Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

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.     

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.     

Fibrillogenesis in tendon healing: an experimental study

The aim of this study was the histochemical, immunohistochemical and ultrastructural analysis of reparative fibrillogenesis in experimental lesions of Achilles' tendon. Subtotal tenotomy of Achilles' tendon was performed in twenty Wistar rats. The scar tissue was analysed 2, 4, 7, 14, 21, 30, 45 and 60 days post-operatively. Histochemical, (resorcin-fuchsin, aldehyde-fuchsin, iron haematoxylin and Fullmer and Lillie's methods) immunohistochemical (antibody against collagen I, II and elastin) and ultrastructural analyses were performed. Three phases in the healing process were distinguished: 1) inflammatory, 2) proliferative, and 3) remodelling phase. The inflammatory phase was characterised by haematoma, fibrin deposition, inflammatory cells, fibroblasts, beginning of collagen fibrillogenesis (200-400 A ? fibrils) and oxytalan fibrils. The proliferative phase was characterised by angiogenesis and fibroblast proliferation. Collagen fibres displayed a random arrangement and had a diameter of 400-600 A. Immature elastic fibres reached maximum tissutal concentration. In the remodelling phase, hypocellularity, normal vascularisation, tendon crimps, collagen fibres (800-1,000 A ?), elastic fibres with increased elastin deposition and reduction in oxytalan fibres were observed. In the course of the healing process collagen and elastic fibre fibrillogenesis exhibited consistent quantitative and qualitative variations (i.e. differences in the type and diameter of fibrils). The present study suggests that, together with other matrix macromolecules, also elastic fibres (oxytalan, elaunin and mature) are synthesised in significantly higher amounts during reparative fibrillogenesis and play a role in cell-matrix interaction.     

Phenotypic modulation and elastin formation of cultured aortic smooth muscle cells grown on and within collagen gels

Aortic smooth muscle cells (SMC) were grown on and within collagen gels and their phenotype and elastin formation were examined. Cells grown on the gels showed multilayer growth with nodule formation. They contained many ribosomes and microfilament bundles with dense bodies. In contrast, cells grown within collagen gels did not form nodules. When collagen gels containing SMC were manually detached from the culture dish, the cells were found to be of the synthetic phenotype with well-developed rough endoplasmic reticulum (ER) and Golgi complexes, but few microfilament bundles. There were numerous elastin deposits in the intercellular spaces. Our findings suggest that the physical property of the substrate affects the phenotype and function of the SMC.     

Smooth muscle cell to elastic lamina connections in developing mouse aorta. Role in aortic medial organization

BACKGROUND: The structural and functional intigration of smooth muscle cells and elastic laminae in the aortic media is not well established. Detailed information concerning normal ultrastructural features of the aortic media will provide a better understanding of the medial changes that occur in vascular diseases such as hypertension and aortic aneurysms. EXPERIMENTAL DESIGN: The ultrastructural development and organization of connections between smooth muscle cells and elastic laminae in the mouse aortic media were studied by light and electron microscopy. RESULTS: Early in development, the smooth muscle cells become linked to the elastic laminae by bundles of microfibrils. These microfibrils become progressively infiltrated with elastin so as to form extensions of elastin from the elastic laminae in the adult media. Each elastin extension spans obliquely from the elastic lamina to the surface of the smooth muscle cell where it attaches in a region of membrane occupied by an intracellular membrane-associated dense plaque. On the cytoplasmic face of the plaque, a contractile filament bundle penetrates and anchors in an orientation similar to that of the extracellular elastin extension. The contractile filament bundle traverses the cell obliquely and anchors in a dense plaque on the opposite side of the cell that is in turn linked to the next elastic lamina by another elastin extension. The extracellular elastin extensions and the intracellular contractile filament bundles thus form a "contractile-elastic unit," a continuous line of structures that links adjacent elastic laminae. The oblique orientation of the contractile-elastic units reverses direction in successive smooth muscle cell layers in a herringbone-like pattern. Thus, tension transmitted to one elastic lamina by the smooth muscle cells on either side results in a uniform force exerted on the elastic lamina in one circumferential direction, that on the adjacent elastic laminae being in the opposite direction. CONCLUSIONS: Results from this study demonstrate the presence of smooth muscle cell to elastic lamina connections that form early in development as contractile-elastic units; basic units of aortic medial ultrastructure. The overall organization of the contractile-elastic units within the aortic media is proposed to provide a means for coordinating contractile and elastic tensions in response to mechanical stresses imposed on the vessel wall.     

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.     

Biochemical characterization of the fibres of collagen and elastin in the aortic wall of SHRSP

1. Vessel wall fragments consisting of collagen, elastin and other insoluble proteins were prepared from the aortas of 6 month old WKY and SHRSP and dead, elderly humans. 2. Prolonged incubations of these fragments with pepsin below or at 30 degrees C resulted in different amounts of insoluble materials containing similar or larger proportions of collagen and other insoluble proteins than the respective vessel fragments. The amounts of the pepsin-insoluble materials obtained from SHRSP were larger than those from WKY but were much smaller than those from elderly humans. 3. The elastins isolated from the vessel fragments were solubilized by pepsin much more effectively than the respective vessel fragments. 4. The pepsin-insoluble materials from WKY were composed of thin mesh-shaped materials, while these materials from both rats did not contain a significant number of distinctive fibrils of collagen, the materials from elderly humans did contain numerous distinctive fibrils of collagen. 5. Large fractions of both the collagen and other proteins in the pepsin-insoluble materials were solubilized by incubation with a crude bacterial collagenase below 30 degrees C or by incubation with pepsin above 40 degrees C where the triple-helical regions of the collagens were unfolded. 6. These results appear to indicate that the aortic wall of SHRSP contains larger amounts of some insoluble components that immobilize the collagen fibrils than that of WKY, but the aortic walls of elderly humans contain much larger amounts of these components than that of SHRSP.     

Interactions of elastin fibers with fibroblasts a time-lapse cinemicrographic study

Adhesion of cells to the extracellular matrix is mediated by structural glycoproteins such as fibronectin and laminin, and also elastonectin, whose role is to ensure binding of elastin fibers to cells. Interactions between elastin fibers and human skin fibroblasts cultured in a Rose chamber were investigated by using cinemicrography to observe elastin fiber attachment, detachment, and displacement over a five-day period. Elastin fiber displacement over the cell layer resulted in aggregation, which was measured using morphometry. The total number of isolated elastin fibers or aggregates decreased between 1 h and 8 h and remained stable thereafter. During the same time interval, significant decreases occurred in the numbers of isolated fibers and small aggregates (perimeter < 0.268 mm; surface area < 894 microns 2), whereas larger aggregates were formed. After 15 hours of interaction, none of the aggregates had a perimeter greater than 0.536 mm, consistent with an increase in aggregate compacting. These data demonstrate that elastin-cell interactions do not occur at random. These interactions may play a pivotal role in morphogenesis and in maintaining the integrity of elastic tissues such as the arterial wall, lungs, and skin.     

Computerized morphometric quantitation of elastin and collagen in SMAS and facial skin and the possible role of fat cells in SMAS viscoelastic properties

Recently, the superficial musculoaponeurotic system (SMAS) was found to be a composite tissue comprising collagen, elastic fibers, and fat cells in an extracellular viscous matrix. Both SMAS and facial skin tissues exhibit viscoelastic properties, but SMAS tissue has delayed stress relaxation. As a consequence, SMAS is viewed as a firmer elastic foundation for the more viscous facial skin. In some patients, a slackening effect of SMAS tissue takes place over a period ranging from weeks to months after tightening. To determine the relative quantity of viscoelastic components and better understand their biomechanical behavior, a quantitative morphometric study of the elastic and collagen fibers in the SMAS and facial skin was conducted. Thirty-four SMAS preparations were taken from 17 patients during either primary face lift operations (12 women) or reoperative face lift procedures (4 women, 1 man), which were performed 4 to 9 months after the original surgery, to examine the elastin and collagen content. For comparison, preauricular skin was also gathered from these patients. The specimens were stained with Weigert's staining to identify elastin and collagen fibers. Using a computerized morphometric analysis, 100 fields of each SMAS and skin specimen were examined. According to our findings, the average percentage of elastin and collagen fibers in SMAS and facial skin was as follows: (1) the percentage of elastin fibers in the SMAS was 4.71 +/- 1.2 (standard error of mean +/- 0.0291); (2) the percentage of elastin fibers in the skin was 6.1 +/- 1.8 (standard error of mean +/- 0.0436); (3) The percentage of collagen fibers in the SMAS was 38.7 +/- 5.9 (standard error of mean +/- 0.1430); and (4) the percentage of collagen fibers in the skin was 48.47 +/- 6.96 (standard error of mean +/- 0.1688). A statistical significance of p < 0.0001 was demonstrated between the collagen and elastin groups. A different percentage of elastin and collagen fibers was found among the 17 patients and within each of them separately. Neither gender nor age differences were found regarding elastin and collagen fiber content. No statistical differences were demonstrated between specimen sources, i.e., whether the operations were primary or reoperative face lift procedures. Findings from previous studies indicate that the cheek has two viscoelastic layers, the skin and the SMAS. The proportional similarity in average percentages of elastin and collagen in SMAS and facial skin cannot explain the relatively delayed stress relaxation effect of the SMAS. Therefore, the fat cells that are found exclusively in the SMAS probably lend a certain degree of firmness to this layer and play a significant role in the long-term efficacy of SMAS surgery.     

Binding of 92 kDa and 72 kDa progelatinases to insoluble elastin modulates their proteolytic activation

92 kDa and 72 kDa gelatinases, two neutral proteinases exhibiting elastinolytic activity and secreted as zymogens by aortic smooth muscle cells, were shown to bind to insoluble elastin. The active form of each enzyme interacted with substrate more avidly than latent form. Once bound to insoluble elastin, 92 kDa progelatinase was totally unaffected by any potential activators tested (tissue kallikrein, neutrophil elastase, plasmin, and stromelysin-1), except aminophenylmercuric acetate (APMA). Binding of 72 kDa progelatinase to insoluble elastin induced a fast autoactivation of the proenzyme followed by its inactivation. This process can be partly inhibited by tissue inhibitor of matrix metalloproteinases-2 (TIMP-2), EDTA and a synthetic inhibitor of matrix metalloproteinases (BB-94). Such an autoactivation process was also partially observed following adsorption of 72 kDa gelatinase to elastin-derived peptides but not to gelatin. Therefore, elastin can act as a template to direct its own proteolysis by 72 kDa gelatinase; such a mechanism could be relevant to the focal elastolysis in the arterial wall during arteriosclerosis.     

The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells

Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.     

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.     

PACAP in the adrenal gland--relationship with choline acetyltransferase, enkephalin and chromaffin cells and effects of immunological sympathectomy

Using indirect immunohistochemistry and immunological sympathectomy pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity (LI) was studied in the adult rat adrenal gland. All PACAP-positive fibres contained choline acetyltransferase (ChAT)-LI and were found in high numbers among noradrenaline chromaffin cells, whereas enkephalin (ENK)/ChAT-immunoreactive (IR) fibres predominantly innervated adrenaline chromaffin cells. After immunological sympathectomy no PACAP-, ChAT- or ENK-IR fibres remained, strongly suggesting a preganglionic origin. A small number of PACAP-IR fibres was also observed in the subcapsular regions both in controls and in sympathectomized animals, presumably representing sensory fibres. These results define a subpopulation of PACAP-containing cholinergic preganglionic fibres in the adult rat adrenal medulla lacking ENK and innervating noradrenaline chromaffin cells. PACAP was also expressed in a few adrenaline chromaffin cells after immunological removal of the preganglionic innervation, suggesting an additional, hormonal role.     

Method of stimulating ovulation rate in Merino ewes may affect conception but not embryo survival

We have recently shown, on young adult rat aorta rings, that elastin peptides induce a dose and endothelium-dependent vasodilation mediated by the 67 kDa subunit of the high affinity elastin-laminin receptor and, at least in part, by EDRF (NO). Here we have studied the effects of elastin peptides at circulating concentrations and below, on noradrenaline-contracted rat aortic rings, as a function of age. First, we have observed that, unlike 2-month-old (2M), 4-6-month-old (4M) and 12-month-old (12M) rat aorta rings, 30-month-old (30M) rat aorta rings were unable to maintain their contraction in long lasting experiments. Secondly, elastin peptides at physiological circulating concentrations (10(-6)-10(-3) mg/ml) induce a dose-dependent vasodilation on 4M rings. By contrast, only higher elastin peptide concentrations (10(-3) mg/ml) were effective on 12M rings, whereas rings from both younger (2M) and older animals (30M) did not respond to elastin peptides. Finally, using lactose and laminin as inhibitors, we have demonstrated that elastin peptide-induced vasodilation on 4M and 12M rings is mediated by the 67 kDa subunit of the elastin-laminin receptor. These experiments suggest that the functional availability of the 67 kDa subunit of the elastin-laminin receptor changes with age. It could be hypothesized that in young animals (0-2M) the reusable shuttle role recently demonstrated for the 67 kDa receptor subunit during elastic fiber formation leads to a major decrease in its availability for signal transduction. On the contrary, in adult animals. (4-12M), when developmental elastogenesis is completed, this subunit is essential for extracellular signal transduction. Inefficiency of this receptor in old animals (30M) can be attributed to its uncoupling from its transduction pathway, as previously shown on human cells. Finally, the age-dependent variations of circulating elastin peptide concentration and elastin-laminin receptor responsiveness to elastin peptides are two independent parameters which could influence the vascular tension regulation.     

Elastin degradation by matrix metalloproteinases. Cleavage site specificity and mechanisms of elastolysis

Insoluble elastin was used as a substrate to characterize the peptide bond specificities of human (HME) and mouse macrophage elastase (MME) and to compare these enzymes with other mammalian metalloproteinases and serine elastases. New amino termini detected by protein sequence analysis in insoluble elastin following proteolytic digestion reveal the P'1 residues in the carboxyl-terminal direction from the scissile bond. The relative proportion of each amino acid in this position reflects the proteolytic preference of the elastolytic enzyme. The predominant amino acids detected by protein sequence analysis following cleavage of insoluble elastin with HME, MME, and 92-kDa gelatinase were Leu, Ile, Ala, Gly, and Val. HME and MME were similar in their substrate specificity and showed a stronger preference for Leu/Ile than did the 92-kDa enzyme. Fibroblast collagenase showed no activity toward elastin. The amino acid residues detected in insoluble elastin following hydrolysis with porcine pancreatic elastase and human neutrophil elastase were predominantly Gly and Ala, with lesser amounts of Val, Phe, Ile, and Leu. There were interesting specificity differences between the two enzymes, however. For both the serine and matrix metalloproteinases, catalysis of peptide bond cleavage in insoluble elastin was characterized by temperature effects and water requirements typical of common enzyme-catalyzed reactions, even those involving soluble substrates. In contrast to what has been observed for collagen, the energy requirements for elastolysis were not extraordinary, consistent with cleavage sites in elastin being readily accessible to enzymatic attack.     

Aorto-intestinal fistula as a possible cause of endoscopically undetermined gastrointestinal hemorrhage

Elastin peptides are present in human blood. As elastin receptors exist on several cell types, especially endothelial cells, this investigation was carried out to study the effect of elastin peptides on vascular tone. For this purpose, rat aortic rings were mounted in an organ bath for isometric tension measurements. Elastin peptides (kappa-elastin) were added in the concentration range of 0.1 ng/ml to 1 microgram/ml, concentrations similar to those found in the circulating blood. In rat aortic rings, precontracted or not with noradrenaline (10(-6) M), elastin peptides induced an endothelium-dependent vasodilation. The pretreatment of aortic rings with N-omega-nitro-L-arginine methyl ester (10(-5) M), an inhibitor of nitric oxide (NO) production, or with indomethacin (10(-5) M), an inhibitor of cyclooxygenase, prevented elastin peptide-induced vasodilation. These findings suggest that elastin peptides act through the synthesis of prostanoids, leading to the production of NO. Moreover, this relaxant effect of elastin peptides was decreased or inhibited when aortic rings were treated with lactose (10(-5) to 10(-2) M) or laminin (10(-6) to 10(-4) mg/ml) whereas lactose or laminin was unable to inhibit acetylcholine-induced vasodilation. These findings suggest that the inhibitory effects of lactose and laminin are specific for elastin peptide receptors and are in agreement with previous studies on these receptors. As there is evidence of the degradation of elastin in several vascular diseases, the concept that elastin peptides may contribute to the control of vascular tone is discussed.     

Manometry of individual segments of the distal esophageal sphincter. Its relation to functional incompetence

The morphology of the arteries in the uterine wall was studied in three multiparous aged mares that had suffered repeated pregnancy failure. The uterine wall arteries exhibited elastosis of the intima or adventitia, or both, resembling "physiological pregnancy sclerosis". In areas affected by elastosis, degeneration of the pre-existing elastic fibres and increased glycosaminoglycans were frequently observed. Newly formed elastic fibres were not evident. Delayed resorption due to disordered metabolic turnover of the elastin was thought to be an important factor in the pathogenesis of the arterial elastosis in the uterine wall.     

Regulation of skeletal muscle perfusion during exercise

OBJECTIVE: To compare the composition and mechanical properties of the newly developed bladder acellular matrix graft (BAMG) with the normal urinary bladder in rat, pig and human. MATERIALS AND METHODS: Rat, pig and human urinary bladders were harvested and divided into control and experimental groups. For the latter, BAMGs were prepared, and light and transmission electron microscopic studies performed. Strips from the normal bladders and the BAMGs (10 in each group) were tested under tension, and the ultimate tensile strength, maximum strain, and elastic modulus were determined from stress/strain curves. RESULTS: Both types I and III collagen, as well as elastic fibres, were observed as major components of the matrix scaffold. There were more collagen type I fibres in the rat than in the pig and human BAMGs, whereas the pig, and particularly the human, both showed higher levels of type III collagen and elastic fibres. These different matrix scaffold patterns were confirmed by electron microscopy. Results from biomechanical testing showed no significant differences for strength, strain or elastic modulus between BAMG and control bladder strips, except in the rat where the maximum strain values were significantly lower. CONCLUSION: There are variations in the acellular matrix structure with similar biomechanical properties between the BAMG and the normal urinary bladder in three different species. These results may underscore the potential of the BAMG. Furthermore, this in vitro model provides a suitable method to study the mechanical properties of the urinary bladder and may serve as a diagnostic tool for various investigations.     

Pharmacologic suppression of experimental abdominal aortic aneurysms: acomparison of doxycycline and four chemically modified tetracyclines

BACKGROUND: Matrix metalloproteinases (MMPs) likely contribute to the degradation of medial elastin in abdominal aortic aneurysms (AAAs), and tetracycline antibiotics exhibit MMP-inhibiting properties. The purpose of this study was to compare the effects of doxycycline and several non-antibiotic chemically modified tetracyclines (CMTs) in a rat model of elastase-induced AAA. METHODS: Fifty-two male Wistar rats underwent intraluminal perfusion of the abdominal aorta with porcine pancreatic elastase. The rats then were treated for 7 days with subcutaneous injections of saline solution, different doses of doxycycline, or 1 of 4 different CMTs. The aortic diameters were measured with microcalipers, and the fixed tissues were examined by means of light microscopy. Gelatin zymography was used to assess the MMP activity in the aortic tissue extracts. RESULTS: The mean aortic diameter in the control group increased by 126% +/- 14% on day 7 (from 1.57 +/- 0.04 mm to 3.54 +/- 0.27 mm; P <.05), and 5 of 6 animals (83%) had AAAs. Doxycycline appeared to inhibit aortic dilatation in a dose-dependent manner, and AAAs did not develop in any animals. Half-maximal effects were observed at a dose of approximately 6 mg/kg/day, and maximal effects were noted at greater than 30 mg/kg/day. No AAAs were observed in the animals that were treated with CMTs at 15 mg/kg/day. Each of the following CMTs exhibited an efficacy that was similar to that of doxycycline (percent inhibition of aortic dilatation vs control; all P <.05): CMT-3 (47.6%), CMT-4 (38.9%), CMT-7 (47.6%), CMT-8 (54.0%), and doxycycline (51.6%). Tissues from saline solution-treated controls exhibited a transmural inflammatory response and marked destruction of the medial elastic lamellae. Tetracycline derivatives limited the disruption of medial elastin without appearing to alter either the inflammatory response or the rat aortic wall production of metallogelatinases. CONCLUSION: Tetracycline derivatives suppress the development of AAAs after elastase-induced aortic injury in the rat. The aneurysm-suppressing effects of doxycycline appear to be dose-dependent and distinct from its antibiotic activities, and they coincide with the structural preservation of medial elastin fibers. Further studies are needed to explore the potential of MMP-inhibiting tetracyclines as a novel pharmacologic strategy for the suppression of aortic aneurysms.