HNE-derived 2-pentylpyrroles are generated during oxidation of LDL, are more prevalent in blood plasma from patients with renal disease or atherosclerosis, and are present in atherosclerotic plaques.

Free radical oxidation of human plasma low-density lipoprotein (LDL) produces 2-pentylpyrrole epitopes that are generated by reaction of 4-hydroxy-2-nonenal (HNE), a product of lipid oxidation, with protein lysyl residues. The HNE-derived 2-pentylpyrrole ("HNE-pyrrole") epitopes were detected with an enzyme-linked immunosorbent assay (ELISA) using antibodies (ON-KLH) raised against protein-bound 2-pentylpyrrole obtained by the reaction of 2-oxononanal (ON) with keyhole limpet hemocyanin (KLH). HNE-pyrrole epitopes in human plasma are not associated primarily with LDL protein, apolipoprotein (apo) B, since only 15% of the total HNE-pyrrole immunoreactivity is removed by immunoprecipitation of apo B. The levels of ON-KLH immunoreactivity detected in human plasma were found to be significantly elevated in renal failure and atherosclerosis patients when compared to those in healthy volunteers. HNE-pyrrole immunoreactivity was also detected in atherosclerotic plaques. The highest levels were associated with extracellular connective tissue. Levels of ON-KLH immunoreactivity in human plasma far exceed levels of free HNE, presumably because of the rapid clearance of free relative to protein-bound HNE. Therefore, HNE-pyrrole epitopes provide a more indelible marker of oxidative injury than levels of free HNE.

Deficiency of P-selectin in a patient with grey platelet syndrome.

Patient B.G. is a 29-yr-old female with a lifelong bleeding disorder characterized clinically by a highly increased bleeding time, menorrhagias, long-lasting bleeding after cuts and tooth extractions and large post-traumatic haematomas. Her coagulation tests were within normal range, platelet count was 140,000-160,000 per microliters, but platelet function was impaired as demonstrated by the absence of collagen-induced aggregation, although no abnormalities were detected in aggregation response to ADP and ristocetin. Morphologically her platelets were characterized by gigantic size-average profile area was about 2.5 times higher than that of control donors, and severe deficiency of alpha-granules-only 16% of their number in control donors. These features taken together indicated the diagnosis of grey platelet syndrome. As has been shown by quantitative immunoblotting, patient's platelets contained small amounts of alpha-granule membrane protein P-selectin-about 15% of that in control donors. The content of plasma membrane glycoproteins IIb-IIIa and Ib was not reduced, suggesting the specific deficiency of alpha-granule membrane protein. Thus, B.G. is the second patient described in the literature (see also Lages et al, J Clin Invest 1991: 87: 919-929) with combined deficiency of alpha-granules and P-selectin.

Centriole modification in human aortic endothelial cells.

The structure of centrioles in endothelial cells of embryonic (22-24 weeks old) and definitive (2, 14-17, and 30-40 years) human aorta in situ and also in aortic endothelial cells dividing in organ and cell cultures (donor age 30-40 years) was studied. It was found that in the endothelial cells from definitive aorta the lengths of mother centrioles vary from 0.5 to 2 microns, whereas the length of daughter centrioles remains constant (0.4-0.5 microns). The distal part of the cylinder of long mother centrioles consists of microtubule doublets. In aorta of donors 30-40 years old in multinucleated cells and in one of 30 single-nucleated cells analyzed, C-shaped long centrioles were seen. These centrioles exhibit a doublet organization along all their length. Mitotic cells in organ and cell culture had a nonequal structure of spindle poles: at one pole, the long mother centriole was seen, while at the other a mother centriole of standard size was found. In such cells of organ culture long centrioles make contact with the remnant of primary cilia until the end of anaphase. In cell culture mitotic cells are also observed containing C-shaped centrioles. In these cells the number of mother centrioles is odd and their number is not equal to the number of daughter centrioles. The possible mechanism for transformation of endothelial centrioles and its role in the control of cell-cycle progression are discussed.

Identification of intimal subendothelial cells from human aorta in primary culture.

Subendothelial cells (SEC) were obtained from the inner intimal layer of adult human aorta by collagenase treatment. SEC were identified in primary culture either as smooth muscle cells by staining with FITC-labeled antisera against human smooth muscle myosin or as macrophages, foam cells and contaminating endothelial cells by their uptake of malondialdehyde treated low density lipoproteins labeled with fluorescent dye 3,3'-dioctadecylindocarbocyanine. Between 1 and 5 days in culture, along with smooth muscle cells (SMC, 38-82%), endothelial cells (0-9%), macrophages and foam cells (2-32%), one more type of cell was found. This cell type resembled SMC in size and shape, but was not stained by antisera to SMC myosin. By ultrastructural criteria these cells were characterized as modulated SMC for they contained prominent rough endoplastic reticulum and Golgi complex together with basement membrane and a large number of plasmalemmal vesicles. Like SMC they reacted with phalloidin and were stained by anti-vimentin but not by anti-desmin monoclonal antibodies. The proportion of such cells varied from 5 to 33% of total cell number and increased in parallel to macrophages and foam cells in vessels with well developed atherosclerotic lesions. We conclude that the applied technique may be used for identification of cultured vascular cells including modulated SMC.

Intimal cells and atherosclerosis. Relationship between the number of intimal cells and major manifestations of atherosclerosis in the human aorta.

The subendothelial intima of human aorta is populated by cells of various shapes. Round and ovoid cells which are lymphocyte- and monocyte-like hematogenous cells account for less than 5% of the cell population. The bulk of the intimal population (over 95%) is made up of cells that can be described as elongated, stellate, elongated with side processes, and irregularly shaped. To identify these morphologic forms, the authors have used target electron microscopy. It has been established that elongated cells devoid of side processes possess all the ultrastructural features of differentiated smooth muscle cells: a developed contractile apparatus in the form of microfilament bundles with dense bodies occupying most of the cytoplasm, basal membrane surrounding the whole of the cell, and micropinocytotic vesicles along the plasma membrane. The other morphologic forms have an ultra-structure that allows us to identify them as so-called modified smooth muscle cells. They differ from the typical smooth muscle cells in that they have fewer contractile structures and a more developed biosynthetic apparatus. Some of stellate and irregular shaped cells are utterly devoid of contractile structures. To quantitate the number of cells of different morphologic forms, the authors used alcoholic-alkaline dissociation of prefixed intima. It was established that the intimal population is multiplied at the site of an atherosclerotic lesion, the number of stellate cells being increased much more substantially, compared with other morphologic cell forms. It was found that an increase in the number of stellate cells is related to such sequelae of atherosclerosis in aorta as intimal thickening, deposition of lipids, and an increased amount of collagen. There was a high positive correlation between the alteration in the stellate cell number occurring in the intima and the above-mentioned parameters (correlation coefficients were 0.732, 0.800 and 0.953, respectively). The correlations between these indexes and the total number of intimal cells or the number of cells belonging to each of the other morphologic forms were not so high. A multivariate analysis gave similar results. Thus, it may be suggested that stellate cells are the principal cell type involved in the disease. This report discusses the origin of stellate and other intimal cells and their role in atherogenesis.

Adult human aortic cells in primary culture: heterogeneity in shape.

Adult human aortic cells have different shapes in situ. To determine whether populations of cultured aortic cells are also polymorphic, a technique for separation of cells from the intimal and medial layers of the human aorta by enzymatic dispersion of the vascular tissue was employed. It was established that aortic cells are polymorphic in primary culture, at least within the first 7 days after seeding. Four main morphological cell types were identified--elongated, asymmetric, polygonal, and stellate. Polygonal and stellate cells are found only in cultures of grossly normal intima. Elongated and asymmetric cells are present in practically all cultures. The ratio of elongated to asymmetric cells in cultures obtained from healthy aortas and atherosclerotic plaques is more or less the same and is approximately 3:1. In cultures of fatty streaks, the portion of asymmetric cells exceeds 50%. With immunofluorescent staining and ultra-structural analysis, cells of all four types were identified as smooth muscle. Possible reasons for the cells polymorphism in primary culture and the prospects of utilizing this culture method in the investigation of cellular aspects of atherogenesis are discussed.

Lipids in cells of atherosclerotic and uninvolved human aorta. I. Lipid composition of aortic tissue and enzyme-isolated and cultured cells.

Phospholipid, triglyceride, cholesterol, and cholesteryl ester contents were measured in unaffected and atherosclerotic areas of human aorta and in a suspension of enzyme-isolated cells from these segments. Aortic tissue and the cells isolated from it, as well as intimal and medial cells, significantly differ in lipid content. As lipoidosis develops in an atherosclerotic lesion, lipids accumulate unevenly in the tissue and cells. In zones of fatty infiltration, lipids accumulate, apparently, mainly inside cells while in the fatty streak and atherosclerotic plaque they predominate in the extracellular space. In a suspension of cells derived from both an atherosclerotic lesion and the underlying media, cholesteryl esters are the main component of excessive fat. In the primary culture of cells enzyme-isolated from unaffected intima, fatty streak, and plaque, the lipid content and composition are retained until Days 12 to 14 and are similar to those of freshly isolated cells.

Cellular composition of atherosclerotic and uninvolved human aortic subendothelial intima. Light-microscopic study of dissociated aortic cells.

Alcoholic-alkaline dissociation was used in the study of cellular composition of human aorta. Cells were isolated from an uninvolved intima and intima with different types of atherosclerotic lesions: fatty infiltration, fatty streak, and atherosclerotic plaque. In the isolated suspension we evaluated the ratio of four previously described morphologic forms of cells: stellate, elongated, elongated with side processes, and flat cells of irregular shape. It was demonstrated that the quota of stellate cells in an atherosclerotic lesion considerably exceeds that of the normal intima. For elongated cells the opposite is true. The other two cell forms are represented in the uninvolved and atherosclerotic intima in approximately equal proportions. Alteration of the ratio of different morphologic forms occurs because of the fact that the number of cells belonging to different morphologic forms increases disproportionately in the lesion zone. Specifically, the number of stellate cells is increased much more substantially, compared with elongated cells.

Heterogeneity of human aortic cells in regard to RNA content.

Cells of human aorta were isolated by dispersing the tissue with collagenase and elastase. The isolated cells were stained in suspension by the acridine orange fluorescent stain. The intensity of red fluorescence (greater than 580 nm) corresponding to the RNA content was measured in each individual cell and registered in a FACS II flow cytofluorometer. It was established that a cell population of human aorta is heterogenous with respect to RNA content. In a population of isolated cells, one can distinguish two subpopulations: (A) small cells with low RNA content, and (B) large cells with high RNA content. The ratio of both cell types varies in intima and media, and different types of atherosclerotic lesions. The share of cells belonging to subpopulation A is lower in media compared to intima. In intima, the number of these cells grows with the degree of atherosclerotic lesion. Possible reasons for the discovered metabolic heterogeneity of human aortic cells and prospects for the application of flow cytofluorometry to a research into cellular mechanisms of atherosclerosis is discussed.

Dissociated cells from different layers of adult human aortic wall.

Cells isolated from a fixed adult human aorta by alcoholic-alkaline dissociation retain their intrinsic shape. They are represented by four major morphological types: stellate, elongated, elongated with side processes, and irregularly shaped cells. All the four types of cells were found in the elastic-hyperplastic intimal sublayer ; elongated and irregularly shaped cells were mainly observed in the musculoelastic sublayer and in the media. Cell density in the atherosclerotic lesion is higher than normal, the number of stellate cells being increased more substantially compared to other cell types. The origin of stellate cells and other morphological cell types, and reasons for their disproportionate accumulation in the atherosclerotic intima are discussed.

Primary cultures of enzyme-isolated cells from normal and atherosclerotic human aorta.

A technique has been developed for isolating cells from the intimal and medial layers of the human aorta by enzymatic dispersion. After mechanical separation of intima, media and adventitia the intima and the media were dispersed by collagenase and elastase. Enzyme-isolated cells seeded in the culture with at a frequency of 30 to 50%. In the primary culture differentiated aortic cells were morphologically heterogenous. It was possible to define four main types of cells according to their shape: polygonal, elongated, asymmetrical and stellate. Polygonal and stellate cells are found only in cultures of grossly normal intima, whereas elongated and asymmetric cells are found in practically all cultures. The ratio of elongated to asymmetric cells in cultures obtained from healthy aorta and atherosclerotic plaque is more or less the same at approximately 3:1. In cultures of fatty streaks the proportion of asymmetric cells exceeds 50%. Using immunofluorescence, all four types of cell were identified as smooth muscle cells. The possible reasons for the cellular polymorphism in primary culture and the prospects of utilizing this culture for the study of cellular aspects of atherosclerosis' pathogenesis are discussed.

Stellate cells in the intima of human aorta. Application of alkaline dissociation method in the analysis of the vessel wall cellular content.

Cells isolated from the fixed human aorta by alkaline dissociation have been studied. Cellular content of normal and atherosclerotic intima is described. Special attention is given to stellate cells that were found in large quantities in all analyzed samples. It is found that in the fatty streak stellate cells are localized nearest to the endothelium but they have no definite localization in the fibrous plaque. In unaffected intima, stellate cells are diffusely localized, and often prevail over other cell types.