Giuliano A. Mottino

Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein. A deep-etch and immunolocalization study of ultrarapidly frozen tissue.

The intima from aortas of normal New Zealand White rabbits was studied 2 hours after infusion of 320 mg human low density lipoprotein (LDL), resulting in a plasma concentration of at least five times and maximally 20 times the values found in normal rabbit serum. The following techniques were used: 1) ultrarapid freezing without chemical fixation, followed by freeze-etching; 2) immunofluorescence microscopy; and 3) postembedding immunogold-labeling electron microscopy. In the latter two methods MB47, a murine monoclonal antibody against human apolipoprotein B, was used as the primary antibody. Control rabbits were infused with the same volume of buffer only. Rotary-shadowed replicas of samples from the LDL-injected rabbits showed the deposition of lipidlike particles in the subendothelial-intimal space that were the size of the injected LDL (23 nm). In focal areas of the intima, groups of 23-nm-sized lipidlike particles and larger lipidlike structures were found enmeshed in the extracellular matrix. Control replicas were essentially free of lipid deposition. Immunofluorescence microscopy of frozen aortic cross sections showed an overall increase in apolipoprotein B in the intima of the LDL-injected rabbits. The presence of apolipoprotein B in the intima was also confirmed by immunoelectron microscopy. These in vivo results show that clustering of LDL-sized particles occurs in the intima within 2 hours of excessive LDL uptake. It also demonstrates the interaction of these LDL-sized particles with the filaments of the extracellular matrix. The clustering of the LDL-sized particles supports the possibility that LDL self-aggregation may occur in vivo and that components of the extracellular matrix are involved in this process.

Ultrastructure of Early Lipid Accumulation in ApoE-Deficient Mice

Apolipoprotein (apo) E-deficient mice develop severe hypercholesterolemia and have lesions that progress from fatty streaks to fibrous plaques distributed in lesion-prone areas throughout the aorta. Lesions develop in apoE-deficient mice on a regular chow diet and will occur faster on a diet higher in cholesterol. Examination of the aortas from these mice on a chow diet by high-resolution, freeze-etch electron microscopy demonstrated lipid retention in the intima by 3 weeks of age. Lipid was retained in the matrix as individual particles between 33 and 48 nm in diameter, aligned along the collagen fibrils and in aggregates consisting of lipid particles with average diameters of 33 and 68 nm. Larger particles seemed to have formed from fusion of smaller particles. Lipid retention was more widespread in 5- and 9-week-old mice. Monocyte attachment to endothelial cells was observed by electron microscopy at 5 weeks of age. The appearance of the intimal lipid was similar to that previously described in rabbit models and suggests that lipid interaction with matrix filaments and subsequent aggregation of lipid particles are critical first steps in the process of foam cell formation.

D-4F decreases brain arteriole inflammation and improves cognitive performance in LDL receptor-null mice on a Western diet

LDL receptor-null mice on a Western diet (WD) have inflammation in large arteries and endothelial dysfunction in small arteries, which are improved with the apolipoprotein A-I mimetic D-4F. The role of hyperlipidemia in causing inflammation of very small vessels such as brain arterioles has not previously been studied. A WD caused a marked increase in the percent of brain arterioles with associated macrophages (microglia) (P < 0.01), which was reduced by oral D-4F but not by scrambled D-4F (ScD-4F; P < 0.01). D-4F (but not ScD-4F) reduced the percent of brain arterioles associated with CCL3/macrophage inflammatory protein-1α (P < 0.01) and CCL2/monocyte chemoattractant protein-1 (P < 0.001). A WD increased (P < 0.001) brain arteriole wall thickness and smooth muscle α-actin, which was reduced by D-4F but not by ScD-4F (P < 0.0001). There was no difference in plasma lipid levels, blood pressure, or arteriole lumen diameter with D-4F treatment. Cognitive performance in the T-maze continuous alternation task and in the Morris Water Maze was impaired by a WD and was significantly improved with D-4F but not ScD-4F (P < 0.05). We conclude that a WD induces brain arteriole inflammation and cognitive impairment that is ameliorated by oral D-4F without altering plasma lipids, blood pressure, or arteriole lumen size.

An ultrastructural study of lipoprotein accumulation in cardiac valves of the rabbit.

Heart valves are composed chiefly of extracellular matrix surrounded by an endothelial cell monolayer and as a result are an excellent model of the intima. Heart valves from rabbits fed a high-cholesterol diet accumulate lipids within the matrix and over time develop fatty streaks similar to those seen in the aorta. In this study we demonstrate that the heart valves (atrioventricular and aortic) can be isolated and used as an in vitro preparation to control and follow low-density lipoprotein (LDL) uptake and deposition. Using thin-section and freeze-etch microscopy we found that LDL rapidly associates with collagen within the extracellular matrix. As it accumulates along the collagen fibers the LDL appears to undergo structural changes in size and surface topography. This association of LDL with collagen may be a key step in lipid aggregation in the intima.

D-4F reduces EO6 immunoreactivity, SREBP-1c mRNA levels, and renal inflammation in LDL receptor-null mice fed a Western diet

LDL receptor-null (LDLR−/−) mice on a Western diet (WD) develop endothelial dysfunction and atherosclerosis, which are improved by the apolipoprotein A-I (apoA-I) mimetic peptide D-4F. Focusing on the kidney, LDLR−/−mice were fed a WD with D-4F or the inactive control peptide scrambled D-4F (ScD-4F) added to their drinking water. The control mice (ScD-4F) developed glomerular changes, increased immunostaining for MCP-1/CCL2 chemokine, increased macrophage CD68 and F4/80 antigens, and increased oxidized phospholipids recognized by the EO6 monoclonal antibody in both glomerular and tublo-interstitial areas. All of these parameters were significantly reduced by D-4F treatment, approaching levels found in wild-type C57BL/6J or LDLR−/− mice fed a chow diet. Sterol-regulatory element binding protein-1c (SREBP-1c) mRNA levels and triglyceride levels were elevated in the kidneys of the control mice (ScD-4F) fed the WD compared with C57BL/6J and LDLR−/− mice on chow (P < 0.001 and P < 0.001, respectively) and compared with D-4F-treated mice on the WD (P < 0.01). There was no significant difference in plasma lipids, lipoproteins, glucose, blood pressure, or renal apoB levels between D-4F- and ScD-4F-treated mice. We conclude that D-4F reduced renal oxidized phospholipids, resulting in lower expression of SREBP-1c, which, in turn, resulted in lower triglyceride content and reduced renal inflammation.

Cardiac excitation-contraction coupling in the absence of Na(+) - Ca2+ exchange.

We investigate cardiac excitation-contraction coupling in the absence of sarcolemmal Na(+) - Ca(2+) exchange using NCX1 knock out mice. Knock out of NCX1 is embryonic lethal, and we measure Ca(2+) transients and contractions in heart tubes from embryos at day 9.5 post coitum. Immunoblot and electron microscopy both indicate that sarcoplasmic reticular membranes are diminished in the knock out (NCX(-/-)) heart tubes. Both Ni(2+) and nifedipine block excitation-contraction coupling in NCX-containing (NCX+) and NCX(-/-) heart tubes indicating an essential role for the L-type Ca(2+) current. Under basal conditions (1Hz stimulation), the NCX(-/-) heart tubes have normal Ca(2+) transients but are unable to maintain homeostasis when Ca(2+) fluxes are increased by various interventions (increased stimulation frequency, caffeine, isoproterenol). In each case, the NCX(-/-) heart tubes respond to the intervention in a more deleterious manner (increased diastolic Ca(2+), decreased Ca(2+) transient) than the NCX+ heart tubes. Expression of the sarcolemmal Ca(2+) pump was not upregulated. The sarcolemmal Ca(2+) pump, however, was able to compensate surprisingly well for the absence of Na(+) - Ca(2+) exchange under basal conditions.