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Biomedical subjects

J C Lewis

Publications and source records attributed to J C Lewis.

At least 19 recordsLinked to original sources
PubMed1992

Ultrastructural localization of peroxidase in atherosclerotic lesions of pigeons.

Atherosclerotic lesions are known to have metabolic alterations which are associated with progressive lipid accumulation. Among the changes, lysosomal enzyme activity has been extensively characterized and at the ultrastructural level has been correlated with the amount of foam cell lipid. In a fashion paralleling lysosomal change, artery wall peroxidase activity is also altered during disease progression. The present study focuses upon the ultrastructural localization of peroxidase activity in atherosclerotic lesions of the aorta and coronary arteries from White Carneau pigeons fed a cholesterol-supplemented (0.3%) diet for 3 years. This resulted in fibrous lesions, rich in smooth muscle cells. The birds were necropsied by perfusion fixation, and peroxidase cytochemistry was carried out using the diaminobenzidine reaction. Peroxidase activity was found within endothelial cells and smooth muscle cells in both the media and intima, but cytochemically demonstrable activity was not found in macrophage foam cells. Peroxidase was localized within the nuclear envelope and endoplasmic reticulum, especially within cells that had lipid inclusions. The degree of peroxidase positivity varied within and among the arteries. In nonlesion regions of the aorta 20% of medial smooth muscle cells was peroxidase positive; the value for coronary artery smooth muscle cells was less. The peroxidase activity within aortic lesions was increased with 44% of intimal smooth muscle cells being positive. Notably, 85-90% of the lipid-containing intimal smooth muscle cells were positive. In contrast, intimal smooth muscle cells in the coronary artery lacked peroxidase reaction product, even in cells containing lipid. We conclude from these studies that aortic lesions contain a cytochemically differentiated subset of lipid-containing, peroxidase-positive smooth muscle cells; but coronary lesions lack a comparable subset of smooth muscle cells.

Animals↗
PubMed1992

Cloned endothelium derived from autoimmune vascular disease retain structural and functional characteristics of normal endothelial cells.

MRL/1pr mice demonstrate anatomic specificity in their development of vasculitis including the small- and medium-sized muscular arteries of the mesentery. To define the functional role of endothelium in vasculitis, we have cloned endothelial cells derived from inflamed small- and medium-sized arteries. Primary cells were derived by enzymatic dispersement and endothelial cells were selected by utilizing a combination of specific culture conditions. Cloned endothelium were developed utilizing limiting dilution cultures supplemented by endothelial cell growth factor. The cloned endothelial cells express many structural features of mature endothelial cells including Factor VIII-RA, non-muscle-specific actin, and Weibel-Palade bodies. Functionally, the clones express functional receptors for the scavenger pathway for LDL metabolism. The cells do not express Class I MHC antigens; however, IFN-beta and IFN-gamma stimulate Class I MHC expression after 24 h, which induces lysis of virus-infected cloned endothelium by Class I-restricted virus-primed T cells. In direct contrast to site-identical vascular smooth muscle cells (VSMCs), endothelial cells do not spontaneously express Class II MHC antigens, nor do they secrete biologically relevant levels of IL-1 unless triggered by lipopolysaccharide. The availability of site-specific cloned endothelium along with cloned VSMCs from autoimmune mice should resolve major experimental controversies involving the pathophysiology of inflammatory vascular disease.

Actins↗
PubMed1992

Platelet morphologic changes and fibrinogen receptor localization. Initial responses in ADP-activated human platelets.

Platelet exposure to agonists results in rapid morphologic changes paralleled by fibrinogen binding and platelet aggregation. The current study used standardized stereology in conjunction with immunogold electron microscopy to correlate the initial morphologic changes with fibrinogen receptor localization on the surfaces of ADP-activated human platelets. A 45% increase in platelet circumference was observed after 3 seconds of activation (P = 0.001). Virtually all of this increase was due to a 13-fold increase in projection membrane, and the projections observed by stereo microscopy at this time were mostly blunt. Both blunt and long projections also accounted for the increase in platelet-platelet contacts at 10 seconds of activation. Immunogold electron microscopy using the monoclonal antibodies P2 and AP-2 against the fibrinogen receptor, glycoprotein IIb/IIIa (GP IIb/IIIa), showed relatively equivalent immunogold densities on projections compared with cell body during 30 seconds of activation. The activation-dependent anti-GP IIb/IIIa monoclonal antibody, 7E3, showed an immunogold density 37% greater on projections compared with cell body (P = 0.0001). Colocalization studies using 7E3 with a polyclonal antifibrinogen antibody showed bound fibrinogen in close proximity to the GP IIb/IIIa localized by 7E3 on projections. These studies support an important role for platelet projections during the earliest stages of fibrinogen binding and ADP-induced aggregation.

Adenosine Diphosphate↗
PubMed1991

Beta VLDL uptake by pigeon monocyte-derived macrophages: correlation of binding dynamics with three-dimensional ultrastructure.

Endocytosis of pigeon beta migrating very-low-density lipoprotein (beta VLDL) by monocyte-derived macrophages (monocyte/macrophages), cultured from Random Bred White Carneau (RBWC) pigeons, occurs by both coated and non-coated regions of the plasma membrane (Henson et al.: Exp. Mol. Pathol. 51:243-263, 1989). Secondary to binding, the beta VLDL is translocated to lysosomes for degradation. Ultimately these events lead to foam cell formation in vitro. Utilizing video-enhanced contrast light microscopy in conjunction with whole mount intermediate-voltage transmission electron microscopy (IVEM) and high-resolution scanning EM, the dynamics of beta VLDL binding have been correlated with ultrastructure. Beta VLDL conjugated to gold colloids was visualized at the surface of living cells by using Allen video-enhanced contrast-differential interference contrast microscopy (AVEC-DIC). Subsequent to AVEC-DIC, direct observation of the identical cells by IVEM and SEM was facilitated through the use of gold finder grids, and these EM observations confirmed identification of the video-observed beta VLDL particles. Upon addition of beta VLDL, pigeon monocyte/macrophages underwent gross morphological changes. These changes were recorded by video as movements at the cytoplasmic periphery, and the movements involved extension of microvilli, expression of retraction fibers, and elaboration of membrane ruffles. When secondarily observed by stereo (3-D) IVEM and SEM, the identification of microvilli, retraction fibers, and membrane ruffles was confirmed and the lipoprotein-gold conjugates were associated with these ligand-induced membrane structures. Beta VLDL-gold conjugates were also associated with pit-like regions at the base of microvilli, while at the base of ruffles, beta VLDL-gold conjugates were located in membrane invaginations and cytoplasmic vesicles.

Animals↗
PubMed1991

Morphologic characteristics of naturally occurring atherosclerosis in the brachiocephalic artery of the pigeon.

Endothelial characteristics and the macrophage foam cell nature of early naturally occurring lesions in the aorta and coronaries of the pigeon have been well characterized. However, the characteristics of pigeon atherosclerosis at other vascular sites have not been extensively studied. The present study was designed to compare atherosclerosis in the brachiocephalic artery with that in the coronaries and aorta. Forty-six White Carneau (WC) pigeons (26 female, 20 male) ranging in age from 2.5 to 7 years were necropsied after fixation under deep anesthesia by perfusion at 160 mm Hg with buffered glutaraldehyde. Arteries stained with Sudan IV for gross evaluation were subsequently processed for SEM and TEM. The occurrence of sudanophilia in the proximal brachiocephalic artery was greater in females (22/26) than in male (2/20). The endothelium, as studied by SEM, was intact over all normal and sudanophilic areas. Cell morphology varied with location in the vessel and gradually changed from polygonally shaped cells with prominent margins and protruding nuclei in the proximal brachiocephalic artery to elongated, flattened cells in distal regions. These regional differences were consistently observed, and did not correlate with age, gender, or areas of lipid accumulation. Unlike lesions in the coronary arteries and at the celiac bifurcation of the aorta, a relative paucity of white blood cells over diffuse sudanophilic areas was observed. This lack of adherent monocytes correlated with lesion ultrastructure. Connective tissue in the intima of the sudanophilic brachiocephalic arteries was disorganized, reflecting both an increase in matrix components and the presence of massive pools of extracellular lipid. Intracellular lipid was minimal and when present was confined to random droplets in the cytoplasm of intimal smooth muscle cells. Monocyte-derived foam cells, characteristic to other vascular beds, were absent from the brachiocephalic artery lesions. These results document differential lesion composition in the WC pigeons and suggest a gender-related susceptibility for brachiocephalic artery atherosclerosis in pigeons.

Animals↗
PubMed1991

Lysosomal lipid accumulation in vascular smooth muscle cells.

Using an inverted culture technique, the accumulation of lipid within vascular smooth muscle cells incubated with lipid droplets was studied. Initially, lipid was found exclusively within cytoplasmic inclusions but, as accumulation continued, lysosomes became the predominant site of lipid storage. After 3 hr of incubation, 84% of lipid was within lysosomes. This lysosomal lipid accumulation produced a tripling of the average size of lysosomes and resulted in lysosomes with complex, multilobed shapes. In contrast, although the number of cytoplasmic inclusions increased with lipid loading, individual inclusions maintained a spherical shape and a consistent diameter of 1-1.3 microns. Concomitant with changes in cellular lipid storage, incubation with lipid droplets induced development of an anastomosing network of acid phosphatase-containing tubules which were spatially related to sites of lysosomal lipid accumulation. Thus lipid accumulation produced ultrastructural alterations in a number of metabolic compartments. Similar alterations in the intracellular compartmentalization of acquired lipid have been demonstrated in foam cells during atherogenesis and have been hypothesized to have profound effects on lipid metabolism and disease progression.

Acetylglucosaminidase↗
PubMed1991

Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells.

Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.

Ammonium Chloride↗
PubMed1991

Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermediate voltage electron microscopy was used to examine monocyte-derived macrophages (monocyte/macrophages) during early in vitro uptake of beta migrating very-low-density lipoproteins (beta VLDL). Lysosomes were identified using acid phosphatase cytochemistry, and in control cells these organelles constituted 3.5% of the total cytoplasmic volume. Both primary and secondary lysosomes were observed. Upon beta VLDL uptake, the total volume of acid-phosphatase-positive organelles increased threefold over 30 minutes, and the reaction product was found in three additional morphologically distinct structures: tubular lysosomes, membrane stacks, and endoplasmic reticulum with widened cisternae. The proportion of the cell occupied by each of the five acid-phosphatase-positive organelles was quantitated at 10 minutes, 30 minutes, 1 hour, and 4 hours of beta VLDL incubation, and their relative abundance was compared with controls that were processed either with no lipoprotein challenge or albumin incubation for 1 hour. Secondary lysosomes compartment volume peaked at 30 minutes; over the ensuing 3.5 hours, however, the reaction progressively shifted to three new membrane-limited locations. Our observations document the complex 3D organization and spacial relationships among the acid-phosphatase-positive structures induced by lipoprotein uptake. The 3D organization patterns for acid-phosphatase-positive lysosomes in lipoprotein-stimulated pigeon monocyte/macrophages were similar in several aspects to the complex lysosomes previously observed in the macrophages of pigeon arterial lesions.

Acid Phosphatase↗
PubMed1990

Image analysis and mathematical correlation between cell number and diameter of colonies in soft agar as a measurement of growth in soft agar.

Automated image analysis was used to measure the colony diameter and estimate the cell number in colonies under conditions for anchorage-independent growth (AIG). The calculated values for the number of AIG cells in a colony of a diameter greater than or equal to 60 microns were decidedly different from the actual values obtained by either automated or manual cell counting. Like animal cells that exhibit AIG, these colonies demonstrating anchorage independence can be enumerated reproducibly. However, the assumption is incorrect that the number of cells (N) in a colony expressing AIG varies directly with a change in the diameter of a colony (d). Whether one counts the number of cells manually in a colony of a definite diameter or arbitrarily uses a diameter function of greater than or equal to 60 microns as a linear distance that indicates the presence of 50 or more cells in a colony, each procedure has its own built-in bias. In procedures that require quantitative data, the most reliable procedure is to standardize the values for diameter of colonies with cell numbers in colonies.

Agar↗
PubMed1990

Early atherogenesis in White Carneau pigeons: effect of a short-term regression diet.

The intracellular distribution of lipid shifts from cytoplasmic to lysosomal localization during the progression of atherosclerosis. It has been suggested that this shift may relate to regressability of lesions. The effects of reducing plasma cholesterol on the regression of early cholesterol-induced atherosclerosis were evaluated. Most small, early lesions disappeared after 5 weeks on the regression regimen. Larger lesions, however, did not change in extent even following 10 weeks regression. Although large lesions were not reduced in size under the regression conditions, total cytoplasmic lipid decreased. Paradoxically, the size of residual intracellular lipid deposits increased. The structural features of these remaining deposits suggest that they were lipid-filled lysosomes. Leukocyte adherence to endothelium, which increases 10- to 20-fold during progression, returned to control levels over most areas of large lesions. Levels of adherent leukocytes remained elevated, however, over small lesions and at the edges of larger lesions. Our data indicate that regression is not a uniform process, but rather, even in early lesions, varies within separate intimal microdomains. In addition, our data suggest that part of the difference may reside in differential partitioning of lipid into lysosomes.

Animals↗
PubMed1990

Fibrinogen and glycoprotein IIb/IIIa localization during platelet adhesion. Localization to the granulomere and at sites of platelet interaction.

Platelet membrane glycoprotein IIb-IIIa plays a focal role in primary hemostasis by serving as the cell surface receptor for fibrinogen. Recent studies by several groups have suggested that GPIIb-IIIa, which is dispersed randomly in the resting cell, undergoes migration leading to receptor clustering after platelet activation. The authors have investigated this activation-dependent relocation of fibrinogen receptors on platelets adherent to a standardized artificial surface. The correlative use of immunogold electron microscopy, ligand-gold binding, and stereo (three-dimensional) electron microscopy (EM) revealed specific localization of fibrinogen and its receptor at points of platelet to platelet interaction. Fibrinogen distribution on the plasma membrane, studied through the use of fibrinogen-gold conjugates with whole-mount adherent platelets, was primarily over the granulomere and at the cell periphery corresponding to sites of platelet-platelet interaction. Compared with the general hyalomere, fibrinogen density over the granulomere and at contact regions was increased 12-fold and 22-fold, respectively, and the specificity of binding at these sites was verified by positive competition with native fibrinogen, one of its degradation products (Fragment D1), and by monoclonal antibodies (HP1-1d and AP-2) specific for GPIIb-IIIa. The distribution of receptor antigens, localized by immunogold EM using antibodies against GPIIb-IIIa, also was localized over the hyalomere, where fibrinogen did not bind. To understand this apparently nonfunctional hyalomere GPIIb-IIIa further, correlative immunocytochemistry was performed using polyclonal and monoclonal antibodies for GPIIb and GPIIIa simultaneously. Colocalization of the antigens was observed consistently over the granulomere and at regions of cell contact, whereas the hyalomere antigens tended to be nonassociated. The studies document GPIIb-IIIa as a function complex at sites of cell interaction where fibrinogen binds.

Cell Communication↗
PubMed1989

Morphological characterization of beta-VLDL and acetylated-LDL binding and internalization by cultured pigeon monocytes.

The ultrastructure of binding, internalization, and translocation of beta-migrating very low density lipoprotein (beta-VLDL) and acetylated low density lipoprotein (Ac-LDL) by cultured pigeon monocytes was examined using lipoprotein-gold conjugates. Through morphometry, differences in the binding and uptake of beta-VLDL-gold and Ac-LDL-gold were documented. Cells exposed to either beta-VLDL-gold or Ac-LDL-gold for 2 hr at 4 degrees C had the label over noncoated regions of the plasma membrane. Upon warming the cells to 37 degrees C for 2 min, 35% of the surface-bound beta-VLDL-gold was within coated pits on the cell surface. Although coated pits occupied less than 2% of the surface, binding of beta-VLDL-gold was 53 times more concentrated in coated pits as compared to noncoated membrane regions. In contrast, Ac-LDL-gold neither bound to coated pits nor relocated into coated regions of the membrane upon warming to 37 degrees C. Both the beta-VLDL-gold and the Ac-LDL-gold were internalized when the cells were rewarmed at 37 degrees C. Most of the internalized gold particles for both lipoproteins were located in electron-lucent vesicles; however, 9% of the intracellular beta-VLDL-gold was observed within coated vesicles at early times. Upon prolonged rewarming (30-90 min), both lipoprotein-gold conjugates were within acid phosphatase-positive lysosomes. Ultimately 83% of the Ac-LDL-gold and 90% of the beta-VLDL-gold were within electron-dense and electron-lucent lysosomes. These results suggested that the receptor-mediated binding and internalization of beta-VLDL and Ac-LDL by pigeon monocyte macrophages proceeded by separate, distinct routes; beta-VLDL by both coated and noncoated pathways while Ac-LDL was internalized exclusively by noncoated mechanisms. Regardless of these internalization differences, both lipoproteins were delivered to lysosomes for degradation.

Acid Phosphatase↗
PubMed1989

Effects of varying dietary fatty acid ratios on plasma lipids and platelet function in the African green monkey.

The influence of varying dietary fatty acid ratios on plasma lipids, platelet function and the potential for thrombosis was evaluated in the African green monkey (Cercopithecus aethiops), an animal model widely used in cardiovascular research. Ten adult animals, 5 males and 5 females, at intervals of 2 months, were fed a series of 7 diets with fatty acid ratios (P:S) ranging from 3:1 to 1:4. Platelet aggregation in vitro, plasma levels of beta-thromboglobulin and platelet factor 4, platelet membrane fatty acid composition and plasma lipids including total cholesterol, HDL and LDL were monitored at the end of each dietary period. Platelet hypersensitivity to ADP aggregation (3 and 10 microM) and plasma beta-thromboglobulin were elevated in both males and females when dietary P:S exceeded 1.5:1 (beta-TG = 45 ng/ml) as compared to control diets either reflecting current North American or that recommended as a desirable dietary goal (P:S = 1:1, beta-TG = 10 ng/ml). Diets enriched in saturated fatty acids (P:S = 1:2) also altered platelet function, but the effects were most consistently observed in female animals (beta-TG = 32 ng/ml). Platelet hypersensitivity was lost and beta-TG levels were at baseline when the animals were returned to the control diets. Platelet sensitivity did not correlate with membrane composition which generally reflected dietary composition. Both the saturated and the polyunsaturated fatty acid enriched diets lowered plasma HDL levels, and the saturated fatty acid diets elevated plasma LDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗