The uptake and degradation of matrix-bound lipoproteins by macrophages require an intact actin Cytoskeleton, Rho family GTPases, and myosin ATPase activity.

A key cellular event in atherogenesis is the interaction of macrophages with lipoproteins in the subendothelium. In vivo, these lipoproteins are bound to matrix and often aggregated, yet most cell-culture experiments explore these events using soluble monomeric lipoproteins. We hypothesized that the internalization and degradation of matrix-retained and aggregated low density lipoprotein (LDL) by macrophages may involve the actin-myosin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL. To explore these ideas, we plated macrophages on sphingomyelinase-aggregated LDL bound to smooth muscle cell-derived matrix in the presence of lipoprotein lipase. The macrophages internalized and degraded the LDL, which was mediated partially by the LDL receptor-related protein. Cytochalasin D and latrunculin A, which block actin polymerization, markedly inhibited the uptake and degradation of matrix-retained LDL but not soluble LDL. Inhibition of Rho family GTPases by Clostridium difficile toxin B blocked the degradation of matrix-retained and aggregated LDL by >90% without any inhibition of soluble LDL degradation. However, specific inhibition of Rho had no effect, suggesting the importance of Rac1 and Cdc42. Degradation of matrix-retained, but not soluble, LDL was also blocked by inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, and myosin ATPase. These findings define fundamental cytoskeletal pathways that may be involved in macrophage foam cell formation in vivo but have been missed by the use of previous cell culture models.

Induction of cellular cholesterol efflux to lipid-free apolipoprotein A-I by cAMP.

In the present study apolipoprotein-mediated free cholesterol (FC) efflux was studied in J774 macrophages having normal cholesterol levels using an experimental design in which efflux occurs in the absence of contributions from cholesteryl ester hydrolysis. The results show that cAMP induces both saturable apolipoprotein (apo) A-I-mediated FC efflux and saturable apo A-I cell-surface binding, suggesting a link between these processes. However, the EC50 for efflux was 5-7-fold lower than the Kd for binding in both control and cAMP-stimulated cells. This dissociation between apo A-I binding and FC efflux was also seen in cells treated for 1 h with probucol which completely blocked FC efflux without affecting apo A-I specific binding. Thus, cAMP-stimulated FC efflux involves probucol-sensitive processes distinct from apo A-I binding to its putative cell surface receptor. FC efflux was also dramatically stimulated in elicited mouse peritoneal macrophages, suggesting that cAMP-regulated apolipoprotein-mediated FC efflux may be important in cholesterol homeostasis in normal macrophages. The presence of a cAMP-inducible cell protein that interacts with lipid-free apo A-I was investigated by chemical cross-linking of 125I-apo A-I with J774 cell surface proteins which revealed a Mr 200 kDa component when the cells were treated with cAMP.

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.

Fatty acid composition of an oral load affects chylomicron size in human subjects.

HDL-phospholipids are determinants in reverse cholesterol transport. They are mostly derived from triacylglycerol (TG)-rich lipoproteins. Chylomicron size is important, therefore, because it is related to the ratio surface phospholipids: core TG and, thus, determines the availability of postprandial phospholipids for transfer to HDL. Eleven healthy young women each ingested four different fat loads supplemented with retinyl palmitate and containing 60 g sunflower oil (SO), oleic-sunflower oil (OSO), mixed oil (MO; (g/kg) linoleic acid 480, oleic acid 380, linolenic acid 13) or beef tallow (BT). At the peak of TG absorption for all loads (4 h) chylomicron diameters, determined by agarose-gel filtration, were larger after SO compared with OSO (P < 0.05) and BT (P = 0.06) and after MO compared with BT (P < 0.05). At 6 h chylomicron size was larger after the vegetable oils compared with BT (P < 0.05 in each case). After each fat load chylomicron size decreased at 6 and 8 h compared with that at 4 h (P < 0.05) except for OSO. Retinyl ester and TG concentrations were lower in chylomicrons after BT than after the other fats but not in the chylomicron-free serum (containing chylomicron remnants), suggesting absorption in the form of very small particles. Compared with the fasting value, the concentration of the Svedberg unit of flotation 20-400 fraction, which contains VLDL and chylomicron remnants, was lower 8 h after MO, the only fat to contain significant amounts of linolenic acid. We conclude that chylomicron size is dependent on the fatty acid composition of ingested fats and the time-course of digestion, being larger for polyunsaturated fatty acid-rich fats and in the early phase of digestion. On the basis of retinyl ester concentration there were no differences between fats in chylomicron-remnant clearance.

Oleic acid-rich fats increase the capacity of postprandial serum to promote cholesterol efflux from Fu5AH cells.

Cell cholesterol efflux to serum is stimulated after an oral fat load. The impact of meal fatty acid composition was explored by measure of serum promoted cholesterol efflux from Fu5AH cells after ingestion of 4 different fats: sunflower (Sf), oleic-sunflower (Ol), a mixed oil (Mx), and beef tallow (Bt). High density lipoprotein (HDL)2 and HDL3 were isolated and analyzed. Cholesterol efflux increased regularly after Ol (P<0.05 at 4 h and P<0.02 at 8 h), and 8 h after Mx (P<0.02) or Bt (P<0.05), but not after Sf. Percent HDL3 phospholipids increased after Ol (P<0.05 at 6 h and P<0.01 at 8 H) and 8 h after Mx (P<0.01). After Ol, variations in efflux and percent phospholipids in HDL3 (but not HDL2) were positively correlated (r=0.929; P=0.007 at 6 h). Using HDL3, efflux increased 6 h after Ol (P<0.05) but not after Sf, and efflux was correlated with HDL3 phospholipid concentration in medium (r=0.913; P=0.011). Thus postprandial increase in cholesterol efflux in influenced by ingested fats in relation to increased phospholipid availability on HDL3. The protective effect of monounsaturated fatty acids against atherogenesis might be partly mediated by an enhanced ability of postprandial serum to accept cell cholesterol.