Regulation of cellular proliferation and intimal formation following balloon injury in atherosclerotic rabbit arteries.

Injury to atherosclerotic arteries induces the expression of growth regulatory genes that stimulate cellular proliferation and intimal formation. Intimal expansion has been reduced in vivo in nonatherosclerotic balloon-injured arteries by transfer of genes that inhibit cell proliferation. It is not known, however, whether vascular cell proliferation can be inhibited after injury in more extensively diseased atherosclerotic arteries. Accordingly, the purpose of this study was to investigate whether expression of recombinant genes in atherosclerotic arteries after balloon injury could inhibit intimal cell proliferation. To test this hypothesis, we examined the response to balloon injury in atherosclerotic rabbit arteries after gene transfer of herpesvirus thymidine kinase gene (tk) and administration of ganciclovir. Smooth muscle cells from hyperlipidemic rabbit arteries infected with adenoviral vectors encoding tk were sensitive to ganciclovir, and bystander killing was observed in vitro. In atherosclerotic arteries, a human placental alkaline phosphatase reporter gene was expressed in intimal and medial smooth muscle cells and macrophages, identifying these cells as targets for gene transfer. Expression of tk in balloon-injured hyperlipidemic rabbit arteries followed by ganciclovir treatment resulted in a 64% reduction in intimal cell proliferation 7 d after gene transfer (P = 0.004), and a 35-49% reduction in internal area 21 d after gene transfer, compared with five different control groups (P < 0.05). Replication of smooth muscle cells and macrophages was inhibited by tk expression and ganciclovir treatment. These findings indicate that transfer of a gene that inhibits cellular proliferation limits the intimal area in balloon-injured atherosclerotic arteries. Molecular approaches to the inhibition of cell proliferation in atherosclerotic arteries constitute a possible treatment for vascular proliferative diseases.

Collagen synthesis in atherosclerosis: too much and not enough.

Fibrillar collagen is a critical component of atherosclerotic lesions. Uncontrolled collagen accumulation leads to arterial stenosis, while excessive collagen breakdown combined with inadequate synthesis weakens plaques thereby making them prone to rupture. This review discusses cellular sources of collagen synthesis in atherosclerosis, local and systemic factors modulating collagen gene expression, as well as temporal and spatial patterns of collagen production in human and experimental atherosclerotic lesions.

The importance of thrombus organization and stellate cell phenotype in collagen I gene expression in human, coronary atherosclerotic and restenotic lesions.

OBJECTIVES: Collagen synthesis is one of the major mechanisms of primary atherosclerotic plaque growth and is likely to be similarly important in restenosis. The patterns of collagen gene expression in human restenosis and associations with thrombosis/hemorrhage have not been described. METHODS: Using human coronary artery samples obtained via the atherectomy catheter, we compared primary plaques (40 specimens) and restenotic lesions (41 specimens) for type I collagen gene expression using immunocytochemistry (SPI.D8 antibody to type I procollagen, an intracellular precursor of mature collagen) with subsequent computer image analysis. RESULTS: Scattered positive cells were identified in specific, non-random patterns. According to logistic regression analyses, type I procollagen gene expression seems to be more closely associated with certain morphological features (organized thrombus, microvessels, regions enriched with stellate cells) than with belonging to a primary vs. a restenotic sample. However, there may be a tendency for restenotic tissue to have slightly higher numbers of type I procollagen-positive cells than primary lesion tissue. CONCLUSIONS: Symptomatic primary and restenotic lesions exhibit similar patterns of type I collagen gene expression. Plaque microvessels and thrombi/hemorrhages (common features of both kinds of advanced lesions) might stimulate collagen synthesis equally well irrelevant to the nature of the lesion.

Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in hypercholesterolemic rabbits.

Tissue factor (TF) expressed in arterial atherosclerotic plaque plays a key role in activating the extrinsic coagulation pathway and triggering acute coronary syndromes. In this study, we developed and characterized a TF-factor (F)VIIa-mediated thrombosis model in rabbits. Balloon catheter-induced endothelial denudation in the femoral artery and a 4-week high cholesterol diet produced a localized atherosclerotic plaque at the injured site. High levels of TF mRNA and TF protein antigen (152 +/- 25 vs. 49 +/- 12 pg mg-1 protein in normal vessels) were detected in these atherosclerotic plaques. Plasma FVII coagulant activity (FVII:C) was significantly increased in the hypercholesterolemic rabbits (36 +/- 1 s) compared with the normal rabbits (44 +/- 1 s, P < 0.0001). Plaque rupture was induced by balloon angioplasty, which resulted in thrombus formation in the injured vessel segment after a brief period of stasis. FVIIai, a specific TF-FVIIa inhibitor, was administered intravenously to rabbits before plaque rupture at 0.3 and 1.0 mg kg-1. FVIIai dose-dependently reduced thrombus mass (14.7 +/- 2.5 and 5.9 +/- 2.2 mg, respectively, vs. 21.6 +/- 1.9 mg in the control group). PD198961, a novel factor Xa inhibitor, and argatroban, a thrombin inhibitor, also dose-dependently inhibited thrombosis. These results indicate that thrombus formation in this model is initiated by the activation of TF-FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVII coagulant activity. This model may be useful for evaluating in vivo efficacy of new antithrombotic drugs, particularly TF-FVIIa inhibitors.

Endothelialized myointimal thickening in the rat aorta as a result of extensive freeze injury.

The regeneration of rat aorta intima and medial layer was examined after extensive freeze injury that caused the death of both endothelial and smooth muscle cells. After 1 month complete re-endothelialization of the denuded area was observed. In parallel, myointimal thickening was formed by the smooth muscle cells (SMC) from the uninjured edges and its thickness increased with time. The SMC of myointimal thickening were stellate in the upper part and elongated near the internal elastic lamina. No regenerative response was seen in the medial layer, consisting only of elastic and collagen fibers, and its thickness was reduced during regeneration. These results show that the regenerative response of medial SMC to extensive freeze injury proceeds in the form of intimal thickening.

Beta-blockers: propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Elimination of propranolol atherogenic effects by papaverine.

The addition of the beta-blockers propranolol, metoprolol, atenolol, pindolol, alprenolol and timolol to a culture of peritoneal macrophages or smooth muscle cells induced an increase in the intracellular cholesterol content. Blood serum obtained from a rabbit after a peroral administration of beta-blockers also induced cholesterol accumulation. This property of drug or blood serum obtained after peroral administration is conventionally referred to as atherogenic potential or atherogenicity. Regular administration of propranolol during a 21-day period evoked stable atherogenicity of rabbit blood serum. This was accompanied by stimulation of manifestations of atherosclerosis in the aorta deendothelialized with a balloon catheter. Propranolol increased neointimal thickening, lipid accumulation, an increase in cell number and in the collagen content. In vitro, the combination of propranolol with papaverine eliminated the atherogenic effect of propranolol which manifested itself as stimulation of cholesterol accumulation in cultured cells. Simultaneous peroral administration of propranolol and papaverine prevented the appearance of serum atherogenicity. Papaverine eliminated neointimal thickening, an increase in cell number and in the lipid and collagen contents evoked by propranolol. Papaverine itself had no effect on these parameters. Thus, the atherogenicity of propranolol as well as capacity of papaverine to eliminate beta-blocker atherogenicity revealed in cell culture was confirmed in vivo. We hope that these results may be useful in the development of new drugs and optimization of antiatherosclerotic drug therapy.

Atherosclerosis-prone branch regions in human aorta: microarchitecture and cell composition of intima.

The microarchitecture and cell composition of intima were studied at the macroscopically unaffected branch regions of human thoracic aorta using en face preparations, scanning and transmission electron microscopy, and immunohistochemistry. The endothelial lining showed a heterogeneous pattern and altered morphology including the areas of deendothelialization covered with platelets and dilated intercellular clefts. Leukocyte adhesion, accumulation of subendothelial macrophages and lymphocytes were characteristic of proximal and lateral zones, while the flow divider showed no significant accumulation of blood cells. Smooth muscle cells (SMCs) on the flow divider were elongated, in a contractile state, contacted side-by-side and did not contain lipid inclusions. In the lateral and proximal zones, intima appeared to be a network of stellate SMCs which were in contact through their processes. Most of the SMCs were in a synthetic state and many of them contained small lipid droplets. The number of procollagen I positive cells and the volume of extracellular components were most significant at the lateral zones rather than at the flow divider. We did not observe any difference in the rate of proliferation. Our results suggest that the intimal layer at the lateral and proximal zones has some distinct structural peculiarities, which provoke the development of initial atherosclerotic lesions at these sites. Such an intimal structure is probably caused by different flow patterns at these zone. However, only the totality of different morphological features exhibited in the area of altered vascular wall shear stress may be considered as a prerequisite for atherosclerotic lesions.

Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma.

BACKGROUND: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions. METHODS: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy. RESULTS: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions. CONCLUSION: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.

Molecular analysis of complex tissues is facilitated by laser capture microdissection: critical role of upstream tissue processing.

Every tissue contains heterogeneous cell populations. Laser capture microdissection (LCM) facilitates cell isolation from complex tissues followed by molecular analysis. LCM entails placing a transparent film over a tissue section or a cytological sample, visualizing the cells microscopically, and selectively adhering the cells of interest to the film with a focused pulse from an infrared laser. The film with the procured cells is then removed from the original sample and placed directly into DNA, RNA, or protein-extraction buffer for processing. LCM has revolutionized molecular analysis of complex tissues because it combines the topographic precision of microscopy with the power of molecular genetics, genomics, and proteomics. However, the success of molecular analysis still depends on the experimental design and requires the understanding of each technical step involved in specimen preparation. This review attempts to rationalize and demystify the choice of various technical options in upstream tissue processing supporting global analytical strategies.

Procollagen production in fresh and cryopreserved aortic valve grafts.

Long-term durability of aortic valve allografts may be enhanced by cellular capacities for regeneration and repair. To evaluate aortic valve graft production of an important structural protein, rat aortic roots were implanted heterotopically into the abdominal aorta of recipient rats. Grafts were either syngeneic or strongly allogeneic, were implanted either fresh or after cryopreservation, and were left in place 2 to 21 days after implantation. A total of 80 aortic valve grafts and the corresponding native aortic valves were examined. The grafts were retrieved and immunocytochemically stained for the presence of procollagen, a precursor to collagen. Regardless of histocompatibility or preservation, grafts exhibited consistent procollagen presence that equaled or exceeded that seen in the corresponding native valves. Positive procollagen staining was predominantly in the aortic wall. The most prominent staining was near the hinge point of the valve leaflets, with no staining in the free portion of the leaflets. Staining with alpha-actin demonstrated vascular smooth muscle in sites remote from the areas positive for procollagen, which suggests that vascular smooth muscle was not responsible for the procollagen production. These findings indicate that cryopreservation is compatible with persistent fibroblast viability and in vivo protein synthesis by both syngeneic and allogeneic aortic valve grafts.

Lipid accumulation in the subendothelial cells of human aortic intima impairs cell-to-cell contacts: A comparative study in situ and in vitro.

We have modified our technique used to examine the three-dimensional cytoarchitecture of human aorta. Using this modification we confirmed the disruption of the contacts among subendothelial cells in the fatty streaks. Thinning and arborization of contact-forming cellular processes were also demonstrated. The incubation of cultured human aortic subendothelial cells with desialylated low-density lipoproteins (LDL), LDL immobilized on latex, and LDL-free latex microspheres induced the alterations in cell-to-cell contacts similar to those occurring in a fatty streak in situ. It is proposed that these alterations may be associated with lipid accumulation and activation of phagocytosis and/or intracellular accumulation of large insoluble particles.

Three-dimensional organization of biological tissues: a new method for investigation using scanning electron microscopy.

A new method is described which enables the visualization of hidden cellular surfaces of biological tissues. The method does not depend on the kind of fixative solution, the duration of the fixation, or the quantity of extracellular matrix. The samples are chemically dissociated with potassium ethoxide until the first individual cells appear in the solution. This initial process is followed by phase contrast microscopy, and remaining tissue is investigated with scanning electron microscopy. The method has proved more effective than conventional enzymatic techniques and is a simple, quick, and effective way to investigate the three-dimensional organization of different normal and pathological tissues.

Stellate cells of aortic intima: I. Human and rabbit.

Stellate cells hitherto accounted exclusively in the innermost elastic-hyperplastic layer were already reported to inhabit human aortic intima. The present paper shows that most of these cells are situated just beneath the endothelium. Stellate cells also appear in the deendothelialization-induced myointimal thickening of rabbit aorta. In the myointimal thickening these cells were revealed in the direct proximity to the endothelium. A conclusion is available that the previously demonstrated polymorphism of human aortic intimal cells may be reproduced in a simple experimental model, which gives new possibilities for the study of the cellular polymorphism in the vessel wall.

Stellate cells of aortic intima: II. Arborization of intimal cells in culture.

The present study analyzed effects of different cAMP-elevators on cell morphology in primary culture of human intimal and medial cells from grossly normal and atherosclerotic areas. In primary culture of human aortic cells adenylate cyclase activator forskolin and other cAMP elevators induced arborization of cells, i.e. they reversibly changed the shape of cells. This resulted in the formation of thin branching processes and in the concentration of cytoplasm around the nucleus. In the culture, the shape of the arborized cells resembled that of stellate ones detected in the aortic intima in situ. The arborization of cells was accompanied by destruction of myofilaments. Due to cAMP elevators' effect, most of the arborized cells were exhibited in the cultures isolated from the elastic-hyperplastic layer of the intima. The number of arborized cells was significantly less in the cultures isolated from the musculo-elastic layer and still lesser in those isolated from media. We failed to reveal any significant difference in the number of arborized cells cultured from fatty streaks, atherosclerotic plaques and grossly normal aortic areas. Obtained results suggest that the previously revealed polymorphism of human aortic intimal cells may be accounted for by the cell shape transformations underlined by the mechanism similar to that of arborization in vitro.

Heterogeneity of smooth muscle cells in embryonic human aorta.

Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for alpha-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.

[An analysis of the endothelial differentiation of the rat aorta during reparative regeneration]. / Analiz differentsirovki éndoteliia aorty krysy v protsesse reparativnoi regeneratsii.

Following freeze-injury the arterial endothelium is able to restore completely its integrity without forming myointimal thickening. In the direction from the center of the former defect to its periphery the specific volume of biosynthetic and bioenergetic apparatus is reduced, specific volume of microvesicles rises, and parajunctional condensations of the microfilaments forms. Adhesion of monocytes to the endothelial surface is detected along with their migration into subendothelial space on all the stages of re-endothelialization.

[Changes in the surface and cytoskeletal apparatus of the endotheliocytes of the rat aorta during division (based on scanning electron microscopic data)]. / Izmeneniia poverkhnosti i oporno-dvigatel'nogo apparata éndoteliotsitov aorty krysy vo vremia deleniia (po dannym skaniruiushchei élektronnoi mikroskopii).

Endothelium of the abdominal aorta of 32 KWR-line rats was exposed to freeze injury. En face preparations were made to look for endothelial cells (EC) on different stages of mitosis. Specimens were dried by critical point technique, and the grid was placed on their surface. Then specimens were investigated in succession with light and scanning electron microscopes. The cytoskeleton of EC was investigated on detergent-extracting preparations. It is shown that the end of phase S of the cell cycle and the beginning of prophase are characterized with the lifting of the nuclear-containing zone. Fine microvilli appear on the EC surface during prophase. The cytoskeleton becomes more structured and polarized. During metaphase, EC becomes spherical, its microvilli are shortened. Fine cytoplasmic shoots are seen to extend from the cell poles to the substratum. The density of the fibrillar structures swiftly rise. During anaphase the EC surface is covered with blebs. During late telophase the surface of dividing EC becomes flatter. Their nuclei are connected with rare bundles of fibrillar structures. Mechanisms of EC surface changes during mitosis and the role of cytoskeletal elements are discussed.

[Cellular orientation and form in the elastic-hyperplastic layer of the human aortic intima in the area of the ostia of the intercostal arteries]. / Orientatsiia i forma kletok élastiko-giperplasticheskogo sloia intimy aorty cheloveka v oblasti ust'ev mezhrebernykh arterii.

Using the Hatcher monolayer method, it has been shown that the aortic intimal smooth muscle cells, disposed on the flow divider of an intercostal artery, have predominantly spindle-like shapes to be oriented perpendicularly to the long axis of the vessel. Unlike, in the proximal and lateral sites of the intercostal ostium the intima is represented by stellate cells lacking any orientation. These distinctions may be presumably determined by the irregular distribution of hemodynamic loading on the aortic intima in different regions around the ostium of an intercostal artery.

[The use of freeze fracturing in combination with light, scanning and transmission electron microscopy for studying the structure of the normal and atherosclerotically altered human aortic intima]. / Ispol'zovanie zamorazhivaniia--skalyvaniia v komplekse so svetovoi, skaniruiushchei i transmissionnoi élektronnoi mikroskopiei dlia issledovaniia struktury normal'noi i aterosleroticheski izmenennoi intimy aorty cheloveka.

Three-dimensional network of smooth muscle cells (SMC) with processes was found in the subendothelial intima of human aorta. The cells were connected with each other through gap junctions. In the direction from the media to the endothelium the number of plasma membrane caveolae increased, their distribution becoming more random. In the fatty streak, the integrity of cellular network was seen destroyed. In the extracellular matrix multilamellar ball-like structures containing large intramembranous particles appeared. In the fibrous plaque, SMCs are completely isolated by connective tissue fibres.

[Changes in the spatial organization and structure of the smooth-muscle cells of the tunica media of the major arteries during lengthwise stretching in situ]. / Izmenenie prostranstvennoi organizatsii i struktury gladkomyshechnykh kletok srednei obolochki magistral'nykh arterii v usloviiakh prodol'nogo rastiazheniia in situ.

It has been shown that in vivo stretching of the arterial wall leads to the loss of cell-cell contacts between medial smooth muscle cells (SMC), and to the change in the cell cooperation type. It is accompanied by the increase in SMC proliferative activity, and by changes in their shape and phenotype, from the contractile to the synthetic one. The increase in the number of extracellular connective tissue structures occurs in the intercellular space.