Measurement of the ultrasonic properties of human coronary arteries in vitro with a 50-MHz acoustic microscope

Ultrasonic attenuation coefficient, wave propagation speed and integrated backscatter coefficient (IBC) of human coronary arteries were measured in vitro over the -6 dB frequency bandwidth (36 to 67 MHz) of a focused ultrasound transducer (50 MHz, focal distance 5.7 mm, f/number 1.7). Corrections were made for diffraction effects. Normal and diseased coronary artery sub-samples (N = 38) were obtained from 10 individuals at autopsy. The measured mean ± SD of the wave speed (average over the entire vessel wall thickness) was 1581.04 ± 53.88 m/s. At 50 MHz, the average attenuation coefficient was 4.99 ± 1.33 dB/mm with a frequency dependence term of 1.55 ± 0.18 determined over the 36- to 67-MHz frequency range. The IBC values were: 17.42 ± 13.02 (sr.m)-1 for thickened intima, 11.35 ± 6.54 (sr.m)-1 for fibrotic intima, 39.93 ± 50.95 (sr.m)-1 for plaque, 4.26 ± 2.34 (sr.m)-1 for foam cells, 5.12 ± 5.85 (sr.m)-1 for media and 21.26 ± 31.77 (sr.m)-1 for adventitia layers. The IBC results indicate the possibility for ultrasound characterization of human coronary artery wall tissue layer, including the situations of diseased arteries with the presence of thickened intima, fibrotic intima and plaque. The mean IBC normalized with respect to the mean IBC of the media layer seems promising for use as a parameter to differentiate a plaque or a thickened intima from a fibrotic intima

Effects of anastomotic angle on vascular tissue responses at end-to-side arterial grafts.

OBJECTIVE: Hemodynamics has been implicated in the late failure of arterial bypass grafts, which frequently occurs at the distal anastomosis site. This study was designed to assess the relationship between local hemodynamics and pathologic responses of the distal anastomosis by manipulation of the angle of anastomosis of the graft, a major determinant of local hemodynamics. METHODS: End-to-side anastomoses of the right carotid to the left carotid arteries of rabbits were performed at anastomotic angles of less than 10 degrees (acute), 45 degrees (intermediate), or 90 degrees (right angle), and then the upstream left carotid arteries were ligated to simulate pathologic occlusion. We examined tissue responses on the wall of the recipient vessel opposite the anastomosis site (the bed), where unusual hemodynamic forces are imposed. RESULTS: Three months after surgery, intimal thickening was observed on the upstream portion of the acute, and more rarely, the intermediate anastomoses only. Medial thinning caused by loss of cells and matrix, and an aneurysm-like dilation, was observed in the right angle and some intermediate anastomoses, but not in the acute anastomoses. En face confocal microscopy at 3 weeks after surgery revealed severe disruption of the internal elastic lamina in all anastomotic models. Zymography and Western immunoblotting demonstrated gelatinolytic activity, caused by expression and activation of MMP-2, that was lowest in the acute anastomoses, higher in the intermediate anastomoses, and highest in the right-angle anastomoses. CONCLUSIONS: We infer that very different pathologic changes to the vessel wall are elicited when local hemodynamics is manipulated by altering the anastomotic branch angle.

Alterations in endothelial F-actin microfilaments in rabbit aorta in hypercholesterolemia.

The current study tests whether hypercholesterolemia influences the distribution of endothelial cell microfilaments during the initiation and growth of fatty streak-type lesions. We classified the lesions occurring over a 20-week period into four types based on the location and extent of macrophage infiltration observed microscopically. The earliest lesion was characterized by leukocytes adherent to the endothelial surface. Minimal lesions were characterized by a few cells in the subendothelium. Intermediate lesions consisted of numerous subendothelial leukocytes in a minimally raised lesion. Advanced fatty streak lesions were elevated, with several layers of leukocytes. The organization of peripheral junctional actin (the dense peripheral band) and of central endothelial cell actin microfilament bundles was studied in each of these lesions by using fluorescent microscopy. We found that in the aorta away from branch sites and in areas away from lesions, the central microfilament distribution was unaffected by hypercholesterolemia. The macrophages entered the wall without any identifiable reorganization in the microfilaments. During the accumulation of subendothelial macrophages in minimal and intermediate lesions, stress fibers were initially increased in comparison to lesion-free areas. In raised advanced lesions, the central microfilaments became thinner and disappeared. However, at flow dividers, where central stress fibers are normally prominent, endothelial cells on the surface of intermediate lesions showed a reduction in central fibers, and peripheral bands became prominent. This finding was associated with changes in cell shape from elongated to cobblestone type. Thus, actin microfilament bundles in endothelial cells underwent substantial changes in distribution during the accumulation of subendothelial macrophages, forming hypercholesterolemia-induced fatty streak-type lesions. These changes may influence endothelial substrate adhesion, permeability, or repair after injury.

Histological and immunohistochemical characteristics of eccentric coronary artery lesions retrieved by atherectomy from cardiac transplant recipients.

The lesions of cardiac allograft vasculopathy are thought to be strongly related to an immune inflammatory process. Little is known about the biology of these eccentric lesions. However, transplant patients may present with focal disease. Coronary atherectomy provides a unique opportunity to study these clinically relevant lesions in surviving transplant patients. In this series we characterized the features of four lesions (two restenotic and two primary) from three cardiac transplant recipients who underwent coronary atherectomy. The histologic characteristics of the lesions were analyzed and immunohistochemistry was used to assess qualitatively the presence of specific markers of inflammation and the extracellular matrix component fibronectin. Histology showed cholesterol clefts, calcium deposits, and foam cells with low to moderate cellularity and moderate to high fibrosis. Interleukin (IL)-1ß was present in two lesions, but tumor necrosis factor (TNF)-α was absent. The adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cellular adhesion molecule (VCAM)-1 and the integrins α5ß1 and α4 were present in all lesions. There was mild to moderate accumulation of fibronectin. Thus, atheroscleroticlike features were present with only low to moderate degrees of immune inflammation. Our findings suggest that eccentric focal plaques in cardiac allograft vasculopathy are less likely to be primarily related to a prominent immune inflammatory process and are similar to atherosclerosis. We speculate that these eccentric lesions that resemble atherosclerosis may be more related to the conventional risk factors for coronary artery disease frequently seen in this population.

Analysis of atherosclerotic plaques obtained by coronary atherectomy: Foam cells correlated positively with subsequent restenosis.

Restenosis following coronary intervention is a complex process the mechanisms of which remains mostly unknown. Tissue obtained by atherectomy is an important means to study restenosis. Previous studies on atherectomy-retrieved tissue have not identified histologic features that correlate with restenosis. We performed an histopathologic evaluation on atherosclerotic plaque tissue obtained by atherectomy from 58 patients, all of whom had a 6-month angiographic follow-up. We identified macrophages and lymphocytes and localized tumor necrosis factor-α expression in the tissue by immunohistochemistry. Histopathology was correlated with late angiographic outcomes. Of 10 histologic features evaluated in the plaque tissue, only the presence of foam cells, identified in paraffin sections, correlated positively with restenosis (p = 0.04). Immunohistochemistry showed that macrophages (p = .07), tumor necrosis factor-α (p = .07), and lymphocytes (p = .14) were more prominent, but not significantly so, in lesions from patients with foam cells and restenosis than in lesions from patients without foam cells or restenosis. Thus the presence of foam cells in primary lesions obtained by atherectomy as identified in paraffin-embedded tissue appears to be a marker for restenosis.

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

BACKGROUND: The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS: Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS: The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS: Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury.

Angiogenesis in atherosclerosis.

This article reviews studies on the structure, complications and pathogenesis of angiogenesis in atherosclerotic plaques. Although important, this topic has not been studied extensively; the authors present a review of the important information available in the literature. Since angiogenesis has profound clinical complications, the focus is on data which provided information to understand the nature of the vascular structures involved and the sequence of events which led to the initiation and growth of these small vessels in the plaque. This review indicates that it is most likely that the capillaries that first form arise from the vasa vasorum. Since the cell biology literature suggests that angiogenesis is regulated by angiogenic factors which stimulate vascular cell migration and proliferation, the known role of several growth factors and inhibitors is reviewed. It is likely that a complex interaction in the atherosclerotic vessel wall results in angiogenesis, and that further study with purified growth factors and other substances is needed to clarify the pathogenesis of this important biological process.

Ultrastructural studies of unstable angina in living man.

Nineteen patients with refractory unstable angina who were undergoing aortocoronary bypass were studied to assess the extent of platelet aggregation present in the microvasculature. Ultrastructural findings on the morphology of cardiac muscle and microvasculature were correlated with the findings on coronary angiograms and thallium scans. There were no significant correlations. The presence of platelet aggregates was identified in four biopsies, two of which had thrombus by angiographic criteria. Biopsy in areas with thallium defects revealed an increased prevalence of white blood cells without acute myocardial infarction. This study confirms the presence of platelet aggregates in patients with unstable angina, albeit at a reduced frequency when compared with autopsy studies.

Repair of the wounded guinea pig tympanic membrane: organization of filamentous actin and spatial cellular reorganization.

Since the role of the epithelial cell in the repair of the wounded tympanic membrane is not well understood, the epithelial cell layers were examined using rhodamine phalloidin to localize F-actin in situ following a full-thickness traumatic perforation. The change in shape of the epithelial cells and the morphological changes in F-actin were characterized. The mucosal cells were remarkably resistant to wounding and their morphology and F-actin distribution remained unchanged. Within 24 hours following perforation, basal cells were prominently stained adjacent to the perforation, whereas the remainder of the drum showed fainter staining similar to non-wounded drums. The basal cells showed a minor shape change in the direction of movement of the surface keratin. By three days, the dense peripheral F-actin staining of basal cells was prominent throughout the entire drum. The suprabasal cells demonstrated a marked shape change by 24 hours following the injury. The normal cobblestone pattern disappeared and cells elongated and were aligned towards the perforation. This occurred first adjacent to the perforation, and by three days re-orientation was present over the entire tympanic membrane. A few fine stress fibers appeared in the suprabasal cells. Histological and electron microscopic evaluation of the areas away from the perforation did not, however, show inflammation or disruption. By one week these changes were resolving and by two weeks the drum, with the exception of the cells in the area of the healed perforation, had returned to normal. The results are discussed with respect to the role of the various cell types in the migratory aspect of tympanic membrane wound repair.

In situ localization of F-actin in the normal and injured guinea-pig tympanic membrane.

Although cell migration is an important function of the epithelial cells of the tympanic membrane (TM), little is known about the distribution of the F-actin cytoskeleton, a contractile protein important in cell motility. The purpose of this experiment was to study the in situ localization of F-actin in the epithelial cells of the TM. F-actin, localized using Rhodamine-phalloidin, was present as a thin cortical band at the margin of both the mucosal cells on the inner side of the drum, and the suprabasal cells of the epidermis. The basal cells showed diffuse circumferential F-actin staining sometimes appearing as short microfilaments. Following a full thickness injury, changes in the distribution of F-actin could be observed with in situ localization. While the diffuse F-actin staining of the basal cells was reduced, both long F-actin microfilament bundles extending parallel to the long axis of the cell and focal aggregates of F-actin were prominent. The suprabasal cells became elongated, and while the F-actin remained localized to the cell margin, faint central F-actin microfilaments were observed. The staining of the mucosal cells remained unchanged. This study showed that the guinea pig TM is a useful model to study the distribution of epithelial F-actin in situ under normal and repair conditions, and that the basal cell layer may be important in regulating migration in the epidermis.

Immunofluorescent microscopy for the identification of human necrotic myocardium.

Human postmortem cardiac muscle was studied by immunofluorescent microscopy. Necrotic cells in acute myocardial infarctions were first identified with the hematoxylin-eosin stain as showing hypereosinophilia and autofluorescence. The results of the immunofluorescence staining showed a marked decrease if not absence of labeling for the Ca+ and Mg+ adenosine triphosphatase (ATPase) and tropomyosin in all necrotic muscle cells within a myocardial infarction. Myocytolytic cells located at the border of the infarct showed a labeling intensity similar to that of normal muscle cells. The use of immunofluorescence localization of muscle-specific proteins can be used as a reliable method to detect myocardial cell necrosis.

In vitro reendothelialization. Microfilament bundle reorganization in migrating porcine endothelial cells.

An experimentally induced wound made in a confluent monolayer culture of porcine thoracic aortic endothelial cells (ECs) was studied 22 hours after wounding using 7-nitrobenz-2-oxa-1,3-diazole (NBD) phallacidin and immunofluorescence microscopy to localize actin and myosin containing microfilament (MF) bundles. ECs extending from the wound edge back toward the confluent monolayer showed a specific change in cell shape and in MF bundle distribution and orientation, which correlated with the cell migration behavior observed using time-lapse cinemicrophotography. The migrating ECs in the first zone, the leading zone, were polygonal to partially elongated in shape, and contained distinct central MF bundles oriented both parallel and perpendicular to the wound edge. The second zone, the elongated zone, was characterized by elongated cells with central MF bundles oriented parallel to the direction of migration. A third zone, the transitional zone, showed nonmigrating polygonal ECs containing prominent central and dense peripheral bands (DPB) of MF bundles. The central MF bundles were oriented randomly with respect to the wound edge. The MF bundles of the confluent resting monolayer were both centrally and peripherally located with the latter being more prominent. The results indicate that the reorientation of central MF bundles and reduction in the peripheral MF bundles are probably important in the reorganization of the cytoskeletal system during the conversion of stationary cells to migrating cells.

The reorganization of cytoskeletal fibre systems in spreading porcine endothelial cells in cultures.

Porcine aortic endothelial cells spreading on a glass substrate undergo characteristic changes in shape which can be classified into four distinct stages. To study the role of the cytoskeleton in cell spreading, we have examined the distribution of microtubules (MT), microfilaments (MF), and intermediate filaments (IF) at each of these stages by using immunofluorescence and antisera specific for tubulin, tropomyosin, myosin, and vimentin. The small round Stage I cells showed diffuse staining with four antisera. In the more flattened spreading Stage II cells, MT and IF were first observed in the perinuclear region while fibres straining positively for tropomyosin and myosin were first seen along the cell margin. Later the MT began to radiate out in all directions from the perinuclear region while the IF became localized in a region on one side of the nucleus. In the very flat Stage III cells with a circular outline, additional MT could be seen along and parallel to cell margin while the IF emanating from the perinuclear region and the tropomyosin and myosin positive fibres became concentrically distributed around the nucleus. In the very flat asymmetric Stage IV cells, both the MT and IF radiated out from the perinuclear region towards the cell periphery while most of the tropomyosin and myosin-positive fibres became reorganized so that they ran parallel to the edges of the cell. In addition several loci from which a number of the tropomyosin and myosin-containing fibres radiated also appeared at this stage. These results indicate that during spreading each of the three major fibre systems undergo extensive and specific reorganization which is well coordinated with changes in cell shape.

Prostaglandin induced shape changes in fibroblasts grown in cell culture.

PGE2 induced shape changes in porcine adventitial fibroblasts grown on glass in low density monolayer cell cultures. Incubation of the cells with PGE2 at concentrations of 100 ng/ml and 1000 ng/ml induced rounding of flat fibroblasts within one hours. The rounded cells had a small rim of cytoplasm around the nucleus and from one to several long thin arborizing cytoplasmic processes extending outward along the substratum. Removal of the PGE2 resulted in transient blebbing of the cell membrane of both the cell body and the processes as the cells returned to their flat normal morphology within one hour. The effect could be inhibited by 1% fetal calf serum. PGF2 alpha did not however induce similar changes. This difference between PGE2 and PGF2 alpha is similar to a report on spreading and migration of mouse peritoneal macrophages, and suggests that under certain conditions PGE2 may have the ability to induce shape changes in cells.

Mechanochemical proteins, cell motility and cell-cell contacts: the localization of mechanochemical proteins inside cultured cells at the edge of an in vitro "wound".

We have examined the distribution of several mechanochemical proteins inside rat A10 cells in monolayer culture, both in sparse cultures and at the edges of in vitro "wounds" in confluent cultures. The proteins examined were actin, myosin, tropomyosin, alpha-actinin, filamin, and tubulin. In each experiment, a pair of these proteins (one of which was usually actin) were examined simultaneously by double fluorescence staining methods. Actin was specificially stained by double fluorescence staining methods. Actin was specifically stained by a method based on heavy meromyosin binding, while the other proteins were specifically stained by indirect immunofluorescence procedures. The most important of the various results described was obtained with cells moving out from the edge of an in vitro wound. Within the flat leading lamella of such a cell, there was an extended region in which myosin was severely depleted or absent compared to the proximal regions of the same cells. By contrast, the other proteins were abundantly present throughout the leading lamella, except for tropomyosin, which was somewhat depleted but not as extensively as myosin. In Nomarski optics, there was no detectable morphological differentiation between the region depleted of myosin and the more proximal portion of the same lamella. While the depletion of myosin from the motile regions of cells does not rule out the involvement of some form of an actomyosin sliding filament mechanism, it suggests that other molecular mechanisms for generating motility be seriously considered.

Ventricular preexcitation syndrome. Accessory left atrioventricular connection and rhabdomyomatous myocardial fibers.

A necropsy study of the heart of a 16-year-old girl with ventricular preexcitation syndrome and supraventricular tachycardia showed an accessory left atrioventricular connection partially within the mitral valve. Both the accessory pathway and the ventricular musculature showed multiple foci of cardiac muscle fibers with rhabdomyomatous features having the characteristic appearance of rhabdomyomatosis. The case is discussed in the context of a literature review of necropsy findings in cases of ventricular preexcitation.

Hodgkin's disease following thorium dioxide angiography.

Hodgkin's disease occurred in a 53-year-old man who, 25 years previously, had undergone cerebral angiography, for which thorium dioxide suspension (Thorotrast) was used. Deposits of throium dioxide were noted in reticuloendothelial cells in various locations. An association between thorium dioxide administration and the subsequent development of malignant tumours and neoplastic hematologic disorders has previously been reported.