Oxidized low-density lipoprotein increases cultured human endothelial cell tissue factor activity and reduces protein C activation

Increasing evidence suggests that the formation of oxidized low-density lipoprotein (Ox-LDL) in vivo is associated with the development of atherosclerotic vascular disease. We investigated the effects of Ox-LDL on two vascular endothelial cell coagulant properties, tissue factor expression, and protein C activation. The Ox-LDL increased human arterial and venous endothelial cell tissue factor activity, with 100 micrograms/ml of Ox-LDL increasing factor activity fourfold. Native LDL modified by incubation with cultured human arterial and venous endothelial cells also induced endothelial cell tissue factor activity. This modification was blocked by coincubation with the antioxidants, probucol or ascorbic acid. It was determined, based on inhibition by known scavenger receptor antagonists (fucoidin, dextran sulfate), that binding of Ox-LDL via the acetyl LDL (scavenger) receptor was partially responsible for the increase in tissue factor expression. Whereas endothelial cell tissue factor expression was increased by incubation with Ox-LDL, protein C activation was reduced approximately 80% by incubating cultured endothelial cells with Ox-LDL. The effect of Ox-LDL on protein C activation was not inhibited by antagonists to the scavenger receptor. These data indicating that an atherogenic lipoprotein can regulate key vascular coagulant activities provide an additional link between vascular disease and thrombosis.     

Expression of host genes during root nodule development in soybeans

Summary Nine unique nodulin cDNA clones from soybean have been characterized with regard to the size of the RNA and the corresponding protein products. Based on the sequence homology between clones C51 and E27 and the multiple RNA species corresponding to clones D41 and E41, it is suggested that some of the nodulin genes represent members of small gene families. The amino acid sequence deduced from the nucleotide sequence of clones C51 and E27 revealed the presence of a signal peptide and no stop transfer signal, typical of membrane proteins, suggesting that the proteins encoded by these clones are localized in organelles and as such probably involved in ureide biosynthesis (Boland et al. 1982; Schubert and Boland 1984). Based on the timing of appearance of RNA corresponding to the nodulin clones and the pattern of their accumulation, at least three sets of nodulin genes are being represented here. Al1 the nodulin RNAs examined were made in Fix^- nodules formed by strain Ag168 (which does not make Cl component of nitrogenase) at a level comparable to that in Fix⁺ nodules and at a very reduced level in Fix^- nodules formed by strain HS124 (which show very few infected cells). It is concluded that all the nodulin genes examined here are induced independent of nitrogenase activity.     

Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans

Summary A cDNA clone (pcPvNGS-01) to glutamine synthetase (GS) mRNA from root nodules of Phaseolus vulgaris showed cross-hybridization to GS and mRNA from soybean root nodules, thus allowing its use as a probe to study the expression of GS genes during root nodule development in soybeans. Hybrid-select translation of root and nodule RNA of soybean with DNA from pcPvNGS-01, followed by 2D gel electrophoresis, showed six peptides in the root and an additional four peptides in the nodule which represent nodule-specific glutamine synthetase (GS_n) gene products. The GS_n gene products appeared for the first time between day 11 and 12 after infection, either concomitant with the onset of nitrogenase activity or immediately following it. The levels of expression of the GS_n and leghemoglobin genes were not affected in young Fix^- nodules formed by Bradyrhizobium japonicum strains that are defective in nitrogenase activity, suggesting that the induction of these two sets of host genes take place independent of nitrogenase activity. However, in Fix^- nodules that are incapable of maintaining the peribacteroid membrane, GS_n gene products were not detected while 1ba, 1bc₂ and 1bc₃ appeared. In both the timing of appearance during root nodule development and the effect of different bacterial mutations on the expression, GS_n genes differ from most other nodulin genes examined (30), suggesting different regulatory mechanisms.     

The effects of second-hand smoke on biological processes important in atherogenesis

Background

Atherosclerosis is the leading cause of death in western societies and cigarette smoke is among the factors that strongly contribute to the development of this disease. The early events in atherogenesis are stimulated on the one hand by cytokines that chemoattract leukocytes and on the other hand by decrease in circulating molecules that protect endothelial cells (ECs) from injury. Here we focus our studies on the effects of "second-hand" smoke on atherogenesis.

Methods

To perform these studies, a smoking system that closely simulates exposure of humans to second-hand smoke was developed and a mouse model system transgenic for human apoB¹⁰⁰ was used. These mice have moderate lipid levels that closely mimic human conditions that lead to atherosclerotic plaque formation.

Results

"Second-hand" cigarette smoke decreases plasma high density lipoprotein levels in the blood and also decreases the ratios between high density lipoprotein and low density lipoprotein, high density lipoprotein and triglyceride, and high density lipoprotein and total cholesterol. This change in lipid profiles causes not only more lipid accumulation in the aorta but also lipid deposition in many of the smaller vessels of the heart and in hepatocytes. In addition, mice exposed to smoke have increased levels of Monocyte Chemoattractant Protein–1 in circulation and in the heart/aorta tissue, have increased macrophages in the arterial walls, and have decreased levels of adiponectin, an EC-protective protein. Also, cytokine arrays revealed that mice exposed to smoke do not undergo the switch from the pro-inflammatory cytokine profile (that develops when the mice are initially exposed to second-hand smoke) to the adaptive response. Furthermore, triglyceride levels increase significantly in the liver of smoke-exposed mice.

Conclusion

Long-term exposure to "second-hand" smoke creates a state of permanent inflammation and an imbalance in the lipid profile that leads to lipid accumulation in the liver and in the blood vessels of the heart and aorta. The former potentially can lead to non-alcoholic fatty liver disease and the latter to heart attacks.     

Automated analysis of FISH and immunohistochemistry images: a review.

Fluorescent in-situ hybridization (FISH) and immunohistochemistry (IHC) constitute a pair of complimentary techniques for detecting gene amplification and overexpression, respectively. The advantages of IHC include relatively cheap materials and high sample durability, while FISH is the more accurate and reproducible method. Evaluation of FISH and IHC images is still largely performed manually, with automated or semiautomated techniques increasing in popularity. Here, we provide a comprehensive review of a number of (semi-) automated FISH and IHC image processing systems, focusing on the algorithmic aspects of each technique. Our review verifies the increasingly important role of such methods in FISH and IHC; however, manual intervention is still necessary in order to resolve particularly challenging or ambiguous cases. In addition, large-scale validation is required in order for these systems to enter standard clinical practice.     

Food for thought: essential fatty acid protects against neuronal deficits in transgenic mouse model of AD

Interactions between environmental and genetic factors may contribute to neurodegenerative disease. In this issue of Neuron, Calon et al. report that a diet low in an essential omega-3 polyunsaturated fatty acid (docosahexaenoic acid) depletes postsynaptic proteins and exacerbates behavioral alterations in a transgenic mouse model of Alzheimer's disease.     

The class A scavenger receptor binds to proteoglycans and mediates adhesion of macrophages to the extracellular matrix

The class A scavenger receptor (SR-A) binds modified lipoproteins and has been implicated in cholesterol ester deposition in macrophages. The SR-A also contributes to cellular adhesion. Using SR-A(+/+) and SR-A(-)/- murine macrophages, we found SR-A expression important for both divalent cation-dependent and -independent adhesion of macrophages to the human smooth muscle cell extracellular matrix. The SR-A mediated 65 and 85% of macrophage adhesion to the extracellular matrix in the presence and absence of serum, respectively. When EDTA was added to chelate divalent cations, the SR-A mediated 90 and 95% of the macrophage adhesion without and with serum, respectively. SR-A-mediated adhesion to the extracellular matrix was prevented by fucoidin, an SR-A antagonist. Biglycan and decorin, proteoglycans of the extracellular matrix, were identified as SR-A ligands. Compared with control cells, Chinese hamster ovary cells expressing the SR-A showed 5- and 6-fold greater cell association (binding and internalization) of (125)I-decorin and -biglycan, respectively. In competition studies, unlabeled proteoglycan or fucoidin competed for binding of (125)I-labeled decorin and -biglycan, and biglycan and decorin competed for the SR-A-mediated cell association and degradation of (125)I-labeled acetylated LDL, a well characterized ligand for the SR-A. These results suggest that the SR-A could contribute to the adhesion of macrophages to the extracellular matrix of atherosclerotic plaques.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.     

Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain

This study was undertaken to determine if apolipoprotein (apo) E-containing lipoproteins and their receptors could provide a system for lipid transport and cholesterol homeostasis in the brain, as they do in other tissues. To accomplish this goal, the lipoproteins in human and canine cerebrospinal fluid (CSF) were characterized, and rat brain and monkey brain were examined for the presence of apoB,E(LDL) receptors. Apolipoprotein E and apoA-I were present in human and canine CSF, but apoB could not be detected. Apo-lipoprotein E and apoA-I were both present on lipoproteins with a density of approximately 1.09 to 1.15 g/ml. In human CSF, the lipoproteins were primarily spherical (approximately 140 A), whereas in canine CSF the lipoproteins were a mixture of discs (200 x 65 A) and spheres (approximately 130 A). Apolipoproteins E and A-I were contained primarily in separate populations of lipoproteins. Although the apoE of CSF was more highly sialylated than plasma apoE, the apoE-containing lipoproteins in canine CSF competed as effectively as canine plasma apoE HDLc for binding of 125I-LDL to the apoB,E(LDL) receptors on human fibroblasts. The presence of apoB,E(LDL) receptors in both rat and monkey brain was demonstrated by immunocytochemistry. Astrocytes abutting on the arachnoid space and pial cells of the arachnoid itself, both of which contact CSF, expressed apoB,E(LDL) receptors. Relatively few receptors were present in the cells of the gray matter of the cortex. Receptors were more prominent on the astrocytes of white matter and in the cells of the brain stem. The expression of apoB,E(LDL) receptors by brain cells and the presence of apoE- and apoA-I-containing lipoproteins in CSF suggest that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues.