The contribution of the macrophage receptor for oxidized LDL to its cellular uptake

Oxidized LDL (Ox-LDL) was shown to be taken up by macrophages via several receptors including the acetyl-LDL(Ac-LDL), the LDL, and the Ox-LDL receptors. Cellular uptake and degradation of Ox-LDL could be dissociated from that of LDL and Ac-LDL as demonstrated by using macrophages that lack the LDL or the Ac-LDL receptors. In J-774 A.1 macrophage-like cell line unlabeled Ox-LDL reduced the 125I-Ox-LDL by up to degradation of 91% whereas unlabeled Ac-LDL and native LDL reduced 125I-Ox-LDL degradation by only 51% and 23%, respectively. Analysis of macrophage degradation of 125I-Ox-LDL in the presence of 30-fold excess concentration of LDL + Ac-LDL (to block uptake of 125I-Ox-LDL via the LDL and the Ac-LDL receptors) revealed that cellular degradation via the Ox-LDL receptor could account for 45% of the macrophage uptake of Ox-LDL.     

MicroRNA-217 ameliorates inflammatory damage of endothelial cells induced by oxidized LDL by targeting EGR1

Molecular and Cellular Biochemistry - Dexmedetomidine (DEX), a highly selective alpha2 adrenergic receptor agonist, is a commonly used anesthetic drug in surgical procedures. Previous...     

A hyperpolarization- and acid-activated nonselective cation current in Xenopus oocytes

Heterologous expression of avariety of membrane proteins in Xenopus oocytes sometimesresults in the appearance of a hyperpolarization-activated inwardcurrent. The nature of this current remains incompletely understood.Some investigators have suggested that this current is a Cl current,whereas others have identified it as a nonselective cation current. Thepurpose of this investigation was to characterize this current in moredetail. The hyperpolarization-activated inward current(I_IN) present in native oocytes wascomposed of a current carried at least partly by Ca and Mg underphysiological ionic conditions plus a Ca-activated Cl current. TheCa-activated Cl current was blocked by chelation of cytosolic Ca with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.When Cl currents were blocked, the cation current could be carried byCa, Mg, or Co, but not appreciably by Ba, Mn, or Cd.I_IN was stimulated by intracellularacidification. The properties of I_IN were quitedifferent from those of the store-operated Ca current. Heterologousexpression of transient receptor potential-like gene product (TRPL),one of the members of the transient receptor potential family ofputative store-operated Ca channels, apparently resulted in alterationof the voltage sensitivity of the endogenous I_IN.

     

Myeloperoxidase-mediated LDL oxidation and endothelial cell toxicity of oxidized LDL: attenuation by (-)-epicatechin

Recent data suggest an inverse epidemiological association between intake of flavanol-rich cocoa products and cardiac mortality. Potential beneficial effect of cocoa may be attributed to flavanol-mediated improvement of endothelial function, as well as to enhancement of bioavailability and bioactivity of nitric oxide in vivo. ( - )-Epicatechin is one bioactive flavanol found in cocoa. This review deals with protective actions of ( - )-epicatechin on two key processes in atherogenesis, oxidation of LDL and damage to endothelial cell by oxidized LDL (oxLDL), with emphasis on data from this laboratory. ( - )-Epicatechin not only abrogates or attenuates LDL oxidation but also counteracts deleterious actions of oxLDL on vascular endothelial cells. These protective actions are only partially shared by other vasoprotective agents such as vitamins C and E or aspirin. Thus, ( - )-epicatechin appears to be a pleiotropic protectant for both LDL and endothelial cells.     

Detrimental effect of oxidized LDL on endothelial arginine metabolism and transportation

The action of oxidatively modified low-density lipoprotein on vascular endothelial cells has been proposed to be a crucial process leading to endothelial dysfunction and atherogenesis. However, the biochemical mechanism for such action is not clear. We have previously shown that arginine uptake and metabolism are major determinants of endothelial function in heart failure and hypertension. In the present study we therefore aimed to assess the effects of oxidized LDL, a major pro-atherogenic molecule, on endothelial l-arginine metabolism and its uptake. Endothelial cells were exposed to oxidized LDL or native LDL for 24h, and the resultant effects on (1) the intracellular content of arginine and its major metabolites including citrulline, N(G)-hydroxy-l-arginine, asymmetric dimethylarginine, symmetric dimethylarginine and ornithine, (2) [3H]-l-arginine uptake and, (3) the pattern of distribution of cationic amino acid transporter 1, the principal l-arginine transporter, by confocal microscopy. Oxidized LDL (100 microg/mL) reduced intracellular arginine and N(G)-hydroxy-l-arginine contents by 56 and 71% (P<0.05), respectively, with a concomitant 205% increase in ADMA (P<0.05). In conjunction, oxidized LDL reduced endothelial uptake of [3H]-arginine by 60%. Furthermore, incubation of endothelial cells with oxLDL led to internalization of cationic amino acid transporter 1. We demonstrate a novel mechanism, reduced l-arginine transport, by which oxidized LDL impairs the ability of the endothelium to generate nitric oxide.     

Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL

Proteoglycans and plasma proteins bound to the endothelial cell glycocalyx are essential for vascular function, but at the same time, they lower capillary tube hematocrit by reducing capillary volume available to flowing blood. Because oxidized low-density lipoproteins (oxLDL) reduce the effective thickness of the glycocalyx (Vink H, Constantinescu AA, and Spaan JAE. Circulation 101: 1500-1502, 2000), we designed the present study to determine whether this is caused by pathological degradation of glycocalyx constituents or increased glycocalyx deformation by elevated shear forces of flowing blood. Capillaries from the right cremaster muscle of 24 hamsters were examined by using intravital microscopy after systemic administration of normal LDL (n = 4), moderate oxLDL (6-h oxidation with CuSO(4), n = 7), severe oxLDL (18-h oxidation, n = 5), and moderate oxLDL plus superoxide dismutase (SOD) and catalase (n = 8). Capillary tube hematocrit increased from 0.16 +/- 0.03 to 0.37 +/- 0.05 and from 0.15 +/- 0.01 to 0.31 +/- 0.03 after moderate oxLDL and severe oxLDL, respectively. These changes were paralleled by increases in red blood cell flux from 8.7 +/- 1.9 to 13.8 +/- 3 and from 10.7 +/- 2.1 to 16.3 +/- 3.2 cells/s after moderate oxLDL and severe oxLDL, respectively, in the absence of changes in anatomic capillary diameter. Red blood cell velocity, as a measure for the shear forces on the glycocalyx, was not affected by oxLDL, whereas tissue pretreatment with SOD and catalase completely abolished the effects of oxLDL on glycocalyx thickness, capillary hematocrit, and red blood cell flux. We conclude that elevation of capillary tube hematocrit by oxLDL reflects degradation of the endothelial glycocalyx by oxygen-derived free radicals.     

Estrogen decreases TNF-alpha and oxidized LDL induced apoptosis in endothelial cells

Apoptosis induced by oxidized low-density lipoproteins (oxLDL) and tumor necrosis factor-alpha (TNF-alpha) is believed to contribute to atherosclerosis and vascular dysfunction. Estrogen treatment reduces apoptosis due to TNF-alpha and we hypothesized that it would also reduce apoptosis due to oxLDL. We also explored the anti-apoptotic mechanisms. We used early passage human umbilical vein endothelial cells (HUVEC) grown in steroid-depleted, red phenol-free medium. Cells were synchronized by starvation for 6h and then treated with oxLDL (75microg/ml) or TNF-alpha (20ng/ml) in the presence of 17-beta-estradiol (E2) (20nM). Apoptosis was analyzed by flow cytometry and caspase-3 cleavage. We also assessed expression of Bcl-2 and Bcl-xL and phosphorylation of BAD. At 6h TNF-alpha induced apoptosis but oxLDL did not; E2 did not affect this TNF-alpha induced apoptosis and there was no change in Bcl-2 or Bcl-xL expression. At 24h both TNF-alpha and oxLDL increased apoptosis and E2 reduced the increase. E2 also increased expression of the anti-apoptotic Bcl-2 and Bcl-xL and increased phosphorylation of proapoptotic BAD which reduces its proapoptotic activity at 1h. However at 24h there was also an increase in total BAD so that the proportion of phosphorylation of BAD decreased. oxLDL induced apoptosis occurs later than that of TNF-alpha. E2 decreased this late phase apoptosis and this likely requires the production of anti-apoptotic proteins.     

Metabolism of oxidized LDL by macrophages

Oxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo.     

Epicatechin protects endothelial cells against oxidized LDL and maintains NO synthase

Intake of flavanol-rich food or beverages was previously shown to ameliorate endothelial function and to enhance bioactivity of nitric oxide with individuals at risk for cardiovascular disease. Here, we examined whether the major dietary flavanol, (-)-epicatechin, counteracts the action of oxidized LDL on endothelial cells, an action considered pivotal for endothelial dysfunction in the pathogenesis of atherosclerosis. Oxidation by myeloperoxidase plus nitrite rendered human LDL cytotoxic towards endothelial cells, more so than oxidation by Cu2+. Oxidized LDL also caused a marked loss of endothelial NO synthase protein which did not occur in the presence of a proteasome inhibitor, lactacystin. Both actions of oxidized LDL, which were not evoked by native LDL, were effectively counteracted by (-)-epicatechin. We conclude that dietary flavanols contribute to protection of the integrity of endothelial cells not only by scavenging free radicals but also by maintaining endothelial NO synthase.     

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.     

Regulation of cell growth by oxidized LDL

The first reports of the influences of oxidized LDL (oxLDL) on cell function pertained to negative effects on cell growth—growth arrest, injury, and toxicity. Since these studies, it has become apparent that sublethal levels of oxLDL cause some, but not all, cells to proliferate. This review highlights the growth-promoting effects of oxLDL rather than its inhibitory or injurious effects. Smooth muscle cells (SMCs) and monocyte-macrophages proliferate after exposure to oxLDL; endothelial cells do not. Scavenger receptors are involved in the proliferative effects on monocyte-macrophages, whereas the effects of oxLDL on SMCs appear to be receptor independent. Lysophosphatidylcholine (lysoPC), and structurally related lipids are among the growth-promoting constituents of oxLDL. OxLDL exerts at least a part of its effects by inducing expression or causing the release of growth factors. OxLDL (or lysoPC) can cause the release of basic fibroblast growth factor (bFGF) from SMCs; oxLDL (or lysoPC) can induce heparin binding EGF-like growth factor (HB-EGF) synthesis and release from macrophages. An imposing array of changes in cytokine and growth factor expression and/or release can be imposed by oxLDL on a wide variety of cell types. These effects and the studies probing the cell signaling events leading to them are described.     

Inability of HDL from abdominally obese subjects to counteract the inhibitory effect of oxidized LDL on vasorelaxation

Abdominal obesity is associated with a decreased plasma concentration of HDL cholesterol and with qualitative modifications of HDL, such as triglyceride enrichment. Our aim was to determine, in isolated aorta rings, whether HDL from obese subjects can counteract the inhibitory effect of oxidized low density lipoprotein (OxLDL) on endothelium-dependent vasodilation as efficiently as HDL from normolipidemic, lean subjects. Plasma triglycerides were 74% higher (P < 0.005) in obese subjects compared with controls, and apolipoprotein A-I (apoA-I) and HDL cholesterol concentrations were 12% and 17% lower (P < 0.05), respectively. HDL from control subjects significantly reduced the inhibitory effect of OxLDL on vasodilation [maximal relaxation (E(max)) = 82.1 +/- 8.6% vs. 54.1 +/- 8.1%; P < 0.0001], but HDL from obese subjects had no effect (E(max) = 47.2 +/- 12.5% vs. 54.1 +/- 8.1%; NS). In HDL from abdominally obese subjects compared with HDL from controls, the apoA-I content was 12% lower (P < 0.05) and the triglyceride-to-cholesteryl ester ratio was 36% higher (P = 0.08)). E(max)(OxLDL + HDL) was correlated with HDL apoA-I content and triglyceride-to-cholesteryl ester ratio (r = 0.36 and r = -0.38, respectively; P < 0.05). We conclude that in abdominally obese subjects, the ability of HDL to counteract the inhibitory effect of OxLDL on vascular relaxation is impaired. This could contribute to the increased cardiovascular risk observed in these subjects.     

The voltage-dependent nonselective cation current in human red blood cells studied by means of whole-cell and nystatin-perforated patch-clamp techniques

Human red blood cells (RBC) can be studied by means of whole-cell and nystatin-perforated patch-clamp techniques. In 85% of the whole-cell experiments (n=86) and 69% of the perforated-patch recordings (n=13), steps to positive potentials, from a holding potential of 0 mV, induced a slow-activating non-inactivating persistent outward current which reverted at about 0 mV. The current activation phase fitted well with a two-component exponential curve. Half-maximal conductance was reached at about 42 mV. Na+ and K+ carried this current, which was not affected by 20 nM charybdotoxin or 20 mM TEA, but was reduced following a partial substitution of extracellular Cl- by tartrate. This current has characteristics similar to the single-channel currents already described in RBC and may be involved in the rapid adaptations of these cells in the circulation.     

The characteristics of action potential and nonselective cation current of cardiomyocytes in rabbit superior vena cava

As a special focus in initiating and maintaining atrial fibrillation (AF), cardiomyocytes in superior vena cava (SVC) have distinctive electrophysiological characters. In this study, we found that comparing with the right atrial (RA) cardiomyoctyes, the SVC cardiomyoctyes had longer APD₉₀ at the different basic cycle lengths; the conduction block could be observed on both RA and SVC cardiomyoctyes. A few of SVC cardiomyoctyes showed slow response action potentials with automatic activity and some others showed early afterdepolarization (EAD) spontaneously. Further more, we found that there are nonselective cation current (I _Ns) in both SVC and RA cardiomyocytes. The peak density of I _Ns in SVC cardiomyocytes was smaller than that in RA cardiomyocytes. Removal of extracellular divalent cation and glucose could increase I _Ns in SVC cardiomyocytes. The agonist or the antagonist of I _Ns may increase or decrease APD. To sum up, some SVC cardiomyocytes possess the ability of spontaneous activity; the difference of transmembrane action potentials between SVC and RA cardiomyocytes is partly because of the different density of I _Ns between them; the agonist or the antagonist of I _Ns can increase or decrease APD leading to the enhancement or reduction of EAD genesis in SVC cardiomyocytes. I _Ns in rabbit myocytes is fairly similar to TRPC3 current in electrophysiological property, which might play an important role in the mechanisms of AF.     

Detoxification of oxidized LDL by transferring its oxidation product(s) to lecithin:cholesterol acyltransferase

In the present study, we isolated modified LCAT (m-LCAT) by hydroxyapatite column chromatography after incubation of crude LCAT (after DEAE SephadexA-50 column chromatography, penultimate step of LCAT purification) with oxidized LDL (oxLDL) at 37 degrees C for 1 h. The activity was found to be about 30% lower than that of native LCAT (n-LCAT). When activity was determined in the presence of oxLDL, m-LCAT was less inhibited than n-LCAT by oxLDL. Treatments of purified LCAT either at 56 degrees C for 30 min, at 100 degrees C for 10 min, or with 6 mM 5-5' -dithiobis-2-nitrobenzoic acid or 9 mM diisopropyl fluorophosphates (each at 37 degrees C for 30 min) resulted in the loss of its cholesterol-esterifying activity. When examined for their ability to detoxify oxLDL, native LCAT and LCAT treated at 56 degrees C for 30 min were found to detoxify oxLDL. These results indicate that oxidation product(s) of LDL is transferred and bound to LCAT in a way that does not depend on its cholesterol-esterifying activity, but rather on the availability of the sulfhydryl group of cysteine residue and the hydroxyl group of serine residue.     

Modulation of Cl-, K+, and nonselective cation conductances by taurine in olfactory receptor neurons of the mudpuppy Necturus maculosus

Odors are transduced by processes that modulate the membrane conductance of olfactory receptor neurons. Olfactory neurons from the aquatic salamander, Necturus maculosus, were acutely isolated without enzymes and studied with a resistive whole-cell method to minimize loss of soluble intracellular constituents. 55 of 224 neurons responded to the test compound taurine at concentrations between 10 nM and 100 microM. Four different conductance changes were elicited by taurine: an increased Cl- conductance (33%), an increased nonselective cation conductance (15%), a decreased Cl- conductance (15%), and a decreased K+ conductance (15%); in addition, responses too small to be characterized were elicited in some neurons. In most cases, taurine appeared to modulate only a single conductance in any particular cell. Modulation of each conductance was dose dependent, and each response ran down quickly in the normal whole-cell mode, presumably due to washout of a diffusible component in the transduction pathway. Modulation of taurine-sensitive conductances caused either inhibitory or excitatory responses. A similar diversity of responses in vivo would produce a complex pattern of electrical activity that could encode the identity and characteristics of an odor.     

Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.     

Norepinephrine-induced sustained inward current in brown fat cells: alpha(1)-mediated by nonselective cation channels

The nature of the sustained norepinephrine-induced depolarization in brown fat cells was examined by patch-clamp techniques. Norepinephrine (NE) stimulation led to a whole cell current response consisting of two phases: a first inward current, lasting for only 1 min, and a sustained inward current, lasting as long as the adrenergic stimulation was maintained. The nature of the sustained current was here investigated. It could be induced by the alpha(1)-agonist cirazoline but not by the beta(3)-agonist CGP-12177A. Reduction of extracellular Cl(-) concentration had no effect, but omission of extracellular Ca(2+) or Na(+) totally eliminated it. When unstimulated cells were studied in the cell-attached mode, some activity of approximately 30 pS nonselective cation channels was observed. NE perfusion led to a 10-fold increase in their open probability (from approximately 0.002 to approximately 0.017), which persisted as long as the perfusion was maintained. The activation was much stronger with the alpha(1)-agonist phenylephrine than with the beta(3)-agonist CGP-12177A, and with the Ca(2+) ionophore A-23187 than with the adenylyl cyclase activator forskolin. We conclude that the sustained inward current was due to activation of approximately 30 pS nonselective cation channels via alpha(1)-adrenergic receptors and that the effect may be mediated via an increase in intracellular free Ca(2+) concentration.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.