Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis.

The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterizes early atherogenesis. Macrophages are also present in more advanced human atherosclerotic plaques and can produce many mediators that may contribute to lesion formation and progression. Macrophage colony-stimulating factor (MCSF) enhances the proliferation and differentiation of monocyte progenitors and is required for the survival and activation of mature monocytes and macrophages. The authors therefore examined the expression of the MCSF gene in cultured human vascular endothelial (EC) and smooth muscle cells (SMC) as well as in atheromatous lesions from rabbits and humans. Growth arrested EC and SMC contain a low level of MCSF mRNA. Bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) induced MCSF mRNA accumulation in a concentration-dependent manner in both EC and SMC. These stimuli induced large increases in MCSF mRNA with peak induction between 4-8 hours after treatment. LPS, IL-1 alpha, and TNF alpha stimulated EC and SMC also showed increased fluorescent antibody staining for MCSF protein and released immunoreactive MCSF in a time-dependent manner. In contrast, phorbol 12-myristate 13-acetate (PMA) was a less potent inducer of MCSF gene expression and iron-oxidized low-density lipoproteins (ox-LDL) did not increase consistently MCSF mRNA or the synthesis and secretion of immunoreactive protein. Northern analysis of mRNA isolated from the atheromatous aorta of rabbits fed a 1% cholesterol diet for 10 weeks showed elevated MCSF mRNA compared with controls. Immunostaining of atheromatous arterial lesions of rabbits demonstrated MCSF protein in association with intimal SMC as well as macrophages. Furthermore, polymerase chain reaction (PCR) analysis of MCSF mRNA in human atheromata showed higher levels than found in nonatherosclerotic arteries and veins. Since the authors found no mRNA for the MCSF receptor, c-fms, in cultured EC or SMC macrophages are likely the primary target for MCSF within atheromatous vessels. The authors therefore investigated the effects of MCSF on monocyte functions related to foam cell development. Treatment of cultured human monocytes with recombinant human MCSF (10(3) U/ml, 72 hr) led to the accumulation of mRNA for the acetyl-LDL (scavenger) receptor and apolipoprotein E (apo E). These studies establish that vascular EC and SMC produce substantial MCSF in response to a variety of stimuli. The local production of MCSF during atherogenesis may contribute to macrophage survival and proliferation or activate specific macrophage functions such as expression of the scavenger receptor and secretion of apo E.     

Expression of very low density lipoprotein receptor mRNA in rabbit atherosclerotic lesions.

The expression of very low density lipoprotein (VLDL) receptor mRNA in atherosclerotic lesions in rabbits was investigated. To examine the expression of the VLDL receptor in the vascular wall, poly(A)+ RNA was isolated from whole aortas of cholesterol-fed New Zealand White (NZW), Watanabe heritable hyperlipidemic (WHHL), and normal NZW rabbits, and then Northern blot analysis was performed. The VLDL receptor mRNA was detected in aortas from both NZW rabbits fed 0.5% cholesterol for 16 weeks and 12-month-old WHHL rabbits, whereas no expression was seen in normal NZW rabbit aortas. To further determine the localization of the VLDL receptor mRNA, in situ hybridization using digoxigenin-labeled riboprobes and immunohistochemistry using monoclonal antibodies against each cell component were performed. Early atherosclerotic lesions, termed fatty streaks, in the NZW rabbits fed 0.5% cholesterol for 4 weeks demonstrated strong expression of the VLDL receptor mRNA by macrophages. The VLDL receptor mRNA was also expressed in more advanced atherosclerotic lesions from both atherogenic animal models. The predominant origin of the VLDL receptor mRNA-positive cells was macrophages, and some intimal smooth muscle cells appeared to express a weak but significant signal in these advanced lesions. Our findings suggest that the VLDL receptor expression may play a role in the development of atherosclerosis.     

Immunohistochemical detection of macrophage-derived foam cells and macrophage colony-stimulating factor in pulmonary atherogenesis of cholesterol-fed rabbits

In order to investigate the role of monocyte/macrophages and their relationship to the expression of macrophage colony-stimulating factor (MCSF) in pulmonary atherosclerosis, lungs were excised from rabbits that had been fed for 60 and 90 days on a diet containing 0.5% cholesterol. In the lungs, fatty streaks and elevated foam cell lesions predominated in the large or medium-sized elastic pulmonary arteries, while massive accumulation of foam cells in the intima of muscular arteries produced marked luminal narrowing and nearly complete occlusion. In these lesions, most of the foam cells were reactive with RbM2, a monoclonal antibody (mAb) against rabbit macrophages, while smooth muscle cell-derived foam cells were detected by mAb against smooth muscle actin in the deeper area of elevated foam cell lesions of elastic arteries. Ultrastructural observation confirmed the presence of monocytes in the intima, their differentiation into macrophages, and their transformation into foam cells in the atherosclerotic lesions. lmmunohistochemical expression of MCSF was demonstrated in the endothelial cells, smooth muscle cells and foam cells. A minor macrophagederived foam cell population was demonstrated to possess a prolif-erative capacity. These data suggest that MCSF is involved in the differentiation of monocytes into macrophages, their transformation into foam cells, and their proliferation during pulmonary atherogenesis.     

Endothelial constitutive nitric oxide synthase protein and mRNA increased in rabbit atherosclerotic aorta despite impaired endothelium-dependent vascular relaxation.

Atherosclerotic arteries are well known to exhibit impaired endothelium-dependent relaxation (EDR), but the exact mechanism of this impairment remains unclear. Recently, endothelial constitutive nitric oxide synthase (ECNOS), which generates nitric oxide and mediates EDR, was cloned, and ECNOS mRNA expression was reported to be modified by various cytokines, lipoproteins, and shear stress. To investigate the expression of ECNOS mRNA and protein in atherosclerotic arteries with impaired EDR, thoracic aortas isolated from Watanabe heritable hyperlipidemic (WHHL) rabbits were examined by using in situ hybridization and immunohistochemistry. Compared with thoracic aortas from Japanese White rabbits, WHHL aortas exhibited significantly impaired EDRs, although both in situ hybridization and immunohistochemistry exhibited enhanced expression of ECNOS mRNA and protein in WHHL aortas. There was no significant relationship between expression of ECNOS mRNA and protein of endothelial cells and age of the examined WHHL aortas. These data suggest that the mechanism of impaired EDR in atherosclerotic arteries is not due to the decrease in ECNOS mRNA and protein.     

Expression of platelet-derived growth factor and c-myc in atherosclerotic lesions in cholesterol-fed chickens: immunohistochemical and in situ hybridization study

Immunohistochemical examination showed no significant expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, PDGF receptors, or of c-myc in the thoracic and abdominal aortas of normal roosters. In cholesterol-fed roosters, intense immunohistochemical reaction for PDGF-B, PDGF receptor, and c-myc was seen in the lipid-rich thickened intimal lesions of the thoracic and abdominal aortas while no significant immunoreaction for PDGF-A was demonstrated in the same lesions. In accordance with immunohistochemical findings, in situ hybridization demonstrated a significant level of expression of PDGF-B, PDGF-A receptor, PDGF-B receptor, and c-myc genes in proliferating intimal cells of the thoracic and abdominal aortas. These results suggest that coordinate actions of PDGF-B and c-myc play an important role in proliferation of intimal cells in the developing atherosclerotic lesions in chickens.     

Lipoprotein degradation and cholesterol esterification in primary cell cultures of rabbit atherosclerotic lesions.

Lipoprotein metabolism and cholesterol accumulation in atherosclerotic lesions was studied using enzymatically isolated primary cell cultures from aortas of rabbits made atherosclerotic by cholesterol feeding. The cultures consisted of macrophages and smooth muscle cells, thus resembling, in composition, fatty streak lesions. The mean (+/- SD) cholesteryl ester content of the dispersed cells was 1059 +/- 445 micrograms/mg cell protein, but it declined steeply during 1 week in primary culture. The uptake of low-density lipoprotein (LDL), beta-migrating very low-density lipoprotein (beta-VLDL), and acetylated LDL (acetyl-LDL), labeled with 125I or with the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine (DiI), was studied in 2-day-old primary cultures. DiI-acetyl-LDL was avidly taken up by the macrophages and, to a lesser extent, by some smooth muscle cells. The uptake of DiI-beta-VLDL by the macrophages was weaker and less homogeneous than that of DiI-acetyl-LDL. The degradation rates of 125I-labeled beta-VLDL, LDL and acetyl-LDL were 135 +/- 54, 195 +/- 20, and 697 +/- 14 ng/mg cell protein/8 hours, respectively. Incubation with unlabeled acetyl-LDL enhanced the incorporation of [3H]oleate into cholesteryl esters and increased the cellular cholesteryl ester content. These results suggest that arterial macrophages and, to some extent, smooth muscle cells from cholesterol-fed rabbits actively metabolize acetyl-LDL and are thus capable of accumulating cholesteryl esters by uptake of modified forms of LDL.     

Expression and Localization of Matrix Metalloproteinase-12 in the Aorta of Cholesterol-Fed Rabbits : Relationship to Lesion Development

Degradation of extracellular matrix (ECM) proteins in the aorta is a critical step for the development of atherosclerosis. Expression of matrix metalloproteinase (MMP)-12 (macrophage elastase), an elastin-degrading proteinase in the MMP family, was investigated in the thoracic aorta of rabbits fed a 1% cholesterol-containing diet for 16 weeks. In the atherosclerotic lesions, MMP-12 was produced abundantly at both the mRNA and protein levels, whereas no expression was observed in the normal rabbit aortas. The principal source of MMP-12 was macrophage foam cells (MFCs) that had infiltrated the atherosclerotic intima; this was demonstrated in both in vitro culture studies of MFCs purified from atherosclerotic lesions and immunohistochemical studies of aortic lesions. Additional biochemical studies using recombinant rabbit MMP-12 revealed that MMP-12 digested elastin, type IV collagen, and fibronectin and also activated MMP-2 and MMP-3. Expression of MMP-12 by human macrophage cell lines was increased by stimulation with acetylated low-density lipoprotein, implying augmentation of MMP-12 production during foam cell formation. Increased expression of MMP-12 in atherosclerotic lesions, concomitant with foam cell generation, which triggers the acceleration of ECM breakdown, is likely to be a critical step in the initiation and progression of the atherosclerotic cascade.     

Immunohistochemical studies of C-reactive protein and apolipoprotein B in inflammatory and arterial lesions

Interactions in vivo between C-reactive protein (CRP) and apolipoprotein B (apo-B)-containing lipoproteins were sought in inflammatory lesions and atherosclerosis. CRP was demonstrated immunohistochemically on the surface of some muscle fibres in locally induced inflammatory lesions in the rabbit, but apoB was not detected in the same distribution. CRP was not detected in catheter-induced aortic endothelial injuries in the rabbit, in arterial lesions containing apoB from cholesterol-fed rabbits, in apoB-containing human fatty streaks or in advanced human atherosclerotic lesions.     

Immunohistochemical localization of apolipoprotein B in human atherosclerotic lesions

A peroxidase-antiperoxidase immunohistochemical method was used to detect the presence of intracellular and extracellular apolipoprotein B (apo B) in paraffin sections of human abdominal aortas and coronary arteries with and without atherosclerotic involvement. The four aortas studied disclosed both intracellular and extracellular apo B in areas in which lipid was found by oil red O staining. Coronary arteries also demonstrated abundant extracellular apo B but failed to disclose intracellular apo B in cells with stainable lipid. Immunohistochemical detection of intracellular apo B in raised aortic lesions but not in cells of coronary lesions suggests that arteries that contain detectable amounts of intracellular apo B correspond to arteries that usually have earlier atherosclerotic involvement.     

Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.

Vascular cell adhesion molecule-1 (VCAM-1), a mononuclear leukocyte adhesion molecule, is expressed in cultured vascular endothelial cells activated by cytokines and is induced in rabbit aortic endothelium in vivo within 1 week after initiation of an atherogenic diet. We now demonstrate that vascular smooth muscle cells can also express VCAM-1 in rabbit atherosclerotic lesions in vivo and in response to cytokines in vitro. Immunohistochemical staining of aortas from rabbits fed a 0.3% cholesterol-containing diet revealed that a portion of smooth muscle cells within intimal foam cell-rich lesions expressed VCAM-1. The intimal VCAM-1-expressing cells localized predominantly in regions above the internal elastic lamina. These VCAM-1-positive cells had the typical spindle shape of smooth muscle cells but had reduced alpha-actin expression in comparison to normal medial smooth muscle cells, and did not bear markers for endothelium, macrophages, and T cells. In culture, rabbit aortic smooth muscle cells expressed VCAM-1 mRNA and protein in a time- and concentration-dependent fashion when exposed to interferon-gamma or Gram-negative bacterial lipopolysaccharide. Cultured human vascular smooth muscle cells also expressed VCAM-1 mRNA and protein in response to lipopolysaccharide, interferon-gamma, and interleukin-4. The monokines interleukin-1 alpha and tumor necrosis factor-alpha did not induce VCAM-1 expression in either rabbit or human vascular smooth muscle cells. Inducible VCAM-1 expression by vascular smooth muscle cells in vivo during hypercholesterolemia and in vitro in response to certain cytokines suggests a broader range of VCAM-1 functions in vascular biology than heretofore appreciated.     

Unesterified cholesterol-rich lipid particles in atherosclerotic lesions of human and rabbit aortas.

Unesterified cholesterol-rich lipid particles were isolated from human and cholesterol-fed rabbit aortas. These particles have been previously reported to constitute the initial lipid deposition in atherosclerotic lesion development. Purification of the particles was accomplished with microfiltration, gel filtration chromatography, and density gradient centrifugation. Particles from both human and rabbit aortas had a density of between 1.02 g/ml and 1.08 g/ml with a peak at d = 1.036 g/ml. These particles had a high molar ratio of unesterified cholesterol to phospholipid (2.4:1 in rabbit, 2.6:1 in human) and a high percentage of their cholesterol in an unesterified form (82% in rabbit, 76% in human). The particles had diameters between 700 and 3000 A and showed unilamellar and multilamellar structures. Freeze-fractured particles had smooth fracture faces and sometimes contained a smooth-surfaced core. Upon incubation with filipin, particles showed typical filipin-sterol complexes, demonstrating the presence of unesterified cholesterol. The particles we have isolated may constitute an early pathologic form of accumulated cholesterol in developing lesions and may represent a degradation product of infiltrated plasma low-density lipoprotein.     

Cytomegalovirus genome and the immediate-early antigen in cells of different layers of human aorta

A number of data suggest that reactivation of cytomegalovirus (CMV) latent in arterial wall cells may contribute to atherogenesis; however, there is no direct evidence available. To address this issue, we have examined, using in situ hybridization or immunohistochemical staining, the frequency of occurrence of cells containing viral genome and of those expressing the IE 70 viral antigen in the endothelial layer and in deeper layers of human aortas with or without visible atherosclerotic lesions. Using endothelial cell cultures or tissue endothelial preparations, we found CMV-hybridizing endothelial cells in 6 of 8 grossly normal aortas and in 16 of 18 lesioned aortas. Antigen-positive endothelial cells were detected in 1 of 5 grossly normal vessels and in 6 of 7 lesioned vessels. Infected endothelial cells were abundant in areas adjacent to orifices of intercostal arteries of grossly normal aortas and in fatty spots of lesioned aortas, but no infected endothelial cells were observed in most plaques examined. In paraffin sections of grossly normal vessels, we detected CMV genome in cells adjacent to lumen and in cells randomly scattered through subendothelial intima and the media; however, no immunoreactive viral protein was found in the same tissue samples. In sections of lesioned vessels, clusters of CMV-hybridizing cells were found in the media in addition to infected cells randomly scattered through the intima and the media. In these samples of lesioned vessels, viral antigen was detected in cells adjacent to lumen and in cells clustered at the intima/media border. We found antigen-positive cells in grossly normal areas of lesioned aortas and in fatty lesions, but not in plaques of the same vessels. The data suggest that accumulation of the immediate-early CMV antigen in cells of endothelial layer and development of antigen-positive cell clusters in deeper layers of vascular wall accompany early atherogenic events in human aorta. Received: 10 June 1999 / Accepted: 16 November 1999     

Ultrastructural localization of endogenous albumin in human aortic tissue by protein A-gold immunocytochemistry.

The presence of endogenous plasma albumin in human aortas was demonstrated by immunocytochemical procedures. The protein A-gold approach, combining high resolution and specificity, was applied at the light and electron microscope levels to determine the in situ cellular and extracellular localization of albumin on tissue sections derived from normal and atherosclerotic human aortas. The distribution of albumin across the aortic wall interstitium was found to be uneven, with low to moderate staining intensities in the aortic subendothelial space, low intensities in the media, and high intensities around the vasa vasorum in the adventitia. Albumin was associated with collagen fibers as well as with the electron-dense material bordering the elastic laminae in both normal and pathologic tissues. Extracellular multilamellar structures were found to be characteristic of the necrotic areas of atherosclerotic aortas. These structures were intensely labeled for albumin, with the labeling being closely associated with the membranes. Numerous smooth muscle cell-derived and monocyte-derived foam cells were present in pathologic tissues, and some of their lysosomal compartments were labeled for albumin, suggesting an internalization and degradation of interstitial albumin by these cells.     

Composition and metabolism of lipid in macrophages from normally fed and cholesterol-fed rabbits

The composition and metabolism of lipid in peritoneal macrophages obtained from normally fed rabbits were compared with those of macrophages obtained from cholesterol-fed rabbits. Macrophages from cholesterol-fed rabbits had a higher cholesterol content and a markedly higher cholesterol ester content than normal macrophages. The increase in cholesterol ester content was most marked for cholesterol oleate and cholesterol linoleate, with the cholesterol ester fatty acid composition of the cholesterol-fed macrophages resembling that of foam cells derived from aortic lesions of similarly cholesterol-fed rabbits. Metabolic differences were also demonstrated between the cells obtained from normal and cholesterol-fed rabbits. In the latter, incorporation of ¹⁴C-labeled acetate into cholesterol was almost completely suppressed whereas in macrophages from normally fed rabbits, ¹⁴C-labeled acetate was incorporated predominantly into cholesterol. Incubation in vitro of normal macrophages for periods up to 20 hr with hyperlipemic serum, however, was not associated with any appreciable suppression of cholesterol synthesis.