Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

Diabetes accelerates smooth muscle accumulation in lesions of atherosclerosis: lack of direct growth-promoting effects of high glucose levels.

In combination with other factors, hyperglycemia may cause the accelerated progression of atherosclerosis in people with diabetes. Arterial smooth muscle cell (SMC) proliferation and accumulation contribute to formation of advanced atherosclerotic lesions. Therefore, we investigated the effects of hyperglycemia on SMC proliferation and accumulation in vivo and in isolated arteries and SMCs by taking advantage of a new porcine model of diabetes-accelerated atherosclerosis, in which diabetic animals are hyperglycemic without receiving exogenous insulin. We show that diabetic animals fed a cholesterol-rich diet, like humans, develop severe lesions of atherosclerosis characterized by SMC accumulation and proliferation, whereas lesions in nondiabetic animals contain fewer SMCs after 20 weeks. However, high glucose (25 mmol/l) does not directly stimulate the proliferation of SMCs in isolated arterial tissue from diabetic or nondiabetic animals, or of cultured SMCs from these animals or from humans. Furthermore, the mitogenic actions of platelet-derived growth factor, IGF-I, or serum are not enhanced by high glucose. High glucose increases SMC glucose metabolism through the citric acid cycle and the pentose phosphate pathway by 240 and 90%, respectively, but <10% of consumed glucose is metabolized through these pathways. Instead, most of the consumed glucose is converted into lactate and secreted by the SMCs. Thus, diabetes markedly accelerates SMC proliferation and accumulation in atherosclerotic lesions. The stimulatory effect of diabetes on SMCs is likely to be mediated by effects secondary to the hyperglycemic state.

Diabetes-induced accelerated atherosclerosis in swine.

Patients with diabetes are at higher risk for atherosclerotic disease than nondiabetic individuals with other comparable risk factors. Studies examining mechanisms underlying diabetes-accelerated atherosclerosis have been limited by the lack of suitable humanoid animal models. In this study, diabetes was superimposed on a well-characterized swine model of atherosclerosis by injection of the beta-cell cytotoxin streptozotocin (STZ), resulting in a >80% reduction in beta-cells and an increase in plasma glucose to diabetic levels. Animals were maintained without exogenous insulin for up to 48 weeks. Plasma glucose and cholesterol levels and lesion extent and severity were quantified in swine with diabetes and hyperlipemia alone and in combination compared with controls. Diabetes had no effect on plasma cholesterol levels, but diabetic/hyperlipemic (D-HL) swine developed hypertriglyceridemia and showed a doubling in aortic sudanophilia over nondiabetic/hyperlipemic (N-HL) swine as early as 12 weeks (47.25 +/- 4.5 vs. 24.0 +/- 4.6%). At 20 weeks, coronary artery stenosis was significantly greater in D-HL than in N-HL animals (86 +/- 10 vs. 46 +/- 8%). Coronary lesions predominantly arose in the first 2-3 cm of the vessels and displayed humanoid morphology. Aortic lesions in D-HL swine had double the cholesterol content of those in N-HL swine, and incorporation of oleate into cholesteryl ester was significantly greater in grossly normal aortic areas of D-HL swine compared with N-HL and was attributed to similar elevated incorporation in monocytes. This large study demonstrates that a model of diabetes with humanoid characteristics, including hypertriglyceridemia and severe, accelerated atherosclerosis can be reproducibly induced and maintained in swine. This model should potentially be of great value in elucidating mechanisms underlying the accelerated atherosclerosis seen in human diabetic individuals.

Visualization of monocyte recruitment into atherosclerotic arteries using fluorescent labelling.

It has been shown that monocytes are present in early atherosclerotic lesions and in mechanically-injured arterial intima, but direct morphological tracing of specific leukocyte populations into such areas has been lacking. A method for FITC-labelling of leukocytes was therefore evaluated for monocyte studies. Monocyte (95% pure) populations were isolated from blood by counterflow centrifugation and labelled by incubation with free fluorescein isothiocyanate 1-hydrochloride (FITC) in Hank's balanced salt solution. FITC-labelled monocytes showed glass adherence, spreading and migration, as well as acid phosphatase positivity and phagocytosis for up to 20 days in tissue culture. For in vivo experiments, hypercholesterolemic (H) and normal (N) swine were bled repeatedly, and monocyte populations were isolated, labelled and reinjected. Labelled cells were found in blood samples. Animals were killed after 9 days, and formaldehyde-fixed and frozen samples of aortae were studied en face and/or sectioned and examined microscopically under fluorescence. FITC-labelled leukocytes could be found adherent to sites of thickened intima but not to normal areas. Labelled cells were also detected within atherosclerotic lesions. These results show the feasibility of the labelling technique and provide direct visualization of monocyte recruitment from the blood into atherosclerotic and lesion-prone areas.

Arterial response to laser operation for removal of atherosclerotic plaques.

The cellular response of normal and atherosclerotic aortic intima after exposure in vivo to a 0.9 mm diameter carbon dioxide laser was examined in hypercholesterolemic swine with light and electron microscopy to evaluate tissue damage, thrombosis, and healing. At energy levels of greater than 5 joules, laser burns appeared as craters less than 1 mm in depth and 2 mm in diameter. Two days after the operation, craters were filled with platelet-fibrin thrombi that did not protrude above the level of adjacent endothelium. The internal elastic lamina was exposed 1 to 2 mm around the crater. This area was surrounded by a ring of densely packed leukocytes at the edge of the normal endothelium. Two weeks after the operation, the depressed crater surface was mostly reendothelialized with small, closely packed endothelial cells. The subjacent thrombus contained numerous phagocytic cells with inclusion of fibrin, erythrocytes, and membranous debris. Proliferative invaginations containing medial smooth muscle cells, mitotic figures, and collagen extended into the pit from the lateral aspects. Eight weeks after the operation, the burned area was still depressed and therefore less occlusive than adjacent lesion areas, and a fibrous cap had formed over the remaining necrotic area. The results suggest that a focused, low-energy carbon dioxide laser can be used to remove focal atherosclerotic plaques from arteries without inducing excessive thrombogenicity. Rapid healing, including reendothelialization and intimal fibrous scarring, with minimal damage to surrounding tissue, was observed.

Removal of surgically induced fibrous arterial plaques by argon ion laser angiosurgery using a multifiber delivery system. An experimental study in the dog.

Removal of intravascular atherosclerotic obstructions by laser irradiation has gained the attention of many investigators, but has proven to be considerably more difficult to accomplish than initially envisioned. We tested, in an animal model, an argon ion laser delivery system that permits control of (1) laser power, (2) exposure time, and (3) laser beam spot size. The study was conducted on surgically, induced focal fibrous plaques in the carotid arteries of nine dogs. Plaque removal, vessel patency, and healing were evaluated angiographically and by light and electron microscopy at intervals up to 60 days after treatment. Results showed that intravascular obstructions could be removed, healing occurred, and vessels remained patent for up to 60 days.

Localization of LDL in arteries: improvements in immunofluorescence procedures.

We have described the development of procedures used to localize LDL in arteries with atherosclerotic lesions from humans and experimental animal models. We have first illustrated data obtained from earlier cryostat studies, described the procedures, given examples of results of LDL localization in sections of paraffin-embedded blocks, and finally outlined the procedures used and examples for LDL localization in epoxy-embedded blocks. Future improvements in resolution of LDL localization will probably require performing electron microscopy on ultrathin sections of epoxy-embedded arteries followed by immunocytochemistry.

Artificial restoration of voice. I: Experiments in phonatory control of the reinnervated canine larynx.

Coordinated electronic pacing of implanted nerve pedicles into paralyzed laryngeal muscles has allowed selective dynamic control of abduction, adduction, and elongation of the vocal cords. Modifications of the original circuit in a cervical muscle model has added fine tuning to basic "all-or-none" pacing. Rehabilitation of phonation illustrated the sophisticated nature of voice and the need for restoration of fine tuning. Five mongrel dogs received nerve-muscle pedicles into the thyroarytenoideus, cricothyroideus, and posterior cricothyroideus after denervation of one hemilarynx. Following appropriate reinnervation time, pedicles and intact recurrent laryngeal nerves were injected with currents of variable amplitudes and pulse widths to achieve graded vocal fold control while air was blown intratracheally towards the glottic chink. Videoscopic and spectral analyses indicated that artificial phonation could be restored to frequencies measured in the normal state. These experiments suggested that rehabilitation of the impaired voice by servocontrol might eventually be feasible.

Immunohistochemical localization of apoprotein B in aortas from hyperlipemic swine. Preferential accumulation in lesion-prone areas.

In hyperlipemic swine, areas of the aortic arch that accumulate intravenously injected Evans blue dye (blue areas) appear to be more susceptible to early atherogenesis than adjacent areas that are devoid of dye uptake (white areas). We used immunofluorescence microscopy to determine the localization of apoprotein-B (apoB) in these blue and white areas, and in intimal cell masses (ICMs) of the abdominal aortas obtained from hyperlipemic and normolipemic swine. The results showed that before aortic lesions were visible grossly or microscopically, extracellular accumulations of apoB occurred preferentially in the thickened intima of blue areas and in ICMs of the abdominal aorta. Normal white areas in the aortic arch and abdominal aortas, and in the aortas obtained from control swine showed negligible immunoreactivity. Thus, the accumulation of apoB at anatomic sites predilected to early atherogenesis lends further evidence linking these lipoproteins with atherogenesis.

The role of the monocyte in atherogenesis: II. Migration of foam cells from atherosclerotic lesions.

A defined role in the atherogenic sequence is proposed for the circulating monocyte. The author has been able to demonstrate a "monocyte clearance system" in which large numbers of circulating monocytes invade the intima of lesion-prone areas in arteries, become phagocytic, and accumulate lipid. A fatty cell lesion results. Once lipid-laden, foam cells migrate back into the bloodstream by crossing the arterial endothelium. The ratio of penetrating monocytes to emerging foam cells decreases as fatty cell lesions develop until a one-to-one ratio is achieved in late fatty cell lesions, which do not progress further. Advanced fibroatherosclerotic plaques in the same animals do not show the same characteristics and have smooth muscle cell involvement. It would appear that advancement of the lesion is at least partially a result of failure of the monocyte clearance system to remove sufficient lipid. The invasion of monocytes and endothelial damage caused by foam cell clearance may, in late fatty lesions, contribute to plaque evolution by introducing growth factors from macrophages and platelets and allowing greater lipid influx. Elucidation of this system was facilitated by the examination of vessels from diet initiation onwards and by the observation of late nonprogressing fatty cell lesions. It is possible that this system exists in other models but has been overlooked by a predilection for the study of advanced lesions that prevails in the literature.

The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions.

In a previous publication the author and his co-workers demonstrated that atherosclerotic lesion development in the aorta of hypercholesterolemic pigs was preceded by intimal penetration of blood-borne mononuclear cells, and that medial smooth muscle cells were not involved in the formation of early fatty lesions in this model. The current study shows that aortic arch lesions do not progress beyond the fatty cell lesion stage for up to 30 weeks of a moderate cholesterol/lard diet, although they become more extensive in area. Mononuclear cells were found adherent to the endothelium, in endothelial junctions, and in the intima during this period, and were ultrastructurally identified as monocytes by the presence of peroxidase-positive granules (peroxisomes) in their cytoplasm. In addition, lesion areas with nonspecific esterase activity correlated well with Sudan IV staining. Intimal monocytes and altered intimal monocytes with an enlarged cytoplasm and containing a few lipid droplets were both shown to be phagocytic by their uptake of ferritin, which had penetrated the intima after intravenous injection. Circulating monocytes and those adherent to the endothelial surface did not contain ferritin in these animals. The results indicate that blood mononuclear cells associated with lesion formation in this model are, in fact, monocytes, which subsequently undergo transformation into macrophage foam cells in fatty streak lesions. The absence of medial cell involvement indicates that monocytes are the major foam cell precursor in these lesions.

Ultrastructural identification of monocyte-derived foam cells in fatty streak lesions.

In a previous publication (1) we demonstrated that atherosclerotic lesion development in the aorta of hypercholesterolemic pigs was preceded by intimal penetration by blood-borne mononuclear cells, and that medial smooth muscle cells were not involved in the development of fatty streak lesions in this model. The current study shows that lesions in the aortic arch do not progress beyond the fatty streak stage up to 30 wk on a hypercholesterolemic diet, and that mononuclear cell involvement continues during this period. Mononuclear cells adherent to the endothelium, or in the intima of lesion areas were identified as monocytes by the presence of peroxidase-positive granules (peroxisomes) in their cytoplasm. As well, "modified" intimal monocytes which contained a few lipid droplets and which were phagocytic, also sometimes contained peroxisomes. The results suggest that monocytes which invade the intima before or during lesion development undergo transformation into macrophage foam cells in fatty streak lesions. The absence of medial cell involvement indicates that monocytes are the major foam cell precursor in these lesions.

Lipid clearance from fatty streak lesions by foam cell migration.

Dietary-induced hypercholesterolemia in swine produces fatty streak lesions in the aortic arch within 15 wks. These lesions, though becoming more extensive in area, do not progress beyond the fatty streak stage up to 30 wks on the diet. The stabilization of these lesions may be attributed, in part, to the extensive migration of foam cells from the lesion into the aortic lumen. Morphological evidence indicates a lumenal movement of these cells. Previous studies (1) indicate that foam cells in these lesions are monocyte-derived. The combined evidence suggests the existence of a monocyte lipid clearance mechanism which may represent an initial response of the blood-vascular system to atherogenesis.

Alteration of endothelial cell surface morphology after experimental aortic coarctation.

The aortic arch of normal swine shows areas of enhanced permeability to proteins which take up intravenously-injected Evans blue (Old blue areas) and adjacent areas of lesser permeability which do not take up dye (white areas). Following 4 wk of coarctation at the ductus scar level (60% occlusion), former white areas demonstrate Evans blue uptake (New blue areas). Microscopic quantitation showed that endothelial cells in new blue areas had a significantly (p less than .001) altered axial ratio (cell length to width) of 1.41 +/- .04 and mean surface area of 704 +/- 35 mu2 compared to the same area in controls (axial ratio 3.30 +/- .09, mean area 545 +/- 38), but were not significantly different from old blue areas (axial ratio 1.39 +/- .03, area 741 +/- 40). Both new and old blue areas contained three times as many dead or injured cells as white areas in controls. Electron microscopy revealed that New blue area endothelium was similar to that of old blue areas, and that focal denudation and platelet adherence was sometimes present in both areas. The results support the belief that the altered characteristics of spontaneous blue areas are due to hemodynamics effects, and demonstrate that arterial occlusion increases endothelial cell surface area and alters cell orientation relative to blood flow, as well as enhancing injury. Such endothelial alterations could potentiate the disease process in partially-occluded arteries.

Evidence for an altered lipid metabolic state in circulating blood monocytes under conditions of hyperlipemia in swine and its implications in arterial lipid metabolism.

Circulating blood monocytes were isolated from normal and hypercholesterolemic swine, and the monocyte lipid compositions and lipid biosynthesis profiles were assessed. The data indicate that monocytes freshly isolated from hyperlipemic swine have increased phospholipid and cholesterol contents and have increased biosynthetic capability for synthesizing phospholipids, triglycerides, and cholesteryl esters, but not cholesterol. The profile of the stimulated lipid synthesis capability is similar to that of the swine aortic intima undergoing atherogenic change. These studies indicate that circulating blood monocytes in hyperlipemic swine, which are known to give rise to intimal foam cells in the early fatty streak lesion, can contribute to altered vessel lipid metabolism without a requirement for in situ modification by wall factors.

Biochemical characterization of neonatal cardiomyocyte development in normotensive and hypertensive rats.

Final cellular replication and functional maturation of the mammalian ventricular myocyte (cardiomyocyte) is known to occur during the first 2 post-natal weeks in rodents, yet the exact temporal sequence of events remains to be clearly established. When a genetic predisposition toward ventricular hypertrophy also manifests its influence during this time period, possible growth aberrations may occur. Using the spontaneously hypertensive rat (SHR) as an animal model, we have determined that biochemical and morphological indices of cardiomyocyte replicative deficits occur during the first 5 to 8 post-natal days relative to the normotensive control strain, Wistar Kyoto (WKY). Reduced ventricular nucleic acid (DNA and RNA) content and disproportionate RNA- and protein-DNA ratios were found in the neonatal SHR. Incorporation of tritiated thymidine into acid precipitable DNA during the first 5 postnatal days was also reduced in the SHR. Morphological evidence of reduced thymidine incorporation was determined autoradiographically at the cellular and nuclear level using isolated cells and nuclei, respectively. The results of our study indicate that SHR cardiomyocyte hyperplasia is reduced during the first 5 to 8 post-natal days when the last "window" of ventricular myocyte cellular replication occurs. Therefore, the maturing ventricle of the neonatal SHR contains fewer cardiomyocytes than its normotensive control and these cells appear to enter a hypertrophic growth phase at an accelerated rate.

Endothelial cell injury in early mild hypercholesterolemia.

Hypercholesterolemia was induced in 6-week-old male swine by feeding them a chow diet supplemented with lard and cholesterol. Aortic samples from areas of spontaneously-differing permeability to proteins, as demarcated by their uptake of Evans blue dye, were prepared and examined using light microscopy and scanning and transmission electron microscopy. At 2, 4, and 6 weeks after diet initiation, cholesterol/lard-fed animals demonstrated leukocyte adhesion to aortic endothelium in both greater (blue) and lesser (white) permeability areas, being greaer in the former. Endothelial cells underlying leukocytes were generally normal in ultrastructural appearance, but showed cytoplasmic uptake of ruthenium red. Endothelial cells in cholesterol/lard-fed animals contained more lysosomes and cytoplasmic filaments than seen in control animals. Blood-derived cells were more frequently seen in the intima of cholesterol/lard-fed animals, and were often observed associated with intercellular junctions in the endothelium.