Mechanobiology of low-density lipoprotein transport within an arterial wall—Impact of hyperthermia and coupling effects

The effects of hyperthermia, coupling attributes and property variations on Low-density lipoprotein (LDL) transport within a multi-layered wall while accounting for the fluid structure interaction (FSI) is analyzed in this work. To understand the potential impact of the hyperthermia process, thermo-induced attributes are incorporated, accounting for the plasma flow, mass transfer, as well as the elastic wall structure. The coupling effect of osmotic pressure, Soret and Dufour diffusion is discussed and their influence on LDL transport is examined, demonstrating that only the Soret effect needs to be accounted for. The effect of thermal expansion on changing the behavior of flow, mass transport, and elastic structure is illustrated and analyzed while incorporating the variations in the effective LDL diffusivity and consumption rate, as well as other dominating parameters. It is shown that hyperthermia results in an enhancement in LDL transport by increasing the concentration levels within the arterial wall.     

Molecular studies of pH-dependent ligand interactions with the low-density lipoprotein receptor

The release of ligand from the low-density lipoprotein receptor (LDLR) has been postulated to involve a "histidine switch"-induced intramolecular rearrangement that discharges bound ligand. A recombinant soluble low-density lipoprotein receptor (sLDLR) was employed in ligand binding experiments with a fluorescently tagged variant apolipoprotein E N-terminal domain (apoE-NT). Binding was monitored as a function of fluorescence resonance energy transfer (FRET) from excited Trp residues in sLDLR to an extrinsic fluorophore covalently attached to Trp-null apoE3-NT. In binding experiments with wild-type (WT) sLDLR, FRET-dependent AEDANS fluorescence decreased as the pH was lowered. To investigate the role of His190, His562, and His586 in sLDLR in pH-dependent ligand binding and discharge, site-directed mutagenesis studies were performed. Compared to WT sLDLR, triple His --> Ala mutant sLDLR displayed attenuated pH-dependent ligand binding and a decreased level of ligand release as a function of low pH. When these His residues were substituted for Lys, the positively charged side chain of which does not ionize over this pH range, ligand binding was nearly abolished at all pH values. When sequential His to Lys mutants were examined, the evidence suggested that His562 and His586 function cooperatively. Whereas the sedimentation coefficient for WT sLDLR increased when the pH was reduced from 7 to 5, no such change occurred in the case of the triple Lys mutant receptor or a His562Lys/His586Lys double mutant receptor. The data support the existence of a cryptic, histidine side chain ionization-dependent alternative ligand that modulates ligand discharge via conformational reorganization.     

Hydrolysis of low-density lipoprotein phospholipids in arterial smooth muscle cells

The hydrolysis of phosphatidylcholine (PC) associated with low-density lipoprotein (LDL) by homogenates of smooth muscle cells from rabbit aorta was studied. 1-Palmitoyl-2-[14C]oleoylPC associated with LDL (LDL-P[14C]OPC) or 1-linoleoyl-2-[14C]linoleoylPC associated with LDL (LDL-L[14C]LPC) was used as the substrate. The optimum pH for the formation of [14C]oleoyllysoPC from LDL-P[14C]OPC and for the formation of [14C]linoleoyllysoPC from LDL-L[14C]LPC was pH 4.5, and pH 4.5 and 7.0, respectively. These activities were designated as phospholipase A1 activities. The optimum pH values for the formation of [14C]oleate from LDL-L[14C]OPC and for the formation of [14C]linoleate from LDL-L[14C]LPC were pH 4.5 and 6.5, and pH 4.5, 6.5 and 8.5, respectively. These activities were designated as phospholipase A2 activities. Ca2+ did not affect acid phospholipase A1 activity, but decreased acid phospholipase A2 activity for the hydrolysis of LDL-L[14C]LPC. When smooth muscle cells were incubated with LDL, both phospholipase A1 and phospholipase A2 activities at pH 4.5 for the hydrolysis of LDL-L[14C]LPC increased significantly. These results indicate that phospholipases A1 and A2, which hydrolyze PC associated with LDL, exist in arterial smooth muscle cells and are involved in the metabolism of LDL incorporated into these cells.     

Dietary modifications of low-density lipoprotein fatty acids in humans: their effect on low-density lipoprotein-fibroblast interactions

The chemical composition and metabolism of low-density lipoproteins (LDLs) in a population of Benedictine nuns were studied after 5-month periods during which the predominant dietary fats were sunflower oil, fluid of palm, peanut oil, milk fats, low erucic acid rapeseed (LEAR) oil, corn oil, olive oil, soybean oil. The population was divided into three groups. The control group (C) included 12 subjects selected at random by taking 2 subjects per age pool among those with plasma cholesterol less than 230 mg/dl. Groups H1 and H2 were selected in the same way among those with plasma cholesterol less than 230 mg/dl. Groups H1 and H2 comprised 6 subjects and differed from each other in the amount of plasma cholesteryl esters, i.e., below and above the mean value of group C. Changes in LDL composition, according to the dietary fat, were associated with changes in LDL catabolism studied in fibroblast cultures, but no significant differences were found between the three groups.     

Effect of extracellular matrix elements on the transport of paclitaxel through an arterial wall tissue mimic

Paclitaxel (PTx) is reported to have an nonuniform steady-state concentration profile in the arterial wall. We utilized epifluorescence microscopy to make precise measurements of fluorescently-labeled PTx (F-PTx) distribution through an in vitro tissue mimic which contained varying concentrations of fibrin, elastin, soybean oil, palmitic acid, and solid glass beads. As little as 0.5 mg/mL of elastin in agarose produced a 50% drop in the measured diffusion coefficient, while as much as 10 mg/mL of fibrin in agarose was required for the same reduction in rate of transport. Because no reduction in the measured diffusion coefficient was observed for solubilized, extracted elastin or unassembled elastin-like polypeptides, the effect was specific to elastic fibers that closely resembled the native elastin network. Collectively, this work identifies a potential source for the high degree of partitioning observed for PTx in native tissue and further develops an in vitro technique for exploring complex tissue-drug interactions.     

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its alteration with the arterial mesenchyma.     

Platelet factor 4 enhances the binding of oxidized low-density lipoprotein to vascular wall cells

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an approximately 10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.     

Effect of low-density lipoprotein on the expression of high affinity Fc gamma receptors

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.     

High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein

Oxidatively modified low-density lipoprotein (LDL), generated as a result of incubation of LDL with specific cells (e.g., endothelial cells, EC) or redox metals like copper, has been suggested to be an atherogenic form of LDL. Epidemiological evidence suggests that higher concentrations of plasma high-density lipoprotein (HDL) are protective against the disease. The effect of HDL on the generation of the oxidatively modified LDL is described in the current study. Incubation of HDL with endothelial cells, or with copper, produced much lower amounts of thiobarbituric acid-reactive products (TBARS) as compared to incubations that contained LDL at equal protein concentrations. Such incubations also did not result in an enhanced degradation of the incubated HDL by macrophages in contrast to similarly incubated LDL. On the other hand, inclusion of HDL in the incubations that contained labeled LDL had a profound inhibitory effect on the subsequent degradation of the incubated LDL by the macrophages while having no effect on the generation of TBARS or the formation of conjugated dienes. This inhibition was not due to the modification of HDL as suggested by the following findings. (A) There was no enhanced macrophage degradation of the HDL incubated with EC or copper alone, together with LDL, despite an increased generation of TBARS. (B) HDL with the lysine groups blocked (acetyl HDL, malondialdehyde (MDA) HDL) was still able to prevent the modification of LDL and (C) acetyl HDL and MDA-HDL competed poorly for the degradation of oxidatively modified LDL. It is suggested that HDL may play a protective role in atherogenesis by preventing the generation of an oxidatively modified LDL. The mechanism of action of HDL may involve exchange of lipid peroxidation products between the lipoproteins.     

Presence of aldehydic epitopes on a minor low-density lipoprotein fraction

A more negatively charged low-density lipoprotein (LDL), named minor LDL (mi-LDL), was separated by ionic exchange chromatography and further characterized. This mi-LDL contained lower amounts of polyunsaturated fatty acids, alpha- or gamma- tocopherol, but higher amounts of lipid hydroperoxides than the major LDL fraction (ma-LDL). We show here for the first time that apoB of mi-LDL is modified by lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Using polyclonal antibodies, generated against 4-HNE- or MDA-LDL and apoB, the ratio of 4-HNE- or MDA-derived epitopes to apoB of mi-LDL and ma-LDL was estimated by means of a solid phase fluorescence immunoassay. The ratio of 4-HNE-derived epitopes to apoB on mi-LDL was fourfold higher, while the ratio of MDA-derived epitopes to apoB was twofold higher, compared with the ratios obtained with ma-LDL. In a competition assay with mi- and ma-LDL, only mi-LDL was an effective competitor to inhibit the immunoreaction of anti-4-HNE-LDL with 4-HNE-LDL (by 24%) and of anti-MDA-LDL with MDA-LDL (by 10%).     

Superoxide anion participation in human monocyte-mediated oxidation of low-density lipoprotein and conversion of low-density lipoprotein to a cytotoxin

Human monocytes, upon activation with opsonized zymosan, altered low-density lipoprotein (LDL) during a 24-h co-incubation, resulting in its oxidation and acquisition of cytotoxic activity against target fibroblast cell lines. Both the oxidation of LDL and its conversion to a cytotoxin were enhanced with time of incubation, with the most substantial changes occurring after 6 h of culture of LDL with activated monocytes. Unactivated monocytes did not mediate either alteration. Superoxide anion (O2-) participated in both the oxidation of LDL and its conversion to a cytotoxin since addition of superoxide dismutase (SOD) at the beginning of the co-incubation inhibited, in a concentration dependent fashion, both the monocyte-mediated oxidation and the monocyte-mediated conversion of LDL to a cytotoxin. As expected, the rate of superoxide anion release was greatest during the respiratory burst, very early in the 24-h incubation (0 to 2 h); however, exposure of LDL to monocytes during the respiratory burst was not required for LDL oxidation. The lower levels of O2- released by the cells hours after the respiratory burst had subsided were sufficient to lead to the initiation of LDL oxidation. Three results indicated that the oxidative modification of LDL into a cytotoxin required O2(-)-independent free radical propagation after O2(-)-dependent initiation. First, oxidation of LDL exposed to the activated, superoxide anion-releasing monocytes for 6 h could be almost completely blocked by the addition at 6 h of the general free radical scavenger butylated hydroxytoluene, but not by SOD. Second, LDL oxidation proceeded even after removal of LDL from the superoxide anion-producing, activated cells after various durations of exposure. Third, the development of substantial levels of lipid peroxidation products and the development of greater cytotoxicity occurred after 6 h of exposure of LDL to activated cells, long after peak O2- release had subsided. These results lead us to conclude that monocyte-mediated oxidation of LDL, leading to its transformation into a cytotoxin, requires release of O2- occurring as a result of activation but not necessarily during the respiratory burst, and also requires O2(-)-independent free radical propagation. The modification of LDL into a potent toxin by activated monocytes may explain the tissue damage in atherosclerotic lesions and other pathologic sites in which inflammatory cells congregate.     

Effect of antioxidant protection by p-coumaric acid on low-density lipoprotein cholesterol oxidation

Mechanisms inwhich p-coumaric acid (CA) acts as an antioxidant are notwell understood. This study investigated whether CA can act as a directscavenger of reactive oxygen species (ROS) and whether it minimizes theoxidation of low-density lipoprotein (LDL). Rats were administered CAin drinking water at low or high doses for 10, 21, and 30 days (uptakeswere 29 and 317 mg/day, respectively). Blood levels of8-epiprostaglandin F₂ were monitored as a marker of LDLoxidation. Oral administration of CA (317 mg/day) for 30 dayssignificantly inhibited LDL oxidation. CA also reduced LDL cholesterollevels in serum but had no effect on levels of high-density lipoproteincholesterol. In vitro studies that used electron spin resonance incombination with spin trapping techniques were used to determine theability of CA to scavenge ROS and alter LDL oxidation. CA effectivelyscavenged ·OH in a dose-dependent manner. IC₅₀ andmaximum velocity for CA scavenging of ·OH were 4.72 µM and 1.2 µM/s, respectively, with a rate constant of 1.8 × 10¹¹ M¹ · s¹. Ourstudies suggest that the antioxidant properties of CA may involve thedirect scavenging of ROS such as ·OH.

     

Effects of flow on LOX-1 and oxidized low-density lipoprotein interactions in brain endothelial cell cultures

Fluid shear stress and uptake of oxidized low-density lipoprotein (ox-LDL) into the vessel wall both contribute to atherosclerosis, but the relationship between shear stress and ox-LDL uptake is unclear. We examined the effects of flow, induced by orbital rotation of bEnd.3 brain endothelial cell cultures for 1 wk, on ox-LDL receptor (LOX-1) protein expression, ox-LDL uptake and ox-LDL toxicity. Orbitally rotated cultures showed no changes in LOX-1 protein expression, ox-LDL uptake or ox-LDL toxicity, compared to stationary cultures. Flow alone does not modify ox-LDL/LOX-1 signaling in bEnd.3 brain endothelial cells in vitro, suggesting that susceptibility of atheroprone vascular sites to lipid accumulation is not due solely to effects of altered flow on endothelium.     

Effect of furosemide on the ionic composition of the arterial wall

Furosemide decreased the intracellular H2O, Cl and K content and Cl concentration of dog carotid arteries incubated in normal or high K Krebs (123 mM). Cl loss exceeded K loss in normal Krebs but was of the same magnitude in high K Krebs. The findings are compatible with the inhibition of a neutral K-Cl coupled uptake.     

Numerical modelling of mass transport in an arterial wall with anisotropic transport properties

Coronary artery disease results in blockages or narrowing of the artery lumen. Drug eluting stents (DES) were developed to replace bare metal stents in an effort to combat re-blocking of the diseased artery following treatment. The numerical models developed within this study focus on representing the changing trends of drug delivery from an idealised DES in an arterial wall with an anisotropic ultra-structure. To reduce the computational burden of solving coupled physics problems, a model reduction strategy was adopted. Particular focus has been placed upon adequately modelling the influence of strut compression as there is a paucity of numerical studies that account for changes in transport properties in compressed regions of the arterial wall due to stent deployment. This study developed an idealised numerical modelling framework to account for the changes in the directionally dependent porosity and tortuosities of the arterial wall as a result of radial strut compression. The results show that depending on the degree of strut compression, trends in therapeutic drug delivery within the arterial wall can be either increased or decreased. The study highlights the importance of incorporating compression into numerical models to better represent transport within the arterial wall and suggests an appropriate numerical modelling framework that could be utilised in more realistic patient specific arterial geometries.     

Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture

To study the mechanism of lipoprotein transport through arterial endothelial cells, porcine endothelial cells were cultured on gelated type I collagen supported by a dacron sheet, and the transport of low density lipoprotein (LDL) labeled with rhodamine B isothiocyanate (RB-LDL) through the cells was measured. Light and scanning electron microscopy showed that the cells on the gel were confluent. There was little RB-LDL transport through the endothelial monolayer at 0 degrees C. RB-LDL transport through the monolayer at 37 degrees C was dose-dependent saturable at 0.4 mg protein/ml. The transport was energy-dependent, since its rate was affected by temperature and was inhibited by a combination of 2-deoxyglucose (50 mM) and NaN3 (10 mM). RB-LDL was shown not to be degraded during transport.