Human Fatty Streaks Research Articles

Abstract 18579: Endocytic Adaptor Protein DAB2 is a Novel Atheroprotective Regulator in Atherosclerosis

Introduction: Endothelial dysfunction and arterial inflammation are critically important for cardiovascular diseases such as atherosclerosis. Of major importance, they are among the predominant culprits that promote the transition from stable to vulnerable atheromas. Our pilot studies revealed Disabled Homolog 2 (Dab2) deficiency in endothelial cells (EC) results in endothelial dysfunction. However, the molecular mechanisms that direct Dab2 to combat endothelial dysfunction during atherosclerosis are completely unknown. Hypothesis: Dab2 prevents the progress of atherosclerosis by maintaining EC function and reducing inflammatory factors. Methods and Results: We examined the expression of endothelial Dab2 in human atherosclerosis patients and ApoE -/- mice fed a western diet (WD) for 12 weeks by immunoflurecent co-staining of VE-cadherin and Dab2 and observed drastically reduced Dab2 expression in the endothelium and intima of human fatty streaks and mouse atherosclerotic aortae. Genetic deletion of Dab2 in the endothelium of ApoE -/- mice on a WD for 12 weeks significantly increased atherosclerosis as revealed by Oil Red O staining of aortic roots and arches, suggesting that Dab2 could be a atheroprotective flow-responsive gene. Similar results were found in WT and EC-iDab2KO mice injected with AAv8-PCSK9 virus. RNA-seq of primary mouse aortic ECs (MAECs) isolated from WT and EC-Dab2iKO mice revealed Dab2 deficiency upregulated many inflammatory genes. qRT-PCR confirmed TNF-α and IL-6 were significantly upregulated in Dab2KO MAECs. Additionally, we observed an increase in Dab2 binding to NEMO in the absence of endocytic adapter proteins epsins via co-IP with anti-Dab2 in WT and epsin-deficient DKO MAECs. We also found three Klf4 binding sites present in the Dab2 gene promoter/enhancer and that overexpression of Klf4 upregulated Dab2 expression in a dosage-dependent manner. Conclusions: Our findings indicated the essential role of Dab2 in protecting the atherogenic endothelium by reducing macrophage accumulation and increasing plaque stability in the aortic roots, which may provide new anti-atherosclerotic therapeutics.

Macrophage Myeloperoxidase Regulation by Granulocyte Macrophage Colony-Stimulating Factor in Human Atherosclerosis and Implications in Acute Coronary Syndromes

Inflammation and oxidative stress contribute to the pathogenesis of many human diseases including atherosclerosis. Advanced human atheroma contains high levels of the enzyme myeloperoxidase that produces the pro-oxidant species, hypochlorous acid (HOCl). This study documents increased numbers of myeloperoxidase-expressing macrophages in eroded or ruptured plaques causing acute coronary syndromes. In contrast, macrophages in human fatty streaks contain little or no myeloperoxidase. Granulocyte macrophage colony-stimulating factor, but not macrophage colony-stimulating factor, selectively regulates the ability of macrophages to express myeloperoxidase and produce HOCl in vitro. Moreover, myeloperoxidase-positive macrophages in plaques co-localized with granulocyte macrophage colony-stimulating factor. Pro-inflammatory stimuli known to be present in human atherosclerotic plaque, including CD40 ligand, lysophosphatidylcholine, or cholesterol crystals, could induce release of myeloperoxidase from HOCl production by macrophages in vitro. HOCl-modified proteins accumulated at ruptured or eroded sites of human coronary atheroma. These results identify granulocyte macrophage colony-stimulating factor as an endogenous regulator of macrophage myeloperoxidase expression in human atherosclerosis and support a particular role for the myeloperoxidase-expressing macrophages in atheroma complication and the acute coronary syndromes.

Oxysterol profiles of normal human arteries, fatty streaks and advanced lesions

Objective: Human atherosclerotic lesions of different stages have quantitative differences in cholesterol and oxysterol content, but information on the oxysterol profile in fatty streaks is limited. This study aims to provide more detailed oxysterol quantification in human fatty streaks, as well as normal aorta and advanced lesions.Methods: A newly adapted method was used, including oxysterol purification by means of a silica cartridge; and it was ensured that artifactual oxysterol formation was kept to a minimum. Cholesterol and oxysterols were estimated by GC and identification confirmed by GC-MS in samples of normal human arterial intima, intima with near-confluent fatty streaks and advanced lesions, in necropsy samples.Results: The oxysterols 7α-hydroxycholesterol, cholesterol-5β, 6β-epoxide, cholesterol-5α, 6α-epoxide, 7β-hydroxycholesterol, 7-ketocholesterol and 27-hydroxycholesterol (formerly known as 26-hydroxy-cholesterol) were found in all the lesions, but were at most very low in the normal aorta, both when related to wet weight and when related to cholesterol. Most components of the normal artery showed some cross-correlation on linear regression analysis, but cross-correlations were weaker in the fatty streaks and advanced lesions. However, in fatty streak there was a marked positive correlation between 27-hydroxycholesterol and cholesterol.Conclusion: The findings confirm that oxysterols are present in fatty streaks and advanced lesions and may arise from different cholesterol oxidation mechanisms, including free radical-mediated oxidation and enzymatic oxidation.

Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the Nε-lysine residues of proteins. The resulting Schiff base can be quantified as Nε-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH3. Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.

Elevated levels of protein-bound p-hydroxyphenylacetaldehyde, an amino-acid-derived aldehyde generated by myeloperoxidase, are present in human fatty streaks, intermediate lesions and advanced atherosclerotic lesions

Reactive aldehydes might have a pivotal role in the pathogenesis of atherosclerosis by covalently modifying low-density lipoprotein (LDL). However, the identities of the aldehyde adducts that form on LDL in vivo are not yet clearly established. We previously demonstrated that the haem protein myeloperoxidase oxidizes proteins in the human artery wall. We also have shown that p-hydroxyphenylacetaldehyde (pHA), the aldehyde that forms when myeloperoxidase oxidizes L-tyrosine, covalently modifies the N(epsilon)-lysine residues of proteins. The resulting Schiff base can be quantified as N(epsilon)-[2-(p-hydroxyphenyl)ethyl]lysine (pHA-lysine) after reduction with NaCNBH(3). Here we demonstrate that pHA-lysine is a marker for LDL that has been modified by myeloperoxidase, and that water-soluble, but not lipid-soluble, antioxidants inhibit the modification of LDL protein. To determine whether myeloperoxidase-generated aldehydes might modify LDL in vivo, we used a combination of isotope-dilution GC-MS to quantify pHA-lysine in aortic tissues at various stages of lesion evolution. We also analysed LDL isolated from atherosclerotic aortic tissue. Comparison of normal and atherosclerotic aortic tissue demonstrated a significant elevation (more than 10-fold) of the reduced Schiff base adduct in fatty streaks, intermediate lesions and advanced lesions compared with normal aortic tissue. Moreover, the level of pHA-lysine in LDL recovered from atherosclerotic aortic intima was 200-fold that in plasma LDL of healthy donors. These results indicate that pHA-lysine, a specific covalent modification of LDL, is generated in human atherosclerotic vascular tissue. They also raise the possibility that reactive aldehydes generated by myeloperoxidase have a role in converting LDL into an atherogenic lipoprotein.

Neutral sphingomyelinase: past, present and future

Sphingomyelin and its metabolic products are now known to have second messenger functions in a variety of cellular signaling pathways. At the epicenter of the sphingomyelin — cell signaling pathway is a family of phospholipases called sphingomyelinases. These enzymes cleave sphingomyelin to produce ceramide and phosphocholine. Ceramide in turn serves as a lipid second messenger that induces a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Neutral sphingomyelinase (N-SMase) is a Mg 2+ sensitive enzyme that can be activated by a host of physiologically relevant and structurally diverse molecules like tumor necrosis factor-α (TNF-α), oxidized human low density lipoproteins (Ox-LDL), and several growth factors. Large amounts of ceramide accumulate in human fatty streaks and plaques along with Ox-LDL, growth factors, and proinflammatory cytokines in human atherosclerosis. A further role of ceramide and N-SMase in atherosclerosis was uncovered by the finding that Ox-LDL and TNF-α stimulated N-SMase activity. In turn, ceramide and/or a homolog serves as an important stress signaling molecule in signal transduction, which leads to apoptosis. Interestingly, an antibody against N-SMase can abrogate Ox-LDL and TNF-α induced apoptosis, and therefore may be useful for additional studies of apoptosis in experimental animals. Overexpression of recombinant human N-SMase in human aortic smooth muscle cells markedly stimulate apoptosis, presumably via the multioligomerization of the ‘death domain’. Since plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke and heart failure. In contrast to these observations in human hepatocytes, TNF-α mediated N-SMase activation did not induce apoptosis. Rather it stimulated the maturation of sterol regulatory element (SRE) binding protein (SREBP-1). Moreover, a cell permeable ceramide was found to reconstitute the phenomenon above in a sterol-independent fashion. These findings provide alternate avenues for therapy of patients with hypercholesterolemia and atherosclerosis. The findings reported here suggests that N-SMase plays important cell regulatory roles and provide an exciting opportunity to further these findings to understand the pathophysiology of human disease states.

Enzymatically modified, nonoxidized LDL induces selective adhesion and transmigration of monocytes and T-lymphocytes through human endothelial cell monolayers.

Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 minutes of incubation. Maximal increases in the number of adherent monocytes (8-fold) and of adherent T lymphocytes (4-fold) were observed after treatment with 50 microg/mL E-LDL. E-LDL was more active than oxidized LDL (ox-LDL), whereas native LDL produced only minor adhesive effects. Both E-LDL and ox-LDL enhanced transmigration of monocytes and of T lymphocytes through endothelial monolayers. Again, E-LDL was more potent than ox-LDL, inducing transmigration to a similar extent as N-formyl-Met-Leu-Phe. In endothelial cells, E-LDL stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cells adhesion molecule-1 (PECAM-1), P-selectin, and E-selectin with distinct kinetics. Analyses with blocking antibodies indicated that ICAM-1 and P-selectin together mediated approximately 70% of cell adhesion, whereas blocking of PECAM-1 had no effect on adhesion but reduced transmigration to less than 50% of controls. E-LDL also upregulated expression of ICAM-1 in human aortic smooth muscle cells, and this correlated with increased adhesion of T lymphocytes. E-LDL is thus able to promote the selective adhesion of monocytes and T lymphocytes to the endothelium, stimulate transmigration of these cells, and foster their retention in the vessel wall by increasing their adherence to smooth muscle cells. These findings underline the potential significance of E-LDL in the pathogenesis of atherosclerosis.

Apoptosis in the atherosclerotic plaque: quantitative and qualitative aspects.

Several laboratories have demonstrated the presence of apoptotic cell death in atherosclerotic plaques. Apoptosis occurs in at least 2 stages. The final "execution" phase, which includes DNA fragmentation, is brief ( approximately 6 hours) and irreversible and can be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique. The TUNEL technique is only selective (rather than specific) for apoptotic nuclei, because these contain a far greater degree of DNA fragmentation than do nonapoptotic nuclei. Nonapoptotic cell nuclei that show high levels of RNA synthesis and splicing can also be labeled. This could explain the large variation in the reported percentages of TUNEL-positive nuclei in the plaques. Therefore, the TUNEL technique should be combined with additional techniques, such as markers of transcription and morphological criteria. Recent studies indicate that human fatty streaks differ from adaptive intimal thickenings by the presence of cells containing pro-apoptotic proteins. However, apoptotic cell death is present only in advanced atherosclerotic plaques that show a dense macrophage infiltration. This indicates that although both smooth muscle cells and macrophages within the human fatty streaks become susceptible to apoptosis, additional factors (mainly macrophage- and lipid-derived factors) are necessary to terminate the cell death pathway.

Apoptosis and related proteins in different stages of human atherosclerotic plaques.

The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions. Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (n=18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (P<0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8+/-3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles. We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques.

Induction of P-selectin by oxidized lipoproteins. Separate effects on synthesis and surface expression.

Leukocyte binding to the endothelium is one of the earliest events in the occurrence of atherosclerosis. Leukocyte adhesion molecules involved in this process have not been definitely identified. We have found that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) for 24 hours caused a 2- to 3-fold increase of P-selectin protein, with little change in P-selectin surface expression. A 15-minute histamine treatment of cells exposed to MM-LDL caused a 50% to 100% increase in P-selectin surface expression compared with cells not treated with the lipoprotein. This increase resulted in a 2-fold increase in binding of leukocytes to the endothelium. Immunostaining of permeabilized HAECs after MM-LDL treatment also revealed a highly reproducible increase in intracellular P-selectin associated with rod-shaped structures, typical of Weibel-Palade bodies. Oxidized phospholipids were shown to be mainly responsible for the action of MM-LDL. This increased P-selectin expression was associated with MM-LDL-induced cAMP elevation. Like histamine, highly oxidized low-density lipoprotein, especially the oxidized fatty acids, caused immediate redistribution of P-selectin to the cell surface followed by reinternalization. Immunohistochemical staining showed that endothelial cells on human fatty streak lesions expressed increased levels of P-selectin compared with nonlesion areas. These studies suggest that P-selectin may play an important role in early recruitment of mononuclear cells to the subendothelium in human atherosclerosis and that oxidized lipoproteins may contribute to the increased expression of this molecule by increasing intracellular stores and causing redistribution to the cell surface.

Mast cells in human fatty streaks and atheromas: implications for intimal lipid accumulation.

Recent immunohistochemical observations on atherosclerotic lesions in human aortas and coronary arteries have revealed that these lesions contain mast cells. Mast cells, better known for their actions in allergic reactions of skin and mucosal surfaces, are filled with cytoplasmic secretory granules that contain histamine, heparin and neutral proteases, notably chymase. When activated these cells degranulate, and in this way may influence lipoprotein metabolism in their immediate environment. Indeed, animal studies have suggested that exocytosed mast cell granules may carry LDL into peritoneal macrophages, and so induce their conversion into foam cells. With immunoelectron microscopic techniques, evidence was obtained suggesting that such a 'granule carrier pathway' may operate in vivo in the human arterial intima.

Chymase in exocytosed rat mast cell granules effectively proteolyzes apolipoprotein AI-containing lipoproteins, so reducing the cholesterol efflux-inducing ability of serum and aortic intimal fluid.

Degranulated mast cells are present in human fatty streaks. Chymase in granules released from degranulated rat serosal mast cells, i.e., in granule remnants, proteolyzes human high density lipoprotein3 (HDL3), and so reduces its ability to induce cholesterol efflux from macrophage foam cells in vitro. In this study we found that remnant chymase, by proteolyzing human serum and human aortic intimal fluid, prevents these two physiologic fluids from effectively inducing cholesterol efflux from cultured macrophage foam cells. Inhibition was strongest when remnants were added to apolipoprotein AI (apoAI)-containing lipoproteins; the remnants had no effect on the weaker efflux produced by apoAI-deficient serum. Western blot analysis showed that granule remnants degrade apoAI in serum and in internal fluid. When released from remnants, chymase lost its ability to proteolyze HDL3 in the presence of serum. Thus, remnant chymase (but not isolated chymase) was able to resist the natural protease inhibitors present in serum and in intimal fluid. The results imply participation of exocytosed mast cell granules in foam cell formation in atherogenesis.

Extracellular mast cell granules carry apolipoprotein B-100-containing lipoproteins into phagocytes in human arterial intima. Functional coupling of exocytosis and phagodytosis in neighboring cells.

In experimental studies in vitro, mast cells have induced uptake of apolipoprotein B-100 (apoB-100)-containing low-density lipoproteins by macrophages, with the subsequent formation of foam cells, the hallmarks of atherosclerosis. Recently, increased numbers of activated, ie, degranulated, mast cells were found to be present in human coronary fatty streaks and atheromas. We therefore sought evidence of a connection between mast cells and foam cell formation in vivo. In electron microscopic studies of human aortic and coronary fatty streaks and atheromas, exocytosed cytoplasmic secretory granules of mast cells were detected in the vicinity of their parent cells. These exocytosed granules had bound apoB-100-containing lipoproteins, as indicated by their positive staining with MB 47, a monoclonal antibody against apoB-100. A smooth muscle cell was observed to be in the process of phagocytosing one such exocytosed granule, and in the vicinity of a degranulated mast cell a foam cell contained an ingested mast cell granule. Therefore, the micrographs show that exocytosed granules of intimal mast cells may contribute to intimal foam cell formation and suggest a role for mast cells in human atherogenesis. More generally, the findings provide evidence that phagocytosis of apoB-100-carrying particles is one mechanism by which lipoproteins enter human arterial intimal cells.

Oxidized LDL, ceroid, and prostaglandin metabolism in human atherosclerosis

A noted histological feature of human atherosclerotic lesions upon dissection is the presence of a ‘pigment’ referred to as ceroid; however the significance of ceroid in human fatty streaks and atherosclerotic plaques is not certain. The research focus to this point has presumed that ceroid synthesis and intracellular accumulation is harmful and may have adverse effects on lesion progression or reversibility. Alternatively, ceroid production may be a defense mechanism employed by cells in the artery wall to prevent uncontrolled synthesis and release of prostaglandins (PGs) or prostaglandin-precursors locally. Export of PGs or PG precursors may promote blood platelet aggregation at the site of export. A feedback inhibition mechanism will arrest the cellular export of prostaglandins, thus ending a potentially disastrous premature clotting event.

Cell type and cell state specific antibodies in the analysis of early lesions of human atherosclerosis.

Studying the cell composition of lesions of human atherosclerosis can yield important information regarding the cellular interactions involved in the pathogenesis of this disease. We have used a panel of monoclonal antibodies, many developed in this laboratory, to study the cell composition of human fatty streaks. Results indicate that the predominant cell type in these lesions is the smooth muscle cell rather than the macrophage. Furthermore, using single and double labeling techniques, it was demonstrated that: (a) no expression of class II HLA antigens by smooth muscle cells could be documented; (b) the beta-chain of the platelet derived growth factor could be demonstrated within some macrophages in a small subset of lesions; and (c) only a very small fraction of the cell population express markers associated with cell proliferation, and those that do are largely macrophages and lymphocytes.

Comparison of CI-976, an ACAT inhibitor, and selected lipid-lowering agents for antiatherosclerotic activity in iliac-femoral and thoracic aortic lesions. A biochemical, morphological, and morphometric evaluation.

Due to the potential importance of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) in the generation of lipid-filled monocytes-macrophages, the ACAT inhibitor CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide) was evaluated relative to selected lipid-lowering agents for their effect on atherosclerotic lesion regression and progression. Atherosclerotic lesions comparable in composition to human fatty streaks were induced by chronic endothelial denudation in the iliac-femoral artery of hypercholesterolemic New Zealand White rabbits before intervention, while naturally occurring fatty streaks developed in the thoracic aorta. CI-976 administered in a hypercholesterolemic diet at a dose that did not lower plasma cholesterol prevented the accumulation of monocytes-macrophages within the preestablished iliac-femoral lesion and reduced the foam cell area by 27-29% relative to the initiation of intervention. CI-976 also blunted the development of thoracic aortic fatty streak-like lesions and decreased the cholesteryl ester enrichment by 46%. CI-976 had no effect on plasma triglycerides and, more importantly, had no effect or decreased liver, iliac-femoral, and thoracic aortic free cholesterol content. Dietary intervention alone increased monocyte-macrophage involvement in the iliac-femoral lesion despite reductions in plasma, liver, and thoracic aortic cholesterol content. Conventional lipid-lowering therapy such as cholestyramine or cholestyramine/niacin required substantial decreases in plasma cholesterol levels to achieve comparable vascular changes. We conclude that inhibition of ACAT within the arterial wall by the potent and specific ACAT inhibitor CI-976, even in the absence of plasma cholesterol lowering, can result in the inhibition of atherosclerotic lesion progression and can enhance regression.

Dietary and mechanically induced rabbit Iliac-femoral atherosclerotic lesions: A chemical and morphologic evaluation

The effect of combining chronic endothelial injury and intermittent meal feeding of a high and low cholesterol, coconut oil, peanut oil diet on plasma lipid and lipoprotein content and on the formation of atherosclerotic lesions within the iliac-femoral artery of rabbits was studied. Alternate feeding of a 1 or 0.1% cholesterol, 3% coconut oil, 3% peanut oil diet for 3 to 14 weeks resulted in a 4- to 11-fold increase in plasma cholesterol with 59 to 79% of the plasma cholesterol eluting in a molecular weight fraction comparable to human low density lipoproteins (LDL). In the iliac-femoral artery, an atherosclerotic intimal lesion with an average cross-sectional area of 0.452 mm 2 was present in 98% of the animals. The lesion was typically eccentric in location and contained both superficial- and deep-intimal lipid-filled monocyte-macrophages, extracellular lipid, smooth muscle cells, and extracellular connective tissue matrix. The relative percent lipid composition of the iliac-femoral lesion was 62% cholesteryl ester, 21% free cholesterol, and 17% phospholipid. Thus, we conclude that the combination of meal feeding a cholesterol/fat diet, dietary regimen and chronic mild endothelial injury in the rabbit results in (1) a diet-induced hypercholesterolemia in which LDL appear to be the predominant lipoprotein; and (2) a lesion within the iliac-femoral artery comparable in histologic and chemical composition to a human fatty streak.

Gene expression in macrophage-rich human atherosclerotic lesions. 15-lipoxygenase and acetyl low density lipoprotein receptor messenger RNA colocalize with oxidation specific lipid-protein adducts.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

A low density lipoprotein-sized particle isolated from human atherosclerotic lesions is internalized by macrophages via a non-scavenger-receptor mechanism.

A lipoprotein particle designated A-LDL, which contains apolipoprotein B (apoB) and which is the size of plasma low density lipoproteins (LDL), was isolated from homogenates of human aortic athersclerotic plaques by a combination of affinity chromatography and gel-filtration. Compared to plasma LDL, A-LDL was more electronegative, its hydrated density was lower and more heterogeneous, and its protein-to-lipid ratio was lower. In addition, apoB in A-LDL was highly degraded, and A-LDL was recognized by mouse peritoneal macrophages (MPM) as indicated by its ability to stimulate cholesterol esterification. Cholesterol esterification was saturable with an apparent Km of 100 micrograms of A-LDL cholesterol/ml. Stimulation of cholesterol esterification was linear with time, leading to extensive accumulation of cholesteryl ester in MPM over a 48-hr time interval. The uptake or degradation of acetyl-LDL (radiolabeled either in the protein with 125I or hydrophobic core with [3H]cholesteryl ether) was markedly decreased by excess unlabeled acetyl-LDL but not by A-LDL, and excess acetyl-LDL did not inhibit the uptake or degradation of labeled A-LDL. However, a 10-fold excess of A-LDL also failed to inhibit the uptake of labeled A-LDL. This finding was consistent with the observation that, unlike the saturable stimulation of cholesterol esterification in MPM induced by A-LDL, the uptake of cholesteryl ether-labeled A-LDL was almost linear over a 0-400 micrograms cholesterol/ml range. This discrepancy between dose response curves for A-LDL, which did not occur for acetyl-LDL, could be eliminated by a 24-hr postincubation period in the absence of lipoprotein, suggesting that A-LDL is catabolized less efficiently than acetyl-LDL following internalization. In summary, we conclude that A-LDL uptake by MPM occurs via a low affinity-high capacity process. Although the uptake of A-LDL is not readily saturated, it is of sufficient affinity to lead to lipid loading of macrophages even when A-LDL is present at relatively low concentrations. If these mechanisms are operative in vivo, they could explain how foam cells in human fatty streak lesions develop.

Lipoprotein metabolism of human and rabbit arterial cells in primary culture

The early atherosclerotic lesion, the fatty streak, consists of cholesteryl ester-containing foam cells originating mainly from monocyte-macrophages and to a lesser extent from smooth muscle cells. In this study, we describe lipoprotein uptake and cholesterol accumulation into enzyme-dispersed primary cell cultures from cholesterol-fed rabbit aortas and human aortic fatty streaks. Uptake of fluorescently labelled acetylated low density lipoprotein (acetyl-LDL) was demonstrable in macrophage-derived foam cells and in many smooth muscle cells in early primary cultures. The uptake of acetyl-LDL led to significantly enhanced cellular esterification of cholesterol. Fluorescent beta-migrating very low density lipoprotein (beta-VLDL) was internalized by a considerable number of lesion macrophages and also by smooth muscle cells. Also beta-VLDL uptake stimulated cholesterol esterification, although the effect was milder than that of acetyl-LDL. These findings lend support to the assumption that, during atherogenesis, arterial macrophages are capable of accumulating cholesteryl esters by receptor-mediated uptake of beta-VLDL and modified LDL. The internalization of modified LDL by smooth muscle cells represents a mechanism potentially contributing to the formation of foam cells in the atherosclerotic lesion.