The cell adhesion molecule E-cadherin is widely expressed in human atherosclerotic lesions

OBJECTIVE: Various cell adhesion molecules are expressed in atherogenesis and the significance of their involvement in atherosclerotic lesion formation is well appreciated. In the present work, we examined whether the Ca(2+)-dependent cell adhesion molecule E-cadherin is also involved in atherogenesis. METHODS: Specimens of carotid artery and aorta were obtained at operation. Expression of E-cadherin was studied by an immunohistochemical method. The nature of E-cadherin-expressing cells was examined by comparative analysis of consecutive sections and by a double immunostaining procedure. An immunohistochemical approach was also applied to examine how the accumulation of oxidised low density lipoproteins (LDL) by intimal cells is associated with E-cadherin expression. RESULTS: No E-cadherin+ cells were found in normal non-atherosclerotic intima but E-cadherin+ cells were present in 96% of the atherosclerotic lesions. In atherosclerotic intima, E-cadherin was expressed by intimal cells showing varying degrees of transformation into foam cells. These E-cadherin+ cells also contained oxidised LDL in their cytoplasm. Differing numbers of CD68+ foam cells (15% to 60%) expressed E-cadherin but all the CD68+ macrophages without signs of transformation into foam cells were negative for E-cadherin. Neither smooth muscle cells nor foam cells of smooth muscle cell origin (smooth muscle alpha-actin+) were found to be positive for E-cadherin. T-cells (CD3+) and endothelial cells (von Willebrand factor+) were also negative for E-cadherin. Only a few vascular dendritic cells (S-100+) expressed E-cadherin and their expression was weak. We also found that a large proportion (40% to 85%) of E-cadherin+ cells did not stain with any cell-type specific markers. CONCLUSIONS: The finding that E-cadherin is expressed in atherosclerotic lesions expands our knowledge of cell adhesion molecules involved in atherogenesis. That E-cadherin is expressed in intimal cells transforming into foam cells suggests that lipid accumulation might be associated with the alteration and reorganisation of cell-to-cell interactions in atherogenesis. The present observations might assist in understanding the mechanisms associated with intracellular lipid accumulation.     

C-reactive protein frequently colocalizes with the terminal complement complex in the intima of early atherosclerotic lesions of human coronary arteries

There is increasing evidence that complement activation may play a role in atherogenesis. Complement proteins have been demonstrated to be present in early atherosclerotic lesions of animals and humans, and cholesterol-induced atherosclerotic lesion formation is reduced in complement-deficient animals. Potential complement activators in atherosclerotic lesions are now a subject matter of debate. C-reactive protein (CRP) is an acute-phase protein that is involved in inflammatory processes in numerous ways. It binds to lipoproteins and activates the complement system via the classic pathway. In this study we have investigated early atherosclerotic lesions of human coronary arteries by means of immunohistochemical staining. We demonstrate here that CRP deposits in the arterial wall in early atherosclerotic lesions with 2 predominant manifestations. First, there is a diffuse rather than a focal deposition in the deep fibroelastic layer and in the fibromuscular layer of the intima adjacent to the media. In this location, CRP frequently colocalizes with the terminal complement complex. Second, the majority of foam cells below the endothelium show positive staining for CRP. In this location, no colocalization with the terminal complement proteins can be observed. Our data suggest that CRP may promote atherosclerotic lesion formation by activating the complement system and being involved in foam cell formation.     

HLA-DM is expressed on the cell surface and colocalizes with HLA-DR and invariant chain in human Langerhans cells

The subcellular distribution patterns of molecules involved in the process of antigen loading [HLA-DR, HLA-DM, and the cytoplasmic and luminal parts of the invariant chain (Ii, CD74)] were investigated in Langerhans cells (LC), both qualitatively and quantitatively. The analysis was performed by immunofluorescence labelling of acetone-fixed vertical cryostat sections from normal human skin specimens and subsequent examination using confocal laser scanning microscopy (CSLM). The intensity-modulated multiple-wavelength scanning (IMS) technique was used to enhance channel separation when scanning dual-labelled specimens. The mean (n = 9) relative epidermal volumes of reactivity were: HLA-DR 8%+/-3%, HLA-DM 6%+/-2%, luminal Ii 6%+/-2%, and cytoplasmic Ii 4%+/-1%. The difference between HLA-DR and the other epitopes was significant at the P<0.001 level. All molecule combinations, except the combination of HLA-DM and luminal Ii (which was not studied), were to various extents colocalized. Experiments performed on unfixed epidermal sheets showed that HLA-DM is present on the cell surface of LC, suggesting that HLA-DM may interact with HLA-DR on the surface to induce peptide loading.     

Expression of ACAT-1 protein in human atherosclerotic lesions and cultured human monocytes-macrophages

The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions. Our results thus support the idea that ACAT-1 plays an important role in differentiating monocytes and in forming macrophage foam cells during the development of human atherosclerosis.     

Plasmodium yoelii YM MAEBL protein is coexpressed and colocalizes with rhoptry proteins

Several human diseases, in particular metabolic disorders, often lead to the accumulation of characteristic metabolites in plasma, urine and cells. The selected diseases of this type include cystinuria and homocystinuria. In the typical laboratory diagnosis of these two diseases, a positive nitroprusside test is followed by quantitative analysis of urine cysteine and homocysteine in order to differentiate between cystinuria and homocystinuria. A sensitive and reproducible assay for total urine cysteine and homocysteine has been developed. The essential steps in the assay include conversion of disulphides to free thiols with tributylphosphine, conjugation of the thiols with 2-chloro-1-methyl pyridinium iodide, separation of S-pyridinium derivatives of cysteine and homocysteine from other endogenous urine thiol derivatives by reversed-phase high-performance liquid chromatography, and detection and quantitation by spectrophotometry. The method has a sensitivity of 4 pmol and is reproducible, intra- and inter-day coefficients of variation are from 1.37 to 4.14% and from 2.38 to 5.01%, respectively. The mean concentration of total urine cysteine and homocysteine in healthy donors (7 men and 7 women) were for women. 92.0 +/- 45.8 and 16.4 +/- 4.8 respectively, and for men 120.9 +/- 46.6 and 21.5 +/- 7.4 nmol/ml, respectively. Total urine homocysteine represents approximately 17.7% of cysteine in the urine of normal individuals.     

A new low density lipoprotein receptor related protein, LRP5, is expressed in hepatocytes and adrenal cortex, and recognizes apolipoprotein E

The isolation and characterization of rabbit and human cDNAs revealed a new low density lipoprotein receptor (LDLR)-related protein (LRP) designated as LRP5. Human LRP5 cDNA encodes a 1, 616-amino acid type I membrane-like protein with three ligand binding repeats in its extracellular region. LDLR-deficient cells transduced by recombinant adenovirus containing human LRP5 exhibited increased binding of apolipoprotein E (apoE)-enriched beta-migrating very low density lipoprotein. Northern blotting and in situ hybridization revealed a high level of LRP5 expression in hepatocytes and the adrenal gland cortex. In LDLR-deficient Watanabe heritable hyperlipidemic rabbits, LRP5 mRNA was increased in the liver and accumulated in cholesterol-laden foam cells of atherosclerotic lesions.     

Low density lipoprotein receptor-negative mice expressing human apolipoprotein B-100 develop complex atherosclerotic lesions on a chow diet: no accentuation by apolipoprotein(a)

We have generated mice with markedly elevated plasma levels of human low density lipoprotein (LDL) and reduced plasma levels of high density lipoprotein. These mice have no functional LDL receptors [LDLR-/-] and express a human apolipoprotein B-100 (apoB) transgene [Tg(apoB+/+)] with or without an apo(a) transgene [Tg(apoa+/-)]. Twenty animals (10 males and 10 females) of each of the following four genotypes were maintained on a chow diet: (i) LDLR-/-, (ii) LDLR-/-;Tg(apoa+/-), (iii) LDLR-/-;Tg(apoB+/+), and (iv)LDLR-/-;Tg(apoB+/+);Tg(apo+/-). The mice were killed at 6 mo, and the percent area of the aortic intimal surface that stained positive for neutral lipid was quantified. Mean percent areas of lipid staining were not significantly different between the LDLR-/- and LDLR-/-;Tg(apoa+/-) mice (1.0 +/- 0.2% vs. 1.4 +/- 0.3%). However, the LDLR-/-;Tg(apoB+/+) mice had approximately 15-fold greater mean lesion area than the LDLR-/- mice. No significant difference was found in percent lesion area in the LDLR-/-;Tg(apoB+/+) mice whether or not they expressed apo(a) [18.5 +/- 2.5%, without lipoprotein(a), Lp(a), vs. 16.0 +/- 1.7%, with Lp(a)]. Histochemical analyses of the sections from the proximal aorta of LDLR-/-;Tg(apoB+/+) mice revealed large, complex, lipid-laden atherosclerotic lesions that stained intensely with human apoB-100 antibodies. In mice expressing Lp(a), large amounts of apo(a) protein colocalized with apoB-100 in the lesions. We conclude that LDLR-/-; Tg(apoB+/+) mice exhibit accelerated atherosclerosis on a chow diet and thus provide an excellent animal model in which to study atherosclerosis. We found no evidence that apo(a) increased atherosclerosis in this animal model.     

Colocalization of iron and ceroid in human atherosclerotic lesions

Although clearly a valid alternative to dismembered pyeloplasty for ureteropelvic junction obstruction in adults, endo(uretero)pyelotomy has not been universally accepted in the general urologic community because of the 10% to 30% inferior success rate and the concern regarding hemorrhagic complications. Identification of prognostic factors for success and complications are therefore of primary importance. The length of stricture, the grade of hydronephrosis, and the level of renal function are well recognized prognostic factors. Crossing vessels also play a role, and in association with the grade of hydronephrosis, they seem to be a major prognosticator of outcome. With attention to such prognostic factors, and appropriate patient selection, results can now approach the gold standard of dismembered pyeloplasty.     

Association of apolipoprotein E with alpha2-macroglobulin in human plasma

Apolipoprotein (apo) E plays a central role in the transport of lipids among different organs and cell types, whereas alpha2-macroglobulin (alpha2M) is responsible for the binding and inactivation of plasma proteases, as well as the transport of various cytokines, growth factors, and hormones. In the present study, evidence is presented for direct binding of apoE with alpha2M in human plasma, based on the observation that two-dimensional non-denaturing gradient gel electrophoretic separation of plasma resulted in co-migration of apoE with alpha2M in a complex intermediate in size (18.5 nm in diameter) between low (LDL) and high density lipoproteins (HDL). ApoE associated with alpha2M could be immunoprecipitated from plasma with anti-human alpha2M antiserum. Purified apoE, labeled with 125I, bound to native and methylamine-activated alpha2M (alpha2M-MA) in vitro in a time- and concentration-dependent manner. ApoE bound to alpha2M-MA with greater affinity than alpha2M. The binding of apoE to both alpha2M and alpha2M-MA did not depend on the presence of lipid. Ingestion of an oral fat load resulted in a reduction in the amount of apoE associated with alpha2M. Sphingomyelin vesicles and very low density lipoproteins (VLDL), but not phosphatidylcholine vesicles or HDL3, inhibited the in vitro binding of 125I-labeled apoE3 to alpha2M and alpha2M-MA. Binding of 125I-labeled apoE3 was also partially inhibited by an excess of platelet-derived growth factor and beta-amyloid protein, but not interferon-gamma. Subjects with an apoE 4/4 phenotype had less apoE associated with alpha2M in plasma than subjects with an apoE 3/3 or 2/2 phenotype, corresponding to reduced in vitro binding of apoE4 with alpha2M or alpha2M-MA. Although the functional significance of apoE binding to alpha2M remains to be determined, the present results demonstrate that: 1) apoE is non-covalently bound to alpha2M in human plasma, 2) alpha2M-MA has a greater capacity to bind apoE than alpha2M, 3) various proteins or lipoproteins known to bind apoE or alpha2M can potentially affect the interaction of apoE with alpha2M, and 4) association of apoE with alpha2M or alpha2M-MA is dependent on apoE phenotype.     

Collagen-synthesizing cells in initial and advanced atherosclerotic lesions of human aorta

Accumulation of extracellular matrix, fibrosis, is regarded to be one of the major manifestations of atherosclerosis. Collagen type I is the predominant matrix component in human atherosclerotic plaques. In this work we have demonstrated procollagen type I expressing cells (PCI-cells) and studied their localization in grossly normal human aorta and atherosclerotic lesions: initial lesions, fatty streaks, fibrolipid lesions (fibrolipid plaque, fibroatheroma), fibrotic lesions (fibrous plaque). PCI-cells were revealed immunocytochemically using SPI.D8 monoclonal antibody against human procollagen type I. We failed to detect PCI-cells in the areas of grossly normal aorta and media underlying atherosclerotic lesions. Positively stained cells were shown in the areas of initial lesions, fatty streaks, fibrolipid and fibrous plaques. The largest amount of PCI-cells was revealed in fatty streaks. These cells were predominantly localized in the preluminal proteoglycan-rich intimal sublayer. Intimal cells in grossly normal regions formed a common cellular network contacting each other with their processes. The cellular network is found to be partly disintegrated in atherosclerotic lesions, which leads to the appearance of isolated cells. The share of isolated PCI-cells localized outside the intimal cellular network was higher in advanced lesions than in the areas of early atherosclerotic lesions. In initial lesions most of PCI-cells were identified as smooth muscle cells using antibodies to smooth muscle alpha-actin. In fatty streaks PCI-expressing smooth muscle cells were fewer in number. Much fewer cells double-stained with anti-alpha-actin and anti-PCI antibodies were found in fibrolipid and fibrous plaques. The proportion of these double stained cells was higher among total number of PCI-cells involved in the cellular network versus PCI-cells outside the network. The results of the study demonstrated that the most active de novo synthesis of interstitial collagen takes place in the regions of atherosclerotic lesions characterized by lipid deposition, which may lead to the further progression of atherosclerotic lesions.     

The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis

Photoreceptor cells of the Drosophila compound eye begin to develop specialized membrane foldings at the apical surface in midpupation. The microvillar structure ultimately forms the rhabdomere, an actin-rich light-gathering organelle with a characteristic shape and morphology. In a P-element transposition screen, we isolated mutations in a gene, bifocal (bif), which is required for the development of normal rhabdomeres. The morphological defects seen in bif mutant animals, in which the distinct contact domains established by the newly formed rhabdomeres are abnormal, first become apparent during midpupal development. The later defects seen in the mutant adult R cells are more dramatic, with the rhabdomeres enlarged, elongated, and frequently split. bif encodes a novel putative protein of 1063 amino acids which is expressed in the embryo and the larval eye imaginal disc in a pattern identical to that of F actin. During pupal development, Bif localizes to the base of the filamentous actin associated with the forming rhabdomeres along one side of the differentiating R cells. On the basis of its subcellular localization and loss-of-function phenotype, we discuss possible roles of Bif in photoreceptor morphogenesis.     

Characterization of apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis

To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was also measured in muscle biopsy samples obtained from seven additional subjects before and after an identical protocol. Rates of insulin stimulated whole-body glucose uptake. Glucose oxidation and muscle glycogen synthesis were 50%-60% lower following the lipid infusion compared with the glycerol infusion and were associated with a approximately 90% decrease in the increment in intramuscular glucose-6-phosphate concentration, implying diminished glucose transport or phosphorylation activity. To distinguish between these two possibilities, intracellular glucose concentration was measured and found to be significantly lower in the lipid infusion studies, implying that glucose transport is the rate-controlling step. Insulin stimulation, during the glycerol infusion, resulted in a fourfold increase in PI 3-kinase activity over basal that was abolished during the lipid infusion. Taken together, these data suggest that increased concentrations of plasma FFA induce insulin resistance in humans through inhibition of glucose transport activity; this may be a consequence of decreased IRS-1-associated PI 3-kinase activity.     

Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein

Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease. All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease. The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors. A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal.     

Observations on bone formation and remodelling in advanced atherosclerotic lesions of human carotid arteries

Immunolocalisation and histochemical techniques were used to examine mineralised bone deposits within late stage atherosclerotic plaques of human carotid arteries. These specimens showed characteristic features of osteogenesis. Large calcifications were often observed in close association with or integrated within mineralised bone. Smooth muscle cells (alpha-actin positive) were often located around osteoid-like matrix, together with focal accumulations of macrophages (CD68 and HAM56 positive). Local accumulations of mast cells (tryptase positive) were consistently observed in close association with the bone. Multinucleated giant cells in close apposition with mineralised bone demonstrated typical osteoclastic morphology, and were positively stained for acid phosphatase and the macrophage marker CD68. Thus, all the normal features of bone formation and resorption were observed in this microcosm of osteogenesis within atherosclerotic plaque; the term 'osteosome' seems appropriate for the structure. These osteosomes have numerous advantages for experimental studies of the various osteogenic factors responsible for bone metabolism, especially following short-term tissue culture. This ex vivo technique was used to demonstrate the distribution and the multiple cellular sources of bone morphogenetic protein 6.     

Monocyte/macrophage recruitment and expression of endothelial adhesion proteins in human atherosclerotic lesions

Since mononuclear cells are recruited in atherosclerotic lesions, the expression of adhesion proteins by the arterial endothelium may play a major role in atherogenesis. The relationships between ICAM-1, E-selectin, and VCAM-1 expression on the arterial endothelium and the presence and degree of maturation of intimal macrophages in human atherosclerotic lesions was investigated. By quantitative double immunostaining with a pan-macrophage-specific monoclonal antibody, HAM-56, and a recently developed monoclonal antibody that is specific for mature macrophages, 3MA-B38, arterial sections were classified as (I) normal, (II) thickened without macrophage infiltration, (III) atherosclerotic with recent macrophage infiltration or (IV) atherosclerotic with infiltration of mature differentiated macrophages. A marked increase in the expression of ICAM-1, E-selectin, and VCAM-1 was observed on endothelial cells adjacent to recently recruited macrophages. Endothelial cells overlying differentiated macrophages exhibited a lower but significant increase in VCAM-1 expression, with no difference in ICAM-1 and E-selectin expression with respect to that observed in endothelium of normal arteries. These findings indicate that the endothelium covering the human arterial wall exhibits different states of activation as reflected by the expression of adhesion proteins, and that intimal monocyte/macrophage recruitment appears to depend on the level of expression of adhesion proteins.     

Survival motor neuron (SMN) protein in rat is expressed as different molecular forms and is developmentally regulated

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by a progressive degeneration of motoneurons in spinal cord and brainstem. The telomeric copy of a duplicated gene termed survival motor neuron (smn), which maps to chromosome 5q13, has been found to be deleted in most patients. The encoded gene product is a novel protein which recently has been shown to accumulate in specific nuclear organelles (gemini of coiled bodies, GEMS), and to play a part in the formation of the spliceosome complex. We have cloned and sequenced the rat smn cDNA. Antibodies generated against an N-terminus peptide recognized a main protein of 32 kDa in immunoblots of rat embryonic tissue extracts. Minor bands of 35 kDa, 45 kDa and, in perinatal muscle, of 24 kDa were also specifically detected, indicating that SMN is expressed as different molecular forms. Subcellular fractionation indicated that the 32 kDa form is mainly soluble, while the 35 kDa and 45 kDa products segregate to the microsomal-mitochondrial fraction. SMN protein is highly regulated during development: expression is high in embryonic tissues (central nervous system, muscle, lung and liver), and then progressively decreases to very low levels in most tissues of the adult. The demonstration of different molecular forms of SMN along with its developmental regulation may help to understand the contribution of this protein in the appearance of SMA phenotype.     

Co-expression of beta-amyloid precursor protein (betaAPP) and apolipoprotein E in cell culture: analysis of betaAPP processing

Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Abeta), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Abeta production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Abeta, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of betaAPP and the secretion of Abeta in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Abeta at concentrations like those in human cerebrospinal fluid. betaAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on betaAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of betaAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Abeta aggregation or clearance under physiologically relevant conditions.