Fusion of neurohypophyseal membranes in vitro

Lesions of the aorta and other organs of copper depleted layers, roosters, day-old chicks and chick embryos were studied by light- and electron microscopy. Copper deficiency results in late and abnormal development of elastic fibres: inhibition of the formation of cross linkages causes a decline of elastin synthesis. The microfibrils constituting the blackbone of the elastic fibre form a honey-comb pattern. The small amount of amorphous elastin is digestible by elastase, the microfibrils are resistant to the action of the enzyme. The aortic elastic fibres are irregularly shaped, broken revealing a honey-comb like pattern. They maintain an increased affinity to PTA. Tears--dissecting aneurysms--arise in the media, vacuoles appear in the heart muscle, haemorrhages and inflammatory changes may arise in other organs.     

Endogenous retroviral sequences present in the human genome

The common carotid arteries of normal adult rats were investigated electron-microscopically after tannic acid fixation. This fixation technique yields a better demonstrability of the structures of the connective tissue, the basal laminae and the surface coat of the cell membrane. The common carotid artery represents a vessel of the elastic type. The intima consists of an endothelium and a narrow gap of connective tissue (0.1-1 micron) which contains single collagenous fibrils and small elastic structures. This space is only occasionally as wide as 3 microns, especially beneath gaps of the internal elastic membrane. In these areas, single cells and structures of densely packed filaments are additionally observed which can neither be attributed to collagenous fibrils nor to elastic fibres. The intima is demarcated from the outside by an internal elastic membrane (1 micron) which shows a number of gaps. The media exhibits 3 to 4 elastic membranes without gaps. Smooth muscle cells of the contractile type stretch in an oblique direction between these membranes, i.e. they are not arranged in a circular or spiral manner. Most of their process-rich ends are inserted directly into the elastic material and not via a basal lamina. Processes from these smooth muscle cells, collagenous fibrils and elastic fibres are seen in the intercellular spaces. The muscle cells are occasionally interlinked by gap junctions. The basal lamina does not surround the muscle cells continuously. The adventitia contains bundles of collagenous fibrils, fibrocytes, a few small vessels and nerves with a perineuronal envelope. Nerves could not be demonstrated in the media. The oblique course of the smooth muscle cells and the insertion into the elastic membranes indicate that these cells do not predominantly contribute to changes in the width of the lumen but also serve the stabilisation and resetting of the elastic membranes. Contraction is probably induced by an opening of stretch-dependent Ca2+ channels. Due to the interlinkage with gap junctions, the muscle cells of one layer respond as a functional unit. Our findings provide a morphological basis for elucidating commonly encountered changes, such as smooth muscle migration through a normally interrupted inner elastic lamina.     

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.     

Transitions in cell organization and in expression of contractile and extracellular matrix proteins during development of chicken aortic smooth muscle: evidence for a complex spatial and temporal differentiation program

Whereas the understanding of the mechanisms underlying skeletal and cardiac muscle development has been increased dramatically in recent years, the understanding of smooth muscle development is still in its infancy. This paper summarizes studies on the ontogeny of chicken smooth muscle cells in the wall of the aorta and aortic arch-derived arteries. Employing immunocytochemistry with antibodies against smooth muscle contractile and extracellular matrix proteins we trace smooth muscle cell patterning from early development throughout adulthood. Comparing late stage embryos to young and adult chickens we demonstrate, for all the stages analyzed, that the cells in the media of aortic arch-derived arteries and of the thoracic aorta are organized in alternating lamellae. The lamellar cells, but not the interlamellar cells, express smooth muscle specific contractile proteins and are surrounded by basement membrane proteins. This smooth muscle cell organization of lamellar and interlamellar cells is fully acquired by embryonic day 11 (ED 11). We further show that, during earlier stages of embryogenesis (ED3 through ED7), cells expressing smooth muscle proteins appear only in the peri-endothelial region of the aortic and aortic arch wall and are organized as a narrow band of cells that does not demonstrate the lamellar-interlamellar pattern. On ED9, infrequent cells organized in lamellar-interlamellar organization can be detected and their frequency increases by ED10. In addition to changes in cell organization, we show that there is a characteristic sequence of contractile and extracellular matrix protein expression during development of the aortic wall. At ED3 the peri-endothelial band of differentiated smooth muscle cells is already positive for smooth muscle alpha actin (alphaSM-actin) and fibronectin. By the next embryonic day the peri-endothelial cell layer is also positive for smooth muscle myosin light chain kinase (SM-MLCK). Subsequently, by ED5 this peri-endothelial band of differentiated smooth muscle cells is positive for alphaSM-actin, SM-MLCK, SM-calponin, fibronectin, and collagen type IV. However, laminin and desmin (characteristic basement membrane and contractile proteins of smooth muscle) are first seen only at the onset of the lamellar-interlamellar cell organization (ED9 to ED10). We conclude that the development of chicken aortic smooth muscle involves transitions in cell organization and in expression of smooth muscle proteins until the adult-like phenotype is achieved by mid-embryogenesis. This detailed analysis of the ontogeny of chick aortic smooth muscle should provide a sound basis for future studies on the regulatory mechanisms underlying vascular smooth muscle development.     

Ultrastructure of arterioles in the cat brain

A total of 110 arterioles were examined in the brains of cats; different sites were studied including the cortex, putamen, pons and crus cerebri. No internal elastic laminae were seen in the subendothelial space, although occasional fragments of elastic material were present in the larger arterioles. The media was composed of one, two or three layers of smooth muscle cells which interlocked in such a way that the vessel wall thickness was constant. Numerous tight junctions were seen between adjacent smooth muscle cells and between the endothelium and smooth muscle cells. Apart from the usual cell organelles, the smooth muscle cells of arterioles had numerous dense patches on the cell surface. The structure of the adventitia varied according to the diameter of the vessel and the site in the brain; it contained adventitial cells, bundles of collagen fibres and nerve fibres. Innervation of arterioles was more constant in the brain stem than in the cortex. Metarterioles had less specialised, atypical smooth muscle cells, a discontinuous media and numerous, extensive myoendothelial tight junctions; they were not innervated by nerve fibres. The diameter of metarterioles was less than 10 micronm whereas that of arterioles was 10-45 micronm. The possible functional aspects of arteriolar innervation are discussed.     

Prevalence of malnutrition in minors under 5 years of age in Tabasco

AIMS: Abdominal aortic aneurysms are characterised by changes in the extracellular matrix of the arterial media, in particular a reduction in elastin concentration. These changes are mediated by increased levels of endogenous matrix metalloproteinases (MMPs). Recently, calcium channel blockers have been shown to increase the proteolytic activity of MMP-2 secreted by vascular smooth muscle cells. It may therefore by hypothesised that calcium antagonists may potentiate the activity of MMPs in aneurysmal disease and thus accelerate AAA expansion. In this study, the ability of amlodipine--a calcium antagonist--to influence elastin degradation, was assessed in a previously described model of aneurysmal disease. METHODS: Porcine aortic segments (n = 8) were pre-incubated in exogenous pancreatic elastase for 24 h prior to culture in standard conditions for 6 days with 10 and 100 micrograms/l amlodipine. Control segments were cultured both with and without amlodipine and without elastase. At the termination of culture MMPs were extracted from the tissue and quantified by a combination of substrate gel enzymography and immunoblotting. The volume fractions of elastin and collagen were determined by stereological analysis of EVG stained sections. RESULTS: Gel enzymography demonstrated significantly increased MMP-9 activity in the amlodipine treated segments, median 4.218 vs. 2.809 arbitrary units (p < 0.01) and this elevated activity was reflected in a significant destruction of medial elastin 27.0 vs. 40.5% (p < 0.05). CONCLUSION: Therapeutic ranges of amlodipine significantly enhanced elastin degradation and potentiated MMP-9 activity within the aortic organ cultures.     

Emergence and distribution of intimal smooth muscle cells in the postnatal rat aorta

The distribution of aortic intimal smooth muscle cells in the normal rat during postnatal development was studied by electron microscopy and by staining with fluorescence-labeled phalloidin. The phenotypes of intimal and medial smooth muscle cells were almost identical at first; however, during development, the former remained synthetic, whereas the latter became contractile. Confocal laser scanning microscopy was utilized to observe intimal and medial cells separately. Intimal smooth muscle cells were rarely observed in neonatal rats, but appeared by 10 days of age and increased during postnatal development. A combination of confocal and conventional fluorescent microscopy clearly demonstrated that the intimal smooth muscle cells were preferentially distributed in: (1) the right-lateral and dorsal wall of the upper thoracic aorta, (2) the left-lateral and ventral wall of distal two-thirds of the descending aorta, and (3) the downstream side of branch orifices. Intimal smooth muscle cells in group (1) were oriented randomly, whereas most in group (2) ran longitudinally. Intimal smooth muscle cells at branches in group (3) ran obliquely from the edges at the downstream side in an upstream direction. They tended to accumulate in regions of the aortic wall considered to be under high tensile stress.     

An operational model of the antigen-antibody interaction

We investigated smooth muscle cell proliferation associated with restenosis after percutaneous transluminal coronary angioplasty (PTCA) in 8 arteries with fragmented internal elastic lamina obtained at autopsy in 7 patients who died between 2 months to 2 years 11 months after coronary angioplasty. The internal elastic lamina fragmentation, measured longitudinally along the blood vessels, measured 6.6 +/- 6.9 mm. Smooth muscle cell proliferation was concentrated around the fragmented internal elastic lamina, extending longitudinally even to unfragmented areas. The proliferation of smooth muscle cells extended for 1.8 +/- 2.2 mm in the proximal portion of the fragmentation, and for 2.0 +/- 2.9 mm in the distal portion. The possibility of new stenoses resulting from smooth muscle cell proliferation at sites adjacent to those subjected to PTCA should be borne in mind when PTCA of the proximal segments of the left anterior descending coronary artery is contemplated.     

Characterization of a monoclonal antibody recognizing a DAG-containing epitope conserved in aphid transmissible potyviruses: evidence that the DAG motif is in a defined conformation

Fetal and postnatal bovine bladders were examined for expression of elastic fiber components by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of fibrillin-1, microfibril-associated glycoprotein (MAGP) and elastin during the fetal period were compared with that of postnatal two year old animals (heifers) and adults. Each bladder was separated into two distinct tissue samples: 1) the outer smooth muscle layer (detrusor) and 2) the inner epithelium (urothelium) lined lamina propria (urotherial-lamina propria). Each of these samples was analyzed separately. Distribution of the elastic fiber components, determined by immunohistochemistry with matrix-specific antibodies, was different depending upon the region of the bladder wall examined and its developmental stage. In particular, MAGP and fibrillin-1 were conspicuously present in the urothelium during the later fetal stages. RNA products of elastic fiber genes were detectable both in the detrusor smooth muscle and urothelial-lamina propria fractions. The highest level of expression occurred in the urothelial-lamina propria fraction during the late second-early third trimester. Elastin expression was different from that of MAGP and fibrillin-1. The highest levels of steady-state elastin mRNA occurred at the earliest developmental stages examined and then progressively decreased through term. A high level of elastin expression occurred within the inner or lamina propria layer of the bladder. Since this layer is the functional capacitance layer within the bladder, its flexibility is likely related to the structural integration of elastin and associated microfibrillar components.     

Long-term ventilation at home in adults with neurological diseases

Protein kinase C (PKC) is now known to play an important physiological role in essentially all cell types. This review will focus on what is known about the kinase in contractile differentiated smooth muscle. Current knowledge on the molecular structure of PKC isoforms will be discussed as they relate to mechanisms of translocation and targeting of the kinase within smooth muscle cells. Studies performed on PKC-dependent signalling pathways in differentiated smooth muscle cells will be discussed with emphasis on studies form our laboratory, especially discussing thin filament linked pathways. Thick filament linked PKC-dependent pathways will be described in more detail elsewhere in this monograph.     

Basic science of abdominal aortic aneurysms: emerging therapeutic strategies for an unresolved clinical problem

Abdominal aortic aneurysms (AAAs) are an increasingly common and potentially lethal condition. Surgical repair of AAA is now yet performed quite safely, yet ruptured AAAs still carry mortality rates of 50% to 70%. Ultrasound screening may help identify unsuspected AAA, thereby allowing elective repair. Because AAAs too small to warrant operation still expand progressively, therapeutic approaches to suppress AAA growth would be welcome. Current concepts indicate that AAAs arise through pathophysiologic process distinct from occlusive atherosclerosis and dominated by degenerative changes in the elastic media. These include marked alterations in elastin and collagen, chronic inflammation, and features of autoimmunity, medial neovascularization, and a decrease in vascular smooth muscle cells. Proteinases associated with mononuclear inflammatory cells, particularly matrix metalloproteinases, likely mediate the degradation of structural proteins in the aortic wall. Experimental studies demonstrate that similar processes occur in an elastase-induced rodent model of AAA, providing a means by which to develop novel therapeutic strategies for this disease. Pharmacologic inhibitors of matrix metalloproteinases act to suppress aortic elastin degradation and limit the growth of experimental AAA in vivo, suggesting at least one approach that may be useful in clinical application. Further developments can be expected to increase knowledge of the pathophysiology underlying aortic aneurysm disease, ultimately providing new therapies for small AAAs based on sound understanding of disease mechanisms.     

Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat

We have characterized matrix metalloproteinase expression in the rat carotid artery after two forms of arterial injury, balloon catheter denudation and nylon filament denudation. Gelatinolytic enzymes with molecular masses of 70 and 62 kD were produced constitutively in the rat carotid. Production of an 88-kD gelatinase was induced after balloon catheter injury, and proteinase production continued during the period of migration of smooth muscle cells from the media to the intima, from 6 hours to 6 days after balloon catheter injury. In addition, a marked increase in 62-kD gelatinolytic activity was observed between 4 and 14 days after arterial injury. Gelatinase activities (88 and 62 kD) were also increased after nylon filament denudation but were markedly less after this injury than after balloon catheter injury. These results suggested a correlation between gelatinase activity and smooth muscle cell migration after arterial injury. Administration of a metalloproteinase inhibitor after balloon catheter injury resulted in a 97% reduction in the number of smooth muscle cells migrating into the intima. Therefore, we hypothesize that gelatinase expression directly facilitates smooth muscle cell migration within the media and into the intima. These results suggest that gelatinases are involved in the vascular smooth muscle cell activation and neointimal formation that characterize arterial tissue remodeling after injury.     

Induction of cell-rich and lipid-rich plaques in a transfilter coculture system with human vascular cells

Cell-to-cell interactions are mainly involved in the control of the proliferation, migration, differentiation and function of different cell types in a wide range of tissues. In the arterial vessel wall, human arterial endothelial cells (haEC) and smooth muscle cells (haSMC) coexist in close contact with each other. In atherogenesis, haSMC can migrate from the media to the subintimal space to form fibromuscular and atheromatous plaques. In the present study, a transfilter coculture system is described, in which the interface between haSMC and confluent or proliferative haEC can be studied in detail. Cells were cocultured on the opposite sides of a porous filter which separates both cell types like the internal elastic lamina in vivo. In cocultures containing proliferative haEC, haSMC growth was significantly stimulated (33.4 +/- 5.7 cells/section, p < 0.05) compared to haSMC monocultures (22.9 +/- 2.5 cells/section) and cocultures containing confluent haEC (15.6 +/- 2.9 cells/section). If confluent haEC were injured mechanically, haSMC growth increased highly significantly (71.3 +/- 16.8 cells/section, p < 0.001). Thus, cell-rich proliferates containing 5-7 layers of haSMC embedded in extracellular matrix were formed after 14 days. On the other hand, after haSMC migration to the endothelial side had occurred, the addition of LDL and monocytes to cocultures with arterial media explants and haEC resulted in the formation of lipid-rich, low-cellular structures. After 28 days, characteristic in vitro plaque growth was induced; the plaque contained a lipid core with predominantly necrotic cells, extracellular lipid accumulations, atypically shaped lipid-loaded haSMC and macrophages, similar to in vivo foam cells, as well as an increased amount of extracellular matrix (collagen I, III and IV). These areas were surrounded by typical fibromuscular caps consisting of smooth muscle alpha-actin-positive haSMC. Finally, the formation of capillaries by haEC could also be observed within these structures.     

Overexpression of P2Y2 purinoceptor in intimal lesions of the rat aorta

Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y2 (P2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.     

Physicochemical properties of arterial elastin and its associated glycoproteins

Microfibrillar glycoproteins are a significant component of vascular elastic tissue, but little is known about their contribution to vascular physiology and pathology. We have investigated some physicochemical properties of the glycoproteins that may be pertinent to these roles. Because of the difficulty in isolating intact glycoproteins in a form and quantity suitable for physicochemical examination, we based our analysis on a comparison of the properties of porcine thoracic aorta and pulmonary artery extracted with GuHCl and collagenase (preparation GC) and after further treatment with dithioerythritol to remove glycoproteins (preparation GC/DTE). Amino acid analysis showed that GC/DTE had the amino acid composition of pure elastin while GC contained a higher proportion of polar amino acids, particularly in the aortic preparation. GC stained with alcian blue, particularly in the intimal region, but GC/DTE did not. GC had a higher water content and a slower viscoelastic response and the circumferential elastic modulus was approximately 50% lower (whether expressed in terms of sample weight or elastin content). Clearly, therefore, the microfibrils do not stiffen the network and may prevent the alignment of elastin fibers in the circumferential direction. Their effect on hydration may arise either because they impose mechanical constraints on the geometry of the network or because they modify the inter- and intramolecular hydrophobic or electrostatic interactions that influence the tissue organization and hydration. Molecular probe measurements of the intrafibrillar pore structure using radiolabeled and fluorescent probes showed that removal of the microfibrils caused a slight decrease in the extrafibrillar water space and a larger decrease in the intrafibrillar water space. Sucrose, a small probe molecule, was able to penetrate most of the intrafibrillar water space when microfibrils were present but was virtually excluded when they were not. Potentiometric titration and radiotracer assays of ion binding both showed that the microfibrils contribute a considerable negative charge (-9 mumoles/g wet tissue in the aortic preparation and -16 mumoles/g wet weight in the pulmonary artery) and increase calcium binding by approximately 30%.     

Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis

This brief review details the structure, nature, and distribution of the fibronexus, and discusses its significance for myofibroblastic differentiation and tumor diagnosis. The fibronexus is a cell surface specialization consisting of intracellular actin filaments and extracellular fibronectin filaments associated with subplasmalemmal plaque material. The fibronexus represents an intercellular junction between myofibroblasts, but in particular is a device for providing contact between myofibroblasts and matrix that mediates continuity between intracellular contractile filaments and extracellular matrix proteins. Immunoelectron microscopy in particular has shown that the intracellular filaments contain actin. The extracellular filaments contain fibronectin and collectively form the fibronectin fibril. The plaque probably contains such proteins as vinculin, talin, alpha-actinin, and integrin. Under appropriate biologic development and fixation conditions, the fibronectin fibril of the fibronexus is characterized by and distinguished from lamina by enhanced density, a rigid appearance, failure to adhere closely to the contours of the cell surface (except focally near the plaque material), and a longitudinally filamentous substructure. Confirmation of the presence of a fibronectin fibril may be obtained by the finding of intense cell surface staining with an antifibronectin antibody. Problems in identifying the fibronexus may be encountered, however, due to poor development and fixation, in which case the filamentous substructure may be inapparent. The fibronexus is such a typical feature of and is often so conspicuous in myofibroblasts that it can be regarded as perhaps essential for the interpretation of myofibroblastic differentiation. Structures with a similar appearance have been documented in fundamentally nonmyofibroblastic cells; these include aortic and scleral spur smooth muscle cells and endothelium. Uncertainties remain in the protein composition of the fibronexus, the nature of its contact with the matrix, and its relationship to similar structures seen in nonmyofibroblastic cells. Immunoelectron microscopy provides a potential means of clarifying some of these questions.     

Mineralocorticoids and hypertension]

The degree of lumen narrowing in advanced lesions correlates poorly with the amount of intimal mass accumulated in the atherosclerotic plaque. As an alternate mechanism of stenosis, we propose that human smooth muscle cells bind to fibrin deposited in the matrix and exert contractile forces to cause a narrowing of the lumen. In the present study we demonstrated in vitro that human newborn aortic smooth muscle cell lines can contract and adhere to fibrin clots composed of either fibronectin-depleted plasma ("plasma") or recombinant fibrin. By using neutralizing antibodies and RGD peptides, we showed that members of the integrin family mediated the interaction between human newborn smooth muscle cells and fibrin. Neutralizing antibodies against the integrin alphavbeta3 (c7E3 Fab and LM609) did not inhibit either plasma clot contraction or recombinant fibrin clot contraction by human newborn smooth muscle cells. In contrast, antibodies against alpha5, beta1, and alpha5/beta1 inhibited contraction of clots composed of either plasma or recombinant fibrin. Anti-alphavbeta3, anti-alphav, anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies inhibited human newborn smooth muscle cell adhesion to plasma clots; however, only anti-alpha5, anti-beta1, and anti-alpha5beta1 antibodies significantly inhibited adhesion to recombinant fibrin. While the linear RGD peptides had no effect, the cyclic peptide penRGD inhibited adhesion to plasma clots and recombinant fibrin. However, it did not block contraction of recombinant fibrin clots. These results suggest that during the interaction of human newborn smooth muscle cell lines with fibrin, alpha5beta1 plays a significant role. This interaction is of potential interest as a target for efforts to block vascular contraction.     

Obstructive lesions of distal mesenteric arteries. A light and electron microscopic study

Two cases of "nonocclusive" intestinal infarction are reported. No thrombosis or significant atherosclerosis was identified and proximal mesenteric arteries were widely patent. However, distal mesenteric arteries were thickened and had pinpoint lumens. Light microscopic findings suggested that this marked luminal narrowing was due to prominent intimal fibromuscular proliferation, medial hypertrophy and mild structural disarray, focal periarterial fibrosis, and transmural elastosis. Electron microscopic findings indicated that the endothelium was normal but the basal lamina was irregularly thickened. The predominant cellular component of the thickened intima consisted of smooth muscle cells, and smooth muscle cells of the media were seen to migrate through an extensively disrupted and degenerated internal elastic lamina. Deposits of young elastic fibers, collagen, and ground substance were also noted, particularly in the intima. The need for careful sectioning and microscopic examination of small distal mesenteric arteries in cases of so-called nonocclusive intestinal infarction is emphasized.     

Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.     

Matrix metalloproteinase-9 (92-kd gelatinase/type IV collagenase equals gelatinase B) can degrade arterial elastin

Degradation of elastic fibers in the arterial walls is an important step in the development of atherosclerosis. To identify the enzyme(s) responsible for the elastinolysis, we have designed an ex vivo model of aortic explants cultured with or without THP-1 cells (human monocyte/macrophage-like cells). After culturing with THP-1 cells for 5 days elastic fibers of the aortic explants were fragmented and lost. With insoluble [3H] elastin as a substrate, elastin-degrading activity could be detected in the culture medium. Zymography in sodium dodecyl sulfate-polyacrylamide gel electrophoresis containing alpha-elastin showed the presence of elastinolytic activity with 92 kd in the medium from the aortic tissue with THP-1 cell cultures, whereas the medium from the aortic tissue without THP-1 cells contained negligible elastinolytic activity. The activity was inhibited by ethylenediamine tetraacetic acid but not by phenylmethane sulfonyl fluoride, N-ethylmaleimide, or pepstatin A, indicating that the enzyme belongs to a class of metalloproteinases. In addition, destruction of the elastic fibers of the aortic explants cultured with THP-1 cells was completely inhibited only by metalloproteinase inhibitors. Immunoblot analyses demonstrated that the proteinase responsible for the elastinolytic activity is matrix metalloproteinase-9 (92-kd gelatinase/type IV collagenase = gelatinase B). Using immunocytochemistry, the metalloproteinase was localized in the THP-1 cells but not in the medial smooth muscle cells. These results suggest that matrix metalloproteinase-9 produced by THP-1 cells is of importance to degradation of elastic fibers in the aortic explants. The role of macrophages in the atherosclerosis is discussed with reference to elastinolysis of the arterial walls.