In contrast, chylomicron clearance and t1/2 were not modified by the diosgenin or bean diets. These results are consistent with the hypothesis that the number and/or affinity of the B/E and A-I receptors, but not E receptors are increased in rats fed biliary cholesterol hypersecretory diets. These studies suggests the presence of a functional inter-relationship between the biliary and sinusoidal pathways of hepatic cholesterol.

Effect of bean intake on biliary lipid secretion and on hepatic cholesterol metabolism in the rat.

We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoprotein casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary bile salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthesized hepatic cholesterol was preferentially channelled to the biliary cholesterol secretory pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.