Search

We performed a systematic analysis of gene expression in arteries and veins by comparing message profiles of macaque aorta and vena cava media using a cDNA array containing 4048 known human genes, approximately 35% of currently named human genes (approximately 11,000). The data show extensive differences in RNA expression in artery versus vein media. Sixty-eight genes had consistent elevation in message expression by the aorta, but none were elevated in the vena cava. The most differentially expressed gene was regulator of G-protein signaling (RGS) 5, at an expression ratio of 46.5+/-12.6 (mean+/-SEM). The data set also contained 2 genes already known to be expressed in the aorta, elastin at 5.0+/-1.4, and the aortic preferentially expressed gene 1 (APEG-1) at 2.3+/-0.6. We chose to analyze RGS5 expression further because of its high level of differential expression in the aorta. Levels of RGS5 mRNA were confirmed by Northern analysis and in situ hybridization. A human tissue RNA dot blot showed that RGS5 message is highest in aorta, followed by small intestine, stomach, and then heart. Northern analysis confirmed that RGS5 expression in human aorta is higher than in any region of the heart. RGS5 is a G-protein signaling regulator of unknown specificity most homologous to RGS4, an inhibitory regulator of pressure-induced cardiac hypertrophy. The expression pattern of the 68 differential genes as a whole is a start toward identifying the molecular phenotypes of arteries and veins on a systematic basis.

X chromosome inactivation studies indicate that smooth muscle cells in human atherosclerosis are monoclonal. Monoclonality could arise either by 1) proliferation of a single cell in the adult intima, eg, by selection or mutation, or 2) proliferation of many cells within a large, pre-existing clonal patch that formed during development. To determine whether clonal expansion occurs concomitantly with plaque growth or as part of normal development, X chromosome inactivation patterns were mapped in microdissected samples of aortic smooth muscle, using the human androgen receptor locus as a marker. As expected, 43% of plaque samples were skewed toward one X chromosome, indicating a monoclonal population. Surprisingly, 25% of normal medial samples and 31% of diffuse intimal thickening samples also were skewed toward one X chromosome, indicating a relatively large patch size. Furthermore, 30% of diffuse intimal thickening and 22% of medial samples showed contiguous regions of 4 mm skewed to the same allele, showing that patch length often exceeded 4 mm. Intima and overlying media typically were skewed to the same allele (73% concordance), suggesting common cells of origin. Because patch size is large in normal arteries, X-inactivation analysis cannot discriminate between a monoclonal and a polyclonal origin of plaque smooth muscle cells. We propose that human arteries grow by expanding coherent smooth muscle clones, with little mixing of adjacent clones. Determining whether plaques arise by clonal expansion will require other approaches, such as analysis of somatic mutations; the finding of large X-inactivation patches raises the possibility that plaques arise from a pre-existing (developmental) clone.

Atherosclerotic plaques contain a large monoclonal population of cells. Monoclonality could arise by somatic mutation, selection of a pre-existing lineage, or expansion of a pre-existing (developmental) clone. To determine the monoclonal cell type in plaque and learn when monoclonality arises, we studied X chromosome inactivation patterns using methylation of the X-linked human androgen receptor gene. Assays based on polymerase chain reaction were performed on samples of known cellular composition, microdissected from histological sections of human arteries. In atherosclerotic vessels, the majority of medial samples (7/11 coronary and 2/3 aortic) showed balanced (paternal and maternal) patterns of X inactivation, indicating polyclonality. In contrast, most samples of plaque smooth muscle cells showed a single pattern of X inactivation (3/4 aortic plaques and 9/11 coronary plaques; P < 0.01 versus media), indicating that plaque smooth muscle cells are monoclonal. Samples of plaque containing inflammatory or endothelial cells showed balanced X inactivation, also demonstrating polyclonality. Multiple plaques from a given patient showed no bias toward one allele, indicating there was no X-linked selection of cells during plaque growth. To determine whether plaques might arise from pre-existing clones (large X inactivation patches), we then studied 10 normal coronaries with diffuse intimal thickening. Six of the ten coronaries showed skewed X inactivation patterns in normal media and intima, suggesting the patch size in normal arteries is surprisingly large. Thus, smooth muscle cells constitute the monoclonal population in atherosclerotic plaques. The finding that normal arteries may have large X inactivation patches raises the possibility that plaque monoclonality may arise by expanding a pre-existing clone of cells rather than generating a new clone by mutation or selection.

Background: To understand which growth factors are important for growth of atherosclerotic plaques, it is necessary to know the factor's relative abundance and how its gene is regulated in relation to cell proliferation. We tested whether platelet-derived growth factor-A (PDGF-A) mRNA levels correlated with cell proliferation in developing aorta, normal adult aorta, and atherosclerotic plaques., Methods and Results: We developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure human PDGF-A mRNA levels in small tissue samples. A mutated PDGF-A synthetic RNA was used as an internal standard to compete with endogenous PDGF-A mRNA for amplification. The assay is highly sensitive and much more precise than routine RT-PCR. Correction for heteroduplex pairing between the endogenous and mutant PCR products correlates precisely with synthetic RNA standards and quantitative Northern blotting. Immunostaining with the proliferation marker (proliferating cell nuclear antigen) showed the following rank order of proliferation: fetal aorta >> atherosclerotic plaque > normal aortic media. PDGF-A mRNA levels, however, did not correlate with proliferation. Normal adult aorta contained the most PDGF-A mRNa (34.0+/-7.6 amol/microgram total RNA). Fetal aortas were intermediate (10.2+/-1.6 amol/microgram total RNA); advanced atherosclerotic plaques contained the least PDGF-A mRNA (0.3+/-0.1 amol/microgram total RNA). PDGF-A protein was readily detectable in normal media by immunostaining. Advanced plaques generally had less cell-associated PDGF-A protein, although A-chain was also detected in plaque matrix., Conclusions: PDGF-A mRNA and protein do not correlate with proliferation among these three groups. The significance of high levels of PDGF-A mRNA in the "quiescent" aortic media is unknown but it clearly does not promote cell replication.

A comparative study of human papillomavirus type 16 E6E7-transfected and normal human aortic smooth muscle cells by morphological, electron microscopic, immunofluorescent, and biochemical analyses demonstrated that the E6E7-expressing cells retained much of the phenotype of normal aortic smooth muscle cells, including expression of smooth muscle markers and appropriate growth responses to PDGF and heparin. These cells differed from normal vascular smooth muscle cells in that they had slightly altered morphology and a higher growth rate that was not due to an autocrine response to secreted PDGF, and they contained more polyribosomes than normal smooth muscle cells.

We describe our studies on the characterization of the cell phenotypes in the wall of the aortic-arch-derived arteries from "late" chicken embryos. Using immunocytochemistry with antibodies against smooth muscle cytoskeletal and basement membrane proteins, we show that the smooth muscle of the aortic-arch-derived arteries from 13- to 19-d-old embryos contains two cell phenotypes organized in interchanging lamellae. One cell phenotype (lamellar cells), but not the other cell phenotype (interlamellar cells), expresses the cytoskeletal proteins desmin and alpha smooth muscle actin (alpha SMactin). Both cell phenotypes express the cytoskeletal protein vimentin. Furthermore, the lamellar cells but not the interlamellar cells are surrounded by the basement membrane proteins laminin and collagen type IV. Performing quail-chick transplantation experiments and using a quail specific antibody, we show that both lamellar and interlamellar cells in the "aortic arches" of a 15-d-old chimeric embryo are derived from neural crest cells. We conclude that the aortic smooth muscle cells from "late" chicken embryos consist of two distinct cell phenotypes which are derived from a common lineage.

Transport parameters that describe a macromolecule entering the arterial wall from plasma can be obtained from concentration profiles of the labeled macromolecule entering the tissue. A new technique has been developed for measuring such concentration profiles, which offers spatial resolution superior to methods that measure profiles of radiolabeled macromolecules by serially sectioning tissue in planes parallel to the endothelium. In addition, this new method preserves cellular organization and tissue structure and permits measurement of concentration profiles underlying focal endothelial injuries or vascular lesions. The technique quantifies the concentration of a protein by measuring associated peroxidase activity. Although the present study was performed using horseradish peroxidase (HRP), the same principles can be applied to other macromolecules linked to HRP or microperoxidase. The colored reaction product of HRP was detected in transverse aortic sections using an image processing system. In the present study, profiles obtained by this new method were validated by comparison with HRP concentration profiles in rat aortas obtained by a serial slicing technique using radiolabeled HRP. We used the technique to measure high-resolution HRP concentration profiles in the intima and media of normal animals. These concentration profiles suggest that the internal elastic lamina acts as a major barrier to transport of macromolecules across the wall of the normal rat aorta. The new method should allow concentration profiles for macromolecules to be quantified in tissue surrounding vessels in the microcirculation, within the thickened intima of large vessels, and across coronary artery walls.

Sympathetic nerves and catecholamines exert growth-promoting trophic influences on arterial smooth muscle in vivo, but the molecular signals mediating these trophic effects are unknown. We report here that the alpha-adrenergic agonist phenylephrine (PE) produced dose-dependent stimulation of platelet-derived growth factor A-chain (PDGF-A) gene expression in rat thoracic aorta via agonist occupancy of alpha 1-adrenergic receptors. Increases in aortic PDGF-A mRNA levels were rapid (maximal at 6 h, 10-fold) and transient. Among seven different tissues studied, PE evoked significant increases in PDGF-A mRNA levels only in the aorta. When periaortic fatty/connective tissues normally adherent to thoracic aorta were examined separately from the remaining aortic vessel wall (endothelium removed), stimulated PDGF-A gene expression was found only in vessel wall (presumably smooth muscle). The physiological alpha-adrenergic agonist norepinephrine also increased aortic PDGF-A mRNA levels. Angiotensin II or endothelin, despite producing blood pressure increases similar to PE, had little or no effect on PDGF-A mRNA abundance in rat aorta. PE-stimulated PDGF-A gene expression was accompanied by increased expression of other growth-related genes including c-fos, c-myc, and ornithine decarboxylase but not DNA synthesis. These results suggest a mechanism for previously described trophic effects of sympathetic nerves and catecholamines on arterial smooth muscle mass, i.e. regulation of growth-related gene expression via alpha 1-adrenergic receptors.

The pattern of early cell movement after an experimental 'wound' and the organization of actin in stationary and moving cultured endothelial cells have been studied by means of: time-lapse photography; indirect immunofluorescence using anti-actin antibodies with and without pretreatment with the actin destabilizing factor present in human plasma; and differential centrifugation and densitometric analysis of stained sodium dodecylsulphate/polyacrylamide gels in order to evaluate the total and relative amounts of G and F-actin. Up to 5 h after a single scratch, movement consists of a coordinate spreading and translocation of a band of about 10 cells from the wound edge. Compared to stationary cells, moving endothelial cells show: no significant changes in the intensity and distribution of immunofluorescent staining with anti-actin antibodies, but an increased sensitivity of cytoplasmic actin, including stress fibres, to the actin-destabilizing factor purified from human plasma; and no significant change in the total amount of actin, but a decreased relative amount of F-actin and a corresponding increased relative amount of G-actin. We conclude that endothelial cell movement in vitro is accompanied by a rapid change in the state of actin organization characterized by an overall decrease in cytoplasmic F-actin.

Although on the average aortic endothelium is a highly stable tissue, available data indicate that there are areas of high, spontaneous, focal cell turnover. This may be significant since the response to experimental endothelial denudation includes thrombosis and smooth muscle proliferation, both components of atherosclerosis lesion formation. Thus questions about mechanisms of endothelial injury and about the role of the repair process in maintenance of endothelial integrity are likely to be important in attempting to understand the pathogenesis of atherosclerosis. Our approach is to ask what factors control endothelial regeneration. Studies include both in vitro and in vivo data. Regeneration in vivo is strongly dependent on the geometry of the vessel; recovery is much more rapid along the vessel axis. This appears to explain the localization of smooth muscle lesions following mechanical removal of the endothelium, since rapidly recovered areas do not develop smooth muscle proliferation. In vitro, regeneration is independent of a requirement for growth factors, implying that the signal for this process is intrinsic to the monolayer. Cytochalasin B inhibits both movement and replication, implying that movement may be required for transition from G0 to G1. Finally, we have discovered that cultured endothelial cells themselves produce a growth factor. This is of potential importance for interactions between smooth muscle cells and endothelial cells in the vascular response to injury.

Endothelial cell-derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat-stable and trypsin-sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen-secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56 degrees C) and were not inactivated by trypsin. Similar to platelet-derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and other criteria.

Aortas of male rabbits (2.0 to 2.5 kg body weight) were denuded of endothelium with a 4F balloon catheter and killed 7, 14, 60, 90, and 180 days later. Aortic endothelial cells regrowth was determined using Evans blue dye and scanning electron microscopy. Early endothelial outgrowth was rapid, but by 14 days the regrowth slowed markedly so that no further increase of endothelial outgrowth was detected at other time periods. Another group of animals received 3H-thymidine 1 hour before death and were killed 4, 14, and 42 days later. The endothelial cells replication in the aortas of these animals was measured from Häutchen preparations. Endothelial cells replication was observed 4 days after balloon injury but was markedly reduced by 14 and 42 days. Intimal smooth muscle cell replication, however, was observed at both these later times. These result demonstrate that in vivo endothelial cells replication stops long before the aorta is repopulated and suggest that some mechanism other than contact between endothelial cells can prevent endothelial cell replication.

Arterial smooth muscle cells become tetraploid with age and hypertension. To further study this phenomenon, neonatal rat aortic smooth muscle cells were placed in cell culture and studied over time. Numerous cells with tetraploid and even octaploid DNA content appeared beginning in primary cultures. These increases in DNA content per cell were determined by quantitative fluorescence microscopy and flow cytometry, and true polyploidy was confirmed by chromosome counts. In contrast, cells from adult rat aortas failed to produce significant polyploid cells over time in culture. In vitro culture of neonatal aortic cells may therefore be a model system for studying the initiation of polyploidy in arterial smooth muscle.

Bovine coagulation factors IX and X bind to independent sites on bovine aortic endothelial cells. Binding studies with cells maintained serum-free showed that there are at least two classes of binding sites for factor IX and factor X with a dissociation constant of 4.9 x 10(-9) M and 2.1 x 10(-8) M for the respective high affinity sites. Ca+2 was required for specific binding and was reversed by addition of EDTA or EGTA. Competition experiments showed that factor IX and factor IXa bind to the same sites, which are different from the factor X binding sites. Neither binding of factor IX or factor X is inhibited by addition of prothrombin or protein C. Indirect immunofluorescence of factor IX indicated that binding was diffuse on the cell surface.

This article reports the ultrastructure of the aortic lining during the repair of mechanically denuded aortic intima in the rat. Three main features were observed: a) Although platelets form a pavement on the exposed components of the aortic intima, platelet thrombi do not form on the denuded surface. b) During the first weeks after injury, a temporary false endothelial lining is formed by modified intimal smooth muscle cells. While the modified smooth muscle cells do not constitute a continuous cell layer, they are like true endothelial cells in that platelets do not adhere to the cell membrane of either cell type. c) A continuous layer of true endothelial cells is formed within 2 months after the original injury. Even after reestablishment of a continuous endothelium, however, abnormalities persist in the form of incompletely formed intercellular junctions. This abnormal endothelium is associated with areas of intimal smooth muscle cell proliferation. These observations are compatible with two alternative interpretations of the role of endothelial injury in the intimal proliferation seen following injury to the vessel wall: a) persistent defects in the endothelium may result in proliferation of underlying arterial smooth muscle cells or b) the proliferation, in converse, may in some manner delay the healing process of the overlying endothelium.

Relief casting of critical point-dried aortic endothelium provides high resolution images of the cell surface, revealing details not readily apparent by scanning electron microscopy or conventional transmission electron microscopy. These surface replicas show the cell junction to be highly interfolded. Caveoli are present, although some cells have many while others have few. Moreover, the caveoli appear as shallow pits, suggesting that the opening of the caveolus is filled or covered. Finally, the replicas show other surface details including microvilli and distinct cell surface textures.

Endothelial cell-conditioned medium contains two classes of factors distinguishable by behavior during dialysis and on specificity for cell type. One species, which diffuses through dialysis tubing with an exclusion limit of 6,000 to 8,000 daltons, supports growth of bovine aortic endothelial (BAE) cells in medium containing a growth limiting concentration of serum (0.2% serum). The production of this material appears to depend upon the presence of serum in the medium being conditioned. The activity increases with time of exposure of BAE cells to serum and with increasing concentration of serum present in the incubation medium. This activity cannot be replaced by exogenous epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin, or thymidine. The second species, the endothelial cell-derived growth factor (ECDGF), is retained by dialysis tubing with an exclusion limit of 6,000 to 8,000 daltons. ECDGF stimulates the growth of smooth muscle cells but does not support BAE cell growth in limiting serum concentrations. unlike the dialyzable species, the production of ECDGF is independent of previous incubation of BAE cell cultures in serum. These studies suggest that BAE cells are able to utilize serum components to produce conditioning factors for their own growth that are distinct from the higher molecular weight ECDGF.

Platelet-derived growth factor (PDGF) is thought to mediate the proliferation of smooth muscle cells in injured arteries, and may be involved in the pathogenesis of atherosclerosis. PDGF-like molecules from non-platelet sources may also play a role in the regulation of cell activity in other circumstances. Transformation of cells by a wide range of oncongenic agents appears to activate a cellular gene encoding a PDGF-like molecule, possibly accounting for the ability of transformed cells to grow without addition of exogenous mitogens. We show here that a molecule (PDGF-c) which can compete with 125I-PDGF for binding to PDGF receptors is secreted by cultured rat aortic smooth muscle cells (rASMC) isolated from 13 to 18-day-old rats (pups) but not from three-month-old animals (adults). Thus, production of PDGF-c appears to be developmentally regulated and may be a factor in the more rapid proliferation of rASMC and synthesis of connective tissue components which occurs during growth of the aorta in vivo.

This paper presents different approaches for studying arterial endothelial cells by scanning electron microscopy (SEM). These include post-fixation silver staining, Ligand-mediated osmium binding, vascular casting, autoradiography and Triton digestion. Post-fixation staining of cells with silver nitrate leads to more specific silver deposits at cell junctions and improved tissue preservation. Endothelium impregnated with osmium bound to ligand (OTO) showed enhanced membrane detail and rendered the tissue conductive. Resin casts of the microvasculature viewed by SEM showed that the endothelial surface of small vessels can now easily be viewed en face. 3H-Thymidine-labeled endothelial cells were recognized using SEM and details of the endothelial cell cytoskeleton were visualized by SEM after Triton digestion. These results demonstrate the versatility of SEM and suggest ways in which the scanning electron microscope can help to better study arterial endothelial cells.

Endothelial cell death and replication was analyzed in the rat aorta with the use of combined IgG immunocytochemistry and 3H-thymidine autoradiography. Dead and replicating cells were clustered in the aortic endothelium of normal rats with a low level of spontaneous cell death and replication. Death and replication were also clustered in rats treated with endotoxin (Escherichia coli lipopolysaccharide [LPS]), a substance which is cytotoxic to endothelial cells in culture. LPS caused a dramatic increase both in endothelial cell death and replication, while no endothelial denudation could be discerned with the use of the 111In-labeled platelet technique. We conclude that endothelial cell death and replication are topographically clustered phenomena, which may imply that cell death leads to a local stimulation of cell replication. Furthermore, LPS induces a massive cell death, which is paralleled by cell replication, yet does not cause any significant increase in denudation. This suggests that the primary effect of LPS on the vessel wall is its cytotoxicity for endothelial cells. Endothelial cell death appears to provide the stimulus for cell replication both in normal and in LPS-treated rats.

The response to injury of the intima of the rat aorta was studied using a nylon monofilament to produce small, defined areas of endothelial denudation. A row of endothelial cells, three to five cells wide, was removed by pulling the tip of the filament along the length of the aorta. Platelet adhered to the exposed subendothelium. After 48 hours, this wound was covered by replicating endothelial cells which were densely packed together. This increased density remained over the site of injury for at least 4 weeks. A circular injury denuded a zone one to two endothelial cells wide. This wound was repopulated by endothelial cells within 8 hours without cell replication. No intimal thickening occurred in the vessels after either injury.

A simple reproducible method is described for isolating pure clonal populations of bovine aortic endothelial cells in culture. Single cells exposed to conditioned medium grow rapidly (clonal cell doubling time approximately 20 h) until a confluent monolayer is reached. This method provides virtually 100% cloning efficiency. In addition, the cell population doubling level of endothelial cell cultures, when continuously exposed to conditioned medium, is increased 40% over controls in fresh medium.

Cell division in the aortic endothelium of the rat is not randomly distributed. Maps of the aortic surface show focal areas where the daily rate of replication may be in excess of 50%. This implies the existence of focal areas of rapid cell growth or rapid cell turnover and other areas where growth or turnover is largely absent.